HNO enhances SERCA2a activity and cardiomyocyte function by promoting redox-dependent phospholamban oligomerization.

AIMS Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca(2+) uptake and myofilament Ca(2+) sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in a redox-dependent manner, improving Ca(2+) handling in isolated myocytes/hearts. RESULTS Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln(-/-)) mice. Compared to WT, pln(-/-) myocytes displayed enhanced resting sarcomere shortening, peak Ca(2+) transient, and blunted β-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca(2+) transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln(-/-) cells/hearts. HNO enhanced SR Ca(2+) uptake in WT but not pln(-/-) SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca(2+)-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available. INNOVATION HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a. CONCLUSIONS PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca(2+) handling in failing hearts.

[1]  A. Shah,et al.  Redox Signaling in Cardiac Physiology and Pathology , 2012, Circulation research.

[2]  D. Wink,et al.  Nitroxyl-Mediated Disulfide Bond Formation Between Cardiac Myofilament Cysteines Enhances Contractile Function , 2012, Circulation research.

[3]  R. Hajjar,et al.  Modulation of Cardiac Contractility by the Phopholamban/SERCA2a Regulatome , 2012, Circulation research.

[4]  G. Hasenfuss,et al.  Cardiac inotropes: current agents and future directions. , 2011, European heart journal.

[5]  D. Kass,et al.  Playing with cardiac "redox switches": the "HNO way" to modulate cardiac function. , 2011, Antioxidants & redox signaling.

[6]  U. Jakob,et al.  Effects of oxidative stress on behavior, physiology, and the redox thiol proteome of Caenorhabditis elegans. , 2011, Antioxidants & redox signaling.

[7]  L. Masterson,et al.  Lethal Arg9Cys phospholamban mutation hinders Ca2+-ATPase regulation and phosphorylation by protein kinase A , 2011, Proceedings of the National Academy of Sciences.

[8]  D. Kass,et al.  Nitroxyl enhances myocyte Ca2+ transients by exclusively targeting SR Ca2+-cycling. , 2010, Frontiers in bioscience.

[9]  A. Trafford,et al.  From the ryanodine receptor to cardiac arrhythmias. , 2009, Circulation journal : official journal of the Japanese Circulation Society.

[10]  D. Mancini,et al.  Calcium upregulation by percutaneous administration of gene therapy in cardiac disease (CUPID Trial), a first-in-human phase 1/2 clinical trial. , 2009, Journal of cardiac failure.

[11]  S. Lancel,et al.  Nitroxyl Activates SERCA in Cardiac Myocytes via Glutathiolation of Cysteine 674 , 2009, Circulation research.

[12]  T. Soares,et al.  Phospholamban modulates the functional coupling between nucleotide domains in Ca-ATPase oligomeric complexes in cardiac sarcoplasmic reticulum. , 2009, Biochemistry.

[13]  Christopher M. Pavlos,et al.  Phospholamban thiols play a central role in activation of the cardiac muscle sarcoplasmic reticulum calcium pump by nitroxyl. , 2008, Biochemistry.

[14]  R. Hajjar,et al.  The cardiac sarcoplasmic/endoplasmic reticulum calcium ATPase: a potent target for cardiovascular diseases , 2008, Nature Clinical Practice Cardiovascular Medicine.

[15]  M. Periasamy,et al.  SERCA2a gene therapy for heart failure: ready for primetime? , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[16]  D. Sanoudou,et al.  The role of SERCA2a/PLN complex, Ca2+ homeostasis, and anti-apoptotic proteins in determining cell fate , 2008, Pflügers Archiv - European Journal of Physiology.

[17]  D. Kass,et al.  Nitroxyl increases force development in rat cardiac muscle , 2007, The Journal of physiology.

[18]  J. Froehlich,et al.  Phospholamban inhibits Ca-ATPase conformational changes involving the E2 intermediate. , 2007, Biochemistry.

[19]  M. Zaccolo,et al.  Nitroxyl Improves Cellular Heart Function by Directly Enhancing Cardiac Sarcoplasmic Reticulum Ca2+ Cycling , 2007, Circulation Research.

[20]  G. Dorn,et al.  The Presence of Lys27 Instead of Asn27 in Human Phospholamban Promotes Sarcoplasmic Reticulum Ca2+-ATPase Superinhibition and Cardiac Remodeling , 2006, Circulation.

[21]  Douglas L Mann,et al.  Mechanisms and models in heart failure: the biomechanical model and beyond. , 2005, Circulation.

[22]  J. Froehlich,et al.  Intermolecular conformational coupling and free energy exchange enhance the catalytic efficiency of cardiac muscle SERCA2a following the relief of phospholamban inhibition. , 2005, Biochemistry.

[23]  S. Cortassa,et al.  Percolation and criticality in a mitochondrial network. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[24]  B. Griffith,et al.  Improved expression and characterization of Ca2+-ATPase and phospholamban in High-Five cells. , 2004, Protein expression and purification.

[25]  Brian O'Rourke,et al.  Synchronized Whole Cell Oscillations in Mitochondrial Metabolism Triggered by a Local Release of Reactive Oxygen Species in Cardiac Myocytes* , 2003, Journal of Biological Chemistry.

[26]  T. Squier,et al.  Phosphorylation by cAMP-dependent protein kinase modulates the structural coupling between the transmembrane and cytosolic domains of phospholamban. , 2003, Biochemistry.

[27]  E. Kranias,et al.  Calcium: Phospholamban: a crucial regulator of cardiac contractility , 2003, Nature Reviews Molecular Cell Biology.

[28]  D. Kass,et al.  Positive inotropic and lusitropic effects of HNO/NO− in failing hearts: Independence from β-adrenergic signaling , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[29]  J. Froehlich,et al.  Phospholamban inhibits Ca2+ pump oligomerization and intersubunit free energy exchange leading to activation of cardiac muscle SERCA2a. , 2003, Annals of the New York Academy of Sciences.

[30]  Steven R Houser,et al.  Is depressed myocyte contractility centrally involved in heart failure? , 2003, Circulation research.

[31]  Harvard Medical School,et al.  Targeting Phospholamban by Gene Transfer in Human Heart Failure , 2002, Circulation.

[32]  U. Schmidt,et al.  Improvement in Survival and Cardiac Metabolism After Gene Transfer of Sarcoplasmic Reticulum Ca2+-ATPase in a Rat Model of Heart Failure , 2001, Circulation.

[33]  D. Kass,et al.  Nitroxyl anion exerts redox-sensitive positive cardiac inotropy in vivo by calcitonin gene-related peptide signaling , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Andrew N. Carr,et al.  Gender influences on sarcoplasmic reticulum Ca2+-handling in failing human myocardium. , 2001, Journal of molecular and cellular cardiology.

[35]  G. Dorn,et al.  Superinhibition of Sarcoplasmic Reticulum Function by Phospholamban Induces Cardiac Contractile Failure* , 2001, The Journal of Biological Chemistry.

[36]  R. Hajjar,et al.  Overwhelming evidence of the beneficial effects of SERCA gene transfer in heart failure. , 2001, Circulation research.

[37]  L. Leinwand,et al.  Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy. , 2001, The Journal of clinical investigation.

[38]  U. Schmidt,et al.  Restoration of contractile function in isolated cardiomyocytes from failing human hearts by gene transfer of SERCA2a. , 1999, Circulation.

[39]  D. Bers,et al.  Ca2+ handling and sarcoplasmic reticulum Ca2+ content in isolated failing and nonfailing human myocardium. , 1999, Circulation research.

[40]  A. Spielman,et al.  Comparison of the kinetic effects of phospholamban phosphorylation and anti-phospholamban monoclonal antibody on the calcium pump in purified cardiac sarcoplasmic reticulum membranes. , 1997, Biochemistry.

[41]  L. Jones,et al.  Functional Co-expression of the Canine Cardiac Ca2+Pump and Phospholamban in Spodoptera frugiperda (Sf21) Cells Reveals New Insights on ATPase Regulation* , 1997, The Journal of Biological Chemistry.

[42]  D. Maclennan,et al.  Phospholamban Inhibitory Function Is Activated by Depolymerization* , 1997, The Journal of Biological Chemistry.

[43]  S. Negash,et al.  Phosphorylation of phospholamban by cAMP-dependent protein kinase enhances interactions between Ca-ATPase polypeptide chains in cardiac sarcoplasmic reticulum membranes. , 1996, Biochemistry.

[44]  N. Alpert,et al.  Alterations in sarcoplasmic reticulum gene expression in human heart failure. A possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. , 1993, Circulation research.

[45]  D. D. Thomas,et al.  Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: electron paramagnetic resonance. , 1991, Biochemistry.

[46]  D. D. Thomas,et al.  Conformational transitions in the calcium adenosinetriphosphatase studied by time-resolved fluorescence resonance energy transfer. , 1989, Biochemistry.

[47]  D. D. Thomas,et al.  Lipid fluidity directly modulates the overall protein rotational mobility of the Ca-ATPase in sarcoplasmic reticulum. , 1988, The Journal of biological chemistry.

[48]  T. Levine,et al.  Calcium uptake by cardiac sarcoplasmic reticulum in human dilated cardiomyopathy. , 1987, Cardiovascular research.

[49]  D. D. Thomas,et al.  Methodology for increased precision in saturation transfer electron paramagnetic resonance studies of rotational dynamics. , 1986, Biophysical journal.

[50]  D. D. Thomas,et al.  Applications of new saturation transfer electron paramagnetic resonance methodology to the rotational dynamics of the Ca-ATPase in sarcoplasmic reticulum membranes. , 1986, Biophysical journal.

[51]  D. D. Thomas,et al.  Temperature dependence of rotational dynamics of protein and lipid in sarcoplasmic reticulum membranes. , 1986, Biochemistry.

[52]  P. A. Lanzetta,et al.  An improved assay for nanomole amounts of inorganic phosphate. , 1979, Analytical biochemistry.

[53]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[54]  A. Gornall,et al.  Determination of serum proteins by means of the biuret reaction. , 1949, The Journal of biological chemistry.