Serine 68 of phospholemman is critical in modulation of contractility, [Ca2+]i transients, and Na+/Ca2+ exchange in adult rat cardiac myocytes

2005 ◽  
Vol 288 (5) ◽  
pp. H2342-H2354 ◽  
Author(s):  
Jianliang Song ◽  
Xue-Qian Zhang ◽  
Belinda A. Ahlers ◽  
Lois L. Carl ◽  
JuFang Wang ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Deletion Mutant ◽  
Cardiac Myocyte ◽  
Contractile Function ◽  
Cardiac Contractility ◽  
Terminal Deletion ◽  
Phosphorylation Sites ◽  
Adult Rat ◽  
Rat Myocytes ◽  
Rat Cardiac Myocytes

Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis and inhibited Na+/Ca2+ exchanger (NCX1). In addition, PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [Ca2+]i transients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum Ca2+-ATPase, Na+-K+-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser62, Ser63, Ser68, and Thr69) were deleted, a partial COOH-terminal deletion mutant in which Ser68 and Thr69 were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser68 or Thr69 in mediating PLM's effect on cardiac contractility. Focusing on Ser68, the Ser68 to Glu mutant was fully effective, the Ser63 to Ala (leaving Ser68 intact) mutant was partially effective, and the Ser68 to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca2+]i transients, and NCX1 currents. Both the Ser63 to Ala and Ser68 to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser68 in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca2+]i transients, and NCX1 activity by PLM is critically dependent on Ser68. We suggest that PLM phosphorylation at Ser68 may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility.

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

2004 ◽  
Vol 286 (4) ◽  
pp. H1322-H1330 ◽  
Author(s):  
M. Ayoub Mirza ◽  
Xue-Qian Zhang ◽  
Belinda A. Ahlers ◽  
Anwer Qureshi ◽  
Lois L. Carl ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Fluorescent Protein ◽  
Contractile Function ◽  
Transient Behavior ◽  
Normal Adult ◽  
Adult Rat ◽  
Rat Hearts ◽  
Rat Myocytes ◽  
Green Fluorescent ◽  
Rat Cardiac Myocytes

Phospholemman (PLM) expression was increased in rat hearts after myocardial infarction (MI). Overexpression of PLM in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis in a manner similar to that observed in post-MI myocytes. In this study, we tested whether PLM downregulation in normal adult rat myocytes resulted in contractility and [Ca2+]i transient changes opposite to those observed in post-MI myocytes. Compared with control myocytes infected with adenovirus (Adv) expressing green fluorescent protein (GFP) alone, myocytes infected with Adv expressing both GFP and rat antisense PLM (rASPLM) had 23% less PLM protein ( P < 0.012) at 3 days, but no differences were found in sarcoplasmic reticulum (SR) Ca2+-ATPase, Na+/Ca2+ exchanger (NCX1), Na+-K+-ATPase, and calsequestrin levels. SR Ca2+ uptake and whole cell capacitance were not affected by rASPLM treatment. Relaxation from caffeine-induced contracture was faster, and NCX1 current amplitudes were higher in rASPLM myocytes, indicating that PLM downregulation enhanced NCX1 activity. In native rat cardiac myocytes, coimmunoprecipitation experiments indicated an association of PLM with NCX1. At 0.6 mM [Ca2+]o, rASPLM myocytes had significantly ( P < 0.003) lower contraction and [Ca2+]i transient amplitudes than control GFP myocytes. At 5 mM [Ca2+]o, both contraction and [Ca2+]i transient amplitudes were higher in rASPLM myocytes. This pattern of contractile and [Ca2+]i transient behavior in rASPLM myocytes was opposite to that observed in post-MI rat myocytes. We conclude that downregulation of PLM in normal rat cardiac myocytes enhanced NCX1 function and affected [Ca2+]i transient and contraction amplitudes. We suggest that PLM downregulation offers a potential therapeutic strategy for ameliorating contractile abnormalities in MI myocytes.

scholarly journals Phospholemman overexpression inhibits Na+-K+-ATPase in adult rat cardiac myocytes: relevance to decreased Na+ pump activity in postinfarction myocytes

2006 ◽  
Vol 100 (1) ◽  
pp. 212-220 ◽  
Author(s):  
Xue-Qian Zhang ◽  
J. Randall Moorman ◽  
Belinda A. Ahlers ◽  
Lois L. Carl ◽  
Douglas E. Lake ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Cardiac Myocytes ◽  
Messenger Rna ◽  
Fluorescent Protein ◽  
Pump Current ◽  
Relative Level ◽  
Adult Rat ◽  
Rat Myocytes ◽  
Rat Cardiac Myocytes ◽  
Rna Levels

Messenger RNA levels of phospholemman (PLM), a member of the FXYD family of small single-span membrane proteins with putative ion-transport regulatory properties, were increased in postmyocardial infarction (MI) rat myocytes. We tested the hypothesis that the previously observed reduction in Na+-K+-ATPase activity in MI rat myocytes was due to PLM overexpression. In rat hearts harvested 3 and 7 days post-MI, PLM protein expression was increased by two- and fourfold, respectively. To simulate increased PLM expression post-MI, PLM was overexpressed in normal adult rat myocytes by adenovirus-mediated gene transfer. PLM overexpression did not affect the relative level of phosphorylation on serine68 of PLM. Na+-K+-ATPase activity was measured as ouabain-sensitive Na+-K+ pump current (Ip). Compared with control myocytes overexpressing green fluorescent protein alone, Ip measured in myocytes overexpressing PLM was significantly ( P < 0.0001) lower at similar membrane voltages, pipette Na+ ([Na+]pip) and extracellular K+ ([K+]o) concentrations. From −70 to +60 mV, neither [Na+]pip nor [K+]o required to attain half-maximal Ip was significantly different between control and PLM myocytes. This phenotype of decreased Vmax without appreciable changes in Km for Na+ and K+ in PLM-overexpressed myocytes was similar to that observed in MI rat myocytes. Inhibition of Ip by PLM overexpression was not due to decreased Na+-K+-ATPase expression because there were no changes in either protein or messenger RNA levels of either α1- or α2-isoforms of Na+-K+-ATPase. In native rat cardiac myocytes, PLM coimmunoprecipitated with α-subunits of Na+-K+-ATPase. Inhibition of Na+-K+-ATPase by PLM overexpression, in addition to previously reported decrease in Na+-K+-ATPase expression, may explain altered Vmax but not Km of Na+-K+-ATPase in postinfarction rat myocytes.

Cardiac myocyte guanosine transport and metabolism

1987 ◽  
Vol 253 (5) ◽  
pp. C645-C651 ◽  
Author(s):  
T. P. Geisbuhler ◽  
D. A. Johnson ◽  
M. J. Rovetto
Keyword(s):  
Cardiac Myocytes ◽  
Rate Constant ◽  
Cardiac Myocyte ◽  
Maximum Velocity ◽  
Guanine Nucleotides ◽  
Inhibition Constant ◽  
Adult Rat ◽  
Adenosine Transport ◽  
Common Carrier ◽  
Rat Cardiac Myocytes

Guanosine transport and metabolism were examined in adult rat cardiac myocytes. Myocytes transported guanosine via saturable [Km = 18 microM, maximum velocity (Vmax) = 3.61 pmol.mg-1.s-1] and nonsaturable (rate constant = 1.47 X 10(-2] processes. The saturable process was inhibited by nitrobenzyl-thioinosine, inosine [inhibition constant (Ki) = 180 microM], and adenosine (Ki = 112 microM). Extracellular guanosine taken up by myocytes was slowly phosphorylated to guanine nucleotides. The majority of guanosine (98%) existed as free intracellular guanosine after 60 s. Countertransport of nucleosides could not be demonstrated in these cells at physiological concentrations in the presence of up to a 10-fold gradient of nucleoside. These studies indicate that adult rat cardiac myocytes can be used to assess myocardial guanosine transport separate from its metabolism. Comparable inhibition of guanosine and adenosine transport by each other and by inosine support the hypothesis that guanosine and adenosine are transported by a common carrier.

Overexpression of phospholemman alters contractility and [Ca2+]i transients in adult rat myocytes

2002 ◽  
Vol 283 (2) ◽  
pp. H576-H583 ◽  
Author(s):  
Jianliang Song ◽  
Xue-Qian Zhang ◽  
Lois L. Carl ◽  
Anwer Qureshi ◽  
Lawrence I. Rothblum ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Contractile Function ◽  
Maximal Contraction ◽  
Western Blots ◽  
Adult Rat ◽  
Rat Myocytes ◽  
Green Fluorescent ◽  
Rat Cardiac Myocytes

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 Ca2+ concentration ([Ca2+]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+/Ca2+ exchanger, SERCA2, and calsequestrin levels in myocytes infected by adenovirus expressing GFP and PLM. At 5 mM extracellular [Ca2+] ([Ca2+]o), maximal contraction amplitudes in PLM-overexpressed myocytes were 24% ( P < 0.005) and [Ca2+]i transient amplitudes were 18% ( P < 0.05) lower than control myocytes. At 0.6 mM [Ca2+]o, however, contraction and [Ca2+]i transient amplitudes were significantly ( P < 0.05) higher in PLM-overexpressed than control myocytes (18% and 42%, respectively); at 1.8 mM [Ca2+]o, the differences in contraction and [Ca2+]i transient amplitudes were narrowed. This pattern of contractile and [Ca2+]itransient 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 Ca2+ fluxes during excitation-contraction.

Phospholemman modulates Na+/Ca2+exchange in adult rat cardiac myocytes

2003 ◽  
Vol 284 (1) ◽  
pp. H225-H233 ◽  
Author(s):  
Xue-Qian Zhang ◽  
Anwer Qureshi ◽  
Jianliang Song ◽  
Lois L. Carl ◽  
Qiang Tian ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Contractile Function ◽  
Resting Membrane Potential ◽  
Adult Rat ◽  
Protein Levels ◽  
Rat Myocytes ◽  
T Tubules ◽  
Green Fluorescent ◽  
Myocyte Contractility ◽  
Rat Cardiac Myocytes

Previous studies have shown that overexpression of phospholemman (PLM) affected contractile function and Ca2+ homeostasis in adult rat myocytes. We tested the hypothesis that PLM modulated Na+/Ca2+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 Ca2+ 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 [Ca2+] ([Ca2+]o), the depressed contraction amplitudes in PLM myocytes were increased towards normal by cooverexpression with NCX1. At 0.6 mM [Ca2+]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+/Ca2+ exchange.

In vivo adenoviral transfer of sorcin reverses cardiac contractile abnormalities of diabetic cardiomyopathy

2004 ◽  
Vol 286 (1) ◽  
pp. H68-H75 ◽  
Author(s):  
Jorge Suarez ◽  
Darrell D. Belke ◽  
Bernd Gloss ◽  
Thomas Dieterle ◽  
Patrick M. McDonough ◽  
...  
Keyword(s):  
Diabetic Cardiomyopathy ◽  
Cardiac Myocyte ◽  
Viral Vector ◽  
Contractile Function ◽  
Cardiac Contractility ◽  
Rat Cardiomyocytes ◽  
Adenoviral Gene Transfer ◽  
Adult Rat ◽  
Adenoviral Transfer

In many types of heart failure cardiac myocyte Ca2+ handling is abnormal because of downregulation of key Ca2+-handling proteins like sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a and ryanodine receptor (RyR)2. The alteration in SERCA2a and RyR2 expression results in altered cytosolic Ca2+ transients, leading to abnormal contraction. Sorcin is an EF-hand protein that confers the property of caffeine-activated intracellular Ca2+ release in nonmuscle cells by interacting with RyR2. To determine whether sorcin could improve the contractile function of the heart, we overexpressed sorcin in the heart of either normal or diabetic mice and in adult rat cardiomyocytes with an adenoviral gene transfer approach. Sorcin overexpression was associated with an increase in cardiac contractility of the normal heart and dramatically rescued the abnormal contractile function of the diabetic heart. These effects could be attributed to an improvement of the Ca2+ transients found in the cardiomyocyte after sorcin overexpression. Viral vector-mediated delivery of sorcin to cardiac myocytes is beneficial, resulting in improved contractile function in diabetic cardiomyopathy.

Isolated cardiac myocytes are sensitized by hypoxia-reoxygenation to neutrophil-released mediators

1994 ◽  
Vol 266 (1) ◽  
pp. H128-H136 ◽  
Author(s):  
M. Buerke ◽  
A. S. Weyrich ◽  
A. M. Lefer
Keyword(s):  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Platelet Activating Factor ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Elastase Inhibitor ◽  
Isolated Cardiac Myocytes ◽  
Rat Myocytes ◽  
Rat Cardiac Myocytes ◽  
Hypoxia Reoxygenation

We exposed isolated rat cardiac myocytes to 20 min of hypoxia followed by 20 min of reoxygenation and observed the effect of supernatants of stimulated neutrophils [polymorphonuclear leukocytes (PMNs)] given at the beginning of reoxygenation. PMN supernatants induced cardiac myocyte injury, which was characterized by a significant (P < 0.01) reduction in cell viability to 53 +/- 3%, vs. 84 +/- 3% in rat myocytes subjected to hypoxia-reoxygenation (H/R) alone. The PMN supernatants also resulted in elevated creatine kinase (CK) activities in the myocyte medium. To examine specific PMN-released mediators that may contribute to this cell death, we studied the effects of hydrogen peroxide (H2O2), elastase, and platelet-activating factor on H/R cardiac myocytes. Incubation of myocytes after hypoxia with 10, 50, and 100 microM H2O2 decreased viability in a concentration-dependent manner (from 83 +/- 2 to 37 +/- 2%; P < 0.01). CK release of H/R myocytes was also significantly increased by 100 microM H2O2 (to 28 +/- 5 from 12 +/- 1% for H/R alone; P < 0.01). Similarly, elastase (5 micrograms/ml) given after hypoxia significantly reduced cardiac myocyte viability during reoxygenation (viability 58 +/- 1 vs. 85 +/- 1% H/R alone; P < 0.05) and increased CK release (to 29 +/- 3 from 11 +/- 1% for H/R alone; P < 0.01), an effect that was abolished by L-680,833, an elastase inhibitor. Unlike H2O2 and elastase, platelet-activating factor had no significant effect on myocyte viability or CK release after H/R.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Down-Regulation of Replication Factor C-40 (RFC40) Causes Chromosomal Missegregation in Neonatal and Hypertrophic Adult Rat Cardiac Myocytes

PLoS ONE ◽  
2012 ◽  
Vol 7 (6) ◽  
pp. e39009 ◽  
Author(s):  
Hirotaka Ata ◽  
Deepa Shrestha ◽  
Masahiko Oka ◽  
Rikuo Ochi ◽  
Chian Ju Jong ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Replication Factor C ◽  
Down Regulation ◽  
Replication Factor ◽  
Adult Rat ◽  
Factor C ◽  
Rat Cardiac Myocytes

Abstract 99: Hypotonic Swelling Promotes Nitric Oxide Release in Cardiac Ventricular Myocytes

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Luis Gonano ◽  
Malena Morell ◽  
Juan I Burgos ◽  
Martin Vila Petroff
Keyword(s):  
Nitric Oxide ◽  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Contractile Function ◽  
Cell Swelling ◽  
Contractile Dysfunction ◽  
Ischemia And Reperfusion ◽  
Hypotonic Solution ◽  
No Release ◽  
Hypotonic Swelling

Cardiac myocyte swelling occurs in multiple pathological situations and in particular contributes to the deleterious effects of ischemia and reperfusion by promoting contractile dysfunction. We investigated whether hypotonic swelling promotes nitric oxide (NO) release in cardiac myocytes and if so, whether it impacts on swelling induced contractile dysfunction. Perfusing rat cardiac myocytes, loaded with the NO sensor DAF-FM, with a hypotonic solution (HS; 217 mOsm), increased cell volume, reduced myocyte contraction and Ca2+ transient amplitude and significantly increased DAF-FM fluorescence. When cells were exposed to the HS supplemented with 2.5 mM of the NO synthase inhibitor L-NAME, cell swelling occurred in the absence of NO release. Swelling-induced NO release was also prevented by the NOS1 inhibitor, Nitroguanidine. In addition, Colchicine (an inhibitor of microtubule polymerization) prevented the increase in DAF-FM fluorescence induced by HS indicating that microtubule integrity is necessary for swelling-induced NO release. The swelling-induced negative inotropic effect was exacerbated in the presence of either L-NAME, Nitroguandine or the guanylate cyclase inhibitor, ODQ, suggesting that NOS1-derived NO provides contractile support via a GMP-dependent mechanism. Indeed, ODQ reduced Ca2+ wave velocity and the HS-induced increment in ryanodine receptor (RyR2) phosphorylation at site Ser2808 suggesting that in the context of hypotonic swelling, cGMP may contribute to preserve contractile function by enhancing SR Ca2+ release. Our findings suggest a novel mechanism for NO release in cardiac myocytes with putative pathophysiological relevance in the context of ischemia and reperfusion, where it may be cardioprotective by reducing the extent of contractile dysfunction associated with hypotonic swelling.

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