Functional adult myocardium in the absence of Na+-Ca2+ exchange: cardiac-specific knockout of NCX1

The excitation-contraction coupling cycle in cardiac muscle is initiated by an influx of Ca2+ through voltage-dependent Ca2+ channels. Ca2+ influx induces a release of Ca2+ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca2+ homeostasis, Ca2+ entry is balanced by efflux mediated by the sarcolemmal Na+-Ca2+ exchanger. In the absence of Na+-Ca2+ exchange, it would be expected that cardiac myocytes would overload with Ca2+. Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na+-Ca2+ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na+-Ca2+ exchanger as measured by both immunoblots and microarray analysis. Ca2+ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca2+ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca2+ influx. Rather than upregulate other Ca2+ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na+-Ca2+ exchanger by limiting Ca2+ influx. The magnitude of Ca2+ transients appears to be maintained by an increased gain of sarcoplasmic reticular Ca2+ release. The myocardium of the NCX1 knockout mice undergoes a remarkable adaptation to maintain near normal cardiac function.     

Alterations in early action potential repolarization causes localized failure of sarcoplasmic reticulum Ca2+ release

Depressed contractility of failing myocytes involves a decreased rate of rise of the Ca2+ transient. Synchronization of Ca2+ release from the junctional sarcoplasmic reticulum (SR) is responsible for the rapid rise of the normal Ca2+ transient. This study examined the idea that spatially and temporally dyssynchronous SR Ca2+ release slows the rise of the cytosolic Ca2+ transient in failing feline myocytes. Left ventricular hypertrophy (LVH) with and without heart failure (HF) was induced in felines by constricting the ascending aorta. Ca2+ transients were measured in ventricular myocytes using confocal line scan imaging. Ca2+ transients were induced by field stimulation, square wave voltage steps, or action potential (AP) voltage clamp. SR Ca2+ release was significantly less well spatially and temporally synchronized in field-stimulated HF versus control or LVH myocytes. Surprisingly, depolarization of HF cells to potentials where Ca2+ currents (ICa) were maximal resynchronized SR Ca2+ release. Correspondingly, decreases in the amplitude of ICa desynchronized SR Ca2+ release in control, LVH, and HF myocytes to the same extent. HF myocytes had significant loss of phase 1 AP repolarization and smaller ICa density, which should both reduce Ca2+ influx. When normal myocytes were voltage clamped with HF AP profiles SR Ca2+ release was desynchronized. SR Ca2+ release becomes dyssynchronized in failing feline ventricular myocytes because of reductions in Ca2+ influx induced in part by alterations in early repolarization of the AP. Therefore, therapies that restore normal early repolarization should improve the contractility of the failing heart.     

Endothelin-1-induced arrhythmogenic Ca2+ signaling is abolished in atrial myocytes of inositol-1,4,5-trisphosphate(IP3)-receptor type 2-deficient mice

Recent studies have suggested that inositol-1,4,5-trisphosphate-receptor (IP3R)-mediated Ca2+ release plays an important role in the modulation of excitation-contraction coupling (ECC) in atrial tissue and the generation of arrhythmias, specifically chronic atrial fibrillation (AF). IP3R type-2 (IP3R2) is the predominant IP3R isoform expressed in atrial myocytes. To determine the role of IP3R2 in atrial arrhythmogenesis and ECC, we generated IP3R2-deficient mice. Our results revealed that endothelin-1 (ET-1) stimulation of wild-type (WT) atrial myocytes caused an increase in basal [Ca2+]i, an enhancement of action potential (AP)-induced [Ca2+]i transients, an improvement of the efficacy of ECC (increased fractional SR Ca2+ release), and the occurrence of spontaneous arrhythmogenic Ca2+ release events as the result of activation of IP3R-dependent Ca2+ release. In contrast, ET-1 did not alter diastolic [Ca2+]i or cause spontaneous Ca2+ release events in IP3R2-deficient atrial myocytes. Under basal conditions the spatio-temporal properties (amplitude, rise-time, decay kinetics, and spatial spread) of [Ca2+]i transients and fractional SR Ca2+ release were not different in WT and IP3R2-deficient atrial myocytes. WT and IP3R2-deficient atrial myocytes also showed a significant and very similar increase in the amplitude of AP-dependent [Ca2+]i transients and Ca2+ spark frequency in response to isoproterenol stimulation, suggesting that both cell types maintained a strong inotropic reserve. No compensatory changes in Ca2+ regulatory protein expression (IP3R1, IP3R3, RyR2, NCX, SERCA2) or morphology of the atria could be detected between WT and IP3R2-deficient mice. These results show that lack of IP3R2 abolishes the positive inotropic effect of neurohumoral stimulation with ET-1 and protects from its arrhythmogenic effects.     

Chronic SR Ca2+-ATPase inhibition causes adaptive changes in cellular Ca2+ transport

Phospholamban, the critical regulator of the cardiac SERCA2a Ca2+ affinity, is phosphorylated at Ser16 and Thr17 during beta-adrenergic stimulation (eg, isoproterenol). To assess the impact of nonphosphorylatable phospholamban, a S16A, T17A double-mutant (DM) was introduced into phospholamban knockout mouse hearts. Transgenic lines expressing DM phospholamban at levels similar to wild types (WT) were identified. In vitro phosphorylation confirmed that DM phospholamban could not be phosphorylated, but produced the same shift in EC50 of SERCA2a for Ca2+ as unphosphorylated WT phospholamban. Rates of basal twitch [Ca2+]i decline were not different in DM versus WT cardiomyocytes. Isoproterenol increased the rates of twitch [Ca2+]i decline in WT, but not DM myocytes, confirming the prominent role of phospholamban phosphorylation in this response. Increased L-type Ca2+ current (ICa) density, with unaltered characteristics, was the major compensation in DM myocytes. Consequently, the normal beta-adrenergic-induced increase in ICa caused larger dynamic changes in absolute ICa density. Isoproterenol increased Ca2+ transients to a comparable amplitude in DM and WT. There were no changes in myofilament Ca2+ sensitivity, or the expression levels and Ca2+ removal activities of other Ca2+-handling proteins. Nor was there evidence of cardiac remodeling up to 10 months of age. Thus, chronic inhibition of SERCA2a by ablation of phospholamban phosphorylation (abolishing its adrenergic regulation) results in a unique cellular adaptation involving greater dynamic ICa modulation. This ICa modulation may partly compensate for the loss in SERCA2a responsiveness and thereby partially normalize beta-adrenergic inotropy in DM phospholamban mice.     

Cellular basis of abnormal calcium transients of failing human ventricular myocytes

Depressed contractility is a central feature of the failing human heart and has been attributed to altered [Ca2+]i. This study examined the respective roles of the L-type Ca2+ current (ICa), SR Ca2+ uptake, storage and release, Ca2+ transport via the Na+-Ca2+ exchanger (NCX), and Ca2+ buffering in the altered Ca2+ transients of failing human ventricular myocytes. Electrophysiological techniques were used to measure and control V(m) and measure I(m), respectively, and Fluo-3 was used to measure [Ca2+]i in myocytes from nonfailing (NF) and failing (F) human hearts. Ca2+ transients from F myocytes were significantly smaller and decayed more slowly than those from NF hearts. Ca2+ uptake rates by the SR and the amount of Ca2+ stored in the SR were significantly reduced in F myocytes. There were no significant changes in the rate of Ca2+ removal from F myocytes by the NCX, in the density of NCX current as a function of [Ca2+]i, ICa density, or cellular Ca2+ buffering. However, Ca2+ influx during the late portions of the action potential seems able to elevate [Ca2+]i in F but not in NF myocytes. A reduction in the rate of net Ca2+ uptake by the SR slows the decay of the Ca2+ transient and reduces SR Ca2+ stores. This leads to reduced SR Ca2+ release, which induces additional Ca2+ influx during the plateau phase of the action potential, further slowing the decay of the Ca2+ transient. These changes can explain the defective Ca2+ transients of the failing human ventricular myocyte.     

Adverse bioenergetic consequences of Na+-Ca2+ exchanger-mediated Ca2+ influx in cardiac myocytes

BACKGROUND: In heart failure, the Na+-Ca2+ exchanger (NCX) is upregulated and mediates Ca2+ influx (instead of efflux) during the cardiac action potential. Although this partly compensates for impaired sarcoplasmic reticulum Ca2+ release and supports inotropy, the energetic consequences have never been considered. Because NCX-mediated Ca2+ influx is rather slow and mitochondrial Ca2+ uptake (which stimulates NADH production by the Krebs cycle) is thought to be facilitated by high Ca2+ gradients in a "mitochondrial Ca2+ microdomain," we speculated that NCX-mediated Ca2+ influx negatively affects the bioenergetic feedback response. Methods and Results- With the use of a patch-clamp-based approach in guinea-pig myocytes, cytosolic and mitochondrial Ca2+ ([Ca2+](c) and [Ca2+](m), respectively) was determined within the same cell after varying Ca2+ influx via L-type Ca2+ channels (I(Ca,L)) or the NCX. The efficiency of mitochondrial Ca2+ uptake, indexed by the slope of plotting [Ca2+](m) against [Ca2+](c) during each Ca2+ transient, was maximal during I(Ca,L)-triggered sarcoplasmic reticulum Ca2+ release. Depletion of sarcoplasmic reticulum Ca2+ load and increased contribution of the NCX to cytosolic Ca2+ influx independently reduced the efficiency of mitochondrial Ca2+ uptake. The upstroke velocity of cytosolic Ca2+ transients closely correlated with the efficiency of mitochondrial Ca2+ uptake. Despite comparable [Ca2+](c), sarcoplasmic reticulum Ca2+ release, but not NCX-mediated Ca2+ influx, led to stimulation of Ca2+-sensitive dehydrogenases of the Krebs cycle. Conclusions- Increased contribution of the NCX to cytosolic Ca2+ transients, which occurs in cardiac myocytes from failing hearts, impairs mitochondrial Ca2+ uptake and the bioenergetic feedback response. This mechanism could contribute to energy starvation of failing hearts.     

T-type Ca2+ current contribution to Ca2+-induced Ca2+ release in developing myocardium

In normal adult-ventricular myocardium, Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is activated via Ca2+ entry through L-type Ca2+ channels. However, embryonic-ventricular myocytes have a prominent T-type Ca2+ current (ICa,T). In this study, the contribution of ICa,T to CICR was determined in chick-ventricular development. Electrically stimulated Ca2+ transients were examined in myocytes loaded with fura-2 and Ca2+ currents with perforated patch-clamp. The results show that the magnitudes of the Ca2+ transient, L-type Ca2+ current (ICa,L) and ICa,T, decline with development with the majority of the decline of transients and ICa,L occurring between embryonic day (ED) 5 and 11. Compared to controls, the magnitude of the Ca2+ transient in the presence of nifedipine was reduced by 41% at ED5, 77% at ED11, and 78% at ED15. These results demonstrated that the overall contribution of ICa,T to the transient was greatest at ED5, while ICa,L was predominate at ED11 and 15. This indicated a decline in the contribution of ICa,T to the Ca2+ transient with development. Nifedipine plus caffeine was added to deplete the SR of Ca2+ and eliminate the occurrence of CICR due to ICa,T. Under these conditions, the transients were further reduced at all three developmental ages, which indicated that a portion of the Ca2+ transients present after just nifedipine addition was due to CICR stimulated by ICa,T. These results indicate that Ca2+ entry via T-type channels plays a significant role in excitation-contraction coupling in the developing heart that includes stimulation of CICR.     

Pharmacological inhibition of na/ca exchange results in increased cellular Ca2+ load attributable to the predominance of forward mode block

Block of Na/Ca exchange (NCX) has potential therapeutic applications, in particular, if a mode-selective block could be achieved, but also carries serious risks for disturbing the normal Ca2+ balance maintained by NCX. We have examined the effects of partial inhibition of NCX by SEA-0400 (1 or 0.3 micromol/L) in left ventricular myocytes from healthy pigs or mice and from mice with heart failure (MLP-/-). During voltage clamp ramps with [Ca2+](i) buffering, block of reverse mode block was slightly larger than of forward mode (by 25+/-5%, P<0.05). In the absence of [Ca2+](i) buffering and with sarcoplasmic reticulum (SR) fluxes blocked, rate constants for Ca2+ influx and Ca2+ efflux were reduced to the same extent (to 36+/-6% and 32+/-4%, respectively). With normal SR function the reduction of inward NCX current (I(NCX)) was 57+/-10% (n=10); during large caffeine-induced Ca2+ transients, it was larger (82+/-3%). [Ca2+](i) transients evoked during depolarizing steps increased (from 424+/-27 to 994+/-127 nmol/L at +10 mV, P<0.05), despite a reduction of I(CaL) by 27%. Resting [Ca2+](i) increased; there was a small decrease in the rate of decline of [Ca2+](i). SR Ca2+) content increased more than 2-fold. Contraction amplitude of field-stimulated myocytes increased in healthy myocytes but not in myocytes from MLP-/- mice, in which SR Ca2+ content remained unchanged. These data provide proof-of-principle that even partial inhibition of NCX results in a net gain of Ca2+. Further development of NCX blockers, in particular, for heart failure, must balance potential benefits of I(NCX) reduction against effects on Ca2+ handling by refining mode dependence and/or including additional targets.     

Proarrhythmia in a non-failing murine model of cardiac-specific Na+/Ca2+ exchanger overexpression: whole heart and cellular mechanisms

The cardiac Na⁺/Ca²⁺ exchanger (NCX) generates an inward electrical current during SR-Ca²⁺ release, thus possibly promoting afterdepolarizations of the action potential (AP). We used transgenic mice 12.5 weeks or younger with cardiomyocyte-directed overexpression of NCX (NCX-Tg) to study the proarrhythmic potential and mechanisms of enhanced NCX activity. NCX-Tg exhibited normal echocardiographic left ventricular function and heart/body weight ratio, while the QT interval was prolonged in surface ECG recordings. Langendorff-perfused NCX-Tg, but not wild-type (WT) hearts, developed ventricular tachycardia. APs and ionic currents were measured in isolated cardiomyocytes. Cell capacitance was unaltered between groups. APs were prolonged in NCX-Tg versus WT myocytes along with voltage-activated K⁺ currents (K_v) not being reduced but even increased in amplitude. During abrupt changes in pacing cycle length, early afterdepolarizations (EADs) were frequently recorded in NCX-Tg but not in WT myocytes. Next to EADs, delayed afterdepolarizations (DAD) triggering spontaneous APs (sAPs) occurred in NCX-Tg but not in WT myocytes. To test whether sAPs were associated with spontaneous Ca²⁺ release (sCR), Ca²⁺ transients were recorded. Despite the absence of sAPs in WT, sCR was observed in myocytes of both genotypes suggesting a facilitated translation of sCR into DADs in NCX-Tg. Moreover, sCR was more frequent in NCX-Tg as compared to WT. Myocardial protein levels of Ca²⁺-handling proteins were not different between groups except the ryanodine receptor (RyR), which was increased in NCX-Tg versus WT. We conclude that NCX overexpression is proarrhythmic in a non-failing environment even in the absence of reduced K_V. The underlying mechanisms are: (1) occurrence of EADs due to delayed repolarization; (2) facilitated translation from sCR into DADs; (3) proneness to sCR possibly caused by altered Ca²⁺ handling and/or increased RyR expression.     

Increased exchange current but normal Ca2+ transport via Na+-Ca2+ exchange during cardiac hypertrophy after myocardial infarction

Hypertrophied and failing cardiac myocytes generally show alterations in intracellular Ca2+ handling associated with changes in the contractile function and arrhythmogenicity. The cardiac Na+-Ca2+ exchange (NCX) is an important mechanism for Ca2+ extrusion and cell relaxation. Its possible involvement in changes of excitation-contraction coupling (EC-coupling) with disease remains uncertain. We analyzed the NCX function in rat ventricular myocytes 5 to 6 months after experimental myocardial infarction (PMI) produced by left coronary artery ligation and from sham-operated (SO) hearts. Caged Ca2+ was dialyzed into the cytoplasm via a patch-clamp pipette and Ca2+ was released by flash photolysis to activate NCX and measure the associated currents (I(NaCa)), whereas [Ca2+]i changes were simultaneously recorded with a confocal microscope. I(NaCa) density normalized to the [Ca2+]i jumps was 2.6-fold higher in myocytes from PMI rats. The level of total NCX protein expression in PMI myocytes was also increased. Interestingly, although the I(NaCa) density in PMI cells was larger, PMI and SO myocytes presented virtually identical Ca2+ transport via the NCX. This discrepancy was explained by a reduced surface/volume ratio (34.8%) observed in PMI cells. We conclude that the increase in NCX density may be a mechanism to maintain the required Ca2+ extrusion from a larger cell to allow adequate relaxation.     

Calcium entry via Na/Ca exchange during the action potential directly contributes to contraction of failing human ventricular myocytes

Prolongation of the Ca2+ transient and action potential (AP) durations are two characteristic changes in myocyte physiology in the failing human heart. The hypothesis of this study is that Ca2+ influx via reverse mode Na+/Ca2+ exchanger (NCX) or via L-type Ca2+ channels directly activates contraction in failing human myocytes while in normal myocytes this Ca2+ is transported into the sarcoplasmic reticulum (SR) to regulate SR Ca2+ stores. METHODS: Myocytes were isolated from failing human (n=6), nonfailing human (n=3) and normal feline hearts (n=9) and whole cell current and voltage clamp techniques were used to evoke and increase the duration of APs (0.5 Hz, 37 degrees C). Cyclopiazonic acid (CPA 10(-6) M), nifedipine (NIF;10(-6) M) and KB-R 7943 (KB-R; 3x10(-6) M) were used to reduce SR Ca2+ uptake, Ca2+ influx via the L-type Ca2+ current and reverse mode NCX, respectively. [Na+)i was changed by dialyzing myocytes with 0, 10 and 20 mM Na(+) pipette solutions. RESULTS: Prolongation of the AP duration caused an immediate prolongation of contraction and Ca2+ transient durations in failing myocytes. The first beat after the prolonged AP was potentiated by 21+/-5 and 27+/-5% in nonfailing human and normal feline myocytes, respectively (P<0.05), but there was no significant effect in failing human myocytes (+5+/-4% vs. steady state). CPA blunted the potentiation of the first beat after AP prolongation in normal feline and nonfailing human myocytes, mimicking the failing phenotype. NIF reduced steady state contraction in feline myocytes but the potentiation of the first beat after AP prolongation was unaltered (21+/-3% vs. base, P<0.05). KB-R reduced basal contractility and abolished the potentiation of the first beat after AP prolongation (2+/-1% vs. steady state). Increasing [Na+]i shortened AP, Ca2+ transient and contraction durations and increased steady state and post AP prolongation contractions. Dialysis with 0 Na+ eliminated these effects. CONCLUSIONS: Ca2+ enters both normal and failing cardiac myocytes during the late portion of the AP plateau via reverse mode NCX. In (normal) myocytes with good SR function, this Ca(2+) influx helps maintain and regulate SR Ca2+ load. In (failing) human myocytes with poor SR function this Ca2+ influx directly contributes to contraction. These studies suggest that the Ca2+ transient of the failing human ventricular myocytes has a higher than normal reliance on Ca2+ influx via the reverse mode of the NCX during the terminal phases of the AP.     

Action potential prolongation in cardiac myocytes of old rats is an adaptation to sustain youthful intracellular Ca2+ regulation

Advanced age in rats is accompanied by reduced expression of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA-2). The amplitudes of intracellular Ca2+ (Ca2+(i)) transients and contractions in ventricular myocytes isolated from old (23-24-months) rats (OR), however, are similar to those of young (4-6-months) rat myocytes (YR). OR myocytes also manifest slowed inactivation of L-type Ca2+ current (I(CaL)) and marked prolongation of action potential (AP) duration. To determine whether and how age-associated AP prolongation preserves the Ca2+(i) transient amplitude in OR myocytes, we employed an AP-clamp technique with simultaneous measurements of I(CaL) (with Na+ current, K+ currents and Ca2+ influx via sarcolemmal Na+-Ca2+ exchanger blocked) and Ca2+(i) transients in OR rat ventricular myocytes dialyzed with the fluorescent Ca2+ probe, indo-1. Myocytes were stimulated with AP-shaped voltage clamp waveforms approximating the configuration of prolonged, i.e. the native, AP of OR cells (AP-L), or with short AP waveforms (AP-S), typical of YR myocytes. Changes in SR Ca2+ load were assessed by rapid, complete SR Ca2+ depletions with caffeine. As expected, during stimulation with AP-S vs AP-L, peak I(CaL) increased, by 21+/-4%, while the I(CaL) integral decreased, by 19+/-3% (P<0.01 for each). Compared to AP-L, stimulation of OR myocytes with AP-S reduced the amplitudes of the Ca2+(i) transient by 31+/-6%, its maximal rate of rise (+dCa2+(i)/dt(max); a sensitive index of SR Ca2+ release flux) by 37+/-4%, and decreased the SR Ca2+ load by 29+/-4% (P<0.01 for each). Intriguingly, AP-S also reduced the maximal rate of the Ca2+(i) transient relaxation and prolonged its time to 50% decline, by 35+/-5% and 33+/-7%, respectively (P<0.01 for each). During stimulation with AP-S, the gain of Ca2+-induced Ca2+ release (CICR), indexed by +dCa2+(i)/dt(max)/I(CaL), was reduced by 46+/-4% vs AP-L (P<0.01). We conclude that the effects of an application of a shorter AP to OR myocytes to reduce +dCa2+(i)/dt(max) and the Ca2+ transient amplitude are attributable to a reduction in SR Ca2+ load, presumably due to a reduced I(CaL) integral and likely also to an increased Ca2+ extrusion via sarcolemmal Na+-Ca2+ exchanger. The decrease in the Ca2+(i) transient relaxation rate in OR cells stimulated with shorter APs may reflect a reduction of Ca2+/calmodulin-kinase II-regulated modulation of Ca2+ uptake via SERCA-2, consequent to a reduced local Ca2+ release in the vicinity of SERCA-2, also attributable to reduced SR Ca2+ load. Thus, the reduction of CICR gain during stimulation with AP-S is the net result of both a diminished SR Ca2+ release and an increased peak I(CaL). These results suggest that ventricular myocytes of old rats utilize AP prolongation to preserve an optimal SR Ca2+ loading, CICR gain and relaxation of Ca2+(i) transients.     

Cardiac submembrane [Na+] transients sensed by Na+-Ca2+ exchange current

Na+ influx via INa during cardiac action potentials can raise bulk [Na+]i by 10 to 15 micromol/L. However, larger rises in submembrane [Na+] ([Na+]sm) local to Na+-Ca2+ exchangers (NCX) could enhance Ca2+ influx via NCX (and Ca2+-induced Ca2+ release). We tested whether INa could increase [Na+]sm, using NCX current (INCX) as a biosensor in rabbit ventricular myocytes (with [Ca2+]i buffered, [Na+]i=10 mmol/L, and other currents blocked). We measured INCX as early as 5 ms after INa. Prior INa activation did not affect INCX at physiological membrane potentials (Em=-100 to +50 mV), but for Em >+50 mV (where INCX is especially sensitive to [Na+]i), INCX shifted outward. At 5 ms and +100 mV, INa shifted INCX outward by 0.23 A/F (corresponding to Delta[Na+]sm=0.24 mmol/L). The effect of INa dissipated with a time constant of approximately 15 ms. Thus, the impact of INa on NCX is almost undetectable at physiological Em and short lived. This suggests that INa effects on excitation-contraction coupling (via outward INCX) are minimal and limited to early during the action potential. However, local Delta[Na+]sm during INa may be 60 times higher than bulk Delta[Na+]i.     

The sarcoplasmic reticulum and the Na+/Ca2+ exchanger both contribute to the Ca2+ transient of failing human ventricular myocytes

Our objective was to determine the respective roles of the sarcoplasmic reticulum (SR) and the Na+/Ca2+ exchanger in the small, slowly decaying Ca2+ transients of failing human ventricular myocytes. Left ventricular myocytes were isolated from explanted hearts of patients with severe heart failure (n=18). Cytosolic Ca2+, contraction, and action potentials were measured by using indo-1, edge detection, and patch pipettes, respectively. Selective inhibitors of SR Ca2+ transport (thapsigargin) and reverse-mode Na+/Ca2+ exchange activity (No. 7943, Kanebo Ltd) were used to define the respective contribution of these processes to the Ca2+ transient. Ca2+ transients and contractions induced by action potentials (AP transients) at 0.5 Hz exhibited phasic and tonic components. The duration of the tonic component was determined by the action potential duration. Ca2+ transients induced by caffeine (Caf transients) exhibited only a phasic component with a rapid rate of decay that was dependent on extracellular Na+. The SR Ca2+-ATPase inhibitor thapsigargin abolished the phasic component of the AP Ca2+ transient and of the Caf transient but had no significant effect on the tonic component of the AP transient. The Na+/Ca2+ exchange inhibitor No. 7943 eliminated the tonic component of the AP transient and reduced the magnitude of the phasic component. In failing human myocytes, Ca2+ transients and contractions exhibit an SR-related, phasic component and a slow, reverse-mode Na+/Ca2+ exchange-related tonic component. These findings suggest that Ca2+ influx via reverse-mode Na+/Ca2+ exchange during the action potential may contribute to the slow decay of the Ca2+ transient in failing human myocytes.     

Ca2+ signaling amplification by oligomerization of L-type Cav1.2 channels

Ca(2+) influx via L-type Ca(v)1.2 channels is essential for multiple physiological processes, including gene expression, excitability, and contraction. Amplification of the Ca(2+) signals produced by the opening of these channels is a hallmark of many intracellular signaling cascades, including excitation-contraction coupling in heart. Using optogenetic approaches, we discovered that Ca(v)1.2 channels form clusters of varied sizes in ventricular myocytes. Physical interaction between these channels via their C-tails renders them capable of coordinating their gating, thereby amplifying Ca(2+) influx and excitation-contraction coupling. Light-induced fusion of WT Ca(v)1.2 channels with Ca(v)1.2 channels carrying a gain-of-function mutation that causes arrhythmias and autism in humans with Timothy syndrome (Ca(v)1.2-TS) increased Ca(2+) currents, diastolic and systolic Ca(2+) levels, contractility and the frequency of arrhythmogenic Ca(2+) fluctuations in ventricular myocytes. Our data indicate that these changes in Ca(2+) signaling resulted from Ca(v)1.2-TS increasing the activity of adjoining WT Ca(v)1.2 channels. Collectively, these data support the concept that oligomerization of Ca(v)1.2 channels via their C termini can result in the amplification of Ca(2+) influx into excitable cells.     

Dual regulation of Ca2+/calmodulin-dependent kinase II activity by membrane voltage and by calcium influx.

Calcium entry through voltage-gated Ca2+ channels is critical in cardiac excitation-contraction coupling and calcium metabolism. In this report, we demonstrate both spatially resolved and temporally distinct effects of Ca2+/calmodulin-dependent protein kinase II (CaMKII) on L-type Ca2+ channel current (ICa) in rat cardiac myocytes. Either depolarization alone or calcium influx can increase the amplitude and slow the inactivation of ICa. The distinct voltage- and Ca(2+)-dependent effects persist with time constants of approximately 1.7 sec and 9 sec, respectively. Both effects are completely abolished by a specific peptide inhibitor of CaMKII. This CaMKII inhibitor also suppresses the prolongation of ICa induced by depolarizing holding potentials. Furthermore, using an antibody specific for the autophosphorylated (activated) CaMKII, we find that this kinase is localized close to sarcolemmal membranes and that the profile of CaMKII activation correlates qualitatively with the changes in ICa under various conditions. Therefore, we conclude that the action of CaMKII on ICa is dually regulated by membrane depolarization and by Ca2+ influx; the latter directly activates CaMKII, whereas the former likely promotes the interaction between constitutive CaMKII and the membrane-channel proteins. These regulatory mechanisms provide positive-feedback control of Ca2+ channels and are probably important in the regulation of cardiac contractility and other intracellular Ca(2+)-regulated processes.     

Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, II: model studies

Ca2+ transients measured in failing human ventricular myocytes exhibit reduced amplitude, slowed relaxation, and blunted frequency dependence. In the companion article (O'Rourke B, Kass DA, Tomaselli GF, K??b S, Tunin R, Marbán E. Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart, I: experimental studies. Circ Res. 1999;84:562-570), O'Rourke et al show that Ca2+ transients recorded in myocytes isolated from canine hearts subjected to the tachycardia pacing protocol exhibit similar responses. Analyses of protein levels in these failing hearts reveal that both SR Ca2+ ATPase and phospholamban are decreased on average by 28% and that Na+/Ca2+ exchanger (NCX) protein is increased on average by 104%. In this article, we present a model of the canine midmyocardial ventricular action potential and Ca2+ transient. The model is used to estimate the degree of functional upregulation and downregulation of NCX and SR Ca2+ ATPase in heart failure using data obtained from 2 different experimental protocols. Model estimates of average SR Ca2+ ATPase functional downregulation obtained using these experimental protocols are 49% and 62%. Model estimates of average NCX functional upregulation range are 38% and 75%. Simulation of voltage-clamp Ca2+ transients indicates that such changes are sufficient to account for the reduced amplitude, altered shape, and slowed relaxation of Ca2+ transients in the failing canine heart. Model analyses also suggest that altered expression of Ca2+ handling proteins plays a significant role in prolongation of action potential duration in failing canine myocytes.     

Transgenic CaMKIIdeltaC overexpression uniquely alters cardiac myocyte Ca2+ handling: reduced SR Ca2+ load and activated SR Ca2+ release

Ca2+/calmodulin-dependent protein kinase II (CaMKII) delta is the predominant cardiac isoform, and the deltaC splice variant is cytoplasmic. We overexpressed CaMKIIdeltaC in mouse heart and observed dilated heart failure and altered myocyte Ca2+ regulation in 3-month-old CaMKIIdeltaC transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca2+]i transient amplitude, and diastolic [Ca2+]i were all reduced by approximately 50% in TG versus WT. This is explained by >50% reduction in SR Ca2+ content in TG versus WT. Peak Ca2+ current (ICa) was slightly increased, and action potential duration was prolonged in TG versus WT. Despite lower SR Ca2+ load and diastolic [Ca2+]i, fractional SR Ca2+ release was increased and resting spontaneous SR Ca2+ release events (Ca2+ sparks) were doubled in frequency in TG versus WT (with prolonged width and duration, but lower amplitude). Enhanced Ca2+ spark frequency was also seen in TG at 4 weeks (before heart failure onset). Acute CaMKII inhibition normalized Ca2+ spark frequency and ICa, consistent with direct CaMKII activation of ryanodine receptors (and ICa) in TG. The rate of [Ca2+]i decline during caffeine exposure was faster in TG, indicating enhanced Na+-Ca2+ exchange function (consistent with protein expression measurements). Enhanced diastolic SR Ca2+ leak (via sparks), reduced SR Ca2+-ATPase expression, and increased Na+-Ca2+ exchanger explain the reduced diastolic [Ca2+]i and SR Ca2+ content in TG. We conclude that CaMKIIdeltaC overexpression causes acute modulation of excitation-contraction coupling, which contributes to heart failure.     

Increased SR Ca2+ cycling contributes to improved contractile performance in SERCA2a-overexpressing transgenic rats

OBJECTIVE: Heart failure is associated with reduced function of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) but increased function of sarcolemmal Na+/Ca2+ exchanger (NCX), leading to decreased SR Ca2+ content and loss of frequency-potentiation of contractile force. We reported that SERCA2a-overexpression in transgenic rat hearts (TG) results in improved contractility. However, it was not clear whether TG have improved contractility due to frequency-dependent improved SR Ca2+ handling. METHODS: Therefore, we characterized TG (n=35) vs. wild-type (WT) control rats (n=39) under physiological conditions (37 degrees C, stimulation rate <8 Hz). Twitch force, intracellular Ca2+ transients ([Ca2+]i), and SR Ca2+ content were measured in isolated muscles. The contribution of transsarcolemmal Ca2+ influx (I(Ca)) through L-type Ca2+ channels (LTCC) and reverse mode NCX (I(Na/Ca)) to Ca2+ cycling were studied in isolated myocytes. RESULTS: With increasing frequency, force increased in TG muscles by 168+/-35% (8 Hz; P<0.05) and SR Ca2+ content increased by maximally 118+/-31% (4 Hz; P<0.05). In WT, there was a flat force-frequency response without changes in SR Ca2+ content. Relaxation parameters of force and [Ca2+]i decay were accelerated at each frequency in TG vs. WT by approximately 10%. At prolonged rest intervals (<240 s), force and SR Ca2+ content increased significantly more in TG. Consequently, absolute SR Ca2+ content measured in myocytes was increased approximately 2-fold in TG. Transsarcolemmal Ca2+ fluxes estimated by I(Ca) (at 0 mV -10.2+/-1.1 vs. -16.9+/-1.3 pA/pF) and I(Na/Ca) (0.17+/-0.02 vs. 0.46+/-0.05 pA/pF) were decreased in TG vs. WT (P<0.05), whereas NCX and LTCC protein expression was only slightly reduced (P=n.s.). CONCLUSION: In summary, SERCA2a-overexpression improved contractility in a frequency-dependent way due to increased SR Ca2+ loading whereas transsarcolemmal Ca2+ fluxes were decreased.     

The Na+/Ca2+ exchange blocker SEA0400 fails to enhance cytosolic Ca2+ transient and contractility in canine ventricular cardiomyocytes

AIMS: This study was designed to evaluate the effects of the Na(+)/Ca(2+) exchange (NCX) inhibitor SEA0400 on Ca(2+) handling in isolated canine ventricular myocytes. METHODS AND RESULTS: Intracellular Ca(2+) ([Ca(2+)](i)) transients, induced by either field stimulation or caffeine flush, were monitored using Ca(2+) indicator dyes. [Ca(2+)](i)-dependent modulation of the inhibitory effect of SEA0400 on NCX was characterized by the changes in Ni(2+)-sensitive current in voltage-clamped myocytes. Sarcoplasmic reticulum (SR) Ca(2+) release and uptake were studied in SR membrane vesicles. Gating properties of single-ryanodine receptors were analysed in lipid bilayers. Ca(2+) sensitivity of the contractile machinery was evaluated in chemically skinned myocytes. In myocytes paced at 1 Hz, neither diastolic [Ca(2+)](i) nor the amplitude of [Ca(2+)](i) transients was significantly altered by SEA0400 up to the concentration of 1 microM, which was shown to inhibit the exchange current. The blocking effect of SEA0400 on NCX decreased with increasing [Ca(2+)](i), and it was more pronounced in reverse than in forward mode operation at every [Ca(2+)](i) examined. The rate of decay of the caffeine-induced [Ca(2+)](i) transients was decreased significantly by 1 microM SEA0400; however, this effect was only a fraction of that observed with 10 mM NiCl(2). Neither SR Ca(2+) release and uptake nor cell shortening and Ca(2+) sensitivity of the contractile proteins were influenced by SEA0400. CONCLUSION: The lack of any major SEA0400-induced shift in Ca(2+) transients or contractility of myocytes can well be explained by its limited inhibitory effect on NCX (further attenuated by elevated [Ca(2+)](i) levels) and a concomitant reduction in Ca(2+) influx due to the predominantly reverse mode blockade of NCX and suppression of L-type Ca(2+) current.