Human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) have been used to screen and characterize drugs and to reveal mechanisms underlying cardiac diseases. However, before hiPSC-CMs can be used as a reliable experimental model, the physiological mechanisms underlying their normal function should be further explored. Accordingly, a major feature of hiPSC-CMs is automaticity, which is regulated by both Ca2+ and membrane clocks. To investigate the mechanisms coupling these clocks, we tested three hypotheses: (1) normal automaticity of spontaneously beating hiPSC-CMs is regulated by local Ca2+ releases (LCRs) and cAMP/PKA-dependent coupling of Ca2+ clock to M clock; (2) the LCR period indicates the level of crosstalk within the coupled-clock system; and (3) perturbing the activity of even one clock can lead to hiPSC-CM–altered automaticity due to diminished crosstalk within the coupled-clock system. By measuring the local and global Ca2+ transients, we found that the LCRs properties are correlated with the spontaneous beat interval. Changes in cAMP-dependent coupling of the Ca2+ and M clocks, caused by a pharmacological intervention that either activates the β-adrenergic or cholinergic receptor or upregulates/downregulates PKA signaling, affected LCR properties, which in turn altered hiPSC-CMs automaticity. Clocks’ uncoupling by attenuating the pacemaker current If or the sarcoplasmic reticulum Ca2+ kinetics, decreased hiPSC-CMs beating rate, and prolonged the LCR period. Finally, LCR characteristics of spontaneously beating (at comparable rates) hiPSC-CMs and rabbit SAN are similar. In conclusion, hiPSC-CM automaticity is controlled by the coupled-clock system whose function is mediated by Ca2+-cAMP-PKA signaling.

In recent years human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) have been used to model inherited as well as acquired cardiac diseases (Itzhaki et al, 2011, 2012; Chauveau et al, 2017; Ben Jehuda et al, 2018; Eisen et al, 2019). Further, hiPSC-CMs are employed to screen and characterize drugs and uncover mechanisms underlying cardiac diseases. However, before hiPSC-CMs can be employed as reliable experimental models, the physiological mechanisms controlling their normal function must be thoroughly explored. Spontaneously beating hiPSC-CMs have attracted enormous interest (Pozo et al., 2022; Morad and Zhang, 2017) because of their ability to generate rhythmic action potentials resembling the sinoatrial node (SAN) cells, which control heart rate. The SAN cell function is orchestrated by an ensemble of surface membrane channels, exchangers, and pumps (the so-called M clock) that are coupled to the sarcoplasmic reticulum (SR; the so-called Ca2+ clock). Spontaneous local Ca2+ release (LCR) from the SR, which activates the M clock to generate action potentials (Lakatta et al, 2010; Neco et al, 2012; Groenke et al, 2013; Yaniv et al, 2013; Tsutsui et al, 2018), and the cAMP/protein kinase A (PKA) activity which regulates mechanisms that drive both clocks (Yaniv et al., 2015; Behar et al., 2016), are the two main signaling cascades that couple these clocks. Thus, in mammalian pacemaker cells, the degree of coupling between the clocks depends on LCR characteristics and the level of cAMP/PKA signaling. Whereas it was shown that the two clocks coexist in human embryonic stem cell–derived cardiomyocytes (Zahanich et al., 2011), human SAN (Tsutsui et al., 2018), and spontaneously beating hiPSC-CMs (Mandel et al., 2012), it is unknown which signal controls their coupling. To investigate the mechanisms that couple these clocks, we tested three hypotheses in hiPSC-CMs: (1) normal automaticity of spontaneously beating hiPSC-CMs is regulated by LCRs and cAMP/PKA-dependent coupling of the Ca2+ and M clocks; (2) the LCR period indicates the level of crosstalk (i.e., the beat interval) within the coupled-clock system; and (3) perturbing the activity of even one clock, can lead to hiPSC-CM dysfunction due to diminished crosstalk within the coupled-clock system. Our main findings were (1) upregulation/downregulation of cAMP-dependent coupling between the clocks influence LCR properties, in turn affecting hiPSC-CMs automaticity; (2) clocks’ uncoupling by attenuating the pacemaker current If or SR Ca2+ kinetics decreased hiPSC-CMs beating rate and prolonged LCR period; and (3) LCR characteristics of spontaneously beating (at comparable rates) hiPSC-CMs and rabbit SAN are similar.

hiPSC generation and differentiation into cardiomyocytes

A skin biopsy was obtained from a 42-yr-old healthy female (clone 24.5) and a foreskin was obtained from a healthy neonatal (clone FSE-5m), as previously characterized and described (Yehezkel et al., 2011; Novak et al., 2015). All donors signed consent forms according to approval #3116 by the Helsinki Committee for experiments on human subjects at Rambam Health Care Campus, Haifa, Israel. hiPSCs were differentiated into cardiomyocytes (hiPSC-CMs) according to the directed differentiation by modulating Wnt/β-catenin signaling, as previously described (Lian et al., 2013).

Ca2+ imaging in single spontaneously beating hiPSC-CMs

Ca2+ images were recorded from 30–60-d-old spontaneously beating hiPSC-CMs. To this end, hiPSC-CM monolayers were dissociated by adding 2 ml/well trypsin-EDTA at room temperature (Biological Industries) followed by incubation at 37°C in 5% CO2 (Forma series 3 water-jacketed CO2 incubator) for 5 min. The contents of each well were collected in a conical tube to which 3 ml RPMI+ medium (RPMI 1640 [Gibco], B-27 [Gibco], and penicillin-streptomycin [Biological Industries]) was added, and was centrifuged (Spectrafuge 6C; Labnet) for 3 min at 800 rpm. The supernatant was aspirated, and the hiPSC-CMs were resuspended in 3 ml RPMI+ medium and reseeded onto the center of a collagen-coated glass-bottom culture dish (MatTek Corporation). The dishes were incubated (37°C, 5% CO2) for a recovery period of 5 d before performing the Ca2+ imaging experiments.

Ca2+ cycling into and out of the cytosol was recorded by monitoring the fluorescence of the Ca2+ indicator dye Fluo-4 AM (Thermo Fisher Scientific) using a LSM880 confocal microscope, as previously described (Davoodi et al., 2017). Dishes with spontaneously beating hiPSC-CMs were loaded with 2.5 μM Flou-4 AM for 20 min in the dark at room temperature and then washed with Tyrode’s solution (at 37°C) containing (in mM) 140 NaCl, 5.4 KCl, 10 HEPES, 2 Na-pyruvate, 10 glucose, 1 MgCl2, and 2 CaCl2 (pH 7.4). Fluorescence was observed by exciting the sample with a 488 nm argon laser and measuring fluorescence emission with LP 505 nm. Ca2+ images were acquired using a 40×/1.2 water immersion lens with line-scan mode (1.22 ms per scan; pixel size, 0.01 µm) oriented along the cell, close to the sarcolemmal membrane.

To identify global and LCRs from the SR and quantify their characteristics, line-scan images were analyzed using a modified version of a semiautomatic Matlab graphic user interface named Sparkalyzer (Davoodi et al., 2017). Briefly, the signal (F) was normalized by the minimal value between beats (F0). Ca2+ transients were semiautomatically detected, and LCRs were manually marked. LCRs were marked as a small increase in Ca2+ signal during the diastolic depolarization that lasted for at least 2 ms, 2 µm length, and at least 1.05 F/F0 height due to the microscope resolution constraints. Only a certain amount of Ca2+ signal can initiate the electrical activity, thus more than one LCR appears at the same beat interval. Beat interval (referred to as the cycle length in Davoodi et al. [2017]) was calculated as the mean beat interval of each cell, and 50 and 90% relaxation time and LCRs parameters were automatically calculated by the software as previously described (Davoodi et al., 2017). The LCR period is referred to as the time between the Ca2+ spark and the former peak (the time elapsed between former AP-induced Ca2+ transient and LCR appearance; Fig. 1; Davoodi et al., 2017). The number of LCRs is the number of detected LCRs in the line-scan Ca2+ image by the user, scaled by the time interval and length of the scan line. The Ca2+ signal of individual LCRs indicating the amount of Ca2+ released by each LCR was calculated using LCR parameters for each LCR as follows: 50% LCR duration × LCR length × normalized amplitude (ms·µm·F/F0).

Drugs

Isoproterenol (ISO), carbachol (CCh), forskolin, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), cyclopiazonic acid (CPA), and hydrochloroquine (HCQ) were purchased from Sigma-Aldrich. IVA was purchased from Toronto Research Chemicals.

Statistics

Data are presented as mean ± SEM. Data were compared using regressions, paired or unpaired Student’s t test, and two-sample Kolmogorov–Smirnov test. Differences were considered statistically significant at P < 0.05 or P < 0.01, as indicated in the respective figures and tables.

Online supplemental material

Fig. S1 shows the classification of control spontaneously beating hiPSC-CMs into two groups based on the average beat interval. Fig. S2 shows average dose-dependent changes in the beat interval in response to forskolin. Fig. S3 shows the percent changes in the beat interval in response to either doses of IVA or CPA and their combination. Fig. S4 shows HCQ-induced changes in global and local Ca2+ properties. Fig. S5 shows a 3-D relationship between hiPSC-CMs, rabbit sinoatrial node cells (SANCs), and rabbit ventricular cells based on LCR properties. Table S1 summarizes the global and local Ca2+ properties of control spontaneously beating hiPSC-CMs. Table S2 summarizes the global and local Ca2+ properties of the two clones used for the experiments. Tables S3, S4, S5, S6, and S7 summarize the effect of ISO and CCh (Table S3), 1 µM ISO (Table S4), forskolin and H-89 (Table S5), IVA and CPA (Table S6), and HCQ (Table S7) on global and local Ca2+ properties in spontaneously beating hiPSC-CMs. Table S8 compares the global and local Ca2+ properties of hiPSC-CMs, rabbit SANCs, and rabbit ventricular cells.

The relationship between LCR characteristics and the basal beat interval

To test the relationship between LCR characteristics and the basal beat interval, Ca2+ transients and LCRs were measured in spontaneously beating hiPSC-CMs. Fig. 1, A–C; and Table S1, respectively, show representative Ca2+ transients and LCRs detected over a wide range of beat intervals and their characteristics. The mean beat interval of hiPSC-CMs derived from Ca2+ line-scan images was 1,354.5 ± 55.7 ms (Fig. 1 D; n = 114), consistent with previously reported hiPSC-CMs beat intervals (Mandel et al., 2012). The high correlation between the LCR period and beat interval (Fig. 1 E; R2 = 0.86) suggests that one is a readout of the other. The LCR period also correlated with 90% time of relaxation (T90; Fig. 1 F), which indicates the 90% replenishment of SR Ca2+ following Ca2+-induced Ca2+ release (Vinogradova et al., 2010). The Gaussian distribution of the beating interval (a Gaussian fitting yielded a mean of 1,024.5 ms and an SD of 373.5 ms; Fig. 1 G) was similar to the LCR period distribution (Kolmogorov–Smirnov test, P < 0.05; Fig. 1 H). In contrast, the T90 distribution (Fig. 1 I) differed from the LCR period and beat interval distributions. Note that no differences were found regarding the beat interval or Ca2+ characteristics between the two clones (Table S2).

To determine whether LCR characteristics are dependent on the beat interval, the data were divided into two groups: hiPSC-CMs with a mean beat interval >2,000 and ≤2,000 ms. The resulting populations (Fig. S1 A) differed in their average T90 (Fig. S1 B), 50% LCR duration (Fig. S1 C), and LCR period (Fig. S1 D). The correlation between LCR period and beat interval (Fig. S1 E) and the LCR period and T90 (Fig. S1 F) was weaker (0.46 and 0.18, respectively) among hiPSC-CMs with beat intervals >2,000 ms. Thus, the LCR period is correlated with the basal beat interval.

LCR characteristics are affected by cAMP/PKA signaling

To determine the effect of altering cAMP/PKA signaling on beat interval and LCR characteristics, we used the non-selective β-adrenergic receptor agonist ISO and the cholinergic receptor agonist CCh. CCh was used at the concentration reported to cause a maximum increase in the beat interval (Nozaki et al., 2017), but that did not stop the spontaneous activity and the appearance of LCRs. ISO concentration was used at a concentration causing the same absolute change in the beat interval as CCh, leading to a decrease in the beat interval (Ben-Ari et al., 2014; Takeda et al., 2021). Of note, the basal beat intervals before drug application are from the same distribution and are not significantly different from each other. Fig. 2, A–D, shows representative examples of the effects of ISO and CCh on the Ca2+ transient. Specifically, 100 nM ISO decreased the beat interval by 22.6 ± 8.9% (n = 7, Fig. 2 E), while 100 nM CCh increased the beat interval by 28.8 ± 14.9% (n = 6, Fig. 2 E). Ca2+ transient characteristics time to peak, 50% relaxation (T50), and T90 were unaffected by ISO, but prolonged by CCh (Table S3). LCR characteristics were affected by ISO and CCh; the absolute changes in the LCR period (Fig. 3 A) and Ca2+ signals of individual LCRs (Fig. 3 B) were similar by either drug perturbation. ISO also increased the normalized LCR signal amplitude but decreased the number of LCRs (Table S3). In contrast, CCh decreased the normalized amplitude and length of LCRs. Finally, ISO and CCh did not alter the tight correlations between the LCR period and beat interval (Fig. 3 C; R2 = 0.86), and between the LCR period and T90 (Fig. 3 D; R2 = 0.59). Of note, a 1 µM ISO affected the beat interval and LCR characteristics (Table S4).

Next, adenylate cyclase was activated by forskolin, and PKA was inhibited by H-89. We used the lowest forskolin concentration that led to maximal shortening of the beat interval (Fig. S2) and the H-89 concentration that caused a similar maximum absolute change in the beat interval. We used the lower forskolin concentration that led to maximal shortening of the beat interval (Fig. S2) and the appropriate H-89 concentration that caused a similar maximum absolute change in the beat interval. Of note, the basal beat intervals before drug application are from the same distribution and are not significantly different from each other. Fig. 4, A–D shows representative examples illustrating the effects of forskolin and H-89 on the Ca2+ transients; H-89 induced bradycardia and arrhythmias in three of the six cells studied. The SD increased for H-89 from 153 ± 56 to 229 ± 56 s. Forskolin decreased the beat interval by 21.9 ± 10.2% (n = 6, Fig. 4 E), while H-89 prolonged the beat interval by 22.3 ± 12.8% (n = 6, Fig. 4 E). Additionally, Ca2+ transient characteristics (time to peak, T50, and T90) and LCR characteristics were unaffected by forskolin and H-89 (Table S5); the LCR period (Fig. 5 A) was decreased by forskolin. The change in the Ca2+ signal of individual LCRs (Fig. 5 B) was increased twofold by forskolin compared with H-89. Forskolin also increased the normalized LCR signal amplitude, while H-89 decreased the normalized amplitude and the number of LCRs and increased LCR duration by 50% (Table S5). Finally, forskolin and H-89 did not affect the tight correlations between the LCR period and beat interval (Fig. 5 C; R2 = 0.81), while the relationship between the LCR period and T90 was reduced (Fig. 5 D; R2 = 0.49).

LCR characteristics are affected by specific clock stimuli

It was demonstrated that the If blocker ivabradine (IVA; 3 µM) blocks only the HCN4 channel (Yaniv et al., 2012). At this concentration, IVA did not affect the T- or L-type or delayed outward potassium current densities (Bois et al., 1996) and SR Ca2+ content. Thus, IVA specifically perturbs the M clock with no direct effect on the Ca2+ clock. On the other hand, the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor CPA has no direct effect on membrane currents (funny and L-type current densities), but only on SR Ca2+ content (Yaniv et al., 2013). The IVA concentration used was previously shown to induce a maximum decrease in beat interval without affecting other channels (T- or L-type or delayed outward potassium current densities) than HCN4 (Chauveau et al., 2017; Yaniv et al., 2013) or the Ca2+ clock (Yaniv et al., 2013). CPA was applied at a concentration causing a similar change in beat interval as IVA but was shown not to affect key channels such as L-type and funny current densities. Fig. 6, A–D, depicts representative examples of the effects of IVA and CPA on Ca2+ transients. 3 µM IVA or 3 µM CPA increased the beat interval by 45.6 ± 7.7% (n = 9, Fig. 6 E) and 48.2 ± 11.7% (n = 7, Fig. 6 E), respectively. Ca2+ time to peak, T50, and T90 were prolonged by both IVA and CPA (Table S6). LCR characteristics were also affected by IVA and CPA; the LCR period (Fig. 7 A) and the Ca2+ signal of individual LCRs (Fig. 7 B) were similarly decreased by IVA and CPA. IVA also decreased the LCR length (Table S6), while CPA decreased the normalized LCR amplitude, as well as the length and number of LCRs. Finally, IVA and CPA did not affect the tight correlations between the LCR period and beat interval (Fig. 7 C; R2 = 0.79). However, the relationship between the LCR period and T90 was not maintained (Fig. 7 D; R2 = 0.54). Note that, when both IVA and CPA were used together, no further increase in the individual effect on the beat interval was measured (Fig. S3; n = 11).

LCR characteristics are affected by HCQ

To demonstrate that LCR characteristics are affected by changes in the beating rate, hiPSC-CMs were treated with HCQ, which causes bradycardia (Capel et al., 2015). We recently showed that HCQ-induced bradycardia was caused by a direct effect on the M clock and an indirect effect on the Ca2+ clock (Segal et al., 2022). Fig. S4, A and B, depicts a representative example of the HCQ effect on Ca2+ transients. Of the three tested HCQ concentrations (1, 3, and 10 µM), only 10 µM increased the beat interval (71.8 ± 13.2%, n = 7; Fig. S4 C). Ca2+ time to peak, T50, and T90 were prolonged by HCQ (Table S7). Further, HCQ increased the LCR period (Fig. S4 D) but decreased the Ca2+ signals of individual LCRs (Fig. S4 E) and their normalized amplitude (Table S7). Finally, the tight correlations between the LCR period and beat interval (Fig. S4 F; R2 = 0.54) and between the LCR period and T90 (Fig. S4 G; R2 = 0.01) were not preserved in the presence of HCQ.

LCR characteristics of hiPSC-CMs are similar to rabbit SAN cells than to rabbit ventricular cardiomyocytes

To explore the similarity between hiPSC-CMs and SAN cell features, we compared LCR characteristics of hiPSC-CMs that beat spontaneously at a beat interval of 1,022.5 ± 10.1 ms with rabbit SAN cells that beat spontaneously at 1,010.3 ± 50 ms and rabbit ventricular cells paced at 1 Hz. Fig. S5 shows the 3-D representation of the relationship between the LCR period, number of LCRs, and 50% LCR duration. The hiPSC-CMs can be separated from the ventricular cells based on the combination of LCR characteristics. We did not compare the properties to atrial cells because we used 30–60-d-old, spontaneously beating hiPSC-CMs, previously shown to present a low occurrence of atrial-like cells (Ben-Ari et al., 2016). Thus, LCR characteristics of spontaneously beating (at comparable rates) hiPSC-CMs and rabbit SAN are similar, as was shown before (Giannetti et al., 2021).

In the present study, we investigated the mechanisms that couple the hiPSC-CM M- and Ca2+ clocks. Drug-induced alterations in Ca2+ or cAMP/PKA signaling affected the spontaneous beat interval and LCRs, supporting the first hypothesis that normal automaticity of spontaneously beating hiPSC-CMs is regulated by Ca2+-cAMP/PKA–dependent coupling of the M- and Ca2+ clocks. Drugs that affect the coupling by disturbing clock mechanisms or their interconnected signaling affected the LCR period and beat interval. The tight correlation between the LCR period and beat interval in response to these drugs supports the hypothesis that the LCR period indicates the level of crosstalk within the coupled-clock system. Finally, disrupting only the M- or Ca2+ clocks by IVA or CPA, respectively, increased the spontaneous beat interval and altered LCR properties, supporting the second hypothesis that perturbing even one clock activity leads to hiPSC-CM dysfunction by decreasing crosstalk within the coupled-clock system.

The LCRs measured in hiPSC-CMs were similar to those reported for other mammals (Lakatta et al., 2010), including humans (Tsutsui et al., 2018). Table S8 presents Ca2+ transient and LCR characteristics of hiPSC-CMs, rabbit SANCs, which beat spontaneously at a similar rate (Davoodi et al., 2017), and paced (1 Hz) ventricular rabbit cells. The T90, the Ca2+ signal of individual LCRs, the LCR period, and the number of LCRs were comparable in hiPSC-CMs and rabbit SANCs. Similarities also existed between the LCR periods of human SANCs and hiPSC-CMs (Tsutsui et al., 2018). However, LCR properties were distinguished from paced ventricular rabbit cells. These results strengthen the hypothesis that spontaneously beating hiPSC-CMs have similar properties as human SANCs.

An increase or decrease in cAMP/PKA-dependent signaling affected the spontaneous activity of hiPSC-CMs. Two methods were used to manipulate the cAMP-dependent clock coupling: (1) autonomic stimuli (ISO or CCh) that affect automaticity by altering adenyl cyclase (AC)-cAMP/PKA–dependent signaling (Figs. 2 and 3) and (2) drugs (forskolin or H-89) that alter cAMP activity (Figs. 4 and 5). In general, ISO and forskolin increased the coupling between the M- and Ca2+ clocks through the increased cAMP-dependent activity of If and increased PKA-dependent activity of M clock–dependent ionic channels (e.g., the L-type Ca2+ channel and slow heart potassium current) and Ca2+ clock–related proteins (ryanodine receptor and phospholamban). Increased clock coupling enhances the interplay between the L-type Ca2+ channel and SR Ca2+ cycling, which lead to changes in LCR parameters (Lyashkov et al., 2018). The net increase in Ca2+ affects the membrane potential through changes in Na+–Ca2+ exchanger (NCX) current (INCX; Bogdanov et al., 2001) and Ca2+-activated SK4 and BK potassium currents (Weisbrod et al., 2013; Lai et al., 2014), which decrease the spontaneous beating interval. CCh and H-89 decreased the coupling between the clocks through a decrease in the interplay between L-type Ca2+ channels and SR Ca2+ cycling, thereby prolonging the spontaneous beat interval. Note, that although LCRs were measured in human SAN (Tsutsui et al., 2018), the mechanisms that control their activity and the LCR period that reports the level of crosstalk within the coupled-clock system were not shown.

This study demonstrated that the magnitude of drug-induced bradycardia is not solely driven by the activity of one channel or one specific clock mechanism, but rather, by the potent crosstalk within the coupled-clock system. The approach used to show this was similar to that applied for SANCs (Yaniv et al., 2013) by CPA, which inhibits SERCA2 Ca2+ pumping but not the M clock directly, or IVA, which inhibits If but does not affect the Ca2+ clock. Both compounds increased beat interval to the same extent and affected LCR characteristics similarly (Figs. 6 and 7). Ca2+ pumping into the SR via SERCA was suppressed by CPA, thereby reducing SR Ca2+ load and subsequently prolonging the LCR period. In turn, the spontaneous LCR Ca2+ signal to activate NCX and Ca2+-activated K channels (SK4 and BK) is reduced and arrived later during the diastolic phase. The resultant decreased feedback between the M- and Ca2+ clocks leads to an increase in the beat interval. In turn, the net Ca2+ influx is reduced, which decreases the interplay between the L-type Ca2+ channel and SR Ca2+ cycling (Lyashkov et al., 2018), leading to further reductions in SR Ca2+ cycling kinetics and prolongation of the LCR period. Similarly, If was inhibited by IVA (Ben-Ari et al., 2014), leading to an increased beat interval, which reduced the net Ca2+ influx and thus the interplay between the L-type Ca2+ channel and SR Ca2+. Reduction in the interaction between altered SR Ca2+ kinetics reduced and delayed the spontaneous LCR ensemble Ca2+ signal to the M clock to activate NCX and Ca2+-activated K channels, subsequently resulting in a further increase in the beat interval. In general, any HCN intervention that increased beat interval will affect the Ca2+ clock. Of note, we employed spontaneous beating hiPSC-CMs that have a higher density of If than young CMs (<30-d-old; Ben-Ari et al., 2016). Thus, we found a substantial effect of IVA on the spontaneous beat interval compared with other groups who used younger hiPSC-CMs (Zhang et al., 2015; Kim et al., 2015).

The current work also demonstrated that the LCR period indicates the level of crosstalk within the coupled-clock system, regardless of the degree of clock coupling perturbation. Longer beat intervals due to spontaneous activity (Fig. S1) and reduced AC-cAMP/PKA activity were associated with a longer LCR period. In contrast, shorter beat intervals resulting from increased AC-cAMP/PKA activity were associated with shorter LCR periods. The relative changes in the LCR period were tightly correlated (R2 = 0.97) to the relative change in the spontaneous beat interval (Fig. 8 A), as was found in rabbit (Vinogradova et al., 2010) and human (Tsutsui et al., 2018) SANCs. It was shown that T90 is an indicator of the rate of SR Ca2+ pumping into the SR (Vinogradova et al., 2010). The fact that the relative changes in the LCR period are tightly correlated (R2 = 0.86) to the relative change in T90 (Fig. 8 B) further strengthens the conclusion that the LCR period indicates the degree of interplay between the L-type Ca2+ channel and SR Ca2+ cycling (Fig. 8 C). Note, the drug that induced bradycardia (forskolin, IVA, CPA, and H-89) decreased the degree of correlation between LCR period and T90.

In addition to IVA, the effect of HCQ (a drug that causes bradycardia; Capel et al., 2015) was determined. HCQ was shown to reduce If, the L-type Ca2+ current, and the rapidly activating potassium current in isolated guinea pig SAN cells (Capel et al., 2015). We found here (Fig. S4) that HCQ-induced bradycardia was associated with a prolonged LCR period and reduced Ca2+ signal of individual LCRs. These results suggest that HCQ affects the spontaneous beating of hiPSC-CMs through changes in the signaling cascades that couple the Ca2+ and M clocks. Note that HCQ reduced the degree of clock coupling to a level where the LCR period is not an indicator of the spontaneous beat interval. Similar phenomena were observed when the physiological spontaneous beat interval was long.

Study limitation

The drug perturbation experiments were conducted on hiPSC-CMs that beat at a rate similar to that of the human heart. We also showed here that the LCR characteristics were comparable between human SAN, which beats at 1–1.5 Hz, and spontaneously beating hiPSC-CMs. It was previously shown that the If density (Giannetti et al., 2021) and action potential characteristics (Ben-Ari et al., 2016) were comparable between human SAN cells and spontaneously beating hiPSC-CMs. However, the exact cardiac origin (i.e., ventricular, sinoatrial, or atrial) of the hiPSC-CMs used in this study was not determined. Future use of SAN cells derived from hiPSCs (Protze et al., 2017) will enable further clarification of the similarities between these cell sources.

David A. Eisner served as editor.

The work was supported by Israel Science Foundation grants ISF 330/19 (Y. Yaniv) and ISF 824/19 (O. Binah), The Rappaport Family Institute for Research in the Medical Science grant 01012020RI (O. Binah), Niedersachsisches Ministerium: Medizinischen Hochschule Hannover grant 11-76251-99-16/14 (O. Binah), and The US-Israel Binational Science Foundation (O. Binah). The funders had no role in study design, data collection or analysis, decision to publish, or preparation of the manuscript.

The authors declare no competing financial interests.

Author contribution: Y. Yaniv conceived and designed the research. S. Mazgaoker performed the experiments and analysis. S. Mazgaoker, I. Weiser-Bitoun, and I. Brosh generated the cells. O. Binah contributed reagents. Y. Yaniv drafted the manuscript. S. Mazgaoker, O. Binah, and Y. Yaniv edited and revised the manuscript. S. Mazgaoker, I. Weiser-Bitoun, I. Brosh, O. Binah, and Y. Yaniv approved the final version.

Behar
,
J.
,
A.
Ganesan
,
J.
Zhang
, and
Y.
Yaniv
.
2016
.
The autonomic nervous system regulates the heart rate through cAMP-PKA dependent and independent coupled-clock pacemaker cell mechanisms
.
Front. Physiol.
7
:
419
.
Ben Jehuda
,
R.
,
B.
Eisen
,
Y.
Shemer
,
L.N.
Mekies
,
A.
Szantai
,
I.
Reiter
,
H.
Cui
,
K.
Guan
,
S.
Haron-Khun
,
D.
Freimark
, et al
.
2018
.
CRISPR correction of the PRKAG2 gene mutation in the patient’s induced pluripotent stem cell-derived cardiomyocytes eliminates electrophysiological and structural abnormalities
.
Heart Rhythm
.
15
:
267
276
.
Ben-Ari
,
M.
,
R.
Schick
,
L.
Barad
,
A.
Novak
,
E.
Ben-Ari
,
A.
Lorber
,
J.
Itskovitz-Eldor
,
M.R.
Rosen
,
A.
Weissman
, and
O.
Binah
.
2014
.
From beat rate variability in induced pluripotent stem cell-derived pacemaker cells to heart rate variability in human subjects
.
Heart Rhythm
.
11
:
1808
1818
.
Ben-Ari
,
M.
,
S.
Naor
,
N.
Zeevi-Levin
,
R.
Schick
,
R.
Ben Jehuda
,
I.
Reiter
,
A.
Raveh
,
I.
Grijnevitch
,
O.
Barak
,
M.R.
Rosen
, et al
.
2016
.
Developmental changes in electrophysiological characteristics of human-induced pluripotent stem cell-derived cardiomyocytes
.
Heart Rhythm
.
13
:
2379
2387
.
Bogdanov
,
K.Y.
,
T.M.
Vinogradova
, and
E.G.
Lakatta
.
2001
.
Sinoatrial nodal cell ryanodine receptor and Na+-Ca2+ exchanger: Molecular partners in pacemaker regulation
.
Circ. Res.
88
:
1254
1258
.
Bois
,
P.
,
J.
Bescond
,
B.
Renaudon
, and
J.
Lenfant
.
1996
.
Mode of action of bradycardic agent, S 16257, on ionic currents of rabbit sinoatrial node cells
.
Br. J. Pharmacol.
118
:
1051
1057
.
Capel
,
R.A.
,
N.
Herring
,
M.
Kalla
,
A.
Yavari
,
G.R.
Mirams
,
G.
Douglas
,
G.
Bub
,
K.
Channon
,
D.J.
Paterson
,
D.A.
Terrar
, and
R.A.B.
Burton
.
2015
.
Hydroxychloroquine reduces heart rate by modulating the hyperpolarization-activated current If: Novel electrophysiological insights and therapeutic potential
.
Heart Rhythm
.
12
:
2186
2194
.
Chauveau
,
S.
,
E.P.
Anyukhovsky
,
M.
Ben-Ari
,
S.
Naor
,
Y.-P.
Jiang
,
P.
Danilo
Jr
,
T.
Rahim
,
S.
Burke
,
X.
Qiu
,
I.A.
Potapova
, et al
.
2017
.
Induced pluripotent stem cell-derived cardiomyocytes provide in vivo biological pacemaker function
.
Circ. Arrhythm. Electrophysiol.
10
:e004508.
Davoodi
,
M.
,
S.
Segal
,
N.
Kirschner Peretz
,
D.
Kamoun
, and
Y.
Yaniv
.
2017
.
Semi-automated program for analysis of local Ca2+ spark release with application for classification of heart cell type
.
Cell Calcium
.
64
:
83
90
.
Eisen
,
B.
,
R.
Ben Jehuda
,
A.J.
Cuttitta
,
L.N.
Mekies
,
Y.
Shemer
,
P.
Baskin
,
I.
Reiter
,
L.
Willi
,
D.
Freimark
,
M.
Gherghiceanu
, et al
.
2019
.
Electrophysiological abnormalities in induced pluripotent stem cell-derived cardiomyocytes generated from Duchenne muscular dystrophy patients
.
J. Cell. Mol. Med.
23
:
2125
2135
.
Giannetti
,
F.
,
P.
Benzoni
,
G.
Campostrini
,
R.
Milanesi
,
A.
Bucchi
,
M.
Baruscotti
,
P.
Dell’Era
,
A.
Rossini
, and
A.
Barbuti
.
2021
.
A detailed characterization of the hyperpolarization-activated “funny” current (If) in human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes with pacemaker activity
.
Pflugers Arch.
473
:
1009
1021
.
Groenke
,
S.
,
E.D.
Larson
,
S.
Alber
,
R.
Zhang
,
S.T.
Lamp
,
X.
Ren
,
H.
Nakano
,
M.C.
Jordan
,
H.S.
Karagueuzian
,
K.P.
Roos
, et al
.
2013
.
Complete atrial-specific knockout of sodium-calcium exchange eliminates sinoatrial node pacemaker activity
.
PLoS One
.
8
:e81633.
Itzhaki
,
I.
,
L.
Maizels
,
I.
Huber
,
L.
Zwi-Dantsis
,
O.
Caspi
,
A.
Winterstern
,
O.
Feldman
,
A.
Gepstein
,
G.
Arbel
,
H.
Hammerman
, et al
.
2011
.
Modelling the long QT syndrome with induced pluripotent stem cells
.
Nature
.
471
:
225
229
.
Itzhaki
,
I.
,
L.
Maizels
,
I.
Huber
,
A.
Gepstein
,
G.
Arbel
,
O.
Caspi
,
L.
Miller
,
B.
Belhassen
,
E.
Nof
,
M.
Glikson
, and
L.
Gepstein
.
2012
.
Modeling of catecholaminergic polymorphic ventricular tachycardia with patient-specific human-induced pluripotent stem cells
.
J. Am. Coll. Cardiol.
60
:
990
1000
.
Kim
,
J.J.
,
L.
Yang
,
B.
Lin
,
X.
Zhu
,
B.
Sun
,
A.D.
Kaplan
,
G.C.L.
Bett
,
R.L.
Rasmusson
,
B.
London
, and
G.
Salama
.
2015
.
Mechanism of automaticity in cardiomyocytes derived from human induced pluripotent stem cells
.
J. Mol. Cell. Cardiol.
81
:
81
93
.
Lai
,
M.H.
,
Y.
Wu
,
Z.
Gao
,
M.E.
Anderson
,
J.E.
Dalziel
, and
A.L.
Meredith
.
2014
.
BK channels regulate sinoatrial node firing rate and cardiac pacing in vivo
.
Am. J. Physiol. Heart Circ. Physiol.
307
:
H1327
H1338
.
Lakatta
,
E.G.
,
V.A.
Maltsev
, and
T.M.
Vinogradova
.
2010
.
A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart’s pacemaker
.
Circ. Res.
106
:
659
673
.
Lian
,
X.
,
J.
Zhang
,
S.M.
Azarin
,
K.
Zhu
,
L.B.
Hazeltine
,
X.
Bao
,
C.
Hsiao
,
T.J.
Kamp
, and
S.P.
Palecek
.
2013
.
Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions
.
Nat. Protoc.
8
:
162
175
.
Lyashkov
,
A.E.
,
J.
Behar
,
E.G.
Lakatta
,
Y.
Yaniv
, and
V.A.
Maltsev
.
2018
.
Positive feedback mechanisms among local Ca releases, NCX, and ICaL ignite pacemaker action potentials
.
Biophys. J.
114
:
1176
1189
.
Mandel
,
Y.
,
A.
Weissman
,
R.
Schick
,
L.
Barad
,
A.
Novak
,
G.
Meiry
,
S.
Goldberg
,
A.
Lorber
,
M.R.
Rosen
,
J.
Itskovitz-Eldor
, and
O.
Binah
.
2012
.
Human embryonic and induced pluripotent stem cell-derived cardiomyocytes exhibit beat rate variability and power-law behavior
.
Circulation
.
125
:
883
893
.
Morad
,
M.
, and
X.H.
Zhang
.
2017
.
Mechanisms of spontaneous pacing: Sinoatrial nodal cells, neonatal cardiomyocytes, and human stem cell derived cardiomyocytes
.
Can. J. Physiol. Pharmacol.
95
:
1100
1107
.
Neco
,
P.
,
A.G.
Torrente
,
P.
Mesirca
,
E.
Zorio
,
N.
Liu
,
S.G.
Priori
,
C.
Napolitano
,
S.
Richard
,
J.P.
Benitah
,
M.E.
Mangoni
, and
A.M.
Gomez
.
2012
.
Paradoxical effect of increased diastolic Ca2+ release and decreased sinoatrial node activity in a mouse model of catecholaminergic polymorphic ventricular tachycardia
.
Circulation
.
126
:
392
401
.
Novak
,
A.
,
L.
Barad
,
A.
Lorber
,
M.
Gherghiceanu
,
I.
Reiter
,
B.
Eisen
,
L.
Eldor
,
J.
Itskovitz-Eldor
,
M.
Eldar
,
M.
Arad
, and
O.
Binah
.
2015
.
Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations
.
J. Cell. Mol. Med.
19
:
2006
2018
.
Nozaki
,
Y.
,
Y.
Honda
,
H.
Watanabe
,
S.
Saiki
,
K.
Koyabu
,
T.
Itoh
,
C.
Nagasawa
,
C.
Nakamori
,
C.
Nakayama
,
H.
Iwasaki
, et al
.
2017
.
CSAHi study-2: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes: Assessment of reference compounds and comparison with non-clinical studies and clinical information
.
Regul. Toxicol. Pharmacol.
88
:
238
251
.
Pozo
,
M.R.
,
G.W.
Meredith
, and
E.
Entcheva
.
2022
.
Human iPSC-Cardiomyocytes as an experimental model to study epigenetic modifiers of electrophysiology
.
Cells
.
11
:
200
.
Protze
,
S.I.
,
J.
Liu
,
U.
Nussinovitch
,
L.
Ohana
,
P.H.
Backx
,
L.
Gepstein
, and
G.M.
Keller
.
2017
.
Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker
.
Nat. Biotechnol.
35
:
56
68
.
Segal
,
S.
,
L.
Arbel-Ganon
,
S.
Mazgaoker
,
M.
Davoodi
, and
Y.
Yaniv
.
2022
.
Increase in Ca2+-activated cAMP/PKA signaling prevents hydroxychloroquine-induced bradycardia of the cardiac pacemaker
.
Front. Physiol.
13
:
839140
.
Takeda
,
M.
,
S.
Miyagawa
,
E.
Ito
,
A.
Harada
,
N.
Mochizuki-Oda
,
M.
Matsusaki
,
M.
Akashi
, and
Y.
Sawa
.
2021
.
Development of a drug screening system using three-dimensional cardiac tissues containing multiple cell types
.
Sci. Rep.
11
:
5654
.
Tsutsui
,
K.
,
O.J.
Monfredi
,
S.G.
Sirenko-Tagirova
,
L.A.
Maltseva
,
R.
Bychkov
,
M.S.
Kim
,
B.D.
Ziman
,
K.V.
Tarasov
,
Y.S.
Tarasova
,
J.
Zhang
, et al
.
2018
.
A coupled-clock system drives the automaticity of human sinoatrial nodal pacemaker cells
.
Sci. Signal.
11
. eaap7608.
Vinogradova
,
T.M.
,
D.X.
Brochet
,
S.
Sirenko
,
Y.
Li
,
H.
Spurgeon
, and
E.G.
Lakatta
.
2010
.
Sarcoplasmic reticulum Ca2+ pumping kinetics regulates timing of local Ca2+ releases and spontaneous beating rate of rabbit sinoatrial node pacemaker cells
.
Circ. Res.
107
:
767
775
.
Weisbrod
,
D.
,
A.
Peretz
,
A.
Ziskind
,
N.
Menaker
,
S.
Oz
,
L.
Barad
,
S.
Eliyahu
,
J.
Itskovitz-Eldor
,
N.
Dascal
,
D.
Khananshvili
, et al
.
2013
.
SK4 Ca2+ activated K+ channel is a critical player in cardiac pacemaker derived from human embryonic stem cells
.
Proc. Natl. Acad. Sci. USA
.
110
:
E1685
E1694
.
Yaniv
,
Y.
,
V.A.
Maltsev
,
B.D.
Ziman
,
H.A.
Spurgeon
, and
E.G.
Lakatta
.
2012
.
The “funny” current inhibition by ivabradine at membrane potentials encompassing spontaneous depolarization in pacemaker cells
.
Molecules
.
17
:
8241
8254
.
Yaniv
,
Y.
,
S.
Sirenko
,
B.D.
Ziman
,
H.A.
Spurgeon
,
V.A.
Maltsev
, and
E.G.
Lakatta
.
2013
.
New evidence for coupled clock regulation of the normal automaticity of sinoatrial nodal pacemaker cells: Bradycardic effects of ivabradine are linked to suppression of intracellular Ca2+ cycling
.
J. Mol. Cell. Cardiol.
62
:
80
89
.
Yaniv
,
Y.
,
A.
Ganesan
,
D.
Yang
,
B.D.
Ziman
,
A.E.
Lyashkov
,
A.
Levchenko
,
J.
Zhang
, and
E.G.
Lakatta
.
2015
.
Real-time relationship between PKA biochemical signal network dynamics and increased action potential firing rate in heart pacemaker cells: Kinetics of PKA activation in heart pacemaker cells
.
J. Mol. Cell. Cardiol.
86
:
168
178
.
Yehezkel
,
S.
,
A.
Rebibo-Sabbah
,
Y.
Segev
,
M.
Tzukerman
,
R.
Shaked
,
I.
Huber
,
L.
Gepstein
,
K.
Skorecki
, and
S.
Selig
.
2011
.
Reprogramming of telomeric regions during the generation of human induced pluripotent stem cells and subsequent differentiation into fibroblast-like derivatives
.
Epigenetics
.
6
:
63
75
Zahanich
,
I.
,
S.G.
Sirenko
,
L.A.
Maltseva
,
Y.S.
Tarasova
,
H.A.
Spurgeon
,
K.R.
Boheler
,
M.D.
Stern
,
E.G.
Lakatta
, and
V.A.
Maltsev
.
2011
.
Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling
.
J. Mol. Cell. Cardiol.
50
:
66
76
.
Zhang
,
X.H.
,
H.
Wei
,
T.
Šarić
,
J.
Hescheler
,
L.
Cleemann
, and
M.
Morad
.
2015
.
Regionally diverse mitochondrial calcium signaling regulates spontaneous pacing in developing cardiomyocytes
.
Cell Calcium
.
57
:
321
336
.
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