Temperature acclimation has no effect on ryanodine receptor expression or subcellular localization in rainbow trout heart

In cardiomyocytes, ryanodine receptors (RYRs) mediate Ca²⁺-induced Ca²⁺-release (CICR) from the sarcoplasmic reticulum (SR) during excitation–contraction (e–c) coupling. In rainbow trout heart, the relative importance of CICR increases with cold-acclimation. Thus, the aim of this study was to investigate the effect of temperature acclimation (4, 11 and 18°C) on RYR intracellular localization and expression density. We used immunocytochemistry to assess intracellular localization in ventricular myocytes and Western blotting to assess RYR expression in both atrial and ventricular tissue. In ventricular myocytes, RYRs were localized peripherally in transverse bands aligning with sarcomeric m-lines and centrally around mitochondria and the nucleus. Localization did not change with temperature acclimation. RYR expression was also unaffected by temperature acclimation. The localization of RYRs at the m-line is similar to neonatal mammalian cardiomyocytes. We suggest this positioning is indicative of myocytes which rely predominantly on transsarcolemmal Ca²⁺-influx, rather than CICR, during e–c coupling.     

Ca2+ Release Events in Cardiac Myocytes Up Close: Insights from Fast Confocal Imaging

The spatio-temporal properties of Ca²⁺ transients during excitation-contraction coupling and elementary Ca²⁺ release events (Ca²⁺ sparks) were studied in atrial and ventricular myocytes with ultra-fast confocal microscopy using a Zeiss LSM 5 LIVE system that allows sampling rates of up to 60 kHz. Ca²⁺ sparks which originated from subsarcolemmal junctional sarcoplasmic reticulum (j-SR) release sites in atrial myocytes were anisotropic and elongated in the longitudinal direction of the cell. Ca²⁺ sparks in atrial cells originating from non-junctional SR and in ventricular myocytes were symmetrical. Ca²⁺ spark recording in line scan mode at 40,000 lines/s uncovered step-like increases of [Ca²⁺]_i. 2-D imaging of Ca²⁺ transients revealed an asynchronous activation of release sites and allowed the sequential recording of Ca²⁺ entry through surface membrane Ca²⁺ channels and subsequent activation of Ca²⁺-induced Ca²⁺ release. With a latency of 2.5 ms after application of an electrical stimulus, Ca²⁺ entry could be detected that was followed by SR Ca²⁺ release after an additional 3 ms delay. Maximum Ca²⁺ release was observed 4 ms after the beginning of release. The timing of Ca²⁺ entry and release was confirmed by simultaneous [Ca²⁺]_i and membrane current measurements using the whole cell voltage-clamp technique. In atrial cells activation of discrete individual release sites of the j-SR led to spatially restricted Ca²⁺ release events that fused into a peripheral ring of elevated [Ca²⁺]_i that subsequently propagated in a wave-like fashion towards the center of the cell. In ventricular myocytes asynchronous Ca²⁺ release signals from discrete sites with no preferential subcellular location preceded the whole-cell Ca²⁺ transient. In summary, ultra-fast confocal imaging allows investigation of Ca²⁺ signals with a time resolution similar to patch clamp technique, however in a less invasive fashion.     

Contribution of mechanical factors to arrhythmogenesis in calcium overloaded cardiomyocytes: model predictions and experiments

It is well-known that Ca²⁺ overload in cardiomyocytes may underlie arrhythmias. However, the possible contribution of mechanical factors to rhythm disturbances in Ca²⁺ overloaded myocytes has not been sufficiently investigated. We used a mathematical model of the electrical and mechanical activity of cardiomyocytes to reveal an essential role of the mechanisms of cardiac mechano-electric feedback in arrhythmogenesis in Ca²⁺ overloaded myocardium. In the model, the following mechanical factors increased Ca²⁺ overload in contracting cardiomyocytes and promoted rhythm disturbances: i) a decrease in the mechanical load for afterloaded contractions; and ii) a decrease in the initial length of sarcomeres for isometric twitches. In exact accordance with the model predictions, in experiments on papillary muscles from the right ventricle of guinea pigs with Ca²⁺ overloaded cardiomyocytes (using 0.5-1 μM of ouabain), we found that emergence of rhythm disturbances and extrasystoles depends on the mechanical conditions of muscle contraction.     

Atrial local Ca signaling and inositol 1,4,5-trisphosphate receptors

In atrial myocytes lacking t-tubules, action potential triggers junctional Ca²⁺ releases in the cell periphery, which propagates into the cell interior. The present article describes growing evidence on atrial local Ca²⁺ signaling and on the functions of inositol 1,4,5-trisphosphate receptors (IP₃Rs) in atrial myocytes, and show our new findings on the role of IP₃R subtype in the regulation of spontaneous focal Ca²⁺ releases in the compartmentalized areas of atrial myocytes. The Ca²⁺ sparks, representing focal Ca²⁺ releases from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR) clusters, occur most frequently at the peripheral junctions in isolated resting atrial cells. The Ca²⁺ sparks that were darker and longer lasting than peripheral and non-junctional (central) sparks, were found at peri-nuclear sites in rat atrial myocytes. Peri-nuclear sparks occurred more frequently than central sparks. Atrial cells express larger amounts of IP₃Rs compared with ventricular cells and possess significant levels of type 1 IP₃R (IP₃R1) and type 2 IP₃R (IP₃R2). Over the last decade the roles of atrial IP₃R on the enhancement of Ca²⁺-induced Ca²⁺ release and arrhythmic Ca²⁺ releases under hormonal stimulations have been well documented. Using protein knock-down method and confocal Ca²⁺ imaging in conjunction with immunocytochemistry in the adult atrial cell line HL-1, we could demonstrate a role of IP₃R1 in the maintenance of peri-nuclear and non-junctional Ca²⁺ sparks via stimulating a posttranslational organization of RyR clusters.     

Regulatory effect of connexin 43 on basal Ca2+ signaling in rat ventricular myocytes

Background

It has been found that gap junction-associated intracellular Ca²⁺ [Ca²⁺]_i disturbance contributes to the arrhythmogenesis and hyperconstriction in diseased heart. However, whether functional gaps are also involved in the regulation of normal Ca²⁺ signaling, in particular the basal [Ca²⁺]_i activities, is unclear.

Methods and Results

Global and local Ca²⁺ signaling and gap permeability were monitored in cultured neonatal rat ventricular myocytes (NRVMs) and freshly isolated mouse ventricular myocytes by Fluo4/AM and Lucifer yellow (LY), respectively. The results showed that inhibition of gap communication by heptanol, Gap 27 and flufenamic acid or interference of connexin 43 (Cx43) with siRNA led to a significant suppression of LY uptake and, importantly, attenuations of global Ca²⁺ transients and local Ca²⁺ sparks in monolayer NRVMs and Ca²⁺ sparks in adult ventricular myocytes. In contrast, overexpression of rat-Cx43 in NRVMs induced enhancements in the above measurements, and so did in HEK293 cells expressing rat Cx43. Additionally, membrane-permeable inositol 1,4,5-trisphosphate (IP₃ butyryloxymethyl ester) and phenylephrine, an agonist of adrenergic receptor, could relieve the inhibited Ca²⁺ signal and LY uptake by gap uncouplers, whereas blockade of IP₃ receptor with xestospongin C or 2-aminoethoxydiphenylborate mimicked the effects of gap inhibitors. More importantly, all these gap-associated effects on Ca²⁺ signaling were also found in single NRVMs that only have hemichannels instead of gap junctions. Further immunostaining/immunoblotting single myocytes with antibody against Cx43 demonstrated apparent increases in membrane labeling of Cx43 and non-junctional Cx43 in overexpressed cells, suggesting functional hemichannels exist and also contribute to the Ca²⁺ signaling regulation in cardiomyocytes.

Conclusions

These data demonstrate that Cx43-associated gap coupling plays a role in the regulation of resting Ca²⁺ signaling in normal ventricular myocytes, in which IP₃/IP₃ receptor coupling is involved. This finding may provide a novel regulatory pathway for mediation of spontaneous global and local Ca²⁺ activities in cardiomyocytes.     

RyR2 Modulates a Ca2+-Activated K+ Current in Mouse Cardiac Myocytes

In cardiomyocytes, Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCCs) binds to and activates RyR2 channels, resulting in subsequent Ca²⁺ release from the sarcoplasmic reticulum (SR) and cardiac contraction. Previous research has documented the molecular coupling of small-conductance Ca²⁺-activated K⁺ channels (SK channels) to VDCCs in mouse cardiac muscle. Little is known regarding the role of RyRs-sensitive Ca²⁺ release in the SK channels in cardiac muscle. In this study, using whole-cell patch clamp techniques, we observed that a Ca²⁺-activated K+ current (I_K,Ca) recorded from isolated adult C57B/L mouse atrial myocytes was significantly decreased by ryanodine, an inhibitor of ryanodine receptor type 2 (RyR2), or by the co-application of ryanodine and thapsigargin, an inhibitor of the sarcoplasmic reticulum calcium ATPase (SERCA) (p<0.05, p<0.01, respectively). The activation of RyR2 by caffeine increased the I_K,Ca in the cardiac cells (p<0.05, p<0.01, respectively). We further analyzed the effect of RyR2 knockdown on I_K,Ca and Ca²⁺ in isolated adult mouse cardiomyocytes using a whole-cell patch clamp technique and confocal imaging. RyR2 knockdown in mouse atrial cells transduced with lentivirus-mediated small hairpin interference RNA (shRNA) exhibited a significant decrease in I_K,Ca (p<0.05) and [Ca²⁺]i fluorescence intensity (p<0.01). An immunoprecipitated complex of SK2 and RyR2 was identified in native cardiac tissue by co-immunoprecipitation assays. Our findings indicate that RyR2-mediated Ca²⁺ release is responsible for the activation and modulation of SK channels in cardiac myocytes.     

Increase of atrial ANP release by 2,3-butanedione monoxime in beating rabbit atria

2,3-Butanedione monoxime (BDM) is a chemical phosphatase and has been known to dissociate mechanical contraction in the excitation–contraction coupling via inhibition of myofibrillar ATPase. BDM has also been found to decrease sarcolemmal L-type Ca²⁺ channel activity and intracellular Ca²⁺ in cardiac myocytes. It has been shown that Ca²⁺ entry via L-type Ca²⁺ channels decreased atrial myocyte atrial natriuretic peptide (ANP) release. The purpose of the present study was to address the effects of BDM in the regulation of ANP release. Experiments were performed in perfused beating rabbit atria. BDM accentuated atrial myocyte ANP release concomitantly with a decrease in atrial stroke volume and pulse pressure in a concentration-dependent manner. The BDM-induced activation of ANP release was attenuated by the treatment with nifedipine, an inhibitor of L-type Ca²⁺ channels. BDM further decreased atrial stroke volume and pulse pressure in the presence of nifedipine. Blockade of function of the sarcoplasmic reticulum with thapsigargin plus ryanodine slightly but not significantly attenuated the BDM-induced activation of ANP release. These data show that BDM is a potent stimulator for the ANP release and also suggest that the mechanism by which BDM activates atrial myocyte ANP release is related to inhibition of the L-type Ca²⁺ channel activity. The present finding also suggests that the effects of ANP released may be considered in an occasion of uncoupling by BDM of the excitation–contraction coupling of cardiomyocytes.     

MURC/CAVIN-4 facilitates store-operated calcium entry in neonatal cardiomyocytes

Intact store-operated calcium entry (SOCE) mechanisms ensure the maintenance of Ca²⁺ homeostasis in cardiomyocytes while their dysregulation promotes the development of cardiomyopathies. To better understand this calcium handling process in cardiomyocytes, we sought to identify unknown protein partners of stromal interaction molecule 1 (STIM1), a main regulatory protein of SOCE. We identified the muscle-related coiled-coil protein (MURC), also known as Cavin-4, as a candidate and showed that MURC interacts with STIM1 in cardiomyocytes. This interaction occurs via the HR1 and ERM domains of MURC and STIM1, respectively. Our results also demonstrated that the overexpression of MURC in neonatal rat ventricular myocytes (NRVM) is sufficient to potentiate SOCE and that its HR1 domain is required to mediate this effect. Interestingly, the R140W-MURC mutant, a missense variant of the HR1 domain associated with human dilated cardiomyopathy, exacerbates the SOCE increase in NRVM. Although the endogenous expression of STIM1 and Ca²⁺ channel Orai1 is not modulated under these conditions, we showed that MURC increases the interaction between these proteins under resting conditions. Our study provides novel evidence that MURC regulates SOCE by interacting with STIM1 in cardiomyocytes. In addition, we identified a first potential mechanism by which the R140W mutation of MURC may contribute to calcium mishandling and the development of cardiomyopathies.     

The progressive effects of a fat enriched diet on ventricular myocyte contraction and intracellular Ca2+ in the C57BL/6J mouse

The C57BL/6J mouse has a genetic susceptibility to develop diabetes when fed with a high-fat, high-sucrose diet. The general characteristics of diet-induced diabetes in this model include progressive development of hyperinsulinaemia, hyperglycaemia, insulin resistance and obesity, features that are frequently observed in the clinical setting. This study investigated the progressive effects of a fat enriched (FE) diet on contraction and intracellular Ca²⁺ in ventricular myocytes from the C57BL/6J mouse. The characteristics of the mice fed with the FE diet compared to mice receiving control diet included progressive increase in the rate of body weight gain, increased fasting blood glucose and time-dependent differences in the disposal of blood glucose after a glucose challenge. The ultrastructure of cardiac myocytes and associated capillaries did not show any gross morphological alteration after 27 weeks of FE diet compared to controls.At 5 months the resting cell length (RCL) and the kinetics of shortening were not significantly altered in ventricular myocytes from mice receiving the FE diet compared to age-matched controls. At 5 and at 7 months the amplitude of shortening was increased in myocytes receiving the FED diet compared to controls. At 7 months the time to half (THALF) relaxation of myocyte contraction was shortened in myocytes from mice receiving the FE diet compared to controls. Mean THALF relaxation in myocytes from mice fed the FE diet was 32.0 ± 1.4 ms (n = 23) compared to 40.2 ± 2.0 ms (n = 27) in controls. Neither resting intracellular Ca²⁺ nor the kinetics or amplitude of the Ca²⁺ transient were altered by FE diet. Differences in myofilament sensitivity to Ca²⁺ might underlie the changes in contractility.     

Oxytocin induces intracellular Ca2+ release in cardiac fibroblasts from neonatal rats

Pituitary neuropeptide oxytocin is increasingly recognised as a cardiovascular hormone, in addition to its many regulatory roles in other organ systems. Studies in atrial and ventricular myocytes from the neonatal and adult rats have identified synthesis of oxytocin and the expression of oxytocin receptors in these cells. In cardiac fibroblasts, the most populous non-myocyte cell type in mammalian heart, the oxytocin receptors have not been described before. In the present study, we have investigated the direct effects of oxytocin on intracellular Ca²⁺ dynamics in ventricular myocytes and fibroblasts from new born rats. In myocytes, oxytocin increased the frequency of spontaneous Ca²⁺ transients and decreased their amplitude. Our data suggest that oxytocin receptors are also present and functional in the majority of cardiac fibroblasts. We used selective oxytocin receptor inhibitor L-371,257 and a number of intracellular Ca ²⁺ release blockers to investigate the mechanism of oxytocin induced Ca²⁺ signalling in cardiac fibroblasts. Our findings suggest that oxytocin induces Ca²⁺ signals in cardiac fibroblasts by triggering endoplasmic reticulum Ca²⁺ release via inositol trisphosphate activated receptors. The functional significance of the oxytocin induced Ca²⁺ signalling in cardiac fibroblasts, especially for their activation into secretory active myofibroblasts, remains to be investigated.     

Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes

Andersen-Tawil syndrome (ATS) is a rare inherited channelopathy. The cardiac phenotype in ATS is typified by a prominent U wave and ventricular arrhythmia. An effective treatment for this disease remains to be established. We reprogrammed somatic cells from three ATS patients to generate induced pluripotent stem cells (iPSCs). Multi-electrode arrays (MEAs) were used to record extracellular electrograms of iPSC-derived cardiomyocytes, revealing strong arrhythmic events in the ATS-iPSC-derived cardiomyocytes. Ca²⁺ imaging of cells loaded with the Ca²⁺ indicator Fluo-4 enabled us to examine intracellular Ca²⁺ handling properties, and we found a significantly higher incidence of irregular Ca²⁺ release in the ATS-iPSC-derived cardiomyocytes than in control-iPSC-derived cardiomyocytes. Drug testing using ATS-iPSC-derived cardiomyocytes further revealed that antiarrhythmic agent, flecainide, but not the sodium channel blocker, pilsicainide, significantly suppressed these irregular Ca²⁺ release and arrhythmic events, suggesting that flecainide's effect in these cardiac cells was not via sodium channels blocking. A reverse-mode Na^+/Ca²⁺exchanger (NCX) inhibitor, KB-R7943, was also found to suppress the irregular Ca²⁺ release, and whole-cell voltage clamping of isolated guinea-pig cardiac ventricular myocytes confirmed that flecainide could directly affect the NCX current (I_NCX). ATS-iPSC-derived cardiomyocytes recapitulate abnormal electrophysiological phenotypes and flecainide suppresses the arrhythmic events through the modulation of I_NCX.     

NAADP influences excitation-contraction coupling by releasing calcium from lysosomes in atrial myocytes

In atrial myocytes, the sarcoplasmic reticulum (SR) has an essential role in regulating the force of contraction as a consequence of its involvement in excitation-contraction coupling (ECC). Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca²⁺ mobilizing messenger that acts to release Ca²⁺ from an acidic store in mammalian cells. The photorelease of NAADP in atrial myocytes increased Ca²⁺ transient amplitude with no effect on accompanying action potentials or the L-type Ca²⁺ current. NAADP-AM, a cell permeant form of NAADP, increased Ca²⁺ spark amplitude and frequency. The effect on Ca²⁺ spark frequency could be prevented by bafilomycin A1, a vacuolar H⁺-ATPase inhibitor, or by disruption of lysosomes by GPN. Bafilomycin prevented staining of acidic stores with LysoTracker red by increasing lysosomal pH. NAADP-AM also produced an increase in the lysosomal pH, as detected by a reduction in LysoSensor green fluorescence. These effects of NAADP were associated with an increase in the amount of caffeine-releasable Ca²⁺ in the SR and may be regulated by β-adrenoceptor stimulation with isoprenaline. These observations are consistent with a role for NAADP in regulating ECC in atrial myocytes by releasing Ca²⁺ from an acidic store, which enhances SR Ca²⁺ release by increasing SR load.     

Stimulus-Dependent Regulation of Nuclear Ca2+ Signaling in Cardiomyocytes: A Role of Neuronal Calcium Sensor-1

In cardiomyocytes, intracellular calcium (Ca²⁺) transients are elicited by electrical and receptor stimulations, leading to muscle contraction and gene expression, respectively. Although such elevations of Ca²⁺levels ([Ca²⁺]) also occur in the nucleus, the precise mechanism of nuclear [Ca²⁺] regulation during different kinds of stimuli, and its relationship with cytoplasmic [Ca²⁺] regulation are not fully understood. To address these issues, we used a new region-specific fluorescent protein-based Ca²⁺ indicator, GECO, together with the conventional probe Fluo-4 AM. We confirmed that nuclear Ca²⁺ transients were elicited by both electrical and receptor stimulations in neonatal mouse ventricular myocytes. Kinetic analysis revealed that electrical stimulation-elicited nuclear Ca²⁺ transients are slower than cytoplasmic Ca²⁺ transients, and chelating cytoplasmic Ca²⁺ abolished nuclear Ca²⁺ transients, suggesting that nuclear Ca²⁺ are mainly derived from the cytoplasm during electrical stimulation. On the other hand, receptor stimulation such as with insulin-like growth factor-1 (IGF-1) preferentially increased nuclear [Ca²⁺] compared to cytoplasmic [Ca²⁺]. Experiments using inhibitors revealed that electrical and receptor stimulation-elicited Ca²⁺ transients were mainly mediated by ryanodine receptors and inositol 1,4,5-trisphosphate receptors (IP3Rs), respectively, suggesting different mechanisms for the two signals. Furthermore, IGF-1-elicited nuclear Ca²⁺ transient amplitude was significantly lower in myocytes lacking neuronal Ca²⁺ sensor-1 (NCS-1), a Ca²⁺ binding protein implicated in IP₃R-mediated pathway in the heart. Moreover, IGF-1 strengthened the interaction between NCS-1 and IP₃R. These results suggest a novel mechanism for receptor stimulation-induced nuclear [Ca²⁺] regulation mediated by IP3R and NCS-1 that may further fine-tune cardiac Ca²⁺ signal regulation.     

Mechanisms by which cytoplasmic calcium wave propagation and alternans are generated in cardiac atrial myocytes lacking T-tubules-insights from a simulation study

This study investigated the mechanisms underlying the propagation of cytoplasmic calcium waves and the genesis of systolic Ca²⁺ alternans in cardiac myocytes lacking transverse tubules (t-tubules). These correspond to atrial cells of either small mammals or large mammals that have lost their t-tubules due to disease-induced structural remodeling (e.g., atrial fibrillation). A mathematical model was developed for a cluster of ryanodine receptors distributed on the cross section of a cell that was divided into 13 elements with a spatial resolution of 2 μm. Due to the absence of t-tubules, L-type Ca²⁺ channels were only located in the peripheral elements close to the cell-membrane surface and produced Ca²⁺ signals that propagated toward central elements by triggering successive Ca²⁺-induced Ca²⁺ release (CICR) via Ca²⁺ diffusion between adjacent elements. Under control conditions, the Ca²⁺ signals did not fully propagate to the central region of the cell. However, with modulation of several factors responsible for Ca²⁺ handling, such as the L-type Ca²⁺ channels (Ca²⁺ influx), SERCA pumps (sarcoplasmic reticulum (SR) Ca²⁺ uptake), and ryanodine receptors (SR Ca²⁺ release), Ca²⁺ wave propagation to the center of the cell could occur. These simulation results are consistent with previous experimental data from atrial cells of small mammals. The model further reveals that spatially functional heterogeneity in Ca²⁺ diffusion within the cell produced a steep relationship between the SR Ca²⁺ content and the cytoplasmic Ca²⁺ concentration. This played an important role in the genesis of Ca²⁺ alternans that were more obvious in central than in peripheral elements. Possible association between the occurrence of Ca²⁺ alternans and the model parameters of Ca²⁺ handling was comprehensively explored in a wide range of one- and two-parameter spaces. In addition, the model revealed a spontaneous second Ca²⁺ release in response to a single voltage stimulus pulse with SR Ca²⁺ overloading and augmented Ca²⁺ influx. This study provides what to our knowledge are new insights into the genesis of Ca²⁺ alternans and spontaneous second Ca²⁺ release in cardiac myocytes that lack t-tubules.     

Cellular Hypertrophy and Increased Susceptibility to Spontaneous Calcium-Release of Rat Left Atrial Myocytes Due to Elevated Afterload

Atrial remodeling due to elevated arterial pressure predisposes the heart to atrial fibrillation (AF). Although abnormal sarcoplasmic reticulum (SR) function has been associated with AF, there is little information on the effects of elevated afterload on atrial Ca²⁺-handling. We investigated the effects of ascending aortic banding (AoB) on Ca²⁺-handling in rat isolated atrial myocytes in comparison to age-matched sham-operated animals (Sham). Myocytes were either labelled for ryanodine receptor (RyR) or loaded with fluo-3-AM and imaged by confocal microscopy. AoB myocytes were hypertrophied in comparison to Sham controls (P<0.0001). RyR labeling was localized to the z-lines and to the cell edge. There were no differences between AoB and Sham in the intensity or pattern of RyR-staining. In both AoB and Sham, electrical stimulation evoked robust SR Ca²⁺-release at the cell edge whereas Ca²⁺ transients at the cell center were much smaller. Western blotting showed a decreased L-type Ca channel expression but no significant changes in RyR or RyR phosphorylation or in expression of Na⁺/Ca²⁺ exchanger, SR Ca²⁺ ATPase or phospholamban. Mathematical modeling indicated that [Ca²⁺]_i transients at the cell center were accounted for by simple centripetal diffusion of Ca²⁺ released at the cell edge. In contrast, caffeine (10 mM) induced Ca²⁺ release was uniform across the cell. The caffeine-induced transient was smaller in AoB than in Sham, suggesting a reduced SR Ca²⁺-load in hypertrophied cells. There were no significant differences between AoB and Sham cells in the rate of Ca²⁺ extrusion during recovery of electrically-stimulated or caffeine-induced transients. The incidence and frequency of spontaneous Ca²⁺-transients following rapid-pacing (4 Hz) was greater in AoB than in Sham myocytes. In conclusion, elevated afterload causes cellular hypertrophy and remodeling of atrial SR Ca²⁺-release.     

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca²⁺ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).¹ Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca²⁺ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca²⁺-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 μM Ca²⁺. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca²⁺ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca²⁺ and Ca²⁺ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca²⁺ transient measurements, performed in these isolated myocytes.     

Channel Activity of Cardiac Ryanodine Receptors (RyR2) Determines Potency and Efficacy of Flecainide and R-Propafenone against Arrhythmogenic Calcium Waves in Ventricular Cardiomyocytes

Flecainide blocks ryanodine receptor type 2 (RyR2) channels in the open state, suppresses arrhythmogenic Ca²⁺ waves and prevents catecholaminergic polymorphic ventricular tachycardia (CPVT) in mice and humans. We hypothesized that differences in RyR2 activity induced by CPVT mutations determines the potency of open-state RyR2 blockers like flecainide (FLEC) and R-propafenone (RPROP) against Ca²⁺ waves in cardiomyocytes. Using confocal microscopy, we studied Ca²⁺ sparks and waves in isolated saponin-permeabilized ventricular myocytes from two CPVT mouse models (Casq2^-/-, RyR2-R4496C^+/-), wild-type (c57bl/6, WT) mice, and WT rabbits (New Zealand white rabbits). Consistent with increased RyR2 activity, Ca²⁺ spark and wave frequencies were significantly higher in CPVT compared to WT mouse myocytes. We next obtained concentration-response curves of Ca²⁺ wave inhibition for FLEC, RPROP (another open-state RyR2 blocker), and tetracaine (TET) (a state-independent RyR2 blocker). Both FLEC and RPROP inhibited Ca²⁺ waves with significantly higher potency (lower IC₅₀) and efficacy in CPVT compared to WT. In contrast, TET had similar potency in all groups studied. Increasing RyR2 activity of permeabilized WT myocytes by exposure to caffeine (150 µM) increased the potency of FLEC and RPROP but not of TET. RPROP and FLEC were also significantly more potent in rabbit ventricular myocytes that intrinsically exhibit higher Ca²⁺ spark rates than WT mouse ventricular myocytes. In conclusion, RyR2 activity determines the potency of open-state blockers FLEC and RPROP for suppressing arrhythmogenic Ca²⁺ waves in cardiomyocytes, a mechanism likely relevant to antiarrhythmic drug efficacy in CPVT.     

Sodium-Calcium Exchange Is Essential for Effective Triggering of Calcium Release in Mouse Heart

In cardiac myocytes, excitation-contraction coupling depends upon sarcoplasmic reticular Ca²⁺ release triggered by Ca²⁺ influx through L-type Ca²⁺ channels. Although Na⁺-Ca²⁺ exchange (NCX) is essential for Ca²⁺ extrusion, its participation in the trigger process of excitation-contraction coupling is controversial. To investigate the role of NCX in triggering, we examined Ca²⁺ sparks in ventricular cardiomyocytes isolated from wild-type (WT) and cardiac-specific NCX knockout (KO) mice. Myocytes from young NCX KO mice are known to exhibit normal resting cytosolic Ca²⁺ and normal Ca²⁺ transients despite reduced L-type Ca²⁺ current. We loaded myocytes with fluo-3 to image Ca²⁺ sparks using confocal microscopy in line-scan mode. The frequency of spontaneous Ca²⁺ sparks was reduced in KO myocytes compared with WT. However, spark amplitude and width were increased in KO mice. Permeabilizing the myocytes with saponin eliminated differences between spontaneous sparks in WT and KO mice. These results suggest that sarcolemmal processes are responsible for the reduced spark frequency and increased spark width and amplitude in KO mice. When myocytes were loaded with 1 mM fluo-3 and 3 mM EGTA via the patch pipette to buffer diadic cleft Ca²⁺, the number of sparks triggered by action potentials was reduced by 60% in KO cells compared to WT cells, despite similar SR Ca²⁺ content in both cell types. When EGTA was omitted from the pipette solution, the number of sparks triggered in KO and WT myocytes was similar. Although the number of sparks was restored in KO cells, Ca²⁺ release was asynchronous. These results suggest that high subsarcolemmal Ca²⁺ is required to ensure synchronous triggering with short spark latency in the absence of NCX. In WT mice, high subsarcolemmal Ca²⁺ is not required for synchronous triggering, because NCX is capable of priming the diadic cleft with sufficient Ca²⁺ for normal triggering, even when subsarcolemmal Ca²⁺ is lowered by EGTA. Thus, reducing subsarcolemmal Ca²⁺ with EGTA in NCX KO mice reveals the dependence of Ca²⁺ release on NCX.