Integrative analysis of calcium cycling in cardiac muscle

The control of intracellular calcium is central to regulation of contractile force in cardiac muscle. This review illustrates how analysis of the control of calcium requires an integrated approach in which several systems are considered. Thus, the calcium content of the sarcoplasmic reticulum (SR) is a major determinant of the amount of Ca(2+) released from the SR and the amplitude of the Ca(2+) transient. The amplitude of the transient, in turn, controls Ca(2+) fluxes across the sarcolemma and thence SR content. This control of SR content influences the response to maneuvers that modify, for example, the properties of the SR Ca(2+) release channel or ryanodine receptor. Specifically, modulation of the open probability of the ryanodine receptor produces only transient effects on the Ca(2+) transient as a result of changes of SR content. These interactions between various Ca(2+) fluxes are modified by the Ca(2+) buffering properties of the cell. Finally, we predict that, under some conditions, the above interactions can result in instability (such as alternans) rather than ordered control of contractility.     

Effects of eicosapentaenoic acid on cardiac SR Ca(2+)-release and ryanodine receptor function

n-3 polyunsaturated fatty acids (PUFAs) can prevent life-threatening arrhythmias but the mechanisms responsible have not been established. There is strong evidence that part of the antiarrhythmic action of PUFAs is mediated through inhibition of the Ca(2+)-release mechanism of the sarcoplasmic reticulum (SR). It has also been shown that PUFAs activate protein kinase A (PKA) and produce effects in the cardiac cell similar to beta-adrenergic stimulation. We have investigated whether the inhibitory effect of PUFAs on the Ca(2+)-release mechanism is caused by direct inhibition of the SR Ca(2+)-release channel/ryanodine receptor (RyR) or requires activation of PKA. Experiments in intact cells under voltage-clamp show that the n-3 PUFA eicosapentaenoic acid (EPA) is able to reduce the frequency of spontaneous waves of Ca(2+)-release while increasing SR Ca(2+) content even when PKA activity is inhibited with H-89. This suggests that the EPA-induced inhibition of SR Ca(2+)-release is not dependent on activation of PKA. Consistent with this, single-channel studies demonstrate that EPA (10-100 microM), but not saturated fatty acids, reduce the open probability (Po) of the cardiac RyR incorporated into phospholipid bilayers. EPA also inhibited the binding of [3H]ryanodine to isolated heavy SR. Our results indicate that direct inhibition of RyR channel gating by PUFAs play an important role in the overall antiarrhythmic properties of these compounds.     

The ryanodine receptor channel as a molecular motif in atrial fibrillation: pathophysiological and therapeutic implications

Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with substantial morbidity and mortality. It causes profound changes in sarcoplasmic reticulum (SR) Ca(2+) homeostasis, including ryanodine receptor channel dysfunction and diastolic SR Ca(2+) leak, which might contribute to both decreased contractile function and increased propensity to atrial arrhythmias. In this review, we will focus on the molecular basis of ryanodine receptor channel dysfunction and enhanced diastolic SR Ca(2+) leak in AF. The potential relevance of increased incidence of spontaneous SR Ca(2+) release for both AF induction and/or maintenance and the development of novel mechanism-based therapeutic approaches will be discussed.     

The RyR2 central domain peptide DPc10 lowers the threshold for spontaneous Ca2+ release in permeabilized cardiomyocytes

OBJECTIVE: In vitro experiments have shown that the ryanodine receptor-2 (RyR2) central domain peptide DPc10 (Gly(2460)-Pro(2495)) mimics channel dysfunction associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) by acting competitively to reduce stabilizing interactions between the N-terminal and central domains. In the present study, DPc10 was used as a tool to establish an adult cell model of the disease and to analyse the underlying mechanisms. METHODS: Rat ventricular myocytes were permeabilized with saponin and perfused with solutions approximating the intracellular milieu containing fluo-3. Sarcoplasmic reticulum (SR) Ca(2+) release was detected using confocal microscopy. DPc10 (10 or 50 microM) was compared with 0.2 mM caffeine, which is known to activate RyR2 and to facilitate Ca(2+)-induced Ca(2+) release (CICR). RESULTS: Introduction of DPc10 induced a transient increase in spark frequency and a sustained rise in resting [Ca(2+)]. Under conditions causing initial Ca(2+) overload of the SR, DPc10 reduced the frequency and amplitude of spontaneous, propagated Ca(2+) release (SPCR). Following equilibration with 10microM DPc10, the cytosolic [Ca(2+)] threshold for SPCR was markedly reduced and the proportion of spontaneously active cells increased. Caffeine induced a similar, transient increase in spark frequency and a reduction in the [Ca(2+)] threshold for SPCR. However, unlike DPc10, caffeine increased SPCR frequency and had no sustained effect on resting [Ca(2+)]. These results suggest that the net effect of DPc10 (and CPVT mutations) on RyR2 function in situ is not only to increase the sensitivity to CICR as caffeine does, but also to potentiate Ca(2+) leakage from the SR. As SPCR can trigger delayed after-depolarisations, the decrease in [Ca(2+)] threshold may contribute to arrhythmias in CPVT patients during exercise or stress.     

Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias

Catecholaminergic polymorphic ventricular tachycardia is a form of exercise-induced sudden cardiac death that has been linked to mutations in the cardiac Ca2+ release channel/ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR). We have shown that catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutations significantly decrease the binding affinity for calstabin-2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. We have proposed that RyR2-mediated diastolic SR Ca2+ leak triggers ventricular tachycardia (VT) and sudden cardiac death. In calstabin-2-deficient mice, we have now documented diastolic SR Ca2+ leak, monophasic action potential alternans, and bidirectional VT. Calstabin-deficient cardiomyocytes exhibited SR Ca2+ leak-induced aberrant transient inward currents in diastole consistent with delayed after-depolarizations. The 1,4-benzothiazepine JTV519, which increases the binding affinity of calstabin-2 for RyR2, inhibited the diastolic SR Ca2+ leak, monophasic action potential alternans and triggered arrhythmias. Our data suggest that calstabin-2 deficiency is as a critical mediator of triggers that initiate cardiac arrhythmias.     

Cardiac alternans do not rely on diastolic sarcoplasmic reticulum calcium content fluctuations

Cardiac alternans are thought to be a precursor to life-threatening arrhythmias. Previous studies suggested that alterations in sarcoplasmic reticulum (SR) Ca2+ content are either causative or not associated with myocyte Ca2+ alternans. However, those studies used indirect measures of SR Ca2+. Here we used direct continuous measurement of intra-SR free [Ca2+] ([Ca2+]SR) (using Fluo5N) during frequency-dependent Ca2+ alternans in rabbit ventricular myocytes. We tested the hypothesis that alternating [Ca2+]SR is required for Ca2+ alternans. Amplitudes of [Ca2+]SR depletions alternated in phase with cytosolic Ca2+ transients and contractions. Some cells showed clear alternation in diastolic [Ca2+]SR during alternans, with higher [Ca2+]SR before the larger SR Ca2+ releases. However, the extent of SR Ca2+ release during the small beats was smaller than expected for the modest decrease in [Ca2+]SR. In other cells, clear Ca2+ alternans was observed without alternations in diastolic [Ca2+]SR. Additionally, alternating cells were observed, in which diastolic [Ca2+]SR fluctuations occurred interspersed by depletions in which the amplitude was unrelated to the preceding diastolic [Ca2+]SR. In all forms of alternans, the SR Ca2+ release rate was higher during large depletions than during small depletions. Although [Ca2+]SR exerts major influence on SR Ca2+ release, alternations in [Ca2+](SR) are not required for Ca2+ alternans to occur. Rather, it seems likely that some other factor, such as ryanodine receptor availability after a prior beat (eg, recovery from inactivation), is of greater importance in initiating frequency-induced Ca2+ alternans. However, once such a weak SR Ca2+ release occurs, it can result in increased [Ca2+]SR and further enhance SR Ca2+ release at the next beat. In this way, diastolic [Ca2+]SR alternans can enhance frequency-induced Ca2+ alternans, even if they initiate by other means.     

Effects of ryanodine and caffeine on contractility, membrane voltage, and calcium exchange in cultured heart cells

To investigate the mechanisms of action of ryanodine and caffeine, changes in mechanical and electrical activity caused by these agents were correlated with alterations in 45Ca fluxes and cell Ca contents in chick embryo ventricular cell monolayer cultures. Ryanodine (10(-10)-10(-5) M) irreversibly decreased contraction amplitude by 10-70% relative to control in a concentration-dependent manner with minimal effects on electrical activity. Ryanodine caused a slight decrease in rapid 45Ca uptake, but no change in total exchangeable calcium content or rapid 45Ca efflux. Caffeine (1-20 mM) caused a transient (less than 10 seconds) 5-12% increase in contraction amplitude followed by a sustained 9-76% decrease in contraction amplitude and a 10 mV decrease in diastolic membrane voltage. Caffeine caused a decrease in rapid 45Ca uptake, a decrease in total exchangeable calcium content, and an increase in rapid 45Ca efflux. These results suggest that caffeine produces a decrease in sarcoplasmic reticulum (SR) Ca2+ uptake, and/or an increase in SR Ca2+ release that eventually depletes the SR of Ca2+, presumably accounting for the negative inotropic effect. The ryanodine effects on contraction are more difficult to account for solely in terms of alterations of transsarcolemmal Ca2+ fluxes and Ca2+ contents. Our data indicate an important role for the SR in excitation-contraction coupling in cultured chick embryo ventricular cells and suggest that SR Ca2+ is part of the rapidly exchanging Ca2+ compartment noted in 45Ca flux studies.     

肌浆网钙泄漏在心脏功能不全中的作用

同步肌浆网Ca2+释放以外的Ca2+释放即肌浆网Ca2+泄漏（Ca2+ leak），包括Ca2+火花（Ca2+ spark）、自发性Ca2+波（spontaneous Ca2+ wave）和其他微小Ca2+释放等。心肌细胞肌浆网Ca2+泄露主要是由II型兰尼碱受体（ryanodine receptor 2,RyR2）介导的，它可以通过减少肌浆网Ca2+的有效释放导致心脏收缩功能障碍；通过升高舒张期胞浆Ca2+引发心脏舒张功能不全，诱发心律失常。此外，肌浆网Ca2+泄露还可以引起因肌浆网Ca2+回摄增多消耗更多的ATP，导致心律失常、心力衰竭等心脏疾病的进一步恶化。     

Doxorubicin-induced calcium release from cardiac sarcoplasmic reticulum vesicles

Doxorubicin, an anthracycline glycoside antibiotic which has been widely used for treatment of several types of cancer (Goormaghtigh and Ruysschaer, 1984), displays a clinically important cardiac toxicity (Young et al., 1981) that can be dissociated from the antitumor activity. Although the main sites of toxicity have been postulated to be on the muscle membranes (Goormaghtigh and Ruysschaer, 1984; Harris and Doroshow, 1985), no information is available for a direct doxorubicin effect on the Ca2+ fluxes in cardiac sarcoplasmic reticulum (SR). Previous studies have shown that micromolar doxorubicin triggers Ca2+ release from skeletal SR vesicles (Zorzato et al., 1985). The objective of this study was to examine the effect of doxorubicin or caffeine on Ca2+ fluxes in cardiac SR in the presence of various Ca2+ release inhibitors. Addition of either doxorubicin (C1/2 = 5 microM), or caffeine (C1/2 = 0.8 mM) triggered Ca2+ release from canine cardiac SR loaded with 45Ca2+ in the presence of 2 mM ATP. The maximal amount of Ca2+ release triggered by doxorubicin (38% of the total loaded Ca2+) was significantly higher than that released by caffeine (25%). Plots of the amount of Ca2+ release triggered by 20 microM doxorubicin or 2 mM caffeine vs. free Ca2+ concentration were a bell-shaped, with maximal Ca2+ release at 0.2 microM Ca2+. Ca2+ release triggered by either 20 microM doxorubicin or 2 mM caffeine was inhibited by ruthenium red (0.1 to 2 microM), ryanodine (1 to 100 microM) or tetracaine (0.1 to 1 mM), whereas 2 mM caffeine did not further activate Ca2+ release triggered by 50 microM doxorubicin, suggesting that the drugs may share the same Ca2+ release channel.     

Potentiation of Ca(2+) release by cADP-ribose in the heart is mediated by enhanced SR Ca(2+) uptake into the sarcoplasmic reticulum

cADP-Ribose (cADPR) is a novel endogenous messenger that is believed to mobilize Ca(2+) from ryanodine-sensitive Ca(2+) stores. Despite intense research, the precise mechanism of action of cADPR remains uncertain, and experimental findings are contradictory. To elucidate the mechanism of cADPR action, we performed confocal Ca(2+) imaging in saponin-permeabilized rat ventricular myocytes. Exposure of the cells to cADPR resulted in a slow (>2 minutes) and steady increase in the frequency of Ca(2+) sparks. These effects on local release events were accompanied by a significant increase in sarcoplasmic reticulum (SR) Ca(2+) content. In comparison, sensitization of ryanodine receptors (RyRs) by caffeine, a true RyR agonist, caused a rapid (<1 second) and transient potentiation of Ca(2+) sparks followed by a decrease in SR Ca(2+) content. When the increase in the SR load was prevented by partial inhibition of the SR Ca(2+) with thapsigargin, cADPR failed to produce any increase in sparking activity. cADPR had no significant impact on activity of single cardiac RyRs incorporated into lipid bilayers. However, it caused a significant increase in the rate of Ca(2+) uptake by cardiac SR microsomes. Our results suggest that the primary target of cADPR is the SR Ca(2+) uptake mechanism. Potentiation of Ca(2+) release by cADPR is mediated by increased accumulation of Ca(2+) in the SR and subsequent luminal Ca(2+)-dependent activation of RyRs.     

Evidence for Ca(2+) activation and inactivation sites on the luminal side of the cardiac ryanodine receptor complex

We have used tryptic digestion to determine whether Ca(2+) can regulate cardiac ryanodine receptor (RyR) channel gating from within the lumen of the sarcoplasmic reticulum (SR) or whether Ca(2+) must first flow through the channel and act via cytosolically located binding sites. Cardiac RyRs were incorporated into bilayers, and trypsin was applied to the luminal side of the bilayer. We found that before exposure to luminal trypsin, the open probability of RyR was increased by raising the luminal [Ca(2+)] from 10 micromol/L to 1 mmol/L, whereas after luminal trypsin exposure, increasing the luminal [Ca(2+)] reduced the open probability. The modification in the response of RyRs to luminal Ca(2+) was not observed with heat-inactivated trypsin, indicating that digestion of luminal sites on the RyR channel complex was responsible. Our results provide strong evidence for the presence of luminally located Ca(2+) activation and inhibition sites and indicate that trypsin digestion leads to selective damage to luminal Ca(2+) activation sites without affecting luminal Ca(2+) inactivation sites. We suggest that changes in luminal [Ca(2+)] will be able to regulate RyR channel gating from within the SR lumen, therefore providing a second Ca(2+)-regulatory effect on RyR channel gating in addition to that of cytosolic Ca(2+). This luminal Ca(2+)-regulatory mechanism is likely to be an important contributing factor in the potentiation of SR Ca(2+) release that is observed in cardiac cells in response to increases in intra-SR [Ca(2+)].     

Dyssynchronous (non-uniform) Ca2+ release in myocytes from streptozotocin-induced diabetic rats

Using biochemical/pharmacological approaches, we previously showed that type 2 ryanodine receptors (RyR2) become dysfunctional in hearts of streptozotocin-induced type 1 diabetic rats. However, the functional consequence of this observation remains incompletely understood. Here we use laser confocal microscopy to investigate whether RyR2 dysfunction during diabetes alters evoked and spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR). After 7-8 weeks of diabetes, steady-state levels of RyR2 remain unchanged in hearts of male Sprague-Dawley rats, but the number of functional receptors decreased by >37%. Interestingly, residual functional RyR2 from diabetic rat hearts exhibited increased sensitivity to Ca(2+) activation (EC(50activation) decreased from 80 microM to 40 microM, peak Ca(2+) activation decreased from 425 microM to 160 microM). When field stimulated, intracellular Ca(2+) release in diabetic ventricular myocytes was dyssynchronous (non-uniform) and this was independent of L-type Ca(2+) currents. Time to peak Ca(2+) increased 3.7-fold. Diabetic myocytes also exhibited diastolic Ca(2+) release and 2-fold higher frequency of spontaneous Ca(2+) sparks, albeit at a lower amplitude. The amplitude of caffeine-releasable Ca(2+) was also lower in diabetic myocytes. RyR2 from diabetic rat hearts exhibited increased phosphorylation at Ser2809 and contained reduced levels of FKBP12.6 (calstablin2). Collectively, these data suggest that RyR2 becomes leaky during diabetes and this defect may be responsible to the reduced SR Ca(2+) load. Diastolic Ca(2+) release could also serve as a substrate for delayed after-depolarizations, contributing to the increased incidence of arrhythmias and sudden cardiac death in type 1 diabetes.     

K201 modulates excitation-contraction coupling and spontaneous Ca2+ release in normal adult rabbit ventricular cardiomyocytes

OBJECTIVES: The drug K201 (JTV-519) increases inotropy and suppresses arrhythmias in failing hearts, but the effects of K201 on normal hearts is unknown. METHODS: The effect of K201 on excitation-contraction (E-C) coupling in normal myocardium was studied by using voltage-clamp and intracellular Ca(2+) measurements in intact cells. Sarcoplasmic reticulum (SR) function was assessed using permeabilised cardiomyocytes. RESULTS: Acute application of <1 micromol/L K201 had no significant effect on E-C coupling. K201 at 1 micromol/L decreased Ca(2+) transient amplitude (to 83+/-7%) without affecting I(Ca,L) or the SR Ca(2+) content. At 3 micromol/L K201 caused a larger reduction of Ca(2+) transient amplitude (to 60+/-7%) with accompanying reductions in I(Ca,L) amplitude (to 66+/-8%) and SR Ca(2+) content (74+/-9%). Spontaneous SR Ca(2+) release during diastole was induced by increasing intracellular [Ca(2+)]. At 1 micromol/L K201 reduced the frequency of spontaneous Ca(2+) release. The effect of K201 on SR-mediated Ca(2+) waves and Ca(2+) sparks was examined in beta-escin-permeabilised cardiomyocytes by confocal microscopy. K201 (1 micromol/L) reduced the frequency and velocity of SR Ca(2+) waves despite no change in SR Ca(2+) content. At 3 micromol/L K201 completely abolished Ca(2+) waves and reduced the SR Ca(2+) content (to approximately 73%). K201 at 1 micromol/L reduced Ca(2+) spark amplitude and frequency. Assays specific to SR Ca(2+)-ATPase and RyR2 activity indicated that K201 inhibited both SR Ca(2+) uptake and release. CONCLUSIONS: K201 modifies E-C coupling in normal cardiomyocytes. A dual inhibitory action on SERCA and RyR2 explains the ability of K201 to suppress spontaneous diastolic Ca(2+) release during Ca(2+) overload without significantly affecting Ca(2+) transient amplitude.     

ATP-dependent effects of halothane on SR Ca2+ regulation in permeabilized atrial myocytes

OBJECTIVE: Previous work suggests that modification of sarcoplasmic reticulum (SR) function may contribute to the cardioprotective effect of halothane during ischaemia and reperfusion. The aim of this study was to investigate the effects of halothane on spontaneous Ca(2+) release from the sarcoplasmic reticulum (Ca(2+) sparks and waves). METHODS: Rat atrial myocytes were permeabilized with saponin and perfused with solutions approximating to the intracellular milieu and containing fluo-3. SR Ca(2+) release was detected using confocal microscopy. RESULTS: In the presence of 5 mM ATP, halothane (0.25-2 mM) had no significant effect on the amplitude or frequency of spontaneous Ca(2+) waves. However, in the presence of 0.05 mM ATP, halothane (0.25-2 mM) induced a concentration-dependent decrease in the amplitude and an increase in the frequency of spontaneous Ca(2+) waves, e.g., 1 mM halothane decreased the amplitude by 34.7+/-3.5% (n=9) and increased the frequency by 67+/-19.9% (n=7). In the presence of 5 mM ATP, 1 mM halothane had no significant effect on the amplitude or frequency of Ca(2+) sparks. When [ATP] was reduced to 0.05 mM, Ca(2+) spark frequency decreased by 67.9+/-14% and the amplitude increased by 27.5+/-4.9% (n=13). Subsequent introduction of halothane (0.5-1 mM) induced a transient burst of Ca(2+) sparks, consistent with ryanodine receptor (RyR) activation. Further experiments showed that the decrease in Ca(2+) spark frequency following ATP depletion was associated with a progressive increase in the SR Ca(2+) content over 1-2 min. This rise in SR Ca(2+) content did not occur when 1 mM halothane was present during ATP depletion. CONCLUSIONS: These data suggest that the sensitivity of the RyR to activation by halothane increases at low [ATP]. In metabolically impaired cells, halothane would be expected to lessen any rise in SR Ca(2+) content and to reduce the amplitude of spontaneous Ca(2+) release. These effects of halothane are considered in relation to the events that occur during ischaemia and reperfusion.     

Deficient ryanodine receptor S-nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes

Altered Ca(2+) homeostasis is a salient feature of heart disease, where the calcium release channel ryanodine receptor (RyR) plays a major role. Accumulating data support the notion that neuronal nitric oxide synthase (NOS1) regulates the cardiac RyR via S-nitrosylation. We tested the hypothesis that NOS1 deficiency impairs RyR S-nitrosylation, leading to altered Ca(2+) homeostasis. Diastolic Ca(2+) levels are elevated in NOS1(-/-) and NOS1/NOS3(-/-) but not NOS3(-/-) myocytes compared with wild-type (WT), suggesting diastolic Ca(2+) leakage. Measured leak was increased in NOS1(-/-) and NOS1/NOS3(-/-) but not in NOS3(-/-) myocytes compared with WT. Importantly, NOS1(-/-) and NOS1/NOS3(-/-) myocytes also exhibited spontaneous calcium waves. Whereas the stoichiometry and binding of FK-binding protein 12.6 to RyR and the degree of RyR phosphorylation were not altered in NOS1(-/-) hearts, RyR2 S-nitrosylation was substantially decreased, and the level of thiol oxidation increased. Together, these findings demonstrate that NOS1 deficiency causes RyR2 hyponitrosylation, leading to diastolic Ca(2+) leak and a proarrhythmic phenotype. NOS1 dysregulation may be a proximate cause of key phenotypes associated with heart disease.     

Ankyrin-B reduction enhances Ca spark-mediated SR Ca release promoting cardiac myocyte arrhythmic activity

Ankyrin-B (AnkB) loss-of-function may cause ventricular arrhythmias and sudden cardiac death in humans. Cardiac myocytes from AnkB heterozygous mice (AnkB(+/-)) show reduced expression and altered localization of Na/Ca exchanger (NCX) and Na/K-ATPase (NKA), key players in regulating [Na](i) and [Ca](i). Here we investigate how AnkB reduction affects cardiac [Na](i), [Ca](i) and SR Ca release. We found reduced NCX and NKA transport function but unaltered [Na](i) and diastolic [Ca](i) in myocytes from AnkB(+/-) vs. wild-type (WT) mice. Ca transients, SR Ca content and fractional SR Ca release were larger in AnkB(+/-) myocytes. The frequency of spontaneous, diastolic Ca sparks (CaSpF) was significantly higher in intact myocytes from AnkB(+/-) vs. WT myocytes (with and without isoproterenol), even when normalized for SR Ca load. However, total ryanodine receptor (RyR)-mediated SR Ca leak (tetracaine-sensitive) was not different between groups. Thus, in AnkB(+/-) mice SR Ca leak is biased towards more Ca sparks (vs. smaller release events), suggesting more coordinated openings of RyRs in a cluster. This is due to local cytosolic RyR regulation, rather than intrinsic RyR differences, since CaSpF was similar in saponin-permeabilized myocytes from WT and AnkB(+/-) mice. The more coordinated RyRs openings resulted in an increased propensity of pro-arrhythmic Ca waves in AnkB(+/-) myocytes. In conclusion, AnkB reduction alters cardiac Na and Ca transport and enhances the coupled RyR openings, resulting in more frequent Ca sparks and waves although the total SR Ca leak is unaffected. This could enhance the propensity for triggered arrhythmias in AnkB(+/-) mice.     

Subcellular calcium dynamics in a whole-cell model of an atrial myocyte

In this study, we present an innovative mathematical modeling approach that allows detailed characterization of Ca(2+) movement within the three-dimensional volume of an atrial myocyte. Essential aspects of the model are the geometrically realistic representation of Ca(2+) release sites and physiological Ca(2+) flux parameters, coupled with a computationally inexpensive framework. By translating nonlinear Ca(2+) excitability into threshold dynamics, we avoid the computationally demanding time stepping of the partial differential equations that are often used to model Ca(2+) transport. Our approach successfully reproduces key features of atrial myocyte Ca(2+) signaling observed using confocal imaging. In particular, the model displays the centripetal Ca(2+) waves that occur within atrial myocytes during excitation-contraction coupling, and the effect of positive inotropic stimulation on the spatial profile of the Ca(2+) signals. Beyond this validation of the model, our simulation reveals unexpected observations about the spread of Ca(2+) within an atrial myocyte. In particular, the model describes the movement of Ca(2+) between ryanodine receptor clusters within a specific z disk of an atrial myocyte. Furthermore, we demonstrate that altering the strength of Ca(2+) release, ryanodine receptor refractoriness, the magnitude of initiating stimulus, or the introduction of stochastic Ca(2+) channel activity can cause the nucleation of proarrhythmic traveling Ca(2+) waves. The model provides clinically relevant insights into the initiation and propagation of subcellular Ca(2+) signals that are currently beyond the scope of imaging technology.     

Ca2+/Calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes

Previous studies in transgenic mice and with isolated ryanodine receptors (RyR) have indicated that Ca2+-calmodulin-dependent protein kinase II (CaMKII) can phosphorylate RyR and activate local diastolic sarcoplasmic reticulum (SR) Ca2+ release events (Ca2+ sparks) and RyR channel opening. Here we use relatively controlled physiological conditions in saponin-permeabilized wild type (WT) and phospholamban knockout (PLB-KO) mouse ventricular myocytes to test whether exogenous preactivated CaMKII or endogenous CaMKII can enhance resting Ca2+ sparks. PLB-KO mice were used to preclude ancillary effects of CaMKII mediated by phospholamban phosphorylation. In both WT and PLB-KO myocytes, Ca2+ spark frequency was increased by both preactivated exogenous CaMKII and endogenous CaMKII. This effect was abolished by CaMKII inhibitor peptides. In contrast, protein kinase A catalytic subunit also enhanced Ca2+ spark frequency in WT, but had no effect in PLB-KO. Both endogenous and exogenous CaMKII increased SR Ca2+ content in WT (presumably via PLB phosphorylation), but not in PLB-KO. Exogenous calmodulin decreased Ca2+ spark frequency in both WT and PLB-KO (K0.5 approximately 100 nmol/L). Endogenous CaMKII (at 500 nmol/L [Ca2+]) phosphorylated RyR as completely in <4 minutes as the maximum achieved by preactivated exogenous CaMKII. After CaMKII activation Ca2+ sparks were longer in duration, and more frequent propagating SR Ca2+ release events were observed. We conclude that CaMKII-dependent phosphorylation of RyR by endogenous associated CaMKII (but not PKA-dependent phosphorylation) increases resting SR Ca2+ release or leak. Moreover, this may explain the enhanced SR diastolic Ca2+ leak and certain triggered arrhythmias seen in heart failure.     

Calcium waves driven by "sensitization" wave-fronts

OBJECTIVE: Cellular Ca(2+) waves are understood as reaction-diffusion systems sustained by Ca(2+)-induced Ca(2+) release (CICR) from Ca(2+) stores. Given the recently discovered sensitization of Ca(2+) release channels (ryanodine receptors; RyRs) of the sarcoplasmic reticulum (SR) by luminal SR Ca(2+), waves could also be driven by RyR sensitization, mediated by SR overloading via Ca(2+) pump (SERCA), acting in tandem with CICR. METHODS: Confocal imaging of the Ca(2+) indicator fluo-3 was combined with UV-flash photolysis of caged compounds and the whole-cell configuration of the patch clamp technique to carry out these experiments in isolated guinea pig ventricular cardiomyocytes. RESULTS: Upon sudden slowing of the SERCA in cardiomyocytes with a photoreleased inhibitor, waves indeed decelerated immediately. No secondary changes of Ca(2+) signaling or SR Ca(2+) content due to SERCA inhibition were observed in the short time-frame of these experiments. CONCLUSIONS: Our findings are consistent with Ca(2+) loading resulting in a zone of RyR 'sensitization' traveling within the SR, but inconsistent with CICR as the predominant mechanism driving the Ca(2+) waves. This alternative mode of RyR activation is essential to fully conceptualize cardiac arrhythmias triggered by spontaneous Ca(2+) release.