Membrane Calcium Activation in Excitation-Contraction Coupling

Depolarization thresholds for eliciting tension and Ca electrogenesis have been compared in isolated crayfish muscle fibers. Just-detectable tensions and Ca spikes induced after treatment with procaine were elicited with intracellularly applied depolarizing currents of fixed duration. Both thresholds were found to increase in a similar manner in fibers exposed to increased concentrations of Ca in the bathing solution or addition of other divalent cations (Mg, Mn, Ni). However, antagonistic effects between divalent cations were also demonstrated. Substitution of increasing amounts of NaSCN for NaCl in the standard saline produced a progressive decrease in both thresholds. The correlation in the change in thresholds for the two processes supports the hypothesis that a change in membrane Ca conductance is an integral step in excitation-contraction coupling.     

Excitation-Contraction Coupling in Crayfish

High-sensitivity recording techniques demonstrate a continuousrelation between the onset and magnitude ot tension and themembrane depolarization that is induced by increasing K in thebathing medium or by intracellularly applied outward currents.This finding is not consistent with the mechanism of signallinge-c coupling by electrotonic spread of a "critical" depolarizationinward along the membrane of the transverse tubular system.It is in accord, however, with the channelled current mechanismthat is based on the known anion-permselectivity of the membranein the terminals of the TTS. The channelled-current model alsopredicts a direct role of Cl and a possible interaction betweenCa and CI in e-c coupling. The initiation and maintenance oftension as well as its magnitude, are in fact dependent uponthe concentrations of Ca and Cl in the medium. Thus, both thesignalling to, and the activation of, the contractile systemappear to be performed by a flow of current in the loop: cellmembrane – cell interior – TTS membrane –TTS channels – exterior, as is envisaged in the channelled-currentmodel of e-c coupling.     

Evidence for Anion-Permselective Membrane in Crayfish Muscle Fibers and Its Possible Role in Excitation-Contraction Coupling

Under certain conditions only, isolated crayfish skeletal muscle fibers change in appearance, becoming grainy, darkening, and seemingly losing their striations. These changes result from development of large vesicles on both sides of the Z-line. The longitudinal sarcoplasmic reticulum remains unaffected. The vesicles are due to swelling of a transverse tubular system (TTS) which is presumably homologous with the T-system tubules of other muscle fibers. The vesiculations occur during efflux of water or on reducing external K or Cl, but only when KCl can leave the fiber. They never result from osmotic, ionic, or electrical changes when KCl cannot leave. Inward currents, applied through a KCl-filled intracellular cathode, also cause the vesiculations. These are not produced when the cathode is filled with K-propionate, nor by outward or longitudinal currents. Thus the transverse tubules swell only when Cl leaves the cell. Accordingly, their membrane is largely or exclusively anion-permselective. These findings also indicate that the TTS forms part of a current loop, connecting with the exterior of the fiber probably through radial tubules (RT) possessing membrane of low conductivity. Thus, part of the current flowing inward across the sarcolemma during activity can return to the exterior through the membrane of the TTS. The structure and properties of the latter offer the possibility for an efficient electrical mechanism to initiate excitation-contraction coupling.     

Ca2+ Sparks Act as Potent Regulators of Excitation-Contraction Coupling in Airway Smooth Muscle

Ca²⁺ sparks are short lived and localized Ca²⁺ transients resulting from the opening of ryanodine receptors in sarcoplasmic reticulum. These events relax certain types of smooth muscle by activating big conductance Ca²⁺-activated K⁺ channels to produce spontaneous transient outward currents (STOCs) and the resultant closure of voltage-dependent Ca²⁺ channels. But in many smooth muscles from a variety of organs, Ca²⁺ sparks can additionally activate Ca²⁺-activated Cl⁻ channels to generate spontaneous transient inward current (STICs). To date, the physiological roles of Ca²⁺ sparks in this latter group of smooth muscle remain elusive. Here, we show that in airway smooth muscle, Ca²⁺ sparks under physiological conditions, activating STOCs and STICs, induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations. Paradoxically, BiMPTs stabilize the membrane potential by clamping it within a negative range and prevent the generation of action potentials. Moreover, blocking either Ca²⁺ sparks or hyperpolarization components of BiMPTs activates voltage-dependent Ca²⁺ channels, resulting in an increase in global [Ca²⁺]_i and cell contraction. Therefore, Ca²⁺ sparks in smooth muscle presenting both STICs and STOCs act as a stabilizer of membrane potential, and altering the balance can profoundly alter the status of excitability and contractility. These results reveal a novel mechanism underlying the control of excitability and contractility in smooth muscle.     

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca²⁺ signaling is a fundamental physiological process in living cells. Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in the striated muscle fibers that appear as highly localized Ca²⁺ release events mediated by ryanodine receptor (RyR) Ca²⁺ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca²⁺ sparks could provide information on the intracellular Ca²⁺ handling properties of healthy and diseased striated muscles. Although Ca²⁺ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca²⁺ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca²⁺ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca²⁺ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca²⁺ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP₃R) and RyR contributes to Ca²⁺ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca²⁺ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).     

Autoradiographic Studies of Intracellular Calcium in Frog Skeletal Muscle

Autoradiographs consisting of a 1000 A thick tissue section and a 1400 A thick emulsion film have been prepared from frog toe muscles labeled with Ca⁴⁵. The muscles had been fixed with an oxalate-containing osmium solution at rest at room temperature, at rest at 4°C, during relaxation following K⁺ depolarization or after prolonged depolarization. From 6 to 39 per cent of K⁺ contracture tension was produced during fixation. The grains in the autoradiographs were always concentrated in the center 0.2 to 0.3 µ of the I band and the region of the overlapping of the thick and thin filaments. The greater the tension produced during fixation, the greater was the concentration in the A band and the smaller the concentration in the I band. Autoradiographs of two muscles fixed by freeze-substitution resembled those of muscles which produced little tension during osmium fixation. Muscles which shortened during fixation produced fewer grains. In the narrow (<2.0 µ) sarcomeres of the shortened muscles, grain density decreased with decreasing sarcomere width. A theoretical analysis of the significance of these grain distributions is proposed and discussed.     

兴奋-收缩偶联中DHPR与RyR的相互作用

兴奋-收缩偶联（E—C coupling）依赖纽胞膜二氢吡啶受体（DHPR）／L型电压门控Ca^2＋通道和肌浆网兰诺定受体（RyR）／Ca^2＋释放通道的相互作用。在骨骼肌细胞中,DHPR与RyRl在结构上二机械偶联,不依赖细胞外Ca^2＋即可激活RyRl;在心肌细胞中,去极化激活DHPR,细胞外Ca^2＋内流,内流的Ca^2＋通过钙诱导钙释放（CICR）机制激活RyR2。最近的研究表明,DHPR与RyR之间的信号转导通常是双向的。DHPR与RyR机械和化学的双向偶联机制调节这两种Ca^2＋通道的效率、精确度和活性。     

The Roles of Calcium in Excitation-Contraction Coupling in Various Muscles of the Frog, Mouse, and Barnacle

In rectus abdominis muscles of the frog the active shorteningprovoked by 15–40 mM K was supported by Ca, Sr, and Ba,but not by Ni, Co, Mn, Cd, or Zn ions. Addition of the lattercations to a solution containing Ca decreased the responsesin a manner suggesting competitive inhibition. The shorteningof the rectus muscle found in divalent cation-free, low K solutionsis abolished by Ni, Co, Mn, and Mg. In rat muscles a transientincrease in the contractural responses to elevated potassiumwas observed when Ni was applied following partial washout ofCa. In single muscle fibers of the barnacle, development oftension was supported by Ca and Sr, and the other divalent cationswere without effect. Retention of Ca⁴⁵ in barnacle resting musclefibers soaked in solutions containing 10 mM Ca for 2 min andsubsequently washed for 10 min was 60 ± 3.1 mµMCa/g, whereas retention of Ca⁴⁵ in contracting muscles similarlyexposed to Ca⁴⁵ was 156 + 17 mµM Ca/g of fresh muscle.The results are compatible with the idea that activation ofcontraction in some types of muscles is due to entry of extracellularCa.     

Voltage-Activated Elementary Calcium Release Events in Isolated Mouse Skeletal Muscle Fibers

The elementary Ca²⁺-release events underlying voltage-activated myoplasmic Ca²⁺ transients in mammalian muscle remain elusive. Here, we looked for such events in confocal line-scan (x,t) images of fluo-3 fluorescence taken from isolated adult mouse skeletal muscle fibers held under voltage-clamp conditions. In response to step depolarizations, spatially segregated fluorescence signals could be detected that were riding on a global increase in fluorescence. These discrete signals were separated using digital filtering in the spatial domain; mean values for their spatial half-width and amplitude were 1.99 ± 0.09 μm and 0.16 ± 0.005 ΔF/F ₀ (n = 151), respectively. Under control conditions, the duration of the events was limited by the pulse duration. In contrast, in the presence of maurocalcine, a scorpion toxin suspected to disrupt the process of repolarization-induced ryanodine receptor (RyR) closure, events uninterrupted by the end of the pulse were readily detected. Overall results establish these voltage-activated low-amplitude local Ca²⁺ signals as inherent components of the physiological Ca²⁺-release process of mammalian muscle and suggest that they result from the opening of either one RyR or a coherently operating group of RyRs, under the control of the plasma membrane polarization.     

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1^−/−) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca²⁺ cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1^−/− compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca²⁺ transient (Δ[Ca²⁺]_i) responses in NOS1^−/− cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca²⁺]_i across the range of pacing frequencies and increased myofilament Ca²⁺ sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca²⁺ reuptake, and restored SR Ca²⁺ content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca²⁺ leak, HYD improves Ca²⁺ cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.     

The Intracellular Site of Calcium Activation of Contraction in Frog Skeletal Muscle

Radioautography has been used to localize ⁴⁵Ca in isotopically labeled frog skeletal muscle fibers which had been quickly frozen during a maintained tetanus, a declining tetanus, or during the period immediately following a tetanus or a contracture. During a tetanus almost all of the myofibrillar ⁴⁵Ca is localized in the region of the sarcomere occupied by the thin filaments. The amount varies with the tension being developed by the muscle. The movement of calcium within the reticulum from the tubular portion to the terminal cisternae during the posttetanic period has a half-time of about 9 sec at room temperature and a Q₁₀ of about 1.7. Repolarization is not necessary for this movement. Evidence is given to support the notion that most calcium efflux from the cell occurs from the terminal cisternae into the transverse tubules.     

Resolved Structural States of Calmodulin in Regulation of Skeletal Muscle Calcium Release

Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca²⁺, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca²⁺, the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca²⁺, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca²⁺ stabilized the closed conformation by a factor of two. We conclude that the Ca²⁺-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca²⁺-dependent structural dynamics of bound CaM.     

Local Control Model of Excitation–Contraction Coupling in Skeletal Muscle

The voltage-activated H⁺ selective conductance of rat alveolar epithelial cells was studied using whole-cell and excised-patch voltage-clamp techniques. The effects of substituting deuterium oxide, D₂O, for water, H₂O, on both the conductance and the pH dependence of gating were explored. D⁺ was able to permeate proton channels, but with a conductance only about 50% that of H⁺. The conductance in D₂O was reduced more than could be accounted for by bulk solvent isotope effects (i.e., the lower mobility of D⁺ than H⁺), suggesting that D⁺ interacts specifically with the channel during permeation. Evidently the H⁺ or D⁺ current is not diffusion limited, and the H⁺ channel does not behave like a water-filled pore. This result indirectly strengthens the hypothesis that H⁺ (or D⁺) and not OH⁻ is the ionic species carrying current. The voltage dependence of H⁺ channel gating characteristically is sensitive to pH_o and pH_i and was regulated by pD_o and pD_i in an analogous manner, shifting 40 mV/U change in the pD gradient. The time constant of H⁺ current activation was about three times slower (τ_act was larger) in D₂O than in H₂O. The size of the isotope effect is consistent with deuterium isotope effects for proton abstraction reactions, suggesting that H⁺ channel activation requires deprotonation of the channel. In contrast, deactivation (τ_tail) was slowed only by a factor ≤1.5 in D₂O. The results are interpreted within the context of a model for the regulation of H⁺ channel gating by mutually exclusive protonation at internal and external sites (Cherny, V.V., V.S. Markin, and T.E. DeCoursey. 1995. J. Gen. Physiol. 105:861–896). Most of the kinetic effects of D₂O can be explained if the pK _a of the external regulatory site is ∼0.5 pH U higher in D₂O.     

Silent Calcium Channels in Skeletal Muscle Fibers of the Crustacean Atya lanipes

The superficial (tonic) abdominal flexor muscles of Atya lanipes do not generate Ca²⁺ action potentials when depolarized and have no detectable inward Ca²⁺ current. These fibers, however, are strictly dependent on Ca²⁺ influx for contraction, suggesting that they depend on Ca²⁺-induced Ca²⁺ release for contractile activation. The nature of the communication between Ca²⁺ channels in the sarcolemmal/tubular membrane and Ca²⁺ release channels in the sarcoplasmic reticulum in this crustacean muscle was investigated. The effects of dihydropyridines on tension generation and the passive electrical response were examined in current-clamped fibers: Bay K 8644 enhanced tension about 100% but did not alter the passive electrical response; nifedipine inhibited tension by about 70%. Sr²⁺ and Ba²⁺ action potentials could be elicited in Ca²⁺-free solutions. The spikes generated by these divalent cations were abolished by nifedipine. As the Sr²⁺ or Ba²⁺ concentrations were increased, the amplitudes of the action potentials and their maximum rate of rise, V _max , increased and tended towards saturation. Three-microelectrode voltage-clamp experiments showed that even at high (138 mm) extracellular Ca²⁺ concentration the channels were silent, i.e., no inward Ca²⁺ current was detected. In Ca²⁺-free solutions, inward currents carried by 138 mm Sr²⁺ or Ba²⁺ were observed. The currents activated at voltages above −40 mV and peaked at about 0 mV. This voltage-activation profile and the sensitivity of the channels to dihydropyridines indicate that they resemble L-type Ca²⁺ channels. Peak inward current density values were low, ca.−33 μA/cm² for Sr²⁺ and −14 μA/cm² for Ba²⁺, suggesting that Ca²⁺ channels are present at a very low density. It is concluded that Ca²⁺-induced Ca²⁺ release in this crustacean muscle operates with an unusually high gain: Ca²⁺ influx through the silent Ca²⁺ channels is too low to generate a macroscopic inward current, but increases sufficiently the local concentration of Ca²⁺ in the immediate vicinity of the sarcoplasmic reticulum Ca²⁺ release channels to trigger the highly amplified release of Ca²⁺ required for tension generation. Received: 5 April 1999/Revised: 15 September 1999     

Intracellular Calcium Movements of Frog Skeletal Muscle during Recovery from Tetanus

Radioautographs of ⁴⁵Ca-labeled frog skeletal muscles have been prepared using freeze-dry and vapor fixation techniques to avoid displacement of the isotope during the preparation of the radioautographs. ⁴⁵Ca has been localized in resting muscles exposed to ⁴⁵Ca Ringer's for 5 min or 5 hr and in isotopically labeled muscles recovering from tetanic stimulation at room temperature or at 4°C. In muscles soaked at rest for 5 min ⁴⁵Ca was present almost exclusively in the terminal cisternae. In all other muscles there were three sites at which the isotope was concentrated: (a) the terminal cisternae, (b) the intermediate cisternae and the longitudinal tubules, and (c) the A band portion of the myofibrils. The terminal cisternae were labeled more rapidly than the myofibrils, but both exchanges were accelerated by electrical stimulation. The amount of ⁴⁵Ca in the longitudinal tubules and the intermediate cisternae decreased with time after a tetanus as the amount in the terminal cisternae increased. It is proposed that electrical stimulation releases calcium from the terminal cisternae and that relaxation occurs from the binding of the released calcium by the longitudinal tubules and the intermediate cisternae. Complete recovery from mechanical activity involves the transport of this bound calcium into the reticulum and its subsequent binding by the terminal cisternae. Resting exchange of calcium occurs primarily between the terminal cisternae and the transverse tubules.     

Role of Ryanodine Receptors in the Assembly of Calcium Release Units in Skeletal Muscle

Abstract. In muscle cells, excitation–contraction (e–c) coupling is mediated by “calcium release units,” junctions between the sarcoplasmic reticulum (SR) and exterior membranes. Two proteins, which face each other, are known to functionally interact in those structures: the ryanodine receptors (RyRs), or SR calcium release channels, and the dihydropyridine receptors (DHPRs), or L-type calcium channels of exterior membranes. In skeletal muscle, DHPRs form tetrads, groups of four receptors, and tetrads are organized in arrays that face arrays of feet (or RyRs). Triadin is a protein of the SR located at the SR–exterior membrane junctions, whose role is not known. We have structurally characterized calcium release units in a skeletal muscle cell line (1B5) lacking Ry₁R. Using immunohistochemistry and freeze-fracture electron microscopy, we find that DHPR and triadin are clustered in foci in differentiating 1B5 cells. Thin section electron microscopy reveals numerous SR–exterior membrane junctions lacking foot structures (dyspedic). These results suggest that components other than Ry₁Rs are responsible for targeting DHPRs and triadin to junctional regions. However, DHPRs in 1B5 cells are not grouped into tetrads as in normal skeletal muscle cells suggesting that anchoring to Ry₁Rs is necessary for positioning DHPRs into ordered arrays of tetrads. This hypothesis is confirmed by finding a “restoration of tetrads” in junctional domains of surface membranes after transfection of 1B5 cells with cDNA encoding for Ry₁R.     

Calsequestrin Is an Inhibitor of Skeletal Muscle Ryanodine Receptor Calcium Release Channels

We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native ryanodine receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of ryanodine receptors to high [Ca²⁺] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of ryanodine receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced ryanodine receptor activity before, but not after, calsequestrin dissociation. A population of ryanodine receptors (∼35%) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native ryanodine receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces ryanodine receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.     

Local Control of Excitation-Contraction Coupling in Human Embryonic Stem Cell-Derived Cardiomyocytes

We investigated the mechanisms of excitation-contraction (EC) coupling in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and fetal ventricular myocytes (hFVMs) using patch-clamp electrophysiology and confocal microscopy. We tested the hypothesis that Ca²⁺ influx via voltage-gated L-type Ca²⁺ channels activates Ca²⁺ release from the sarcoplasmic reticulum (SR) via a local control mechanism in hESC-CMs and hFVMs. Field-stimulated, whole-cell [Ca²⁺]_i transients in hESC-CMs required Ca²⁺ entry through L-type Ca²⁺ channels, as evidenced by the elimination of such transients by either removal of extracellular Ca²⁺ or treatment with diltiazem, an L-type channel inhibitor. Ca²⁺ release from the SR also contributes to the [Ca²⁺]_i transient in these cells, as evidenced by studies with drugs interfering with either SR Ca²⁺ release (i.e. ryanodine and caffeine) or reuptake (i.e. thapsigargin and cyclopiazonic acid). As in adult ventricular myocytes, membrane depolarization evoked large L-type Ca²⁺ currents (I _Ca) and corresponding whole-cell [Ca²⁺]_i transients in hESC-CMs and hFVMs, and the amplitude of both I _Ca and the [Ca²⁺]_i transients were finely graded by the magnitude of the depolarization. hESC-CMs exhibit a decreasing EC coupling gain with depolarization to more positive test potentials, “tail” [Ca²⁺]_i transients upon repolarization from extremely positive test potentials, and co-localized ryanodine and sarcolemmal L-type Ca²⁺ channels, all findings that are consistent with the local control hypothesis. Finally, we recorded Ca²⁺ sparks in hESC-CMs and hFVMs. Collectively, these data support a model in which tight, local control of SR Ca²⁺ release by the I _Ca during EC coupling develops early in human cardiomyocytes.