No abstract

Previous studies showed increased phospholemman (PLM) mRNA after myocardial infarction (MI) in rats (Sehl PD, Tai JTN, Hillan KJ, Brown LA, Goddard A, Yang R, Jin H, and Lowe DG. Circulation 101: 1990-1999, 2000). We tested the hypothesis that, in normal adult rat cardiac myocytes, PLM overexpression alters contractile function and cytosolic Ca(2+) concentration ([Ca(2+)](i)) homeostasis in a manner similar to that observed in post-MI myocytes. Compared with myocytes infected by control adenovirus expressing green fluorescent protein (GFP) alone, Western blots indicated a 41% increase in PLM expression after 72 h (P < 0.001) but no changes in Na(+)/Ca(2+) exchanger, SERCA2, and calsequestrin levels in myocytes infected by adenovirus expressing GFP and PLM. At 5 mM extracellular [Ca(2+)] ([Ca(2+)](o)), maximal contraction amplitudes in PLM-overexpressed myocytes were 24% (P < 0.005) and [Ca(2+)](i) transient amplitudes were 18% (P < 0.05) lower than control myocytes. At 0.6 mM [Ca(2+)](o), however, contraction and [Ca(2+)](i) transient amplitudes were significantly (P < 0.05) higher in PLM-overexpressed than control myocytes (18% and 42%, respectively); at 1.8 mM [Ca(2+)](o), the differences in contraction and [Ca(2+)](i) transient amplitudes were narrowed. This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-overexpressed myocytes mimics that observed in post-MI rat myocytes. We suggest that PLM overexpression observed in post-MI myocytes may partly account for contractile abnormalities by perturbing Ca(2+) fluxes during excitation-contraction.

Previous studies have shown that overexpression of phospholemman (PLM) affected contractile function and Ca(2+) homeostasis in adult rat myocytes. We tested the hypothesis that PLM modulated Na(+)/Ca(2+) exchanger (NCX1) activity. PLM was overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. After 72 h, the half-time of relaxation from caffeine-induced contracture, an estimate of forward NCX1 activity, was prolonged 1.8-fold (P < 0.003) in myocytes overexpressing PLM compared with control myocytes overexpressing green fluorescent protein alone. Reverse NCX1 current (3 Na(+) out:1 Ca(2+) in) was significantly (P < 0.0001) lower in PLM myocytes, especially at more positive voltages. Immunofluorescence demonstrated colocalization of PLM and NCX1 to the plasma membrane and t-tubules. Resting membrane potential, action potential amplitude and duration, myocyte size, and NCX1 and calsequestrin protein levels were not affected by PLM overexpression. At 5 mM extracellular [Ca(2+)] ([Ca(2+)](o)), the depressed contraction amplitudes in PLM myocytes were increased towards normal by cooverexpression with NCX1. At 0.6 mM [Ca(2+)](o), the supranormal contraction amplitudes in PLM myocytes were reduced by cooverexpression with NCX1. We conclude that PLM modulated myocyte contractility partly by inhibiting Na(+)/Ca(2+) exchange.

adult rat myocyte culture; patch clamp; fura-2; edge detection; excitation-contraction coupling PHOSPHOLEMMAN (PLM), a 72-amino acid membrane phosphoprotein with a single transmembrane domain (13), belongs to the FXYD gene family of small ion transporter regulators (18). In the heart and skeletal muscle, PLM is a major sarcolemmal substrate for protein kinases A and C (9, 14). Recent studies suggest that PLM regulates both Na ϩ -K ϩ -ATPase (4, 25) and Na ϩ /Ca 2ϩ exchanger (NCX1) (24) activities in cardiac muscle. In rat hearts that have survived myocardial infarction (MI), expression of PLM mRNA was increased twofold as early as 3 days after MI and remained elevated for at least 2 wk after MI (15). Interestingly, overexpression of PLM in normal adult rat cardiac myocytes affected myocyte contractility and cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) transients (17) in a pattern similar to the changes observed in post-MI rat myocytes (2,22). It is noteworthy that in post-MI rat myocytes, Na ϩ -dependent Ca 2ϩ uptake in sarcolemmal (SL) vesicles (5), NCX1 currents (27), and Na (6) were depressed. On the basis of the above observations, an attractive hypothesis is that overexpression of PLM in post-MI rat myocytes, by inhibiting two major SL ion transporters, resulted in abnormal [Ca 2ϩ ] i homeostasis and altered contractility. Downregulating PLM would therefore offer a rational approach to ameliorating contractile abnormalities post-MI. There are no published studies on the effects of PLM downregulation in cardiac tissues. The present study was undertaken to test the hypothesis that in adult rat cardiac myocytes, PLM downregulation alters cardiac myocyte contraction, [Ca 2ϩ ] i transient dynamics, and Na ϩ /Ca 2ϩ exchanger function. METHODSMyocyte isolation and culture. The protocol for myocyte isolation was approved by the Institutional Animal Care and Use Committee. Cardiac myocytes were isolated from the septum and left ventricular (LV) free wall of male Sprague-Dawley rats (ϳ280 g) as previously described (3). Isolated myocytes were seeded on laminin-coated coverslips and cultured with serum-free medum 199 (Earle's salts without L-glutamine and NaHCO3) supplemented with creatine, carnitine, taurine, and NaHCO 3 (17, 26). After 2 h, media were changed to remove nonadherent myocytes. Six hours after isolation, cultured myocytes were electrically paced {1 Hz, extracellular Ca 2ϩ concentration ([Ca 2ϩ ]o) ϭ 1.8 mM} (17,26). Culture media were changed daily over the course of experiments. Under continuous pacing culture conditions, we have previously demonstrated that myocyte contractility did not decline for at least 72 h (17).Construction of recombinant replication-deficient adenovirus expressing antisense PLM. The basic protocol has been described by He et al. (7). Initially, the coding sequence of dog heart PLM together with 5Ј-untranslated and 3Ј-untranslated sequences (13, 17) was

No abstract