Moderate heart dysfunction in mice with inducible cardiomyocyte-specific excision of the Serca2 gene (original) (raw)
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Journal of Biological Chemistry, 2000
The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca 2؉ affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately ϳ20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calciumdependent calcium uptake measurements showed that the maximal velocity of Ca 2؉ uptake was not changed, but the apparent pump affinity for Ca 2؉ (K 0.5) was increased in SERCA2b transgenic mice (0.199 ؎ 0.011 M) compared with wild-type control mice (0.269 ؎ 0.012 M, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.
Journal of Biological Chemistry, 2000
A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca 2؉-ATPase isoform 2 (SERCA2) gene, in which SERCA2 protein levels are reduced by ϳ35%, was used to investigate the effects of decreased SERCA2 level on intracellular Ca 2؉ homeostasis and contractile properties in isolated cardiomyocytes. When compared with wild-type controls, SR Ca 2؉ stores and Ca 2؉ release in myocytes of SERCA2 heterozygous mice were decreased by ϳ40-60% and ϳ30-40%, respectively, and the rate of myocyte shortening and relengthening were each decreased by ϳ40%. However, the rate of Ca 2؉ transient decline () was not altered significantly, suggesting that compensation was occurring in the removal of Ca 2؉ from the cytosol. Phospholamban, which inhibits SERCA2, was decreased by ϳ40% in heterozygous hearts, and basal phosphorylation of Ser-16 and Thr-17, which relieves the inhibition, was increased ϳ2and 2.1-fold. These results indicate that reduced expression and increased phosphorylation of phospholamban provides compensation for decreased SERCA2 protein levels in heterozygous heart. Furthermore, both expression and current density of the sarcolemmal Na ؉-Ca 2؉ exchanger were up-regulated. These results demonstrate that a decrease in SERCA2 levels can directly modify intracellular Ca 2؉ homeostasis and myocyte contractility. However, the resulting deficit is partially compensated by alterations in phospholamban/SERCA2 interactions and by up-regulation of the Na ؉-Ca 2؉ exchanger.
Proceedings of the National Academy of Sciences, 2004
Transient elevations of cytosolic Ca 2؉ are a common mechanism of cellular signaling. In striated muscle, the sarco(endo)plasmic reticulum Ca 2؉ ATPase (SERCA) plays an important role in terminating Ca 2؉ transients by returning cytosolic Ca 2؉ to intracellular stores. Stored Ca 2؉ can then be released again for subsequent signaling. We down-regulated SERCA2 gene expression in cultured cardiac myocytes by means of endogenous transcription of small interfering RNA encoded by an exogenous cDNA template. The cDNA template was delivered by adenovirus vector. Reduction of SERCA expression in all myocytes in culture was documented by immunochemistry, real-time RT-PCR, and determination of ATP-dependent Ca 2؉ transport. The reduction of SERCA2 expression was associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na ؉ ͞Ca 2؉ exchanger, indicating that intracellular store deficiency was compensated for by Ca 2؉ fluxes through the plasma membrane. In fact, SERCA silencing was followed by increased transcription of Na ؉ ͞ Ca 2؉ exchanger, TRPC4, TRPC5, and related transcriptional factors, such as stimulating protein 1, myocyte enhancer factor 2, and nuclear factor of activated cells 4, through activation of calcineurin. This finding demonstrates that the observed compensation occurs through transcriptional crosstalk and the remodeling of Ca 2؉ signaling pathways. The wide significance of this regulatory mechanism is related to its general involvement in Ca 2؉ signaling dynamics and in cardiac development and hypertrophy.
Journal of Biological Chemistry, 1999
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.
AJP: Heart and Circulatory Physiology, 2014
Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2 transports Ca2+ from the cytosol into the sarcoplasmic reticulum of cardiomyocytes and is essential for maintaining myocardial Ca2+ handling and thus the mechanical function of the heart. SERCA2 is a major ATP consumer in excitation-contraction coupling but is regarded to contribute to energetically efficient Ca2+ handling in the cardiomyocyte. Previous studies using cardiomyocyte-specific SERCA2 knockout (KO) mice have demonstrated that decreased SERCA2 activity reduces the Ca2+ transient amplitude and induces compensatory Ca2+ transport mechanisms that may lead to more inefficient Ca2+ transport. In this study, we examined the relationship between left ventricular (LV) function and myocardial O2 consumption (MV̇o2) in ex vivo hearts from SERCA2 KO mice to directly measure how SERCA2 elimination influences mechanical and energetic features of the heart. Ex vivo hearts from SERCA2 KO hearts developed mechanical dysfunction at 4 wk an...
Regulation and rate limiting mechanisms of Ca2+ ATPase (SERCA2) expression in cardiac myocytes
Molecular and Cellular Biochemistry, 2012
Involvement of the calcineurin/NFAT pathway in transcription of cardiac sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) was demonstrated (Prasad and Inesi, Am J Physiol Heart Circ Physiol 300(1):H173-H180, 2011) by upregulation of SERCA2 following calcineurin (CN) activation by cytosolic Ca(2+), and downregulation of SERCA2 following CN inhibition with cyclosporine (CsA) or CN subunits gene silencing. We show here that in cultured cardiac myocytes, competitive engagement of the CN/NFAT pathway is accompanied by downregulation of SERCA2 and Ca(2+) signaling alterations. In fact, SERCA2 downregulation occurs following infection of myocytes with adenovirus vectors carrying luciferase or SERCA1 cDNA under control of NFAT-dependent promoters, but not under control of CMV promoters that do not depend on NFAT. SERCA2 downregulation is demonstrated by comparison with endogenous transcription and protein expression standards such as GAPDH and actin, indicating prominent SERCA2 involvement by the CN/NFAT pathway. Transcription of genes involved in hypertrophy, triggered by adrenergic agonist or by direct protein kinase C (PKC) activation with phorbol 12-myristate 13-acetate (PMA), is also prominently dependent on CN/NFAT. This is demonstrated by CN inhibition with CsA, CN subunits gene silencing with siRNA, displacement of NFAT from CN with 9,10-Dihydro-9,10[1&amp;amp;amp;#39;,2&amp;amp;amp;#39;]-benzenoanthracene-1,4-dione (INCA-6), and myocyte infection with vectors carrying luciferase cDNA under control of NFAT-dependent promoter. We show here that competitive engagement of the CN/NFAT pathway by endogenous genes involved in hypertrophy produces downregulation of SERCA2, reduction of Ca(2+) transport and inadequate Ca(2+) signaling. It is most interesting that, in the presence of adrenergic agonist, specific protein kinase C (PKC) inhibition with 3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (Gö 6983) prevents development of hypertrophy and maintains adequate SERCA2 levels and Ca(2+) signaling.
Cell Calcium, 2003
Adult SERCA2 b/b mice expressing the non-muscle Ca 2+ transport ATPase isoform SERCA2b in the heart instead of the normally predominant sarcomeric SERCA2a isoform, develop mild concentric ventricular hypertrophy with impaired cardiac contractility and relaxation [Circ. Res. 89 (2001) 838]. Results from a separate study on transgenic mice overexpressing SERCA2b in the normal SERCA2a context were interpreted to show that SERCA2b and SERCA2a are differentially targeted within the cardiac sarcoplasmic reticulum (SR) [J. Biol. Chem. 275 ]. Since a different subcellular distribution of SERCA2b could underlie alterations in Ca 2+ handling observed in SERCA2 b/b , we wanted to compare SERCA2b distribution in SERCA2 b/b with that of SERCA2a in wild-type (WT). Using confocal microscopy on immunostained fixed myocytes and BODIPY-thapsigargin-stained living cells, we found that in SERCA2 b/b mice SERCA2b is correctly targeted to cardiac SR and is present in the same SR regions as SERCA2a and SERCA2b in WT. We conclude that there is no differential targeting of SERCA2a and SERCA2b since both are found in the longitudinal SR and in the SR proximal to the Z-bands. Therefore, alterations in Ca 2+ handling and the development of hypertrophy in adult SERCA2 b/b mice do not result from different SERCA2b targeting.