Myosin binding protein-C modulates loaded sarcomere shortening in rodent permeabilized cardiac myocytes

The heart’s pumping capacity is determined by myofilament-loaded shortening and power output since the heart always works against an afterload to eject blood. Power is work done per unit time and measured by the product of force and velocity. At a molecular level, these contractile properties are linked to the number of attached cross-bridges and their cycling rate, and many signaling pathways modulate these factors. For instance, myofilaments are first activated by intracellular Ca²⁺, which binds to thin filaments and permits strong binding of myosin to actin (Solaro and Van Eyk, 1996; Vibert et al., 1997; Xu et al., 1999; Pirani et al., 2006). These strongly bound heads further activate thin filaments (McKillop and Geeves, 1993) and undergo force-generating transitions. In addition to canonical thin filament regulation, more recent evidence shows thick filament regulation of cardiac contraction (Reconditi et al., 2017; Brunello et al., 2020). Thick filaments transition between OFF and ON structural states as a function of stress (Linari et al., 2015; Irving, 2017; Brunello et al., 2023; Marcucci, 2023), thereby providing a mechanosensing mechanism that matches the cross-bridge number and myofilament shortening and power to the external load.

While several studies have investigated force–velocity relationships in cardiac muscle preparations (Sonnenblick, 1962; Brenner, 1986; Chiu et al., 1987, 1989; Sweitzer and Moss, 1993; McDonald, 2000a, 2011), fewer have examined loaded shortening and power at the level of the sarcomere (Daniels et al., 1984; De Tombe and Ter Keurs, 1990; McDonald et al., 1998; Korte and McDonald, 2007), which is the functional unit of cardiac myofilaments. The first aim of this study was to quantify the shortening velocity relationship between cardiac myocyte preparation length (ML) and sarcomere length (SL) during load clamps in rodent permeabilized cardiac myocytes.

The second aim was to investigate the role of the cardiac myosin binding protein-C (cMyBP-C) in regulating loaded shortening at both the ML and SL levels. In brief, cMyBP-C is a flexible 140–150 kDa protein localized to seven to nine transverse stripes spaced ∼43 nm apart in each half-thick filament in the sarcomere region termed the C-zone (Starr and Offer, 1971; Pepe and Drucker, 1975; Craig and Offer, 1976; Hartzell and Sale, 1985; Bennett et al., 1986; Lee et al., 2015). cMyBP-C consists of multiple immunoglobin-like and fibronectin-like domains, numbered C0–C10 (Flashman et al., 2004; Barefield and Sadayappan, 2010; Harris et al., 2011). In all isoforms, there is a conserved MyBP-C specific domain (i.e., the M-domain) between C1 and C2, which contains one to four serines that can be phosphorylated by PKA (Barefield and Sadayappan, 2010; Ackermann and Kontrogianni-Konstantopoulos, 2011; Harris et al., 2011). In hearts, cMyBP-C is phosphorylated with β-adrenergic stimulation, which correlates with faster cardiac muscle twitch dynamics (Jeacocke and England, 1980; Hartzell, 1984; Tong et al., 2008).

Regarding function, cMyBP-C appears to regulate several myocardial contractile properties. For instance, ablation of cMyBP-C decreases Ca²⁺ sensitivity of force (Harris et al., 2002; Palmer et al., 2004), speeds both isometric contraction rates and maximal velocities of shortening (Korte et al., 2003; Palmer et al., 2004; Stelzer et al., 2006b; de Lange et al., 2013; Moss et al., 2015; Giles et al., 2021), and augments stretch activation (Stelzer et al., 2006a) in permeabilized strips of ventricular tissue. In addition, ablation of cMyBP-C speeds loaded shortening and increases power output in permeabilized cardiac myocyte preparations (Korte et al., 2003; Hanft et al., 2021). Interestingly, permeabilized cardiac myocyte preparations lacking cMyBP-C exhibit unique force–velocity and consequent power–load relationships, whereby loaded shortening and power are inordinately elevated at higher loads (Korte et al., 2003; Hanft et al., 2021), which implicates a role for cMyBP-C in load sensing. We addressed whether previously reported faster ML-loaded shortening, especially at high loads, also occurs at the sarcomere level in permeabilized cardiac myocyte preparations from transgenic mice lacking cMyBP-C.

Last, we addressed the hypothesis that cMyBP-C regulates loaded shortening and power output by modulating cross-bridge availability. We tested this hypothesis by characterizing force–velocity and power–load relationships in mouse permeabilized cardiac myocyte preparations that were deficient in cMyBP-C at both the ML and SL level before and after treatment with the small molecule, mavacamten, a myosin cross-bridge inhibitor.

Compositions of relaxing and activating solutions were as follows: 7 mM EGTA, 1 mM free Mg²⁺, 20 mM imidazole, 4 mM MgATP, 14.5 mM creatine phosphate, pH 7.0, various Ca²⁺ concentrations between 10⁻⁹ M (relaxing solution) and 10^−4.5 M (maximal Ca²⁺ activating solution), and sufficient KCl to adjust ionic strength to 180 mM. The final concentrations of each metal, ligand, and metal–ligand complex were determined with a computer program (Fabiato, 1988). Preceding each Ca²⁺ activation, myocyte preparations were immersed for 30 s in a solution of reduced Ca²⁺–EGTA buffering capacity, which was identical to a normal relaxing solution except that EGTA was reduced to 0.5 mM. This protocol resulted in more rapid development of steady-state force during subsequent activation and helped preserve the striation pattern. The relaxing solution contained 2 mM EGTA, 5 mM MgCl₂, 4 mM ATP, 10 mM imidazole, and 100 mM KCl at pH 7.0 with the addition of a protease inhibitor cocktail (Set I Calbiochem). For mavacamten experiments, 0.5 µM mavacamten was added to all pCa solutions resulting in a reduction of maximal force by ∼40%, in agreement with previous studies (Awinda et al., 2020, 2021; Sewanan et al., 2021; George et al., 2023). For experiments in the presence of 0.5 µM mavacamten, the submaximal calcium concentration was determined by selecting the pCa solution with mavacamten that elicited as close to 50% maximal force obtained in pCa 4.5 also containing mavacamten.

All procedures involving animal use were performed according to the Animal Care and Use Committee of the University of Missouri. Male Sprague–Dawley rats (6 wk of age) (N = 5) were obtained from Envigo, housed in groups of two, and provided access to food and water ad libitum. Male and female (2–4-mo old) transgenic cMyBP-C wildtype (WT) (N = 5) and knock-out (KO) mice (N = 5) (both with background strain: 129S1/Svlm) were also used for permeabilized cardiac myocyte mechanical measurements. The cMyBP-C KO mouse line was originally established in the Laboratory of Animal Resource in the UW School of Medicine and Public Health at the University of Wisconsin-Madison and was re-derived at the University of Missouri Mutant Mouse Resource and Research Center. This cMyBP-C KO transgenic mouse line has been previously well characterized (Harris et al., 2002; Korte et al., 2003; Stelzer et al., 2006a, 2007; Luther et al., 2008; Tong et al., 2008, 2015; Chen et al., 2012; Colson et al., 2012; Rosas et al., 2015). All procedures involving animals were performed in accordance with the Animal Care and Use Committee of the University of Wisconsin and the University of Missouri.

Permeabilized cardiac myocyte preparations were obtained by mechanical disruption of rodent hearts as described previously (McDonald, 2000a). Rodents were anaesthetized by the inhalation of isoflurane (20% vol/vol in olive oil), and hearts were excised and rapidly placed in an ice-cold relaxing solution. The left ventricle was separated from the right ventricle and dissected from the atria, cut into 2–3 mm pieces, and further disrupted for 5 s in a Waring blender. The resulting suspension of cardiac myocyte preparations was centrifuged for 105 s at 165 ×g, after which the supernatant fluid was discarded. The myocyte preparations were permeabilized by suspending the cell pellet for 2 min in 0.3% ultrapure Triton X-100 (Pierce Chemical Co.) in cold relaxing solution. The permeabilized myocyte preparations were washed twice with cold relaxing solution, suspended in 10 ml of relaxing solution, and kept on ice for the experimental day.

The experimental apparatus for mechanical measurements of permeabilized myocyte preparations has been described previously (McDonald, 2000a). A permeabilized cardiac myocyte preparation was mounted between a force transducer and motor by placing the ends of a myocyte into stainless steel troughs (25 gauge), and the ends were secured by overlaying a 0.5-mm length of 4-0 monofilament nylon suture (Ethicon, Inc.) and tightening the suture into the troughs with loops of 10-0 monofilament (Ethicon, Inc.) (Fig. 1 A and Fig. S1; permeabilized cardiac myocyte preparation characteristics are provided in Table 1). The attachment procedure was performed under a stereomicroscope (90× zoom) using finely shaped forceps (McDonald, 2000a).

Before mechanical measurements, the experimental apparatus was mounted on the stage of an inverted microscope (model IX-70, Olympus Instrument Co., with a 40× objective [Olympus UWD 40, 0.55 N.A.]). Force measurements were made using a capacitance-gauge transducer (Model 403-sensitivity of 20 mV/mg [plus a 10× amplifier] and resonant frequency of 600 Hz; Aurora Scientific, Inc.). Length changes were introduced using a DC torque motor (model 308, Aurora Scientific, Inc.) driven by voltage commands from a personal computer via a 16-bit D/A converter (AT-MIO-16E-1; National Instruments Corp.). Force and length signals were digitized at 1 kHz and stored on a personal computer using LabView for Windows (National Instruments Corp.). SL was monitored (at 1 kHz) using an IonOptix SarcLen system (IonOptix), which was done with a fast Fourier transform algorithm of the video image of the myocyte.

The protocol to obtain force, rate of force, force–velocity, and power–load measurements has been described in detail (McDonald, 2000b; Hinken and McDonald, 2004; Korte and McDonald, 2007; Hanft et al., 2021), and all measurements were done at 16 ± 1°C. Once attached, the relaxed permeabilized cardiac myocyte preparation was adjusted to an SL of ∼2.25 µm, and passive tension was assessed by slacking the preparation in pCa 9.0 solution. To measure ML- and SL-loaded shortening and power output, the following protocol was used. An attached myocyte was transferred into maximal Ca²⁺ activating solution (pCa 4.5), allowed to develop force to a plateau after which it was rapidly slacked by 15–20% ML, held for 15–20 ms and then rapidly re-stretched, and subsequent force redevelopment trace acquired. The myocyte was then transferred to a submaximal Ca²⁺ activating solution that yielded approximately half-maximal Ca²⁺ activated force and then a series of subisometric force clamps were applied to determine isotonic shortening velocities. The isotonic force was maintained using a servo system for 100–250 ms, and ML and SL changes during this time were monitored (Fig. 1, Video 1 and Video 2 show examples of force clamps and corresponding ML and SL traces). Following the force clamp, the myocyte was slackened to a near-zero force to estimate the relative load sustained during the isotonic shortening, after which the myocyte was re-extended to its starting length. The myocytes were kept in submaximal Ca²⁺ activating solution for 3–5 min during which 10–20 force clamps were performed. A final force measurement was made in pCa 4.5 solution, and if it was found to be below 70% of initial maximum tension, data from that myocyte were discarded. To assess the effects of mavacamten, the same protocol was repeated in the presence of 0.5 µM mavacamten.

Curve fitting was performed using a customized program written in Qbasic, as well as commercial software (SigmaPlot).

Slopes of the ML versus SL shortening velocity relationships were calculated by linear regression. Contractile properties before and after mavacamten treatment were compared by paired tests. The experimental data between groups was compared by one-way ANOVA. A Student–Newman–Keuls post-hoc test assessed differences between the means. P < 0.05 was accepted as a statistically significant difference. N = number of animals and n = number of permeabilized cardiac myocyte preparations. Values are expressed as means ± SEM.

Fig. S1 shows video images of a rat permeabilized ventricular cardiac myocyte preparation (pink rectangle signifies region of interest [ROI] for SL tracking) during relaxation (pCa 9.0) and maximal Ca²⁺ activation (pCa 4.5). Video 1 displays videos of load clamps in rodent permeabilized ventricular cardiac myocyte preparations during approximately half-maximal Ca²⁺ activation; SL was monitored at 240 Hz. Video 2 displays videos of load clamps in rodent permeabilized ventricular cardiac myocyte preparations during approximately half-maximal Ca²⁺ activation; SL was monitored at 1 kHz.

The first objective was to systematically compare ML and SL shortening during load clamps. For these experiments, we used rat permeabilized cardiac myocyte preparations to simultaneously track ML and SL over a range of load clamps during half-maximal Ca²⁺ activations. Fig. 2 A shows representative ML, SL, and force traces during load clamps of ∼75%, 50%, and 25% isometric force for the rat permeabilized cardiac myocyte preparations. Fig. 2 B shows the cumulative force–velocity relationship and power–load curve at both ML and SL levels for five rat cardiac myocyte preparations. Fig. 2 B inset shows the relationship between ML and SL velocities for the rat permeabilized cardiac myocyte preparations. ML and SL traces were closely matched as SL velocities were ∼77% of ML velocities, derived from the slope (0.77) of the ML to SL relationship. The 95% confidence intervals of the relationship had a lower limit of 0.74 and upper limit of 0.80, and there is slightly more variation at higher velocities, i.e., lower loads. The ML to SL relationship had an r² value of 0.98 (Fig. 2 B inset). This ML to SL relationship indicates that the majority of ML shortening comprises sarcomere shortening and sarcomere shortening in the demarcated ROI, which encompassed ∼30% of the cardiac myocyte preparation width and largely represents cardiac myocyte preparation shortening during load clamps at half-maximal Ca²⁺ activation.

The second aim was to interrogate cMyBP-C’s role in regulating loaded shortening at both the ML and SL levels. We have previously observed relatively faster-loaded shortening and increased power at high loads, at least at the ML level, in permeabilized cardiac myocyte preparations lacking cMyBP-C (Korte et al., 2003; Hanft et al., 2021). We initially addressed whether the previously reported faster ML-loaded shortening (especially at high loads) also occurs at the sarcomere level by comparing permeabilized cardiac myocyte preparations obtained from transgenic mice with and without cMyBP-C. For these experiments, force–velocity and power–load relationships were characterized in cMyBP-C WT and cMyBP-C KO mouse permeabilized cardiac myocyte preparations, respectively, at both the ML and SL levels. Fig. 3 A shows representative ML, SL, and force traces during three different load clamps for a representative cMyBP-C WT mouse permeabilized cardiac myocyte preparation. Fig. 3 B shows the force–velocity relationship and power–load curve at both ML and SL levels for cMyBP-C WT mouse cardiac myocyte preparations (n = 5). The Fig. 3 B inset shows the relationship between ML and SL velocities for cMyBP-C WT mouse permeabilized cardiac myocyte preparations. For cMyBP-C WT, SL velocities were ∼67% of ML velocities since the slope of the ML to SL relationship was 0.67. The 95% confidence intervals had a lower limit of 0.60 and an upper limit of 0.73. The ML to SL relationship had an r² value of 0.86 (Fig. 3 B, inset). Similar experiments for cMyBP-C KO permeabilized cardiac myocyte preparations are shown in Fig. 4. Fig. 4 A shows representative ML, SL, and force traces during three different load clamps for a representative cMyBP-C KO mouse permeabilized cardiac myocyte preparation. Fig. 4 B shows the force–velocity relationship and power–load curve at both ML and SL levels for cMyBP-C KO mouse cardiac myocyte preparations (n = 5). Fig. 4 B inset shows the relationship between ML and SL velocities for the cMyBP-C KO mouse permeabilized cardiac myocyte preparations. The slope of the ML to SL relationship was 0.72 with 95% confidence intervals having a lower limit of 0.65 and an upper limit of 0.76. The ML to SL relationship had an r² value of 0.89 (Fig. 4 B, inset). Importantly, cMyBP-C KO cardiac myocyte preparations exhibited disproportionately high sarcomere shortening velocities at high loads (Fig. 4 B), a finding consistent with previous reports that tracked solely ML (Korte et al., 2003; Hanft et al., 2021). This indicates that the previously observed fast velocities at high loads in cMyBP-C KO myocytes were a function of sarcomere shortening as opposed to other factors such as variations in permeabilized myocyte preparation end compliance, i.e., compliance arising from attaching the ends of the myocyte preparation to our apparatus (illustrated in Fig. 1 A).

The third aim was to test the hypothesis that cMyBP-C regulates loaded shortening and power output by modulating cross-bridge availability. We addressed this aim by characterizing force–velocity and power–load relationships in cMyBP-C WT and KO mouse permeabilized cardiac myocyte preparations before and during treatment with the small molecule, mavacamten, a myosin cross-bridge inhibitor. Table 1 shows that mavacamten (0.5 µM) reduced maximal force, tension, and rates of force development, and decreased the myofilaments’ sensitivity to activator calcium (i.e., greater [Ca²⁺] was needed to attain approximately half-maximal tension) similarly in cMyBP-C WT and KO cardiac myocyte preparations. Fig. 5 A and Fig. 6 A show representative ML, SL, and force traces during three different load clamps of a cMyBP-C WT and KO mouse permeabilized cardiac myocyte preparation, respectively, in presence of mavacamten. Fig. 5 B and Fig. 6 B show the cumulative force–velocity relationship and power–load curve with mavacamten for a cMyBP-C WT and KO mouse cardiac myocyte preparation, respectively. Fig. 5 B inset shows the relationship between ML and SL velocities for cMyBP-C WT mouse permeabilized cardiac myocyte preparations with mavacamten. SL velocities were ∼73% of ML velocities, with the slope of the ML to SL relationship being 0.73 and the 95% confidence intervals having a lower limit of 0.68 and an upper limit of 0.78. The ML to SL relationship had an r² value of 0.94 (Fig. 5 B, inset). Fig. 6 B inset shows the relationship between ML and SL velocities for cMyBP-C KO mouse permeabilized cardiac myocyte preparations after mavacamten. SL velocities were ∼75% of ML velocities derived from the slope of the ML to SL relationship. The 95% confidence intervals had a lower limit of 0.71 and an upper limit of 0.79. The ML to SL relationship had an r² value of 0.93 (Fig. 6 B, inset). Fig. 7, A and B, shows cumulative force–velocity relationships before and with 0.5 µM mavacamten for cMyBP-C WT and KO cardiac myocyte preparations, respectively. Fig. 7 C shows cumulative power–load relationships before and with mavacamten for cMyBP-C WT and KO cardiac myocytes. In cMyBP-C KO myocytes, mavacamten normalized power output (Fig. 7 C) by shifting the power–load curves toward cMyBP-C WT permeabilized cardiac myocyte preparations. This was most obvious at high loads (shaded area in Fig. 7 C). Interestingly, mavacamten also reduced normalized power output in cMyBP-C WT cardiac myocytes, with the effect being greatest at high loads (Fig. 7 C). As follows, Fig. 8 compares relative power at loads between 65% and 85% isometric tension (Po) for all groups. Relative power at high loads was significantly higher in cMyBP-C KO cardiac myocytes compared with all other groups. In contrast, cMyBP-C WT cardiac myocytes treated with mavacamten exhibited significantly lower relative power at high loads compared with all other groups. In summary, these results indicate that mavacamten reduces loaded shortening velocity and power and suggest that cMyBP-C regulates sarcomere-loaded shortening by modulating cross-bridge availability, especially when sarcomeres are working against high loads relative to isometric tension.

A goal of this study was to systematically quantify the relationship between ML and SL during loaded shortening in mammalian permeabilized cardiac myocyte preparations. We report that ML and SL are closely, but not entirely, matched during load clamps and there is more variation in the ML:SL relationship at lower loads, i.e., higher shortening velocities. We also report that cMyBP-C regulates loaded shortening velocity by normalizing SL shortening velocity, especially at high loads, by seemingly modulating cross-bridge availability.

While some studies have reported SL shortening in intact (Daniels et al., 1984; De Tombe and Ter Keurs, 1990, 1991; De Tombe and ter Keurs, 1992) and permeabilized (McDonald et al., 1998; Korte and McDonald, 2007) cardiac muscle preparations, no studies, to our knowledge, have systematically quantified the relationship between ML and SL during loaded shortening in rodent permeabilized cardiac myocytes. We report that SL velocities were ∼70–80% of ML velocities during approximately half-maximal Ca²⁺ activation of rat and mouse permeabilized cardiac myocyte preparations. While the ML:SL relationships are highly correlated, the question arises as to why ML and SL are not matched to unity. Several factors may contribute to the offset, i.e., slightly faster ML velocities compared with SL velocities. First, while end compliance of these preparations is low (<3%) (McDonald et al., 1998), recoil shortening of compliant ends likely contributes to the offset. Second, the ROI of the striation pattern consists of only ∼30% of myocyte preparation width, so striation heterogeneity across the myocyte preparation is not monitored, thus, sarcomere heterogeneity also likely contributes to the offset. Third, there are other cellular/sarcomere components that likely contribute to non-unity ML versus SL shortening; these include cytoskeleton proteins, mitochondrial components, Z-discs, and titin. For example, it is plausible that increased passive tension may better unify ML and SL relationships; this remains to be assessed. Overall, our results show that ML and SL loaded shortening are closely, but not perfectly matched, and, thus, implicate that ML shortening is a strong quantitative and qualitative surrogate for sarcomere behavior during loaded shortening, at least during approximately half-maximal Ca²⁺ activation of rodent permeabilized cardiac myocyte preparations at 16 ± 1°C.

The second goal of this study was to interrogate the role that cMyBP-C plays in regulating loaded sarcomere shortening and power output. MyBP-C is a relatively abundant protein in striated muscle with 1 mol of MyBP-C per 9 mol of myosin. cMyBP-C has been localized to seven to nine transverse stripes spaced ∼43 nm apart in each half-thick filament in the region that is termed the C-zone (Pepe and Drucker, 1975; Craig and Offer, 1976; Bennett et al., 1986; Lee et al., 2015). Several different models have been proposed for MyBP-C’s organization in the sarcomere based on in vitro binding, electron microscopy, and x-ray diffraction studies. For example, Moolman-Smook et al. (2002) proposed that MyBP-C molecules form a collar around the backbone of the thick filament. It was speculated that the collar packs the thick filament backbone more tightly and restricts actin–myosin interactions, whereas the release of the collar loosens the backbone and enhances cross-bridge formation. Consistent with this model, both ablation of cMyBP-C and myofilaments containing phospho-mimetic cMyBP-C yield an apparent transfer of mass away from the thick filaments toward the thin filaments as quantified by greater I_I,I/I_I.0 intensity ratios from x-ray diffraction patterns (Colson et al., 2007). More recent immuno-EM and cryo-EM studies found an axial orientation of the C-terminal domains, suggesting that domains C10–C7 run parallel along the thick filament surface, and the N-terminus (C6–C0) extends toward the thin filaments (Lee et al., 2015; Dutta et al., 2023; Tamborrini et al., 2023). Interestingly, in vitro studies show the N-terminal region including the M-domain binds myosin at both its S2–S1 junction and the S1 neck region (Starr and Offer, 1978; Ababou et al., 2008; Bhuiyan et al., 2012). Interestingly, the N-terminus regions of MyBP-C also bind actin (with micromolar affinity) in vitro (Rybakova et al., 2011; van Dijk et al., 2014) and N-terminal binding of MyBP-C to both myosin (S2) and actin are reduced by PKA phosphorylation (Shaffer et al., 2009; Weith et al., 2012a, 2012b). Recent in vitro experiments have shown that filament sliding is slowed as thin filaments translocate into the C-zone of native thick filaments, and this process is modulated by cMyBP-C phosphorylation (Previs et al., 2012). Further, 3D reconstruction of electron micrographs of thin filaments decorated with cMyBP-C fragments indicate that cMyBP-C N-terminal domains compete with tropomyosin for the subdomain 1 of actin, implicating cMyBP-C directly interacts with actin; this interaction may assist in the activation of the thin filament (Whitten et al., 2008; Mun et al., 2014) and/or create a drag force that opposes shortening (Walcott et al., 2015; Robinett et al., 2019). Consistent with this, it was observed that transgenic expression of a mutant cMyBP-C with a point mutation (leu348pro) in the M domain, which increases the binding affinity of cMyBP-C to actin, increased Ca²⁺ sensitivity of force and slowed loaded shortening velocities (Bezold et al., 2013; Bezold, K.L., et al. 2014. Circulat. Res. Abstract 93. https://doi.org/10.1161/res.115.suppl_1.93). Together, these results provide the scientific premise that MyBP-C can both constrain myosin cross-bridges and impose an internal load in the sarcomere by binding to actin.

In this study, we tested the hypothesis that cMyBP-C regulates loaded shortening and power output by modulating cross-bridge availability. We characterized force–velocity and power–load relationships in mouse permeabilized cardiac myocyte preparations that were deficient in cMyBP-C at both the ML and SL levels before and after treatment with the small molecule, mavacamten, a myosin cross-bridge inhibitor. We found that ablation of cMyBP-C shifted force–velocity relationships and power–load curves upward at high loads at both the SL and ML levels, a finding consistent with our previous work at the ML level only (Korte et al., 2003; Hanft et al., 2021). Interestingly, we have measured force–velocity and power–load relationships under a plethora of conditions but have only observed inordinately elevated shortening velocities and power output at high loads in cMyBP-C KO mouse permeabilized cardiac myocyte preparations (McDonald, 2000a; Herron et al., 2001; Hinken and McDonald, 2004; Hinken et al., 2006, 2012; Korte and McDonald, 2007; McDonald et al., 2020; Hanft et al., 2021). This finding (observed only in cMyBP-C KO mouse permeabilized cardiac myocyte preparations) appears to involve increased cross-bridge availability since mavacamten, a cross-bridge inhibitor, normalized force–velocity and power–load relationships, especially at high loads. These results implicate cMyBP-C as a sarcomeric load sensor, which adjusts the number of cycling cross-bridges to match the load. This concept is consistent with high-resolution x-ray diffraction studies, whereby load increases the thick filament transitions to the ON state, thereby unlocking the additional cross-bridges required to sustain shortening and myosin cross-bridges in the cMyBP-C zone (C-zone), which are most active during after-loaded contractions (Brunello et al., 2020). It seems plausible that cMyBP-C could sense load via mechanotransduction through thick filaments, where cMyBP-C resides, or via cMyBP-C connections between thick and thin filaments. Altogether, cMyBP-C has the potential as a load sensor that matches sarcomere/myocardial power to system-level hemodynamics. This load sensor postulate necessitates additional experimental testing at multiple scales including isolated working hearts. The impact of cMyBP-C acting as a molecular load sensor is underscored by the fact that ∼30% of all familial hypertrophic cardiomyopathic (FHC) cases are linked to cMyBP-C, and alterations in cMyBP-C phosphorylation levels are implicated in cardiac stress pathways during normal physiologic signaling and pathological states including acquired heart failure (Harris et al., 2011; Kuster et al., 2012; Sadayappan and de Tombe, 2012; Gupta and Robbins, 2014; Hanft et al., 2017).

The data underlying Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, and Fig. 8 are available in the published article. All original force clamp traces are available from the corresponding author upon reasonable request.

Henk L. Granzier served as editor.

This work was supported by a National Heart, Lung, and Blood Institute grant (R01-HL148785 to K.S. McDonald). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author contributions: K.S. McDonald: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing, T.J. Kalogeris: Writing - review & editing, A.B. Veteto: Conceptualization, Data curation, Investigation, Methodology, Resources, Software, Validation, Writing - review & editing, D.J. Davis: Data curation, Methodology, Writing - review & editing, L.M. Hanft: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Validation, Visualization, Writing - original draft, Writing - review & editing.