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Influence of different culture conditions on sarcoplasmic reticular calcium transport in isolated neonatal rat cardiomyocytes

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Abstract

This study investigates sarcoplasmic reticulum (SR) calcium-(Ca2+) transport ATPase (SERCA2a) and phospholamban (PLB) in cultured spontaneously contracting neonatal rat cardiomyocytes (CM) to ascertain the function of both SR proteins under various culture conditions. The two major SR proteins were readily detectable in cultured CM by immunofluorescent microscopy using specific anti-SERCA2 and anti-PLB antibodies. Double labeling technique revealed that PLB-positive CM also labeled with anti-SERCA2. Coexpression of SERCA2 and PLB in CM was supported by measurement of cell homogenate oxalate-supported Ca2+ uptake which was completely inhibited by thapsigargin and stimulated by protein kinase A-catalyzed phosphorylation. Under serum-free conditions, incubation of CM with the SERCA2a expression modulator 3,3′,5-triiodo-L-thyronine (100 nM, 72 h) resulted in elevated Ca2+ uptake of +33%. Specific Ca2+ uptake activity was not altered if insulin was omitted from the serum-free culture medium but total SR Ca2+ transport activity was reduced under this culture condition. The results indicate that primary culture of spontaneously contracting neonatal rat CM can be employed as a useful model system for investigating both short- and long-term mechanisms determining the Ca2+ re-uptake function of the SR under defined culture conditions.

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References

  1. Katz AM, Takenaka H, Watras J: The sarcoplasmic reticulum. In: HA Fozzard, E Haber, RB Jennings, AM Katz, HE Morgan (eds). The Heart and Cardiovascular System. Raven Press, New York, 1986, pp 731–746

    Google Scholar 

  2. Lompré A-M, Lambert F, Lakatta EG, Schwartz K: Expression of sarcoplasmic reticulum Ca2+-ATPase and calsequestrin genes in rat heart during ontogenic development and aging. Circ Res 69: 1380–1388, 1991

    PubMed  Google Scholar 

  3. Freestone N, Singh J, Krause E-G, Vetter R: Early postnatal changes in sarcoplasmic reticulum calcium transport function in spontaneously hypertensive rats. Mol Cell Biochem 164/164: 57–66, 1996

    Google Scholar 

  4. Vetter R, Studer R, Reinecke H, Kolar F, Ostadalova I, Drexler H: Reciprocal changes in the postnatal expression of the sarcolemmal Na+-Ca2+-exchanger and SERCA2 in rat heart. J Mol Cell Cardiol 27: 1689–1701, 1995

    PubMed  Google Scholar 

  5. Maciel LM, Polikar R, Rohrer D, Popovich BK, Dillmann WH: Ageinduced decreases in the messenger RNA coding for the sarcoplasmic reticulum Ca2+-ATPase of the rat heart. Circ Res 67: 230–234, 1990

    PubMed  Google Scholar 

  6. Arai M, Otsu K, Maclennan DH, Periasamy M: Regulation of sarcoplasmic reticulum gene expression during cardiac and skeletal muscle development. Am J Physiol 262: C614–C620, 1992

    PubMed  Google Scholar 

  7. Komuro I, Kurabayashi M, Shibazaki Y, Takaku F, Yazaki Y: Molecular cloning and characterization of a Ca2+ + Mg2+-dependent adenosine triphosphatase from rat cardiac sarcoplasmic reticulum. Regulation of its expression by pressure overload and developmental stage. J Clin Invest 83: 1102–1108, 1989

    PubMed  Google Scholar 

  8. Levitsky D, de la Bastie D, Schwartz K, Lompré A-M: Ca2+-ATPase and function of sarcoplasmic reticulum during cardiac hypertrophy. Am J Physiol 261: 23–26, 1991

    PubMed  Google Scholar 

  9. Schwartz K, Carrier L, Lompré A-M, Mercadier JJ, Boheler KR: Contractile proteins and sarcoplasmic reticulum calcium-ATPase gene expression in the hypertrophied and failing heart. Basic Res Cardiol 87: 285–290, 1992

    PubMed  Google Scholar 

  10. de la Bastie D, Levitsky D, Rappaport L, Mercadier J-J, Marotte F, Wisnewsky C, Brovkovich V, Schwartz K, Lompré A-M: Function of the sarcoplasmic reticulum and expression of its Ca2+-ATPase gene in pressure overload-induced cardiac hypertrophy in the rat. Circ Res 66: 554–564, 1990

    PubMed  Google Scholar 

  11. Mercadier JJ, Lompré A-M, Duc P, Boheler KR, Fraysse JB, Wisnewsky C, Allen PD, Komajda M, Schwartz K: Altered sarcoplasmic reticulum Ca2+-ATPase gene expression in the human ventricle during end-stage heart failure. J Clin Invest 85: 305–309, 1990

    PubMed  Google Scholar 

  12. Arai M, Matsui H, Periasamy M: Sarcoplasmic reticulum gene expression in cardiac hypertrophy and heart failure. Circ Res 74: 555–564, 1994

    PubMed  Google Scholar 

  13. Lammerich A, Günther J, Pfitzer G, Storch E, Vetter R: Alterations of cardiac contractile function are related to changes in membrane calcium transport in spontaneously hypertensive rats. J Hypertens 13: 1313–1324, 1995

    PubMed  Google Scholar 

  14. Hasenfuss G, Reinecke H, Studer R, Meyer M, Pieske B, Holtz J, Holubarsch C, Posival H, Just H, Drexler H: Relation between myocardial function and expression of sarcoplasmic reticulum Ca2+-ATPase in failing and nonfailing human myocardium. Circ Res 75: 434–442, 1994

    PubMed  Google Scholar 

  15. Vetter R, Kott M, Rupp H: Differential influences of carnitine palmitoyltransferase-I inhibition and hyperthyroidism on cardiac growth and sarcoplasmic reticulum phosphorylation. Eur Heart J 15 (Suppl D): 31–37, 1994

    PubMed  Google Scholar 

  16. Karczewski P, Bartel S, Krause EG: Differential sensitivity to isoprenaline of troponin I and phospholamban phosphorylation in isolated rat hearts. Biochem J 266: 115–122, 1990

    PubMed  Google Scholar 

  17. Wegener AD, Simmerman HK, Lindemann JP, Jones LR: Phospholamban phosphorylation in intact ventricles. Phosphorylation of serine 16 and threonine 17 in response to β-adrenergic stimulation. J Biol Chem 264: 11468–11474, 1989

    PubMed  Google Scholar 

  18. Tada M, Katz AM: Phosphorylation of the sarcoplasmic reticulum and sarcolemma. Annu Rev Physiol 44: 401–423, 1982

    PubMed  Google Scholar 

  19. Lindemann JP, Watanabe AM: Phosphorylation of phospholamban in intact myocardium. Role of Ca2+-calmodulin-dependent mechanisms. J Biol Chem 260: 4516–4525, 1985

    PubMed  Google Scholar 

  20. Engelmann GL, McTiernan C, Gerrity RG, Samarel AM: Serum-free primary cultures of neonatal rat cardiomyocytes: Cellular and molecular applications. Technique 2: 279–291, 1990

    Google Scholar 

  21. Halle W, Wollenberger A: Differentiation and behavior of isolated embryonic and neonatal heart cells in a chemically defined medium. Am J Cardiol 25: 292–299, 1970

    PubMed  Google Scholar 

  22. Wallukat G, Wollenberger A: Supersensitivity to β-adrenoceptor stimulation evoked in cultured neonatal rat heart myocytes by L(+)-lactate and pyruvate. J Auton Pharmacol 13: 1–14, 1993

    PubMed  Google Scholar 

  23. Werdan K, Erdmann E: Preparation and culture of embryonic and neonatal heart muscle cells: Modification of transport activity. Meth Enzymol 173: 634–662, 1989

    PubMed  Google Scholar 

  24. Healy GM, Parker RC: An improved chemically defined basal medium (CMRL-1415) for newly explanted mouse embryo cells. J Cell Biol 30: 531–538, 1966

    PubMed  Google Scholar 

  25. Kaasik A, Paju K, Vetter R, Seppet EK: Thyroid hormones increase the contractility but suppress the effect of β-adrenergic agonist by decreasing phospholamban expression in rat atria. Cardiovasc Res 35: 106–112, 1997

    PubMed  Google Scholar 

  26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275, 1951

    PubMed  Google Scholar 

  27. Feher JJ, Briggs FN, Hess ML: Characterization of cardiac sarcoplasmic reticulum from ischemic myocardium: Comparison of isolated sarcoplasmic reticulum with unfractionated homogenates. J Mol Cell Cardiol 12: 427–432, 1980

    PubMed  Google Scholar 

  28. Solaro RJ, Briggs FN: Estimating the functional capabilities of sarcoplasmic reticulum in cardiac muscle. Circ Res 34: 531–540, 1974

    PubMed  Google Scholar 

  29. Vetter R, Rupp H: CPT-1 inhibition by etomoxir has a chamber-related action on cardiac sarcoplasmic reticulum and isomyosins. Am J Physiol 267: H2091–H2099, 1994

    PubMed  Google Scholar 

  30. Lompré A-M, de la Bastie D, Boheler KR, Schwartz K: Chacterization and expression of the rat heart sarcoplasmic reticulum Ca2+-ATPase mRNA. FEBS Lett 249: 35–41, 1989

    PubMed  Google Scholar 

  31. Lytton J, Westlin M, Hanley MR: Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps. J Biol Chem 266: 17067–17071, 1991

    PubMed  Google Scholar 

  32. Seppet EK, Kadaya LY, Hata T, Kallikorm AP, Saks VA, Vetter R, Dhalla NS: Thyroid control over membrane processes in rat heart. Am J Physiol 261: 66–71, 1991

    PubMed  Google Scholar 

  33. Rohrer D, Dillmann WH: Thyroid hormone markedly increases the mRNA coding for sarcoplasmic reticulum Ca2+-ATPase in the rat heart. J Biol Chem 263: 6941–6944, 1988

    PubMed  Google Scholar 

  34. Arai M, Otsu K, Maclennan DH, Alpert NR, Periasamy M: Effect of thyroid hormone on the expression of messenger RNA encoding sarcoplasmic reticulum proteins. Circ Res 69: 266–276, 1991

    PubMed  Google Scholar 

  35. Rohrer DK, Hartong R, Dillmann WH: Influence of thyroid hormone and retinoic acid on slow sarcoplasmic reticulum Ca2+ ATPase and myosin heavy chain α gene expression in cardiac myocytes. Delineation of cis-active DNA elements that confer responsiveness to thyroid hormone but not to retinoic acid. J Biol Chem 266: 8638–8646, 1991

    PubMed  Google Scholar 

  36. Bartel S, Willenbrock R, Haase H, Karczewski P, Wallukat G, Dietz R, Krause EG: Cyclic GMP-mediated phospholamban phosphorylation in intact cardiomyocytes. Biochem Biophys Res Commun 214: 75–80, 1995

    PubMed  Google Scholar 

  37. Karczewski P, Vetter R, Holtzhauer M, Krause EG: Indirect technique for the estimation of cAMP-dependent and Ca2+/calmodulin-dependent phospholamban phosphorylation state in canine heart in vivo. Biomed Biochim Acta 45: S227–S231, 1986

    PubMed  Google Scholar 

  38. Kolar F, Seppet EK, Vetter R, Prochazka J, Grunermel J, Zilmer K, Ostadal B: Thyroid control of contractile function and calcium handling in neonatal rat heart. Pflügers Arch 421: 26–31, 1992

    Google Scholar 

  39. Dillmann WH: Biochemical basis of thyroid hormone action in the heart. Am J Med 88: 626–630, 1990

    PubMed  Google Scholar 

  40. Beekman RE, Van Hardeveld C, Simonides WS: On the mechanism of the reduction by thyroid hormone of β-adrenergic relaxation rate stimulation in rat heart. Biochem J 259: 229–236, 1989

    PubMed  Google Scholar 

  41. Penpargkul S, Fein F, Sonnenblick EH, Scheurer J: Depressed cardiac sarcoplasmic reticular function from diabetic rats. J Mol Cell Cardiol 13: 303–309, 1981

    PubMed  Google Scholar 

  42. Ganguly PK, Pierce GN, Dhalla KS, Dhalla NS: Defective sarcoplasmic reticular calcium transport in diabetic cardiomyopathy. Am J Physiol 244: E528–E535, 1983

    PubMed  Google Scholar 

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Authors and Affiliations

  1. Max Delbrück Center for Molecular Medicine (MDC), Berlin-Buch, Germany

    Roland Vetter, Monika Kott & Wolfgang Schulze

  2. Institute of Clinical Pharmacology and Toxicology, Free University of Berlin, Berlin, Germany

    Roland Vetter

  3. Molecular Cardiology Laboratory, Philipps University Marburg, Marburg

    Heinz Rupp

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  1. Roland Vetter
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  2. Monika Kott
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  3. Wolfgang Schulze
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  4. Heinz Rupp
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Vetter, R., Kott, M., Schulze, W. et al. Influence of different culture conditions on sarcoplasmic reticular calcium transport in isolated neonatal rat cardiomyocytes. Mol Cell Biochem 188, 177–185 (1998). https://doi.org/10.1023/A:1006850724830

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