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.

2؉transport pump whose expression is regulated during skeletal and cardiac muscle development and in response to various pathophysiological and hormonal states. Employing transient transfection analyses in Sol8 muscle cells, we have identified two positive regulatory regions, one distal (؊1810 base pair (bp) to ؊1110 bp) and one proximal (؊284 bp to ؊72 bp), within the SERCA2 promoter. The proximal promoter region from ؊284 bp to ؊80 bp was shown to confer muscle-specific expression to a heterologous promoter in Sol8 cells. This region is highly GC-rich containing the consensus sequence for four Sp1 elements (GGGCGG) and three Sp1-like elements (GGGAGG). DNase I footprint analysis with Sol8 nuclear extracts and purified Sp1 protein showed the protection of the seven Sp1 binding sites. In addition, site-directed mutagenesis of the Sp1 consensus sites demonstrated that Sp1 sites are essential for the muscle-specific expression of the SERCA2 promoter. Furthermore, we demonstrate that cotransfection of an Sp1 expression vector together with SERCA2-CAT constructs can up-regulate SERCA2 promoter activity. These results imply that the Sp1 transcription factor plays an important role in the transcriptional regulation of SERCA2 within muscle cells.

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