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.     

Na+/Ca2+ exchange current (I(Na/Ca)) and sarcoplasmic reticulum Ca2+ release in catecholamine-induced cardiac hypertrophy

OBJECTIVE: Catecholamines that accompany acute physiological stress are also involved in mediating the development of hypertrophy and failure. However, the cellular mechanisms involved in catecholamine-induced cardiac hypertrophy, particularly Ca2+ handling, are largely unknown. We therefore investigated the effects of cardiac hypertrophy, produced by isoprenaline, on I(Na/Ca) and sarcoplasmic reticulum (SR) function in isolated myocytes. METHODS: I(Na/Ca) was studied in myocytes from Wistar rats, using descending (+80 to -110 mV) voltage ramps under steady state conditions. Myocytes were also loaded with fura-2 and either field stimulated or voltage clamped to assess [Ca2+]i and SR Ca2+ content. RESULTS: Ca2+-dependent, steady state I(Na/Ca) density was increased in hypertrophied myocytes (P<0.05). Ca2+ release from the SR was also increased, whereas resting [Ca2+]i and the rate of decline of [Ca2+]i to control levels were unchanged. SR Ca2+ content, estimated by using 10.0 mmol/l caffeine, was also significantly increased in hypertrophied myocytes, but only when myocytes were held and stimulated from their normal resting potential (-80 mV) but not from -40 mV. However, the rate of decline of caffeine-induced Ca2+ transients or I(Na/Ca) was not significantly different between control and hypertrophied myocytes. Ca2+-dependence of I(Na/Ca), examined by comparing the slope of the descending phase of the hysteresis plots of I(Na/Ca) vs. [Ca2+]i, was also similar in the two groups of cells. CONCLUSION: Data show that SR Ca2+ release and SR Ca2+ content were increased in hypertrophied myocytes, despite an increase in the steady state I(Na/Ca) density. The observation that increased SR function occurred only when myocytes were stimulated from -80 mV suggests that Na+ influx may play a role in altering Ca2+ homeostasis in hypertrophied cardiac muscle, possibly through increased reverse Na+/Ca2+ exchange, particularly at low stimulation frequencies.     

Reduced myocardial sarcoplasmic reticulum Ca(2+)-ATPase protein expression in compensated primary and secondary human cardiac hypertrophy.

Pathological intracellular calcium handling has been proposed to underlie the alterations of contractile behavior in hypertrophied myocardium. However, the myocardial protein expression of intracellular calcium transport proteins in compensated human left ventricular hypertrophy has not yet been studied. We investigated septal myocardial specimens of patients suffering from hypertrophic obstructive cardiomyopathy (n=14) or from acquired aortic valve stenosis (n=11) undergoing myectomy or aortic valve replacement, respectively. For comparison, we studied non-hypertrophied myocardium of six non-failing hearts which could not be transplanted for technical reasons. The myocardial density of the calcium release channel of the sarcoplasmic reticulum (SR) was determined by(3)H-ryanodine binding. Myocardial contents of SR Ca(2+)-ATPase, phospholamban, calsequestrin and Na(+)/Ca(2+)-exchanger were analysed by Western blot analysis. The myocardial SR calcium release channel density was not significantly different in hypertrophied and non-failing human myocardium. In both hypertrophic obstructive cardiomyopathy and in aortic valve stenosis, SR Ca(2+)-ATPase expression was reduced by about 30% compared to non-failing myocardium (P<0.05), whereas the expression of phospholamban, calsequestrin, and the Na(+)/Ca(2+)-exchanger was unchanged. The decrease of SR Ca(2+)-ATPase expression was still observable when related to its regulatory protein phospholamban or to the myosin content of the homogenates (P<0.05). Furthermore, the SR Ca(2+)-ATPase expression was inversely correlated to the septum thickness assessed by echocardiography, but not to age, cardiac index or outflow tract gradient. In primary as well as in secondary hypertrophied human myocardium, the expression of SR Ca(2+)-ATPase is reduced and inversely related to the degree of the hypertrophy. The diminished SR Ca(2+)-ATPase expression might result in reduced Ca(2+)reuptake into the SR and might contribute to altered contractile behavior in hypertrophied human myocardium.     

Ca(2+) signaling in cardiac myocytes overexpressing the alpha(1) subunit of L-type Ca(2+) channel

Voltage-gated L-type Ca(2+) channels (LCCs) provide Ca(2+) ingress into cardiac myocytes and play a key role in intracellular Ca(2+) homeostasis and excitation-contraction coupling. We investigated the effects of a constitutive increase of LCC density on Ca(2+) signaling in ventricular myocytes from 4-month-old transgenic (Tg) mice overexpressing the alpha(1) subunit of LCC in the heart. At this age, cells were somewhat hypertrophic as reflected by a 20% increase in cell capacitance relative to those from nontransgenic (Ntg) littermates. Whole cell I(Ca) density in Tg myocytes was elevated by 48% at 0 mV compared with the Ntg group. Single-channel analysis detected an increase in LCC density with similar conductance and gating properties. Although the overexpressed LCCs triggered an augmented SR Ca(2+) release, the "gain" function of EC coupling was uncompromised, and SR Ca(2+) content, diastolic cytosolic Ca(2+), and unitary properties of Ca(2+) sparks were unchanged. Importantly, the enhanced I(Ca) entry and SR Ca(2+) release were associated with an upregulation of the Na(+)-Ca(2+) exchange activity (indexed by the half decay time of caffeine-elicited Ca(2+) transient) by 27% and SR Ca(2+) recycling by approximately 35%. Western analysis detected a 53% increase in the Na(+)-Ca(2+) exchanger expression but no change in the abundance of ryanodine receptor (RyR), SERCA2, and phospholamban. Analysis of I(Ca) kinetics suggested that SR Ca(2+) release-dependent inactivation of LCCs remains intact in Tg cells. Thus, in spite of the modest cardiac hypertrophy, the overexpressed LCCs form functional coupling with RyRs, preserving both orthograde and retrograde Ca(2+) signaling between LCCs and RyRs. These results also suggest that a modest but sustained increase in Ca(2+) influx triggers a coordinated remodeling of Ca(2+) handling to maintain Ca(2+) homeostasis.     

Net Ca2+ influx and sarcoplasmic reticulum Ca2+ uptake in resting single myocytes of the rat heart: comparison with guinea-pig

We tested Shattock and Bers' (1989) hypothesis according to which in rat cardiac myocytes net Ca2+ influx during diastole via Na/Ca exchange provides the main route of entry of Ca2+ available for activation of contractions. We used injections of caffeine into the close vicinity of the single, isolated rat or guinea-pig ventricular myocytes in order to release Ca2+ from sarcoplasmic reticulum (SR). The cells responded to caffeine with a transient contracture, the amplitude of which was regarded as a relative index of SR Ca2+ content. Application of caffeine deprived the SR of Ca2+. This was manifested by a very small (rat) or absent (guinea-pig) contractile response to the second application of caffeine and by a decrease of the amplitude of the first post caffeine contraction to 8 +/- 3% (rat) or to 16 +/- 6% (guinea-pig) of control. In the rat myocytes SR deprived of Ca2+ was able to recover its Ca2+ store even in the resting cell. This was indicated by the time dependent recovery of contractile response to the second application of caffeine and of the amplitude of the post-caffeine electrically evoked contractions. The recovery of post-caffeine contractile responses was completely inhibited by Ca2+ free solution, by 5.0 mM Ni+ and by low K+ (1.0 mM) hyperpolarising solution superfused from the first application of caffeine or during rest. The recovery was enhanced by superfusion of the cells with low Na+ (50%) solution. These results show that there is a considerable net Ca2+ influx by means of Na/Ca exchange and then the SR Ca2+ uptake in the resting rat myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arrhythmogenesis and contractile dysfunction in heart failure: Roles of sodium-calcium exchange, inward rectifier potassium current, and residual beta-adrenergic responsiveness

Ventricular arrhythmias and contractile dysfunction are the main causes of death in human heart failure (HF). In a rabbit HF model reproducing these same aspects of human HF, we demonstrate that a 2-fold functional upregulation of Na(+)-Ca(2+) exchange (NaCaX) unloads sarcoplasmic reticulum (SR) Ca(2+) stores, reducing Ca(2+) transients and contractile function. Whereas beta-adrenergic receptors (beta-ARs) are progressively downregulated in HF, residual beta-AR responsiveness at this critical HF stage allows SR Ca(2+) load to increase, causing spontaneous SR Ca(2+) release and transient inward current carried by NaCaX. A given Ca(2+) release produces greater arrhythmogenic inward current in HF (as a result of NaCaX upregulation), and approximately 50% less Ca(2+) release is required to trigger an action potential in HF. The inward rectifier potassium current (I(K1)) is reduced by 49% in HF, and this allows greater depolarization for a given NaCaX current. Partially blocking I(K1) in control cells with barium mimics the greater depolarization for a given current injection seen in HF. Thus, we present data to support a novel paradigm in which changes in NaCaX and I(K1), and residual beta-AR responsiveness, conspire to greatly increase the propensity for triggered arrhythmias in HF. In addition, NaCaX upregulation appears to be a critical link between contractile dysfunction and arrhythmogenesis.     

Increased Na+ concentration and altered Na/K pump activity in hypertrophied canine ventricular cells

OBJECTIVE: To investigate whether hypertrophy in the dog with chronic atrioventricular block (CAVB) alters [Na+]i and Na/K-pump function of ventricular myocytes. METHODS: We measured the [Na+]i dependence of the Na/K pump current, I(p). This relation was used as a calibration curve for [Na+]i based on I(p). We measured I(p) at the time of access and extrapolated [Na+] at the pump sites, i.e. subsarcolemmal [Na+], [Na+](subs), from the calibration curve. RESULTS: The extrapolated [Na+](subs) was significantly higher in CAVB (7.9 vs. 3.2 mM in control). The [Na+]i dependence of I(p) in CAVB myocytes was shifted to the right (range of [Na+](i): 0-20 mM). In resting cells, the I(p), i.e. steady state Na+ efflux, which matches Na+ influx, was higher in CAVB (0.25+/-0.02 vs. 0.47+/-0.06 pA/pF, P<0.05). Maximal I(p) density was not different, and DHO sensitivity was not altered. CONCLUSIONS: Hypertrophy in CAVB cells is associated with increased [Na+](subs). This results from an increase in Na+ influx, and a decreased sensitivity of I(p) for Na+ in the range of [Na+]i studied. There is no evidence for a decrease in total pump capacity or for a functional Na/K-ATPase isoform shift. The rise in Na+ contributes to the contractile adaptation and preservation of sarcoplasmic reticulum Ca2+ content at the low heart rates of the dog with CAVB.     

Sarcoplasmic reticulum Ca(2+) release causes myocyte depolarization. Underlying mechanism and threshold for triggered action potentials

Spontaneous sarcoplasmic reticulum (SR) Ca(2+) release causes delayed afterdepolarizations (DADs) via Ca(2+)-induced transient inward currents (I:(ti)). However, no quantitative data exists regarding (1) Ca(2+) dependence of DADs, (2) Ca(2+) required to depolarize the cell to threshold and trigger an action potential (AP), or (3) relative contributions of Ca(2+)-activated currents to DADs. To address these points, we evoked SR Ca(2+) release by rapid application of caffeine in indo 1-AM-loaded rabbit ventricular myocytes and measured caffeine-induced DADs (cDADs) with whole-cell current clamp. The SR Ca(2+) load of the myocyte was varied by different AP frequencies. The cDAD amplitude doubled for every 88+/-8 nmol/L of Delta[Ca(2+)](i) (simple exponential), and the Delta[Ca(2+)](i) threshold of 424+/-58 nmol/L was sufficient to trigger an AP. Blocking Na(+)-Ca(2+) exchange current (I(Na/Ca)) by removal of [Na](o) and [Ca(2+)](o) (or with 5 mmol/L Ni(2+)) reduced cDADs by >90%, for the same Delta[Ca(2+)](i). In contrast, blockade of Ca(2+)-activated Cl(-) current (I(Cl(Ca))) with 50 micromol/L niflumate did not significantly alter cDADs. We conclude that DADs are almost entirely due to I(Na/Ca), not I(Cl(Ca)) or Ca(2+)-activated nonselective cation current. To trigger an AP requires 30 to 40 micromol/L cytosolic Ca(2+) or a [Ca(2+)](i) transient of 424 nmol/L. Current injection, simulating I(ti)s with different time courses, revealed that faster I:(ti)s require less charge for AP triggering. Given that spontaneous SR Ca(2+) release occurs in waves, which are slower than cDADs or fast I(ti)s, the true Delta[Ca(2+)](i) threshold for AP activation may be approximately 3-fold higher in normal myocytes. This provides a safety margin against arrhythmia in normal ventricular myocytes.     

Age and hypertrophy alter the contribution of sarcoplasmic reticulum and Na+/Ca2+ exchange to Ca2+ removal in rat left ventricular myocytes

Age and hypertension contribute significantly to cardiac morbidity and mortality, however the importance of each during the progression of hypertrophy is unclear. This investigation examined the effect of age and hypertension on Ca(2+) handling in rat ventricular myocytes by comparing a genetic model of hypertension and cardiac hypertrophy (spontaneously hypertensive rat, SHR) with its normotensive control (Wistar-Kyoto rat, WKY) at 5 and 8 months of age. Experiments were performed on single left ventricular myocytes isolated from SHR or WKY hearts. Intracellular Ca(2+) was measured optically using fura-2 or fluo-3. SHR myocytes had a significantly larger cell width and volume and a significantly decreased cell length/width ratio at 5 and 8 months compared to normotensive controls. Age had no effect on cell length, width, volume or the length/width ratio. Ca(2+) transient amplitude, sarcoplasmic reticulum (SR) Ca(2+) content and contraction amplitude were unaffected by age or hypertrophy. However at 8 months the contribution of the SR to Ca(2+) uptake during relaxation decreased, with a concomitant increase in the contribution of Na(+)/Ca(2+) exchanger (NCX) function to relaxation, in SHR and WKY myocytes. The incidence of non-synchronous SR Ca(2+) release decreased with age but not hypertrophy in SHR and WKY myocytes. These results show that the changes in Ca(2+) handling observed during progression of mild hypertrophy in SHR are the same as those that occur during ageing in normotensive control animals and can, therefore, be ascribed to maturation rather than hypertrophy.     

Sarcoplasmic reticulum Ca2+ refilling controls recovery from Ca2+-induced Ca2+ release refractoriness in heart muscle

In cardiac muscle Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca2+ influx via L-type Ca2+ channels. At present, the mechanisms underlying termination of SR Ca2+ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca2+ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca2+ release, we analyzed Na+-Ca2+ exchange currents reflecting cytosolic Ca2+ signals induced by UV-laser flash-photolysis of caged Ca2+. Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, beta-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by approximately 2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 micromol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca2+ refilling mediated by the SR Ca2+-pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca2+ sensitivity of CICR appears to be regulated by SR Ca2+ content, possibly resulting from a change in the steady-state Ca2+ sensitivity and in the gating kinetics of the SR Ca2+ release channels (ryanodine receptors). During Ca2+ release, the concomitant reduction in Ca2+ sensitivity of the ryanodine receptors might also underlie Ca2+ spark termination by deactivation.     

Upregulated Na/Ca exchange is involved in both contractile dysfunction and arrhythmogenesis in heart failure

Systolic heart failure (HF) is characterized by reduced systolic function and often by arrhythmias. We studied a rabbit model of HF (induced by combined aortic insufficiency and stenosis) which shows both contractile dysfunction and arrhythmogenesis. In this model we find an ~100 % increase in Na/Ca exchange (NaCaX) expression at the level of mRNA, protein and function, but only a modest decrease in SR Ca-ATPase (~24 %, only detectable in cellular function). This combination results in a 40 % reduction in SR Ca content in HF, which is sufficient to explain the 40 % reduction in twitch Ca transients and 30 – 38 % decrease in contractile function in this HF model. When stimulated by isoproterenol the SR Ca load readily reaches the threshold for spontaneous SR Ca release (this threshold Ca load is unchanged in HF). This SR Ca release activates a transient inward current (I _ti) carried exclusively by NaCaX. For a given SR Ca release there is greater I _ti in HF (due to higher NaCaX). We also find a 49 % decrease in the inward rectifier potassium current (I _K1), which allows greater depolarization for a given I _ti. Thus, higher NaCaX and lower I _K1 greatly increase the likelihood that an SR Ca release-induced delayed afterdepolarization (DAD) will trigger an arrhythmogenic action potential. We conclude that NaCaX contributes in major ways to both contractile dysfunction (by reducing SR Ca) and increased propensity for triggered arrhythmias (by increasing I _ti and DADs).     

Possible mechanism responsible for mechanical dysfunction of ischemic myocardium: a role of oxygen free radicals

It has been proposed that a major target organelles damaged by the ischemic process, probably by the oxygen free radicals generated, is the portion of the excitation-contraction coupling system that regulates Ca2+ delivery (the sarcoplasmic reticulum and sarcolemma) to the contractile proteins. We tested this hypothesis by studying the effect of in vitro generation of oxygen free radicals from xanthine-xanthine oxidase system or dihydroxyfumarate (DHF)/Fe3+-ADP system on Ca2+ flux behavior of canine cardiac sarcoplasmic reticulum (SR); sarcolemmal (Na+, K+)-ATPase and Na+-Ca2+ exchange activities; and myofibrillar (Ca2+, Mg2+)-ATPase activity. Generation of oxygen free radicals by xanthine oxidase acting on xanthine as a substrate increased the passive Ca2+ efflux and decreased intravesicular Ca2+ with no effect on active Ca2+ influx (Ca2+-ATPase) of SR vesicles. Similar exposure of sarcolemmal vesicles to xanthine plus xanthine oxidase stimulated Na+-Ca2+ exchange activity. When sarcolemmal vesicles were incubated with DHF plus Fe3+-ADP, (Na+, K+)-ATPase activity was decreased. It is postulated that the SR Ca2+ efflux pathways but not catalytic activity of the Ca2+ pump and sarcolemmal (Na+, K+)-ATPase involving Na+-Ca2+ exchange activity are altered by oxygen free radicals, and such changes may partly account for the occurrence of intracellular Ca2+ overload during the course of myocardial ischemia. Interestingly, oxygen free radicals from xanthine-xanthine oxidase system had no effect on myofibrillar pCa-ATPase curve. From this set of observations we would hypothesize that the SR and sarcolemma may be the principal target organelles of oxygen free radicals attack in the ischemic injury and not the contractile proteins per se.     

Sarcoplasmic reticulum Ca content, sarcolemmal Ca influx and the genesis of arrhythmias in isolated guinea-pig cardiomyocytes

Early afterdepolarizations are seen during the repolarization phases of the action potential and delayed afterdepolarizations appear later, usually following complete repolarization of the cell membrane potential. Both forms of afterdepolarization are linked to the occurrence of aftercontractions, seem to play a role in the generation of ventricular arrhythmias and are believed to be the result of abnormalities of intracellular Ca handling. Suggestions for the mechanisms responsible vary from both types of afterpotential being mediated by Ca release from the sarcoplasmic reticulum, to early afterdepolarization formation being due to reactivation of the L-type sarcolemmal Ca channels during the action potential. We tried to assess the functional importance of the sarcoplasmic reticulum or Ca influx in the development of afterpotentials and abnormal contractile activity in guinea-pig cardiac myocytes. Ca influx was increased using isoproterenol, Bay K8644 or increasing extracellular [Ca]. Sarcoplasmic reticulum Ca content was measured using rapid cooling contractures or caffeine-induced Na/Ca exchange current and the sarcoplasmic reticulum was inhibited using caffeine or thapsigargin. Aftercontractions associated with either early or delayed afterdepolarizations could be induced by increasing Ca influx. The increased Ca influx produced increases in sarcoplasmic reticulum Ca content and aftercontractions were associated with a larger SR Ca content. However, the sarcoplasmic reticulum was no more loaded with Ca when aftercontractions occurred than when aftercontractions did not occur. Preventing Ca sequestration by the sarcoplasmic reticulum inhibited the formation of aftercontractions. The results suggest that alterations to both Ca influx and sarcoplasmic reticulum Ca content are required to produce aftercontractions.     

Crosstalk between L-type Ca2+ channels and the sarcoplasmic reticulum: alterations during cardiac remodelling

In the cardiac dyad, sarcolemmal L-type Ca(2+) channels (LCCs) and sarcoplasmic reticulum (SR) Ca(2+) release channels (RyR) are structurally in close proximity. This organization provides for an efficient functional coupling, tuning SR Ca(2+) release for optimal contraction of the myocyte. Given that LCC are regulated by the prevailing [Ca(2+)], this structural organization is the setting for feedback mechanisms and crosstalk. A defective coupling of Ca(2+) influx via LCC to activation of RyR has been implicated in reduced SR Ca(2+) release in heart failure. Both functional changes in LCC properties and structural re-organization of LCC in T-tubules could be involved. LCC are regulated by cytosolic Ca(2+), and crosstalk with SR Ca(2+) handling occurs on a long-term basis, i.e. during steady-state changes in heart rate, on an intermediate-term basis, i.e. on a beat-to-beat basis during sudden rate changes, and on a very short- or immediate-term basis, i.e. during a single heartbeat. We review the properties and consequences of these different feedback mechanisms and the changes in heart failure and cardiac hypertrophy that have thus far been studied.     

Cardiac-specific overexpression of Galphaq alters excitation-contraction coupling in isolated cardiac myocytes.

Transgenic mice with cardiac-specific overexpression of G alpha q exhibit a biochemical and physiological phenotype of load-independent cardiac hypertrophy with contractile dysfunction. To elucidate the cellular basis for altered contractility, we measured cellular contraction, Ca(2+)transients, and l -type Ca(2+)channel currents (I(Ca)) in left ventricular (LV) myocytes isolated from non transgenic (NT) controls or G alpha q hearts. Although baseline contractile function (% shortening) and the amplitude of Ca(2+)transients in G alpha q myocytes were similar to NT myocytes, the rates of cellular shortening and relengthening and the duration of Ca(2+)transients were prolonged in G alpha q myocytes. Myocytes from G alpha q hearts had larger cell capacitance but no change in I(Ca)density, voltage-dependence of activation and inactivation. The responses of I(Ca)to dihydropyridine drugs and a membrane permeable cAMP analog, 8-(4-chlorophenylthio) cAMP, were not altered; however, the time course of I(Ca)inactivation was significantly slower in G alpha q myocytes compared to NT myocytes. The kinetic difference in inactivation was abolished when Ba(2+)was used as the charge carrier or when the sarcoplasmic reticulum (SR) Ca(2+)was depleted by ryanodine, suggesting that Ca(2+)-dependent inactivation is reduced in G alpha q myocytes due to altered SR Ca(2+)release. Consistent with this hypothesis, the function of SR as assessed by the maximal Ca(2+)uptake rates and the apparent affinity of SR Ca(2+)-ATPase for Ca(2+)was reduced in ventricles of G alpha q heart. These results suggest that the reduced SR function contributes to the depressed contractility associated with this form of cardiac hypertrophy.     

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

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

Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure

Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin-dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF.     

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.     

Coordinated control of cell Ca(2+) loading and triggered release from the sarcoplasmic reticulum underlies the rapid inotropic response to increased L-type Ca(2+) current

The aim of this study was to investigate how sarcoplasmic reticulum (SR) Ca(2+) content and systolic Ca(2+) are controlled when Ca(2+) entry into the cell is varied. Experiments were performed on voltage-clamped rat and ferret ventricular myocytes loaded with fluo-3 to measure intracellular Ca(2+) concentration ([Ca(2+)](i)). Increasing external Ca(2+) concentration ([Ca(2+)](o)) from 1 to 2 mmol/L increased the amplitude of the systolic Ca(2+) transient with no effect on SR Ca(2+) content. This constancy of SR content is shown to result because the larger Ca(2+) transient activates a larger Ca(2+) efflux from the cell that balances the increased influx. Decreasing [Ca(2+)](o) to 0.2 mmol/L decreased systolic Ca(2+) but produced a small increase of SR Ca(2+) content. This increase of SR Ca(2+) content is due to a decreased release of Ca(2+) from the SR resulting in decreased loss of Ca(2+) from the cell. An increase of [Ca(2+)](o) has two effects: (1) increasing the fraction of SR Ca(2+) content, which is released on depolarization and (2) increasing Ca(2+) entry into the cell. The results of this study show that the combination of these effects results in rapid changes in the amplitude of the systolic Ca(2+) transient. In support of this, the changes of amplitude of the transient occur more quickly following changes of [Ca(2+)](o) than following refilling of the SR after depletion with caffeine. We conclude that the coordinated control of increased Ca(2+) entry and greater fractional release of Ca(2+) is an important factor in regulating excitation-contraction coupling.