SERCA2 Levels Research Articles

Alteration of myocardial sarcoplasmic reticulum Ca2+‐ATPase and Na+–Ca2+exchanger expression in human left ventricular volume overload

Reduced myocardial contractility is often attributed to altered Ca(2+) transients and expression of Ca(2+)-ATPase of the SR (SERCA) and Na+/Ca(2+)exchanger (NCX) genes. To assess myocardial expression of SERCA and NCX protein levels in left ventricular (LV) remodelling due to chronic severe mitral regurgitation (MR). Myocardial expression of SERCA/NCX in biopsy specimens obtained during mitral surgery was assessed in 36 MR patients with LV remodelling and plasma neurohumoral/cytokine activation and in four non-failing hearts (NFH). Myocardial protein levels of SERCA were significantly (20%) lower in the MR group than in NFH group (p=0.016). No significant changes in NCX were observed. However, a lack of homogeneity with regard to SERCA/NCX proteins was observed. Moreover, SERCA was negatively correlated with BNP (r=-0.49, p=0.02), TNFalpha (r=-0.68, p=0.0005) and IL-6 (r=-0.52, p=0.02), whereas NCX was only negatively correlated with TNFalpha (r=-0.62, p=0.002). MR patients showed wide variations in SERCA/NCX protein expression. Myocardial protein levels of SERCA were significantly lower in the MR population. Moreover, a correlation between BNP, cytokines (IL-6, TNFalpha) and the expression of SERCA/NCX proteins was observed.

The Role of the Calcium Sensing Receptor in Regulating Intracellular Calcium Handling in Human Epidermal Keratinocytes

Calcium is critical for controlling the balance of proliferation and differentiation in epidermal keratinocytes. We previously reported that the calcium sensing receptor (CaR) is required for mediating Ca2+ signaling and extracellular Ca2+ (Ca2+(o))-induced differentiation. In this study, we investigated the mechanism by which CaR regulates intracellular Ca2+ (Ca2+(i)) and its role in differentiation. Membrane fractionation, fluorescence immunolocalization, and co-immunoprecipitation studies were performed to assess potential interactions between CaR and other regulators of Ca2+ stores and channels. We found that the glycosylated form of CaR forms a complex with phospholipase C gamma1, IP3 receptor (IP3R), and the Golgi Ca2+-ATPase, secretory pathway Ca2+-ATPase 1, in the trans-Golgi. Inactivation of the endogenous CaR gene by adenoviral expression of a CaR antisense cDNA inhibited Ca2+(i) response to Ca2+(o), decreased Ca2+(i) stores, decreased Ca2+(o)-induced differentiation, but augmented store-operated channel activity and Ca2+ uptake by intracellular organelles. Our results indicate that CaR regulates keratinocyte differentiation in part by modulating Ca2+(i) stores via interactions with Ca2+ pumps and channels that regulate those stores.

Angiotensin-converting enzyme inhibitor improves force and Ca2+-frequency relationships in myocytes from rats with heart failure

Objectives — The objectives were to study the effects of an ACE inhibitor on the force-frequency relationship (FFR) and its possible mechanism in isolated failing myocytes.Methods and results — Male Wistar rats were randomized into a heart failure group treated with perindopril (CHF-T, 3mg.kg-1.d-1), a heart failure group without treatment (CHF-C) and a sham-operated group (PS). Heart failure was induced by constriction of the abdominal aorta. All groups were further followed up for 12weeks. Left ventricular myocytes were isolated. Cell-shortening fraction (FS) and intracellular calcium transients were measured at different frequency field stimulations. A negative force-frequency relationship (FFR) was found in the CHF-C group compared with the positive-negative biphasic FFR in the PS group, and a flat FFR in the CHF-T group. Intracellular Ca2+ frequency relationships (CaFRs) were positive-negative biphasic in both the PS and CHF-T groups, whereas a negative CaFR was found in the CHF-C group. Regardless of the stimulation frequency, FS significantly correlated with [Ca2+]imax in the PS or CHF-C groups. Compared to the PS group, protein levels of SERCA2 significantly decreased and NCX1 increased in the CHF-C group. In the CHF-T group, these changes were reduced.Conclusion —The ACE inhibitor could improve the impaired FFR of isolated failing myocytes.This effect was possibly mediated via ameliorating the disturbance of CaFR.

Tight interplay between the Ca 2+ affinity of the cardiac SERCA2 Ca 2+ pump and the SERCA2 expression level

A reduced activity of the sarcoplasmic reticulum Ca 2+ pump SERCA2a is a hallmark of cardiac dysfunction in heart failure. In SERCA2 b/ b mice, the normal SERCA2a isoform is replaced by SERCA2b, displaying a higher Ca 2+ affinity. This elicited decreased cardiac SERCA2 expression and cardiac hypertrophy. Here, the interplay was studied between the increased Ca 2+ affinity and a reduced expression of the pump and its role in the cardiac remodeling was investigated. First, SERCA2 b/ 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 Ca 2+ affinity. Moreover, the higher SERCA2 content did not prevent hypertrophy. Second, we studied heterozygous SERCA2 b/ 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 SERCA2 b/ 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 Ca 2+ affinity (controlled by PLB). This compensatory response may be important to prevent cardiac remodeling.

Cardiac Hypertrophy and Reduced Contractility in Hearts Deficient in the Titin Kinase Region

Titin is a giant protein crucial for the assembly and elasticity of the sarcomere. Recently, titin has been linked to signal transduction through its kinase domain, which has been proposed to sense mechanical load. We developed a knockout in which expression of M-line-deficient titin can be induced in adult mice and investigated the role of the titin kinase region in cardiac function. Isolated heart experiments revealed that in titin M-line-deficient mice, the contractile response to beta-adrenergic agonists and extracellular calcium is reduced. However, the Ca2+ sensitivity and cooperativity of activation of skinned cardiac muscle were unchanged. In knockout mice, calcium transients showed a reduced rate of calcium uptake, and expression analysis showed reduced levels of calmodulin, phospholamban, and SERCA2. Ultimately, knockout mice developed cardiac hypertrophy and heart failure, which involves protein kinase C signal transduction but not the mitogen-activated protein kinase pathway. The titin kinase region emerges as a regulator of contractile function through effects on calcium handling and hypertrophy through protein kinase signal transduction. These novel functions of titin might provide a rationale for future therapeutic approaches to attenuate or reverse symptoms of heart failure.

Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 +/- 0.48 vs food-restricted group = 4.84 +/- 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 +/- 0.44 vs food-restricted group = 7.96 +/- 0.45, and control = 1.52 +/- 0.06 vs food-restricted group = 1.53 +/- 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

Lithium suppresses epidermal SERCA2 and PMR1 levels in the rat

Autosomal dominant mutations in the genes encoding the calcium ATPases SERCA2 and PMRI/SPCA1 cause the genodermatoses Darier disease (DD) and Hailey-Hailey disease (HHD), respectively. Recent observations indicated that the level of the pathogenic proteins greatly decreases in the affected areas of the epidermis in these disorders. Here we addressed how lithium, a recognized exacerbating factor in Darier disease, affects the epidermal expression of SERCA2 and PMR1/SPCA1 in the rat as a model. Standard histologic and immunohistochemical methods were utilized in 3 lithium-treated and 3 control animals. A significant suppression of epidermal SERCA2 and PMR1 levels were observed as a result of lithium therapy in addition to marked qualitative and quantitative changes in the stratum corneum and the granular layer of the epidermis in the treated animals. Our findings suggest that exacerbating factors in calcium ATPase disorders of the skin suppress epidermal SERCA2 and PMR1 levels, further decreasing the already haploinsufficient protein expression to a potentially critical level in Darier disease and Hailey-Hailey disease, respectively. Lithium therapy should specifically be avoided not only in Darier disease, but Hailey-Hailey disease as well.

Maintaining serum response factor activity in the older heart equal to that of the young adult is associated with better cardiac response to isoproterenol stress

To understand the effect of transcription regulation in modulating cardiac aging, we sought to study the role of serum response factor (SRF), a key transcription factor in the heart that is normally increased with senescence and also in congestive heart failure. A Tet-Off gene expression system was used for cardiac-specific over-expression of a mutant SRF protein. In these binary transgenic mice, there is no age-related increase in SRF protein expression; in fact, there appeared to be a mild reduction of SRF protein (Mild-R SRF Tg). The older, middle-aged (15 mo) Mild-R SRF Tg mice appeared healthier and were better able to maintain their left ventricular systolic pressure (LVSP) in response to moderate â-adrenergic stimulation compared with age-matched Non-Tg mice, which demonstrated a negative ionotropic response. The Mild-R SRF Tg hearts had lower mRNA expression of BNP (p < 0.05), and the sodium calcium exchanger (p < 0.05), compared to Non-Tg. Mild-R SRF Tg had higher mRNA levels of SERCA2 (p < 0.05) and ryanodine receptor 2 (p < 0.05) compared to Non-Tg hearts. These findings suggest that preventing the age-associated increase in SRF is associated with better preserved intracellular calcium handling and functional response to stress; it might be advantageous for the older adult heart. This mouse model could be helpful in elucidating the molecular mechanisms underlying certain age-related changes in cardiac reserve capacity and response to stress.

Reduced myocardial expression of calcium handling protein in patients with severe chronic mitral regurgitation☆☆☆

Left ventricle (LV) function was shown to be a principal determinant of morbidity and mortality in both uncorrected and surgically corrected mitral regurgitation (MR). However, the cellular mechanisms that develop in the LV remodeling secondary to volume overload in chronic severe MR is still not well defined. In single ventricular myocyte, a reduced contraction and slowed relaxation have been mainly attributed to defective intracellular Ca2+ currents. Between several Ca2+ handling proteins, sarcoplasmic reticulum Ca2+-ATPase 2 (SERCA2) expression and activity determines not only the extent and rate of relaxation, but also the rate and amplitude of contraction. The aim of the study was to determine whether modifications of SERCA2 gene expression occurs in LV wall remodeling process secondary to chronic severe MR. The LV samples were obtained from 12 patients presented LV wall remodeling (LV: diastolic/systolic diameter-70+/-7 mm vs 46+/-10 mm; diastolic/systolic volume-260+/-65 ml vs 102+/-68 ml) due to chronic, severe MR. Expressions of SERCA2 isoforms-SERCA2a and 2b mRNAs were estimated by semiquantitative RT-PCR and normalized to GAPDH. The protein levels of SERCA2 were determined by Western blot after normalization to actin. Results were compared with samples from non-failing human hearts (NFH). On SERCA2 mRNA levels, important reduction on both SERCA isoforms SERCA2a (-40%) and SERCA2b (-49%) compared to NFH, together with significant correlation between isoforms (r = 0.89; p = 0.01) were observed. SERCA2 protein levels were decreased (-38%) in MR compared to NFH. Also significant correlations between SERCA2a/2b and SERCA2 protein expression (r = 0.83, p = 0.017; r = 0.68, p = 0.05, respectively) were observed. Moreover, a negative correlation between protein levels of SERCA2 (r = -0.64, p = 0.053) and left ventricular diastolic diameter was observed. In chronic volume overload the down-regulation of SERCA2a and 2b at the mRNA and SERCA2 protein levels exist. Moreover, protein levels of SERCA2 tend to correlate to the grade of left ventricular diastolic dilatation and suggest an important role LV remodeling.

Relevance of Brain Natriuretic Peptide in Preload-Dependent Regulation of Cardiac Sarcoplasmic Reticulum Ca 2+ ATPase Expression

In heart failure (HF), ventricular myocardium expresses brain natriuretic peptide (BNP). Despite the association of elevated serum levels with poor prognosis, BNP release is considered beneficial because of its antihypertrophic, vasodilating, and diuretic properties. However, there is evidence that BNP-mediated signaling may adversely influence cardiac remodeling, with further impairment of calcium homeostasis. We studied the effects of BNP on preload-dependent myocardial sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression. In rabbit isolated muscle strips stretched to high preload and shortening isotonically over 6 hours, the SERCA/glyceraldehyde phosphate dehydrogenase mRNA ratio was enhanced by 168% (n=8) compared with unloaded preparations (n=8; P<0.001). Recombinant human BNP at a concentration typically found in end-stage HF patients (350 pg/mL) abolished SERCA upregulation by stretch (n=9; P<0.0001 versus BNP free). Inhibition of cyclic guanosine 3',5' monophosphate (cGMP)-phosphodiesterase-5 mimicked this effect, whereas inhibition of cGMP-dependent protein kinase restored preload-dependent SERCA upregulation in the presence of recombinant human BNP. Furthermore, in myocardium from human end-stage HF patients undergoing cardiac transplantation (n=15), BNP expression was inversely correlated with SERCA levels. Moreover, among 23 patients treated with left ventricular assist devices, significant SERCA2a recovery occurred in those downregulating BNP. Our data indicate that preload stimulates SERCA expression. BNP antagonizes this mechanism via guanylyl cyclase-A, cGMP, and cGMP-dependent protein kinase. This novel action of BNP to uncouple preload-dependent SERCA expression may adversely affect contractility in patients with HF.

Understanding Diastolic Heart Failure With Preserved Ejection Fraction

Congestive heart failure (HF) is a frequent reason for hospital admission. Fifty percent of HF hospitalizations have normal ejection fraction (EF), 1 referred to as diastolic HF (DHF). In contrast, patients with systolic HF (SHF) have reduced EF. The DHF phenotype describes patients with signs and symptoms of HF, normal EF, normal left ventricular (LV) volume, hypertension with increased relative wall thickness, LV diastolic dysfunction, and predominantly elderly women. 1,2 Etiology of DHF Longstanding hypertension may result in significant myocardial hypertrophy, appearance of a smaller ventricular chamber, increased ventricular stiffness, and delayed relaxation. Increased ventricular stiffness requires increased pressure to maintain normal filling in the presence of normal or reduced chamber volume.3 Although DHF has not been studied as extensively as SHF, limited data obtained from patients hospitalized for DHF suggest that demographic characteristics, comorbidities, and pathology differ from those with SHF. Despite these differences, DHF is similar to SHF with regard to volume overload, reduced exercise capacity, impaired quality of life, and long-term mortality.1

The Focal Nature of Darier's Disease Lesions: Calcium Pumps, Stress, and Mutation?

Haploinsufficiency of the ATP2A2 gene product, SERCA2, underlies most cases of Darier's disease. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase isoform 2 (SERCA2) is an intracellular Ca2+ pump that replenishes ER Ca2+, and it seems likely that the disease manifests in stress-induced lesions because SERCA levels become limiting as extra demands are made on the pump in times of stress. However, Müller and colleagues (2006) present a radical new proposal invoking somatic mutation as the basis for Darier lesions. Using a novel animal model for depleted keratinocyte SERCA-gated Ca2+ stores, the authors show that keratinocytes from Darier-like lesions retain their distinctive phenotype after culture, suggesting heritable defects. Mechanistically linking stress, calcium levels, mutation, and disease pathogenesis is complicated, and the proposal is likely to be controversial. However, recent reports of age- and stress-dependent tumor formation in the mouse model for SERCA2 haploinsufficiency (ATP2A2 heterozygous mouse) support the proposal that deficiency in SERCA-gated ER Ca2+ replenishment may be linked to mutation accumulation.

Phospholamban Ablation Rescues SR Ca2+ Loading But Not Cardiac Function In CaMKIIλC Transgenic Mice

Ca2+/calmodulin dependent kinase II (CaMKII) is a key candidate for controlling phosphorylation and function of Ca2+ regulatory proteins of the sarcoplasmic reticulum (SR), including the ryanodine receptors (RyR2) and phospholamban (PLB). The predominant form of CaMKII in the heart is the λ isoform. Previous work from our laboratory has shown that transgenic (TG) mice overexpressing CaMKIIλC in the heart develop dilated cardiomyopathy due to increased phosphorylation of the RyR2 and enhanced SR Ca2+ leak. The associated lowering of SR Ca2+ load likely contributes to decreased ventricular contractility. Since PLB limits SR Ca2+ uptake, we hypothesized that PLB ablation would recover SR Ca2+ load and rescue the heart failure phenotype. Accordingly, we crossed PLB −/ − mice with CaMKIIλ TG and found surprisingly, that the phenotype was exaggerated in the double mutant (DM) mice. DM showed severe dilated cardiomyopathy and contractile failure despite Ca2+ load repletion, and normalized twitch Ca2+ transients relative to TG. There was no significant change in expression levels of calsequestrin, SERCA, and Na+/Ca2+exchanger, major Ca2+ handling proteins. RyR2 phosphorylation at known CaMKII sites (Ser2815 and 2809) was not greater in DM vs TG mice. However, SR Ca2+ sparks were increased, presumably due to the normalized Ca2+ load. Elevated Ca2+ and CaMKII have been implicated in cardiomyocyte apoptosis and preliminary studies suggest an increase in TUNEL positive cells in DM. We are currently testing the hypothesis that increased apoptosis contributes to the more severe phenotype in DM vs TG.

Exercise training restores cardiac dysfunction and altered sarcoplasmic calcium reuptake in a genetic model of cardiomyopathy

We tested the hypothesis that exercise training (ET) would restore cardiac dysfunction and intracellular calcium regulation (ICR) in mice lacking α2A/α2C adrenoceptors(DKO). We studied a cohort of DKO and their wild type (WT) controls from 3–5 mo of age. Exercise training (ET) consisted of 8-wk swimming session. Exercise capacity was measured using a graded treadmill protocol. Heart rate (HR) was determined by tail cuff. Fractional shortening (FS) by echocardiography. Cardiac sympathetic tone (CST) was estimated by pharmacological blockade. The protein expression of cardiac SERCA2a, phospholamban (PLN), phospho-Ser16-PLN, Na+/Ca2+ exchanger (NCX), ryanodine receptor (RYR) and phospho-RYR2 were analyzed using Western blotting technique. At 5 mo of age, DKO displayed exercise intolerance and higher HR associated with significantly increased CST. In addition, DKO presented lower FS than WT mice. The impaired FS in DKO was paralleled by a reduction of SERCA2a and NCX expression. Conversely, phospho-RYR2 was increased in DKO when compared with WT mice. ET prevented exercise intolerance, and it restored baseline HR, FS and CST. The improved FS was associated with restored SERCA and phospho-RYR2 expression levels. Indeed, ET increased SERCA/PLB ratio in 5 mo-old DKO mice. Collectively we provide direct evidence for the beneficial effects of ET on cardiac function of DKO associated with improved ICR. Supported by FAPESP#02/04588-8 and 04/00745-7.

The Presence of Lys27 Instead of Asn27 in Human Phospholamban Promotes Sarcoplasmic Reticulum Ca 2+ -ATPase Superinhibition and Cardiac Remodeling

Phospholamban (PLN) is an inhibitor of the Ca2+ affinity of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2). The amino acid sequence of PLN is highly conserved, and although all species contain asparagine (Asn), human PLN is unique in containing lysine (Lys) at amino acid 27. Human PLN was introduced in the null background. Expression of human PLN, at similar levels to mouse wild-type PLN, resulted in significant decreases in the affinity of SERCA2 for Ca2+, attributed to unique spatial conformation of this PLN form and increases in its monomeric active unit compared with mouse PLN. The increased inhibition by human PLN was associated with attenuated cardiac contractility in the intact-animal, organ, and cardiomyocyte levels and with depressed calcium kinetics. These inhibitory effects could not be fully reversed even on maximal isoproterenol stimulation. There were no alterations in the expression levels of SERCA2, calsequestrin, ryanodine receptor, and FKBP12, although the sodium/calcium exchanger and the L-type Ca2+ channel expression levels were upregulated. The depressed function resulted in increased heart/body weight ratios and phosphorylation levels of Akt, p38, and Erk1/2. Human PLN may play a more inhibitory role than that of other species in Ca2+ cycling. Expression of human PLN in the mouse is compensated by alterations in Ca2+-handling proteins and cardiac remodeling in an effort to normalize cardiac contractility. Thus, the unique amino acid sequence of human PLN may be critical in maintaining a high cardiac reserve, which is of paramount importance in the regulation of human cardiac function.

Targeted Overexpression of Sarcolipin in the Mouse Heart Decreases Sarcoplasmic Reticulum Calcium Transport and Cardiac Contractility

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca(2+) ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by beta-adrenergic agonists.

Increased phospholamban phosphorylation limits the force–frequency response in the MLP–/– mouse with heart failure

Reduced Ca(2+) release from the sarcoplasmic reticulum (SR) and a negative force-frequency relation characterize end-stage human heart failure. The MLP(-/-) mouse with dilated cardiomyopathy is used as a model to explore novel therapeutic interventions but the alterations in Ca(2+) handling in MLP(-/-) remain incompletely understood. We studied [Ca(2+)](i) in left ventricular myocytes from MLP(-/-) and WT mice (3-4 months old; whole-cell voltage clamp, 30 degrees C). At 1 Hz stimulation, the amplitude of [Ca(2+)](i) transients was similar. However, in contrast to WT, at higher frequencies the [Ca(2+)](i) transient amplitude declined in MLP(-/-) and there was no increase in SR Ca(2+) content. Unexpectedly, the decline of [Ca(2+)](i) was faster in MLP(-/-) than in WT (at 1 Hz, tau of 80 +/- 9 vs. 174 +/- 29 ms, P < 0.001) and the frequency-dependent acceleration of the decline was abolished suggesting an enhanced basal SERCA activity. Indeed, the Ca(2+) affinity of SR Ca(2+) uptake in homogenates was higher in MLP(-/-), with the maximal uptake rate similar to WT. Phosphorylation of phospholamban in MLP(-/-) was increased (2.3-fold at Ser(16) and 2.9-fold at the Thr(17) site, P < 0.001) with similar SERCA and total phospholamban protein levels. On increasing stimulation frequency to 4 Hz, WT, but not MLP(-/-), myocytes had a net gain of Ca(2+), suggesting inadequate Ca(2+) sequestration in MLP(-/-). In conclusion, increased baseline phosphorylation of phospholamban in MLP(-/-) leads to a reduced reserve for frequency-dependent increase of Ca(2+) release. This represents a novel paradigm for altered Ca(2+) handling in heart failure, underscoring the importance of phosphorylation pathways.

Calcium Uptake and Release through Sarcoplasmic Reticulum in the Inferior Oblique Muscles of Patients with Inferior Oblique Overaction

We characterized and compared the characteristics of Ca2+ movements through the sarcoplasmic reticulum of inferior oblique muscles in the various conditions including primary inferior oblique overaction (IOOA), secondary IOOA, and controls, so as to further understand the pathogenesis of primary IOOA. Of 15 specimens obtained through inferior oblique myectomy, six were from primary IOOA, 6 from secondary IOOA, and the remaining 3 were controls from enucleated eyes. Ryanodine binding assays were performed, and Ca2+ uptake rates, calsequestrins and SERCA levels were determined. Ryanodine bindings and sarcoplasmic reticulum Ca2+ uptake rates were significantly decreased in primary IOOA (p<0.05). Western blot analysis conducted to quantify calsequestrins and SERCA, found no significant difference between primary IOOA, secondary IOOA, and the controls. Increased intracellular Ca2+ concentration due to reduced sarcoplasmic reticulum Ca2+ uptake may play a role in primary IOOA.

Effects of simvastatin on cardiac performance and expression of sarco-plasmic reticular calcium regulatory proteins in rat heart

To investigate the effect of simvastatin on the cardiac contractile function and the alteration of gene and protein expression of the sarcoplasmic calcium regulatory proteins, including sarcoplasmic reticulum Ca2+-ATPase (SERCA), phospholamban (PLB), and ryanodine receptor 2 (RyR2) in rat hearts. Langendorff-perfused rat hearts were subjected to 60-min perfusion with different concentrations of simvastatin (1, 3, 10, 30, or 100 microml/L), and the parameters of cardiac function such as left ventricular developed pressure (LVDP), +dp/dtmax, and -dp/dtmax were determined. The cultured neonatal rat ventricular cardiomyocytes were incubated with simvastatin (1, 3, 10, 30, and 100 micromol/L) for 1 h or 24 h. The levels of SERCA, PLB, and RyR2 expression were measured by reverse transcription-polymerase chain reaction and Western blot. Cytotoxic effect of simvastatin on ventricular cardiomyocytes was assessed by the MTT colorimetric assay. LVDP, +dp/dtmax, and -dp/dtmax of hearts were increased significantly after treatment with simvastatin 3, 10, and 30 micromol/L. In simvastatin-treated isolated hearts, the levels of mRNA expression of SERCA and RyR2 were elevated compared with the control (P<0.05), while the mRNA expression of PLB did not change. After the cultured neonatal rat ventricular cardiomyocytes were incubated with 3, 10, 30, and 100 mumol/L simvastatin for 1 h, SERCA and RyR2 mRNA expressions of cardiomyocytes rose, but there was no alteration in protein expressions. However, with the elongation of simvastatin treatment to 24 h, the protein expression of SERCA and RyR2 were also elevated. Additionally, simvastatin (1-30 micromol/L) had no influence on cell viability of cultured cardiac myocytes, but simvastatin 100 micromol/L inhibited the cell viability. Simvastatin improved cardiac performance accompanied by the elevation of SERCA and RyR2 gene and protein expression.

Effects of sarcoplasmic reticulum Ca2+-ATPase overexpression in postinfarction rat myocytes

Previous studies in adult myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated abnormal contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) homeostasis and decreased sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) expression and activity, but sarcoplasmic reticulum Ca(2+) leak was unchanged. In the present study, we investigated whether SERCA2 overexpression in MI myocytes would restore contraction and [Ca(2+)](i) transients to normal. Compared with sham-operated hearts, 3-wk MI hearts exhibited significantly higher left ventricular end-diastolic and end-systolic volumes but lower fractional shortening and ejection fraction, as measured by M-mode echocardiography. Seventy-two hours after adenovirus-mediated gene transfer, SERCA2 overexpression in 3-wk MI myocytes did not affect Na(+)-Ca(2+) exchanger expression but restored the depressed SERCA2 levels toward those measured in sham myocytes. In addition, the reduced sarcoplasmic reticulum Ca(2+) uptake in MI myocytes was improved to normal levels by SERCA2 overexpression. At extracellular Ca(2+) concentration of 5 mM, the subnormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were restored to normal by SERCA2 overexpression. However, at 0.6 mM extracellular Ca(2+) concentration, the supernormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were exacerbated by SERCA2 overexpression. We conclude that SERCA2 overexpression was only partially effective in ameliorating contraction and [Ca(2+)](i) transient abnormalities in our rat model of ischemic cardiomyopathy. We suggest that other Ca(2+) transport pathways, e.g., Na(+)-Ca(2+) exchanger, may also play an important role in contractile and [Ca(2+)](i) homeostatic abnormalities in MI myocytes.