In steady state, the Ca content of the sarcoplasmic reticulum (SR) of cardiac myocytes is determined by a balance among influx and efflux pathways. The SR Ca content may be limited mainly by the ATP-supplied chemical potential that is inherent in the gradient between SR and cytosol. That is, forward Ca pumping from cytosol to SR may be opposed by energetically conservative reverse pumping dependent on intra-SR free [Ca]. On the other hand, SR Ca loading may be limited by dissipative pathways (pump slippage and/or pump-independent leak). To assess how SR Ca content is limited, we loaded voltage-clamped ferret ventricular myocytes cumulatively with known amounts of Ca via L-type Ca channels (ICa), using Na-free solutions to prevent Na/Ca exchange. We then measured the maximal resulting caffeine-released SR Ca content under control conditions, as well as when SR Ca pumping was accelerated by isoproterenol (1 micro M) or slowed by thapsigargin (0.2-0.4 micro M). Under control conditions, SR Ca content reached a limit of 137 micro mol.liter cytosol-1 (nonmitochondrial volume) when measured by integrating caffeine-induced Na/Ca exchange currents lintegraINaCaXdt) and of 119 micro mol.liter cytosol-1 when measured using fluorescence signals dependent on changes in cytosolic free Ca ([Ca]i). When Ca-ATPase pumping rate was slowed 39% by thapsigargin, the maximal SR Ca content decreased by 5 (integralINaCaXdt method) or 23% (fluorescence method); when pumping rate was increased 74% by isoproterenol, SR Ca content increased by 10% (fluorescence method) or 20% (integralINaCaXdt method). The relative stability of the SR Ca load suggests that dissipative losses have only a minor influence in setting the SR Ca content. Indeed, it appears that the SR Ca pump in intact cells can generate a [Ca] gradient approaching the thermodynamic limit.

The diastolic membrane potential (Vm) can be hyperpolarized or depolarized by various factors such as hyperkalemia or hypokalemia in the long term, or by delayed afterdepolarizations in the short term. In this study, we investigate how Vm affects Ca sparks and waves. We use a physiologically detailed mathematical model to investigate individual factors that affect Ca spark generation and wave propagation. We focus on the voltage range of -90 ∼ -70 mV, which is just below the Vm for sodium channel activation. We find that Vm depolarization promotes Ca wave propagation and hyperpolarization prevents it. This finding is directly validated in voltage clamp experiments with Ca waves using isolated rat ventricular myocytes. Ca transport by the sodium-calcium exchanger (NCX) is determined by Vm as well as Na and Ca concentrations. Depolarized Vm reduces NCX-mediated efflux, elevating [Ca]i, and thus promoting Ca wave propagation. Moreover, depolarized Vm promotes spontaneous Ca releases that can cause initiation of multiple Ca waves. This indicates that during delayed afterdepolarizations, Ca release units (CRUs) interact with not just the immediately adjacent CRUs via Ca diffusion, but also further CRUs via fast (∼0.1 ms) changes in Vm mediated by the voltage and Ca-sensitive NCX. This may contribute significantly to synchronization of Ca waves among multiple cells in tissue.

Rationale

Intercellular uncoupling and Ca²⁺ (Ca) mishandling can initiate triggered ventricular arrhythmias. Spontaneous Ca release activates inward current which depolarizes membrane potential (V_m) and can trigger action potentials in isolated myocytes. However, cell-cell coupling in intact hearts limits local depolarization and may protect hearts from this arrhythmogenic mechanism. Traditional optical mapping lacks the spatial resolution to assess coupling of individual myocytes.

Objective

We investigate local intercellular coupling in Ca-induced depolarization in intact hearts, using confocal microscopy to measure local V_m and intracellular [Ca] simultaneously.

Methods and results

We used isolated Langendorff-perfused hearts from control (CTL) and heart failure (HF) mice (HF induced by transaortic constriction). In CTL hearts, 1.4% of myocytes were poorly synchronized with neighboring cells and exhibited asynchronous (AS) Ca transients. These AS myocytes were much more frequent in HF (10.8% of myocytes, P<0.05 versus CTL). Local Ca waves depolarized V_m in HF but not CTL hearts, suggesting weaker gap junction coupling in HF-AS versus CTL-AS myocytes. Cell-cell coupling was assessed by calcein fluorescence recovery after photobleach during intracellular [Ca] recording. All regions in CTL hearts exhibited faster calcein diffusion than in HF, with HF-AS myocyte being slowest. In HF-AS, enhancing gap junction conductance (with rotigaptide) increased coupling and suppressed V_m depolarization during Ca waves. Conversely, in CTL hearts, gap junction inhibition (carbenoxolone) decreased coupling and allowed Ca wave-induced depolarizations. Synchronization of Ca wave initiation and triggered action potentials were observed in HF hearts and computational models.

Conclusions

Well-coupled CTL myocytes are effectively voltage-clamped during Ca waves, protecting the heart from triggered arrhythmias. Spontaneous Ca waves are much more common in HF myocytes and these AS myocytes are also poorly coupled, enabling local Ca-induced inward current of sufficient source strength to overcome a weakened current sink to depolarize V_m and trigger action potentials.

Key points

[Ca²⁺ ]_i enhanced rabbit ventricular slowly activating delayed rectifier K⁺ current (I_Ks ) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K⁺ current (I_Kr ) amplitude and voltage dependence were unaffected by high [Ca²⁺ ]_i . When measuring or simulating I_Ks during an action potential, I_Ks was not different during a physiological Ca²⁺ transient or when [Ca²⁺ ]_i was buffered to 500 nm.

Abstract

The slowly activating delayed rectifier K⁺ current (I_Ks ) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca²⁺ ([Ca²⁺ ]_i ) and β-adrenergic receptor (β-AR) stimulation modulate I_Ks amplitude and kinetics, but details of these important I_Ks regulators and their interaction are limited. We assessed the [Ca²⁺ ]_i dependence of I_Ks in steady-state conditions and with dynamically changing membrane potential and [Ca²⁺ ]_i during an AP. I_Ks was recorded from freshly isolated rabbit ventricular myocytes using whole-cell patch clamp. With intracellular pipette solutions that controlled free [Ca²⁺ ]_i , we found that raising [Ca²⁺ ]_i from 100 to 600 nm produced similar increases in I_Ks as did β-AR activation, and the effects appeared additive. Both β-AR activation and high [Ca²⁺ ]_i increased maximally activated tail I_Ks , negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well-established mathematical model of the rabbit myocyte. In both AP-clamp experiments and simulations, I_Ks recorded during a normal physiological Ca²⁺ transient was similar to I_Ks measured with [Ca²⁺ ]_i clamped at 500-600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca²⁺ ]_i regulates I_Ks amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca²⁺ ]_i , in the submembrane or junctional cleft space, is not required to maximize [Ca²⁺ ]_i -dependent I_Ks activation during normal Ca²⁺ transients.

The cardiac sarcolemmal Na-Ca exchanger (NCX) is allosterically regulated by [Ca](i) such that when [Ca](i) is low, NCX current (I(NCX)) deactivates. In this study, we used membrane potential (E(m)) and I(NCX) to control Ca entry into and Ca efflux from intact cardiac myocytes to investigate whether this allosteric regulation (Ca activation) occurs with [Ca](i) in the physiological range. In the absence of Ca activation, the electrochemical effect of increasing [Ca](i) would be to increase inward I(NCX) (Ca efflux) and to decrease outward I(NCX). On the other hand, Ca activation would increase I(NCX) in both directions. Thus, we attributed [Ca](i)-dependent increases in outward I(NCX) to allosteric regulation. Ca activation of I(NCX) was observed in ferret myocytes but not in wild-type mouse myocytes, suggesting that Ca regulation of NCX may be species dependent. We also studied transgenic mouse myocytes overexpressing either normal canine NCX or this same canine NCX lacking Ca regulation (Delta680-685). Animals with the normal canine NCX transgene showed Ca activation, whereas animals with the mutant transgene did not, confirming the role of this region in the process. In native ferret cells and in mice with expressed canine NCX, allosteric regulation by Ca occurs under physiological conditions (K(mCaAct) = 125 +/- 16 nM SEM approximately resting [Ca](i)). This, along with the observation that no delay was observed between measured [Ca](i) and activation of I(NCX) under our conditions, suggests that beat to beat changes in NCX function can occur in vivo. These changes in the I(NCX) activation state may influence SR Ca load and resting [Ca](i), helping to fine tune Ca influx and efflux from cells under both normal and pathophysiological conditions. Our failure to observe Ca activation in mouse myocytes may be due to either the extent of Ca regulation or to a difference in K(mCaAct) from other species. Model predictions for Ca activation, on which our estimates of K(mCaAct) are based, confirm that Ca activation strongly influences outward I(NCX), explaining why it increases rather than declines with increasing [Ca](i).

Niemann-Pick type C1 (NPC1) protein is essential for the transport of externally derived cholesterol from lysosomes to other organelles. Deficiency of NPC1 underlies the progressive NPC1 neurodegenerative disorder. Currently, there are no curative therapies for this fatal disease. Given the Ca²⁺ hypothesis of neurodegeneration, which posits that altered Ca²⁺ dynamics contribute to neuropathology, we tested if disease mutations in NPC1 alter Ca²⁺ signaling and neuronal plasticity. We determine that NPC1 inhibition or disease mutations potentiate store-operated Ca²⁺ entry (SOCE) due to a presenilin 1 (PSEN1)-dependent reduction in ER Ca²⁺ levels alongside elevated expression of the molecular SOCE components ORAI1 and STIM1. Associated with this dysfunctional Ca²⁺ signaling is destabilization of neuronal dendritic spines. Knockdown of PSEN1 or inhibition of the SREBP pathway restores Ca²⁺ homeostasis, corrects differential protein expression, reduces cholesterol accumulation, and rescues spine density. These findings highlight lysosomes as a crucial signaling platform responsible for tuning ER Ca²⁺ signaling, SOCE, and synaptic architecture in health and disease.

Cardiomyocyte Na⁺ and Ca²⁺ mishandling, upregulated Ca²⁺/calmodulin-dependent kinase II (CaMKII), and increased reactive oxygen species (ROS) are characteristics of various heart diseases, including heart failure (HF), long QT (LQT) syndrome, and catecholaminergic polymorphic ventricular tachycardia (CPVT). These changes may form a vicious cycle of positive feedback to promote cardiac dysfunction and arrhythmias. In HF rabbit cardiomyocytes investigated in this study, the inhibition of CaMKII, late Na⁺ current (I_NaL), and leaky ryanodine receptors (RyRs) all attenuated the prolongation and increased short-term variability (STV) of action potential duration (APD), but in age-matched controls these inhibitors had no or minimal effects. In control cardiomyocytes, we enhanced RyR leak (by low [caffeine] plus isoproterenol mimicking CPVT) which markedly increased STV and delayed afterdepolarizations (DADs). These proarrhythmic changes were significantly attenuated by both CaMKII inhibition and mitochondrial ROS scavenging, with a slight synergy with I_NaL inhibition. Inducing LQT by elevating I_NaL (by Anemone toxin II, ATX-II) caused markedly prolonged APD, increased STV, and early afterdepolarizations (EADs). Those proarrhythmic ATX-II effects were largely attenuated by mitochondrial ROS scavenging, and partially reduced by inhibition of CaMKII and pathological leaky RyRs using dantrolene. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) bearing LQT3 mutation SCN5A N406K, dantrolene significantly attenuated cell arrhythmias and APD prolongation. Targeting critical components of the Na⁺-Ca²⁺-CaMKII-ROS-I_NaL arrhythmogenic vicious cycle may exhibit important on-target and also trans-target effects (e.g., I_NaL and RyR inhibition can alter I_NaL-mediated LQT3 effects). Incorporating this vicious cycle into therapeutic strategies provides novel integrated insight for treating cardiac arrhythmias and diseases.