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Atrial fibrillation (AF), the most common arrhythmia, has been associated with different electrophysiological, molecular, and structural alterations in atrial cardiomyocytes. Therefore, more studies are required to elucidate the genetic and molecular basis of AF. Various genome-wide association studies (GWAS) have strongly associated different single nucleotide polymorphisms (SNPs) with AF. One of these GWAS identified the rs13376333 risk SNP as the most significant one from the 1q21 chromosomal region. The rs13376333 risk SNP is intronic to the KCNN3 gene that encodes for small conductance calcium-activated potassium channels type 3 (SK3). However, the functional electrophysiological effects of this variant are not known. SK channels represent a unique family of K+ channels, primarily regulated by cytosolic Ca2+ concentration, and different studies support their critical role in the regulation of atrial excitability and consequently in the development of arrhythmias like AF. Since different studies have shown that both upregulation and downregulation of SK3 channels can lead to arrhythmias by different mechanisms, an important goal is to elucidate whether the rs13376333 risk SNP is a gain-of-function (GoF) or a loss-of-function (LoF) variant. A better understanding of the functional consequences associated with these SNPs could influence clinical practice guidelines by improving genotype-based risk stratification and personalized treatment. Although research using native human atrial cardiomyocytes and animal models has provided useful insights, each model has its limitations. Therefore, there is a critical need to develop a human-derived model that represents human physiology more accurately than existing animal models. In this context, research with human induced pluripotent stem cells (hiPSC) and subsequent generation of cardiomyocytes derived from hiPSC (hiPSC-CMs) has revealed the underlying causes of various cardiovascular diseases and identified treatment opportunities that were not possible using in vitro or in vivo studies with animal models. Thus, the ability to generate atrial cardiomyocytes and atrial tissue derived from hiPSCs from human/patients with specific genetic diseases, incorporating novel genetic editing tools to generate isogenic controls and organelle-specific reporters, and 3D bioprinting of atrial tissue could be essential to study AF pathophysiological mechanisms. In this review, we will first give an overview of SK-channel function, its role in atrial fibrillation and outline pathophysiological mechanisms of KCNN3 risk SNPs. We will then highlight the advantages of using the hiPSC-CM model to investigate SNPs associated with AF, while addressing limitations and best practices for rigorous hiPSC studies.

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease and often results in cardiac remodeling and an increased incidence of sudden cardiac arrest (SCA) and death, especially in youth and young adults. Among thousands of different variants found in HCM patients, variants of TNNT2 (cardiac troponin T-TNNT2) are linked to increased risk of ventricular arrhythmogenesis and sudden death despite causing little to no cardiac hypertrophy. Therefore, studying the effect of TNNT2 variants on cardiac propensity for arrhythmogenesis can pave the way for characterizing HCM in susceptible patients before sudden cardiac arrest occurs. In this study, a TNNT2 variant, I79N, was generated in human cardiac recombinant/reconstituted thin filaments (hcRTF) to investigate the effect of the mutation on myofilament Ca2+ sensitivity and Ca2+ dissociation rate using steady-state and stopped-flow fluorescence techniques. The results revealed that the I79N variant significantly increases myofilament Ca2+ sensitivity and decreases the Ca2+ off-rate constant (k off). To investigate further, a heterozygous I79N+/- TNNT2 variant was introduced into human-induced pluripotent stem cells using CRISPR/Cas9 and subsequently differentiated into ventricular cardiomyocytes (hiPSC-CMs). To study the arrhythmogenic properties, monolayers of I79N+/- hiPSC-CMs were studied in comparison to their isogenic controls. Arrhythmogenesis was investigated by measuring voltage (V m) and cytosolic Ca2+ transients over a range of stimulation frequencies. An increasing stimulation frequency was applied to the cells, from 55 to 75 bpm. The results of this protocol showed that the TnT-I79N cells had reduced intracellular Ca2+ transients due to the enhanced cytosolic Ca2+ buffering. These changes in Ca2+ handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. While wild-type (WT) hiPSC-CMs were accurately entrained to frequencies of at least 150 bpm, the I79N hiPSC-CMs demonstrated clear patterns of alternans for both V m and Ca2+ transients at frequencies >75 bpm. Lastly, a transcriptomic analysis was conducted on WT vs. I79N+/- TNNT2 hiPSC-CMs using a custom NanoString codeset. The results showed a significant upregulation of NPPA (atrial natriuretic peptide), NPPB (brain natriuretic peptide), Notch signaling pathway components, and other extracellular matrix (ECM) remodeling components in I79N+/- vs. the isogenic control. This significant shift demonstrates that this missense in the TNNT2 transcript likely causes a biophysical trigger, which initiates this significant alteration in the transcriptome. This TnT-I79N hiPSC-CM model not only reproduces key cellular features of HCM-linked mutations but also suggests that this variant causes uncharted pro-arrhythmic changes to the human action potential and gene expression.

Atrial fibrillation (AF), the most common arrhythmia, has been associated with different electrophysiological, molecular, and structural alterations in atrial cardiomyocytes. Therefore, more studies are required to elucidate the genetic and molecular basis of AF. Various genome-wide association studies (GWAS) have strongly associated different single nucleotide polymorphisms (SNPs) with AF. One of these GWAS identified the rs13376333 risk SNP as the most significant one from the 1q21 chromosomal region. The rs13376333 risk SNP is intronic to the KCNN3 gene that encodes for small conductance calcium-activated potassium channels type 3 (SK3). However, the functional electrophysiological effects of this variant are not known. SK channels represent a unique family of K+ channels, primarily regulated by cytosolic Ca2+ concentration, and different studies support their critical role in the regulation of atrial excitability and consequently in the development of arrhythmias like AF. Since different studies have shown that both upregulation and downregulation of SK3 channels can lead to arrhythmias by different mechanisms, an important goal is to elucidate whether the rs13376333 risk SNP is a gain-of-function (GoF) or a loss-of-function (LoF) variant. A better understanding of the functional consequences associated with these SNPs could influence clinical practice guidelines by improving genotype-based risk stratification and personalized treatment. Although research using native human atrial cardiomyocytes and animal models has provided useful insights, each model has its limitations. Therefore, there is a critical need to develop a human-derived model that represents human physiology more accurately than existing animal models. In this context, research with human induced pluripotent stem cells (hiPSC) and subsequent generation of cardiomyocytes derived from hiPSC (hiPSC-CMs) has revealed the underlying causes of various cardiovascular diseases and identified treatment opportunities that were not possible using in vitro or in vivo studies with animal models. Thus, the ability to generate atrial cardiomyocytes and atrial tissue derived from hiPSCs from human/patients with specific genetic diseases, incorporating novel genetic editing tools to generate isogenic controls and organelle-specific reporters, and 3D bioprinting of atrial tissue could be essential to study AF pathophysiological mechanisms. In this review, we will first give an overview of SK-channel function, its role in atrial fibrillation and outline pathophysiological mechanisms of KCNN3 risk SNPs. We will then highlight the advantages of using the hiPSC-CM model to investigate SNPs associated with AF, while addressing limitations and best practices for rigorous hiPSC studies. [ABSTRACT FROM AUTHOR]

Aims Long QT syndrome type 2 (LQTS2) is associated with inherited variants in the cardiac human ether-à-go-go-related gene (hERG) K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR–Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant and its phenotypic rescue by using the Type 1 hERG activator, RPR260243. Methods and results The above approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean action potential duration (APD90). However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of APD90 during rate transitions, and increases the incidence of 2:1 block. During paired S1–S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness. Conclusion Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs. [ABSTRACT FROM AUTHOR]

Cardiac troponin C (cTnC) is the critical Ca2+‐sensing component of the troponin complex. Binding of Ca2+ to cTnC triggers a cascade of conformational changes within the myofilament that culminate in force production. Hypertrophic cardiomyopathy (HCM)‐associated TNNC1 variants generally induce a greater degree and duration of Ca2+ binding, which may underly the hypertrophic phenotype. Regulation of contraction has long been thought to occur exclusively through Ca2+ binding to site II of cTnC. However, work by several groups including ours suggest that Mg2+, which is several orders of magnitude more abundant in the cell than Ca2+, may compete for binding to the same cTnC regulatory site. We previously used isothermal titration calorimetry (ITC) to demonstrate that physiological concentrations of Mg2+ may decrease site II Ca2+‐binding in both N‐terminal and full‐length cTnC. Here, we explore the binding of Ca2+ and Mg2+ to cTnC harbouring a series of TNNC1 variants thought to be causal in HCM. ITC and thermodynamic integration (TI) simulations show that A8V, L29Q and A31S elevate the affinity for both Ca2+ and Mg2+. Further, L48Q, Q50R and C84Y that are adjacent to the EF hand binding motif of site II have a more significant effect on affinity and the thermodynamics of the binding interaction. To the best of our knowledge, this work is the first to explore the role of Mg2+ in modifying the Ca2+ affinity of cTnC mutations linked to HCM. Our results indicate a physiologically significant role for cellular Mg2+ both at baseline and when elevated on modifying the Ca2+ binding properties of cTnC and the subsequent conformational changes which precede cardiac contraction. [ABSTRACT FROM AUTHOR]

Long QT interval syndrome (LQTS) type 1 (LQT1) has been reported to arise from mutations in the S3 domain of KCNQ1, but none of the seven S3 mutations in the literature have been characterized with respect to trafficking or biophysical deficiencies. Surface channel expression was studied using a proteinase K assay for KCNQ1 D202H/ N, I204F/M, V205M, S209F, and V215M coexpressed with KCNE1 in mammalian cells. In each case, the majority of synthesized channel was found at the surface, but mutant [I.sub.Ks]. current density at +100 mV was reduced significantly for S209F, which showed ~75% reduction over wild type (WT). All mutants except S209F showed positively shifted [V.sub.I/2]'s of activation and slowed channel activation compared with WT ([V.sub.1/2] = +17.7 [+ or -] 2.4 mV and [[tau].sub.activation] of 729 ms at +20 mV; n = 18). Deactivation was also accelerated in all mutants versus WT (126 [+ or -] 8 ms at -50 mV; n = 27), and these changes led to marked loss of repolarizing currents during action potential clamps at 2 and 4 Hz, except again S209F. KCNQ1 models localize these naturally occurring S3 mutants to the surface of the helices facing the other voltage sensor transmembrane domains and highlight inter-residue interactions involved in activation gating. V207M, currently classified as a polymorphism and facing lipid in the model, was indistinguishable from WT [I.sub.Ks]. We conclude that S3 mutants of KCNQ1 cause LQTS predominantly through biophysical effects on the gating of [I.sub.Ks], but some mutants also show protein stability/trafficking defects, which explains why the kinetic gain-of-function mutation S209F causes LQT1. doi/ 10.1085/jgp.200910351

Members of the [Na.sup.2+]/[Ca.sup.2+] exchanger (NCX) family are important regulators of cytosolic [Ca.sup.2+] in myriad tissues and are highly conserved across a wide range of species. Three distinct NCX genes and numerous splice variants exist in mammals, many of which have been characterized in a variety of heterologous expression systems. Recently, however, we discovered a fourth NCX gene (NCX4), which is found exclusively in teleost, amphibian, and reptilian genomes. Zebrafish (Danio rerio) NCX4a encodes for a protein of 939 amino acids and shows a high degree of identity with known NCXs. Although knockdown of NCX4a activity in zebrafish embryos has been shown to alter left-right patterning, it has not been demonstrated that NCX4a functions as a NCX. In this study, we 1) demonstrated, for the first time, that this gene encodes for a novel NCX; 2) characterized the tissue distribution of zebrafish NCX4a; and 3) evaluated its kinetic and transport properties. While ubiquitously expressed, the highest levels of NCX4a expression occurred in the brain and eyes. NCX4a exhibits modest levels of [Na.sup.+]-dependent inactivation and requires much higher levels of regulatory [Ca.sup.2+] to activate outward exchange currents. NCX4a also exhibited extremely fast recovery from [Na.sup.+]-dependent inactivation of outward currents, faster than any previously characterized wild-type exchanger. While this result suggests that the [Na.sup.+]-dependent inactive state of NCX4a is far less stable than in other NCX family members, this exchanger was still strongly inhibited by 2 [micro]M exchanger inhibitory peptide. We demonstrated that a new putative member of the NCX gene family, NCX4a, encodes for a NCX with unique functional properties. These data will be useful in understanding the role that NCX4a plays in embryological development as well as in the adult, where it is expressed ubiquitously. calcium homeostatis; [Na.sup.+]/[Ca.sup.2+] exchanger; development

It is commonly accepted that L-type [Ca.sup.2+] channel-mediated [Ca.sup.2+]-induced [Ca.sup.2+] release (CICR) is the dominant mode of excitation-contraction (E-C) coupling in the adult mammalian heart and that there is no appreciable CICR in neonates. However, we have observed that cell contraction in the neonatal heart was significantly decreased after sarcoplasmic reticulum (SR) [Ca.sup.2+] depletion with caffeine. Therefore, the present study investigated the developmental changes of CICR in rabbit ventricular myocytes at 3, 10, 20, and 56 days of age. We found that the inhibitory effect of the L-type [Ca.sup.2+] current ([I.sub.Ca]) inhibitor nifedipine (Nif; 15 [micro]M) caused an increasingly larger reduction of [Ca.sup.2+] transients on depolarization in older age groups [from ~15% in 3-day-old (3d) myocytes to ~90% in 56-day-old (56d) myocytes]. The remaining [Ca.sup.2+] transient in the presence of Nif in younger age groups was eliminated by the inhibition of [Na.sup.+]/[Ca.sup.2+] exchanger (NCX) with the subsequent addition of 10 [micro]M KB-R7943 (KB-R). Furthermore, [Ca.sup.2+] transients were significantly reduced in magnitude after the depletion of SR [Ca.sup.2+] with caffeine in all age groups, although the effect was significantly greater in the older age groups (from ~40% in 3d myocytes up to ~70% in 56d myocytes). This SR [Ca.sup.2+]-sensitive [Ca.sup.2+] transient in the earliest developmental stage was insensitive to Nil but was sensitive to the subsequent addition of KB-R, indicating the presence of NCX-mediated CICR that decreased significantly with age (from ~37% in 3d myocytes to ~0.5% in 56d myocytes). In contrast, the/ca-mediated CICR increased significantly with age (from ~10% in 3d myocytes to ~70% in 56d myocytes). The CICR gain as estimated by the integral of the CICR [Ca.sup.2+] transient divided by the integral of its [Ca.sup.2+] transient trigger was smaller when mediated by NCX (~1.0 for 3d myocytes) than when mediated by [I.sub.Ca] (~3.0 for 56d myocytes). We conclude that the lower-efficiency NCX-mediated CICR is a predominant mode of CICR in the earliest developmental stages that gradually decreases as the more efficient L-type [Ca.sup.2+] channel-mediated CICR increases in prominence with ontogeny. excitation-contraction coupling; [Na.sup.+]/[Ca.sup.2+] exchanger; L-type [Ca.sup.2+] channels; sarcoplasmic reticulum

While it has been reported that a sparse sarcoplasmic reticulum (SR) and a low SR [Ca.sup.2+] pump density exist at birth, we and others have recently shown that significant amounts of [Ca.sup.2+] are stored in the neonatal rabbit heart SR. Here we try to determine developmental changes in SR [Ca.sup.2+] loading mechanisms and [Ca.sup.2+] pump efficacy in rabbit ventricular myocytes. SR [Ca.sup.2+] loading ([load.sub.SR]) and [k.sub.0.5] ([Ca.sup.2+] concentration at half-maximal SR [Ca.sup.2+] uptake) were higher and lower, respectively, in younger age groups. Inhibition of the L-type calcium current ([I.sub.Ca]) with 15 [micro]M nifedipine dramatically reduced [load.sub.SR] in older but not in younger age groups. In contrast, subsequent inhibition of the [Na.sup.+]/[Ca.sup.2+] exchanger (NCX) with l0 [micro]M KB-R7943 strongly reduced [load.sub.SR] in the younger but not the older age groups. Accordingly, the time integral of the inward NCX current (tail [I.sub.NCX]) elicited on repolarization was highly sensitive to nifedipine in the older groups and sensitive to KB-R7943 in the younger groups. Interestingly, slow SR loading took place in the presence of both nifedipine and KB-R7943 in all age groups, although it was less prominent in the older groups. We conclude that the SR loading capacity at the earliest postnatal stages is at least as large as that of adult myocytes. However, reverse-mode NCX plays a prominent role in SR [Ca.sup.2+] loading at early postnatal stages while [I.sub.Ca] is the main source of SR [Ca.sup.2+] loading at late postnatal and adult stages. sarco(endo)plasmic reticulum [Ca.sup.2+] pump; sarcoplasmic reticulum [Ca.sup.2+] loading; cytosolic [Ca.sup.2+] concentration; L-type [Ca.sup.2+] channel; [Na.sup.2+]/[Ca.sup.2+] exchanger; neonate cardiomyocytes; store-operated [Ca.sup.2+] channel

L-type [Ca.sup.2+] channel-mediated, [Ca.sup.2+]-induced [Ca.sup.2+] release (CICR) is the dominant mode of excitation-contraction (E-C) coupling in the mature mammalian myocardium but is thought to be absent in the fetal and newborn mammalian myocardium. Furthermore, the characteristics and contributors of E-C coupling at the earliest developmental stages are poorly understood. In this study, we measured [[sup.3]H](+)PN200-110 dihydropyridine binding capacity, functionality and expression of the L-type [Ca.sup.2+] channel, and cytosolic [[Ca.sup.2+]] ([[[Ca.sup.2+]].sub.i]) at various developmental stages (3, 6, 10, 20, and 56 days old) to characterize ontogenetic changes in E-C coupling. We found that 1) the whole cell L-type [Ca.sup.2+] channel peak current ([I.sub.Ca]) density increased slightly in parallel with cell growth, but the current-voltage relationship, the steady-state activation, and the maximum DHP binding and binding affinity did not exhibit significant developmental changes; 2) sarcoplasmic reticuhun [Ca.sup.2+] dependence of inactivation rates of L-type [Ca.sup.2+] channel and peak of [I.sub.Ca] density were only observed after 10 days of age, which temporally coincides with transverse (T)-tubule formation; 3) the relationship between [[[Ca.sup.2+]].sub.i] and voltage changed from a linear relationship at the earliest developmental stages to a 'bell-shaped' relationship at the later developmental stages, presumably corresponding to a switch from reverse-mode Na/Ca exchange-dependent to [I.sub.Ca]-dependent E-C coupling; and 4) the expression of two different splice variants of [Ca.sub.v] 1.2, IVS3A and IVS3B, switched from predominantly IVS3A at the earliest stages to IVS3B at the later developmental stages. Our data suggest that whereas the density of functional dihydropyridine receptors (DHPRs) increases only slightly during ontogeny, the enhancement of functional coupling between DHPR and ryanodine receptor is dramatic between the second and third weeks after birth. Furthermore, we found that the differential expression of splice variants during development temporally correlated with the appearance of [I.sub.Ca]-dependent E-C coupling and T-tubule formulation. neonate myocardium; contractility; excitation-contraction coupling; dihydropyridine receptor

Store-operated [Ca.sup.2+] entry (SOCE), which is [Ca.sup.2+] entry triggered by the depletion of intracellular [Ca.sup.2+] stores, has been observed in many cell types, but only recently has it been suggested to occur in cardiomyocytes. In the present study, we have demonstrated SOCE-dependent sarcoplasmic reticulum (SR) [Ca.sup.2+] loading ([load.sub.SR]) that was not altered by inhibition of L-type [Ca.sup.2+] channels, reverse mode Na+/[Ca.sup.2+] exchange (NCX), or nonselective cation channels. In contrast, lowering the extracellular [Ca.sup.2+] to 0 mM or adding either 0.5 mM [Zn.sup.2+] or the putative store-operated channel (SOC) inhibitor SKF-96365 (100 [micro]M) inhibited [load.sup.SR] at rest. Interestingly, inhibition of forward mode NCX with 30 [micro]M KB-R7943 stimulated SOCE significantly and resulted in enhanced [load.sup.SR]. In addition, manipulation of the extracellular and intracellular [Na.sup.+] concentrations further demonstrated the modulatory role of NCX in SOCE-mediated SR [Ca.sup.2+] loading. Although there is little knowledge of SOCE in cardiomyocytes, the present results suggest that this mechanism, together with NCX, may play an important role in SR [Ca.sup.2+] homeostasis. The data reported herein also imply the presence of microdomains unique to the neonatal cardiomyocyte. These findings may be of particular importance during open heart surgery in neonates, in which uncontrolled SOCE could lead to SR [Ca.sup.2+] overload and arrhythmogenesis. cardiac ontogeny; cardiac excitation-contraction coupling; calcium homeostasis

Much less is known about the contributions of the [Na.sup.+]/[Ca.sup.2+] exchanger (NCX) and sarcoplasmic reticulum (SR) [Ca.sup.2+] pump to cell relaxation in neonatal compared with adult mammalian ventricular myocytes. Based on both biochemical and molecular studies, there is evidence of a much higher density of NCX at birth that subsequently decreases during the next 2 wk of development. It has been hypothesized, therefore, that NCX plays a relatively more important role for cytosolic [Ca.sup.2+] decline in neonates as well as, perhaps, a role in excitation-contraction coupling in reverse mode. We isolated neonatal ventricular myocytes from rabbits in four different age groups: 3, 6, 10, and 20 days of age. Using an amphotericin-perforated patch-clamp technique in fluo-3-loaded myocytes, we measured the caffeine-induced inward NCX current ([I.sub.NCX]) and the [Ca.sup.2+] transient. We found that the integral of [I.sub.NCX], an indicator of SR [Ca.sup.2+] content, was greatest in myocytes from younger age groups when normalized by cell surface area and that it decreased with age. The velocity of [Ca.sup.2+] extrusion by NCX ([V.sub.NCX]) was linear with [[Ca.sup.2+]] and did not indicate saturation kinetics until [[Ca.sup.2+]] reached 1-3 [micro]M for each age group. There was a significantly greater time delay between the peaks of [I.sub.NCX] and the [Ca.sup.2+] transient in myocytes from the youngest age groups. This observation could be related to structural differences in the subsarcolemmal microdomains as a function of age. ontogeny of cardiac excitation-contraction coupling; sodium/calcium exchanger; cytosolic calcium concentration; subsarcolemmal calcium concentration; sarcoplasmic reticulum calcium content

Whole cell patch clamp and intracellular [Ca.sup.2+] transients in trout atrial cardiomyocytes were used to quantify calcium release from the sarcoplasmic reticulum (SR) and examine its dependency on the [Ca.sup.2+] trigger source. Short depolarization pulses (2-20 ms) elicited large caffeine-sensitive tail currents. The [Ca.sup.2+] carried by the caffeine-sensitive tail current after a 2-ms depolarization was 0.56 amol [Ca.sup.2+]/pF, giving an SR [Ca.sup.2+] release rate of 279 amol [Ca.sup.2+]*[p.sup.F-1]* [s.sup.-1] or 4.3 mM/s. Depolarizing cells for 10 ms to different membrane potentials resulted in a local maximum of SR [Ca.sup.2+] release, intracellular [Ca.sup.2+] transient, and cell shortening at 10 reV. Although 100 [micro]M CD[Cl.sub.2] abolished this local maximum, it had no effect on SR [Ca.sup.2+] release elicited by a depolarization to 110 or 150 reV, and the SR [Ca.sup.2+] release was proportional to the membrane potential in the range -50 to 150 mV with 100 [micro]M CD[Cl.sub.2]. Increasing the intracellular [Na.sup.+] concentration ([Na.sup.+]) from 10 to 16 mM enhanced SR [Ca.sup.2+] release but reduced cell shortening at all membrane potentials examined. In the absence of TTX, SR [Ca.sup.2+] release was potentiated with 16 mM but not 10 mM pipette [Na+]. Comparison of the total sarcolemmal [Ca.sup.2+] entry and the [Ca.sup.2+] released from the SR gave a gain factor of 18.6 [- or +] 7.7. Nifedipine (Nil) at 10 [micro]M inhibited L-type [Ca.sup.2+] current ([I.sub.ca]) and reduced the time integral of the tail current by 61%. The gain of the Nif-sensitive SR [Ca.sup.2+] release was 16.0 [+ or -] 4.7. A 2-ms depolarization still elicited a contraction in the presence of Nif that was abolished by addition of 10 mM Ni[Cl.sub.2]. The gain of the Nilinsensitive but Ni[Cl.sub.2]-sensitive SR [Ca.sup.2+] release was 14.8 [+ or -] 7.1. Thus both reverse-mode [Na.sup.+]/[Ca.sup.2+] exchange (NCX) and [I.sub.ca] can elicit [Ca.sup.2+] release from the SR, but [I.sub.ca] is more efficient than reverse-mode NCX in activating contraction. This difference may be due to extrusion of a larger fraction of The [Ca.sup.2+] released from the SR by reverse-mode NCX rather than a smaller gain for NCX-induced [Ca.sup.2+] release. [Ca.sup.2+] current; [Ca.sup.2+] transients; caffeine; cardiac; excitation-contraction coupling