Regulation of cardiac excitation-contraction coupling: a cellular update

The primary purpose of this paper is to present a basic overview of some "relatively" new ideas related to the regulation of cardiac performance and underlying excitation-contraction (EC) coupling that have yet to be incorporated to textbooks currently used for introductory graduate-level physiology courses. Within the context of cardiac EC coupling, this review incorporates information on microdomains and local control theory, with particular emphasis on the role of Ca(2+) sparks as a key regulatory component of ventricular myocyte contraction dynamics. Recent information pertaining to Ca(2+) release mechanisms specific to the sarcoplasmic reticulum is also presented, as well as the idea of the ryanodine receptor as a macromolecular signaling complex. Because of the potential relationship to maladaptive functional responses under conditions of cardiovascular pathology, the regulatory role of cardiac adrenergic and additional G protein-coupled receptors known to regulate cardiac function is included, and fundamental concepts related to intracellular signaling are discussed. Finally, information on the roles of vascular and cardiac nitric oxide as an important regulator of cardiac performance is included to allow students to begin to think about the ubiquitous role of nitric oxide in the regulation of the cardiovascular system. An important point of emphasis is that whole organ cardiac dynamics can be traced back to the cellular events regulating intracellular Ca(2+) homeostasis and as such provides an important conceptual framework from which the students can begin to think about whole organ physiology in health and disease.     

Intracellular Ca(2+) dynamics and the stability of ventricular tachycardia

Ventricular fibrillation (VF), the major cause of sudden cardiac death, is typically preceded by ventricular tachycardia (VT), but the mechanisms underlying the transition from VT to VF are poorly understood. Intracellular Ca(2+) overload occurs during rapid heart rates typical of VT and is also known to promote arrhythmias. We therefore studied the role of intracellular Ca(2+) dynamics in the transition from VT to VF, using a combined experimental and mathematical modeling approach. Our results show that 1) rapid pacing of rabbit ventricular myocytes at 35 degrees C led to increased intracellular Ca(2+) levels and complex patterns of action potential (AP) configuration and the intracellular Ca(2+) transients; 2) the complex patterns of the Ca(2+) transient arose directly from the dynamics of intracellular Ca(2+) cycling, and were not merely passive responses to beat-to-beat alterations in AP; 3) the complex Ca(2+) dynamics were simulated in a modified version of the Luo-Rudy (LR) ventricular action potential with improved intracellular Ca(2+) dynamics, and showed good agreement with the experimental findings in isolated myocytes; and 4) when incorporated into simulated two-dimensional cardiac tissue, this action potential model produced a form of spiral wave breakup from VT to a VF-like state in which intracellular Ca(2+) dynamics played a key role through its influence on Ca(2+)-sensitive membrane currents such as I(Ca), I(NaCa), and I(ns(Ca)). To the extent that spiral wave breakup is useful as a model for the transition from VT to VF, these findings suggest that intracellular Ca(2+) dynamics may play an important role in the destabilization of VT and its degeneration into VF.     

Pacemaker activity and ionic currents in mouse atrioventricular node cells

It is well established that Pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of I(Na) by tetrodotoxin (TTX) or of I(Ca,L) by isradipine abolished AVNCs pacemaker activity. TTX-resistant (I(Nar)) and TTX-sensitive (I(Nas)) Na(+) currents were recorded in mouse AVNCs, as well as T-(I(Ca,T)) and L-type (I(Ca,L)) Ca(2+) currents I(Ca,L) density was lower than in SANCs (51%). The density of the hyperpolarization-activated current, (I(f)) and that of the fast component of the delayed rectifier current (I(Kr)) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of I(f) by 3 μM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for I(f) in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K(+) current (I(to)). In contrast, no detectable slow component of the delayed rectifier current (I(Ks)) could be recorded in AVNCs. The lower densities of I(f) and I(Ca,L), as well as higher expression of I(Kr) in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs.     

Dynamical analysis of the calcium signaling pathway in cardiac myocytes based on logarithmic sensitivity analysis

Many cellular functions are regulated by the Ca(2+) signal which contains specific information in the form of frequency, amplitude, and duration of the oscillatory dynamics. Any alterations or dysfunctions of components in the calcium signaling pathway of cardiac myocytes may lead to a diverse range of cardiac diseases including hypertrophy and heart failure. In this study, we have investigated the hidden dynamics of the intracellular Ca(2+) signaling and the functional roles of its regulatory mechanism through in silico simulations and parameter sensitivity analysis based on an experimentally verified mathematical model. It was revealed that the Ca(2+) dynamics of cardiac myocytes are determined by the balance among various system parameters. Moreover, it was found through the parameter sensitivity analysis that the self-oscillatory Ca(2+) dynamics are most sensitive to the Ca(2+) leakage rate of the sarcolemmal membrane and the maximum rate of NCX, suggesting that these two components have dominant effects on circulating the cytosolic Ca(2+).     

Diastolic calcium release controls the beating rate of rabbit sinoatrial node cells: numerical modeling of the coupling process

Recent studies employing Ca2+ indicators and confocal microscopy demonstrate substantial local Ca2+ release beneath the cell plasma membrane (subspace) of sinoatrial node cells (SANCs) occurring during diastolic depolarization. Pharmacological and biophysical experiments have suggested that the released Ca2+ interacts with the plasma membrane via the ion current (INaCa) produced by the Na+/Ca2+ exchanger and constitutes an important determinant of the pacemaker rate. This study provides a numerical validation of the functional importance of diastolic Ca2+ release for rate control. The subspace Ca2+ signals in rabbit SANCs were measured by laser confocal microscopy, averaged, and calibrated. The time course of the subspace [Ca2+] displayed both diastolic and systolic components. The diastolic component was mainly due to the local Ca2+ releases; it was numerically approximated and incorporated into a SANC cellular electrophysiology model. The model predicts that the diastolic Ca2+ release strongly interacts with plasma membrane via INaCa and thus controls the phase of the action potential upstroke and ultimately the final action potential rate.     

Species- and tissue-dependent effects of NO and cyclic GMP on cardiac ion channels

Biochemical studies have established the presence of a NO pathway in the heart, including sources of NO and various effectors. Several cardiac ion channels have been shown to be modified by NO, such as L-type Ca(2+), ATP-sensitive K(+), and pacemaker f-channels. Some of these effects are mediated by cGMP, through the activity of three main proteins: the cGMP-dependent protein kinase (PKG), the cGMP-stimulated phosphodiesterase (PDE2) and the cGMP-inhibited PDE (PDE3). Other effects appear independent of cGMP, as for instance the NO modulation of the ryanodine receptor-Ca(2+) channel. In the case of the cardiac L-type Ca(2+) channel current (I(Ca,L)), both cGMP-dependent and cGMP-independent effects have been reported, with important tissue and species specificity. For instance, in rabbit sinoatrial myocytes, NO inhibits the beta-adrenergic stimulation of I(Ca,L) through activation of PDE2. In cat and human atrial myocytes, NO potentiates the cAMP-dependent stimulation of I(Ca,L) through inhibition of PDE3. In rabbit atrial myocytes, NO enhances I(Ca,L) in a cAMP-independent manner through the activation of PKG. In ventricular myocytes, NO exerts opposite effects on I(Ca,L): an inhibition mediated by PKG in mammalian myocytes but by PDE2 in frog myocytes; a stimulation attributed to PDE3 inhibition in frog ventricular myocytes but to a direct effect of NO in ferret ventricular myocytes. Finally, NO can also regulate cardiac ion channels by a direct action on G-proteins and adenylyl cyclase.     

Beyond Bowditch: the convergence of cardiac chronotropy and inotropy

The ability of the heart to acutely beat faster and stronger is central to the vertebrate survival instinct. Released neurotransmitters, norepinephrine and epinephrine, bind to beta-adrenergic receptors (beta-AR) on pacemaker cells comprising the sinoatrial node, and to beta-AR on ventricular myocytes to modulate cellular mechanisms that govern the frequency and amplitude, respectively, of the duty cycles of these cells. While a role for sarcoplasmic reticulum Ca(2+) cycling via SERCA2 and ryanodine receptors (RyR) has long been appreciated with respect to cardiac inotropy, recent evidence also implicates Ca(2+) cycling with respect to chronotropy. In spontaneously beating primary sinoatrial nodal pacemaker cells, RyR Ca(2+) releases occurring during diastolic depolarization activate the Na(+)-Ca(2+) exchanger (NCX) to produce an inward current that enhances their diastolic depolarization rate, and thus increases their beating rate. beta-AR stimulation synchronizes RyR activation and Ca(2+) release to effect an increased beating rate in pacemaker cells and contraction amplitude in myocytes: in pacemaker cells, the beta-AR stimulation synchronization of RyR activation occurs during the diastolic depolarization, and augments the NCX inward current; in ventricular myocytes, beta-AR stimulation synchronizes the openings of unitary L-type Ca(2+) channel activation following the action potential, and also synchronizes RyR Ca(2+) releases following depolarization, and in the absence of depolarization, both leading to the generation of a global cytosolic Ca(i) transient of increased amplitude and accelerated kinetics. Thus, beta-AR stimulation induced synchronization of RyR activation (recruitment of additional RyRs to fire) and of the ensuing Ca(2+) release cause the heart to beat both stronger and faster, and is thus, a common mechanism that links both the maximum achievable cardiac inotropy and chronotropy.     

Calcium-associated mechanisms in gut pacemaker activity

A considerable body of evidence has revealed that interstitial cells of Cajal (ICC), identified with c-Kit-immunoreactivity, act as gut pacemaker cells, with spontaneous Ca(2+) activity in ICC as the probable primary mechanism. Namely, intracellular (cytosolic) Ca(2+) oscillations in ICC periodically activate plasmalemmal Ca(2+)-dependent ion channels and thereby generate pacemaker potentials. This review will, thus, focus on Ca(2+)-associated mechanisms in ICC in the gastrointestinal (GI) tract, including auxiliary organs.     

Anion channels influence ECC by modulating L-type Ca(2+) channel in ventricular myocytes.

Anion channels are extensively expressed in the heart, but their roles in cardiac excitation-contraction coupling (ECC) are poorly understood. We, therefore, investigated the effects of anion channels on cardiac ventricular ECC. Edge detection, fura 2 fluorescence measurements, and whole cell patch-clamp techniques were used to measure cell shortening, the intracellular Ca(2+) transient, and the L-type Ca(2+) current (I(Ca,L)) in single rat ventricular myocytes. The anion channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid reversibly inhibited the Ca(2+) transients and cell shortening in a dose-dependent manner. Comparable results were observed when the majority of the extracellular Cl(-) was replaced with the relatively impermeant anions glutamate (Glt(-)) and aspartate (Asp(-)). NPPB and niflumic acid or the Cl(-) substitutes did not affect the resting intracellular Ca(2+) concentration but significantly inhibited I(Ca,L). In contrast, replacement of extracellular Cl(-) with the permeant anions NO, SCN(-), and Br(-) supported the ECC and I(Ca,L), which were still sensitive to blockade by NPPB. Exposure of cardiac ventricular myocytes to a hypotonic bath solution enhanced the amplitude of cell shortening and supported I(Ca,L), whereas hypertonic stress depressed the contraction and I(Ca,L). Moreover, cardiac contraction was completely abolished by NPPB (50 microM) under hypotonic conditions. It is concluded that a swelling-activated anion channel may be involved in the regulation of cardiac ECC through modulating L-type Ca(2+) channel activity.     

Ginsenoside Re suppresses electromechanical alternans in cat and human cardiomyocytes

Ginseng botanicals are increasingly used as complementary or alternative medicines for a variety of cardiovascular diseases, yet little is known about their cellular actions in cardiac muscle. Electromechanical alternans (EMA) is a proarrhythmic cardiac abnormality that results from disturbances of intracellular Ca(2+) homeostasis. This study sought to determine whether a purified ginsenoside extract of ginseng, Re, exerts effects to suppress EMA and to gain insight into its mechanism of action. Alternans was induced by electrically pacing cardiomyocytes at room temperature. Re (> or = 10 nM) reversibly suppressed EMA recorded from cat ventricular and atrial myocytes and Langendorff-perfused cat hearts. In cat ventricular myocytes, Re reversibly suppressed intracellular Ca(2+) concentration ([Ca(2+)](i)) transient alternans. Re exerted no significant effects on baseline action potential configuration or sarcolemmal L-type Ca(2+) current (I(Ca,L)), Na(+) current, or total K(+) conductance. In human atrial myocytes, Re suppressed mechanical alternans and exerted no effect on I(Ca,L). In cat ventricular myocytes, Re increased [Ca(2+)](i) transient amplitude and decreased sarcoplasmic reticulum (SR) Ca(2+) content, resulting in an increase in fractional SR Ca(2+) release. In SR microsomes isolated from cat ventricles, Re had no effect on SR Ca(2+) uptake. Re increased the open probability of ryanodine receptors (RyRs), i.e., SR Ca(2+)-release channels, isolated from cat ventricles and incorporated into planar lipid bilayers. We concluded that ginsenoside Re suppresses EMA in cat atrial and ventricular myocytes, cat ventricular muscle, and human atrial myocytes. The effects of Re are not mediated via actions on sarcolemmal ion channels or action potential configuration. Re acts via a subcellular mechanism to enhance the opening of RyRs and thereby overcome the impaired SR Ca(2+) release underlying EMA.     

Contractility of ventricular myocytes is well preserved despite altered mechanisms of Ca2+ transport and a changing pattern of mRNA in aged type 2 Zucker diabetic fatty rat heart

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The objective of the study was to investigate ventricular myocyte shortening, intracellular Ca(2+) signalling and expression of genes encoding cardiac muscle proteins in the aged Zucker diabetic fatty (ZDF) rat. There was a fourfold elevation in non-fasting blood glucose in ZDF rats (478.43 ± 29.22 mg/dl) compared to controls (108.22 ± 2.52 mg/dl). Amplitude of shortening, time to peak (TPK) and time to half (THALF) relaxation of shortening were unaltered in ZDF myocytes compared to age-matched controls. Amplitude and THALF decay of the Ca(2+) transient were unaltered; however, TPK Ca(2+) transient was prolonged in ZDF myocytes (70.0 ± 3.2 ms) compared to controls (58.4 ± 2.3 ms). Amplitude of the L-type Ca(2+) current was reduced across a wide range of test potentials (-30 to +40 mV) in ZDF myocytes compared to controls. Sarcoplasmic reticulum Ca(2+) content was unaltered in ZDF myocytes compared to controls. Expression of genes encoding cardiac muscle proteins, membrane Ca(2+) channels, and cell membrane ion transport and intracellular Ca(2+) transport proteins were variously altered. Myh6, Tnnt2, Cacna2d3, Slc9a1, and Atp2a2 were downregulated while Myl2, Cacna1g, Cacna1h, and Atp2a1 were upregulated in ZDF ventricle compared to controls. The results of this study have demonstrated that preserved ventricular myocyte shortening is associated with altered mechanisms of Ca(2+) transport and a changing pattern of genes encoding a variety of Ca(2+) signalling and cardiac muscle proteins in aged ZDF rat.     

Minor contribution of cytosolic Ca2+ transients to the pacemaker rhythm in guinea pig sinoatrial node cells

The question of the extent to which cytosolic Ca(2+) affects sinoatrial node pacemaker activity has been discussed for decades. We examined this issue by analyzing two mathematical pacemaker models, based on the "Ca(2+) clock" (C) and "membrane clock" (M) hypotheses, together with patch-clamp experiments in isolated guinea pig sinoatrial node cells. By applying lead potential analysis to the models, the C mechanism, which is dependent on potentiation of Na(+)/Ca(2+) exchange current via spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) during diastole, was found to overlap M mechanisms in the C model. Rapid suppression of pacemaker rhythm was observed in the C model by chelating intracellular Ca(2+), whereas the M model was unaffected. Experimental rupturing of the perforated-patch membrane to allow rapid equilibration of the cytosol with 10 mM BAPTA pipette solution, however, failed to decrease the rate of spontaneous action potential within ～30 s, whereas contraction ceased within ～3 s. The spontaneous rhythm also remained intact within a few minutes when SR Ca(2+) dynamics were acutely disrupted using high doses of SR blockers. These experimental results suggested that rapid disruption of normal Ca(2+) dynamics would not markedly affect spontaneous activity. Experimental prolongation of the action potentials, as well as slowing of the Ca(2+)-mediated inactivation of the L-type Ca(2+) currents induced by BAPTA, were well explained by assuming Ca(2+) chelation, even in the proximity of the channel pore in addition to the bulk cytosol in the M model. Taken together, the experimental and model findings strongly suggest that the C mechanism explicitly described by the C model can hardly be applied to guinea pig sinoatrial node cells. The possible involvement of L-type Ca(2+) current rundown induced secondarily through inhibition of Ca(2+)/calmodulin kinase II and/or Ca(2+)-stimulated adenylyl cyclase was discussed as underlying the disruption of spontaneous activity after prolonged intracellular Ca(2+) concentration reduction for >5 min.     

Cardiac excitation-contraction coupling: role of membrane potential in regulation of contraction

The steps that couple depolarization of the cardiac cell membrane to initiation of contraction remain controversial. Depolarization triggers a rise in intracellular free Ca(2+) which activates contractile myofilaments. Most of this Ca(2+) is released from the sarcoplasmic reticulum (SR). Two fundamentally different mechanisms have been proposed for SR Ca(2+) release: Ca(2+)-induced Ca(2+) release (CICR) and a voltage-sensitive release mechanism (VSRM). Both mechanisms operate in the same cell and may contribute to contraction. CICR couples the release of SR Ca(2+) closely to the magnitude of the L-type Ca(2+) current. In contrast, the VSRM is graded by membrane potential rather than Ca(2+) current. The electrophysiological and pharmacological characteristics of the VSRM are strikingly different from CICR. Furthermore, the VSRM is strongly modulated by phosphorylation and provides a new regulatory mechanism for cardiac contraction. The VSRM is depressed in heart failure and may play an important role in contractile dysfunction. This review explores the operation and characteristics of the VSRM and CICR and discusses the impact of the VSRM on our understanding of cardiac excitation-contraction coupling.     

Caveolin-3 regulates protein kinase A modulation of the Ca(V)3.2 (alpha1H) T-type Ca2+ channels

Voltage-gated T-type Ca(2+) channel Ca(v)3.2 (α(1H)) subunit, responsible for T-type Ca(2+) current, is expressed in different tissues and participates in Ca(2+) entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Ca(v)3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca(2+) in different cells. We examined the localization and regulation of the Ca(v)3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Ca(v)3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Ca(v)3.1 and Ca(v)3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Ca(v)3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Ca(v)3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Ca(v)3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (I(Ca,T)) and adenovirus (AdCa(v)3.2)-mediated increase in peak Ca(v)3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of I(Ca,T) by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Ca(v)3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca(2+) signaling during normal and pathological processes.     

Dynamical description of sinoatrial node pacemaking: improved mathematical model for primary pacemaker cell

We developed an improved mathematical model for a single primary pacemaker cell of the rabbit sinoatrial node. Original features of our model include 1) incorporation of the sustained inward current (I(st)) recently identified in primary pacemaker cells, 2) reformulation of voltage- and Ca(2+)-dependent inactivation of the L-type Ca(2+) channel current (I(Ca,L)), 3) new expressions for activation kinetics of the rapidly activating delayed rectifier K(+) channel current (I(Kr)), and 4) incorporation of the subsarcolemmal space as a diffusion barrier for Ca(2+). We compared the simulated dynamics of our model with those of previous models, as well as with experimental data, and examined whether the models could accurately simulate the effects of modulating sarcolemmal ionic currents or intracellular Ca(2+) dynamics on pacemaker activity. Our model represents significant improvements over the previous models, because it can 1) simulate whole cell voltage-clamp data for I(Ca,L), I(Kr), and I(st); 2) reproduce the waveshapes of spontaneous action potentials and ionic currents during action potential clamp recordings; and 3) mimic the effects of channel blockers or Ca(2+) buffers on pacemaker activity more accurately than the previous models.     

Phospholemman expression is high in the newborn rabbit heart and declines with postnatal maturation

Phospholemman (PLM) is a small sarcolemmal protein that modulates the activities of Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger (NCX), thus contributing to the maintenance of intracellular Na(+) and Ca(2+) homeostasis. We characterized the expression and subcellular localization of PLM, NCX, and the Na(+)/K(+)-ATPase alpha1-subunit during perinatal development. Western blotting demonstrates that PLM (15kDa), NCX (120kDa), and Na(+)/K(+)-ATPase alpha-1 (approximately 100kDa) proteins are all more than 2-fold higher in ventricular membrane fractions from newborn rabbit hearts (1-4-day old) compared to adult hearts. Our immunocytochemistry data demonstrate that PLM, NCX, and Na(+)/K(+)-ATPase are all expressed at the sarcolemma of newborn ventricular myocytes. Taken together, our data indicate that PLM, NCX, and Na(+)/K(+)-ATPase alpha-1 proteins have similar developmental expression patterns in rabbit ventricular myocardium. Thus, PLM may have an important regulatory role in maintaining cardiac Na(+) and Ca(2+) homeostasis during perinatal maturation.     

On the footsteps of Triadin and its role in skeletal muscle

Calcium is a crucial element for striated muscle function. As such, myoplasmic free Ca2+ concentration is delicately regulated through the concerted action of multiple Ca2+ pathways that relay excitation of the plasma membrane to the intracellular contractile machinery. In skeletal muscle, one of these major Ca2+ pathways is Ca2+ release from intracellular Ca2+ stores through type-1 ryanodine receptor/Ca2+ release channels (RyR1), which positions RyR1 in a strategic cross point to regulate Ca2+ homeostasis. This major Ca2+ traff ic point appears to be highly sensitive to the intracellular environment, which senses through a plethora of chemical and protein-protein interactions. Among these modulators, perhaps one of the most elusive is Triadin, a musclespecif ic protein that is involved in many crucial aspect of muscle function. This family of proteins mediates complex interactions with various Ca2+ modulators and seems poised to be a relevant modulator of Ca2+ signaling in cardiac and skeletal muscles. The purpose of this review is to examine the most recent evidence and current understanding of the role of Triadin in muscle function, in general, with particular emphasis on its contribution to Ca2+ homeostasis.     

Proton-sensing Ca2+ binding domains regulate the cardiac Na+/Ca2+ exchanger

The cardiac Na(+)/Ca(2+) exchanger (NCX) regulates cellular [Ca(2+)](i) and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C(2) regulatory domains, Ca(2+) binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, I(NCX), whereas [H(+)](i) was varied using an ammonium chloride "rebound" method at constant extracellular pH 7.4. At pH(i) = 7.2 and [Ca(2+)](i) < 120 nM, I(NCX) was less than 4% that of its maximally Ca(2+)-activated value. I(NCX) increases steeply at [Ca(2+)](i) between 130-150 nM with a Hill coefficient (n(H)) of 8.0 ± 0.7 and K(0.5) = 310 ± 5 nM. At pH(i) = 6.87, the threshold of Ca(2+)-dependent activation of I(NCX) was shifted to much higher [Ca(2+)](i) (600-700 nM), and the relationship was similarly steep (n(H) = 8.0±0.8) with K(0.5) = 1042 ± 15 nM. The V(max) of Ca(2+)-dependent activation of I(NCX) was not significantly altered by low pH(i). The Ca(2+) affinities for CBD1 (0.39 ± 0.06 μM) and CBD2 (K(d) = 18.4 ± 6 μM) were exquisitely sensitive to [H(+)], decreasing 1.3-2.3-fold as pH(i) decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C(2) regulatory domains CBD1 and CBD2.     

Sodium accumulation in SERCA knockout-induced heart failure

In cardiomyocytes, a major decrease in the level of sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) can severely impair systolic and diastolic functions. In mice with cardiomyocyte-specific conditional excision of the Serca2 gene (SERCA2 KO), end-stage heart failure developed between four and seven weeks after gene deletion combined with [Na(+)](i) elevation and intracellular acidosis. In this study, to investigate the underpinning changes in Ca(2+) dynamics and metabolic homeostasis, we developed data-driven mathematical models of Ca(2+) dynamics in the ventricular myocytes of the control, four-week, and seven-week SERCA2 knockout (KO) mice. The seven-week KO model showed that elevated [Na(+)](i) was due to increased Na(+) influxes through the Na(+)/Ca(2+) exchanger (NCX) and the Na(+)/H(+) exchanger, with the latter exacerbated by intracellular acidosis. Furthermore, NCX upregulation in the seven-week KO model resulted in increased ATP consumption for ion transport. Na(+) accumulation in the SERCA KO due to NCX upregulation and intracellular acidosis potentially play a role in the development of heart failure, by initiating a reinforcing cycle involving: a mismatch between ATP demand and supply; an increasingly compromised metabolism; a decreased pH(i); and, finally, an even greater [Na(+)](i) elevation.