Nuclear Ca2+ regulates cardiomyocyte function.

In the heart, cytosolic Ca(2+) signals are well-characterized events that participate in the activation of cell contraction. In contrast, nuclear Ca(2+) contribution to cardiomyocyte function remains elusive. Here, we examined functional consequences of buffering nuclear Ca(2+) in neonatal cardiomyocytes. We report that cardiomyocytes contain a nucleoplasmic reticulum, which expresses both ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (InsP(3)R), providing a possible way for active regulation of nuclear Ca(2+). Adenovirus constructs encoding the Ca(2+) buffer protein parvalbumin were targeted to the nucleus with a nuclear localization signal (Ad-PV-NLS) or to the cytoplasm with a nuclear exclusion signal (Ad-PV-NES). A decrease in the amplitude of global Ca(2+) transients and RyR-II expression, as well as an increase in cell beating rate were observed in Ad-PV-NES and Ad-PV-NLS cells. When nuclear Ca(2+) buffering was imposed nuclear enlargement, increased calcineurin expression, NFAT translocation to the nucleus and subcellular redistribution of atrial natriuretic peptide were observed. Furthermore, prolongation of action potential duration occurred in adult ventricular myocytes. These results suggest that nuclear Ca(2+) levels underlie the regulation of specific protein targets and thereby modulate cardiomyocyte function. The local nuclear Ca(2+) signaling and the structures that control it constitute a novel regulatory motif in the heart.     

Tight interplay between the Ca2+ affinity of the cardiac SERCA2 Ca2+ pump and the SERCA2 expression level

A reduced activity of the sarcoplasmic reticulum Ca2+ pump SERCA2a is a hallmark of cardiac dysfunction in heart failure. In SERCA2b/b mice, the normal SERCA2a isoform is replaced by SERCA2b, displaying a higher Ca2+ affinity. This elicited decreased cardiac SERCA2 expression and cardiac hypertrophy. Here, the interplay was studied between the increased Ca2+ affinity and a reduced expression of the pump and its role in the cardiac remodeling was investigated. First, SERCA2b/b mice were crossed with SERCA2b transgenes to boost cardiac SERCA2b expression. However, the enforced expression of SERCA2b was spontaneously countered by an increased inhibition by phospholamban (PLB), reducing the pump's Ca2+ affinity. Moreover, the higher SERCA2 content did not prevent hypertrophy. Second, we studied heterozygous SERCA2b/WT mice, which also express lower SERCA2 levels compared to wild-type. Hypertrophy was not observed. In heterozygotes, SERCA2b expression was specifically suppressed, explaining the reduced SERCA2 content. The SERCA2b/WT model strikingly differs from the homozygote models because SERCA2a (not SERCA2b) is the major isoform and because the inhibition of the pump by PLB is decreased instead of being increased. Thus, a tight correlation exists between the SERCA2 levels and Ca2+ affinity (controlled by PLB). This compensatory response may be important to prevent cardiac remodeling.     

Regulation of calpain activity in rat brain with altered Ca2+ homeostasis

Activation of calpain occurs as an early event in correlation with an increase in [Ca2+]i induced in rat brain upon treatment with a high salt diet for a prolonged period of time. The resulting sequential events have been monitored in the brain of normal and hypertensive rats of the Milan strain, diverging for a constitutive alteration in the level of [Ca2+]i found to be present in nerve cells of hypertensive animals. After 2 weeks of treatment, the levels of the plasma membrane Ca2+-ATPase and of native calpastatin are profoundly decreased. These degradative processes, more pronounced in the brain of hypertensive rats, are progressively and efficiently compensated in the brain of both rat strains by different incoming mechanisms. Along with calpastatin degradation, 15-kDa still-active inhibitory fragments are accumulated, capable of efficiently replacing the loss of native inhibitor molecules. A partial return to a more efficient control of Ca2+ homeostasis occurs in parallel, assured by an early increase in the expression of Ca2+-ATPase and of calpastatin, both producing, after 12 weeks of a high salt (sodium) diet, the restoration of almost original levels of the Ca2+ pump and of significant amounts of native inhibitor molecules. Thus, conservative calpastatin fragmentation, associated with an increased expression of Ca2+-ATPase and of the calpain natural inhibitor, has been demonstrated to occur in vivo in rat brain. This represents a sequential adaptive response capable of overcoming the effects of calpain activation induced by a moderate long term elevation of [Ca2+]i.     

Altered Ca2+ homeostasis and impaired mitochondrial function in cardiomyopathy

This work investigates whether purine metabolism and release is related to cardioprotection with hyperkalemia and hypothermia. Langendorff guinea-pig hearts were used to either monitor metabolism during ischemia or to measure functional recovery, myocardial injury and release of purine during reperfusion. Hearts underwent 30 min ischemia using one of the following protocols: control (normothermic buffer), hyperkalaemia (high-potassium buffer), hypothermia (20°C) and hyperkalemia + hypothermia. At the end of 30 min ischemia, hyperkalemia was associated with similar metabolic changes (rise in purine and lactate and fall in adenine nucleotides) to control group. Accumulation of purine was due to a rise in inosine, xanthine and hypoxanthine and was largely prevented by hypothermia and hyperkalemia + hypothermia. Upon reperfusion, there was a time-dependent release of all purine, lactate and AMP. A fast (peak in less than 20 sec) release of inosine, xanthine, hypoxanthine and lactate was highest in control followed by hyperkalemia then hypothermia and little release in hyperkalemia + hypothermia. Adenosine and AMP release was slow (peak at 3 min), only significant in control and was likely to be due to sarcolemmal disruption as the profile followed lactate dehydrogenase release. Recovery (left ventricular developed pressure) was 63% control, 82% hyperkalemia, 77% hypothermia and 98% for hyperkalemia + hypothermia. The loss of purine during reperfusion but not their production during ischemia is related to cardioprotection with hyperkalemia. The possibility that the consequences of hyperkalemia modulate a sodium-dependent purine efflux, is discussed. The reduced loss of purine in hypothermia or in hyperkalemia + hypothermia is likely to be due to a lower metabolic activity during ischemia.     

New perspectives on the role of SERCA2's Ca2+ affinity in cardiac function

Cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (SR) Ca2+ transport ATPase (SERCA2a). The SR Ca2+ -uptake activity not only determines the speed of Ca(2+) removal during relaxation, but also the SR Ca2+ content and therefore the amount of Ca2+ released for cardiomyocyte contraction. The Ca2+ affinity is the major determinant of the pump's activity in the physiological Ca2+ concentration range. In the heart, the affinity of the pump for Ca2+ needs to be controlled between narrow borders, since an imbalanced affinity may evoke hypertrophic cardiomyopathy. Several small proteins (phospholamban, sarcolipin) adjust the Ca2+ affinity of the pump to the physiological needs of the cardiomyocyte. It is generally accepted that a chronically reduced Ca2+ affinity of the pump contributes to depressed SR Ca2+ handling in heart failure. Moreover, a persistently lower Ca2+ affinity is sufficient to impair cardiomyocyte SR Ca2+ handling and contractility inducing dilated cardiomyopathy in mice and humans. Conversely, the expression of SERCA2a, a pump with a lower Ca2+ affinity than the housekeeping isoform SERCA2b, is crucial to maintain normal cardiac function and growth. Novel findings demonstrated that a chronically increased Ca2+ affinity also may trigger cardiac hypertrophy in mice and humans. In addition, recent studies suggest that some models of heart failure are marked by a higher affinity of the pump for Ca2+, and hence by improved cardiomyocyte relaxation and contraction. Depressed cardiomyocyte SR Ca2+ uptake activity may therefore not be a universal hallmark of heart failure.     

Cytosolic Ca2+ homeostasis is a constitutive function of the V-ATPase in Saccharomyces cerevisiae

The vacuole is the major site of intracellular Ca(2+) storage in yeast and functions to maintain cytosolic Ca(2+) levels within a narrow physiological range via a Ca(2+) pump (Pmc1p) and a H(+)/Ca(2+) antiporter (Vcx1p) driven by the vacuolar H(+)-ATPase (V-ATPase). We examined the function of the V-ATPase in cytosolic Ca(2+) homeostasis by comparing responses to a brief Ca(2+) challenge of a V-ATPase mutant (vma2Delta) and wild-type cells treated with the V-ATPase inhibitor concanamycin A. The kinetics of the Ca(2+) response were determined using transgenic aequorin as an in vivo cytosolic Ca(2+) reporter system. In wild-type cells, the V-ATPase-driven Vcx1p was chiefly responsible for restoring cytosolic Ca(2+) concentrations after a brief pulse. In cells lacking V-ATPase activity, brief exposure to elevated Ca(2+) compromised viability, even when there was little change in the final cytosolic Ca(2+) concentration. vma2Delta cells were more efficient at restoring cytosolic [Ca(2+)] after a pulse than concanamycin-treated wild-type cells, suggesting long term loss of V-ATPase triggers compensatory mechanisms. This compensation was dependent on calcineurin, and was mediated primarily by Pmc1p.     

Ca2+ transport ATPase isoforms SERCA2a and SERCA2b are targeted to the same sites in the murine heart

Adult SERCA2(b/b) mice expressing the non-muscle Ca2+ transport ATPase isoform SERCA2b in the heart instead of the normally predominant sarcomeric SERCA2a isoform, develop mild concentric ventricular hypertrophy with impaired cardiac contractility and relaxation [Circ. Res. 89 (2001) 838]. Results from a separate study on transgenic mice overexpressing SERCA2b in the normal SERCA2a context were interpreted to show that SERCA2b and SERCA2a are differentially targeted within the cardiac sarcoplasmic reticulum (SR) [J. Biol. Chem. 275 (2000) 24722]. Since a different subcellular distribution of SERCA2b could underlie alterations in Ca2+ handling observed in SERCA2(b/b), we wanted to compare SERCA2b distribution in SERCA2(b/b) with that of SERCA2a in wild-type (WT). Using confocal microscopy on immunostained fixed myocytes and BODIPY-thapsigargin-stained living cells, we found that in SERCA2(b/b) mice SERCA2b is correctly targeted to cardiac SR and is present in the same SR regions as SERCA2a and SERCA2b in WT. We conclude that there is no differential targeting of SERCA2a and SERCA2b since both are found in the longitudinal SR and in the SR proximal to the Z-bands. Therefore, alterations in Ca2+ handling and the development of hypertrophy in adult SERCA2(b/b) mice do not result from different SERCA2b targeting.     

A diversity of SERCA Ca2+ pump inhibitors

The SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) is probably the most extensively studied membrane protein transporter. There is a vast array of diverse inhibitors for the Ca2+ pump, and many have proved significant in helping to elucidate both the mechanism of transport and gaining conformational structures. Some SERCA inhibitors such as thapsigargin have been used extensively as pharmacological tools to probe the roles of Ca2+ stores in Ca2+ signalling processes. Furthermore, some inhibitors have been implicated in the cause of diseases associated with endocrine disruption by environmental pollutants, whereas others are being developed as potential anticancer agents. The present review therefore aims to highlight some of the wide range of chemically diverse inhibitors that are known, their mechanisms of action and their binding location on the Ca2+ ATPase. Additionally, some ideas for the future development of more useful isoform-specific inhibitors and anticancer drugs are presented.     

New N-aryl-N-alkyl-thiophene-2-carboxamide compound enhances intracellular Ca2+ dynamics by increasing SERCA2a Ca2+ pumping

The type 2a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) plays a central role in the intracellular Ca²⁺ homeostasis of cardiac myocytes, pumping Ca²⁺ from the cytoplasm into the sarcoplasmic reticulum (SR) lumen to maintain relaxation (diastole) and prepare for contraction (systole). Diminished SERCA2a function has been reported in several pathological conditions, including heart failure. Therefore, development of new drugs that improve SERCA2a Ca²⁺ transport is of great clinical significance. In this study, we characterized the effect of a recently identified N-aryl-N-alkyl-thiophene-2-carboxamide (or compound 1) on SERCA2a Ca²⁺-ATPase and Ca²⁺ transport activities in cardiac SR vesicles, and on Ca²⁺ regulation in a HEK293 cell expression system and in mouse ventricular myocytes. We found that compound 1 enhances SERCA2a Ca²⁺-ATPase and Ca²⁺ transport in SR vesicles. Fluorescence lifetime measurements of fluorescence resonance energy transfer between SERCA2a and phospholamban indicated that compound 1 interacts with the SERCA-phospholamban complex. Measurement of endoplasmic reticulum Ca²⁺ dynamics in HEK293 cells expressing human SERCA2a showed that compound 1 increases endoplasmic reticulum Ca²⁺ load by enhancing SERCA2a-mediated Ca²⁺ transport. Analysis of cytosolic Ca²⁺ dynamics in mouse ventricular myocytes revealed that compound 1 increases the action potential-induced Ca²⁺ transients and SR Ca²⁺ load, with negligible effects on L-type Ca²⁺ channels and Na⁺/Ca²⁺ exchanger. However, during adrenergic receptor activation, compound 1 did not further increase Ca²⁺ transients and SR Ca²⁺ load, but it decreased the propensity toward Ca²⁺ waves. Suggestive of concurrent desirable effects of compound 1 on RyR2, [³H]-ryanodine binding to cardiac SR vesicles shows a small decrease in nM Ca²⁺ and a small increase in μM Ca²⁺. Accordingly, compound 1 slightly decreased Ca²⁺ sparks in permeabilized myocytes. Thus, this novel compound shows promising characteristics to improve intracellular Ca²⁺ dynamics in cardiomyocytes that exhibit reduced SERCA2a Ca²⁺ uptake, as found in failing hearts.     

Ca2+ homeostasis in unstimulated platelets

Unstimulated platelets maintain a low cytosolic free Ca2+ concentration and a steep plasma membrane Ca2+ gradient. The mechanisms that are required have not been completely defined. In the present studies, 45Ca2+ was used to examine the kinetics of Ca2+ exchange in intact unstimulated platelets. Quin2 was used to measure the cytosolic free Ca2+ concentration. Under steady-state conditions, the maximum rate of Ca2+ exchange across the platelet plasma membrane, 2 pmol/10(8) platelets/min, was observed at extracellular free Ca2+ concentrations 20-fold less than in plasma. Two intracellular exchangeable Ca2+ pools were identified. The size of the more rapidly exchanging pool (t 1/2, 17 min) and the cytosolic free Ca2+ concentration were relatively unaffected by large changes in the extracellular Ca2+ concentration. In contrast, the size of the more slowly exchanging Ca2+ pool (t 1/2, 300 min) varied with the extracellular Ca2+ concentration, which suggests that it is physically as well as kinetically distinct from the rapidly exchangeable Ca2+ pool. The locations of the Ca2+ pools were determined by differential permeabilization of 45Ca2+-loaded platelets with digitonin. 45Ca2+ in the rapidly exchanging pool was released with lactate dehydrogenase, which suggests that it is located in the cytosol. 45Ca2+ in the slowly exchanging pool was released with markers for both the dense tubular system and mitochondria, but inhibition of mitochondrial Ca2+ uptake with carbonyl cyanide m-chlorophenylhydrazone had no effect on the size of the slowly exchangeable Ca2+ pool or the cytosolic free Ca2+ concentration. In contrast, addition of metabolic inhibitors (KCN plus carbonyl cyanide m-chlorophenylhydrazone plus deoxyglucose) or trifluoperazine caused a decrease in the size of the slowly exchangeable Ca2+ pool and an increase in the cytosolic free Ca2+ concentration. These observations suggest that Ca2+ homeostasis in unstimulated platelets is maintained by limiting Ca2+ influx from plasma, actively promoting Ca2+ efflux, and sequestering Ca2+ within an internal site, which is most likely the dense tubular system and not mitochondria.     

Expression of SERCA isoform with faster Ca2+ transport properties improves postischemic cardiac function and Ca2+ handling and decreases myocardial infarction

Myocardial ischemia-reperfusion (I/R) injury is associated with contractile dysfunction, arrhythmias, and myocyte death. Intracellular Ca(2+) overload with reduced activity of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is a critical mechanism of this injury. Although upregulation of SERCA function is well documented to improve postischemic cardiac function, there are conflicting reports where pharmacological inhibition of SERCA improved postischemic function. SERCA2a is the primary cardiac isoform regulating intracellular Ca(2+) homeostasis; however, SERCA1a has been shown to substitute SERCA2a with faster Ca(2+) transport kinetics. Therefore, to further address this issue and to evaluate whether SERCA1a expression could improve postischemic cardiac function and myocardial salvage, in vitro and in vivo myocardial I/R studies were performed on SERCA1a transgenic (SERCA1a(+/+)) and nontransgenic (NTG) mice. Langendorff-perfused hearts were subjected to 30 min of global ischemia followed by reperfusion. Baseline preischemic coronary flow and left ventricular developed pressure were significantly greater in SERCA1a(+/+) mice compared with NTG mice. Independent of reperfusion-induced oxidative stress, SERCA1a(+/+) hearts demonstrated greatly improved postischemic (45 min) contractile recovery with less persistent arrhythmias compared with NTG hearts. Morphometry showed better-preserved myocardial structure with less infarction, and electron microscopy demonstrated better-preserved myofibrillar and mitochondrial ultrastructure in SERCA1a(+/+) hearts. Importantly, intraischemic Ca(2+) levels were significantly lower in SERCA1a(+/+) hearts. The cardioprotective effect of SERCA1a was also observed during in vivo regional I/R with reduced myocardial infarct size after 24 h of reperfusion. Thus SERCA1a(+/+) hearts were markedly protected against I/R injury, suggesting that expression of SERCA 1a isoform reduces postischemic Ca(2+) overload and thus provides potent myocardial protection.     

Effects of peroxynitrite on sarco/endoplasmic reticulum Ca2+ pump isoforms SERCA2b and SERCA3a

Sarco(endo)plasmic reticulum Ca²⁺ (SERCA) pumps are important for cell signaling. Three different genes, SERCA1, 2, and 3, encode these pumps. Most tissues, including vascular smooth muscle, express a splice variant of SERCA2 (SERCA2b), whereas SERCA3a is widely distributed in tissues such as vascular endothelium, tracheal epithelium, mast cells, and lymphoid cells. SERCA2b protein is readily inactivated by peroxynitrite that may be formed during cardiac ischemia reperfusion or during immune response after infection. Here, we compared the peroxynitrite sensitivity of SERCA2b and SERCA3a by using microsomes prepared from HEK-293T cells overexpressing the pumps. We incubated the microsomes with different concentrations of peroxynitrite and determined Ca²⁺ uptake, Ca²⁺-Mg²⁺-ATPase, Ca²⁺-dependent formation of acylphosphate intermediate, and protein mobility in Western blots. Ca²⁺ uptake, Ca²⁺-Mg²⁺-ATPase, and Ca²⁺-dependent formation of acylphosphate intermediate were inactivated for both SERCA2b and SERCA3a, but the latter was more resistant to the inactivation. Western blots showed that SERCA2b and SERCA3a proteins oligomerized after treatment with peroxynitrite, but each with a slightly different pattern. Compared with monomers, the oligomers may be less efficient in forming the acylphosphate intermediate and in conducting the remainder of the steps in the reaction cycle. We conclude that the resistance of SERCA3a to peroxynitrite may aid the cells expressing them in functioning during exposure to oxidative stress. free radicals; Ca²⁺-Mg²⁺-ATPase; ischemia; coronary artery; vascular smooth muscle; sarco(endo)plasmic reticulum Ca²⁺ pumps     

Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum

Recent data have revealed an unexpected role of Bcl-2 in modulating the steady-state levels and agonist-dependent fluxes of Ca(2+) ions. Direct monitoring of endoplasmic reticulum (ER) Ca(2+) concentration with recombinant probes reveals a lower state of filling in Bcl-2-overexpressing cells and a higher leak rate from the organelle. The broader set of indirect data using cytosolic probes reveals a more complex scenario, as in many cases no difference was detected in the Ca(2+) content of the intracellular pools. At the same time, Ca(2+) signals have been shown to affect important checkpoints of the apoptotic process, such as mitochondria, thus tuning the sensitivity of cells to various challenges. In this contribution, we will review (i) the data on the effect of Bcl-2 on [Ca(2+)](er), (ii) the functional significance of the Ca(2+)-signalling alteration and (iii) the current insight into the possible mechanisms of this effect.     

Plasticity and adaptation of Ca2+ signaling and Ca2+-dependent exocytosis in SERCA2(+/-) mice

Darier's disease (DD) is a high penetrance, autosomal dominant mutation in the ATP2A2 gene, which encodes the SERCA2 Ca2+ pump. Here we have used a mouse model of DD, a SERCA2(+/-) mouse, to define the adaptation of Ca2+ signaling and Ca2+-dependent exocytosis to a deletion of one copy of the SERCA2 gene. The [Ca2+]i transient evoked by maximal agonist stimulation was shorter in cells from SERCA2(+/-) mice, due to an up-regulation of specific plasma membrane Ca2+ pump isoforms. The change in cellular Ca2+ handling caused approximately 50% reduction in [Ca2+]i oscillation frequency. Nonetheless, agonist-stimulated exocytosis was identical in cells from wild-type and SERCA2(+/-) mice. This was due to adaptation in the levels of the Ca2+ sensors for exocytosis synaptotagmins I and III in cells from SERCA2(+/-) mice. Accordingly, exocytosis was approximately 10-fold more sensitive to Ca2+ in cells from SERCA2(+/-) mice. These findings reveal a remarkable plasticity and adaptability of Ca2+ signaling and Ca2+-dependent cellular functions in vivo, and can explain the normal function of most physiological systems in DD patients.     

Regulation of SERCA Ca2+ pump expression by cytoplasmic Ca2+ in vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) express three isoforms of the sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) pump; SERCA2b predominates (91%), whereas SERCA2a (6%) and SERCA3 (3%) are present in much smaller amounts. Treatment with thapsigargin (Tg) or A-23187 increased the level of mRNA encoding SERCA2b four- to fivefold; SERCA3 increased about 10-fold, whereas SERCA2a was unchanged. Ca2+ chelation prevented the Tg-induced SERCA2b increase, whereas Ca2+ elevation itself increased SERCA2b expression. These responses were discordant with those of 78-kDa glucose-regulated protein/immunoglobulin-binding protein (grp78/BiP), an endoplasmic reticulum stress-response protein. SERCA2b mRNA elevation was much larger than could be accounted for by the observed increase in message stability. The induction of SERCA2b by Tg did not require protein synthesis, nor was it affected by inhibitors of calcineurin, protein kinase C, Ca2+/calmodulin-dependent protein kinase, or tyrosine protein kinases. Treatment with the nonselective protein kinase inhibitor H-7 prevented Tg-induced SERCA2b expression from occurring, whereas another nonselective inhibitor, staurosporine, was without effect. We conclude that changes in cytosolic Ca2+ control the expression of SERCA2b in VSMC via a mechanism involving a currently uncharacterized, H-7-sensitive but staurosporine-insensitive, protein kinase.     

The SERCA3-type of organellar Ca2+pumps

Of all the SERCA pumps, SERCA3 was the latest to be described and the least well known. Its primary structure deviates more than usual from the other members of the SERCA family. It is not known whether its remarkably low affinity for Ca²⁺ (K_0.5 > 1M) observed upon expression in the COS cell system occurs also in its normal cellular context. SERCA3 is particularly expressed at high levels in different types of blood cells and related cells like platelets, lymphocytes, mast cells and arterial endothelial cells. It is also found in cerebellar Purkinje neurons. The physiological significance of this expression pattern remains unknown.     

Mitochondrial Ca2+ homeostasis in health and disease

Although it has long been known that mitochondria possess a complex molecular repertoire for accumulating and releasing Ca2+, only in recent years has a large body of data demonstrated that these organelles promptly respond to Ca(2+)-mediated cell stimulations. In this contribution, we will review the principles of mitochondrial Ca2+ homeostasis and its signaling role in different physiological and pathological conditions.