The sarco(endo)plasmic reticulum Ca 2؉ -ATPase isoform 2 (SERCA2) gene encodes both SERCA2a, the cardiac sarcoplasmic reticulum Ca 2؉ pump, and SERCA2b, which is expressed in all tissues. To gain a better understanding of the physiological functions of SERCA2, we used gene targeting to develop a mouse in which the promoter and 5 end of the gene were eliminated. Mating of heterozygous mutant mice yielded wild-type and heterozygous offspring; homozygous mutants were not observed. RNase protection, Western blotting, and biochemical analysis of heart samples showed that SERCA2 mRNA was reduced by ϳ45% in heterozygous mutant hearts and that SERCA2 protein and maximal velocity of Ca 2؉ uptake into the sarcoplasmic reticulum were reduced by ϳ35%. Measurements of cardiovascular performance via transducers in the left ventricle and right femoral artery of the anesthetized mouse revealed reductions in mean arterial pressure, systolic ventricular pressure, and the absolute values of both positive and negative dP/dt in heterozygous mutants. These results demonstrate that two functional copies of the SERCA2 gene are required to maintain normal levels of SERCA2 mRNA, protein, and Ca 2؉ sequestering activity, and that the deficit in Ca 2؉ sequestering activity due to the loss of one copy of the SERCA2 gene impairs cardiac contractility and relaxation. The SERCA21 gene encodes two Ca 2ϩ -transporting ATPases, SERCA2a and SERCA2b, which differ in their C-terminal sequences as a result of alternative splicing (1-3). SERCA2a is expressed at highest levels in heart, where it plays a central role in cardiomyocyte Ca 2ϩ handling required for excitation/ contraction coupling (reviewed in Ref. 4). Contraction is initiated by an increase in Ca 2ϩ concentrations around the myofibrils, which occurs as Ca 2ϩ is released from the SR or enters the cell via channels in the sarcolemma. SERCA2a pumps Ca 2ϩ out of the cytosol and into the SR, thereby contributing to the low diastolic Ca 2ϩ levels required for relaxation and replenishing Ca 2ϩ stores needed for the next contraction. SERCA2a serves a similar function in slow twitch skeletal muscle and is also expressed in some smooth muscles (5). In contrast to the limited tissue distribution and organ-specific function of SERCA2a, SERCA2b is expressed in all tissues and it has been suggested that it plays an essential housekeeping role (1), although it undoubtedly serves some organ-specific functions as well.An important role for SERCA2a in cardiac function is well established. Recent studies have shown that the levels of SERCA2a are decreased in several animal models of cardiac hypertrophy and human heart failure (reviewed in Refs. 6 and 7). However, it is currently unknown whether, and to what extent, the reductions in SERCA2a levels contribute to altered contractile function. In addition, it is unclear whether there are homeostatic mechanisms within the cardiac myocyte that are capable of sensing perturbations in the levels of pump activity and adjusting its expression accordingly. These are i...

Abstract-Cardiac hypertrophy and heart failure are known to be associated with a reduction in Ca 2ϩ -ATPase pump levels of the sarcoplasmic reticulum (SR). To determine whether, and to what extent, alterations in Ca 2ϩ pump numbers can affect contraction and relaxation parameters of the heart, we have overexpressed the cardiac SR Ca 2ϩ -ATPase specifically in the mouse heart using the ␣-myosin heavy chain promoter. Analysis of 2 independent transgenic lines demonstrated that sarco(endo)plasmic reticulum Ca 2ϩ -ATPase isoform (SERCA2a) mRNA levels were increased 3.88Ϯ0.4-fold and 7.90Ϯ0.2-fold over those of the control mice. SERCA2a protein levels were increased by 1.31Ϯ0.05-fold and 1.54Ϯ0.05-fold in these lines despite high levels of mRNA, suggesting that complex regulatory mechanisms may determine the SERCA2a pump levels. The maximum velocity of Ca 2ϩ uptake (V max ) was increased by 37%, demonstrating that increased pump levels result in increased SR Ca 2ϩ uptake function. However, the apparent affinity of the SR Ca 2ϩ -ATPase for Ca 2ϩ remains unchanged in transgenic hearts. To evaluate the effects of overexpression of the SR Ca 2ϩ pump on cardiac contractility, we used the isolated perfused work-performing heart model. The transgenic hearts showed significantly higher myocardial contractile function, as indicated by increased maximal rates of pressure development for contraction (ϩdP/dt) and relaxation (-dP/dt), together with shortening of the normalized time to peak pressure and time to half relaxation. Measurements of intracellular free calcium concentration and contractile force in trabeculae revealed a doubling of Ca 2ϩ transient amplitude, with a concomitant boost in contractility. The present study demonstrates that increases in SERCA2a pump levels can directly enhance contractile function of the heart by increasing SR Ca 2ϩ transport. (Circ Res. 1998;83:1205-1214.)Key Words: sarcoplasmic reticulum Ca 2ϩ -ATPase Ⅲ transgenic mice Ⅲ Ca 2ϩ uptake Ⅲ working heart model T he sarcoplasmic reticulum (SR) plays a central role in the contraction-and-relaxation cycle of the heart by regulating intracellular calcium (Ca 2ϩ ) concentrations (reviewed in Reference 1). Ca 2ϩ release from the SR via the ryanodine receptor initiates muscle contraction, whereas Ca 2ϩ reuptake into the lumen of the SR leads to muscle relaxation. The Ca 2ϩ uptake function of the SR is driven by an ATP-dependent Ca 2ϩ transport pump, the sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA). Molecular cloning analyses have identified a family of SERCA pumps encoded by 3 highly homologous genes (SERCA1, SERCA2, and SERCA3). [2][3][4][5][6][7][8] The SERCA2 gene encodes 2 isoforms, SERCA2a and SERCA2b, which differ at the COOH terminus as a result of alternative splicing (SERCA2a comprises 4 amino acids, and SERCA2b comprises 49 amino acids). [5][6][7] SERCA2a is the primary SERCA isoform expressed in the heart and is also present in slow-twitch skeletal muscle, smooth muscle, and fetal fast-twitch muscle. 9,10 In the rat heart, SERCA2a expr...

In this study, we investigated whether the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ transport pump (SERCA1a) can functionally substitute the cardiac SERCA2a isoform and how its overexpression affects cardiac contractility. For this purpose, we generated transgenic (TG) mice that specifically overexpress SERCA1a in the heart, using the cardiac-specific alpha-myosin heavy chain promoter. Ectopic expression of SERCA1a resulted in a 2.5-fold increase in the amount of total SERCA protein. At the same time, the level of the endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin, and tropomyosin, remained unchanged. The steady-state level of SERCA phosphoenzyme intermediate was increased 2.5-fold, and the maximal velocity of Ca2+ uptake was increased 1.7-fold in TG hearts, demonstrating that the overexpressed protein is functional. Although the basal cytosolic calcium signal was decreased by 38% in TG cardiomyocytes, the amplitude of cytosolic calcium signal was increased by 71.8%. The rate of calcium resequestration was also increased in TG myocytes, which was reflected by a 51.6% decrease in the normalized time to 80% decay of calcium signal. This resulted in considerably increased peak rates of myocyte shortening and relengthening (50.0% and 66.6%, respectively). Cardiac functional analysis using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in TG hearts (41.9% and 39.5%, respectively). The time to peak pressure and the time to half-relaxation were shorter (29.1% and 32.7%, respectively). In conclusion, our study demonstrates that the SERCA1a pump can functionally substitute endogenous SERCA2a, and its overexpression significantly enhances Ca2+ transport and contractile function of the myocardium. These results also demonstrate that the SERCA pump level is a critical determinant of cardiac contractility.

We used an exon-specific gene-targeting strategy to generate a mouse model deficient only in the SM-B myosin isoform. Here we show that deletion of exon-5B (specific for SM-B) in the gene for the heavy chain of smooth muscle myosin results in a complete loss of SM-B myosin and switching of splicing to the SM-A isoform, without affecting SM1 and SM2 myosin content. Loss of SM-B myosin does not affect survival or cause any overt smooth muscle pathology. Physiological analysis reveals that absence of SM-B myosin results in a significant decrease in maximal force generation and velocity of shortening in smooth muscle tissues. This is the first in vivo study to demonstrate a functional role for the SM-B myosin isoform. We conclude that the extra seven-residue insert in the surface loop 1 of SM-B myosin is a critical determinant of crossbridge cycling and velocity of shortening.

We recently generated a transgenic (TG) mouse model in which the fast-twitch skeletal muscle sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA1a) is overexpressed in the heart. Ectopic overexpression of SERCA1a results in remodeling of the cardiac SR containing 80% SERCA1a and 20% endogenous SERCA2a with an ∼2.5-fold increase in the total amount of SERCA protein (E. Loukianov et al. Circ. Res. 83: 889–897, 1998). We have analyzed the Ca2+ transport properties of membranes from SERCA1a TG hearts in comparison to control hearts. Our data show that the maximal velocity of SR Ca2+ transport was significantly increased (∼1.9-fold) in TG hearts, whereas the apparent affinity of the SERCA pump for Ca2+ was not changed. Addition of phospholamban antibody in the Ca2+ uptake assays increased the apparent affinity for Ca2+ to the same extent in TG and non-TG (NTG) hearts, suggesting that phospholamban regulates the SERCA1a pump in TG hearts. Analysis of SERCA enzymatic properties in TG hearts revealed that the SERCA pump affinity for ATP, the Hill coefficient, the pH dependence of Ca2+ uptake, and the effect of acidic pH on Ca2+ transport were similar to those of NTG hearts. Interestingly, the rate constant of phosphoenzyme decay (turnover rate of SERCA enzyme) was also very similar between TG and NTG hearts. Together these findings suggest that 1) the SERCA1a pump can functionally substitute for SERCA2a and is regulated by endogenous phospholamban in the heart and 2) SERCA1a exhibits several enzymatic properties similar to those of SERCA2a when expressed in a cardiac setting.

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Findings from our limited sample size suggest that peripubertal environmental exposures are associated with sperm DNA methylation in young adults.

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