Abstract
Ca2+ signaling plays an essential role in several functions of cardiac myocytes. Transient rises and reductions of cytosolic Ca2+, permitted by the sarcoplasmic reticulum Ca2+ ATPase (SERCA2) and other proteins, control each cycle of contraction and relaxation. Here we provide a practical method for isolation of neonatal rat cardiac myocytes and measurement of Ca2+ transients in cultured cardiac myocytes, yielding information on kinetic resolution of the transients, variations of cytosolic Ca2+ concentrations, and adequacy of intracellular Ca2+ stores. We also provide examples of experimental perturbations that can be used to assess the contribution of SERCA2 to Ca2+ signaling.
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References
Carafoli E (2002) Calcium signaling: a tale for all seasons. Proc Natl Acad Sci 99:1115–1122
Clapham DE (2007) Calcium signaling. Cell 131:1047–1058
Bers DM (2008) Calcium cycling and signaling in cardiac myocytes. Ann Rev Physiol 70:23-49
Inesi G, Ebashi S, Watanabe S (1964) Preparation of vesicular relaxing factor from bovine heart tissue. Am J Physiol 207:1339–1344
Zarain-Herzberg A, MacLennan DH, Periasamy M (1990) Characterization of rabbit cardiac sarco(endo)plasmic reticulum Ca2+-ATPase gene. J Biol Chem 265:4670–4677
Lytton J, Westlin M, Burk SE, Shull GE, MacLennan DH (1992) Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps. J Biol Chem 267:14483–14489
Dally S, Bredoux R, Corvazier E, Anderson JP, Clausen JD,Dode L, Fanchaouy M, Gelebart P, Monceau V, Del Monte F, Gwathmey JK, Hajjar R, Chaabane C, Bobe R, Raies A, Enouf J (2006) Ca2+-ATPases in non-failing and failing heart: evidence for a novel cardiac sarco/endoplasmic reticulum Ca2+-ATPase 2 isoform (SERCA2). Biochem J 395:249–258
Prasad AM, Inesi G (2009) Effects of thapsigargin and phenylephrine on calcineurin and protein kinase C signaling functions in cardiac myocytes. Am J Physiol Cell Physiol 296:C992–C1002
Dani AM, Cittadini A, Inesi G (1979) Calcium transport and contractile activity in dissociated mammalian heart cells. Am J Physiol 237:C147–C155
Cavagna M, O’Donnell JM, Sumbilla C, Inesi G, Klein MG (2000) Exogenous Ca2+-ATPase isoform effects on Ca2+ transients of embryonic chicken and neonatal rat cardiac myocytes. J Physiol 528:53–63
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450
Flesch M, Schwinger RH, Schnabel P, Schiffer F, van Gelder I, Bavendiek U, Südkamp M, Kuhn-Regnier F, Böhm M (1996) Sarcoplasmic reticulum Ca2+ATPase and phospholamban mRNA and protein levels in end-stage heart failure due to ischemic or dilated cardiomyopathy. J Mol Med 74:321–332
Kirby MS, Sagara Y, Gaa S, Inesi G, Lederer WJ, Rogers TB (1992) Thapsigargin inhibits contraction and Ca2+ transient in cardiac cells by specific inhibition of the sarcoplasmic reticulum Ca2+ pump. J Biol Chem 267:12545–12551
Prasad AM, Ma H, Sumbilla C, Lee DI, Klein MG, Inesi G (2007) Phenylephrine hypertrophy, Ca2+-ATPase (SERCA2), and Ca2+ – signaling in neonatal rat cardiac myocytes. Am J Physiol Cell Physiol 292:C2269–C2275
Periasamy M, Reed TD, Liu LH, Ji Y, Loukianov E, Paul RJ, Nieman ML, Riddle T, Duffy JJ, Doetschman T, Lorenz JN, Shull GE (1999) Impaired cardiac performance in heterozygous mice with a null mutation in the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene. J Biol Chem 274:2556–2562
Seth M, Sumbilla C, Mullen SP, Lewis D, Klein MG, Hussain A, Soboloff J, Gill DL, Inesi G (2004) Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes. Proc Natl Acad Sci USA 101:16683–16688
He H, Giordano FJ, Hilal-Dandan R, Choi DJ, Rockman HA, McDonough PM, Bluhm WF, Meyer M, Sayen MR, Swanson E, Dillman WH (1997) Overexpression of the rat sarcoplasmic reticulum Ca2+ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation. J Clin Invest 100:380–389
Baker DL, Hashimoto K, Grupp IL, Ji Y, Reed T, Loukianov E, Grupp G, Bhagwhat A, Hoit B, Walsh R, Marban E, Periasamy M (1998) Targetedoverexpression of the sarcoplasmic reticulum Ca2+ATPase increases cardiac contractility in transgenic mouse hearts. Circ Res 83:1205–1214
Loukianov E, Ji Y, Grupp IL, Kirkpatrick DL, Baker DL, Loukianova T, Grupp G, Lytton J, Walsh RA, Periasamy M (1998) Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase Circ Res 83:889–897
Hajjar RJ, Kang JX, Gwathmey JK, Rosenzweig A (1997) Physiological effects of adenoviral gene transfer of sarcoplasmic reticulum calcium ATPase in isolated rat myocytes. Circulation 95:423–429
Lundblad A, Gonzalez-Serratos H, Inesi G, Swanson J, Paolini P (1986) Patterns of sarcomere activation, temperature dependence, and effect of ryanodine in chemically skinned cardiac fibers. J Gen Physiol 87:885–905
Acknowledgment
This and related work were supported by National Institutes of Health Grant NHBLI RO301-69830.
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Prasad, A.M., Inesi, G. (2012). Analysis of Calcium Transients in Cardiac Myocytes and Assessment of the Sarcoplasmic Reticulum Ca2+-ATPase Contribution. In: DiMario, J. (eds) Myogenesis. Methods in Molecular Biology, vol 798. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-343-1_24
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DOI: https://doi.org/10.1007/978-1-61779-343-1_24
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