Effects of phospholemman downregulation on contractility and [Ca²⁺]_itransients in adult rat cardiac myocytes

Abstract

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

Secondary structure, orientation, and oligomerization of phospholemman, a cardiac transmembrane protein

“…It is phosphorylated by protein kinase A and C at different sites in the cytoplasmic domain (Palmer et al 1991), which suggests that PLM may be an important control point in the function of heart cells. The biological importance of PLM is further emphasized by the fact that PLM exhibits a close similarity to a number of other newly identified membrane proteins, including CHIF, RIC, and MAT-8, that form a new superfamily of membrane proteins with a single transmembrane domain (Sweadner and Rael 2000).The function of PLM is not completely understood, yet recent evidence suggests that PLM may function as a regulator of the Na + /K + -ATPase (Silverman et al 2005) and that it also colocalizes with the Na + /Ca 2+ -exchanger (Mirza et al 2004). Electrical measurements of PLM in artificial lipid bilayers and oocytes show that PLM facilitates the membrane flux of ions (Chen et al 1999) and taurine transport (Moorman et al 1995).…”

Identification of an Endogenous Inhibitor of the Cardiac Na+/Ca2+ Exchanger, Phospholemman

“…Immunoreactivity was detected using an enhanced chemiluminescence kit, BioMax XAR film (Eastman Kodak Co., Rochester, NY), and a developer. Na ϩ /Ca 2ϩ Exchange Current (I NaCa ) Measurements-Whole-cell patch clamp recordings were performed at 30°C as described previously (12)(13)(14)(15) ] i to equilibrate with those present in pipette solution. A descending voltage ramp (from ϩ100 to Ϫ120 mV; 500 mV/s) was immediately followed by an ascending voltage ramp (from Ϫ120 to ϩ100 mV; 500 mV/s).…”

“…Currents were derived from measurements during the descending voltage ramp. I NaCa was defined as the difference current measured in the absence and presence of Cd 2ϩ (12). Currents were filtered at 1 kHz, and data were acquired at 2 kHz.…”

Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+-K+-ATPase

“…This small 72-amino acid integral membrane phosphoprotein with a single transmembrane domain was initially proposed to be involved in cell volume regulation in noncardiac tissues (7). In the mammalian heart, PLM regulates the activities of both Na ϩ -K ϩ -ATPase (8,16,26) and the cardiac Na ϩ /Ca 2ϩ exchanger (NCX1) (14,17).…”