A novel interaction mechanism accounting for different acylphosphatase effects on cardiac and fast twitch skeletal muscle sarcoplasmic reticulum calcium pumps. (1/4498)

In cardiac and skeletal muscle Ca2+ translocation from cytoplasm into sarcoplasmic reticulum (SR) is accomplished by different Ca2+-ATPases whose functioning involves the formation and decomposition of an acylphosphorylated phosphoenzyme intermediate (EP). In this study we found that acylphosphatase, an enzyme well represented in muscular tissues and which actively hydrolyzes EP, had different effects on heart (SERCA2a) and fast twitch skeletal muscle SR Ca2+-ATPase (SERCA1). With physiological acylphosphatase concentrations SERCA2a exhibited a parallel increase in the rates of both ATP hydrolysis and Ca2+ transport; in contrast, SERCA1 appeared to be uncoupled since the stimulation of ATP hydrolysis matched an inhibition of Ca2+ pump. These different effects probably depend on phospholamban, which is associated with SERCA2a but not SERCA1. Consistent with this view, the present study suggests that acylphosphatase-induced stimulation of SERCA2a, in addition to an enhanced EP hydrolysis, may be due to a displacement of phospholamban, thus to a removal of its inhibitory effect.  (+info)

Expression of skeletal muscle sarcoplasmic reticulum calcium-ATPase is reduced in rats with postinfarction heart failure. (2/4498)

OBJECTIVE: To determine whether heart failure in rats is associated with altered expression of the skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA). METHODS: SERCA protein and mRNA were examined in the soleus muscles of eight female rats with heart failure induced by coronary artery ligation, six weeks after the procedure (mean (SEM) left ventricular end diastolic pressure 20.4 (2.2) mm Hg) and in six sham operated controls by western and northern analyses, respectively. RESULTS: SERCA-2a isoform protein was reduced by 16% (112 000 (4000) v 134 000 (2000) arbitrary units, p < 0.001), and SERCA-2a messenger RNA was reduced by 59% (0.24 (0. 06) v 0.58 (0.02) arbitrary units, p < 0.001). Although rats with heart failure had smaller muscles (0.54 mg/g v 0.66 mg/g body weight), no difference in locomotor activity was observed. CONCLUSIONS: These results may explain the previously documented abnormalities in calcium handling in skeletal muscle from animals with the same model of congestive heart failure, and could be responsible for the accelerated muscle fatigue characteristic of patients with heart failure.  (+info)

Ca-releasing action of beta, gamma-methylene adenosine triphosphate on fragmented sarcoplasmic reticulum. (3/4498)

beta,gamma-Methylene adenosine triphosphate (AMPOPCP) has two effects on fragmented sarcoplasmic reticulum (FSR), i.e., inhibition of the rate of Ca uptake and the induction of Ca release from FSR filled with Ca. The Ca release brought about by AMPOPCP has many features in common with the mechanism of Ca-induced Ca release: i) it is inhibited by 10 mM procaine; ii) the amount of Ca release increases with increase in the extent of saturation of FSR with Ca; iii) increase of the Ca concentration in the extent of saturation of FSR with Ca; iii) increase of the Ca concentration in the medium facilitates the release of Ca. However, no facilitation of Ca release upon decrease of Mg concentration in the medium is observable. AMPOPCP and caffeine potentiate each other remarkably in their Ca-releasing action, irrespective of the kind of substrate. From the mode of action of AMPOPCP on the rate of Ca uptake, the amount of phosphorylated intermediate (EP), and the effect on Sr release, it is suggested that the state of the FSR-ATP complex is crucial for Ca-induced Ca release.  (+info)

Mutations of Arg198 in sarcoplasmic reticulum Ca2+-ATPase cause inhibition of hydrolysis of the phosphoenzyme intermediate formed from inorganic phosphate. (4/4498)

Arg198 of sarcoplasmic reticulum Ca2+-ATPase was substituted with lysine, glutamine, glutamic acid, alanine, and isoleucine by site-directed mutagenesis. Kinetic analysis was performed with microsomal membranes isolated from COS-1 cells which were transfected with the mutated cDNAs. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was determined by first phosphorylating the Ca2+-ATPase with 32Pi and then diluting the sample with non-radioactive Pi. This rate was reduced substantially in the mutant R198Q, more strongly in the mutants R198A and R1981, and most strongly in the mutant R198E, but to a much lesser extent in R198K. The reduction in the rate of dephosphorylation was consistent with the observed decrease in the turnover rate of the Ca2+-ATPase accompanied by the steady-state accumulation of the ADP-insensitive phosphoenzyme formed from ATP. These results indicate that the positive charge and high hydrophilicity of Arg198 are critical for rapid hydrolysis of the ADP-insensitive phosphoenzyme.  (+info)

A repetitive mode of activation of discrete Ca2+ release events (Ca2+ sparks) in frog skeletal muscle fibres. (5/4498)

1. Ca2+ release events (Ca2+ 'sparks'), which are believed to arise from the opening of a sarcoplasmic reticulum (SR) Ca2+ release channel or a small cluster of such channels that act as a release unit, have been measured in single, frog (Rana pipiens) skeletal muscle fibres. 2. Under conditions of extremely low rates of occurrence of Ca2+ sparks we observed, within individual identified triads, repetitive Ca2+ release events which occurred at a frequency more than 100-fold greater than the prevailing average event rate. Repetitive sparks were recorded during voltage-clamp test depolarizations after a brief (0.3-2 s) repriming interval in fibres held at 0 mV and in chronically depolarized, 'notched' fibres. 3. These repetitive events are likely to arise from the re-opening of the same SR Ca2+ release channel or release unit operating in a repetitive gating mode ('rep-mode'), rather than from the random activation of multiple, independent channels or release units within a triad. A train of rep-mode events thus represents a series of Ca2+ sparks arising from a single location within the fibre. Rep-mode events are activated among different triads in a random manner after brief repriming. The frequency of repetitive events among all identified events during voltage-clamp depolarization to 0 mV after brief repriming was 3.9 +/- 1.3 %. The occurrence of repetitive events was not related to exposure of the fibre to laser illumination. 4. The events observed within a rep-mode train exhibited a relatively uniform amplitude. Analysis of intervals between identified events in triads exhibiting rep-mode trains indicated similar variations of fluorescence as in neighbouring, quiescent triads, suggesting there was not a significant number of small, unidentified events at the triads exhibiting rep-mode activity. 5. The distribution of rep-mode interspark intervals exhibited a paucity of events at short intervals, consistent with the need for recovery from inactivation before activation of the next event in a repetitive train. The mean interspark interval of repetitive sparks during voltage-clamp depolarizations was 88 +/- 5 ms, and was independent of membrane potential. 6. The individual Ca2+ sparks within a rep-mode train were similar in average amplitude and spatiotemporal extent to singly occurring sparks, suggesting a common mechanism for termination of the channel opening(s) underlying both types of events. The average properties of the sparks did not vary during a train. The relative amplitude of a spark within a rep-mode was not correlated with its rise time. 7. Repetitive Ca2+ release events represent a mode of gating of SR Ca2+ release channels which may be significant during long depolarizations and which may be influenced by the biochemical state of the SR ryanodine receptor Ca2+ release channels.  (+info)

Local control models of cardiac excitation-contraction coupling. A possible role for allosteric interactions between ryanodine receptors. (6/4498)

In cardiac muscle, release of activator calcium from the sarcoplasmic reticulum occurs by calcium- induced calcium release through ryanodine receptors (RyRs), which are clustered in a dense, regular, two-dimensional lattice array at the diad junction. We simulated numerically the stochastic dynamics of RyRs and L-type sarcolemmal calcium channels interacting via calcium nano-domains in the junctional cleft. Four putative RyR gating schemes based on single-channel measurements in lipid bilayers all failed to give stable excitation-contraction coupling, due either to insufficiently strong inactivation to terminate locally regenerative calcium-induced calcium release or insufficient cooperativity to discriminate against RyR activation by background calcium. If the ryanodine receptor was represented, instead, by a phenomenological four-state gating scheme, with channel opening resulting from simultaneous binding of two Ca2+ ions, and either calcium-dependent or activation-linked inactivation, the simulations gave a good semiquantitative accounting for the macroscopic features of excitation-contraction coupling. It was possible to restore stability to a model based on a bilayer-derived gating scheme, by introducing allosteric interactions between nearest-neighbor RyRs so as to stabilize the inactivated state and produce cooperativity among calcium binding sites on different RyRs. Such allosteric coupling between RyRs may be a function of the foot process and lattice array, explaining their conservation during evolution.  (+info)

Cellular mechanisms of altered contractility in the hypertrophied heart: big hearts, big sparks. (7/4498)

To investigate the cellular mechanisms for altered Ca2+ homeostasis and contractility in cardiac hypertrophy, we measured whole-cell L-type Ca2+ currents (ICa,L), whole-cell Ca2+ transients ([Ca2+]i), and Ca2+ sparks in ventricular cells from 6-month-old spontaneously hypertensive rats (SHRs) and from age- and sex-matched Wistar-Kyoto and Sprague-Dawley control rats. By echocardiography, SHR hearts had cardiac hypertrophy and enhanced contractility (increased fractional shortening) and no signs of heart failure. SHR cells had a voltage-dependent increase in peak [Ca2+]i amplitude (at 0 mV, 1330+/-62 nmol/L [SHRs] versus 836+/-48 nmol/L [controls], P<0.05) that was not associated with changes in ICa,L density or kinetics, resting [Ca2+]i, or Ca2+ content of the sarcoplasmic reticulum (SR). SHR cells had increased time of relaxation. Ca2+ sparks from SHR cells had larger average amplitudes (173+/-192 nmol/L [SHRs] versus 109+/-64 nmol/L [control]; P<0.05), which was due to redistribution of Ca2+ sparks to a larger amplitude population. This change in Ca2+ spark amplitude distribution was not associated with any change in the density of ryanodine receptors, calsequestrin, junctin, triadin 1, Ca2+-ATPase, or phospholamban. Therefore, SHRs with cardiac hypertrophy have increased contractility, [Ca2+]i amplitude, time to relaxation, and average Ca2+ spark amplitude ("big sparks"). Importantly, big sparks occurred without alteration in the trigger for SR Ca2+ release (ICa,L), SR Ca2+ content, or the expression of several SR Ca2+-cycling proteins. Thus, cardiac hypertrophy in SHRs is linked with an alteration in the coupling of Ca2+ entry through L-type Ca2+ channels and the release of Ca2+ from the SR, leading to big sparks and enhanced contractility. Alterations in the microdomain between L-type Ca2+ channels and SR Ca2+ release channels may underlie the changes in Ca2+ homeostasis observed in cardiac hypertrophy. Modulation of SR Ca2+ release may provide a new therapeutic strategy for cardiac hypertrophy and for its progression to heart failure and sudden death.  (+info)

The sarcoplasmic reticulum and the Na+/Ca2+ exchanger both contribute to the Ca2+ transient of failing human ventricular myocytes. (8/4498)

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.  (+info)