LAUSR

AIMS A key event in the regulation of cardiac contraction and relaxation is the phosphorylation of phospholamban (PLN) that relieves the inhibition of the sarco/endoplasmic Ca2+-ATPase SERCA2a. PLN exists in an equilibrium between monomers and pentamers. While only monomers can inhibit SERCA2a by direct interaction, the functional role of pentamers is still unclear. This study investigates the functional consequences of PLN pentamerization. METHODS AND RESULTS We generated transgenic mouse models expressing either a PLN mutant that cannot form pentamers (TgAFA-PLN) or wildtype PLN (TgPLN) in a PLN-deficient background. TgAFA-PLN hearts demonstrated 3-fold stronger phosphorylation of monomeric PLN, accelerated Ca2+ cycling of cardiomyocytes and enhanced contraction and relaxation of sarcomeres and whole hearts in vivo. All of these effects were observed under baseline conditions and abrogated upon inhibition of protein kinase A (PKA). Mechanistically, far western kinase assays revealed that PLN pentamers are phosphorylated by PKA directly and independent of any subunit exchange for free monomers. In vitro-phosphorylation of synthetic PLN demonstrated that pentamers even provide a preferred PKA substrate and compete with monomers for the kinase, thereby reducing monomer phosphorylation and maximizing SERCA2a inhibition. However, β-adrenergic stimulation induced strong PLN monomer phosphorylation in TgPLN hearts and sharp acceleration of cardiomyocyte Ca2+ cycling and hemodynamic values that now were indistinguishable from TgAFA-PLN and PLN-KO hearts. The pathophysiological relevance of PLN pentamerization was evaluated using transverse aortic constriction (TAC) to induce left ventricular pressure overload. Compared to TgPLN, TgAFA-PLN mice demonstrated reduced survival after TAC, impaired cardiac hemodynamics, failure to respond to adrenergic stimulation, higher heart weight, and increased myocardial fibrosis. CONCLUSIONS The findings show that PLN pentamerization greatly impacts on SERCA2a activity as it mediates the full range of PLN effects from maximum inhibition to full release of SERCA2a. function. This regulation is important for myocardial adaptation to sustained pressure overload. TRANSLATIONAL PERSPECTIVE Pentamerization of PLN adds to the regulation of cardiac contractile function and facilitates myocardial transition to an energy saving mode during resting phases. Thus, PLN pentamers would protect cardiomyocytes from energetic deficits, and they improve stress adaptation of the heart as shown for sustained pressure overload in this study. Strategies that target PLN pentamerization promise therapeutic potential in the treatment of myocardial maladaptation to stress as well as cardiac pathologies associated with altered monomer-to-pentamer ratios, e.g., cardiomyopathies due to PLN mutations, certain types of heart failure, and aged hearts.