Aims

Contraction and relaxation of the heart is controlled by periodical changes of cytosolic calcium concentrations. The calcium ATPase SERCA2a initiates relaxation by removal of calcium from the cytosol. SERCA2a activity is regulated by phospholamban (PLN).

Besides PLN monomers that inhibit SERCA2a activity, PLN is known to form pentamers that do not possess any inherent SERCA2a-inhibitory potential. The present study examines the functional relevance of PLN pentamers for cardiomyocyte calcium regulation and sarcomere mechanics as well as whole heart function at baseline and under conditions of increased cardiac workload.

Methods and Results

We generated transgenic mouse models expressing either wildtype PLN (TgPLN) or a pentamer-deficient PLN mutant (TgAFA-PLN) in a genetic PLN knockout background in a cardiomyocyte-specific manner. In these mouse models, we examined the functional consequences of PLN pentamerization under basal conditions and after induction of chronic left ventricular pressure overload by transverse aortic constriction (TAC).

TgPLN and TgAFA-PLN mice were born at Mendelian ratios and hearts remained morphologically indistinguishable from wild-types over a period of >6 months. TAC of 8 week-old mice induced increased mortality in TgAFA-PLN mice compared to TgPLN. Furthermore, surviving TgAFA-PLN mice showed excessive cardiac fibrosis, a greater increase of heart weight, impaired left ventricular hemodynamics and a blunted response to adrenergic stimulation. These findings clearly pointed towards a protective role of PLN pentamers under conditions of chronically increased cardiac workload.

Biochemical analyses revealed ~3-fold stronger phosphorylation of SERCA2a-inhibitory monomers in TgAFA-PLN hearts at baseline and after TAC. Phosphorylation of PLN by protein kinase A (PKA) abolishes SERCA2a inhibition. Consequently, TgAFA-PLN showed faster kinetics of calcium cycling and sarcomere contraction/relaxation compared to TgPLN in isolated cardiomyocytes, and pharmacological PKA inhibition abolished these differences. Using far western kinase assays, we further showed that PLN pentamers constitute a direct PKA substrate that competes with PLN monomers for the active sites of the kinase, thereby reducing monomer phosphorylation. Importantly, we found no differences in expression and phosphorylation of key calcium-regulatory proteins other than PLN.

Hemodynamic measurements showed that our findings in single cardiomyocytes of TgAFA-PLN and TgPLN mice translated to whole heart function in vivo in that TgAFA-PLN showed enhanced contraction and relaxation kinetics under basal conditions. These differences were abrogated upon intravenous application of the beta-adrenergic receptor agonist dobutamine. Likewise, calcium cycling and sarcomere kinetics of TgPLN, which were slower under basal conditions, showed a stronger increase upon beta-adrenergic stimulation and became indistinguishable from TgAFA-PLN as well as PLN-KO hearts. Thus, PLN pentamers did not restrict PLN inactivation upon beta-adrenergic stimulation.

Conclusion

The findings demonstrate that PLN pentamerization is indispensable for maximizing SERCA2a inhibition under basal conditions without restricting PLN inactivation upon beta-adrenergic activation. The extended dynamic range of SERCA2a regulation and the increased sensitivity of the heart to adrenergic stimulation by PLN pentamers seem to play an important role in myocardial adaptation to sustained pressure overload.