Cardiac Sarcoplasmic Reticulum Calcium Pump Research Articles

Ginkgo biloba extract EGB761 protects against aging-associated diastolic dysfunction in cardiomyocytes of D-galactose-induced aging rat.

The aim of the present study was to make use of the artificially induced aging model cardiomyocytes to further investigate potential anti-aging-associated cellular diastolic dysfunction effects of EGB761 and explore underlying molecular mechanisms. Cultured rat primary cardiomyocytes were treated with either D-galactose or D-galactose combined with EGB761 for 48 h. After treatment, the percentage of cells positive for SA-β-gal, AGEs production, cardiac sarcoplasmic reticulum calcium pump (SERCA) activity, the myocardial sarcoplasmic reticulum calcium uptake, and relative protein levels were measured. Our results demonstrated that in vitro stimulation with D-galactose induced AGEs production. The addition of EGB761 significantly decreased the number of cells positive for SA-β-gal. Furthermore, decreased diastolic [Ca2+]i, curtailment of the time from the maximum concentration of Ca2+ to the baseline level and increased reuptake of Ca2+ stores in the SR were also observed. In addition, the level of p-Ser16-PLN protein as well as SERCA was markedly increased. The study indicated that EGb761 alleviates formation of AGEs products on SERCA2a in order to mitigate myocardial stiffness on one hand; on other hand, improve SERCA2a function through increase the amount of Ser16 sites PLN phosphorylation, which two hands finally led to ameliorate diastolic dysfunction of aging cardiomyocytes.

The Role of Phospholamban Cysteines in the Activation of the Cardiac Sarcoplasmic Reticulum Calcium Pump by Nitroxyl

Phospholamban (PLN) is an integral membrane protein that regulates the Ca2+ pump (SERCA2a) in cardiac sarcoplasmic reticulum (CSR). Phosphorylation of PLN in response to β-adrenergic stimulation enhances cardiac inotropy by increasing CSR Ca2+ uptake. Nitroxyl (HNO), a new candidate drug therapy for congestive heart failure, improves overall cardiovascular function by increasing Ca2+ release and re-uptake in CSR through a direct interaction with RyR2 and SERCA2a, respectively. Using insect cell ER microsomes expressing SERCA2a +/− PLN (WT and Cys → Ala mutant) we have shown that activation of SERCA2a by HNO is PLN-dependent and entails covalent modification of PLN cysteines. Although HNO stimulates SERCA2a activity by uncoupling PLN from SERCA2a, the role of the cysteine residues in the activation mechanism is not completely understood. We propose that HNO, a thiol oxidant, modifies one or more of the three PLN cysteine residues (C36, C41, C46), affecting the regulatory potency of PLN toward SERCA2a. Examples include intra-molecular disulfide cross-links within single PLN molecules or inter-molecular disulfide cross-links between PLN molecules or PLN and SERCA2a. To test this hypothesis, we have constructed a series of PLN mutants containing single, double and triple cysteine substitutions (alanine replacing cysteine). Each of these mutant PLNs will be co-expressed with SERCA2a in insect cells and cell microsomes will be treated with Angeli's salt (an HNO donor) to determine which cysteine residue(s) are essential for activation monitored by enzyme assay and fluorescence spectroscopy of SERCA2a. The results show that intermolecular PLN disulfides play a minor role in activation by HNO. Studies with the Cys → Ala mutations will be useful in determining which cysteine pairs in PLN contribute to intramolecular disulfide cross-links leading to the relief of PLN inhibition and SERCA2a activation.

Stimulation of Cardiac Sarcoplasmic Reticulum Calcium Pump by Acylphosphatase: RELATIONSHIP TO PHOSPHOLAMBAN PHOSPHORYLATION

Ca2+ transport by cardiac sarcoplasmic reticulum is tightly coupled with the enzymatic activity of Ca2+-dependent ATPase, which forms and decomposes an intermediate phosphoenzyme. Heart sarcoplasmic reticulum Ca2+ pump is regulated by cAMP-dependent protein kinase (PKA) phospholamban phosphorylation, which results in a stimulation of the initial rates of Ca2+ transport and Ca2+ ATPase activity. In the present studies we found that acylphosphatase from heart muscle, used at concentrations within the physiological range, actively hydrolyzes the phosphoenzyme of cardiac sarcoplasmic reticulum Ca2+ pump, with an apparent Km on the order of 10(-7) M, suggesting an high affinity of the enzyme for this special substrate. In unphosphorylated vesicles acylphosphatase enhanced the rate of ATP hydrolysis and Ca2+ uptake with a concomitant significant decrease in apparent Km for Ca2+ and ATP. In vesicles whose phospholamban was PKA-phosphorylated, acylphosphatase also stimulated the rate of Ca2+ uptake and ATP hydrolysis but to a lesser extent, and the Km values for Ca2+ and ATP were not significantly different with respect to those found in the absence of acylphosphatase. These findings suggest that acylphosphatase, owing to its hydrolytic effect, accelerates the turnover of the phosphoenzyme intermediate with the consequence of an enhanced activity of Ca2+ pump. It is known that phosphorylation of phospholamban results in an increase of the rate at which the phosphoenzyme is decomposed. Thus, as discussed, a competition between phospholamban and acylphosphatase effect on the phosphoenzyme might be proposed to explain why the stimulation induced by this enzyme is less marked in PKA-phosphorylated than in unphosphorylated heart vesicles.

Dependence of cardiac sarcoplasmic reticulum calcium pump activity on the phosphorylation status of phospholamban.

The application of electrophoretic resolution of the different phosphorylation species of pentameric phospholamban as a measure of phosphorylation stoichiometry was examined and verified. This enabled a critical evaluation of a number of issues central to current models of calcium pump regulation in cardiac sarcoplasmic reticulum. The phospholamban content of numerous preparations was calculated from 32P incorporation at a given stoichiometry, and compared with the respective calcium pump concentration (derived by comparison with a Coomassie-stained calibration curve of the fast-twitch skeletal muscle isozyme). A relationship of 2 mol of phospholamban:1 mol of ATPase resulted (phospholamban monomer:ATPase monomer), which was maintained throughout all vesicle subpopulations. The precise mechanism of coupling of phospholamban phosphorylation to calcium pump stimulation was probed, with particular emphasis on the individual contributions of each phosphorylated species (P1 to P5). This relationship could be adequately explained in three ways: (i) each phosphorylation event contributed equally to calcium pump stimulation; (ii) P1 and P2 were incapable of stimulating calcium pump activity, but full stimulation occurred upon generation of species P3; or (iii) the phosphospecies P1 was without effect on basal calcium pump activity, but successive phosphorylations contributed equally to stimulation. Finally, the functional implication of dual site phosphorylation of phospholamban (cAMP- and the endogenous calmodulin-dependent kinases) was examined. No change in calcium pump activity accompanied the second tier of phosphorylation over that achieved by the first.

Modulation by Polyelectrolytes of Canine Cardiac Microsomal Calcium Uptake and the Possible Relationship to Phospholamban

Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in canine cardiac microsomes were found to be stimulated by heparin and various other polyanions. Prior treatment of the microsomes with the ionophores alamethicin or A23187 produced no change in the extent of stimulation of the ATPase activity by heparin yet eliminated net calcium uptake. This finding and a lack of change in the stoichiometric ratio of mol of calcium transported/mol of ATP hydrolyzed (calcium:ATP) suggest that the effect of heparin is on the calcium pump rather than on a parallel calcium efflux pathway. Certain polycationic compounds including poly-L-arginine and histone inhibited both cardiac and fast skeletal muscle microsomal calcium uptake and also produced no change in the stoichiometric ratio of calcium to ATP. Several lines of evidence indicate that the polyanionic compounds tested stimulate calcium uptake by interacting with phospholamban, the putative phosphorylatable regulator of the cardiac sarcoplasmic reticulum calcium pump, whereas polycationic compounds appear to interact with the pump. (i) Heparin stimulated calcium uptake to the same extent as protein kinase A or trypsin, whereas prior phosphorylation or tryptic cleavage of phospholamban from the membrane abolished the stimulatory effect of heparin. (ii) Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in fast skeletal muscle microsomes, which lack phospholamban, were unaffected by heparin. (iii) Purified cardiac (Ca2+ + Mg2+)-ATPase activity was no longer stimulated by heparin yet was still inhibited by polycationic compounds. The heparin-induced stimulation of calcium uptake was dependent on the pH and ionic strength of the heparin-containing preincubation medium, hence electrostatic interactions appear to play a significant role in heparin's stimulatory action. The data are consistent with an inhibitory role of the positively charged cytoplasmic domain of phospholamban with respect to calcium pump activity and the relief of the inhibition upon reduction in phospholamban's positive charge by phosphorylation or binding of polyanions.

Mechanism of the stimulation of cardiac sarcoplasmic reticulum calcium pump by calmodulin.

Calmodulin has been shown to stimulate the initial rates of Ca2+-uptake and Ca2+-ATPase in cardiac sarcoplasmic reticulum, when it is present in the reaction assay media for these activities. To determine whether the stimulatory effect of calmodulin is mediated directly through its interaction with the Ca2+-ATPase, or indirectly through phosphorylation of phospholamban by an endogenous protein kinase, two approaches were taken in the present study. In the first approach, the effects of calmodulin were studied on a Ca2+-ATPase preparation, isolated from cardiac sarcoplasmic reticulum, which was essentially free of phospholamban. The enzyme was preincubated with various concentrations of calmodulin at 0 degrees C and 37 degrees C, but there was no effect on the Ca2+-ATPase activity assayed over a wide range of [Ca2+] (0.1-10 microM). In the second approach, cardiac sarcoplasmic reticulum vesicles were prephosphorylated by an endogenous protein kinase in the presence of calmodulin. Phosphorylation occurred predominantly on phospholamban, an oligomeric proteolipid. The sarcoplasmic reticulum vesicles were washed prior to assaying for Ca2+ uptake and Ca2+-ATPase activity in order to remove the added calmodulin. Phosphorylation of phospholamban enhanced the initial rates of Ca2+-uptake and Ca2+-ATPase, and this stimulation was associated with an increase in the affinity of the Ca2+-pump for calcium. The EC50 values for calcium activation of Ca2+-uptake and Ca2+-ATPase were 0.96 +/- 0.03 microM and 0.96 +/- 0.1 microM calcium by control vesicles, respectively. Phosphorylation decreased these values to 0.64 +/- 0.12 microM calcium for Ca2+-uptake and 0.62 +/- 0.11 microM calcium for Ca2+-ATPase. The stimulatory effect was associated with increases in the apparent initial rates of formation and decomposition of the phosphorylated intermediate of the Ca2+-ATPase. These findings suggest that calmodulin regulates cardiac sarcoplasmic reticulum function by protein kinase-mediated phosphorylation of phospholamban.

Role of phospholamban in regulating cardiac sarcoplasmic reticulum calcium pump.

Cardiac sarcoplasmic reticulum plays a critical role in the excitation-contraction cycle and hormonal regulation of heart cells. Catecholamines exert their ionotropic action through the regulation of calcium transport into the sarcoplasmic reticulum. Cyclic 3'-5'-adenosine monophosphate (cAMP) causes the cAMP-dependent protein kinase to phosphorylate the regulatory protein phospholamban, which results in the stimulation of calcium transport. Calmodulin also phosphorylates phospholamban by a calcium-dependent mechanism. We have reported the isolation and purification of phospholamban with low deoxycholate (DOC) concentrations (5 X 10(-6) M). We have also reported the isolation and purification of Ca2+ + Mg2+-ATPase with a similar procedure. Both phospholamban and Ca2+ + Mg2+-ATPase retained their native properties associated with sarcoplasmic reticulum vesicles. Further, we have shown that the removal of phospholamban from membranes of sarcoplasmic reticulum vesicles uncouples Ca2+-uptake from ATPase without any effect on Ca2+ + Mg2+-ATPase activity or Ca2+ efflux. Phospholamban appears to be the substrate for both the Ca2+-calmodulin system and the cAMP-dependent protein kinase system. It is found that the phosphorylation of phospholamban by the Ca2+-calmodulin system is required for the normal basal level of Ca2+ transport, and that the phosphorylation of phospholamban at another site by the cAMP-dependent protein kinase system causes the stimulation of Ca2+-transport above the basal level. The functional effects of the phosphorylation of phospholamban by cAMP-dependent protein kinase system are expressed only after the phosphorylation of phospholamban with Ca2+-calmodulin system. We propose a model for the cardiac Ca2+ + Mg2+-ATPase, whereby the enzyme is normally uncoupled from Ca2+ uptake. The enzyme becomes coupled to Ca2+ transport after the first site of phospholamban is phosphorylated with the Ca2+-calmodulin system. When the second site of phospholamban is phosphorylated with cAMP-dependent protein kinase both Ca2+ transport and ATPase are stimulated and phospholamban becomes inaccessible to DOC solubilization and trypsin.

Phospholamban, the regulator of the cardiac sarcoplasmic reticulum calcium pump, does not copurify with the Ca 2+-ATPase enzyme

Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3′,5′-monophosphate (cAMP)-dependent and by Ca 2+-calmodulin-dependent protein kinases on an M r 22000 protein called phospholamban. Both types of phosphorylation are associated with an increase in the initial rate of Ca 2+ transport. Thus, phospholamban appears to be a regulator for the calcium pump in cardiac sarcoplasmic reticulum. However, there is conflicting evidence as to the degree of association of the Ca 2+-ATPase with its regulator, phospholamban. In this study, we report that phospholamban does not copurify with a Ca 2+-ATPase preparation of high specific activity. Although 32P-labeled phospholamban is solubilized in the same fraction as the Ca 2+-ATPase from cardiac sarcoplasmic reticulum, it dissociates from the Ca 2+ pump during subsequent purification steps. Our isolation procedure results in an increase of over 4-fold in the specific activity of the Ca 2+-ATPase, but a decrease of 2.5-fold in the specific activity of 32P i-phosphoester bonds (pmol P i/mg). Furthermore, the purified Ca 2+-ATPase enzyme preparation is not a substrate for protein kinase in vitro to any significant extent. These data indicate that phospholamban does not copurify with the Ca 2+-ATPase from cardiac sarcoplasmic reticulum. Isolation of a Ca 2+-ATPase preparation essentially free of phospholamban will aid in future kinetic studies designed to elucidate similarities and differences in the Ca 2+-ATPase parameters from cardiac and skeletal muscle (which is known not to contain phospholamban).

Phosphorylation and the control of calcium fluxes.

Cell activation, e.g. stimulus-contraction or stimulus-secretion coupling, is brought about by a 100-fold increase in cytosolic free Ca2+ concentration from 0.1 to 10 microM, upon release of Ca2+ from intrareticular or extracellular stores along the concentration gradient. A return to steady state is achieved by either Na+-Ca2+ exchange or ATP-dependent Ca2+ transport against the concentration gradient. Both processes, Ca2+ influx and Ca2+ efflux, are regulated by sophisticated covalent mechanisms. The positive inotropic effect of adrenalin is mediated by the cyclic-AMP-dependent phosphorylation of cardiac sarcolemmal proteins, among which calciductin is the major phosphate acceptor. Upon cyclic-AMP-dependent phosphorylation, the slow Ca2+ channel is activated 3.5 time above its basal low-conductance state, and retains its characteristics, competition by divalent metals, inhibition by La3+ and Ca2+ entry blockers. The adrenalin-induced abbreviation of systole is also explained in terms of the dual phosphorylation of the cardiac sarcoplasmic reticulum calcium pump activator, phospholamban, by cyclic-AMP-dependent protein kinase on the one hand and Ca2+-calmodulin-dependent phospholamban kinase on the other. Calciductin and phospholamban are closely similar acidic proteolipids. A phospholamban-like protein is also found in platelet Ca2+-accumulating vesicles, where its cyclic-AMP-dependent phosphorylation doubles the rate of Ca2+ efflux. These observations raise the possibility that calcium fluxes are regulated by phosphorylation of membrane-bound proteolipids. More generally, phosphorylation modulates K+, Na+ and Ca2+ fluxes through membranes, i.e. the general excitability properties of the cell.

25] Covalent regulation of the cardiac sarcoplasmic reticulum calcium pump: Purification and properties of phospholamban, a substrate of cAMP-dependent protein kinase and Ca2+-calmodulin-dependent phospholamban kinase

The rate of calcium uptake by the cardiac sarcoplasmic reticulum Ca 2+ , Mg 2+ -ATPase is regulated by both cAMP-dependent phosphorylation and Ca 2+ -calmodulin-dependent phosphorylation. The target of this covalent concerted regulation is phospholamban, a proteolipid of M r 11,000. The short stretch of phospholamban exposed at the cytosolic side of the membrane contains both sites of phosphorylation, perhaps in a double-headed configuration. The membrane-bound kinase acts as a sensor of the cytosolic free Ca 2+ concentration. Phospholamban seems to be strongly associated with its calmodulin-dependent kinase and with the calcium pump. The canine cardiac sarcoplasmic reticulum (SR) is prepared from dog hearts arrested in diastole after pentobarbital injection. Both the Ca 2+ -calmodulin - and cAMP-dependent phosphorylations are followed. Each type of phosphorylation can be studied without interference from the other: the cAMP-dependent phosphorylation is performed in the presence of ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA) and catalytic subunit (C subunit) and in the absence of calmodulin, whereas the Ca 2+ -CaM-dependent phosphorylation is done in the presence of Ca 2+ , PKI, and exogenous calmodulin and in the absence of cAMP or added C subunit.

Ca2+/calmodulin-dependent phospholamban kinase from cardiac sarcoplasmic reticulum is distinct from phosphorylase kinase and forms a regulatory complex with phospholamban and the Ca2+-ATPase.

We recently reported that phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump, is phosphorylated by both cAMP-dependent protein kinase and a membrane-bound, Ca2+/calmodulin-dependent phospholamban kinase. Phospholamban kinase and glycogen phosphorylase b kinase share the same substrate specificity. They differ however in that phospholamban kinase exhibits an absolute requirement for exogenous calmodulin. In line with the latter observation, phospholamban kinase is shown in this report to be inhibited by fluphenazine. Lower concentrations of the drug induced an activation of the kinase, presumably by hydrophobic interaction with either membrane phospholipids or integral proteins. Also, phospholamban kinase was found to be totally insensitive to antibodies elicited against phosphorylase kinase. Since antipsychotic drugs fail to inhibit the delta-subunit-dependent activity of phosphorylase kinase, the above findings confirm that the two kinases are distinct molecular entities. After detergent solubilization of the sarcoplasmic reticulum, the phospholamban-ATPase complex remains a substrate for phospholamban kinase activity, which retains the ability to catalyze the phosphorylation of exogenous phosphorylase b. However, the Ca2+ dependence is entirely lost upon solubilization and no kinase activity is retained on calmodulin-Sepharose in the presence of Ca2+ ions. Phospholamban and phosphorylase kinase activities copurify with the pump-phospholamban complex upon fractionation of the solubilized proteins by density gradient ultracentrifugation, suggesting a tight interaction between the ATPase, its activator, and the phospholamban kinase. A tentative schematic representation of this supramolecular assembly is based upon the results described in this and preceding papers.

The cyclic AMP-dependent modulation of cardiac sarcolemmal slow calcium channels

The epinephrine-induced abbreviation of systole is well explained by the cAMP-dependent phosphorylation of phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump, and of the inhibitory component of the troponin complex. In contrast, the molecular mechanism of the β-agonist inotropic effects remained unclear until the recent finding that the depolarization-induced Ca 2+ uptake by cardiac sarcolemmal vesicles, the in vitro equivalent of the slow Ca 2+ channel, is activated upon cAMP-dependent phosphorylation of a sarcolemmal integral protein, calciductin [ 34]. This paper provides additional evidence for this mechanism by showing a linear correlation between calciductin phosphorylation and voltage-dependent Ca 2+ uptake. At no detectable radiolabelling with ( 32P)-phosphate, the latter is still ca 28% of its maximal value, suggesting the possibility of residual unlabeled protein-bound phosphate. Calciductin is an excellent substrate of cAMP-dependent protein kinase, since low levels of its catalytic subunit ( ca 6% of the subunit liberated after complete dissociation of the enzyme) are capable of submaximally phosphorylating calciductin and activating Ca 2+ uptake. The Ca 2+ channels of phosphorylated vesicles are similar to those of non-phosphorylated vesicles and of other excitable cells in their inhibition by the calcium antagonist verapamil, lanthanum and other divalent cations. In contrast to the voltage-dependent Ca 2+ uptake, Na + Ca 2+ exchange was not modulated by cAMP-dependent phosphorylation. Sarcolemmal proteolipids were entirely extracted by acidic organic solvent mixtures and purified by high performance liquid chromatography. They made up 2% (w/w) of sarcolemmal proteins and contained essentially calciductin (97%) together with a 14 000 to 16 000 dalton component (3%). Sarcolemmal proteolipids were phosphorylated on seryl residues only. Their amino acid composition indicated the presence of a large number of acidic and hydrophobic residues, accounting for the behaviour of calciductin as an acidic integral proteolipid, similar to phospholamban.

Calcium-calmodulin-dependent phosphorylations in the control of muscular contraction

Muscular contraction is triggered by the increase in free calcium concentration and modulated by cyclic nucleotide-dependent phosphorylation. Beside a direct trigger of sarcomeric muscle contraction through binding of troponin C, calcium ions trigger or modulate contractility through calcium-calmodulin-dependent myosin light chain kinases, and increase the rate of relaxation through the calmodulin-dependent phosphorylation of phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump. In both cases, a concerted regulation by calcium and cyclic nucleotides is observed. Hyperactivation of the calcium pump is brought about by additional phosphorylation of phospholamban by cAMP-dependent protein kinase. Similarly myofibrillar myosin light chain kinases from smooth and skeletal muscles are substrates of the cAMP-dependent protein kinase. The calmodulin-dependent protein kinases are probably organized into supramolecular regulatory complexes.

Phospholamban, activator of the cardiac sarcoplasmic reticulum calcium pump. Physicochemical properties and diagonal purification.

Phospholamban, activator of the cardiac sarcoplasmic reticulum calcium pump. Physicochemical properties and diagonal purification.