Activity Of Sarcoplasmic Reticulum Ca2 Research Articles

Cl-IB-MECA protects against doxorubicin induced cardiotoxicity in rats

Cardiotoxicity that is associated with doxorubicin (DOX) treatment limits the therapeutic efficiency of this drug. Using cultured cardiomyocytes, we found that the A3 adenosine selective agonist Cl-IB-MECA (2-chloro-N(6)-(3-iodobenzyl) adenosine-5′-N-methyluronamide) reduces DOX cardiotoxicity. We wished to determine if the protection demonstrated by Cl-IB-MECA attenuates cardiac depression in experiments in-vivo and to validate the combined effects of Dox and Cl-IB-MECA on sarcoplasmic reticulum (SR) and intra-mitochondrial Ca2+ accumulation. Control, DOX, Cl-IB-MECA and DOX+Cl-IB-MECA rat groups were tested. DOX (2.5 mg/kg i.p.) and Cl-IB-MECA (33 μg/kg i.v.) were injected 6 times on alternate days. Left ventricular functions (LVF), SR Ca2+ uptake, cytosolic and intra mitochondrial calcium and release were measured at the end of the injection period and 2 weeks post-injection period. Echocardiography data have shown that left ventricular end systolic diameter was higher and fractional shortening was significantly lower in DOX treated animals compared to the control groups (35%, 54%) (p<0.01). Improved LVF resulted when DOX was combined with Cl-IB-MECA. At the cellular level, DOX increased Ca2+ levels in the cytosol and mitochondria through diminishing SR Ca2+ reuptake. The addition of Cl-IB-MECA to DOX significantly improved SERCA2a efficiency. DOX, combined with Cl-IB-MECA resulted in improved cardiac hemodynamic function. This protection is partially caused through the decrease of calcium overload and activation of SR Ca2+ uptake.

Role of Phospholamban in Cyclic GMP Mediated Signaling in Cardiac Myocytes

We tested the hypothesis that the negative functional effects of cyclic GMP on cardiac myocytes were mediated through phospholamban (PLB) and activation of sarcoplasmic reticulum Ca²⁺-ATPase. Using ventricular myocytes from wild type (WT, n=10) and PLB knockout (PLB-KO, n=10) mouse hearts, functional changes were measured using a video edge detector at baseline and after 10^-6, 10^-5M 8-bromo-cyclic GMP (cGMP), 10^-8, 10^-7M C-type natriuretic peptide (CNP), or 10^-6, 10^-5M S-nitroso-N-acetyl-penicillamine (SNAP, nitric oxide donor). Changes in cytosolic Ca²⁺ concentration were assessed in fura 2-loaded WT and PLB-KO myocytes. Cyclic GMP dependent phosphorylation analysis was also performed in WT and PLB-KO myocytes. 8-bromo-cGMP 10^-5M caused a significant decrease in %shortening (3.6±0.2% to 2.3±0.1%) in WT, but little change in PLB-KO myocytes (3.4±0.1% to 3.2±0.2%). Similarly, CNP and SNAP reduced %shortening of WT, but not PLB-KO myocyte. Changes in other contractile parameters such as maximum rate of shortening and relaxation were consistent with the changes in % shortening. Intracellular Ca²⁺ transients changed similarly to cell contractility in WT and PLB-KO myocytes treated with cGMP and CNP; i.e. Ca²⁺ transients decreased with cGMP or CNP in WT myocytes, but were unchanged in PLB-KO myocytes. cGMP dependent phosphorylation analysis showed that some proteins were phosphorylated by cGMP to a lesser extent in PLB-KO compared with WT myocytes, suggesting impaired cGMP-kinase function in PLB-KO cardiac myocytes. These results indicated that cGMP-induced reductions in cardiac myocyte function were at least partially mediated through the action of phospholamban.

The inotropic effect of cardioactive glycosides in ventricular myocytes requires Na+–Ca2+ exchanger function

Glycoside-induced cardiac inotropy has traditionally been attributed to direct Na(+)-K(+)-ATPase inhibition, causing increased intracellular [Na(+)] and consequent Ca(2+) gain via the Na(+)-Ca(2+) exchanger (NCX). However, recent studies suggested alternative mechanisms of glycoside-induced inotropy: (1) direct activation of sarcoplasmic reticulum Ca(2+) release channels (ryanodine receptors; RyRs); (2) increased Ca(2+) selectivity of Na(+) channels (slip-mode conductance); and (3) other signal transduction pathways. None of these proposed mechanisms requires NCX or an altered [Na(+)] gradient. Here we tested the ability of ouabain (OUA, 3 microm), digoxin (DIG, 20 microm) or acetylstrophanthidin (ACS, 4 microm) to alter Ca(2+) transients in completely Na(+)-free conditions in intact ferret and cat ventricular myocytes. We also tested whether OUA directly activates RyRs in permeabilized cat myocytes (measuring Ca(2+) sparks by confocal microscopy). In intact ferret myocytes (stimulated at 0.2 Hz), DIG and ACS enhanced Ca(2+) transients and cell shortening during twitches, as expected. However, prior depletion of [Na(+)](i) (in Na(+)-free, Ca(2+)-free solution) and in Na(+)-free solution (replaced by Li(+)) the inotropic effects of DIG and ACS were completely prevented. In voltage-clamped cat myocytes, OUA increased Ca(2+) transients by 48 +/- 4% but OUA had no effect in Na(+)-depleted cells (replaced by N-methyl-d-glucamine). In permeabilized cat myocytes, OUA did not change Ca(2+) spark frequency, amplitude or spatial spread (although spark duration was slightly prolonged). We conclude that the acute inotropic effects of DIG, ACS and OUA (and the effects on RyRs) depend on the presence of Na(+) and a functional NCX in ferret and cat myocytes (rather than alternate Na(+)-independent mechanisms).

Effects of combination of irbesartan and perindopril on calcineurin expression and sarcoplasmic reticulum Ca2+-ATPase activity in rat cardiac pressure-overload hypertrophy

To observe effects of angiotensin (Ang) II receptor antagonist (AT1) irbesartan and angiotensin-converting enzyme (ACE) inhibitor perindopril on rat myocardium calcineurin expression and sarcoplasmic reticulum Ca(2+)-ATPase activity in the model of pressure-overload cardiac hypertrophy. Forty male adult Sprague Dawley rats were divided into 5 groups. One group was treated by sham operation; four groups were myocardium hypertrophy cases caused by banding aortic above renal artery. Drugs were given one week after operation. Group 1: sham group, rats (n=8) were gavaged with normal saline 2 ml/(kg.d) (ig); Group 2: control group, rats (n=8) were treated with normal saline 2 ml/(kg.d) (ig); Group 3: rats (n=8) were given perindopril 2 mg/(kg.d) (ig); Group 4: rats (n=8) were treated with irbesartan 20 mg/(kg.d) (ig); Group 5: rats (n=8) were given irbesartan 20 mg/(kg.d) plus perindopril 2 mg/(kg.d) (ig). Morphometric determination, calcineurin expression and sarcoplasmic reticulum Ca(2+)-ATPase activity were done at the end of 6 week of drug intervention. Expression of calcineurin in myocardium was detected by immunohistochemistry. Left ventricular mass index (LVMI), transverse diameter of myocardial cell (TDM), calcineurin activity were remarkably decreased after drug intervention and this decrease was most remarkable in the combination drug therapy group. Sarcoplasmic reticulum Ca(2+)-ATPase activity was increased after drug intervention, especially in the combined drug therapy group. Calcineurin expression in myocardium was remarkably decreased after drug intervention. LVMI was positively correlated with TDM and calcineurin, negatively correlated with sarcoplasmic reticulum Ca(2+)-ATPase. These data suggest that irbesartan and perindopril inhibit cardiac hypertrophy through the increased activity of sarcoplasmic reticulum Ca(2+)-ATPase and decreased expression of calcineurin. Their combination had better effects on regressing of ventricular hypertrophy.

Effects of reduced glycogen on structure and in vitro function of rat sarcoplasmic reticulum Ca2+-ATPase

The aim of this study was to examine the effects of reduced glycogen concentration on sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity in rat fast-twitch muscles. In the first experiment, the gastrocnemius (GAS) muscle from one leg was removed, followed by starvation for 24-72 h, after which the remaining GAS was removed. Intra-animal comparisons revealed that starvation caused a 25% reduction (P<0.05) in the glycogen concentration but no change in SR Ca(2+)-ATPase activity in the GAS. In the second experiment, the SR was purified from a mixture of the GAS and vastus lateralis muscles. In half of the samples obtained from each animal, glycogen was extracted from the SR by treatment with glucoamylase. Treatment resulted in a 94.1 and 70.2% decrease (P<0.01) in glycogen and glycogen phosphorylase, respectively, and a 41.5% increase (P<0.05) in a fluorescein isothiocyanate (FITC) binding to SR Ca(2+)-ATPase. On the other hand, SR Ca(2+)-ATPase activity and the affinity of the enzyme for ATP were unaltered. These results do not implicate depletion of muscle glycogen as a contributor to impaired SR Ca(2+)-ATPase activity as measured in vitro. Therefore, it is concluded that muscle glycogen does not influence exercise tolerance and work productivity in working muscles by modulating the structure of protein involved in Ca(2+) sequestering. Furthermore, it is suggested that the FITC binding assay may be inappropriate as a method for examining the mechanisms for the altered activity of SR Ca(2+)-ATPase.

Influence of N-dodecyl- N,N-dimethylamine N-oxide on the activity of sarcoplasmic reticulum Ca 2+-transporting ATPase reconstituted into diacylphosphatidylcholine vesicles: Effects of bilayer physical parameters

Sarcoplasmic reticulum Ca-transporting ATPase (EC 3.6.1.38) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 °C, the specific activity of ATPase displays a maximum (31.5 ± 0.8 IU/mg) for dioleoylphosphatidylcholine (diC18 : 1PC) and decreases progressively for both shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydration and lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstituted into dierucoylphosphatidylcholine (diC22 : 1PC), the zwitterionic surfactant N-dodecyl- N,N-dimethylamine N-oxide (C12NO) stimulates the ATPase activity from 14.2 ± 0.6 to 32.5 ± 0.8 IU/mg in the range of molar ratios C12NO : diC22 : 1PC = 0 ÷ 1.2. In dilauroylphosphatidylcholines (diC12 : 0PC) and diC18 : 1PC, the effect of C12NO is twofold—the ATPase activity is stimulated at low and inhibited at high C12NO concentrations. In diC18 : 1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO : diC18 : 1PC ≤ 1.3 in bilayers, where the bilayer thickness estimated by SANS decreases by 0.4 ± 0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increases indicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N +–P − accompanied by a variation of the local membrane dipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO : diC18 : 1PC = 1.3 ÷ 2.5, where mixed tubular micelles are detected by SANS in C12NO + diC18 : 1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes in dipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca 2+-ATPase activity.

43. Targeting the Biology of Heart Disease: Engineered Zinc Finger Protein Repressors of Phospholamban as a Potential Therapy for Congestive Heart Failure

Improper calcium handling of the heart is a hallmark of patients with congestive heart failure (CHF). Because calcium is critical for cardiac contractility, proteins that regulate calcium homeostasis are potential targets treating CHF. Phospholamban (PLN) decreases contractility by inhibiting the activity of Sarcoplasmic Reticulum Ca2+ ATPase 2 isoform A (SERCA2a); an increased PLN/SERCA2a ratio is often found in CHF patients. Recent studies have demonstrated that ablation or inhibition of PLN function can improve cardiac contractile properties in animal models of CHF, suggesting that down-regulation of PLN may improve cardiac function in CHF patients. Importantly, inhibition of PLN enhances calcium handling without activating b-adrenergic pathways, which is known to have many side-effects and increase mortality. The development of small-molecule inhibitors of PLN function has so far been unsuccessful, largely due to the difficulty of inhibiting protein-protein interactions (such as that between PLN and SERCA2a) using small molecules. On the other hand, approaches that aim to block the expression of PLN may provide a superior means of achieving the desired therapeutic effect.

Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca(2+) channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca(2+) channel opening rather than the domain switch itself.

A novel plasma membrane Ca 2+-pump inhibitor: caloxin 1A1

The plasma membrane Ca 2+–Mg 2+-ATPase is a Ca 2+-pump that expels Ca 2+ from cells. Here we report caloxin 1A1–a novel peptide inhibitor (K i=100 μM) of plasma membrane Ca 2+-pump–obtained by screening a cysteine bridge-constrained random peptide library for binding to the first extracellular domain of plasma membrane Ca 2+-pump. Dithiothreitol removed the inhibition indicating that the constraint imposed by the cysteine bridge is required for the inhibition. Caloxin 1A1 also inhibited the fast twitch sarcoplasmic reticulum Ca 2+–Mg 2+-ATPase although weakly. Glutathione dimers (containing a cysteine bridge) inhibited the Ca 2+–Mg 2+-ATPase activity of sarcoplasmic reticulum Ca 2+–Mg 2+-ATPase, but not that of plasma membrane Ca 2+-pump. Caloxin 1A1 stabilised Ca 2+-dependent formation of the acid stable 140-kDa acylphosphate which is a partial reaction of this enzyme. Thus caloxin 1A1 inhibits the plasma membrane Ca 2+-pump by perturbing the first extracellular domain indicating that the transmembrane domains 1 and 2 play a role in its reaction cycle. This finding is consistent with rearrangements that occur in transmembrane helices 1 and 2 during reaction cycle of sarcoplasmic reticulum Ca 2+-pump. Caloxin 1A1 caused an increase in cytosolic Ca 2+ concentration in endothelial cells.

Effect of Astragalosides on Intracellular Calcium Overload in Cultured Cardiac Myocytes of Neonatal Rats

Astragalosides were the main active components from a native Chinese herb Astragalus membranaceus. Recent studies have shown that Astragalosides have a protective effect on myocardial injury in rats. The present study was designed to investigate the effect of Astragalosides on intracellular calcium overload and sarcoplasmic reticulum calcium load (SR Ca2+ load) in cultured cardiac myocytes from neonatal rats. Astragalosides (100 microg/ml) were incubated in the presence of isoproterenol (ISO) (10(-5) M) for 72 hours in cardiomyocytes. Metoprorol (10(-6) M), a beta1-selective antagonist, was cultured in the same condition as Astragalosides. The result showed that intracellular calcium concentration ([Ca2+]i) and SR Ca2+ load increased in ISO-treated cardiac myocytes as compared to control (P < 0.01). Astragalosides prevented ISO-induced increase in [Ca2+]i and SR Ca2+ load. Metoprolol also inhibited those increase. The mRNA expression and activity of sarcoplasmic reticulum Ca2+ ATPase (SERCA) were enhanced following ISO treatment in cardiac myocytes, and these increases were inhibited by Astragalosides or metoprolol (P < 0.05). The decrease of superoxide dismutase (SOD) activity and the elevation of intracellular maleic dialdehyde (MDA) were observed after ISO treatment in cardiac myocytes. Both Astragalosides and metoprolol restored the SOD activity and reduced the level of MDA. We conclude that Astragalosides have the effects on reducing [Ca2+]i and SR Ca2+ load, enhancing free radical removal and decreasing lipid peroxidation in ISO-treated cardiomyocytes, which might account for their protective effect on myocardial injury.

Role of the Sequence Surrounding Predicted Transmembrane Helix M4 in Membrane Association and Function of the Ca2+ Release Channel of Skeletal Muscle Sarcoplasmic Reticulum (Ryanodine Receptor Isoform 1)

The role of the sequence surrounding M4 in ryanodine receptors (RyR) in membrane association and function was investigated. This sequence contains a basic, 19-amino acid M3/M4 loop, a hydrophobic 44-49 amino acid sequence designated M4 (or M4a/M4b), and a hydrophilic M4/M5 loop. Enhanced green fluorescent protein (EGFP) was inserted into RyR1 and truncated just after the basic sequence, just after M4, within the M4/M5 loop, just before M5 and just after M5. The A52 epitope was inserted into RyR2 and truncated just after M4a. Analysis of these constructs ruled out a M3/M4 transmembrane hairpin and narrowed the region of membrane association to M4a/M4b. EGFP inserted between M4a and M4b in full-length RyR2 was altered conformationally, losing fluorescence and gaining trypsin sensitivity. Although it was accessible to an antibody from the cytosolic side, tryptic fragments were membrane-bound. The expressed protein containing EGFP retained caffeine-induced Ca(2+) release channel function. These results suggest that M4a/M4b either forms a transmembrane hairpin or associates in an unorthodox fashion with the cytosolic leaflet of the membrane, possibly involving the basic M3/M4 loop. The expression of a mutant RyR1, Delta4274-4535, deleted in the sequence surrounding both M3 and M4, restored robust, voltage-gated L-type Ca(2+) currents and Ca(2+) transients in dyspedic myotubes, demonstrating that this sequence is not required for either orthograde (DHPR activation of sarcoplasmic reticulum Ca(2+) release) or retrograde (RyR1 increase in DHPR Ca(2+) channel activity) signals of excitation-contraction coupling. Maximal amplitudes of L-currents and Ca(2+) transients with Delta4274-4535 were larger than with wild-type RyR1, and voltage-gated sarcoplasmic reticulum Ca(2+) release was more sensitive to activation by sarcolemmal voltage sensors. Thus, this region may act as a negative regulatory module that increases the energy barrier for Ca(2+) release channel opening.

Gene Therapy in Congestive Heart Failure

Although research and development of gene therapy have encountered several difficulties in recent years, they have also begun to show potential for the treatment of acquired and inherited human diseases. Gene therapy may have wide applicability for the treatment of numerous cardiovascular diseases, including congestive heart failure (CHF), ischemic heart disease, and cardiomyopathy. CHF remains a leading cause of death and morbidity in industrialized nations, and the number of patients with CHF continues to increase as the population ages. Pharmacological therapies control symptoms, reduce left ventricular (LV) dilation, improve function, and reduce mortality. These therapies control the disease but do not cure it. Currently, however, the growing knowledge of molecular mechanisms involved in myocardial dysfunction has allowed the identification of potential therapeutic targets designed with the aim of curing CHF. Several studies in animal models have provided hope that gene therapy may be one of the future strategies for the treatment of clinical CHF. In these studies, myocardial gene transfer has been used to target at least 3 different biological pathways that play a crucial role in the pathophysiology of CHF: Intracellular calcium signaling, β1-adrenergic receptor (β1-AR) signaling, and antiapoptotic signaling. The activity of sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) is decreased in failing myocardium, and this results in abnormal Ca2+ handling.1 In an animal model of heart failure, Miyamoto et al2 showed that in vivo overexpression of SERCA2a, achieved by catheter injection of an adenovirus carrying the SERCA2a gene into the aortic root, restored systolic and diastolic function to normal levels. In another study, Muller et al3 analyzed the chronic overexpression of SERCA2a in both normal and pressure-overloaded hearts (induced by abdominal aortic banding of transgenic rats). Overexpression of SERCA2a resulted in a …

Antibody probe study of Ca2+ channel regulation by interdomain interaction within the ryanodine receptor.

N-terminal and central domains of ryanodine receptor 1 (RyR1), where many reported malignant hyperthermia (MH) mutations are localized, represent putative channel regulatory domains. Recent domain peptide (DP) probe studies led us to the hypothesis that these domains interact to stabilize the closed state of channel (zipping), while weakening of domain-domain interactions (unzipping) by mutation de-stabilizes the channel, making it leaky to Ca2+ or sensitive to the agonists of RyR1. As shown previously, DP1 (N-terminal domain peptide) and DP4 (central domain peptide) produced MH-like channel activation/sensitization effects, presumably by peptide binding to sites critical to stabilizing domain-domain interactions and resultant loss of conformational constraints. Here we report that polyclonal anti-DP1 and anti-DP4 antibodies also produce MH-like channel activation and sensitization effects as evidenced by about 4-fold enhancement of high affinity [3H]ryanodine binding to RyR1 and by a significant left-shift of the concentration-dependence of activation of sarcoplasmic reticulum Ca2+ release by polylysine. Fluorescence quenching experiments demonstrate that the accessibility of a DP4-directed, conformationally sensitive fluorescence probe linked to the RyR1 N-terminal domain is increased in the presence of domain-specific antibodies, consistent with the view that these antibodies produce unzipping of interacting domains that are of hindered accessibility to the surrounding aqueous environment. Our results suggest that domain-specific antibody binding induces a conformational change resulting in channel activation, and are consistent with the hypothesis that interacting N-terminal and central domains are intimately involved in the regulation of RyR1 channel function.

Ouabain increases sarcoplasmic reticulum calcium release in cardiac myocytes.

The inotropic and toxic effects of cardiac glycosides are thought to be related to their ability to inhibit the Na,K-ATPase. We examined the effects of ouabain and its analogs on sarcoplasmic reticulum (SR) Ca(2+) release in intact cat ventricular myocytes under Na(+)-free conditions and in myocytes in which the sarcolemma was permeabilized using saponin so that cytoplasmic ionic composition was fixed by the bath solutions. We also compared ouabain actions in cat myocytes to those in rat myocytes because the latter is considered to be a glycoside-insensitive species. In intact cat myocytes (Na(+)-free conditions), spontaneous Ca(2+) sparks were prolonged and frequency, amplitude and width were reduced by exposure to ouabain (3 microM). Nearly identical results were obtained with its analogs dihydroouabain or ouabagenin (10 microM). The frequency of spontaneous Ca(2+) waves was also reduced by ouabain. In contrast, ouabain (100 microM) had negligible effects on sparks and waves in rat myocytes in Na(+)-free conditions, consistent with the decreased sensitivity to cardiac glycosides observed in this species. In cat myocytes permeabilized with saponin (0.01%), ouabain (>or=50 nM) decreased spark frequency and increased background SR Ca(2+) leak only when the SR was well loaded (free [Ca(2+)] = 275 nM) and not when SR load was low (free [Ca(2+)] = 50 nM). Similar effects were observed in rat myocytes only when ouabain concentration was 1 microM. These results suggest that the cellular actions of cardiac glycosides may include a direct effect on SR Ca(2+) release, possibly through activation of SR Ca(2+) release channels (ryanodine receptors). In addition, these results are consistent with the idea that direct activation of SR Ca(2+) release is dependent on the extent of SR Ca(2+) load, with elevated load increasing sensitivity of the channel release mechanism to activation by glycoside.

Transgenic rat hearts overexpressing SERCA2a show improved contractility under baseline conditions and pressure overload.

The activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) is reduced in the failing myocardium. Therefore, transfer of SERCA2a cDNA is considered as a therapeutical approach. The aim of this study was analysis of the long-term effect of SERCA2a overexpression in normal as well as pressure overload challenged myocardium of transgenic rats. Independent transgenic rat lines were established expressing the rat SERCA2a cDNA specifically in the myocardium resulting in increased SERCA2a protein levels by 30-70%. Simultaneous measurements of isometric contraction and calcium transients were carried out in right ventricular papillary muscle preparations. Hemodynamic parameters were measured in hearts of unchallenged rats as well as 10 weeks after pressure overload induced by abdominal aortic banding. Analysis of calcium handling and contractile parameters in isolated right ventricular papillary muscles revealed significant shortening of intracellular calcium transients and half maximal relaxation times (RT(50)). Assessing myocardial contractility in working heart preparations, both transgenic rat lines revealed elevated left ventricular pressure, improved systolic and diastolic parameters, attenuated negative force-frequency relation, and a dose-dependent beta-adrenergic effect. Aortic banding resulted in reduction of left ventricular pressure and worsening of contraction and relaxation parameters with no differences in mortality in both transgenic (+dP/dt 3084+/-96 vs. 3938+/-250 mmHg/s; RT(50) 47.0+/-1.2 vs. 36.7+/-1.4 ms) and wild-type rats (+dP/dt 2695+/-86 vs. 3297+/-122 mmHg/s; RT(50) 53.0+/-1.6 vs. 44.1+/-1.4). SERCA2a overexpressing hearts revealed improved hemodynamic parameters compared to wild-type controls. Acceleration of isovolumetric relaxation characterized by the index Tau was directly correlated to SERCA2a protein concentrations. Overexpression of SERCA2a protein results in a positive inotropic effect under baseline conditions remaining preserved under pressure overload without affecting mortality. Therefore therapeutic transfer of SERCA2a may become a potential approach for gene therapy of congestive heart failure. Moreover, transgenic SERCA2a rats will be useful for studies of long-term SERCA2a overexpression in further cardiovascular disease models.

Cardiac-type EC-Coupling in Dysgenic Myotubes Restored with Ca 2+ Channel Subunit Isoforms α1C and α1D Does not Correlate with Current Density

Ca 2+-induced Ca 2+-release (CICR)—the mechanism of cardiac excitation-contraction (EC) coupling—also contributes to skeletal muscle contraction; however, its properties are still poorly understood. CICR in skeletal muscle can be induced independently of direct, calcium-independent activation of sarcoplasmic reticulum Ca 2+ release, by reconstituting dysgenic myotubes with the cardiac Ca 2+ channel α 1C (Ca V1.2) subunit. Ca 2+ influx through α 1C provides the trigger for opening the sarcoplasmic reticulum Ca 2+ release channels. Here we show that also the Ca 2+ channel α 1D isoform (Ca V1.3) can restore cardiac-type EC-coupling. GFP- α 1D expressed in dysgenic myotubes is correctly targeted into the triad junctions and generates action potential-induced Ca 2+ transients with the same efficiency as GFP- α 1C despite threefold smaller Ca 2+ currents. In contrast, GFP- α 1A, which generates large currents but is not targeted into triads, rarely restores action potential-induced Ca 2+ transients. Thus, cardiac-type EC-coupling in skeletal myotubes depends primarily on the correct targeting of the voltage-gated Ca 2+ channels and less on their current size. Combined patch-clamp/fluo-4 Ca 2+ recordings revealed that the induction of Ca 2+ transients and their maximal amplitudes are independent of the different current densities of GFP- α 1C and GFP- α 1D. These properties of cardiac-type EC-coupling in dysgenic myotubes are consistent with a CICR mechanism under the control of local Ca 2+ gradients in the triad junctions.

Interleukin-2 increases activity of sarcoplasmic reticulum Ca2+-ATPase, but decreases its sensitivity to calcium in rat cardiomyocytes.

To further explore the role of interleukin-2 (IL-2) in cardiac function, we investigated its effects on the intracellular calcium transient and the activity of sarcoplasmic reticulum (SR) Ca2+-ATPase in rat cardiomyocytes. IL-2 (200 U/ml) decreased the amplitude of electrically stimulated and caffeine-induced intracellular Ca2+ transients in ventricular myocytes, but increased the end-diastolic calcium level. IL-2 did not affect the sarcolemmal L-type Ca2+ channel activity. The activity of SR Ca2+-ATPase from IL-2-treated hearts increased in a dose-dependent manner, but the sarcolemmal Ca2+-ATPase activity did not change. After incubation of SR with ATP, the activity of SR Ca2+-ATPase from IL-2-treated hearts increased much more than that in the control group. The responsiveness of SR Ca2+-ATPase from IL-2-perfused hearts to the free calcium concentration was inhibited. The Ca2+ uptake and Ca2+ content were reduced in the SR vesicles prepared from IL-2-treated rat heart. Pretreatment with the kappa-opioid receptor antagonist nor-binaltorphimine (10 nM) attenuated the effect of IL-2 on the SR Ca2+-ATPase activity, SR Ca2+ uptake, and Ca2+ content. The activity of Ca2+-ATPase in SR isolated from untreated hearts did not change when IL-2 and SR were coincubated. Thus, we conclude that the decreased calcium transient induced by IL-2 results from reduced SR calcium release, which is due to decreased SR Ca2+ uptake mediated by cardiac kappa-opioid receptors, but not from reduced activity of the sarcolemmal L-type calcium channel.

Vasodilation by the Calcium-mobilizing Messenger Cyclic ADP-ribose

In artery smooth muscle, adenylyl cyclase-coupled receptors such as beta-adrenoceptors evoke Ca(2+) signals, which open Ca(2+)-activated potassium (BK(Ca)) channels in the plasma membrane. Thus, blood pressure may be lowered, in part, through vasodilation due to membrane hyperpolarization. The Ca(2+) signal is evoked via ryanodine receptors (RyRs) in sarcoplasmic reticulum proximal to the plasma membrane. We show here that cyclic adenosine diphosphate-ribose (cADPR), by activating RyRs, mediates, in part, hyperpolarization and vasodilation by beta-adrenoceptors. Thus, intracellular dialysis of cADPR increased the cytoplasmic Ca(2+) concentration proximal to the plasma membrane in isolated arterial smooth muscle cells and induced a concomitant membrane hyperpolarization. Smooth muscle hyperpolarization mediated by cADPR, by beta-adrenoceptors, and by cAMP, respectively, was abolished by chelating intracellular Ca(2+) and by blocking RyRs, cADPR, and BK(Ca) channels with ryanodine, 8-amino-cADPR, and iberiotoxin, respectively. The cAMP-dependent protein kinase A antagonist N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked hyperpolarization by isoprenaline and cAMP, respectively, but not hyperpolarization by cADPR. Thus, cADPR acts as a downstream element in this signaling cascade. Importantly, antagonists of cADPR and BK(Ca) channels, respectively, inhibited beta-adrenoreceptor-induced artery dilation. We conclude, therefore, that relaxation of arterial smooth muscle by adenylyl cyclase-coupled receptors results, in part, from a cAMP-dependent and protein kinase A-dependent increase in cADPR synthesis, and subsequent activation of sarcoplasmic reticulum Ca(2+) release via RyRs, which leads to activation of BK(Ca) channels and membrane hyperpolarization.

Altered sarcoplasmic reticulum function in rat diaphragm after high-intensity exercise.

The present study examined the effects of acute high-intensity exercise on Ca(2+) uptake and release rates and Ca(2+)-adenosine triphosphatase (ATPase) activity of the sarcoplasmic reticulum (SR) from the costal diaphragm. The rats were run on a treadmill at an estimated requirement of 100% of maximal O2 consumption until fatigued (average time to exhaustion: 4.79 min). Muscle lactate and inorganic phosphate after exercise were increased by 65% (P < 0.05) and 35% (P < 0.05), respectively. With exercise, Ca(2+) uptake and release, which were detected in homogenates using the Ca(2+) fluorescent dye indo-1, were decreased by 24% (P < 0.05) and 22% (P < 0.05), respectively. The reduction in Ca(2+) uptake was paralleled by decreased activity of SR Ca(2+)-ATPase in both the absence and presence of Ca(2+) ionophore. These findings demonstrate that, in the diaphragm as well as in the locomotor muscles that have been explored in previous studies, the attenuations of the SR function is brought about by acute high-intensity exercise. These changes in the SR of the diaphragm may contribute, at least in part, to deteriorations in exercise tolerance and work productivity resulting from repetitive physical activities.

Luminal Ca 2+ and the activity of sarcoplasmic reticulum Ca 2+ pumps modulate histamine-induced all-or-none Ca 2+ release in smooth muscle cells

We have studied histamine (HA)-evoked intracellular Ca 2+ release in single, freshly isolated myocytes from the guinea pig urinary bladder. Short applications of histamine (5 s) produced a thapsigargin (TG)-sensitive transient increase in intracellular calcium concentration ([Ca 2+] i). It was established that histamine and caffeine (Caff) released Ca 2+ from the same intracellular stores in these cells. Reducing the Ca 2+ content of internal stores by incubating cells with U-73343 or cyclopiazonic acid (CPA) inhibited the histamine-evoked Ca 2+ release in 69% and 60% of cells, respectively. Under these conditions, all cells released Ca 2+ in response to either caffeine or acetylcholine (ACh). However, decreasing internal Ca 2+ stores by removing external Ca 2+ inhibited histamine-induced Ca 2+ mobilization in only 22% of cells. A similar small fraction of cells was inhibited when sarcoplasmic reticulum (SR) Ca 2+ pumps were quickly blocked to avoid a significant reduction of luminal Ca 2+. In conclusion, lowering the luminal Ca 2+ content in combination with an impairment of the SR Ca 2+ pump activity significantly diminishes the ability of histamine to evoke an all-or-none intracellular Ca 2+ release.