A three-dimensional simulation model of cardiomyocyte integrating excitation-contraction coupling and metabolism

Recent studies have revealed that Ca²⁺ not only regulates the contraction of cardiomyocytes, but can also function as a signaling agent to stimulate ATP production by the mitochondria. However, the spatiotemporal resolution of current experimental techniques limits our investigative capacity to understand this phenomenon. Here, we created a detailed three-dimensional (3D) cardiomyocyte model to study the subcellular regulatory mechanisms of myocardial energetics. The 3D cardiomyocyte model was based on the finite-element method, with detailed subcellular structures reproduced, and it included all elementary processes involved in cardiomyocyte electrophysiology, contraction, and ATP metabolism localized to specific loci. The simulation results were found to be reproducible and consistent with experimental data regarding the spatiotemporal pattern of cytosolic, intrasarcoplasmic-reticulum, and mitochondrial changes in Ca²⁺; as well as changes in metabolite levels. Detailed analysis suggested that although the observed large cytosolic Ca²⁺ gradient facilitated uptake by the mitochondrial Ca²⁺ uniporter to produce cyclic changes in mitochondrial Ca²⁺ near the Z-line region, the average mitochondrial Ca²⁺ changes slowly. We also confirmed the importance of the creatine phosphate shuttle in cardiac energy regulation. In summary, our 3D model provides a powerful tool for the study of cardiac function by overcoming some of the spatiotemporal limitations of current experimental approaches.     

Regulation of the RyR channel gating by Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Ryanodine receptors (RyRs) are the Ca²⁺ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca²⁺ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca²⁺. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca²⁺ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca²⁺ at these sites to inhibit RyRs and this plays an important role in modulating their Ca²⁺-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca²⁺ and Mg²⁺ and how these mechanisms control Ca²⁺ release in excitation-contraction coupling and cardiac pacemaking.     

CaMKII‐dependent ryanodine receptor phosphorylation mediates sepsis‐induced cardiomyocyte apoptosis

Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis‐induced apoptosis. Wild‐type (WT) CASP mice hearts showed an increase in apoptosis respect to WT‐Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3‐I) were protected against sepsis‐induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca²⁺ release, prevented apoptosis in WT‐CASP. To examine whether CaMKII‐dependent RyR2 phosphorylation mediates diastolic Ca²⁺ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation‐dependent enhancement in diastolic SR Ca²⁺ release leading to mitochondrial Ca²⁺ overload, mitochondrial Ca²⁺ retention capacity was reduced in mitochondria isolated from WT‐CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene‐treated mice. We conclude that in sepsis, CaMKII‐dependent RyR2 phosphorylation results in diastolic Ca²⁺ release from SR which leads to mitochondrial Ca²⁺ overload and apoptosis.     

Distinct Functional Roles of Cardiac Mitochondrial Subpopulations Revealed by a 3D Simulation Model

Experimental characterization of two cardiac mitochondrial subpopulations, namely, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), has been hampered by technical difficulties, and an alternative approach is eagerly awaited. We previously developed a three-dimensional computational cardiomyocyte model that integrates electrophysiology, metabolism, and mechanics with subcellular structure. In this study, we further developed our model to include intracellular oxygen diffusion, and determined whether mitochondrial localization or intrinsic properties cause functional variations. For this purpose, we created two models: one with equal SSM and IFM properties and one with IFM having higher activity levels. Using these two models to compare the SSM and IFM responses of [Ca²⁺], tricarboxylic acid cycle activity, [NADH], and mitochondrial inner membrane potential to abrupt changes in pacing frequency (0.25–2 Hz), we found that the reported functional differences between these subpopulations appear to be mostly related to local [Ca²⁺] heterogeneity, and variations in intrinsic properties only serve to augment these differences. We also examined the effect of hypoxia on mitochondrial function. Under normoxic conditions, intracellular oxygen is much higher throughout the cell than the half-saturation concentration for oxidative phosphorylation. However, under limited oxygen supply, oxygen is mostly exhausted in SSM, leaving the core region in an anoxic condition. Reflecting this heterogeneous oxygen environment, the inner membrane potential continues to decrease in IFM, whereas it is maintained to nearly normal levels in SSM, thereby ensuring ATP supply to this region. Our simulation results provide clues to understanding the origin of functional variations in two cardiac mitochondrial subpopulations and their differential roles in maintaining cardiomyocyte function as a whole.     

Mitochondria in cardiomyocyte Ca signaling

Ca²⁺ signaling is of vital importance to cardiac cell function and plays an important role in heart failure. It is based on sarcolemmal, sarcoplasmic reticulum and mitochondrial Ca²⁺ cycling. While the first two are well characterized, the latter remains unclear, controversial and technically challenging.In mammalian cardiac myocytes, Ca²⁺ influx through L-type calcium channels in the sarcolemmal membrane triggers Ca²⁺ release from the nearby junctional sarcoplasmic reticulum to produce Ca²⁺ sparks. When this triggering is synchronized by the cardiac action potential, a global [Ca²⁺]_i transient arises from coordinated Ca²⁺ release events. The ends of intermyofibrillar mitochondria are located within 20 nm of the junctional sarcoplasmic reticulum and thereby experience a high local [Ca²⁺] during the Ca²⁺ release process. Both local and global Ca²⁺ signals may thus influence calcium signaling in mitochondria and, reciprocally, mitochondria may contribute to the local control of calcium signaling. In addition to the intermyofibrillar mitochondria, morphologically distinct mitochondria are also located in the perinuclear and subsarcolemmal regions of the cardiomyocyte and thus experience a different local [Ca²⁺].Here we review the literature in regard to several issues of broad interest: (1) the ultrastructural basis for mitochondrion – sarcoplasmic reticulum cross-signaling; (2) mechanisms of sarcoplasmic reticulum signaling; (3) mitochondrial calcium signaling; and (4) the possible interplay of calcium signaling between the sarcoplasmic reticulum and adjacent mitochondria.Finally, this review discusses experimental findings and mathematical models of cardiac calcium signaling between the sarcoplasmic reticulum and mitochondria, identifies weaknesses in these models, and suggests strategies and approaches for future investigations.     

Inhibiting Na+/K+ ATPase Can Impair Mitochondrial Energetics and Induce Abnormal Ca2+ Cycling and Automaticity in Guinea Pig Cardiomyocytes

Cardiac glycosides have been used for the treatment of heart failure because of their capabilities of inhibiting Na⁺/K⁺ ATPase (NKA), which raises [Na⁺]_i and attenuates Ca²⁺ extrusion via the Na⁺/Ca²⁺ exchanger (NCX), causing [Ca²⁺]_i elevation. The resulting [Ca²⁺]_i accumulation further enhances Ca²⁺-induced Ca²⁺ release, generating the positive inotropic effect. However, cardiac glycosides have some toxic and side effects such as arrhythmogenesis, confining their extensive clinical applications. The mechanisms underlying the proarrhythmic effect of glycosides are not fully understood. Here we investigated the mechanisms by which glycosides could cause cardiac arrhythmias via impairing mitochondrial energetics using an integrative computational cardiomyocyte model. In the simulations, the effect of glycosides was mimicked by blocking NKA activity. Results showed that inhibiting NKA not only impaired mitochondrial Ca²⁺ retention (thus suppressed reactive oxygen species (ROS) scavenging) but also enhanced oxidative phosphorylation (thus increased ROS production) during the transition of increasing workload, causing oxidative stress. Moreover, concurrent blocking of mitochondrial Na⁺/Ca²⁺ exchanger, but not enhancing of Ca²⁺ uniporter, alleviated the adverse effects of NKA inhibition. Intriguingly, NKA inhibition elicited Ca²⁺ transient and action potential alternans under more stressed conditions such as severe ATP depletion, augmenting its proarrhythmic effect. This computational study provides new insights into the mechanisms underlying cardiac glycoside-induced arrhythmogenesis. The findings suggest that targeting both ion handling and mitochondria could be a very promising strategy to develop new glycoside-based therapies in the treatment of heart failure.     

A computational model of pig ventricular cardiomyocyte electrophysiology and calcium handling: Translation from pig to human electrophysiology

The pig is commonly used as an experimental model of human heart disease, including for the study of mechanisms of arrhythmia. However, there exist differences between human and porcine cellular electrophysiology: The pig action potential (AP) has a deeper phase-1 notch, a longer duration at 50% repolarization, and higher plateau potentials than human. Ionic differences underlying the AP include larger rapid delayed-rectifier and smaller inward-rectifier K⁺-currents (I_Kr and I_K1 respectively) in humans. AP steady-state rate-dependence and restitution is steeper in pigs. Porcine Ca²⁺ transients can have two components, unlike human. Although a reliable computational model for human ventricular myocytes exists, one for pigs is lacking. This hampers translation from results obtained in pigs to human myocardium. Here, we developed a computational model of the pig ventricular cardiomyocyte AP using experimental datasets of the relevant ionic currents, Ca²⁺-handling, AP shape, AP duration restitution, and inducibility of triggered activity and alternans. To properly capture porcine Ca²⁺ transients, we introduced a two-step process with a faster release in the t-tubular region, followed by a slower diffusion-induced release from a non t-tubular subcellular region. The pig model behavior was compared with that of a human ventricular cardiomyocyte (O’Hara-Rudy) model. The pig, but not the human model, developed early afterdepolarizations (EADs) under block of I_K1, while I_Kr block led to EADs in the human but not in the pig model. At fast rates (pacing cycle length = 400 ms), the human cell model was more susceptible to spontaneous Ca²⁺ release-mediated delayed afterdepolarizations (DADs) and triggered activity than pig. Fast pacing led to alternans in human but not pig. Developing species-specific models incorporating electrophysiology and Ca^2+-handling provides a tool to aid translating antiarrhythmic and arrhythmogenic assessment from the bench to the clinic.     

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.     

Role of myocardial viscoelasticity in disturbances of electrical and mechanical activity in calcium overloaded cardiomyocytes: mathematical modeling

Cardiomyocyte Ca²⁺ overload is closely linked to cardiac arrhythmias. We have earlier shown in a mathematical model that myocardium mechanical activity may contribute to rhythm disturbances induced by Ca²⁺ overload in cardiomyocytes with reduced Na⁺-K⁺ pump work (Sulman et al., 2008). The same model is used here to address possible contribution of the passive mechanical properties of cardiac muscle (i.e. myocardial viscous and elastic properties) to the arrhythmogenesis. In a series of contractions at regular pacing rate of 75 beats/min a model with higher viscosity demonstrated essentially earlier appearance of extrasystoles due to a faster cardiomyocyte Ca²⁺ loading up to a level triggering spontaneous Ca²⁺ releases from the sarcoplasmic reticulum. The model predicts that myocardial elasticity also may affect arrhythmogenesis in cardiomyocytes overloaded with Ca²⁺. Contribution of the mechanical properties of the myocardial tissue to the arrhythmia has been analyzed for wide ranges of both viscosity and elasticity coefficients. The results suggest that myocardial viscoelastic properties may be a factor affecting Ca²⁺ handling in cardiomyocytes and contributing to cardiac mechano-electric feedback in arrhythmogenesis.     

Superresolution Modeling of Calcium Release in the Heart

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cellular contraction during cardiac excitation-contraction coupling. The fundamental element of CICR in the heart is the calcium (Ca²⁺) spark, which arises from a cluster of ryanodine receptors (RyR). Opening of these RyR clusters is triggered to produce a local, regenerative release of Ca²⁺ from the sarcoplasmic reticulum (SR). The Ca²⁺ leak out of the SR is an important process for cellular Ca²⁺ management, and it is critically influenced by spark fidelity, i.e., the probability that a spontaneous RyR opening triggers a Ca²⁺ spark. Here, we present a detailed, three-dimensional model of a cardiac Ca²⁺ release unit that incorporates diffusion, intracellular buffering systems, and stochastically gated ion channels. The model exhibits realistic Ca²⁺ sparks and robust Ca²⁺ spark termination across a wide range of geometries and conditions. Furthermore, the model captures the details of Ca²⁺ spark and nonspark-based SR Ca²⁺ leak, and it produces normal excitation-contraction coupling gain. We show that SR luminal Ca²⁺-dependent regulation of the RyR is not critical for spark termination, but it can explain the exponential rise in the SR Ca²⁺ leak-load relationship demonstrated in previous experimental work. Perturbations to subspace dimensions, which have been observed in experimental models of disease, strongly alter Ca²⁺ spark dynamics. In addition, we find that the structure of RyR clusters also influences Ca²⁺ release properties due to variations in inter-RyR coupling via local subspace Ca²⁺ concentration ([Ca²⁺]_ss). These results are illustrated for RyR clusters based on super-resolution stimulated emission depletion microscopy. Finally, we present a believed-novel approach by which the spark fidelity of a RyR cluster can be predicted from structural information of the cluster using the maximum eigenvalue of its adjacency matrix. These results provide critical insights into CICR dynamics in heart, under normal and pathological conditions.     

Diversity of mitochondrial Ca signaling in rat neonatal cardiomyocytes: Evidence from a genetically directed Ca probe,mitycam-E31Q

I_Ca-gated Ca²⁺ release (CICR) from the cardiac SR is the main mechanism mediating the rise of cytosolic Ca²⁺, but the extent to which mitochondria contribute to the overall Ca²⁺ signaling remains controversial. To examine the possible role of mitochondria in Ca²⁺ signaling, we developed a low affinity mitochondrial Ca²⁺ probe, mitycam-E31Q (300–500 MOI, 48–72 h) and used it in conjunction with Fura-2AM to obtain simultaneous TIRF images of mitochondrial and cytosolic Ca²⁺ in cultured neonatal rat cardiomyocytes. Mitycam-E31Q staining of adult feline cardiomyocytes showed the typical mitochondrial longitudinal fluorescent bandings similar to that of TMRE staining, while neonatal rat cardiomyocytes had a disorganized tubular or punctuate appearance. Caffeine puffs produced rapid increases in cytosolic Ca²⁺ while simultaneously measured global mitycam-E31Q signals decreased more slowly (increased mitochondrial Ca²⁺) before decaying to baseline levels. Similar, but oscillating mitycam-E31Q signals were seen in spontaneously pacing cells. Withdrawal of Na⁺ increased global cytosolic and mitochondrial Ca²⁺ signals in one population of mitochondria, but unexpectedly decreased it (release of Ca²⁺) in another mitochondrial population. Such mitochondrial Ca²⁺ release signals were seen not only during long lasting Na⁺ withdrawal, but also when Ca²⁺ loaded cells were exposed to caffeine-puffs, and during spontaneous rhythmic beating. Thus, mitochondrial Ca²⁺ transients appear to activate with a delay following the cytosolic rise of Ca²⁺ and show diversity in subpopulations of mitochondria that could contribute to the plasticity of mitochondrial Ca²⁺ signaling.     

Moment closure for local control models of calcium-induced calcium release in cardiac myocytes

In prior work, we introduced a probability density approach to modeling local control of Ca²⁺-induced Ca²⁺ release in cardiac myocytes, where we derived coupled advection-reaction equations for the time-dependent bivariate probability density of subsarcolemmal subspace and junctional sarcoplasmic reticulum (SR) [Ca²⁺] conditioned on Ca²⁺ release unit (CaRU) state. When coupled to ordinary differential equations (ODEs) for the bulk myoplasmic and network SR [Ca²⁺], a realistic but minimal model of cardiac excitation-contraction coupling was produced that avoids the computationally demanding task of resolving spatial aspects of global Ca²⁺ signaling, while accurately representing heterogeneous local Ca²⁺ signals in a population of diadic subspaces and junctional SR depletion domains. Here we introduce a computationally efficient method for simulating such whole cell models when the dynamics of subspace [Ca²⁺] are much faster than those of junctional SR [Ca²⁺]. The method begins with the derivation of a system of ODEs describing the time-evolution of the moments of the univariate probability density functions for junctional SR [Ca²⁺] jointly distributed with CaRU state. This open system of ODEs is then closed using an algebraic relationship that expresses the third moment of junctional SR [Ca²⁺] in terms of the first and second moments. In simulated voltage-clamp protocols using 12-state CaRUs that respond to the dynamics of both subspace and junctional SR [Ca²⁺], this moment-closure approach to simulating local control of excitation-contraction coupling produces high-gain Ca²⁺ release that is graded with changes in membrane potential, a phenomenon not exhibited by common pool models. Benchmark simulations indicate that the moment-closure approach is nearly 10,000-times more computationally efficient than corresponding Monte Carlo simulations while leading to nearly identical results. We conclude by applying the moment-closure approach to study the restitution of Ca²⁺-induced Ca²⁺ release during simulated two-pulse voltage-clamp protocols.     

Oxidative Stress and Ca2+ Release Events in Mouse Cardiomyocytes

Cellular oxidative stress, associated with a variety of common cardiac diseases, is well recognized to affect the function of several key proteins involved in Ca²⁺ signaling and excitation-contraction coupling, which are known to be exquisitely sensitive to reactive oxygen species. These include the Ca²⁺ release channels of the sarcoplasmic reticulum (ryanodine receptors or RyR2s) and the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Oxidation of RyR2s was found to increase the open probability of the channel, whereas CaMKII can be activated independent of Ca²⁺ through oxidation. Here, we investigated how oxidative stress affects RyR2 function and SR Ca²⁺ signaling in situ, by analyzing Ca²⁺ sparks in permeabilized mouse cardiomyocytes under a broad range of oxidative conditions. The results show that with increasing oxidative stress Ca²⁺ spark duration is prolonged. In addition, long and very long-lasting (up to hundreds of milliseconds) localized Ca²⁺ release events started to appear, eventually leading to sarcoplasmic reticulum (SR) Ca²⁺ depletion. These changes of release duration could be prevented by the CaMKII inhibitor KN93 and did not occur in mice lacking the CaMKII-specific S2814 phosphorylation site on RyR2. The appearance of long-lasting Ca²⁺ release events was paralleled by an increase of RyR2 oxidation, but also by RyR-S2814 phosphorylation, and by CaMKII oxidation. Our results suggest that in a strongly oxidative environment oxidation-dependent activation of CaMKII leads to RyR2 phosphorylation and thereby contributes to the massive prolongation of SR Ca²⁺ release events.     

Fructose modulates cardiomyocyte excitation-contraction coupling and Ca²⁺ handling in vitro

Background

High dietary fructose has structural and metabolic cardiac impact, but the potential for fructose to exert direct myocardial action is uncertain. Cardiomyocyte functional responsiveness to fructose, and capacity to transport fructose has not been previously demonstrated.

Objective

The aim of the present study was to seek evidence of fructose-induced modulation of cardiomyocyte excitation-contraction coupling in an acute, in vitro setting.

Methods and Results

The functional effects of fructose on isolated adult rat cardiomyocyte contractility and Ca²⁺ handling were evaluated under physiological conditions (37°C, 2 mM Ca²⁺, HEPES buffer, 4 Hz stimulation) using video edge detection and microfluorimetry (Fura2) methods. Compared with control glucose (11 mM) superfusate, 2-deoxyglucose (2 DG, 11 mM) substitution prolonged both the contraction and relaxation phases of the twitch (by 16 and 36% respectively, p<0.05) and this effect was completely abrogated with fructose supplementation (11 mM). Similarly, fructose prevented the Ca²⁺ transient delay induced by exposure to 2 DG (time to peak Ca²⁺ transient: 2 DG: 29.0±2.1 ms vs. glucose: 23.6±1.1 ms vs. fructose +2 DG: 23.7±1.0 ms; p<0.05). The presence of the fructose transporter, GLUT5 (Slc2a5) was demonstrated in ventricular cardiomyocytes using real time RT-PCR and this was confirmed by conventional RT-PCR.

Conclusion

This is the first demonstration of an acute influence of fructose on cardiomyocyte excitation-contraction coupling. The findings indicate cardiomyocyte capacity to transport and functionally utilize exogenously supplied fructose. This study provides the impetus for future research directed towards characterizing myocardial fructose metabolism and understanding how long term high fructose intake may contribute to modulating cardiac function.     

Distribution and Function of Cardiac Ryanodine Receptor Clusters in Live Ventricular Myocytes

The cardiac Ca²⁺ release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca²⁺ sparks showed that Ca²⁺ sparks originated exclusively from RyR2 clusters. Ca²⁺ sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca²⁺ level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.     

Molecular Determinants of Calpain-dependent Cleavage of Junctophilin-2 Protein in Cardiomyocytes

Junctophilin-2 (JP2), a membrane-binding protein that provides a structural bridge between the plasmalemma and sarcoplasmic reticulum, is essential for precise Ca²⁺-induced Ca²⁺ release during excitation-contraction coupling in cardiomyocytes. In animal and human failing hearts, expression of JP2 is decreased markedly, but the molecular mechanisms underlying JP2 down-regulation remain incompletely defined. In mouse hearts, ischemia/reperfusion injury resulted in acute JP2 down-regulation, which was attenuated by pretreatment with the calpain inhibitor MDL-28170 or by transgenic overexpression of calpastatin, an endogenous calpain inhibitor. Using a combination of computational analysis to predict calpain cleavage sites and in vitro calpain proteolysis reactions, we identified four putative calpain cleavage sites within JP2 with three N-terminal and one C-terminal cleavage sites. Mutagenesis defined the C-terminal region of JP2 as the predominant calpain cleavage site. Exogenous expression of putative JP2 cleavage fragments was not sufficient to rescue Ca²⁺ handling in JP2-deficient cardiomyocytes, indicating that cleaved JP2 is non-functional for normal Ca²⁺-induced Ca²⁺ release. These data provide new molecular insights into the posttranslational regulatory mechanisms of JP2 in cardiac diseases.     

Transport of Ca2+ from Sarcoplasmic Reticulum to Mitochondria in Rat Ventricular Myocytes

Studies with electron microscopy have shown that sarcoplasmic reticulum (SR) andmitochondria locate close to each other in cardiac muscle cells. We investigated the hypothesis thatthis proximity results in a transient exposure of mitochondrial Ca²⁺ uniporter (CaUP) to highconcentrations of Ca²⁺ following Ca²⁺ release from the SR and thus an influx of Ca²⁺into mitochondria. Single ventricular myocytes of rat were skinned by exposing them to aphysiological solution containing saponin (0.2 mg/ml). Cytosolic Ca²⁺ concentration ([Ca²⁺]_c)and mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) were measured with fura-2 and rhod2,respectively. Application of caffeine (10 mM) induced a concomitant increase in[Ca²⁺]_c and [Ca²⁺]_m.Ruthenium red, at concentrations that block CaUP but not SR release, diminished thecaffeine-induced increase in [Ca²⁺]_m but not[Ca²⁺]_c. In the presence of 1 mM BAPTA, a Ca²⁺ chelator,the caffeine-induced increase in [Ca²⁺]_m was reduced substantially less than [Ca²⁺]_c. Moreover,inhibition of SR Ca²⁺ pump with two different concentrations of thapsigargin caused anincrease in [Ca²⁺]_m, which was related to the rate of [Ca²⁺]_c increase. Finally, electronmicroscopy showed that sites of junctions between SR and T tubules from which Ca²⁺ is released,or Ca²⁺ release units, CRUs, are preferentially located in close proximity to mitochondria.The distance between individual SR Ca²⁺ release channels (feet or ryanodine receptors) isvery short, ranging between approximately 37 and 270 nm. These results are consistent withthe idea that there is a preferential coupling of Ca²⁺ transport from SR to mitochondria incardiac muscle cells, because of their structural proximity.     

Modeling Local X-ROS and Calcium Signaling in the Heart

Stretching single ventricular cardiac myocytes has been shown experimentally to activate transmembrane nicotinamide adenine dinucleotide phosphate oxidase type 2 to produce reactive oxygen species (ROS) and increase the Ca²⁺ spark rate in a process called X-ROS signaling. The increase in Ca²⁺ spark rate is thought to be due to an increase in ryanodine receptor type 2 (RyR2) open probability by direct oxidation of the RyR2 protein complex. In this article, a computational model is used to examine the regulation of ROS and calcium homeostasis by local, subcellular X-ROS signaling and its role in cardiac excitation-contraction coupling. To this end, a four-state RyR2 model was developed that includes an X-ROS-dependent RyR2 mode switch. When activated, [Ca²⁺]_i-sensitive RyR2 open probability increases, and the Ca²⁺ spark rate changes in a manner consistent with experimental observations. This, to our knowledge, new model is used to study the transient effects of diastolic stretching and subsequent ROS production on RyR2 open probability, Ca²⁺ sparks, and the myoplasmic calcium concentration ([Ca²⁺]_i) during excitation-contraction coupling. The model yields several predictions: 1) [ROS] is produced locally near the RyR2 complex during X-ROS signaling and increases by an order of magnitude more than the global ROS signal during myocyte stretching; 2) X-ROS activation just before the action potential, corresponding to ventricular filling during diastole, increases the magnitude of the Ca²⁺ transient; 3) during prolonged stretching, the X-ROS-induced increase in Ca²⁺ spark rate is transient, so that long-sustained stretching does not significantly increase sarcoplasmic reticulum Ca²⁺ leak; and 4) when the chemical reducing capacity of the cell is decreased, activation of X-ROS signaling increases sarcoplasmic reticulum Ca²⁺ leak and contributes to global oxidative stress, thereby increases the possibility of arrhythmia. The model provides quantitative information not currently obtainable through experimental means and thus provides a framework for future X-ROS signaling experiments.     

Membrane topology and membrane retention of the ryanodine receptor calcium release channel

The ryanodine receptor (RyR) is a Ca²⁺ release channel located in the sarcoplasmic/endoplasmic reticulum (ER) membrane and plays a critical role in excitation-contraction coupling of skeletal and cardiac muscles. RyR normally exists in a tetrameric structure and contains two functional domains: a carboxyl-terminal hydrophobic domain that contains the conduction pore of the Ca²⁺ release channel, and a large amino-terminal domain that contains sites responsible for channel regulation. Recent studies involving mutagenesis and heterologous expression have helped unravel the structure-function relationship of RyR, including transmembrane topology and intracellular localization of the Ca²⁺-release channel. The carboxyl-terminal portion of RyR contains the putative transmembrane segments and is sufficient to form a functional Ca²⁺-release channel. The amino-terminal region of the protein contains sites responsible for regulation by endogenous modulators such as Ca²⁺ and Mg²⁺ and by exogenous ligands such as caffeine. The membrane topology of RyR appears to contain an even number (four or six) of transmembrane segments with a ion selectivity filter present within a region residing between the last two segments, similar to potassium channel, whose atomic structure was described recently. The transmembrane segments also contain sequences that are responsible for localization of RyR in the endoplasmic reticulum, and this sequence is highly conserved in IP₃ receptors, which also function as Ca²⁺-release channels.     

Regulation of mitochondrial Ca<Superscript>2+</Superscript> and its effects on energetics and redox balance in normal and failing heart

Ca²⁺ has been well accepted as a signal that coordinates changes in cytosolic workload with mitochondrial energy metabolism in cardiomyocytes. During increased work, Ca²⁺ is accumulated in mitochondria and stimulates ATP production to match energy supply and demand. The kinetics of mitochondrial Ca²⁺ ([Ca²⁺]_m) uptake remains unclear, and we review the debate on this subject in this article. [Ca²⁺]_m has multiple targets in oxidative phosphorylation including the F1/FO ATPase, the adenine nucleotide translocase, and Ca²⁺-sensitive dehydrogenases (CaDH) of the tricarboxylic acid (TCA) cycle. The well established effect of [Ca²⁺]_m is to activate CaDHs of the TCA cycle to increase NADH production. Maintaining NADH level is not only critical to keep a high oxidative phosphorylation rate during increased cardiac work, but is also necessary for the reducing system of the cell to maintain its reactive oxygen species (ROS) —scavenging capacity. Further, we review recent data demonstrating the deleterious effects of elevated Na⁺ in cardiac pathology by blunting [Ca²⁺]_m accumulation.