Altered contractility and [Ca2+]i homeostasis in phospholemman-deficient murine myocytes: role of Na+/Ca2+ exchange.
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Journal: 2007/January - American Journal of Physiology - Heart and Circulatory Physiology
ISSN: 0363-6135
Abstract:
Phospholemman (PLM) regulates contractility and Ca(2+) homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice backbred to a pure congenic C57BL/6 background. Cell length, cell width, and whole cell capacitance were not different between wild-type and PLM-null myocytes. Compared with wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na(+)/Ca(2+) exchanger, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, alpha(1)-subunit of Na(+)-K(+)-ATPase, and calsequestrin levels in PLM-null myocytes. At 5 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), contraction and cytosolic [Ca(2+)] ([Ca(2+)](i)) transient amplitudes and SR Ca(2+) contents in PLM-null myocytes were significantly (P < 0.0004) higher than wild-type myocytes, whereas the converse was true at 0.6 mM [Ca(2+)](o). This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na(+)/Ca(2+) exchanger. Indeed, we have previously reported that Na(+)/Ca(2+) exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotropy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged (P < 0.04) in PLM-null myocytes. Whole cell Ca(2+) current densities were similar between wild-type and PLM-null myocytes, as were the fast- and slow-inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and Ca(2+) fluxes, likely by modulating Na(+)/Ca(2+) exchange activity.
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Am J Physiol Heart Circ Physiol 291(5): H2199-H2209
PMID: 16751288

Altered contractility and [Ca<sup>2+</sup>]<sub>i</sub> homeostasis in phospholemman-deficient murine myocytes: Role of Na<sup>+</sup>/Ca<sup>2+</sup> exchange

Department of Cellular and Molecular Physiology and
Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania;
Cardiovascular Division, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia; and
Weis Center for Research, Geisinger Medical Center, Danville, Pennsylvania
Address Correspondence To: Joseph Y. Cheung, M.D., Ph.D., Department of Cellular &amp; Molecular Physiology, Milton S. Hershey Medical Center, MC-H166, Hershey, PA 17003, Tel. (717) 531-5748, Fax. (717) 531-7667, Email: ude.usp@1cyj
A.L. Tucker and J. Song contributed equally to this study.
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Abstract

Phospholemman (PLM), a 72-amino acid sarcolemmal protein, has been shown to regulate contractility and Ca homeostasis in cardiac myocytes, likely via its modulatory influence on Na/Ca exchanger and Na-K-ATPase. In this study, we characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice back-bred to a pure congenic C57BL/6 background. There were no differences in cell length, cell width and whole cell capacitance between wild-type and PLM-null myocytes. Compared to wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na/Ca exchanger, sarcoplasmic reticulum (SR) Ca-ATPase, α1-subunit of Na-K-ATPase and calsequestrin levels in PLM-null myocytes. At 5mM extracellular [Ca] concentration ([Ca]o), contraction and cytosolic [Ca] ([Ca]i) transient amplitudes and SR Ca contents in PLM-null myocytes were significantly (p<0.0004) higher than wild-type myocytes. At 0.6 mM [Ca]o, however, contraction and [Ca]i transient amplitudes and SR Ca contents were significantly lower in PLM-null than wild-type myocytes. At 1.8 mM [Ca]o, the differences in contraction and [Ca]i transient amplitudes were no longer apparent. This pattern of contractile and [Ca]i transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na/Ca exchanger. Indeed, we have previously reported that Na/Ca exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotropy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged (p<0.04) in PLM-null myocytes. Whole cell Ca current densities were similar between wild-type and PLM-null myocytes, as were the fast and slow inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and Ca fluxes, likely by modulating Na/Ca exchange activity.

Keywords: heart, mouse, knockout, fura-2, patch-clamp
Abstract

References

  • 1. Adachi-Akahane S, Lu L, Li Z, Frank JS, Philipson KD, Morad MCalcium signaling in transgenic mice overexpressing cardiac Na(+)-Ca2+ exchanger. J Gen Physiol. 1997;109:717–729.[Google Scholar]
  • 2. Ahlers BA, Zhang XQ, Moorman JR, Rothblum LI, Carl LL, Song J, Wang J, Geddis LM, Tucker AL, Mounsey JP, Cheung JYIdentification of an endogenous inhibitor of the cardiac Na+/Ca2+ exchanger, phospholemman. J Biol Chem. 2005;280:19875–19882.[PubMed][Google Scholar]
  • 3. Armoundas AA, Hobai IA, Tomaselli GF, Winslow RL, O'Rourke BRole of sodium-calcium exchanger in modulating the action potential of ventricular myocytes from normal and failing hearts. Circ Res. 2003;93:46–53.[Google Scholar]
  • 4. Bassani JW, Bassani RA, Bers DMRelaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol. 1994;476:279–293.[Google Scholar]
  • 5. Beguin P, Crambert G, Guennoun S, Garty H, Horisberger JD, Geering KCHIF, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the gamma-subunit. Embo J. 2001;20:3993–4002.[Google Scholar]
  • 6. Beguin P, Crambert G, Monnet-Tschudi F, Uldry M, Horisberger JD, Garty H, Geering KFXYD7 is a brain-specific regulator of Na,K-ATPase alpha 1-beta isozymes. Embo J. 2002;21:3264–3273.[Google Scholar]
  • 7. Bers DM, Berlin JRKinetics of [Ca]i decline in cardiac myocytes depend on peak [Ca]i. Am J Physiol. 1995;268:C271–277.[PubMed][Google Scholar]
  • 8. Braz JC, Gregory K, Pathak A, Zhao W, Sahin B, Klevitsky R, Kimball TF, Lorenz JN, Nairn AC, Liggett SB, Bodi I, Wang S, Schwartz A, Lakatta EG, DePaoli-Roach AA, Robbins J, Hewett TE, Bibb JA, Westfall MV, Kranias EG, Molkentin JDPKC-alpha regulates cardiac contractility and propensity toward heart failure. Nat Med. 2004;10:248–254.[PubMed][Google Scholar]
  • 9. Crambert G, Fuzesi M, Garty H, Karlish S, Geering KPhospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties. Proc Natl Acad Sci U S A. 2002;99:11476–11481.[Google Scholar]
  • 10. Davis CE, Patel MK, Miller JR, John JE, 3rd, Jones LR, Tucker AL, Mounsey JP, Moorman JREffects of phospholemman expression on swelling-activated ion currents and volume regulation in embryonic kidney cells. Neurochem Res. 2004;29:177–187.[PubMed][Google Scholar]
  • 11. Despa S, Bossuyt J, Han F, Ginsburg KS, Jia LG, Kutchai H, Tucker AL, Bers DMPhospholemman-phosphorylation mediates the beta-adrenergic effects on Na/K pump function in cardiac myocytes. Circ Res. 2005;97:252–259.[PubMed][Google Scholar]
  • 12. Fuller W, Eaton P, Bell JR, Shattock MJIschemia-induced phosphorylation of phospholemman directly activates rat cardiac Na/K-ATPase. Faseb J. 2004;18:197–199.[PubMed][Google Scholar]
  • 13. Henderson SA, Goldhaber JI, So JM, Han T, Motter C, Ngo A, Chantawansri C, Ritter MR, Friedlander M, Nicoll DA, Frank JS, Jordan MC, Roos KP, Ross RS, Philipson KDFunctional adult myocardium in the absence of Na+-Ca2+ exchange: cardiac-specific knockout of NCX1. Circ Res. 2004;95:604–611.[PubMed][Google Scholar]
  • 14. Jia LG, Donnet C, Bogaev RC, Blatt RJ, McKinney CE, Day KH, Berr SS, Jones LR, Moorman JR, Sweadner KJ, Tucker ALHypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice. Am J Physiol Heart Circ Physiol. 2005;288:H1982–1988.[PubMed][Google Scholar]
  • 15. Kowdley GC, Ackerman SJ, Chen Z, Szabo G, Jones LR, Moorman JRAnion, cation, and zwitterion selectivity of phospholemman channel molecules. Biophys J. 1997;72:141–145.[Google Scholar]
  • 16. Licata A, Aggarwal R, Robinson RB, Boyden PFrequency dependent effects on Cai transients and cell shortening in myocytes that survive in the infarcted heart. Cardiovasc Res. 1997;33:341–350.[PubMed][Google Scholar]
  • 17. Mahmmoud YA, Cramb G, Maunsbach AB, Cutler CP, Meischke L, Cornelius FRegulation of Na,K-ATPase by PLMS, the phospholemman-like protein from shark: molecular cloning, sequence, expression, cellular distribution, and functional effects of PLMS. J Biol Chem. 2003;278:37427–37438.[PubMed][Google Scholar]
  • 18. Mirza MA, Zhang XQ, Ahlers BA, Qureshi A, Carl LL, Song J, Tucker AL, Mounsey JP, Moorman JR, Rothblum LI, Zhang TS, Cheung JYEffects of phospholemman downregulation on contractility and [Ca(2+)]i transients in adult rat cardiac myocytes. Am J Physiol Heart Circ Physiol. 2004;286:H1322–1330.[PubMed][Google Scholar]
  • 19. Nuss HB, Houser SRSodium-calcium exchange-mediated contractions in feline ventricular myocytes. Am J Physiol Heart Circ Physiol. 1992;263:H1161–1169.[PubMed][Google Scholar]
  • 20. Palmer CJ, Scott BT, Jones LRPurification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. J Biol Chem. 1991;266:11126–11130.[PubMed][Google Scholar]
  • 21. Ruknudin A, He S, Lederer WJ, Schulze DHFunctional differences between cardiac and renal isoforms of the rat Na+-Ca2+ exchanger NCX1 expressed in Xenopus oocytes. J Physiol 529 Pt. 2000;3:599–610.[Google Scholar]
  • 22. Sakurai K, Norota I, Tanaka H, Kubota I, Tomoike H, Endo MNegative inotropic effects of angiotensin II, endothelin-1 and phenylephrine in indo-1 loaded adult mouse ventricular myocytes. Life Sci. 2002;70:1173–1184.[PubMed][Google Scholar]
  • 23. Shattock MJ, Bers DM. Rat vs. rabbit ventricle: Ca flux and intracellular Na assessed by ion-selective microelectrodes. Am J Physiol. 1989;256:C813–822.[PubMed]
  • 24. Silverman BZ, Fuller W, Eaton P, Deng J, Moorman JR, Cheung JY, James AF, Shattock MJSerine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase. Cardiovasc Res. 2005;65:93–103.[PubMed][Google Scholar]
  • 25. Song J, Zhang XQ, Ahlers BA, Carl LL, Wang J, Rothblum LI, Stahl RC, Mounsey JP, Tucker AL, Moorman JR, Cheung JYSerine 68 of phospholemman is critical in modulation of contractility, [Ca2+]i transients, and Na+/Ca2+ exchange in adult rat cardiac myocytes. Am J Physiol Heart Circ Physiol. 2005;288:H2342–2354.[PubMed][Google Scholar]
  • 26. Song J, Zhang XQ, Carl LL, Qureshi A, Rothblum LI, Cheung JYOverexpression of phospholemman alters contractility and [Ca(2+)](i) transients in adult rat myocytes. Am J Physiol Heart Circ Physiol. 2002;283:H576–583.[PubMed][Google Scholar]
  • 27. Sweadner KJ, Rael EThe FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression. Genomics. 2000;68:41–56.[PubMed][Google Scholar]
  • 28. Tadros GM, Zhang XQ, Song J, Carl LL, Rothblum LI, Tian Q, Dunn J, Lytton J, Cheung JYEffects of Na(+)/Ca(2+) exchanger downregulation on contractility and [Ca(2+)](i) transients in adult rat myocytes. Am J Physiol Heart Circ Physiol. 2002;283:H1616–1626.[PubMed][Google Scholar]
  • 29. Therien AG, Goldshleger R, Karlish SJ, Blostein RTissue-specific distribution and modulatory role of the gamma subunit of the Na,K-ATPase. J Biol Chem. 1997;272:32628–32634.[PubMed][Google Scholar]
  • 30. Walaas SI, Czernik AJ, Olstad OK, Sletten K, Walaas OProtein kinase C and cyclic AMP-dependent protein kinase phosphorylate phospholemman, an insulin and adrenaline-regulated membrane phosphoprotein, at specific sites in the carboxy terminal domain. Biochem J. 1994;304 ( Pt 2):635–640.[Google Scholar]
  • 31. Yao A, Su Z, Nonaka A, Zubair I, Lu L, Philipson KD, Bridge JH, Barry WHEffects of overexpression of the Na+-Ca2+ exchanger on [Ca2+]i transients in murine ventricular myocytes. Circ Res. 1998;82:657–665.[PubMed][Google Scholar]
  • 32. Zhang LQ, Zhang XQ, Musch TI, Moore RL, Cheung JYSprint training restores normal contractility in postinfarction rat myocytes. J Appl Physiol. 2000;89:1099–1105.[PubMed][Google Scholar]
  • 33. Zhang LQ, Zhang XQ, Ng YC, Rothblum LI, Musch TI, Moore RL, Cheung JYSprint training normalizes Ca(2+) transients and SR function in postinfarction rat myocytes. J Appl Physiol. 2000;89:38–46.[PubMed][Google Scholar]
  • 34. Zhang XQ, Ahlers BA, Tucker AL, Song J, Wang J, Moorman JR, Mounsey JP, Carl LL, Rothblum LI, Cheung JY. Phospholemman inhibition of the cardiac Na+/Ca2+ exchanger. Role of phosphorylation. J Biol Chem. 2006;281:7784–7792.
  • 35. Zhang XQ, Moore RL, Tillotson DL, Cheung JYCalcium currents in postinfarction rat cardiac myocytes. Am J Physiol. 1995;269:C1464–1473.[PubMed][Google Scholar]
  • 36. Zhang XQ, Moorman JR, Ahlers BA, Carl LL, Lake DE, Song J, Mounsey JP, Tucker AL, Chan YM, Rothblum LI, Stahl RC, Carey DJ, Cheung JYPhospholemman Overexpression Inhibits Na+-K+-ATPase in Adult Rat Cardiac Myocytes: Relevance to Decreased Na+ Pump Activity in Post-infarction Myocytes. J Appl Physiol. 2006;100:212–220.[Google Scholar]
  • 37. Zhang XQ, Ng YC, Moore RL, Musch TI, Cheung JYIn situ SR function in postinfarction myocytes. J Appl Physiol. 1999;87:2143–2150.[PubMed][Google Scholar]
  • 38. Zhang XQ, Qureshi A, Song J, Carl LL, Tian Q, Stahl RC, Carey DJ, Rothblum LI, Cheung JYPhospholemman modulates Na+/Ca2+ exchange in adult rat cardiac myocytes. Am J Physiol Heart Circ Physiol. 2003;284:H225–233.[PubMed][Google Scholar]
  • 39. Zhang XQ, Song J, Rothblum LI, Lun M, Wang X, Ding F, Dunn J, Lytton J, McDermott PJ, Cheung JYOverexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes. Am J Physiol Heart Circ Physiol. 2001;281:H2079–2088.[PubMed][Google Scholar]
  • 40. Zhang XQ, Zhang LQ, Palmer BM, Ng YC, Musch TI, Moore RL, Cheung JYSprint training shortens prolonged action potential duration in postinfarction rat myocyte: mechanisms. J Appl Physiol. 2001;90:1720–1728.[PubMed][Google Scholar]
  • 41. Zhou YY, Wang SQ, Zhu WZ, Chruscinski A, Kobilka BK, Ziman B, Wang S, Lakatta EG, Cheng H, Xiao RPCulture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology. Am J Physiol Heart Circ Physiol. 2000;279:H429–436.[PubMed][Google Scholar]
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