Precise matching of energy substrate delivery to local metabolic needs is essential for the health and function of all tissues. Here, we outline a mechanistic framework for understanding this critical process, which we refer to as electro-metabolic signaling (EMS). All tissues exhibit changes in metabolism over varying spatiotemporal scales and have widely varying energetic needs and reserves. We propose that across tissues, common signatures of elevated metabolism or increases in energy substrate usage that exceed key local thresholds rapidly engage mechanisms that generate hyperpolarizing electrical signals in capillaries that then relax contractile elements throughout the vasculature to quickly adjust blood flow to meet changing needs. The attendant increase in energy substrate delivery serves to meet local metabolic requirements and thus avoids a mismatch in supply and demand and prevents metabolic stress. We discuss in detail key examples of EMS that our laboratories have discovered in the brain and the heart, and we outline potential further EMS mechanisms operating in tissues such as skeletal muscle, pancreas, and kidney. We suggest that the energy imbalance evoked by EMS uncoupling may be central to cellular dysfunction from which the hallmarks of aging and metabolic diseases emerge and may lead to generalized organ failure states—such as diverse flavors of heart failure and dementia. Understanding and manipulating EMS may be key to preventing or reversing these dysfunctions.

Introduction

From the exquisitely elegant, computationally intense functions of the brain to the error-free pumping of the heart across activity levels, there is a constant requirement that energy is supplied to tissues and waste products are removed in an efficient manner. At the cellular level, the maintenance of ionic gradients across the plasma membrane, protein synthesis and transport, the processes of cell division, growth, and repair (Buttgereit and Brand, 1995), and the diverse operations beyond these are all inextricably tied to the ready availability of local energy substrates. As these substrates are delivered via the bloodstream, it makes all cells in all tissues critically dependent on an optimized vascular function to direct blood to areas of metabolic need at exactly the right moment. Understanding exactly how this task is achieved throughout the body has engaged cardiovascular biologists since the earliest descriptions of the circulatory system (Young, 1929; Harvey, 1957). Now with the advent of modern molecular, electrophysiological, and imaging techniques, substantial progress has been made in determining the structure and function of the vascular system at high resolution (Blinder et al., 2013; Tykocki et al., 2017; Vanlandewijck et al., 2018; Hariharan et al., 2020; Kirst et al., 2020; Jackson, 2022; Meng et al., 2022). Together, these data enable precision measurements of blood flow control spanning the molecular to the systems levels. However, we still have an incomplete understanding of how blood flow through tissues with highly varied energetic demands is mapped to metabolic needs at the organ, local network, and individual cell levels.

The EMS hypothesis

A diverse set of electro-metabolic signaling (EMS) mechanisms operate throughout organ systems to precisely match local energy demand with supply via capillary electrical signaling to tightly control blood flow.

In this article, we set out the concept of EMS as a framework to garner a new understanding of local blood flow control mechanisms, which we have been developing over a number of years (Lederer et al., 1996; Longden et al., 2017; Zhao et al., 2020a). We hypothesize that this provides the core mechanistic link that enables the matching of energy demand to the delivery of increased blood flow across all organ systems (Fig. 1). Here, we outline the theoretical basis of this unifying EMS hypothesis and underscore the technical developments that are needed to accelerate progress in studying this phenomenon. The core predictions made by the EMS hypothesis are:

  • Energy-intensive processes in all organ systems should couple to local hyperemia, as has been established for brain, heart, and skeletal muscle among others.

  • The signaling mechanisms that couple energy usage to blood flow should satisfy the hallmarks of EMS, which we outline below.

  • EMS mechanisms operating in different organ systems should share the same basis (Fig. 1) but may be tailored to suit the particular spatiotemporal energy needs of the local tissue.

  • Uncoupling of EMS should disrupt the matching of energy supply to local demand and thereby may precipitate a range of tissue dysfunction.

We explore these predictions in detail and outline key outstanding questions that need to be addressed to subject this hypothesis to rigorous attempts at falsification.

Energy substrate supply and metabolism

The nanobattery adenosine triphosphate (ATP) stores energy within its hydrolyzable phosphate–phosphate bonds that can be tapped through serial chemical reactions to release energy to power work throughout the cell. These reactions yield adenosine diphosphate (ADP) and subsequently adenosine monophosphate (AMP), which can then be further processed into adenosine. Most often, however, ADP is rapidly phosphorylated back to ATP to recharge the battery. Thus, cellular processes are primarily fueled by the free energy liberated from the transfer of phosphoryl groups from ATP to acceptor molecules. The breaking of the phosphoanhydride bonds between these groups (yielding ADP and inorganic phosphate—Pi—if from ATP) enables the work that undergirds the full gamut of complex cellular behaviors, ranging from motility and contraction to ion transport, secretion, and the diverse reactions in cellular signaling. All these processes are thus reliant on a range of metabolic enzymes and mitochondrial functions, and the preferred carbon source utilized for metabolism on a moment-to-moment basis differs between tissues. In the brain, glucose is most heavily used (Mergenthaler et al., 2013) while the heart often prioritizes lipid sources (Goldberg et al., 2012).

Diverse energy buffers are also available, including glycogen stored in the liver which can be mobilized to generate more glucose on an as-needed basis. Circulating glucose is taken up into cells via facilitated diffusion enabled by glucose transporters (GLUTs) and then catabolized through the subsequent reactions of glycolysis, the tricarboxylic acid (TCA) cycle (Arnold and Finley, 2023), and oxidative phosphorylation (Bonora et al., 2012; Fig. 2). The complete oxidation of a single glucose molecule through these processes is estimated to yield the formation of around 30 ATP molecules, although this number varies between sources and may be modified as further information becomes available (Rich, 2003).

Initially, glycolysis yields two ATP molecules during the hydrolysis of a glucose molecule to two pyruvates. The latter can then enter the TCA cycle in the mitochondria after conversion to the high-energy intermediate acetyl coenzyme A (acetyl-CoA) to produce the coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2), which in turn act as electron donors for the electron transport chain (ETC) in the inner mitochondrial membrane, wherein the flow of electrons through the protein complexes comprising the ETC culminate in the coupling of the electrochemical proton (H+) gradient to oxidative phosphorylation, generating ATP via the activity of ATP synthase (Fig. 2). In a system replete with energy substrates, each mitochondrial ATP synthase produces in excess of 100 ATP per second through this process, which can be increased to >400 ATP per second under catalytically optimal conditions (Matsuno-Yagi and Hatefi, 1988). In intact cells, these processes ultimately maintain a cytoplasmic ATP concentration in the millimolar range (Greiner and Glonek, 2021), which is typically several orders of magnitude higher than that of ADP (free [ADP] in heart tissue has been estimated at 15 µM [Weiss et al., 1992], in the brain at 12 µM [Roth and Weiner, 1991; Tabula Muris Consortium, 2020], and in resting skeletal muscle at 6 µM [Roth and Weiner, 1991]) and orders of magnitude higher still than that of AMP (estimated at 71 nM in brain and 5 nM in muscle [Roth and Weiner, 1991], and 250 nM in heart [Airan et al., 2009]). The TCA cycle is the linkage point for the oxidation of all energy substrates, including fatty acids, lactate, ketone bodies, and amino acids, which are through various mechanisms processed to acetyl-CoA, which is then used to produce ATP through the mechanisms described above (Arnold and Finley, 2023).

Ultimately the substrates and the oxygen (O2) required to power the ETC are delivered in the blood via the plasma and its O2-laden red blood cells (RBCs), respectively, which are directed through the incredibly elaborate vascular networks of all tissues (Box 1). To avoid deficits in energy supply, substrate delivery via the blood needs to be matched precisely to the activity levels of organs and muscle at the most macroscopic levels, all the way down to that of individual cells, while also being rapidly and continually modulated to meet fluctuating local energetic needs. Extensive work has uncovered the molecular mechanisms through which the contractile state of smooth muscle cells (SMCs) in arteries and arterioles is regulated to control perfusion (Segal et al., 1989; Segal and Duling, 1989; McCarron and Halpern, 1990; Brayden and Nelson, 1992; Daut et al., 1994; Knot and Nelson, 1995, 1998; Little et al., 1995a, 1995b; Nelson et al., 1995; Bonev and Nelson, 1996; Dora et al., 1997, 2003; Haas and Duling, 1997; Beach et al., 1998; Edwards et al., 1998; Porter et al., 1998; Welsh and Segal, 1998; Jaggar et al., 2000; Jaggar, 2001; Heppner et al., 2002; Ledoux et al., 2008; Xi et al., 2008; Figueroa and Duling, 2009; Hannah et al., 2011; Mufti et al., 2010; Takeda et al., 2011; Bagher et al., 2012; Dabertrand et al., 2012, 2013; Sonkusare et al., 2012, 2014, 2016; Straub et al., 2012, 2014; Tran et al., 2012; Abd El-Rahman et al., 2013; Gonzales et al., 2014; Harraz et al., 2014; Mercado et al., 2014; Earley and Brayden, 2015; Sullivan et al., 2015; Wilson et al., 2015; Garland et al., 2017; McCarron et al., 2017; Tajada et al., 2017; Lee et al., 2018; Hald and Welsh, 2020; Ottolini et al., 2020; Mironova et al., 2023), which is governed by control of the membrane potential and intracellular Ca2+ levels. More recent work is now revealing the role of the endothelial cells (ECs) of capillaries and their closely associated pericytes calling for blood flow increases and in the fine-tuning of the incoming flow of blood (Beach et al., 1998; McGahren et al., 1998; Hall et al., 2014; Longden et al., 2017, 2021; Harraz et al., 2018a, 2022; Lamb et al., 2018; Gonzales et al., 2020; Moshkforoush et al., 2020; Zhao et al., 2020a; Hartmann et al., 2021; Rosehart et al., 2021; Thakore et al., 2021; Zambach et al., 2021; Hariharan et al., 2022; Klug et al., 2023).

Box 1

Vascular organization and cell types

The vasculature is broadly segmented into arteries/arterioles, capillaries, and venules/veins, which coordinate distinct aspects of blood delivery. Arteries and arterioles are composed of an inner lining of endothelial cells (ECs) that directly interface with the blood to convert luminal signals into a form that can be integrated by the overlying smooth muscle cells (SMCs; Fig. B1). The latter are the key contractile cells that, through actin–myosin crossbridge cycling, determine the diameter of the lumen and thus the flow of blood flow into the capillaries. Control of SMC membrane potential and intracellular Ca2+ are central to this process.

Figure B1.

Vascular organization and cell types. (A) Scanning electron micrograph (SEM) of a bifurcating arteriole covered in contractile smooth muscle cells (SMCs; individual cells denoted in orange). Scale bar: 50 µm. (B) SEM of a contractile pericyte and its processes (pink) enwrapping a capillary. Note the clear distinction from SMC morphology. Scale bar: 11.5 µm. A and B adapted with permission from Rodriguez-Baeza et al. (1998). (C) SEM of brain capillaries showing the sequential branch ordering of vessels up to the fifth order (green). Note this system is not perfect, as some anastomosing vessels could be labeled as two distinct orders (*). Scale bar: 50 µm. Adapted with permission from Krucker et al. (2006). (D) SEM of the choroid vasculature showing lobular vascular architecture which makes it impossible to accurately assign branch orders (e.g., green vessels). 65× magnification. Adapted with permission from Miodoński and Jasiński (1979).

Figure B1.

Vascular organization and cell types. (A) Scanning electron micrograph (SEM) of a bifurcating arteriole covered in contractile smooth muscle cells (SMCs; individual cells denoted in orange). Scale bar: 50 µm. (B) SEM of a contractile pericyte and its processes (pink) enwrapping a capillary. Note the clear distinction from SMC morphology. Scale bar: 11.5 µm. A and B adapted with permission from Rodriguez-Baeza et al. (1998). (C) SEM of brain capillaries showing the sequential branch ordering of vessels up to the fifth order (green). Note this system is not perfect, as some anastomosing vessels could be labeled as two distinct orders (*). Scale bar: 50 µm. Adapted with permission from Krucker et al. (2006). (D) SEM of the choroid vasculature showing lobular vascular architecture which makes it impossible to accurately assign branch orders (e.g., green vessels). 65× magnification. Adapted with permission from Miodoński and Jasiński (1979).

Close modal

In a number of vascular beds, capillaries are typically referred to by “branch order” in which the final SMC-covered arteriole is taken as the zero-order vessel, and the initial capillary emanating from this is the first-order capillary. From here, branch numbering increases sequentially each time the capillaries branch, regardless of vessel orientation or diameter. However, this classification system is difficult to apply in vascular beds in which the capillaries are highly anastomosing, such as heart, where this hierarchical ordering quickly breaks down by dint of the many reciprocal connections between adjacent capillaries (Longden et al., 2023; Fig. B1). The capillaries are composed of exceptionally thin (∼100 nm) ECs that wrap around themselves once to form a lumen just wide enough to allow RBCs to squeeze through in single file, experiencing significant mechanical forces as they go, and are covered by the cell bodies and processes of a range of pericytes.

Using the brain as an example, at least three pericyte subtypes have been distinguished based on their locations in the capillary bed, their relative expression of α-smooth muscle actin (α-SMA), and the appearance of their processes, designated ensheathing (or contractile) pericytes, mesh pericytes, and thin-strand pericytes (Grant et al., 2019; Gonzales et al., 2020; Hariharan et al., 2020; Ratelade et al., 2020). Contractile pericytes (Fig. B1) cover the majority of the parenchyma-facing surface of the first to approximately the fourth-order capillary ECs found immediately downstream of SMC-enwrapped penetrating arterioles and heavily express α-SMA but have a distinct morphology to SMCs. Mesh pericytes are found immediately adjacent to the last contractile pericytes of a given capillary offshoot and exhibit intermediate levels of vessel coverage and are α-SMA-negative. Finally, thin-strand pericyte (Hariharan et al., 2020) coverage begins at approximately fifth-order branches and continues until the capillaries merge with the venular system (Grant et al., 2019). Each thin-strand pericyte has a unique domain of extensive, exquisitely fine processes that stretch along the capillary surface for up to ∼300 µm (Berthiaume et al., 2018). Compared with contractile pericytes, thin-strand pericytes express very low levels of α-SMA (Alarcon-Martinez et al., 2018; Vanlandewijck et al., 2018), suggesting that their contributions to blood flow control mechanistically, in contrast with the rapid and dynamic regulation of the diameter of the underlying capillary orchestrated by their upstream contractile counterparts (Hill et al., 2015; Gonzales et al., 2020; Zambach et al., 2021). Indeed, recently, the contractile abilities of thin-strand pericytes were explored using optogenetic actuators and were revealed to be comparatively slower and smaller in magnitude, yet still capable of exerting a significant influence on blood flow (Hartmann et al., 2021). Detailed studies of pericyte morphology and organization in other tissues are currently awaiting investigators’ attention.

RBCs transit the capillary bed, releasing their oxygen to the tissue as they go and are accompanied by nutrient-rich plasma from which energy substrates are extracted and these are drained back toward the heart for recirculation initially in small venules and then in larger veins. The veins consist of the same components as the arterioles, but here the smooth muscle is disorganized and non-contractile (Hill et al., 2015), and as such, these vessels do not substantially change their diameter. In most tissues, luminal valves prevent the backflow of blood and pooling but these are absent from the brain where gravity assists drainage.

The milieu and signaling environments of the various organ systems differ vastly throughout an organism, and thus the question arises of how the continuous body-wide arterio–capillary–venous network correctly integrates the thousands of data streams arising from the diverse activities of local cells with its relatively limited toolkit of protein sensors and actuators. Here, we propose that the solutions to this problem lie in a set of sophisticated yet generalized signaling mechanisms that ultimately convert increases in local energy demand into electrical signaling throughout the vasculature and take center stage in matching blood flow to metabolic needs in all tissues—a process we refer to as electro-metabolic signaling or EMS (Fig. 1). We outline this concept in detail and provide examples which we have elucidated in the brain and the heart, along with key experimental hallmarks that can be used to implicate this process in other tissues. We explore possibilities for the breakdown of EMS, which may play an important role in a wide range of disease contexts that involve mismatches between energy supply and demand, and we detail the essential questions that need to be addressed to build a complete view of the mechanistic landscape of EMS.

KATP channels are the key EMS transducers linking the cellular metabolic state to an electrical signal

Central to EMS is the conversion of metabolic information into electrical information that can be propagated through the “wires” of the vasculature, and ATP-sensitive potassium (K+; KATP) channels are uniquely suited to perform this task. Accordingly, we review below the key biophysical features of these transducers, with a central focus on the vascular isoform of this channel which makes an important contribution to EMS in the brain, before discussing mechanisms through which KATP channels may be engaged in response to changing the metabolic state of the tissue.

KATP channels lie at the center of the EMS signaling network and serve as the primary transducer that ties the internal cellular metabolic state to the membrane potential. These channels thus can convert changes in the local metabolic environment into electrical messages that can be transmitted over long distances (typically with the aid of Kir2.1 channels, as detailed below) to the contractile cells of the vasculature (i.e., SMCs, and the contractile pericytes of the proximal regions of the capillary bed; Box 1).

The pore-forming subunits of KATP channels are members of the Kir channel family (Inagaki et al., 1995; Hibino et al., 2010). Currents through this family of channels depend on the driving force for K+ (i.e., membrane potential [Em] minus EK). Accordingly, when Em is experimentally held at voltages negative to EK (which does not occur under physiological conditions), Kir channels conduct large, ohmic inward currents, but when Em is positive to EK (as in healthy physiological conditions), outward currents with varying degrees of inward rectification are observed according to the channel subtype, causing the current–voltage relationship to deviate from ohmic linearity. Rectification ranges from strong, in which only a small amount of current flows from the interior of the cell to the exterior at potentials positive to EK—as is the case for Kir2.1 channels which feature prominently in electrical conduction, described below—to weak, where rectification begins only at very positive potentials, tens of millivolts from EK, as is the case for KATP channels (Hibino et al., 2010).

The weakly rectifying current–voltage profile of KATP channels results mainly from intracellular Mg2+ block at positive potentials (more positive than approximately −20 mV [Horie et al., 1987]). The key property of these channels is that they operate as exquisite molecular transducers coupling complex changes in intracellular energy state to membrane electrical activity. This stems from the unique sensitivity of KATP channels to the ratio of intracellular ATP to ADP, being initially named for the fact that when ATP levels increase, their K+ conductance is inhibited (Noma, 1983). The basic structure of a functional KATP channel is a hetero–octameric complex composed of four Kir6.x subunits (either Kir6.1 or Kir6.2, encoded by KCNJ8 and KCNJ11, respectively) and four sulphonyl urea receptor (SUR) subunits (SUR1 or SUR2, encoded by ABCC8 or ABCC9, respectively; Clement et al., 1997; Inagaki et al., 1997; Shyng and Nichols, 1997). Different Kir6.x/SURx subunit combinations create KATP channels with differing nucleotide sensitivities and biophysical characteristics. The resultant diversity of KATP channels are widely expressed in cardiac myocytes (Noma, 1983), pancreatic β-cells (Cook and Hales, 1984), skeletal muscle (Spruce et al., 1985), neurons (Liss and Roeper, 2001; Ballanyi, 2004; Martínez-François et al., 2018), as well as vascular smooth muscle (Standen et al., 1989; Beech et al., 1993; Sancho et al., 2022), endothelial cells (ECs; Aziz et al., 2017; Sancho et al., 2022), and pericytes (He et al., 2018; Vanlandewijck et al., 2018; Hariharan et al., 2020, 2022; Sancho et al., 2022).

The most well-studied KATP isotype in pancreatic β-cells is formed by Kir6.2 and SUR1 subunits (Noma, 1983; Inagaki et al., 1995). In contrast, the “vascular” form of the KATP channel found in SMCs, ECs, and pericytes is formed from Kir6.1 and SUR2 (Nelson and Quayle, 1995; Seino and Miki, 2003; He et al., 2018; Vanlandewijck et al., 2018). SUR2 undergoes a range of alternative splicing events (Chutkow et al., 1999), which modulate the nucleotide-binding properties and pharmacology of the channel in its final form (Reimann et al., 2000). The variant known as SUR2B, resulting from the excision of the final 129 coding base pairs plus 48 from the 3′ untranslated region, is thought to be the predominant isoform found in the vasculature (Chutkow et al., 1999). In the heart, a combination of Kir6.2/SUR2A gives rise to KATP channels that have distinct properties from those in the vasculature and other locations. A number of recent studies have solved the structures of several KATP channel types at high-resolution by leveraging advances in single particle cryo-electron microscopy (cryo-EM; Cheng, 2015; Cheng et al., 2015), including a hamster SUR1–mouse/rat Kir6.2 hybrid complex and a human SUR1–Kir6.2 complex in both closed and open states (Lee et al., 2017; Li et al., 2017; Martin et al., 2017; Zhao and Mackinnon, 2021). Recently, Sung et al. (2021) provided the first cryo-EM structures for the vascular Kir6.1/SUR2B KATP channel in the presence of ATP and bound by the inhibitory compound glibenclamide. This study determined that the architecture of the vascular isoform of the channel exhibits key structural and conformational features that are not observed in pancreatic KATP channels prepared under similar conditions (Martin et al., 2017; Wu et al., 2018), such as a displaced cytoplasmic domain as detailed below, which may confer distinct properties at the functional level that influence how these different channel subtypes contribute to EMS.

Both Kir6.1 and Kir6.2 subunits are composed of two transmembrane helices bridged by an extracellular loop, which in tetrameric assemblies generates the narrow portion of the pore that comprises the highly conserved K+ selectivity filter, accompanied by cytoplasmic N- and C-terminal domains (Nichols, 2006). The hallmark feature of KATP channels is their regulation of Em in response to changes in intracellular nucleotide concentrations (Table 1), which bind at several locations throughout the channel complex. Micromolar ATP inhibits KATP channels through a direct interaction with the Kir6.x subunit. Residues R50 and K185 are essential for this ATP inhibition in Kir6.2, and these positive charges are conserved at equivalent positions in Kir6.1, but absent in most other ATP-insensitive Kir family members (Trapp et al., 2003).

Because cytosolic intracellular ATP concentration is relatively high under physiological conditions, typically in the range of 1–2 mM in SMCs (Larcombe-McDouall et al., 1999; Gribble et al., 2000) and 5–10 mM in cardiac myocytes (Greiner and Glonek, 2021), this can exert a powerful suppressive influence on basal channel activity and is counterbalanced by the influences of other interacting molecules and signaling modifications outlined below (Huang et al., 2019). Kir6.2 variants typically exhibit higher free ATP sensitivity than Kir6.1-containing channels (Table 1), and the recent structure provided by Sung and colleagues reveals subtle differences that may contribute to this distinction. In Kir6.1-containing channels, the cytoplasmic domain of the pore-forming subunit is oriented ∼5.8 Å further away from the membrane and rotated counterclockwise relative to its Kir6.2 counterpart, allowing for fewer close residue interactions with ATP than are seen in the Kir6.2 ATP binding site. This <1 nm difference in cytoplasmic domain positioning is thus a possible contributory factor to the lower ATP sensitivity of the vascular KATP isoform (Yamada et al., 1997; Satoh et al., 1998).

The large accompanying SUR subunits are members of the ATP-binding cassette (ABC) membrane protein family and have 17 TM regions (Fig. 3). These regions are grouped into three transmembrane domains: TMD0 (comprising TM1–TM5), TMD1 (TM6–TM11), and TMD2 (TM12–TM17). TMD0 is bound to the channel α subunit via a bridging cytosolic loop referred to as L0, which plays a crucial role in channel gating and activity, likely by transmitting signals to the pore-forming subunit through conformational rearrangements (Babenko and Bryan, 2003; Masia et al., 2007; McClenaghan et al., 2018). Each SUR subunit contains two nucleotide-binding domains (NBDs): one between TMD1 and TMD2 (termed NBD1) and another in the C-terminus following TMD2 (termed NBD2; Hibino et al., 2010; Lee et al., 2017; Li et al., 2017; Martin et al., 2017). These NBDs are key sites for nucleotide regulation and allow the interactions of magnesium (Mg2+)-bound adenosine diphosphate (Mg-ADP) and Mg-ATP (as well as other Mg-nucleotides) to produce a key stimulatory effect on KATP channel activity through NBD dimerization resulting in channel opening. Here, Mg-ATP binding to NBD1 in conjunction with Mg-ADP binding to NBD2 promotes dimerization, which leads to channel opening through conformational changes mediated via a structural element connecting NBD1 to TMD2 known as the N1–T2 linker. At its C-terminal end, this linker contains 15 glutamate/aspartate residues, forming a region known as the ED domain. Molecular dynamics analyses suggest that when MgADP is absent, electrostatic interaction between NBD2 and the Kir6.1 cytoplasmic domain acts as an autoinhibitory motif, preventing NBD2 dimerization with NBD1 and avoiding unregulated activation—consistent with low spontaneous activity observed for the vascular form of KATP channels (Fujita and Kurachi, 2000). The binding of MgADP to NBD2 alters the shape of the ED domain, a reorganization that allows the rotation of NBD2 toward NBD1 and thus promotes dimerization and the transmission of a conformational change that opens the channel.

A striking difference between the cardiac and vascular forms of the KATP channel lies in the contrasting sensitivities of channel activity to the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). In Kir6.2-containing KATP channels (Hilgemann and Ball, 1996; Baukrowitz et al., 1998), the presence of PIP2 decreases the affinity of the channel for ATP >100-fold and dramatically increases channel open probability in the absence of nucleotides (Shyng and Nichols, 1998). In contrast, the Kir6.1 form of the channel possesses a higher affinity for PIP2, with PIP2 binding so tightly as to render channel activity insensitive to physiological fluctuations of this lipid, and it has thus been suggested that this higher affinity may underlie the more stable openings characteristic of vascular KATP channels (MacGregor et al., 2002; Quinn et al., 2003). In the context of EMS, these observations collectively suggest that the mechanisms that engage KATP channels in cells expressing Kir6.2-containing KATP channels, such as cardiac myocytes, may differ from Kir6.1-expressing vascular cells. Indeed in the heart, with its exceptionally high intracellular ATP levels (∼5–10 mM [Greiner and Glonek, 2021]), modulation of membrane PIP2 levels might play an important role in creating the conditions for KATP channel engagement. In the vasculature, by contrast, PIP2 concentration-related contributions are much less likely, and it may be that changes in ATP:ADP ratio come to the fore in this instance, or that other modulatory events such as coincident channel phosphorylation are important for KATP activation.

In this vein, a stimulatory effect of protein kinase A (PKA) has been observed for the activity of Kir6.1-containing channels. PKA is activated by membrane Gs-coupled GPCRs that convert ATP into cyclic AMP via adenylyl cyclase, which in turn promotes PKA engagement. PKA phosphorylation of multiple sites (Ser-1351, Ser-1387) on the pore-forming and regulatory subunits of the KATP channel is known to produce a profound increase in channel open probability (Shi et al., 2007). Similarly, PKG promotes KATP activation in the neuronal (Kir6.2/SUR1) and cardiac (Kir6.2/SUR2A) forms of the channel (Han et al., 2001; Chai and Lin, 2010; Chai et al., 2011), and vascular (Kir6.1/SUR2B) KATP channels can also be engaged to generate membrane hyperpolarization by stimuli activating guanylyl cyclase-cGMP signaling (Kubo et al., 1994; Murphy and Brayden, 1995), which acts through PKG. In contrast, several consensus protein kinase C (PKC) sites in the distal C-terminus of Kir6.1 have been identified that mediate KATP inhibition. PKC is activated downstream of diacylglycerol (DAG), which is produced as a result of GqPCR engagement and PIP2 hydrolysis. Therefore, activation of any of the plethora of GqPCRs that are expressed in pericytes (Hariharan et al., 2020) or cardiac myocytes (Litviňuková et al., 2020) and other tissues is predicted to inhibit KATP channel activity in the cell. When the serine residues that are the targets of PKC phosphorylation (Ser-354, Ser-379, Ser-385, Ser-391, and Ser-397) are mutated individually, moderate reductions in PKC inhibition are observed, whereas combined mutation of these residues produces greater effects, implying that the degree of phosphorylation of KATP channels by PKC produces a graded inhibition (Shi et al., 2008), allowing fine-tuning of activity.

In addition to these canonical signaling events, a number of other modifications are known to affect KATP channel trafficking and activity, including glycosylation (Conti et al., 2002), nitrosylation (Kawano et al., 2009), myristoylation (Lu et al., 2006), and palmitoylation (Yang et al., 2020). Combined, these modes of KATP regulation represent a rich and diverse molecular toolkit for modifying KATP channel activity and density to influence the membrane potential of the cell in response to a range of extracellular signals and across broad spatiotemporal domains.

Kir2 channels are central to transmission of electrical signals

Strong inward-rectifier K+ channels of the Kir2 family are widely expressed and in the vasculature are functionally expressed in the plasma membranes of capillary (Longden et al., 2017; Harraz et al., 2018a, 2018b) and arteriolar (Ahn et al., 2017; Sonkusare et al., 2016) ECs and in SMCs (Longden et al., 2014; Sonkusare et al., 2016). Three Kir2 channel subtypes (Kir2.1–2.3, encoded by KCNJ2, KCNJ12, and KCNJ4) have been identified and share similar structural and biophysical features, including their near complete block by intracellular Mg2+ and polyamines as Vm is progressively depolarized from EK, and sensitivity to blockade by external barium (Ba2+) ions (Hibino et al., 2010). The crystal structure of Kir2.2 (Tao et al., 2009) and more recently a cryo-EM structure of Kir2.1 (Fernandes et al., 2022) have been solved, revealing stunning details of these channels (Fig. 3).

Similar to the pore-forming region of the KATP channels described above, Kir2 channels are formed of tetramers of α subunits, each of which has two transmembrane helices (termed M1 and M2) that are connected via pore-forming loops and intracellular N- and C-termini and lack any accompanying accessory subunits (Fig. 3). The interaction of these channels with PIP2 is critical for their activity, and readers are referred to extensive discussion of PIP2 as a key modulator of ion channel activity in elegant recent works for more detail (Hille et al., 2015; Harraz et al., 2020). PIP2 binds to the Kir2.1 channel with a KD of ∼3 µM, and it has been proposed that such binding leads to a conformational change in a conserved 15 amino-acid cytoplasmic motif known as the “G-loop” that leads to channel gating (Fernandes et al., 2022).

The other major interaction of Kir2 channels that is essential for understanding their function is their intracellular block by polyamines and Mg2+ ions which underlies their strongly rectifying behavior. This has been attributed to two negatively charged regions of the pore around residue D172 in the M2 helix (Lu and MacKinnon, 1994) and the E224 and E299 residues close to the G-loop (Kubo and Murata, 2001). The rings of negative charge that these residues impart to the channel pore attract positively charged blocking particles, which in turn is thought to impair K+ permeation by hindering ion access to the selectivity filter (Fernandes et al., 2022). As this blockade by polyamines and Mg2+ depends on voltage, it can as a matter of course be relived through membrane hyperpolarization toward EK. Hyperpolarization progressively lowers the probability that the pore is occluded and thereby permits K+ exit from the cell down the electrochemical gradient to generate outward hyperpolarizing K+ currents. Accordingly, there is a self-amplifying effect inherent to Kir currents wherein the outward currents permitted by hyperpolarization generate further hyperpolarization and thereby promote further relief of voltage-dependent channel blockade (Longden and Nelson, 2015). A similar effect can be achieved through the elevation of external K+, which causes a rightward shift in the current–voltage profile of the channel. In the context of the vasculature, where resting Vm is between −30 and −40 mV, this rightward shift allows for relief of the voltage-dependent block of the channel by promoting the relief of the channel block at these potentials. We and others have extensively reviewed the biophysical properties of Kir2 channels, and thus readers are directed to these articles for further information on these topics (Hibino et al., 2010; Longden and Nelson, 2015).

We argue below that the outward currents through Kir2 channels generated by membrane hyperpolarization, alongside their K+ sensitivity, are critical for signal transmission from capillaries during EMS.

The hallmarks of EMS

At its core, the input–output pattern of EMS is reflected in the conversion of the level of local metabolic demand in a tissue into electrical signaling throughout the vasculature (Fig. 1). Below, we discuss three key stages that define this phenomenon: (1) the generation of metabolic signals and the array of possible forms they may take; (2) the sensing and transduction mechanisms that generate vascular electrical signals, along with the propagation mechanisms for these signals from capillaries to arterioles; and (3) the homeostatic adjustment of blood flow to deliver energy substrates to satisfy local metabolic needs.

Stage I: Elevated metabolic activity is the initiating input to EMS

The first hallmark of EMS is that it is driven by an elevation of metabolic demand in the tissue (Fig. 1). This naturally results in an array of complex intra- and extracellular modifications which may act as triggering factors to initiate the adaptive blood flow changes provided by EMS. Given the wide range of potential energy sources and the enormous complexity of the cell’s metabolic pathways, there exists a vast array of biochemical signatures that could act as the initiation event for EMS in different tissues. Here, we focus our discussion on currently known EMS signals and exciting unexplored possibilities.

Decreased substrate availability as an EMS trigger

Perhaps operating most directly as an initiating factor, a decrease in local energy substrate availability due to uptake or depletion during activity may act as a primary driver of EMS. Indeed, we recently demonstrated that this is the case for brain glucose, where capillary pericytes sense and respond to local glucose availability (Hariharan et al., 2022), and similar mechanisms may exist for other energy substrates.

Many cells express glucose transporters (GLUTs) to directly take up glucose from their environment. GLUTs are facilitative transporters that utilize the gradient of glucose to direct it to locations of relatively low concentration and have varying kinetics and affinities which can be modulated by intracellular signaling pathways, such that glucose uptake can be modified in an activity-dependent manner (Thorens and Mueckler, 2010). For example, the membrane density of GLUT4 proteins can be rapidly adjusted in response to signaling events. In muscle and fat cells, GLUT4 is under the direct control of insulin signaling, the elevation of which results in rapid membrane insertion of reserve pools of this transporter stored in submembrane vesicles, thereby rapidly augmenting the glucose import capacity of the cell (Stöckli et al., 2011). Increasing glucose import via such mechanisms can lead to an increase in the breakdown and oxidation of glucose by active cells, and this can in turn rapidly change local external glucose concentrations.

Brain regional glucose has been observed to fall ∼30% from its resting level of ∼1 mM during a maze task (McNay et al., 2001), presumably due to its rapid uptake by neurons (Lundgaard et al., 2015; Li et al., 2023) or local astrocytes (Pellerin and Magistretti, 1994; Rouach et al., 2008), with onset on the order of seconds (Gold and Korol, 2012). Interestingly, in aged animals, depletion of local glucose during the same task is even more extreme, reaching a nadir of ∼50% below resting levels during task performance (McNay and Gold, 2001), which could further increase metabolic strain on highly active neurons. In younger animals, the drop in glucose is typically sustained for some time during task performance and then corrected back to baseline levels (McNay et al., 2001), suggesting perhaps that the initial dearth of glucose could act as a signal to the vasculature to indicate local metabolic activation and prompt the initiation of EMS to correct the supply deficit.

Tight control of local glucose is essential in the brain, which operates as an on-demand system relying almost exclusively on incoming glucose transported across the blood–brain barrier by the heavily expressed GLUTs, in particular the GLUT1 isoform (Deng et al., 2014), located in the endothelium and astrocytic endfeet (Mergenthaler et al., 2013). A prevailing view is that glucose is taken up into astrocytic endfeet during neuronal activity via GLUT1 transporters prompted by glutamatergic signaling and glutamate uptake at the tripartite synapse, and this promotes glycolysis leading to the generation of pyruvate. This generated pyruvate is then converted to lactate (by lactate dehydrogenase [LDH] 5) in astrocytes, which is passed via monocarboxylate transporters (MCTs 4 and 1) into the extracellular space, and MCT2 on neurons then permits its uptake into these cells. It is suggested that the lactate accumulated in neurons is then converted by resident LDH1 into pyruvate which can enter the TCA cycle to ultimately generate ATP via oxidative phosphorylation (Mergenthaler et al., 2013). This mechanism has been termed the astrocyte-neuron lactate shuttle (ANLS). However, recent studies have questioned the extent to which neurons depend on astrocytes to provide lactate as a fuel via this mechanism, as opposed to taking up glucose directly from their immediate environment and processing this with their own metabolic machinery (Chuquet et al., 2010; Lundgaard et al., 2015). Indeed, GLUT3, which has a higher glucose transport rate than GLUT1 (Simpson et al., 2008), is expressed by neurons to enable their direct uptake of glucose from the surrounding parenchyma, and this has led to a counterview in which neurons process their own glucose for glycolysis and oxidative phosphorylation (Patel et al., 2014; Lundgaard et al., 2015; Peng et al., 2021), in the process releasing lactate into the extracellular space (Dienel, 2012). Further work is needed to establish exactly how glucose is handled by these two cell types.

The brain also maintains a diminutive reservoir of glycogen (Choi and Gruetter, 2003; Öz et al., 2003, 2015; DiNuzzo, 2019), which accounts for a low percentage of total glucose metabolism during normal operations and is at least 20 times smaller than reserves stored in muscle, and 50–100 times lower than that sequestered in the liver (Öz et al., 2007; Obel et al., 2012). Thus, while the brain appears to depend directly on local glucose on an as-needed basis, in systems with significant glycogen reserves these may be mobilized as glucose levels fall to provide sustained buffering for energy production, and thus EMS may turn on over a longer and more gradual time scale or may depend on other triggering factors.

Intriguingly, several GLUT isoforms are expressed directly in SMCs and pericytes, including transcripts for GLUT1, GLUT3, and GLUT4 (He et al., 2018; Vanlandewijck et al., 2018). The relative activity of these transporters could influence the sensitivity of EMS mechanisms embedded in the vasculature. For example, if GLUT4 transporters are active in the membrane as opposed to sequestered in reserve pools this could reduce the sensitivity of vascular EMS mechanisms to glucose fluctuations by maintaining higher intracellular ATP levels. These possibilities for tuning EMS and their consequences for blood flow control are intriguing avenues for future investigation.

Cytosolic ATP as a driver of EMS

The major mechanism through which the energy substrates that are taken into a cell are converted into ATP is mitochondrial oxidative phosphorylation. This process will set the bulk ATP level in the cytosol, which is typically within the millimolar range, as noted above. Although many mechanisms exist to protect and maintain cytosolic ATP concentrations, it is possible that subtle fluctuations in bulk ATP could drive EMS via KATP channel activity.

The organization, function, and regulation of KATP channels are likely to reflect the EMS mechanisms deployed by a given cell type. At one extreme are the cardiac ventricular myocytes, wherein the KATP channel density is extremely high (∼10 channels/µm2, corresponding to around 50,000 channels per cell; Nichols and Lederer, 1990). This high expression of the KATP channel is appropriate for the 5–10 mM cytosolic ATP concentration estimated from NMR data (Greiner and Glonek, 2021) when considered alongside the low IC50 for ATP at the cardiac isoform of the KATP channel (∼21–100 µM; Table 1; Foster and Coetzee, 2016). Having thousands of KATP channels in the membrane will increase sensitivity to small dips in ATP, which will likely increase their open probability only marginally. Given the large single channel current (∼80 pS; Foster and Coetzee, 2016), a single channel opening is capable of deflecting the membrane potential substantially, and thus this could thus tightly couple small changes in ATP to EMS mechanisms in the heart.

Similar measurements are needed to inform our understanding of the possibilities for KATP channel control throughout the vasculature in pericytes, SMCs, and ECs (and of course also in other cell types expressing KATP channels), as channel membrane density and intracellular ATP levels and dynamics are thought to be key parameters that contribute to EMS control mechanisms. For example, at the opposite extreme to cardiac myocytes would be a small cell working as a metabolic sentinel to influence local blood flow by directly modulating electrical activity in the vascular system, like capillary pericytes in the brain (Hariharan et al., 2022). Such cells would benefit from having local intracellular machinery to make and control ATP directly around the channel in a submembrane region of the cell. Very tight coupling between extracellular substrate availability and submembrane ATP would thereby allow tight and responsive control of the KATP channel to rapidly produce hyperpolarizing electrical signals, a concept we explore in more detail below.

Glycolytic-KATP channel complexes: Potential EMS signaling microdomains responding to localized changes intracellular energy status

Upon entering the cell cytosol glucose is broken down via a series of 10 enzymatic reactions to produce ATP, pyruvate, and NADH. Intriguingly, a range of glycolytic enzymes, including aldolase A, pyruvate kinase, triosephosphate isomerase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been found to be physically associated with KATP channels in cardiac membranes in studies using a combination of two-hybrid screening, GST pulldown and immunoprecipitation (Dhar-Chowdhury et al., 2005; Jovanović et al., 2005; Hong et al., 2011). More recently, a glycolytic metabolon regulating the KATP channel has been described in pancreatic membranes (Ho et al., 2023). These close associations may provide a direct means through which highly localized metabolism of glucose in the submembrane environment could be directly coupled to KATP activity.

Such local organization to enable compartmentalized communication could be critically important for tight control of KATP activity, as even during vigorous activity global ATP levels are closely safeguarded (Balaban, 1986) and thus the global concentration change in a typical cell is minimal. Many intracellular homeostatic mechanisms have evolved to preserve this high concentration of available intracellular ATP. Examples abound: creatine kinase, found in skeletal and heart muscle, in the brain, and in the mitochondria of various tissues, plays an important role in producing creatine-phosphate when ATP demands are low, which acts as an immediately available energy reservoir that can be drawn upon to generate ATP from ADP during periods of intense work (Yaniv et al., 2010; Metallo and Vander Heiden, 2013); phosphofructokinase-1 (PFK-1), a rate-limiting enzyme that performs the third reaction in the glycolytic chain (Jenkins et al., 2011), is allosterically controlled—when ATP is replete, PFK-1 is inhibited, but as energy availability falls within the cell and ADP and AMP levels rise its activity is promoted with the effect of buffering ATP levels by modulating glycolytic rate which can be tuned over a 100-fold range (Voet et al., 2016). There are many further examples of regulatory processes that operate to protect ATP availability, and as such it is unlikely that a decrease in intracellular ATP per se operates widely as a trigger of EMS mechanisms. (Although, note the example of the high density of KATP channels in cardiac membranes in the preceding section, which offers a simple emergent solution to the problem of sensing subtle changes in ATP.)

As intracellular ADP and AMP levels are typically several orders of magnitude below ATP in a cell at rest, their relative variance is much more dramatic during cellular activity. Indeed, as working muscle ramps its ATP turnover rate, ADP levels have been estimated to increase as much as fivefold (Allen et al., 1997) and AMP levels even more so (McConell et al., 2020). The combination of relatively stable ATP levels as a backdrop against substantial relative changes in ADP thus decreases the ATP:ADP ratio—a key intracellular energy parameter that influences a wide range of intracellular metabolic activities which may be more likely to be a key EMS cue. Recently, the development of a highly sensitive reporter of ATP:ADP ratio, PercevalHR (Tantama et al., 2013), has allowed for the first single-cell resolution observations of changes in this metabolic indicator. Intriguingly, this sensor revealed that decreases in external glucose around neurons within the physiological range result in a substantial decrease in ATP:ADP ratio. Electrical activity was also directly linked to changes in ratio, and these were directly linked to hyperpolarization of the membrane through the engagement of KATP channels (Tantama et al., 2013). As such, activity-induced changes in ATP:ADP ratio primarily resulting from relative changes in ADP concentrations likely represent a more labile metabolic parameter than bulk ATP that can be rapidly converted into robust electrical activity in cells expressing KATP channels, which could be a central trigger for blood flow increases in EMS. In vascular cells expressing KATP for example, it is anticipated that the ATP:ADP ratio will tilt in favor of channel activation (i.e., higher ADP, lower ATP) during local substrate depletion in the tissue to generate electrical signals and promote hyperemia.

Returning to the observation that KATP channels physically associate with glycolytic machinery, the substrates of aldolase and pyruvate kinase were both shown to suppress KATP channel activity in excised membrane patches (Hong et al., 2011), and these observations are buttressed by similar findings in vesicles containing KATP channels derived from small intestine epithelial cells (Dubinsky et al., 1998). It thus follows that the glycolytic machinery of the cell may constitute part of a macromolecular signaling complex organized around KATP channels that tightly regulates their activity. A decrease in glycolysis could trigger a rapid increase in microdomain ADP around the channel (and potentially a fall in submembrane ATP), which could increase KATP activity. Accordingly, the KATP channel in certain situations may be set up to sense a functionally compartmentalized ATP:ADP ratio in the submembrane region around the channel, as opposed to that of the bulk cytoplasm. Such a privileged environment would presumably serve to amplify subtle changes in metabolism and enable the KATP channel to be rapidly responsive. A number of studies also suggest that the sodium/potassium (Na+/K+) ATPase is associated with this membrane complex of glycolytic enzymes and KATP channels, which may add a further dimension to this organization (Urbach et al., 1996; Schultz, 1997; Dubinsky et al., 1998; Sepp et al., 2014): ATP from microdomain glycolysis could fuel pumping activity to restore Na+ and K+ levels after membrane potential deviations. Such increases in Na+/K+ ATPase activity would be expected to affect the local ATP:ADP ratio and increase KATP channel activity. While it is yet to be determined whether such highly localized glycolytic-KATP channel complexes contribute to EMS, this is an intuitive and appealing possibility.

Beyond glucose, variations in the availability of other energy substrates could trigger EMS through similar principles but the involvement of distinct molecular players. The heart, in contrast to the brain, has been described as “omnivorous,” in that it generates ATP from an array of substrates (Wende et al., 2017). Under normal conditions in healthy adults, free fatty acid oxidation takes center-stage in heart metabolism, generating acetyl-coA, which is then utilized to produce ATP via oxidative phosphorylation. In contrast, catabolism of glucose makes only a small contribution to cardiac energy under working conditions (e.g., only 5% of ATP is produced by glycolysis), but this balance may shift when the heart is under stress. Further contributions to heart metabolism are provided by amino acids, ketone bodies, and circulating lactate (Ritterhoff and Tian, 2017).

The kidney also has incredibly high energy demands, turning over large amounts of ATP to enable its roles in filtering waste from the blood, reabsorbing nutrients, and regulating pH and blood pressure. This organ exhibits regional dependence on energy substrate preference, with the proximal tubules in the renal cortex relying on the oxidation of free fatty acids to generate large amounts of ATP to accommodate the high level of water and solute transport occurring at this location (indeed, the complete β-oxidation of a fatty acid molecule is more energetically lucrative than glucose oxidation—for example, the 16-carbon fatty acid palmitate will yield 106 ATP versus ∼32 ATP per glucose molecule). The lower O2 environment of the medulla and distal tubules relies instead on glucose for glycolysis (Alsahli and Gerich, 2017; Bhargava and Schnellmann, 2017). These variances in energy preferences across organs, locally within tissues, and under different circumstances thus raise the possibility that fluctuations in local availability of a range of energy substrates may alter intracellular energy status to then activate EMS in a substrate-tailored and location-dependent manner.

Metabolically driven release of mediators

Increased metabolic activity may also drive the release of canonical vasodilatory mediators from active tissue. These may include (but are not limited to) adenosine, which is a direct product of the breakdown of ATP via ADP and AMP and is released in the brain (Wall and Dale, 2008; Wu et al., 2023), skeletal muscle (Hellsten et al., 1998), and heart during increased activity (Hori and Kitakaze, 1991); K+ which, while not a direct metabolic byproduct, is released during action potentials and thus accurately reflects local activity in electrically active tissues (Ballanyi et al., 1996; Rasmussen et al., 2019); and NO, a diffusible gas which is produced in response to Ca2+ elevations in metabolically active tissues such as the brain (Garthwaite, 2019) and skeletal muscle (Stamler and Meissner, 2001). The documentation of metabolically driven mediators is underway in a range of tissues, and a full accounting of these will be necessary to gain a complete understanding of the diverse EMS mechanisms throughout tissues, several of which may operate in parallel to ensure the fidelity of energy delivery in response to changes in local metabolism. One mediator that is a direct product of metabolism and is therefore well suited for EMS is lactate.

Lactate as a signaling molecule

Pyruvate generated during glycolysis can be metabolized by LDH enzymes to lactic acid which ionizes to form lactate, at the same time generating NAD+. This is a reversible reaction in which lactate and NAD+ can also be converted into NADH and pyruvate. In the brain, the reversibility of this reaction forms the basis of the ANLS model, although observations of lactate flux favoring brain exit to the circulation have questioned the extent to which this mechanism is favored for neuronal ATP generation (Dienel, 2012). Nevertheless, lactate levels in the brain clearly do increase during activity (Prichard et al., 1991; Hu and Wilson, 1997a, 1997b), and interestingly the activity of KATP channels in neocortical neurons has been shown to be modulated by the level of extracellular lactate, with its accumulation inhibiting KATP channels composed of Kir6.2 and SUR1 subunits after its transport into cells and subsequent metabolism, thus depolarizing the cell membrane and increasing firing rate (Karagiannis et al., 2021). Intriguingly, activation of vascular KATP channels by intracellular lactate has been observed in SMCs (Han et al., 1993), resulting instead in membrane hyperpolarization. These disparate observations raise the need for further work to determine the precise fate of lactate in the brain and delineate the responses of different cells of the neurovascular unit to local extra- and intracellular lactate levels and establish underlying mechanisms.

One consistent observation, however, is that accumulation of lactate causes vasodilation of local arterioles and arteriole-proximate capillaries through several mechanisms including modulation of contractile pericyte Ca2+ (Yamanishi et al., 2006), nitric oxide (NO)–dependent activation of vascular KATP channels (Hein et al., 2006), and the actions of extracellular prostaglandin E2 (Gordon et al., 2008). While further work is needed to determine the relative contributions of these mechanisms to blood flow control in arterioles and throughout the capillary bed, these observations suggest that the increases in lactate that occur in the brain during concerted activity can be sensed by the vasculature and converted into hyperemia, thus fitting the profile of an EMS signaling molecule.

Beyond the flow of brain lactate into the vascular system, the levels of lactate in the blood are strongly correlated to the level of lactate in working muscle, and a major fate of this molecule is to be transported to the liver where it is used to synthesize glucose or glycogen through the processes of gluconeogenesis and glyconeogenesis (Rui, 2014). However, it is also evident that circulating lactate can be directly taken up by cells as a carbon source (Quistorff et al., 2020) and this raises the possibility of whole-organism activity and energy state regulating EMS broadly throughout a number of tissues. For example, during intense whole-animal activity, the release of lactate could act as an EMS trigger in skeletal muscle as noted in the previous section, and the increase in circulating lactate could also drive EMS through its import into the heart (Duncker and Bache, 2008) and brain, if the blood concentration is sufficiently high enough to favor influx (Quistorff et al., 2020). Such a mechanism for lactate triggering coordinated blood flow increases across multiple organs could serve to rapidly optimize energy delivery during activity and represents an intriguing avenue for further exploration.

Gases as metabolic indicators: pO2 and pCO2

Oxidative metabolic pathways converge on the generation of acetyl-coA in the mitochondria for further metabolic processing via the TCA cycle to generate electron donors for the ETC, with O2 acting as the ultimate electron acceptor resulting in its reduction to H2O. This process of electron transfer generates a large H+ gradient across the inner mitochondrial membrane, culminating in the generation of ATP through the activity of the ATP synthase (Fig. 2). As oxidative phosphorylation is dependent on O2, its local partial pressure (pO2) can dictate the energy production means of the cell and thus exert influence over the metabolic state.

It is well established that hypoxia acts as a potent vasodilatory stimulus, and seminal studies have demonstrated that arterial diameter can be bidirectionally modulated as an inverse function of local pO2 (Jackson, 2016). However, as has been extensively discussed elsewhere (Taggart and Wray, 1998; Jackson, 2016), the precise molecular mechanisms through which decreases in pO2 trigger dilations are not known. Is also unknown whether hypoxia engages electrical signaling in the capillary bed, but it seems possible that similar mechanisms to those evoking arteriolar smooth muscle hyperpolarization may have evolved in this region of the vasculature to facilitate communication upstream to control arteriole and proximal capillary diameter and regulate blood flow in response to local O2 fluctuations.

In the brain, state-of-the-art measurements of pO2 have recently challenged the controversial view that hyperemia-induced increases in local pO2 are preceded by an initial dip (Kim et al., 2000; Logothetis, 2000; Buxton, 2001; Lindauer et al., 2001b; Vanzetta and Grinvald, 2001; Thompson et al., 2003, 2004; Kasischke et al., 2004; Dunn et al., 2005; Offenhauser et al., 2005; Sirotin et al., 2009; Hu and Yacoub, 2012; Ma et al., 2016; Aydin et al., 2022), which has been postulated to be driven by increased neuronal metabolism during activity. Instead, recent data support the view that O2 tension is initially unchanged by the initiation of neuronal activity and is then followed by a large influx in O2 during hyperemia (Aydin et al., 2022). Furthermore, classic positron-emission tomography experiments have convincingly demonstrated that activity-evoked increases in neuronal activity are not associated with significant increases in O2 consumption but are accompanied by increased glucose utilization—leading to the hypothesis that aerobic glycolysis predominates as a mechanism to rapidly supply energy during sudden increases in brain activity (Raichle and Mintun, 2006). Accordingly, it appears unlikely that changes in local pO2 would operate as a trigger of EMS in this organ. However, in tissues that routinely experience local or global periods of anaerobic work (e.g., exercising skeletal muscle), pO2 could be an important driver of EMS. Taking the example of skeletal muscle further, the energetic demands imposed by intensive work here lead to periods in which O2 demand outstrips supply. In addition to the immediate and rapid ATP supply provided by the skeletal muscle phosphagen system, glycolysis predominates during such periods due to the rate at which it can be rapidly ramped up to produce ATP. Mitochondrial respiration is comparatively slow as a means of energy production (Baker et al., 2010), and the decline in intracellular pO2 imposed by the high energetic demand imposed by intense exercise (Richardson et al., 1995; Wagner, 2012) will reduce the ability of this system to supply ATP to meet local needs. During such periods, decreased intracellular pO2 may act as a direct trigger of EMS by increasing the production or availability of feed-forward hyperpolarizing/vasodilating molecules such as prostaglandins and NO (Jackson, 2016). However, the mechanisms for O2-dependent production of these mediators are not clear and require further experimental attention, but it has been speculated that prostaglandin production may involve O2-dependent oxidation and inhibition of upstream cyclooxygenase enzymes (Jackson, 2016).

An interesting layer of pO2-mediated blood flow control can also be envisioned for extended bouts of hypoxia, where transcriptional changes evoked by the engagement of hypoxia-inducible factor (HIF-1; Majmundar et al., 2010; Yuan et al., 2013) could serve to tune EMS elements and modulate the sensitivity of this system. HIF-1 consists of HIF-1α and HIF-1β subunits, which respond to hypoxia by modulating the expression of >100 downstream genes (Weidemann and Johnson, 2008; Masoud and Li, 2015). Notably, hypoxia has been associated with HIF-1–mediated upregulation of Kir6.1/SUR2 KATP channel expression (Raeis et al., 2010). A resultant increase in functional protein at the membrane surface could thus serve to amplify EMS responses in the affected tissue. Moreover, HIF-mediated increases in Kir2.1 (central to EMS signal transmission, see below) have also been reported (Yamamura et al., 2018). Further work is needed to survey the tissue locations and circumstances in which drops in pO2 act to promote vasodilation and to establish the precise molecular mechanisms through which this leads ultimately to SMC membrane hyperpolarization and vasodilation. Exploring the full impact of hypoxia on EMS signaling pathways is thus an intriguing avenue for experimental investigation.

An obligate byproduct of aerobic metabolic activity is CO2, which is produced during the cyclical processing of citrate to oxaloacetate during the TCA cycle. As a gas, CO2 diffuses from its point source of production and is ultimately removed from the blood. Accordingly, CO2 readily passes through the SMCs, ECs, and pericytes of the vasculature, which may have intrinsic mechanisms to detect local pCO2. In addition to binding to deoxygenated RBCs and being secreted via ventilation, a major fate of CO2 is to be processed by the large family of carbonic anhydrase metalloenzymes, which catalyze the interconversion of CO2 and H2O to carbonic acid (H2CO3) which can, in turn, dissociate to form bicarbonate ions (HCO3) and protons (H+). Normally, pCO2 in the tissue is tightly controlled at around 40 mmHg (Patel and Sharma, 2023). During an increase in oxidative metabolic activity, the increase in CO2 generated can result in an increase in H+ concentration, decreasing pH. The cerebral circulation is remarkably sensitive to CO2-induced acidification, with most vessels exhibiting rapid dilation to acidosis through multiple mechanisms detailed below and as such, hypercapnia produces robust blood flow responses in the brain and appears to play a major role in the process of neurovascular coupling (Hosford et al., 2022). At the arteriolar level, a central mechanism through which this occurs is the conversion of cytosolic Ca2+ waves to Ca2+ sparks in SMCs which activate large-conductance Ca2+ activated K+ (BK) channels to hyperpolarize the membrane (Dabertrand et al., 2012), likely through a decrease in the open probability of ryanodine receptors (RyRs) in the sarcoplasmic reticulum (Wray and Burdyga, 2010). H+ ions can also decrease the activity of L-type voltage-dependent Ca2+ channels (VDCCs; West et al., 1992), increase the generation of NO (Lindauer et al., 2001a), and stimulate KATP channels (Wang et al., 2003). An exception to these responses in the cerebral circulation is the arterioles of the brainstem retrotrapezoid nucleus, a key chemosensory region responsible for regulating ventilation in response to changes in blood pH (Cleary et al., 2020). Here, CO2 drives a decrease in vessel diameter, which is thought to concentrate the CO2/H+ stimulus for chemosensing neurons, thus ensuring a more robust increase in ventilation to clear excess CO2.

The circulation in a range of peripheral organs also exhibits sensitivity to hypercapnia. In the coronary vasculature, stimulation of NO generation is thought to play an important role in mediating dilation in response to increased pCO2 (Heintz et al., 2005; Tzou et al., 2007), and similar mechanisms have been suggested in the brachial artery (de Matthaeis et al., 2014). In the mesenteric arteries of the intestines, hypercapnic vasodilation has been attributed to KATP channel engagement promoting membrane hyperpolarization (Wang et al., 2003). In skeletal muscle, as discussed above, acidosis and increases in CO2 up to 10% cause vasodilation and elevate blood flow (Lamb et al., 1966; Charter et al., 2018). In the kidney, which plays a central role in acid-base balance of the blood stream, hypercapnia instead induces vasoconstrictor responses and decreases renal blood flow through mechanisms that have not been fully clarified but appear to involve activation of the renal nerve (Norman et al., 1970; Chapman et al., 2020). Thus, in the context of EMS CO2 likely plays distinct tissue-dependent roles, with many organs (brain, heart, skeletal muscle) equipped to respond to increases in CO2/H+ and resultant acidosis by generating membrane hyperpolarization to increase blood flow.

Redox state

Reactive oxygen species (ROS) are highly reactive O2-containing molecules that are generated during oxidative phosphorylation in mitochondria through electron leak, resulting in the reduction of molecular O2, and by NADPH oxidases in the cytosol. ROS play important physiological signaling roles within cells and common species include superoxide anion radicals (O2·), hydroxyl radicals (·OH), and nonradical oxidants such as hydrogen peroxide (H2O2) and singlet oxygen (1O2). In addition to their roles in varied processes such as signal transduction and control of gene expression (Nose, 2000; Forman et al., 2010; Checa and Aran, 2020), during heightened activity ROS production can exceed the capacity of antioxidant mechanisms in the cell, allowing these molecules to build to disruptive levels. This accumulation damages DNA and proteins and interferes with the operation of important signaling pathways such as NO-mediated communication (Förstermann, 2010; Checa and Aran, 2020). Redox homeostasis is thus a critical factor in ensuring the continued smooth functioning of cells on a moment-to-moment basis.

Interestingly, important roles for NADPH oxidases 2 and 4 (NOX2; NOX4)—key sources of cytosolic ROS—in endothelial-dependent vasodilation have been reported. In cerebral arteries, endothelial NOX2 is associated with transient receptor potential ankyrin 1 (TRPA1) channels, which respond to NOX2-derived ROS with Ca2+ signals that couple to EC small- and intermediate-conductance Ca2+-activated K+ channels to produce vasodilation (Sullivan et al., 2015). Importantly, TRPA1 channels in capillary endothelial cells have also recently been shown to contribute the capillary-to-arteriole electrical signaling in the brain (Thakore et al., 2021), raising the possibility for ROS-initiated long-distance communication within the vasculature. NOX4 has also been observed in ECs (and also appears to be expressed by some pericytes [He et al., 2018; Vanlandewijck et al., 2018]) and has been proposed to drive membrane hyperpolarization through the generation of H2O2 (Ray et al., 2011), which promotes the activity of KATP and Ca2+-activated K+ channels (Bychkov et al., 1999; Matoba et al., 2000; Dantzler et al., 2019). As H2O2 is able to readily permeate membranes, it could also act as an intercellular indicator of redox status and as a signaling molecule, although its brief half-life likely limits its active range prior to decomposition (Lennicke et al., 2015; Ledo et al., 2022). These observations support the possibility that changes in tissue redox state brought about by increases in oxidative metabolism or membrane NADPH oxidase activity may couple directly to electrical activity, which in the vasculature could be translated into increases in blood flow. The resultant incoming blood might not only provide further energy substrates for metabolism but could also serve a homeostatic function by delivering circulating antioxidant molecules to active tissues to redress redox balance. Experiments involving direct measurements of ROS levels during tissue activity changes, and manipulation of their levels in conjunction with blood flow measurements are needed to test these ideas which likely necessitates the development of new and more sensitive indicators.

The foregoing provides a non-exhaustive outline of known and unexplored but potentially important EMS triggers. Following the triggering event, a membrane electrical signal must next be generated that can ultimately influence blood flow.

Stage II: Chemical-to-electrical transduction and propagation mechanisms underpin EMS

Upon detection of biochemical changes resulting from elevated metabolic activity in a tissue, the second stage of EMS centers on the conversion of this initiating factor into an electrical signal in the vasculature that can propagate to reach from the initiation site through multiple vessel branches to contractile mural cells (Box 1). Alternatively, the process of electrical signal generation could also occur directly in arterioles if the initiating cells are suitably proximal, although, in general, this is likely to be secondary, simply due to the relative paucity of spatial coverage by arterioles and the much more extensive arborization of the capillaries that forms the bulk of the vasculature throughout all tissues (Fig. 4). This close association with working cells thus ideally positions capillaries as the primary sensors and responders to local activity (Gould et al., 2017).

K+ channels as signal generators

In many excitable cells, electrical signaling takes the form of regenerative, propagating depolarizations that characterize action potentials (e.g., cardiac myocytes, neurons, pancreatic β cells, and gut smooth muscle) generated by Na+- or Ca2+-permeable channels. In the vasculature, the organization of electrical signaling could be conceived of as being “inverted,” with the dominant role played by hyperpolarizations generated through K+ channel activity. Below, we outline studies supporting this conception of electrical signaling throughout the vasculature.

KATP channels generate signals in response to energy deficits

Recently, we found that KATP channels take center stage in generating electrical signals in capillaries, which are then transmitted over long distances during EMS in the brain and heart. In the brain, the pericytes found on the abluminal wall of the capillaries greatly extend the sensory capabilities of the vessels by imbuing them with the ability to directly respond to the availability of local glucose (see Stage I, above) and likely a broad range of other signals through their repertoire of G protein-coupled receptors (GPCRs) and ion channels (Hariharan et al., 2020). Analysis of recent brain single-cell RNAseq data revealed that KATP channels account for nearly half of the total expression of all ion channel genes in these pericytes (Hariharan et al., 2020) and acts as a reliable marker of these cells in mice (He et al., 2018; Vanlandewijck et al., 2018). These channels are also expressed by human pericytes (Crouch et al., 2022; Yang et al., 2022; although note that not all studies find enrichment of KATP genes in humans [Song et al., 2020]) and have recently been shown to be functional in mouse brain and retinal pericytes (Sancho et al., 2022). We thus examined the role of these channels in pericyte-mediated control of blood flow and strikingly, our studies suggest that the electrical signal resulting from the activation of KATP channels in a single pericyte is capable of exerting robust control of upstream capillaries covered by contractile pericytes and arterioles and can be triggered by a fall in local glucose availability (Hariharan et al., 2022). This KATP channel-dependent basis for EMS extends to other tissues, with a variant having initially been detected by us in the heart (Zhao et al., 2020a), where KATP channels positioned in cardiac myocytes are electrically coupled to adjacent capillary ECs. Here, as the work of cardiac myocytes increases, impacting intracellular energy status, KATP channel open probability increases, and this drives membrane hyperpolarization which is likely passed via a small number of gap junctions from the cardiac myocyte to its surrounding capillaries. From here, the signal can spread throughout the vasculature to modulate blood flow (Longden et al., 2023). Similar mechanisms may also operate in other tissues in which KATP channels play important signaling roles such as the pancreas or skeletal muscle. Accordingly, we posit that KATP channels activated by a wide variety of metabolic signals (see Stage I) operate as the central node in the EMS signaling network generating electrical signals to influence blood flow.

In addition to KATP channels, other K+ channels known to be expressed throughout the vasculature might also contribute to electrical signal generation. For example, in the arteriolar endothelium, small- and intermediate-conductance Ca2+-activated K+ channels are engaged during Ca2+ elevations. The resultant hyperpolarization can be transmitted to the underlying SMCs via gap junction-containing myo-endothelial projections—specialized points of contact between ECs and SMCs through the fenestrated internal elastic lamina of these vessels. These channels may thus also contribute to EMS as signal generators or boosters, and further work is needed to address whether this is the case. Yet other K+ channel isoforms in the endothelium, pericytes, and smooth muscle (e.g., KV and BK channels in the latter [Brayden and Nelson, 1992; Straub et al., 2009; Jackson, 2017]) could also contribute to this process. It is also possible that some capillary conducted responses may be fully or partially Ca2+-mediated. For example, recent work (Thakore et al., 2021) has shown TRPA1-initiated Ca2+ signals spreading throughout capillaries—via pannexin hemichannel release of ATP in turn activating P2X receptors on endothelial cells—at ∼35 µm/s, ultimately activating SK and IK channels which then generate electrical signals for the upstream arteriole.

Kir2.1 channels mediate signal propagation through the vasculature

Once a hyperpolarizing electrical signal has been generated in response to metabolic activity, it must be transmitted—in some cases over long distances—to reach upstream capillaries and arterioles covered with contractile cells (Box 1) to drive their relaxation. In the brain, we initially characterized this process in capillary ECs, which express the strong-inward rectifier K+ (Kir2.1) channel (Longden and Nelson, 2015; Longden et al., 2017; Harraz et al., 2018a; Moshkforoush et al., 2020). This channel responds directly to both increases in external K+ and, importantly, hyperpolarization (Longden and Nelson, 2015). These stimuli can relieve the voltage-dependent block of Kir2.1 channels by the positively charged intracellular polyamines (such as spermine and putrescine) and Mg2+ that produce their characteristic inwardly rectifying current–voltage relationship. The blocking process here likely results from a combination of charge screening through the interaction of polyamines with key residues in the channels’ inner vestibule outlined above, lowering the K+ permeation rate, along with direct pore occlusion at more depolarized potentials (Xie et al., 2002). The process of blockade relief by K+ elevation and/or an initial hyperpolarization can generate a feed-forward effect that—if it overcomes the background depolarizing conductances of the cell (Tsoukias et al., 2007)—can rapidly drive the membrane potential to close to the K+ equilibrium potential (EK) through K+ efflux (Longden and Nelson, 2015). These properties of Kir2.1 and its expression throughout the capillary endothelium form the basis for a minimal regenerative electrical signaling system that allows for the long-range transmission of hyperpolarizations. Conceptually, injection of sufficient current into an endothelial cell at any point in a capillary network could be passed via gap junctions (likely composed of connexins 43 and 45; He et al., 2018; Vanlandewijck et al., 2018; Hariharan et al., 2020) into adjacent endothelial cells where it can regenerate itself through the unblocking of resident Kir2.1 channels in the next cell. In principle, this process can generate electrical signals that can traverse long vascular distances to reach the upstream supplying arteriole to drive (Fig. 4). Evidence for such a capillary-to-arteriole signaling system has now been provided by a number of studies in both brain and heart (Longden et al., 2017, 2023; Zhao et al., 2020a; Hariharan et al., 2022), and similar findings have emerged in skeletal muscle (Lamb et al., 2018). Moreover, studies in arterioles have provided evidence for long-range conduction at this level of the vascular bed (Emerson and Segal, 2001), and these electrical signals can be readily transferred from ECs to SMCs (Yamamoto et al., 1999; Sonkusare et al., 2016). It is likely that similar mechanisms operate in other tissues and further studies are needed to address this possibility.

The heart is distinct from the brain in that direct current injection from the cardiac myocyte through a sparse set of gap junctions could provide a low-pass filtered current source for hyperpolarizing or depolarizing changes in capillary voltage. Direct measurements of voltage are needed in all cell types, and implementing novel imaging tools such as genetically encoded voltage indicators to enable these measurements is therefore essential.

Together, the K+ channels that act as signal generators for EMS—primarily KATP channels—pass signals on to the Kir2.1 channels that mediate distance transmission of electrical signals through the vascular network to enable the precise delivery of blood to the tissue in the third and final stage of this process.

Stage III: Hyperpolarizing electrical signaling adjusts blood flow as the homeostatic output of EMS

After its transmission through the capillary network, an electrical signal will arrive at vascular branches covered with contractile cells—either contractile pericytes in the proximal branches of the capillary bed or SMCs covering arterioles and arteries. The mechanisms governing contractile pericyte contraction and relaxation have not been elucidated in detail, but given their expression of α-SMA (Box 1) and the presence of functional VDCCs (Gonzales et al., 2020), these are likely to be relatively similar to those of upstream SMCs, which we focus on here (although it is noteworthy that differences in the ion channel complement and relative expression of several proteins have been reported between these two cell types which likely contribute to functional differences [Gonzales et al., 2020; Ratelade et al., 2020]).

In SMCs, the interplay between membrane potential and intracellular Ca2+ is critically important. An electrical signal arriving at the arteriolar endothelium can be readily transferred to the smooth muscle via gap junction-containing myoendothelial projections, as noted above. This handover of hyperpolarization in turn will close L-type voltage-gated Ca2+ channels (VGCCs) embedded in the sarcolemma, which in turn decreases global intracellular Ca2+. In SMCs, global Ca2+ levels are sensed by calmodulin, which influences the activity of myosin light chain kinase, which transfers phosphates from ATP to the myosin head group to enable its interaction with actin fibers in the process of actin–myosin crossbridge cycling. Accordingly, a fall in Ca2+ in the SMC resulting from VGCC closure will interrupt this process, leading to a relaxation of the contractile machinery. This process playing out through many cells along the length of the vessel in response to a vasodilatory stimulus leads to dilation of the arteriole and an increase in blood flow.

The magnitude and reach of an arriving electrical signal will determine the resolution of the blood flow response. Here, the steep relationship between membrane potential and SMC tone will dictate the size of the resultant diameter change. In pressurized cerebral arteries, SMC membrane potential is roughly −40 mV under resting conditions (Knot and Nelson, 1998), which closely aligns also with the resting potentials of capillary endothelial cells and pericytes (Hariharan et al., 2022; Sancho et al., 2022). At −60 mV, these arteries are almost maximally dilated, and thus arriving signals with a magnitude within this 20-mV range produce a nearly linear effect on vessel diameter which exponentially amplifies blood flow as a product of the fourth power of the vessel radius, as described by the Hagen-Poiseuille equation. This voltage–diameter relationship is well supported in cerebral arteries and has also been explored in detail in arteries from hamster cheek pouch and intestine (Xia and Duling, 1998) and skeletal muscle (Wölfle et al., 2011). Indeed, in the latter, vessels are maximally constricted at approximately −35 mV and maximally dilated at approximately −50 mV, further illustrating the incredibly sensitive relationship between vessel diameter and membrane voltage (Kotecha and Hill, 2005). It will be important to fully elucidate these relationships for arterioles controlling blood flow to capillary beds in each tissue of interest, as differences in expression and environment may influence their range, linearity, and steepness which consequently shapes the hemodynamic response.

If a signal is transmitted from capillaries in a metabolically active area to a feed arteriole that has multiple capillary offshoots along its length, the bulk increase in blood flow through the artery in response to its dilation could be expected to be distributed throughout all capillaries emanating from this vessel (Fig. 5). In contrast, if a signal reaches only the contractile pericyte-covered capillary nearest to its point of origin (i.e., reaches the proximal capillaries but not the arteriole itself), the blood flow increase resulting from the dilating capillary would be expected to be much smaller and more localized, being a product of the dilating capillary diverting blood away from other areas of the network by modifying the path-of-least-resistance for flow (and absent a global increase in blood flow elicited by dilating the feed arteriole). Thus, the resolution of the blood delivery through the capillary network (i.e., the number of branches experiencing hyperemia) is predicted to be a function of the reach (i.e., length constant) and magnitude of electrical signals, assuming no changes in other ongoing processes (Fig. 5). However, the α-SMA-expressing precapillary sphincters found at the point of transition from arteriole to capillary in multiple capillary beds including those of the brain (He et al., 2010; Grubb et al., 2020), heart (Anderson and Anderson, 1980), and skeletal muscle (Rhodin, 1967) also likely play an important role in controlling hyperemic resolution at this level of the vascular tree through moment-to-moment modification of their contractile state. An electrical signal arriving from a capillary tree in an active area of tissue is expected to open the gateway sphincter as it passes into the arteriole, while other sphincters controlling flow to other non-electrically active capillary networks fed by the same arteriole remain closed. This would accordingly promote a preferential distribution of blood to the capillaries in the active region downstream of the relaxing sphincter. As long as the sphincters controlling the access of blood to other capillary offshoots remain constricted at the same time, this should amplify blood flow to the electrically active capillaries while minimizing the perturbation of flow through capillaries in areas that are not currently generating EMS signals. There are likely even more mechanisms for modifying flow pathways through the capillaries. For example, signals inducing slow or tonic constrictions of thin-strand pericytes (Fernández-Klett et al., 2010; Hartmann et al., 2021) may selectively narrow certain vessels to alter the path of least resistance through the capillary bed over longer time scales. Furthermore, data indicating that contractile pericytes located at branch points can independently regulate perfusion through the daughter branches they cover suggest that the regulation of blood distribution is highly complex and controlled at multiple levels (Gonzales et al., 2020). Fully studying all such possibilities will be necessary to arrive at a model that captures the complexity of the regulation of blood flow through capillary networks. It is also interesting to consider that electrical signals arriving at the venous side of the vasculature could be an important component of EMS. Detailed information on the flow of electrical signals through three-dimensional vascular networks in combination with dynamic tissue activity, vessel diameter, and blood flow measurements is thus a critically important development that is needed to fully understand exactly how these blood delivery issues are handled across tissues and under varying metabolic loads.

The shape of the blood flow response will be influenced thus by the length constant of an electrical signal, and also its magnitude and duration. In vivo, the duration of arriving electrical signals will likely differ depending on the tissue, the type of activity for which blood is being supplied, and the specific molecular setup of the local vasculature, among other factors. Broadly, electrical signaling could operate in either analog or digital modes. In the brain, present data suggest that the electrical signaling component of EMS in response to local activity most closely resembles a digital electrical signal (Fig. 4), which propagates in a regenerative fashion through the vasculature to drive upstream dilation and increase blood flow. These signals have rapid on- and off-kinetics and their duration is tied closely to the length of the stimulus. This digital signal analogy is supported by data indicating an all-or-none property of electrical signaling through brain capillaries, in that stimuli below K+ concentrations of 7 mM are ineffective in changing arteriole diameter, whereas K+ concentrations above this concentration evoke near-maximal dilation of the upstream arteriole (Longden et al., 2017). This behavior can be explained from the perspective of the Kir channel by introducing a bistable membrane potential with points of stability at EK when channels are fully active and the resting membrane potential when they are closed (Moshkforoush et al., 2020). Similarly, the transfer of signals along lengths of arterial tissue has been studied in detail, and this work reveals the conduction of electrical signals along the endothelium which is largely sustained at distances of up to 2 mm, suggesting an active regeneration mechanism (Emerson and Segal, 2000, 2001), which is readily transferred laterally to the smooth muscle (Yamamoto et al., 1999).

In other tissues and in other tissue activity scenarios, a form of analog signaling could predominate. From a biophysical perspective, such signaling would be by its nature non-regenerative and non-propagative and could instead be organized around the electrotonic conduction of potentials according to the cable properties of the local vessels. Possible molecular configurations that would enable such a signal would be a capillary bed that receives electrical input via gap junctions from either parenchymal cells or overlying pericytes acting as signal generators, while at the same time lacking a high density of Kir channels in the ECs. Without a regenerative mechanism to amplify and propagate the signal, a voltage change would instead exponentially decay as a function of vessel distance, the degree of membrane leak, and the size of other “background” conductances. EMS operating in this mode would thus give rise to signals that are a more direct function of the amplitude and duration of the initiating metabolic stimulus and would as a result likely operate over slower and more local time scales and shape the blood flow response accordingly (Fig. 5).

The incoming blood that results from these dilations initiated by EMS performs multiple homeostatic functions. In addition to the delivery of energy substrates and O2, CO2, lactate, and other waste products generated during metabolic activity are removed (Fig. 1), and in the process pH and temperature of the tissue may be regulated also. Accordingly, the increase in metabolic activity in the tissue is compensated for and deviations in intracellular energy state (i.e., changes in ATP:ADP ratio) are reversed, effectively matching energy demand to supply and ensuring continued optimal function of the tissue.

Taken together, the three stages of the EMS hypothesis outlined above provide a mechanistic framework enabling the accurate and timely coupling of energy supply through blood flow to local metabolic needs in highly active tissues. We postulate that EMS variants operate in all tissues with high energy consumption (e.g., brain, heart, kidney, skeletal muscle) and likely also in other areas and that this precise matching helps to ensure the health and function of local cells on a moment-to-moment basis across long periods of time.

EMS uncoupling as a mediator of aging

We posit that EMS plays a key role in matching the energetic demands of varied physiological processes with precise moment-to-moment energy substrate delivery via the blood. It thus follows that processes that disrupt EMS would be detrimental to overall tissue function, stemming from an inability to meet ongoing energetic demands. Aging is one such process that could degrade EMS and is characterized by a panoply of functional changes that result in overall organismal decline. A number of hallmarks of the aging process have been defined and these include genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, inflammation, dysbiosis, and alterations to intercellular communication (López-Otín et al., 2023).

It is possible that age-related alterations to intercellular signaling (for example, due to the loss of cardiac and vascular KATP channel function that has been documented in older animals [Yang et al., 2016]) could be a proximate cause that deranges the metabolic sensing or signal transmission aspects of EMS, which would then be expected to contribute further to the aging process. It is not difficult to conceive that the reduced substrate delivery emanating from impaired blood flow regulation due to loss of EMS could then play into mitochondrial dysfunction and thus exert an outsized impact on cellular energy status. Indeed, many of the aging hallmarks outlined above depend on precise control of energy generation and delivery. For example, altered substrate availability will affect the activity of the mechanistic target of the rapamycin (MTOR) complex which modulates the activity of a range of transcription factors influencing the expression of elements that contribute to a huge number of core cellular activities, including protein synthesis and proteasome activity, autophagy, and metabolism (López-Otín et al., 2023). Accordingly, it seems feasible that age-related declines in EMS could play a key role in aging through knock-on effects on mechanisms that are at the heart of the aging process. By preventing EMS degradation over long time spans—for example through the development of gerotherapies that preserve EMS sensing, transduction, and propagation mechanisms—it may be possible to preserve overall tissue and organ function and slow aging.

EMS uncoupling as a driver of organ failure states

Our hypothesis places EMS at the center of blood flow modulation mechanisms in a range of tissues. Extending from the foregoing, it follows that disease process-related breakdown of EMS mechanisms could be an initiating factor in the mismatch between energy supply and demand which then precipitates metabolic failure states that compromise organ function and lead to further pathology.

Organ function is intricately intertwined with metabolism, and thus pathologies that disrupt metabolic function have wide-ranging and frequently disastrous effects. Metabolic syndrome is highly prevalent, and in the US is estimated to affect ∼35% of the population (Hirode and Wong, 2020). It consists of a group of conditions that encompasses obesity, hypertension, and dyslipidemia among others which greatly increase the risk of developing diabetes, heart disease, and stroke, and is overall linked to early death (Eckel et al., 2010). A major factor that promotes the development of metabolic syndrome is a high-fat diet, and interestingly, this has been associated with the impairment of a wide range of elements that comprise the EMS mechanisms we have identified. Specifically, a high-fat diet has been shown to impair vascular KATP channel function (Fan et al., 2008, 2009) and endothelial Kir2.1 function (Fancher et al., 2020; Ahn et al., 2022), the two key EMS elements involved in the transduction of metabolic signals to changes in membrane electrical activity and transmission of electrical signals throughout the vasculature, respectively. Moreover, vascular smooth muscle undergoes a range of changes in obesity including an increase in the density of voltage-gated Ca2+ channels which enhances contraction (Owen et al., 2013), along with a decrease in BK channel function (Borbouse et al., 2009), promoting hypertension and a decrease in blood flow.

Such changes would be expected to weaken EMS by reducing the ability to generate substantial electrical signals, while also diminishing their propagation throughout the vasculature. Weaker signals arriving at the arteriolar level would also encounter a higher basal contractile state, resulting from a higher level of intracellular Ca2+ at a given membrane potential. This is predicted to further limit the ability of the arriving hyperpolarization to drive a significant decrease in the level of intracellular Ca2+ to drive dilations and increase blood flow to metabolically active regions. Accordingly, functional disruption of EMS could be an early event that compounds issues locally within the affected tissue driven by insufficient energy substrate delivery. Exploration of these possibilities will reveal the extent of the contribution of EMS disruption to a range of pathologies in organs throughout the body.

The pancreas plays a central role in energy balance by controlling blood glucose levels through insulin secretion. As blood glucose levels rise (e.g., postprandially) insulin secretion increases, which promotes glucose utilization, uptake, and storage. Efficient microvascular function and tight control of blood flow are accordingly essential to pancreatic function, and impairment of pericyte and capillary signaling as well as structural microvascular abnormalities are early events in the pathogenesis of diabetes. As such, the loss of blood flow control in pancreatic islets could be a key event that impacts nutrient sensing, hormone release, and the timely control of glycemic status (Almaça et al, 2018; Gonçalves et al, 2023). The resulting derangements in insulin signaling and blood glucose levels in turn will have a range of further effects that could compromise efficient energy matching to local metabolic demands and contribute to multiorgan functional decline.

Heart failure is associated with metabolic changes that lead to loss of efficient energy generation that contributes to profound impairment of cardiac output. Accordingly, blood flow is disrupted throughout the body, further compounding any existing energetic issues and creating novel problems associated with a lack of proper substrate delivery. Due to these far-reaching ramifications, heart failure is perceived as a complex, multiorgan syndrome with metabolic failure as the central driving force (Rosano and Vitale, 2018). Under normal circumstances, the heart predominantly oxidizes free fatty acids but also uses glucose to produce the ATP needed for optimal function. In heart failure, insulin resistance is thought to limit glucose utilization and favor the use of free fatty acids for ketogenesis. This leads to a reduction in the production of ATP in the heart, which in turn contributes to a decline in the efficiency of cardiac output. This is interwoven with other abnormal processes such as structural changes and an increase in oxidative stress (Rosano and Vitale, 2018). After the onset of cardiac failure, activation of the sympathetic nervous system and renin–angiotensin system may also trigger an increase in cytokines, angiotensin II, and free fatty acids. The latter damages the pancreas, impairing insulin regulation of blood glucose and disrupting glucose handling by skeletal muscle. The elevated angiotensin II can also drive vasoconstriction—further impairing blood delivery—and hampered endothelial function has been noted in this context (Ashrafian et al., 2007; Giannitsi et al., 2019). Activation of the angiotensin AT1 receptor also produces ROS in the vessel wall which could contribute to signaling processes under normal conditions but may disrupt components of local EMS mechanisms when out of balance with antioxidants (Nickenig and Harrison, 2002). Against this backdrop, impairment of EMS may contribute to disturbed metabolic activity in the heart and therefore the reduction of cardiac output and its downstream effects. For example, disruption of KATP-mediated electrical signaling from cardiac myocytes to ECs (Zhao et al., 2020a) would be predicted to impair the tight matching of blood flow to energy demand and therefore contribute to cardiac dysfunction. Further work is needed to explore this possibility and the potential disruption of other aspects of EMS in the context of heart disease.

Disruption of cerebral blood flow is thought to be one of the earliest factors on the path to dementia, in some cases being detectable decades before the onset of cognitive decline (Iturria-Medina et al., 2016). This has been suggested as a watershed moment that leads to a range of compounding consequences that ultimately lead to neuronal dysfunction, death, and cognitive decline. Indeed, it has been postulated that once a critical threshold of cerebral hypoperfusion has been crossed, a panoply of cellular issues ensue throughout the neurovascular unit stemming from this disrupted energy balance (de la Torre, 2000a). These include the degeneration of pericytes (Sagare et al., 2013; Halliday et al., 2016) and profound alterations to endothelial (Wang et al., 2018) and smooth muscle cells (Chabriat et al., 2009), leading, in turn, to further hemodynamic impairments, increased generation of free radicals by starved mitochondria, damage of cellular macromolecules, loss of control over membrane potential and ionic imbalance due to Na+/K+ ATPase dysfunction, derangements in Ca2+ handling, impacts on gene expression, and impaired protein production, posttranslational modification, and trafficking (de la Torre, 2000b). Together, these mounting problems initially cause neuronal dysfunction and lead ultimately to the death of the cell. As EMS provides an essential link matching blood flow to neuronal metabolic needs, subtle functional disturbances in this process could divorce capillary electrical signaling from local metabolism and thus lead to mismatching between neuronal energy supply and demand, driving the advancement of the cascade of issues outlined above. Importantly, such disruptions could be initiated in the brain capillaries themselves or could arise as the result of dysfunction in other organs (e.g., pancreas and heart) leading to inefficiencies in energy substrate supply that disrupt brain metabolism. Studies taking an integrated multiorgan view of the cascading effects that result from blood flow impairment throughout the body are thus essential to explore the impacts of energy substrate delivery impairment on integrated physiological functioning.

Outstanding questions and future directions

Our recent studies have revealed overlapping yet distinct EMS mechanisms that operate in both the brain (Hariharan et al., 2022; Longden et al., 2023) and heart (Zhao et al., 2020a; Longden et al., 2023). Work in these systems offers a platform to deepen our understanding EMS in these and other organs, and here we outline the key outstanding questions at the present juncture and the technological developments that will be necessary to gain a full understanding of this system and its contributions to health and disease.

Key questions to unlock a deeper understanding of EMS

What is the full range of organ systems that utilize EMS to match blood flow to cellular metabolic demands?

As noted above, our work has revealed EMS mechanisms in the brain and heart, but other organs with high energy demands remain unexplored. Of particular note, the kidneys rank alongside the heart as the joint-top organs with the highest resting energy expenditure (Wang et al., 2010). The brain consumes around half as much ATP “at rest,” followed closely by the liver, with skeletal muscle and adipose tissue trailing these and accounting for only a small fraction of basal metabolic rate. Of course, both individual brain regions and skeletal muscles engage in dramatic changes in their activity levels (i.e., when engaging in computations or contractions, respectively). Accordingly, it is important to investigate in detail the different types of EMS mechanisms operating in these tissues and beyond, where variations and modifications may provide insight into local metabolic regulation. For example, it is possible that EMS is absent in a tissue like the liver, which maintains a large store of glycogen to draw upon to meet its energy needs when glucose levels in the blood are low (Rui, 2014). In the kidney, the glomerular filtration rate is homeostatically controlled and is influenced by blood flow, increases in which drive filtration, greater reabsorption, and increased metabolic demands. Investigating whether other EMS mechanisms are at play in the kidney at the capillary and arteriolar levels may yield further useful insights. By extension, surveying the operation of EMS and its, similarities, differences, and ramifications throughout all organs are important avenues for future work.

What is the full range of metabolic triggers of EMS?

Current data suggest important roles for intracellular energy state (i.e., ATP:ADP ratio) resulting from increased work and decreased local energy substrate availability as triggering factors in EMS. Moving forward, it will be important to determine the full range of factors produced in response to metabolic deviations in eliciting electrical signaling through the vasculature and their detailed mechanisms. For example, investigating the relationship between lactate and capillary electrical signaling is an important and tractable set of experiments that is within easy reach. Measurements of O2 tension in vivo are more difficult but have been made possible by the development of elegant phosphorescent lifetime imaging approaches (Sakadzić et al., 2010). Improvements in the time resolution of these measurements and their wider application will offer deeper insights into the relationship between local O2 fluctuations and blood flow. Given the huge complexity of biochemical pathways, the number of potential factors that could engage EMS is vast, and mechanisms could be tailored to elicit electrical responses from metabolic processes that produce a wide array of metabolites and byproducts. Efforts to understand the breadth of these metabolic signaling mechanisms and the specific mechanisms through which they produce electrical signaling in the vasculature will thus reveal insights into EMS in a range of contexts.

What is the full range of mechanisms that convert metabolic load to blood flow elevations at the arteriolar and capillary levels?

Here, we have considered EMS mechanisms primarily from the standpoint of propagated hyperpolarizations from capillaries to arterioles. As discussed above, different mechanisms underlying these signals may be tailored to suit the particular blood flow needs of a given tissue. For example, the kidney requires constant blood flow to enable optimal filtration, and thus feedback mechanisms to maintain blood flow at a homeostatic set-point may dominate in this organ. In contrast, heart, brain, and skeletal muscle all exhibit rapid changes in activity and metabolism across various spatiotemporal domains and thus the EMS mechanisms that have evolved are likely to be specialized to meet their individual blood flow requirements.

Although in general arteriole coverage is sparser than the reach of the capillary bed, it will be important alongside work in capillaries to understand the EMS mechanisms operating directly in arterioles at the levels of the arteriolar endothelium and in SMCs, which may have unique mechanisms to sense local metabolic changes.

How does EMS breakdown contribute to aging and disease?

Given the suspected widespread nature of EMS signaling, the potential for contribution to aging and to a range of disease processes is extensive. Aging is characterized by a general decline in function during adulthood. Given the essential need of all organ systems to match metabolic needs with energy delivery through tightly controlled blood flow, it is possible that disruption of EMS makes a key contribution to the aging process. Here, a decline in the efficiency of EMS mechanisms could lead to a gradually widening gap between energy demand and delivery, which in turn is expected to influence essentially all aspects of cellular health. Extending this to disease processes, contexts for EMS disruption that are of particular interest are disorders involving a metabolic component such as diabetes, metabolic syndrome, Alzheimer’s disease, and vascular dementia, and also rarer inherited metabolic diseases such as Niemann-Pick disease, Tay-Sachs disease, or porphyria. Accordingly, understanding how EMS contributes to both aging and disease and finding ways to restore or boost flagging EMS mechanisms may help to extend the health span of aging individuals and may offer novel approaches to treat diseases of metabolism that impact a wide range of organ systems.

Technological developments to enable breakthroughs in understanding EMS

To enable deeper insight into the organization and operation of EMS mechanisms described above, key technological developments are needed that will expand sensing capabilities and augment the resolution of imaging approaches. Together, these will allow us to measure EMS in increasingly complex and holistic scenarios.

Development of approaches to directly visualize EMS in tissue volumes

At present, multiphoton imaging methods are best suited to imaging hemodynamics and EMS processes occurring deep within tissues. However, several technical hurdles make visualization of EMS processes challenging. In particular, standard multiphoton systems are typically restricted to time-lapse imaging of single planes at video rate using resonant and galvanometer scanning mirrors to guide rapid laser rastering or acquisition of small volumes at a few hertz using piezoelectric objective drives. To be able to fully understand EMS, advances enabling video rate imaging of electrical signaling in large tissue volumes are required, thus capturing the dynamics occurring in highly convoluted vascular networks. A number of recent technical achievements in multiphoton imaging make it feasible to overcome these issues in the near future. In particular, the development of FACED microscopy and its extension to blood flow imaging enables kilohertz imaging of single planes and megahertz imaging of single lines. These approaches are fast enough to capture the dynamics of even the fastest moving RBCs moving through the largest arteries and enable for the first time the precise measurement of blood flow (i.e., RBC flux and velocity) without the need to completely collapse the spatial dimension to a single line. The application of Bessel-focused beams has enabled the rapid imaging of considerably larger volumes than were previously accessible (Fan et al., 2020a), and the development of SCAPE imaging has also enabled high-speed dynamic volumetric imaging in vivo (Bouchard et al., 2015; Voleti et al., 2019). The widespread adoption of these exciting techniques will enable a more detailed understanding of the vascular dynamics of various organ systems, and the volume of data these approaches generate will necessarily spur the development of further automated techniques for analysis.

Accompanying these hardware developments is the need to develop novel models to enable direct visualization of the EMS process. Thus, the generation of mouse lines that express state-of-the-art voltage sensors such as ASAP (Evans et al., 2023) or opto-patch (Fan et al., 2020b) variants in pericytes, ECs, and SMCs to enable direct visualization of electrical signaling occurring throughout the vasculature of an organism are essential. These approaches can be combined with other mouse lines and viral transfection approaches to enable the visualization of local activity, metabolic processes, and measurements in the vasculature (Zhao et al., 2020b) throughout tissues of interest.

Approaches to visualize the activity of energy substrate availability and metabolic pathway activity in real time

Until recently, few options have existed to visualize metabolic processes directly, and available techniques required combined measurements from many cells to obtain low temporal resolution readouts. However, in recent years advances in probe development now promise great insights into metabolic imaging in vivo, in real time, and with single-cell resolution. Indeed, a range of recently developed substrate, metabolite, and energy status sensors are poised to enable visualization of cell metabolism in unprecedented detail. For example, the recently developed glucose sensor, green glifon (Mita et al., 2019), has an excellent spatiotemporal resolution, allowing the real-time measurement of glucose uptake in single cells. Combination of this with viral vectors that allow for specific expression in cells of interest will enable real-time monitoring of glucose availability and uptake during various scenarios (e.g., brain region activation or increased heart contraction). Alongside this glucose sensor, fluorescent protein-based probes for lactate and pyruvate have also been developed (Harada et al., 2020), and thus their deployment in various cell types should allow the visualization of glycolytic flux and the flow of lactate produced during metabolism. The development of phosphorescent probes with lifetimes that are sensitive to the local concentration of O2 is also now enabling the visualization of vessel and tissue oxygenation. These studies are updating traditional models of O2 diffusion into the tissue such as the Krogh cylinder by providing novel insights into the dynamics of O2 consumption in working tissue. Improvements in the spatiotemporal resolution of these imaging techniques and their deployment in in vivo studies throughout the body will add layers of detail to our understanding of O2 delivery and utilization (Sakadzić et al., 2010; Parpaleix et al., 2013; Lyons et al., 2016; Aydin et al., 2022).

Operating at the opposite end of the spectrum of substrate consumption is PercevalHR, a probe for intracellular energy status. This allows real-time measurement of ATP:ADP ratio and has been deployed in neurons during activation with glucose, KCl, or electrical stimulation and provides excellent resolution revealing how these stimuli alter the ratio of high energy molecules within the cell (Tantama et al., 2013). Notably, several of these probes are two-photon compatible and so should be deployable in vivo to allow the observation of how these different aspects of metabolism intersect with one another in a systems context. Ongoing developments in the field of redox biology, such as the development of the novel redox probe FROG/B (Sugiura et al., 2020), also promise to overcome the limitations of current probes which makes their direct application and interpretation in vivo challenging. Further work to produce imaging tools with superior signal-to-noise ratio and tailored subcellular localization in organelles will undoubtedly yield deeper insights into this exciting area for further development.

Visualization of the nanoscale organization of EMS complexes

A further priority is determining the specific molecular organization of the signaling components that enable EMS in different cell types and systems. Considering KATP channels as the central node of currently known EMS pathways, further work is needed to establish the specific membrane localization of these channels in different cells and to elucidate the specific macromolecular signaling complexes that they occupy. In brain pericytes, for example, important questions to answer in the near future are whether the KATP channels are primarily found in the cell body or in thin-strand processes, or whether they are evenly distributed throughout the plasma membrane. Insights into the molecular partners with which these channels most closely associate, be that the enzymes of the glycolytic machinery (Dhar-Chowdhury et al., 2005), PKA, and A-kinase anchoring proteins (Hayabuchi et al., 2001), or as yet undetermined signaling partners, will help in the determination of the precise intracellular mechanisms through which these channels are engaged. An appealing possibility in this context is that the KATP channel could occupy a macromolecular signaling complex positioned in or very close to peg-socket junctions between pericytes and endothelial cells (Ornelas et al., 2021), permitting the immediate transfer of charge generated by KATP channels into the underlying endothelium for optimal electrical signaling upstream to arterioles. Further technical developments are needed to probe EMS at this level of detail, but answering nanoscale questions will offer insight that likely will be essential for understanding how EMS falters in aging and disease and will be needed for finding potential solutions to rectifying such issues.

Summary and conclusions

In summary, we outline a hypothesis for EMS as a generalized blood flow control mechanism operating across tissues with a wide range of spatiotemporal energy needs. Specifically, EMS can be initiated by a range of metabolic changes, including those of substrate availability, altered intracellular energy status, and byproducts of metabolism. Through the activity of K+ channels and KATP channels in particular, metabolic cues are converted into electrical signals in the vasculature which are then conducted along the capillary and arteriolar endothelium to produce relaxation of upstream SMCs and contractile pericytes and increase blood flow to areas of metabolic need. It is possible that disruption of EMS in aging and in disease states with metabolic and vascular components is a key event that leads to a mismatch between energy supply and demand and participates actively in the development of cellular dysfunction and nominally irreversible tissue damage. Accordingly, targeting EMS for the development of novel therapeutics may provide the means to protect blood flow and energy delivery to tissues, thereby counteracting the metabolic deficits that arise during aging (Amorim et al., 2022), and helping to restore energy balance in diseases ranging from Alzheimer’s and dementia to diabetes, metabolic syndrome, heart failure, and chronic kidney disease.

As such, research aimed at fully elucidating the physiological mechanisms of EMS and visualizing its contributions to blood flow control in complex three-dimensional time-resolved networks is essential. Equally important are investigations of mechanisms by which the EMS may be damaged during aging and disease. Likewise, therapeutic repair or restoration of damaged EMS components to ameliorate or fix this damage may provide partial or complete repair in diverse degenerative diseases.

David A. Eisner served as editor.

Support for this work was provided to T.A. Longden by the National Institutes of Health National Institutes on Aging (1R01AG066645) and Neurological Disorders and Stroke, (5R01NS115401, and 1DP2NS121347) and by the American Heart Association (19IPLOI34660108) and to. W.J. Lederer by the National Institutes of Heart, Lung and Blood (R01 HL142290), Allergy and Infectious Diseases (U19 AI090959), General Medical Sciences (GM140822), and by special funds from BioMET at the University of Maryland School of Medicine.

Author contributions: All authors reviewed the manuscript and approved its submission.

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