Activity Of Sarcoplasmic Reticulum Ca2 Research Articles

Mutations in proteins involved in E-C coupling and SOCE and congenital myopathies.

In skeletal muscle, Ca2+ necessary for muscle contraction is stored and released from the sarcoplasmic reticulum (SR), a specialized form of endoplasmic reticulum through the mechanism known as excitation-contraction (E-C) coupling. Following activation of skeletal muscle contraction by the E-C coupling mechanism, replenishment of intracellular stores requires reuptake of cytosolic Ca2+ into the SR by the activity of SR Ca2+-ATPases, but also Ca2+ entry from the extracellular space, through a mechanism called store-operated calcium entry (SOCE). The fine orchestration of these processes requires several proteins, including Ca2+ channels, Ca2+ sensors, and Ca2+ buffers, as well as the active involvement of mitochondria. Mutations in genes coding for proteins participating in E-C coupling and SOCE are causative of several myopathies characterized by a wide spectrum of clinical phenotypes, a variety of histological features, and alterations in intracellular Ca2+ balance. This review summarizes current knowledge on these myopathies and discusses available knowledge on the pathogenic mechanisms of disease.

B-type natriuretic peptide and its role in altering Ca2+-regulatory proteins in heart failure-mechanistic insights.

Heart failure (HF) is a worldwide disease with high levels of morbidity and mortality. The pathogenesis of HF is complicated and involves imbalances in hormone and electrolyte. B-type natriuretic peptide (BNP) has served as a biomarker of HF severity, and in recent years, it has been used to treat the disease, thanks to its cardio-protective effects, such as diuresis, natriuresis, and vasodilatation. In stage C/D HF, symptoms are severe despite elevated BNP. Disturbances in Ca2+ homeostasis are often a dominating feature of the disease, causing Ca2+-regulatory protein dysfunction, including reduced expression and activity of sarcoplasmic reticulum Ca2+-ATPase2a (SERCA2a), impaired ryanodine receptors (RYRs) function, intensive Na+-Ca2+ exchanger (NCX), and downregulation of S100A1. The relationship between natriuretic peptides (NPs) and Ca2+-regulatory proteins has been widely studied and represents important mechanisms in the etiology of HF. In this review, we present evidence that BNP may regulate Ca2+-regulatory proteins, in particular, suppressing SERCA2a and S100A1 expression. However, relationships between BNP and other Ca2+-regulatory proteins remain vague.

Schekwanglupaside C, a new lupane saponin from Schefflera kwangsiensis, is a potent activator of sarcoplasmic reticulum Ca2+-ATPase

Schefflera kwangsiensis Merr. ex H.L. Li (Araliaceae) is a widely used traditional Chinese medicine for pain management in the clinic. In the present study, we isolated a previously undescribed lupane saponin, designated as schekwanglupaside C (Sch C) from the ethanolic extract of S. kwangsiensis. The structure of Sch C was determined by comprehensive spectroscopic and spectrometric analyses and chemical degradation. In primary cultured cortical neurons, Sch C altered the pattern of spontaneous Ca2+ oscillation (SCO) with a slight increase in the frequency of SCO right after addition and a gradual decrease in the frequency and amplitude of SCO, that dynamic change mimicked by an activator of sarcoplasmic reticulum Ca2+-ATPase (SERCA). The IC50 values for Sch C suppression of the frequency and amplitude of SCO were 1.75 and 2.51 μM, respectively. Furthermore, we demonstrated that Sch C is a potent SERCA activator (EC50 = 1.20 μM). Given the pivotal role of SERCA in the progression of neuropathic pain and neurodegenerative diseases, Sch C represents a new drug lead compound to develop the treatment of neuropathic pain and Alzheimer's disease.

Nitroxyl (HNO) targets phospholamban cysteines 41 and 46 to enhance cardiac function.

Nitroxyl (HNO) positively modulates myocardial function by accelerating Ca2+ reuptake into the sarcoplasmic reticulum (SR). HNO-induced enhancement of myocardial Ca2+ cycling and function is due to the modification of cysteines in the transmembrane domain of phospholamban (PLN), which results in activation of SR Ca2+-ATPase (SERCA2a) by functionally uncoupling PLN from SERCA2a. However, which cysteines are modified by HNO, and whether HNO induces reversible disulfides or single cysteine sulfinamides (RS(O)NH2) that are less easily reversed by reductants, remain to be determined. Using an 15N-edited NMR method for sulfinamide detection, we first demonstrate that Cys46 and Cys41 are the main targets of HNO reactivity with PLN. Supporting this conclusion, mutation of PLN cysteines 46 and 41 to alanine reduces the HNO-induced enhancement of SERCA2a activity. Treatment of WT-PLN with HNO leads to sulfinamide formation when the HNO donor is in excess, whereas disulfide formation is expected to dominate when the HNO/thiol stoichiometry approaches a 1:1 ratio that is more similar to that anticipated in vivo under normal, physiological conditions. Thus, 15N-edited NMR spectroscopy detects redox changes on thiols that are unique to HNO, greatly advancing the ability to detect HNO footprints in biological systems, while further differentiating HNO-induced post-translational modifications from those imparted by other reactive nitrogen or oxygen species. The present study confirms the potential of HNO as a signaling molecule in the cardiovascular system.

Action of Iron Nitrosyl Complexes, NO Donors, on the Activity of Sarcoplasmic Reticulum Ca2+-ATPase and Cyclic Guanosine Monophosphate Phosphodiesterase.

The effect of iron nitrosyl complexes, NO donors, of a general formula [Fe2(L)2(NO)4] with functional sulfur-containing ligands (L-3-nitro-phenol-2-yl, 4-nitro-phenol-2-yl, or 1-methyl-tetrazol-5-yl) on the activity of sarcoplasmic reticulum Ca2+-ATPase and cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) was studied. The test complexes uncoupled the hydrolytic and transport functions of Ca2+- ATPase, thus disturbing the balance of Ca2+ ions in cells, which may affect the formation of thrombi and adhesion of metastatic cells to the endothelium of capillaries. They also inhibited the activity of cGMP PDE, thereby contributing to the accumulation of the second messenger cGMP. The studied iron nitrosyl complexes can be considered as potential drugs.

Pycnogenol Cytotoxicity in Pancreatic INS-1E β cells Induced by Calcium Dysregulation.

Natural standardized flavonoid extract from the bark of Pinus pinaster, Pycnogenol (Pyc), was recently found to decrease intensively the activity of sarcoplasmic reticulum Ca2+ -ATPase of rabbit skeletal muscle (SERCA1). On the basis of this inhibitory effect in a cell-free system and similarities of SERCA1 to its other isoforms, proapoptotic properties of Pyc may be expected in cellular systems. Pycnogenol (40-100μg/mL) induced a concentration-dependent decrease of the viability of pancreatic INS-1E β cells associated with induction of apoptosis. In addition, intracellular Ca2+ level increase was found along with reduction of protein expression level of SERCA2b and impairment of insulin secretion by β cells. These facts indicate that Pyc may induce apoptosis by impairment of calcium homeostasis. Copyright © 2017 John Wiley & Sons, Ltd.

Luteolin Enhances Sarcoplasmic Reticulum Ca2+-ATPase Activity through p38 MAPK Signaling thus Improving Rat Cardiac Function after Ischemia/Reperfusion

Background/Aims: A major challenge for current therapeutic strategies against ischemia/reperfusion (I/R) is the lack of effective drugs. Considering luteolin enhances the activity of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) to improve the systolic/diastolic function of rat hearts and cardiomyocytes during the I/R process, we studied the regulatory function of the p38 MAPK pathway in this protective mechanism. Methods: Isolated cardiomyocytes and perfused hearts were separately divided into five groups and used to investigate I/R. The phosphorylation of p38 and phospholamban (p-PLB), the levels and activity of SERCA2a and the levels of proteins related to apoptosis were measured. Apoptotic cells were assessed using the TUNEL assay. Single-cell shortening, Ca²⁺ transients, and the decay of the mitochondrial membrane potential (Δψm) were detected. Results: The p38 MAPK pathway was activated during the I/R process, and inhibiting it with SB203580 promoted p-PLB, which enhanced the activity of SERCA2a and relieved the calcium overload to promote the recovery of the Δψm and reduce cardiomyocyte apoptosis in I/R. Luteolin also suppressed the activation of the p38 MAPK pathway and showed cardioprotective effects during I/R injury. Conclusions: We conclude that luteolin enhances SERCA2a activity to improve systolic/diastolic function during I/R in rat hearts and cardiomyocytes by attenuating the inhibitive effects of the p38 pathway on p-PLB.

An Emerging Strategy of Gene Therapy for Cardiac Disease

Heart disease is the major cause of morbidity and mortality worldwide.1,2 The current therapeutic approaches for heart failure are limited because postnatal cardiomyocytes have little regenerative capacity. Therefore, a new strategy needs to be established to improve the cardiac function. Article, see p 1147 Gene therapy is one of the most attractive new therapeutic strategies. Several kinds of cardiac gene therapy have so far been reported. Jeffrey M. Isner’s group reported that the administration of plasmid vectors encoding cDNA of the 165-amino acid isoform of vascular endothelial growth factor induced angiogenesis in patients with ischemic heart disease and with ischemic limbs.3,4 The effect of vascular endothelial growth factor gene therapy is mediated by the paracrine of cytokines (Figure, A). Figure. Schematic presentation of 3 types of gene therapies. Another type of gene therapy targets cardiomyocytes. The expression and activity of sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) have been observed to decrease in cardiomyocytes in a failing heart, and the overexpression of SERCA2a could restore the cardiac function in heart failure by improving calcium handling in the cardiomyocytes (Figure, B). Clinical trials for SERCA2A gene therapy were conducted for patients with heart failure, and positive results have been reported.5,6 A new strategy for gene therapy …

Cardiac resynchronization therapy (CRT): Clinical trials, guidelines, and target populations

Over the last 10 years, several large, well-designed clinical trials have firmly established the role of cardiac resynchronization therapy (CRT) as a recommended treatment strategy for moderate-to-severe heart failure (HF). A review of the relevant results from the MUSTIC, MIRACLE, CONAK-CD, and MIRACLE ICD trials reveals that in patients with New York Heart Association (NYHA) class III-IV HF, CRT produces consistent improvements in quality of life, functional status, and exercise capacity while also providing strong evidence for reverse remodeling and diminished functional mitral regurgitation, resulting in reductions in both HF hospitalizations and all-cause morbidity and mortality. In patients with earlier NYHA class I-II HF, the benefit of CRT has been more controversial. The principal ongoing challenges addressed in this article include the substantial 30% of patients who receive a CRT device but fail to respond, the wide variations in how to define "response" vs "nonresponse," and how to identify patients who will benefit from CRT, especially narrow QRS (<120 ms), those with right bundle branch block, and those with mild-to-moderate (NYHA class I-II) HF. An important result of this uncertainty is the lack of a good sense of the optimal rate of CRT implantation, making consideration of the data reviewed in this article crucial for identifying important gaps of knowledge and mechanisms of action that need to be studied in the near future.

Waveless mammalian muscle

Waveless mammalian muscle

Abnormal Termination of Ca 2+ Release Is a Common Defect of RyR2 Mutations Associated With Cardiomyopathies

Naturally occurring mutations in the cardiac ryanodine receptor (RyR2) have been associated with both cardiac arrhythmias and cardiomyopathies. It is clear that delayed afterdepolarization resulting from abnormal activation of sarcoplasmic reticulum Ca2+ release is the primary cause of RyR2-associated cardiac arrhythmias. However, the mechanism underlying RyR2-associated cardiomyopathies is completely unknown. In the present study, we investigate the role of the NH2-terminal region of RyR2 in and the impact of a number of cardiomyopathy-associated RyR2 mutations on the termination of Ca2+ release. The 35-residue exon-3 region of RyR2 is associated with dilated cardiomyopathy. Single-cell luminal Ca2+ imaging revealed that the deletion of the first 305 NH2-terminal residues encompassing exon-3 or the deletion of exon-3 itself markedly reduced the luminal Ca2+ threshold at which Ca2+ release terminates and increased the fractional Ca2+ release. Single-cell cytosolic Ca2+ imaging also showed that both RyR2 deletions enhanced the amplitude of store overload-induced Ca2+ transients in HEK293 cells or HL-1 cardiac cells. Furthermore, the RyR2 NH2-terminal mutations, A77V, R176Q/T2504M, R420W, and L433P, which are associated with arrhythmogenic right ventricular displasia type 2, also reduced the threshold for Ca2+ release termination and increased fractional release. The RyR2 A1107M mutation associated with hypertrophic cardiomyopathy had the opposite action (i.e., increased the threshold for Ca2+ release termination and reduced fractional release). These results provide the first evidence that the NH2-terminal region of RyR2 is an important determinant of Ca2+ release termination, and that abnormal fractional Ca2+ release attributable to aberrant termination of Ca2+ release is a common defect in RyR2-associated cardiomyopathies.

Study of dilated cardiomyopathy and phospholamban

It is discovered that dilated cardiomyopathy (DCM) is not only affected by environmental factors, but also has a significant genetic basis. Phospholamban ( PLN ), one of these related genes, is a prominent regulator of myocardial contractility, It is an important regulatory factor for heart muscle contraction. It can regulate the activity of sarcoplasmic reticulum Ca2+ ATPase ( SERCA2a), and inhibit SERCA2a activity through restricting the Ca2+ flow in myocardial cells. The activity of SERCA2a can be improved by lowering the expression of PLNmRNA, and the affinity of SERCA2a is strengthened, so that the heart's activity is improved. Key words: Dilated cardiomyopathy; Phospholamban; Ca2 + ATPase; Cardiac resynchronization therapy

Taxol, a microtubule stabilizer, prevents ischemic ventricular arrhythmias in rats.

Microtubule integrity is important in cardio-protection, and microtubule disruption has been implicated in the response to ischemia in cardiac myocytes. However, the effects of Taxol, a common microtubule stabilizer, are still unknown in ischemic ventricular arrhythmias. The arrhythmia model was established in isolated rat hearts by regional ischemia, and myocardial infarction model by ischemia/reperfusion. Microtubule structure was immunohistochemically measured. The potential mechanisms were studied by measuring reactive oxygen species (ROS), activities of oxidative enzymes, intracellular calcium concentration ([Ca2+]i) and Ca2+ transients by using fluorometric determination, spectrophotometric assays and Fura-2-AM and Fluo-3-AM, respectively. The expression and activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) was also examined using real-time polymerase chain reaction, Western blot and pyruvate/Nicotinamide adenine dinucleotide-coupled reaction. Our data showed that Taxol (0.1, 0.3 and 1 μM) effectively reduced the number of ventricular premature beats and the incidence and duration of ventricular tachycardia. The infarct size was also significantly reduced by Taxol (1 μM). At the same time, Taxol preserved the microtubule structure, increased the activity of mitochondrial electron transport chain complexes I and III, reduced ROS levels, decreased the rise in [Ca2+]i and preserved the amplitude and decay times of Ca2+ transients during ischemia. In addition, SERCA2a activity was preserved by Taxol during ischemia. In summary, Taxol prevents ischemic ventricular arrhythmias likely through ameliorating abnormal calcium homeostasis and decreasing the level of ROS. This study presents evidence that Taxol may be a potential novel therapy for ischemic ventricular arrhythmias.

Structure-Based Design of Phospholamban Mutants for Gene Therapy

We are using structural biology, molecular biology, physiology, gene delivery and preclinical testing to develop an optimized gene-therapy approach for the treatment of heart failure (HF). A key abnormality in HF is defective function of sarcoplasmic reticulum (SR). Deficient Ca2+ uptake during relaxation, associated with a decrease in activity of SR Ca2+-ATPase (SERCA2a), has been identified in failing hearts from both humans and animal models. SERCA activity is regulated by phospholamban (PLB), a small membrane protein that partially inhibits SERCA in the absence of β-adrenergic phosphorylation of PLB. Our approach combining structure-function analysis of PLB and the direct detection of PLB-SERCA interactions relies on (a) fluorescence to show that SERCA inhibition can be relieved without dissociation of PLB, (b) magnetic resonance to define the structural dynamics of PLB's functional interaction with SERCA, and (c) establishing structure-based computational design principles to produce PLB mutants (PLBM) binding tightly to SERCA but having decreased inhibitory potency. We have obtained evidence that non-inhibitory PLB mutants S16E or L31A are capable of competing with WT-PLB to reverse SERCA inhibition. We are currently testing the physical and functional interaction between SERCA and optimized PLBM. Based on positive in vitro results, rAAV is used to test PLBM in rodent and porcine HF models for in vivo efficacy.

Age-related changes in contraction and relaxation of rat diaphragm

Age-related changes of physiological and biochemical properties were examined in the diaphragm muscle, which has particularly high activation compared to that of other skeletal muscles. The diaphragm from 10-week-, 50-week- and 100-week-old male Wistar rats were used to measure in vitro isometric contractile properties, sarcoplasmic reticulum (SR) Ca2+-ATPase activity, and myosin heavy chain (MHC) isoform composition. Although there were no significant differences in specific twitch tension of the diaphragm among the groups, there was significant reduction in specific tetanic tension in the 50-week to 100-week groups. The contraction time and 1/2 relaxation time of twitch contraction extended with aging, and significant differences were found between 10-week-old and 100-week-old diaphragms. Regarding the activity of SR Ca2+-ATPase, the pattern of age-related change was similar to that in the 1/2 relaxation time and there was a significant difference between 10-week-old and 100-week-old diaphragms. There was a significant increase in the relative composition of the MHC I isoform in 100-week-diaphragms compared to that in 10-week-old diaphragms and a concomitant decrease in the relative composition of fast myosin was noted. These findings demonstrated that older diaphragms have slower contraction and relaxation speeds, and these alterations were attributed to changes in SR Ca2+-ATPase activity and MHC isoform composition.

Photon radiation-induced structural and functional changes in the myocardium of hypertensive spontaneously hypertensive rats

Hypertensive SHR rates were irradiated with orange-red light using a Korobkov photon light-emitting diode matrix with a maximum radiation at 612 nm; irradiation was performed daily for 1 h for 13 days. After the course of irradiation, the rhythmoinothropic characteristics of the cardiac papillary muscle significantly improved. Morphological analysis revealed active rearrangement in myocytes, which were observed primarily in the structure of the sarcoplasmic reticulum (SR), whose relative area increased more than twice compared to the control. Apparently, photon therapy of hypertensive rats normalizes calcium homeostasis in myocytes and improves the calcium-transport function of SR. The normalization of structural and functional characteristics of the myocardium with hypertensive rates may result from an increase in the SR buffer capacity and activation of SR Ca2+-ATPase. Furthermore, qualitative and quantitative changes in the proportion of capillaries, myofibrils, and mitochondria in myocytes indicate the development of adaptive-compensatory processes leading to the activation of biosynthetic processes and an increase in the energy potential of the myocardium.

Cold Tolerance in Hypothyroid Rabbits: Role of Skeletal Muscle Mitochondria and Sarcoplasmic Reticulum Ca2+ ATPase Isoform 1 Heat Production

Brown adipose tissue (BAT) is involved in rat and mice thermoregulation, and heat produced by BAT depends on the concerted action of thyroid hormones and catecholamines. Little is known about cold-induced thermogenesis in mammals that have little or no BAT, such as rabbits. In these animals, thermogenesis primarily occurs in skeletal muscle. In this work, we have studied the effect of cold acclimation (4 C for 10 d) in normal and hypothyroid rabbits. It is known that hypothyroid rats die after a few hours of cold exposure. We now show that, different from rats, hypothyroid rabbits sustain their body temperature and survive after 10 d cold exposure. When compared with rabbits kept at room temperature, the muscles of cold-exposed rabbits showed a dark red color characteristic of oxidative muscle fibers. According to this pattern, we observed that in both normal and hypothyroid rabbits, cold exposure promotes an increase in oxygen consumption by skeletal muscle mitochondria. Moreover, in red muscle, cold acclimation induces an increase in the expression and activity of sarcoplasmic reticulum Ca(2+) ATPase isoform 1 (SERCA1), one of the muscle enzymes involved in heat production. We conclude that rabbit cold tolerance is probably related to increased muscle oxidative metabolism and heat production by SERCA1 and that these changes are not completely dependent on normal thyroid function.

L-type Ca2+ channels in the heart: Structure and regulation

This review analyzes the structure and regulation mechanisms of voltage-dependent L-type Ca(2+) channel in the heart. L-type Ca(2+) channels in the heart are composed of four different polypeptide subunits, and the pore-forming subunit alpha(1) is the most important part of the channel. In cardiac myocytes, Ca(2+) enter cell cytoplasm from extracellular space mainly through L-type Ca(2+) channels; these channels are very important system in heart Ca(2+) uptake regulation. L-type Ca(2+) channels are responsible for the activation of sarcoplasmic reticulum channels (RyR2) and force of muscle contraction generation in heart; hence, activity of the heart depends on L-type Ca(2+) channels. Phosphorylation of channel-forming subunits by different kinases is one of the most important ways to change the activity of L-type Ca(2+) channel. Additionally, the activity of L-type Ca(2+) channels depends on Ca(2+) concentration in cytoplasm. Ca(2+) current in cardiac cells can facilitate, and this process is regulated by phosphorylation of L-type Ca(2+) channels and intracellular Ca(2+) concentration. Disturbances in cellular Ca(2+) transport and regulation of L-type Ca(2+) channels are directly related to heart diseases, life quality, and life span.

S100A1 Binds to the Calmodulin-binding Site of Ryanodine Receptor and Modulates Skeletal Muscle Excitation-Contraction Coupling

S100A1, a 21-kDa dimeric Ca2+-binding protein, is an enhancer of cardiac Ca2+ release and contractility and a potential therapeutic agent for the treatment of cardiomyopathy. The role of S100A1 in skeletal muscle has been less well defined. Additionally, the precise molecular mechanism underlying S100A1 modulation of sarcoplasmic reticulum Ca2+ release in striated muscle has not been fully elucidated. Here, utilizing a genetic approach to knock out S100A1, we demonstrate a direct physiological role of S100A1 in excitation-contraction coupling in skeletal muscle. We show that the absence of S100A1 leads to decreased global myoplasmic Ca2+ transients following electrical excitation. Using high speed confocal microscopy, we demonstrate with high temporal resolution depressed activation of sarcoplasmic reticulum Ca2+ release in S100A1-/- muscle fibers. Through competition assays with sarcoplasmic reticulum vesicles and through tryptophan fluorescence experiments, we also identify a novel S100A1-binding site on the cytoplasmic face of the intact ryanodine receptor that is conserved throughout striated muscle and corresponds to a previously identified calmodulin-binding site. Using a 12-mer peptide of this putative binding domain, we demonstrate low micromolar binding affinity to S100A1. NMR spectroscopy reveals this peptide binds within the Ca2+-dependent hydrophobic pocket of S100A1. Taken together, these data suggest that S100A1 plays a significant role in skeletal muscle excitation-contraction coupling, primarily through specific interactions with a conserved binding domain of the ryanodine receptor. This warrants further investigation into the use of S100A1 as a therapeutic target for the treatment of both cardiac and skeletal myopathies.

Sarcoplasmic reticulum Ca2+ release channel complex and automatism: A matter of fine tuning

See article by Dirksen et al. [10] (pages 69–78) and Faber and Rudy [15] (pages 79–88) in this issue. In heart muscle, excitation contraction coupling (ECC) relies on proper release of Ca2+ from the sarcoplasmic reticulum (SR), which is accomplished by the activation of SR Ca2+ release channels (also known as ryanodine receptors, RyR) upon Ca2+ binding, a process termed calcium-induced calcium release [1]. The spontaneous activation of a cluster of RyRs produces local Ca2+ release seen under confocal fluorescence microscopy as minuscule flashes of light called Ca2+ sparks, the elementary Ca2+ release events [2]. SR release units are never totally quiescent. They keep on releasing Ca2+ randomly even during diastole, when sparks are rare in time and spatially separable [3]. During ECC, thousands of intracellular release events are synchronized by Ca2+ influx during the action potential, which causes both spatial and temporal overlap of elementary events in a way to produce a global intracellular transient increase in Ca2+ concentration ([Ca2+] i ) that initiates cell contraction. For relaxation to occur, [Ca2+] i must be reduced to the resting levels. This is accomplished by a number of transport systems that compete for Ca2+, mainly the SR Ca2+-ATPase and the Na+/Ca2+exchanger (NCX) [3]. The amount of Ca2+ released is strongly regulated by the SR Ca2+ content, both during systole [4] and diastole [5], so that release ceases at critically low SR loads, a condition in which a significant amount of Ca2+ (more than 40% of maximum load) can still be found in the organelle [4]. SR Ca2+ content, in turn, is regulated under normal physiological conditions by … *Centro de Engenharia Biomedica/UNICAMP, Caixa Postal 6040, 13084-971 Campinas, SP, Brazil. Tel.: +55 19 3521 9287; fax: +55 19 3289 3346. bassani{at}ceb.unicamp.br