Clusters of calcium release channels harness the Ising phase transition to confine their elementary intracellular signals.

Intracellular Ca signals represent a universal mechanism of cell function. Messages carried by Ca are local, rapid, and powerful enough to be delivered over the thermal noise. A higher signal-to-noise ratio is achieved by a cooperative action of Ca release channels such as IP3 receptors or ryanodine receptors arranged in clusters (release units) containing a few to several hundred release channels. The channels synchronize their openings via Ca-induced Ca release, generating high-amplitude local Ca signals known as puffs in neurons and sparks in muscle cells. Despite the positive feedback nature of the activation, Ca signals are strictly confined in time and space by an unexplained termination mechanism. Here we show that the collective transition of release channels from an open to a closed state is identical to the phase transition associated with the reversal of magnetic field in an Ising ferromagnet. Our simple quantitative criterion closely predicts the Ca store depletion level required for spark termination for each cluster size. We further formulate exact requirements that a cluster of release channels should satisfy in any cell type for our mapping to the Ising model and the associated formula to remain valid. Thus, we describe deterministically the behavior of a system on a coarser scale (release unit) that is random on a finer scale (release channels), bridging the gap between scales. Our results provide exact mapping of a nanoscale biological signaling model to an interacting particle system in statistical physics, making the extensive mathematical apparatus available to quantitative biology.

Mechanisms of Calcium Leak from Cardiac Sarcoplasmic Reticulum Revealed by Statistical Mechanics.

Heart muscle contraction is normally activated by a synchronized Ca release from sarcoplasmic reticulum (SR), a major intracellular Ca store. However, under abnormal conditions, Ca leaks from the SR, decreasing heart contraction amplitude and increasing risk of life-threatening arrhythmia. The mechanisms and regimes of SR operation generating the abnormal Ca leak remain unclear. Here, we employed both numerical and analytical modeling to get mechanistic insights into the emergent Ca leak phenomenon. Our numerical simulations using a detailed realistic model of the Ca release unit reveal sharp transitions resulting in Ca leak. The emergence of leak is closely mapped mathematically to the Ising model from statistical mechanics. The system steady-state behavior is determined by two aggregate parameters: the analogs of magnetic field (h) and the inverse temperature (ß) in the Ising model, for which we have explicit formulas in terms of SR [Ca] and release channel opening and closing rates. The classification of leak regimes takes the shape of a phase ß-h diagram, with the regime boundaries occurring at h = 0 and a critical value of ß (ß∗) that we estimate using a classical Ising model and mean field theory. Our theory predicts that a synchronized Ca leak will occur when h > 0 and ß >ß∗, and a disordered leak occurs when ß <ß∗ and h is not too negative. The disorder leak is distinguished from synchronized leak (in long-lasting sparks) by larger Peierls contour lengths, an output parameter reflecting degree of disorder. Thus, in addition to our detailed numerical model approach, we also offer an instantaneous computational tool using analytical formulas of the Ising model for respective ryanodine receptor parameters and SR Ca load that describe and classify phase transitions and leak emergence.

Electrophysiological heterogeneity of pacemaker cells in the rabbit intercaval region, including the SA node: insights from recording multiple ion currents in each cell.

Cardiac pacemaker cells, including cells of the sinoatrial node, are heterogeneous in size, morphology, and electrophysiological characteristics. The exact extent to which these cells differ electrophysiologically is unclear yet is critical to understanding their functioning. We examined major ionic currents in individual intercaval pacemaker cells (IPCs) sampled from the paracristal, intercaval region (including the sinoatrial node) that were spontaneously beating after enzymatic isolation from rabbit hearts. The beating rate was measured at baseline and after inhibition of the Ca2+ pump with cyclopiazonic acid. Thereafter, in each cell, we consecutively measured the density of funny current ( If), delayed rectifier K+ current ( IK) (a surrogate of repolarization capacity), and L-type Ca2+ current ( ICa,L) using whole cell patch clamp. The ionic current densities varied to a greater extent than previously appreciated, with some IPCs demonstrating very small or zero If . The density of none of the currents was correlated with cell size, while ICa,L and If densities were related to baseline beating rates. If density was correlated with IK density but not with that of ICa,L. Inhibition of Ca2+ cycling had a greater beating rate slowing effect in IPCs with lower If densities. Our numerical model simulation indicated that 1) IPCs with small (or zero) If or small ICa,L can operate via a major contribution of Ca2+ clock, 2) If-Ca2+-clock interplay could be important for robust pacemaking function, and 3) coupled If- IK function could regulate maximum diastolic potential. Thus, we have demonstrated marked electrophysiological heterogeneity of IPCs. This heterogeneity is manifested in basal beating rate and response to interference of Ca2+ cycling, which is linked to If. NEW & NOTEWORTHY In the present study, a hitherto unrecognized range of heterogeneity of ion currents in pacemaker cells from the intercaval region is demonstrated. Relationships between basal beating rate and L-type Ca2+ current and funny current ( If) density are uncovered, along with a positive relationship between If and delayed rectifier K+ current. Links are shown between the response to Ca2+ cycling blockade and If density.

Stabilization of diastolic calcium signal via calcium pump regulation of complex local calcium releases and transient decay in a computational model of cardiac pacemaker cell with individual release channels.

Intracellular Local Ca releases (LCRs) from sarcoplasmic reticulum (SR) regulate cardiac pacemaker cell function by activation of electrogenic Na/Ca exchanger (NCX) during diastole. Prior studies demonstrated the existence of powerful compensatory mechanisms of LCR regulation via a complex local cross-talk of Ca pump, release and NCX. One major obstacle to study these mechanisms is that LCR exhibit complex Ca release propagation patterns (including merges and separations) that have not been characterized. Here we developed new terminology, classification, and computer algorithms for automatic detection of numerically simulated LCRs and examined LCR regulation by SR Ca pumping rate (Pup) that provides a major contribution to fight-or-flight response. In our simulations the faster SR Ca pumping accelerates action potential-induced Ca transient decay and quickly clears Ca under the cell membrane in diastole, preventing premature releases. Then the SR generates an earlier, more synchronized, and stronger diastolic LCR signal activating an earlier and larger inward NCX current. LCRs at higher Pup exhibit larger amplitudes and faster propagation with more collisions to each other. The LCRs overlap with Ca transient decay, causing an elevation of the average diastolic [Ca] nadir to ~200 nM (at Pup = 24 mM/s). Background Ca (in locations lacking LCRs) quickly decays to resting Ca levels (<100 nM) at high Pup, but remained elevated during slower decay at low Pup. Release propagation is facilitated at higher Pup by a larger LCR amplitude, whereas at low Pup by higher background Ca. While at low Pup LCRs show smaller amplitudes, their larger durations and sizes combined with longer transient decay stabilize integrals of diastolic Ca and NCX current signals. Thus, the local interplay of SR Ca pump and release channels regulates LCRs and Ca transient decay to insure fail-safe pacemaker cell operation within a wide range of rates.

Long-term HIF-1α transcriptional activation is essential for heat-acclimation-mediated cross tolerance: mitochondrial target genes.

An important adaptive feature of heat acclimation (HA) is the induction of cross tolerance against novel stressors (HACT) Reprogramming of gene expression leading to enhanced innate cytoprotective features by attenuating damage and/or enhancing the response of "help" signals plays a pivotal role. Hypoxia-inducible factor-1α (HIF-1α), constitutively upregulated by HA (1 mo, 34°C), is a crucial transcription factor in this program, although its specific role is as yet unknown. By using a rat HA model, we studied the impact of disrupting HIF-1α transcriptional activation [HIF-1α:HIF-1ß dimerization blockade by intraperitoneal acriflavine (4 mg/kg)] on its mitochondrial gene targets [phosphoinositide-dependent kinase-1 (PDK1), LON, and cyclooxygenase 4 (COX4) isoforms] in the HA rat heart. Physiological measures of cardiac HACT were infarct size after ischemia-reperfusion and time to rigor contracture during hypoxia in cardiomyocytes. We show that HACT requires transcriptional activation of HIF-1α throughout the course of HA and that this activation is accompanied by two metabolic switches: 1) profound upregulation of PDK1, which reduces pyruvate entry into the mitochondria, consequently increasing glycolytic lactate production; 2) remodeling of the COX4 isoform ratio, inducing hypoxic-tolerant COX4.2 dominance, and optimizing electron transfer and possibly ATP production during the ischemic and hypoxic insults. LON and COX4.2 transcript upregulation accompanied this shift. Loss of HACT despite elevated expression of the cytoprotective protein heat shock protein-72 concomitantly with disrupted HIF-1α dimerization suggests that HIF-1α is essential for HACT. The role of a PDK1 metabolic switch is well known in hypoxia acclimation but not in the HA model and its ischemic setting. Remodeling of COX4 isoforms by environmental acclimation is a novel finding.

RyR-NCX-SERCA local cross-talk ensures pacemaker cell function at rest and during the fight-or-flight reflex.

RATIONALE: A recent study published in Circulation Research by Gao et al used sinoatrial node (SAN)-targeted, incomplete Ncx1 knockout in mice to explore the role of the Na(+)/Ca(2+) exchanger (NCX) in cardiac pacemaker. The authors concluded that NCX is required for increasing sinus rates, but not for maintaining resting heart rate. This conclusion was based, in part, on numeric model simulations performed by Gao et al that reproduced their experimental results of unchanged action potentials in the knockout SAN cells. The authors, however, did not simulate the NCX current (INCX), that is, the subject of the study. OBJECTIVE: We extended numeric examinations to simulate INCX in their incomplete knockout SAN cells that is crucial to interpret the study results. METHODS AND RESULTS: INCX and Ca(2+) dynamics were simulated using different contemporary numeric models of SAN cells. We found that minimum diastolic Ca(2+) levels and INCX amplitudes generated by remaining NCX molecules (only 20% of control) remained almost unchanged. Simulations using a new local Ca(2+) control model indicate that these powerful compensatory mechanisms involve complex local cross-talk of Ca(2+) cycling proteins and NCX. Specifically, lower NCX expression facilitates Ca(2+)-induced Ca(2+) release and larger local Ca(2+) releases that stabilize diastolic INCX. Further reduction of NCX expression results in arrhythmia and halt of automaticity. CONCLUSIONS: Remaining NCX molecules in the incomplete knockout model likely produce almost the same diastolic INCX as in wild-type cells. INCX contribution is crucially important for both basal automaticity of SAN cells and during the fight-or-flight reflex.

Modern perspectives on numerical modeling of cardiac pacemaker cell.

Cardiac pacemaking is a complex phenomenon that is still not completely understood. Together with experimental studies, numerical modeling has been traditionally used to acquire mechanistic insights in this research area. This review summarizes the present state of numerical modeling of the cardiac pacemaker, including approaches to resolve present paradoxes and controversies. Specifically we discuss the requirement for realistic modeling to consider symmetrical importance of both intracellular and cell membrane processes (within a recent "coupled-clock" theory). Promising future developments of the complex pacemaker system models include the introduction of local calcium control, mitochondria function, and biochemical regulation of protein phosphorylation and cAMP production. Modern numerical and theoretical methods such as multi-parameter sensitivity analyses within extended populations of models and bifurcation analyses are also important for the definition of the most realistic parameters that describe a robust, yet simultaneously flexible operation of the coupled-clock pacemaker cell system. The systems approach to exploring cardiac pacemaker function will guide development of new therapies such as biological pacemakers for treating insufficient cardiac pacemaker function that becomes especially prevalent with advancing age.

Mechanisms of beat-to-beat regulation of cardiac pacemaker cell function by Ca²âº cycling dynamics.

Whether intracellular Ca(2+) cycling dynamics regulate cardiac pacemaker cell function on a beat-to-beat basis remains unknown. Here we show that under physiological conditions, application of low concentrations of caffeine (2-4 mM) to isolated single rabbit sinoatrial node cells acutely reduces their spontaneous action potential cycle length (CL) and increases Ca(2+) transient amplitude for several cycles. Numerical simulations, using a modified Maltsev-Lakatta coupled-clock model, faithfully reproduced these effects, and also the effects of CL prolongation and dysrhythmic spontaneous beating (produced by cytosolic Ca(2+) buffering) and an acute CL reduction (produced by flash-induced Ca(2+) release from a caged Ca(2+) buffer), which we had reported previously. Three contemporary numerical models (including the original Maltsev-Lakatta model) failed to reproduce the experimental results. In our proposed new model, Ca(2+) releases acutely change the CL via activation of the Na(+)/Ca(2+) exchanger current. Time-dependent CL reductions after flash-induced Ca(2+) releases (the memory effect) are linked to changes in Ca(2+) available for pumping into sarcoplasmic reticulum which, in turn, changes the sarcoplasmic reticulum Ca(2+) load, diastolic Ca(2+) releases, and Na(+)/Ca(2+) exchanger current. These results support the idea that Ca(2+) regulates CL in cardiac pacemaker cells on a beat-to-beat basis, and suggest a more realistic numerical mechanism of this regulation.

Elementary intracellular Ca signals approximated as a transition of release channel system from a metastable state.

Cardiac muscle contraction is initiated by an elementary Ca signal (called Ca spark) which is achieved by collective action of Ca release channels in a cluster. The mechanism of this synchronization remains uncertain. We approached Ca spark activation as an emergent phenomenon of an interactive system of release channels. We constructed a weakly lumped Markov chain that applies an Ising model formalism to such release channel clusters and probable open channel configurations and demonstrated that spark activation is described as a system transition from a metastable to an absorbing state, analogous to the pressure required to overcome surface tension in bubble formation. This yielded quantitative estimates of the spark generation probability as a function of various system parameters. We performed numerical simulations to find spark probabilities as a function of sarcoplasmic reticulum Ca concentration, obtaining similar values for spark activation threshold as our analytic model, as well as those reported in experimental studies. Our parametric sensitivity analyses also showed that the spark activation threshold decreased as Ca sensitivity of RyR activation and RyR cluster size increased.

A novel conceptual model of heart rate autonomic modulation based on a small-world modular structure of the sinoatrial node.

The present view on heartbeat initiation is that a primary pacemaker cell or a group of cells in the sinoatrial node (SAN) center paces the rest of the SAN and the atria. However, recent high-resolution imaging studies show a more complex paradigm of SAN function that emerges from heterogeneous signaling, mimicking brain cytoarchitecture and function. Here, we developed and tested a new conceptual numerical model of SAN organized similarly to brain networks featuring a modular structure with small-world topology. In our model, a lower rate module leads action potential (AP) firing in the basal state and during parasympathetic stimulation, whereas a higher rate module leads during ß-adrenergic stimulation. Such a system reproduces the respective shift of the leading pacemaker site observed experimentally and a wide range of rate modulation and robust function while conserving energy. Since experimental studies found functional modules at different scales, from a few cells up to the highest scale of the superior and inferior SAN, the SAN appears to feature hierarchical modularity, i.e., within each module, there is a set of sub-modules, like in the brain, exhibiting greater robustness, adaptivity, and evolvability of network function. In this perspective, our model offers a new mainframe for interpreting new data on heterogeneous signaling in the SAN at different scales, providing new insights into cardiac pacemaker function and SAN-related cardiac arrhythmias in aging and disease.

Disorder in Ca2+ release unit locations confers robustness but cuts flexibility of heart pacemaking.

Excitation-contraction coupling kinetics is dictated by the action potential rate of sinoatrial-nodal cells. These cells generate local Ca releases (LCRs) that activate Na/Ca exchanger current, which accelerates diastolic depolarization and determines the pace. LCRs are generated by clusters of ryanodine receptors, Ca release units (CRUs), residing in the sarcoplasmic reticulum. While CRU distribution exhibits substantial heterogeneity, its functional importance remains unknown. Using numerical modeling, here we show that with a square lattice distribution of CRUs, Ca-induced-Ca-release propagation during diastolic depolarization is insufficient for pacemaking within a broad range of realistic ICaL densities. Allowing each CRU to deviate randomly from its lattice position allows sparks to propagate, as observed experimentally. As disorder increases, the CRU distribution exhibits larger empty spaces and simultaneously CRU clusters, as in Poisson clumping. Propagating within the clusters, Ca release becomes synchronized, increasing action potential rate and reviving pacemaker function of dormant/nonfiring cells. However, cells with fully disordered CRU positions could not reach low firing rates and their ß-adrenergic-receptor stimulation effect was substantially decreased. Inclusion of Cav1.3, a low-voltage activation L-type Ca channel isoform into ICaL, strongly increases recruitment of CRUs to fire during diastolic depolarization, increasing robustness of pacemaking and complementing effects of CRU distribution. Thus, order/disorder in CRU locations along with Cav1.3 expression regulates pacemaker function via synchronization of CRU firing. Excessive CRU disorder and/or overexpression of Cav1.3 boosts pacemaker function in the basal state, but limits the rate range, which may contribute to heart rate range decline with age and disease.

The paradigm shift: Heartbeat initiation without "the pacemaker cell".

The current dogma about the heartbeat origin is based on "the pacemaker cell," a specialized cell residing in the sinoatrial node (SAN) that exhibits spontaneous diastolic depolarization triggering rhythmic action potentials (APs). Recent high-resolution imaging, however, demonstrated that Ca signals and APs in the SAN are heterogeneous, with many cells generating APs of different rates and rhythms or even remaining non-firing (dormant cells), i.e., generating only subthreshold signals. Here we numerically tested a hypothesis that a community of dormant cells can generate normal automaticity, i.e., "the pacemaker cell" is not required to initiate rhythmic cardiac impulses. Our model includes 1) non-excitable cells generating oscillatory local Ca releases and 2) an excitable cell lacking automaticity. While each cell in isolation was not "the pacemaker cell", the cell system generated rhythmic APs: The subthreshold signals of non-excitable cells were transformed into respective membrane potential oscillations via electrogenic Na/Ca exchange and further transferred and integrated (computed) by the excitable cells to reach its AP threshold, generating rhythmic pacemaking. Cardiac impulse is an emergent property of the SAN cellular network and can be initiated by cells lacking intrinsic automaticity. Cell heterogeneity, weak coupling, subthreshold signals, and their summation are critical properties of the new pacemaker mechanism, i.e., cardiac pacemaker can operate via a signaling process basically similar to that of "temporal summation" happening in a neuron with input from multiple presynaptic cells. The new mechanism, however, does not refute the classical pacemaker cell-based mechanism: both mechanisms can co-exist and interact within SAN tissue.

Functional Heterogeneity of Cell Populations Increases Robustness of Pacemaker Function in a Numerical Model of the Sinoatrial Node Tissue.

Each heartbeat is initiated by specialized pacemaker cells operating within the sinoatrial node (SAN). While individual cells within SAN tissue exhibit substantial heterogeneity of their electrophysiological parameters and Ca cycling, the role of this heterogeneity for cardiac pacemaker function remains mainly unknown. Here we investigated the problem numerically in a 25 × 25 square grid of connected coupled-clock Maltsev-Lakatta cell models. The tissue models were populated by cells with different degree of heterogeneity of the two key model parameters, maximum L-type Ca current conductance (g CaL ) and sarcoplasmic reticulum Ca pumping rate (P up ). Our simulations showed that in the areas of P up -g CaL parametric space at the edge of the system stability, where action potential (AP) firing is absent or dysrhythmic in SAN tissue models populated with identical cells, rhythmic AP firing can be rescued by populating the tissues with heterogeneous cells. This robust SAN function is synergistic with respect to heterogeneity in g CaL and P up and can be further strengthened by clustering of cells with similar properties. The effect of cell heterogeneity is not due to a simple summation of activity of intrinsically firing cells naturally present in heterogeneous SAN; rather AP firing cells locally and critically interact with non-firing/dormant cells. When firing cells prevail, they recruit many dormant cells to fire, strongly enhancing overall SAN function; and vice versa, prevailing dormant cells suppress AP firing in cells with intrinsic automaticity and halt SAN function. The transitions between firing and non-firing states of the system are sharp, resembling phase transitions in statistical physics. Furthermore, robust function of heterogeneous SAN tissue requires weak cell coupling, a known property of the central area of SAN where cardiac impulse emerges; stronger cell coupling reduces AP firing rate and ultimately halts SAN automaticity at the edge of stability.

Synchronization of stochastic Ca²(+) release units creates a rhythmic Ca²(+) clock in cardiac pacemaker cells.

In sinoatrial node cells of the heart, beating rate is controlled, in part, by local Ca²(+) releases (LCRs) from the sarcoplasmic reticulum, which couple to the action potential via electrogenic Na(+)/Ca²(+) exchange. We observed persisting, roughly periodic LCRs in depolarized rabbit sinoatrial node cells (SANCs). The features of these LCRs were reproduced by a numerical model consisting of a two-dimensional array of stochastic, diffusively coupled Ca²(+) release units (CRUs) with fixed refractory period. Because previous experimental studies showed that ß-adrenergic receptor stimulation increases the rate of Ca²(+) release through each CRU (dubbed I(spark)), we explored the link between LCRs and I(spark) in our model. Increasing the CRU release current I(spark) facilitated Ca²(+)-induced-Ca²(+) release and local recruitment of neighboring CRUs to fire more synchronously. This resulted in a progression in simulated LCR size (from sparks to wavelets to global waves), LCR rhythmicity, and decrease of LCR period that parallels the changes observed experimentally with ß-adrenergic receptor stimulation. The transition in LCR characteristics was steeply nonlinear over a narrow range of I(spark), resembling a phase transition. We conclude that the (partial) periodicity and rate regulation of the "Calcium clock" in SANCs are emergent properties of the diffusive coupling of an ensemble of interacting stochastic CRUs. The variation in LCR period and size with I(spark) is sufficient to account for ß-adrenergic regulation of SANC beating rate.

Beat-to-beat Ca(2+)-dependent regulation of sinoatrial nodal pacemaker cell rate and rhythm.

Whether intracellular Ca(2+) regulates sinoatrial node cell (SANC) action potential (AP) firing rate on a beat-to-beat basis is controversial. To directly test the hypothesis of beat-to-beat intracellular Ca(2+) regulation of the rate and rhythm of SANC we loaded single isolated SANC with a caged Ca(2+) buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca(2+). Prior to introduction of the caged Ca(2+) buffer, spontaneous local Ca(2+) releases (LCRs) during diastolic depolarization were tightly coupled to rhythmic APs (r²=0.9). The buffer markedly prolonged the decay time (T50) and moderately reduced the amplitude of the AP-induced Ca(2+) transient and partially depleted the SR load, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slower and dysrhythmic. When Ca(2+) was acutely released from the caged compound by flash photolysis, intracellular Ca(2+) dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca(2+) dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that intracellular Ca(2+) regulates normal SANC automaticity on a beat-to-beat basis.

Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling.

There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca(2+) imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like Local Ca(2+)Releases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca(2+)current (I(CaL)) induces a global Ca(2+) release (CICR), depleting the Ca(2+) content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca(2+)release of multiple LCRs throughout the cell. The LCRs generate an inward Na(+)/Ca(2+)exchanger (NCX) current (absent in Na(+)-free solution) that ignites the next AP. Interfering with SR Ca(2+) cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na(+)-free solution), or I(CaL) (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca(2+) loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic "pacemaker-like" cells. SR Ca(2+) loading and LCR activity could be also increased with a selective activation of SR Ca(2+) pumping by a phospholamban antibody. We conclude that SR Ca(2+) loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. I(CaL) synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca(2+) release and NCX current. Rhythmic ESC automaticity can be achieved by boosting "coupling" factors, such as cAMP/PKA signaling, that enhance interactions between SR and sarcolemma.

Heat acclimation and exercise training interact when combined in an overriding and trade-off manner: physiologic-genomic linkage.

Combined heat acclimation (AC) and exercise training (EX) enhance exercise performance in the heat while meeting thermoregulatory demands. We tested the hypothesis that different stress-specific adaptations evoked by each stressor individually trigger similar cardiac alterations, but when combined, overriding/trade-off interactions take place. We used echocardiography, isolated cardiomyocyte imaging and cDNA microarray techniques to assay in situ cardiac performance, excitation-contraction (EC) coupling features, and transcriptional programs associated with cardiac contractility. Rat groups studied were controls (sedentary 24°C); AC (sedentary, 34°C, 1 mo); normothermic EX (treadmill at 24°C, 1 mo); and heat-acclimated, exercise-trained (EXAC; treadmill at 34°C, 1 mo). Prolonged heat exposure decreased heart rate and contractile velocity and increased end ventricular diastolic diameter. Compared with controls, AC/EXAC cardiomyocytes demonstrated lower l-type Ca(2+) current (I(CaL)) amplitude, higher Ca(2+) transient (Ca(2+)T), and a greater Ca(2+)T-to-I(CaL) ratio; EX alone enhanced I(CaL) and Ca(2+)T, whereas aerobic training in general induced cardiac hypertrophy and action potential elongation in EX/EXAC animals. At the genomic level, the transcriptome profile indicated that the interaction between AC and EX yields an EXAC-specific molecular program. Genes affected by chronic heat were linked with the EC coupling cascade, whereas aerobic training upregulated genes involved with Ca(2+) turnover via an adrenergic/metabolic-driven positive inotropic response. In the EXAC cardiac phenotype, the impact of chronic heat overrides that of EX on EC coupling components and heart rate, whereas EX regulates cardiac morphometry. We suggest that concerted adjustments induced by AC and EX lead to enhanced metabolic and mechanical performance of the EXAC heart.

Ca sparks do not explain all ryanodine receptor-mediated SR Ca leak in mouse ventricular myocytes.

Diastolic Ca leak from the sarcoplasmic reticulum (SR) of ventricular myocytes reduces the SR Ca content, stabilizing the activity of the SR Ca release channel ryanodine receptor for the next beat. SR Ca leak has been visualized globally using whole-cell fluorescence, or locally using confocal microscopy, but never both ways. When using confocal microscopy, leak is imaged as "Ca sparks," which are fluorescent objects generated by the local reaction-diffusion of released Ca and cytosolic indicator. Here, we used confocal microscopy and simultaneously measured the global ryanodine-receptor-mediated leak rate (J(leak)) and Ca sparks in intact mouse ventricular myocytes. We found that spark frequency and J(leak) are correlated, as expected if both are manifestations of a common phenomenon. However, we also found that sparks explain approximately half of J(leak). Our strategy unmasks the presence of a subresolution (i.e., nonspark) release of potential physiological relevance.

Ca(2+)-dependent components of inactivation of unitary cardiac L-type Ca(2+) channels.

A Ca(2+) ion-dependent inactivation (CDI) of L-type Ca(2+) channels (LCC) is vital in limiting and shaping local Ca(2+) ion signalling in a variety of excitable cell types. However, under physiological conditions the unitary LCC properties that underlie macroscopic inactivation are unclear. Towards this end, we have probed the gating kinetics of individual cardiac LCCs recorded with a physiological Ca(2+) ion concentration (2 mM) permeating the channel, and in the absence of channel agonists. Upon depolarization the ensemble-averaged LCC current decayed with a fast and a slow exponential component. We analysed the unitary behaviour responsible for this biphasic decay by means of a novel kinetic dissection of LCC gating parameters. We found that inactivation was caused by a rapid decrease in the frequency of LCC reopening, and a slower decline in mean open time of the LCC. In contrast, with barium ions permeating the channel ensemble-averaged currents displayed only a single, slow exponential decay and little time dependence of the LCC open time. Our results demonstrate that the fast and slow phases of macroscopic inactivation reflect the distinct time courses for the decline in the frequency of LCC reopening and the open dwell time, both of which are modulated by Ca(2+) influx. Analysis of the evolution of CDI in individual LCC episodes was employed to examine the stochastic nature of the underlying molecular switch, and revealed that influx on the order of a thousand Ca(2+) ions may be sufficient to trigger CDI. This is the first study to characterize both the unitary kinetics and the stoichiometry of CDI of LCCs with a physiological Ca(2+) concentration. These novel findings may provide a basis for understanding the mechanisms regulating unitary LCC gating, which is a pivotal element in the local control of Ca(2+)-dependent signalling processes.

Physiologic gating properties of unitary cardiac L-type Ca2+ channels.

The contraction of adult mammalian ventricular cardiomyocytes is triggered by the influx of Ca(2+) ions through sarcolemmal L-type Ca(2+) channels (LCCs). However, the gating properties of unitary LCCs under physiologic conditions have remained elusive. Towards this end, we investigated the voltage-dependence of the gating kinetics of unitary LCCs, with a physiologic concentration of Ca(2+) ions permeating the channel. Unitary LCC currents were recorded with 2mM external Ca(2+) ions (in the absence of LCC agonists), using cell-attached patches on K-depolarized adult rat ventricular myocytes. The voltage-dependence of the peak probability of channel opening (Po vs. Vm) displayed a maximum value of 0.3, a midpoint of -12 mV, and a slope factor of 8.5. The maximum value for Po of the unitary LCC was significantly higher than previously assumed, under physiologic conditions. We also found that the mean open dwell time of the unitary LCC increased twofold with depolarization, ranging from 0.53+/-0.02 ms at -30 mV to 1.08+/-0.03 ms at 0 mV. The increase in mean LCC open time with depolarization counterbalanced the decrease in the single LCC current amplitude; the latter due to the decrease in driving force for Ca(2+) ion entry. Thus, the average amount of Ca(2+) ions entering through an individual LCC opening ( approximately 300-400 ions) remained relatively constant over this range of potentials. These novel results establish the voltage-dependence of unitary LCC gating kinetics using a physiologic Ca(2+) ion concentration. Moreover, they provide insight into local Ca(2+)-induced Ca(2+) release and a more accurate basis for mathematical modeling of excitation-contraction coupling in cardiac myocytes.