Phospholamban is the regulator of the Ca'+-ATPase in cardiac sarcoplasmic reticulum (SR), and it has been suggested to be an important determinant in the inotropic responses of the heart to 8-adrenergic stimulation. To determine the role of phospholamban in vivo, the gene coding for this protein was targeted in murine embryonic stem cells, and mice deficient in phospholamban were generated. The phospholamban-deficient mice showed no gross developmental abnormalities but exhibited enhanced myocardial performance without changes in heart rate. The time to peak pressure and the time to half-relaxation were significantly shorter in phospholamban-deficient mice compared with their wild-type homozygous littermates as assessed in work-performing mouse heart preparations under identical venous returns, afterloads, and heart rates. The first derivatives of intraventricular pressure (±dP/dt) were also significantly elevated, and this was associated with an increase in the affinity of the SR Ca +-ATPase for Ca`in the phospholamban-deficient hearts. Baseline levels of these parameters in the phospholamban-deficient hearts were equal to those observed in hearts of wild-type littermates maximally stimulated with the (-agonist isoproterenol. These findings indicate that phospholamban acts as a critical repressor of basal myocardial contractility and may be the key phosphoprotein in mediating the heart's contractile responses to f-adrenergic agonists. (Circ Res. 1994; 75:401-409.) Key Words * phospholamban * gene targeting . sarcoplasmic reticulum * cardiac contractility * l3-agonists C ardiac 8-adrenergic stimulation is associated with increases in the force of contraction and in the rates of rise and fall of force. These changes are mediated by increases in cAMP levels, which lead to phosphorylation of key regulatory proteins that may act as effectors of the adrenergic stimulation. One of these phosphoproteins is phospholamban, the regulator of the Ca`+-ATPase in cardiac sarcoplasmic reticulum (SR). Dephosphorylated phospholamban is an inhibitor of the Ca2`-ATPase activity, and phosphorylation relieves this inhibition.' The inhibition has been suggested to involve physical direct interaction between the two proteins,23 followed by conformational changes in the SR Ca2`-ATPase.

It is well accepted that inhibition of the Na,K-ATPase in the heart, through effects on the Na/Ca exchanger, raises the intracellular Ca2+ concentration and strengthens cardiac contraction. However, the contribution that individual isoforms make to this calcium regulatory role is unknown. Assessing the phenotypes of mouse hearts with genetically reduced levels of Na,K-ATPase alpha 1 or alpha 2 isoforms clearly demonstrates different functional roles for these isoforms in vivo. Heterozygous alpha 2 hearts are hypercontractile as a result of increased calcium transients during the contractile cycle. In contrast, heterozygous alpha 1 hearts are hypocontractile. The different functional roles of these two isoforms are further demonstrated since inhibition of the alpha 2 isoform with ouabain increases the contractility of heterozygous alpha 1 hearts. These results definitively illustrate a specific role for the alpha 2 Na,K-ATPase isoform in Ca2+ signaling during cardiac contraction.

Tropomyosins comprise a family of actin-binding proteins that are central to the control of calcium-regulated striated muscle contraction. To understand the functional role of tropomyosin isoform differences in cardiac muscle, we generated transgenic mice that overexpress striated muscle-specific ␤-tropomyosin in the adult heart. Nine transgenic lines show a 150-fold increase in ␤-tropomyosin mRNA expression in the heart, along with a 34-fold increase in the associated protein. This increase in ␤-tropomyosin message and protein causes a concomitant decrease in the level of ␣-tropomyosin transcripts and their associated protein. There is a preferential formation of the ␣␤-heterodimer in the transgenic mouse myofibrils, and there are no detectable alterations in the expression of other contractile protein genes, including the endogenous ␤-tropomyosin isoform. When expression from the ␤-tropomyosin transgene is terminated, ␣-tropomyosin expression returns to normal levels. No structural changes were observed in these transgenic hearts nor in the associated sarcomeres. Interestingly, physiological analyses of these hearts using a work-performing model reveal a significant effect on diastolic function. As such, this study demonstrates that a coordinate regulatory mechanism exists between ␣-and ␤-tropomyosin gene expression in the murine heart, which results in a functional correlation between ␣-and ␤-tropomyosin isoform content and cardiac performance.

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...

The ␣ -myosin heavy chain ( ␣ -MyHC) is the major contractile protein expressed in the myocardium of adult mice. We have produced mice carrying a null mutation of ␣ -MyHC by homologous recombination in murine ES cells. Homozygous null animals die between 11 and 12 d in utero of gross heart defects, while ␣ -MyHC ϩ / Ϫ heterozygotes survive and appear externally normal. The presence of a single functional ␣ -MyHC ϩ allele in heterozygous animals results in reduced levels of the transcript and protein as well as fibrosis and alterations in sarcomeric structure. Examination of heart function using a working heart preparation revealed severe impairment of both contractility and relaxation in a subset of the ␣ -MyHC ϩ / Ϫ animals. Thus, two ␣ -MyHC ϩ alleles are necessary for normal cardiac development, and hemizygosity for the normal allele can result in altered cardiac function. ( J. Clin. Invest. 1996Invest. . 98:1906Invest. -1917