Effects of mutant and antisense RNA of phospholamban on SR Ca2+-ATPase activity and cardiac myocyte contractility

Background-The delayed cardiac relaxation in failing hearts has been attrib uted to a reduced activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA2), Phospholamban (PLB) inhibits SERCA2 activity and is therefore a potential t arget to improve the cardiac performance in heart failure. Methods and Results-Mutants of PLB (Adv/mPLB) ol antisense RNA of PLB (Adv/ asPLB) was expressed in cardiac myocytes by recombinant adenovirus, and the ir effects on SERCA2 activity and myocyte contractility were studied. One m PLB, K3E/R14E, pentamerized with endogenous PLB in neonatal myocytes and re sulted in a 45% increase in the affinity of SERCA2 for Ca2+ and 27% faster diastolic Ca2+ decline as determined by SR Ca-45 uptake assays and by indo I-facilitated Ca2+ transient measurement, respectively. Edge-detection anal ysis of adult myocyte contractility showed a 74% increase in fractional sho rtening, accompanied by 115% increase in velocity of relengthening and 25% decrease in time to half-maximal relengthening. In parallel, infection of n eonatal cardiac myocytes by Adv/asPLB decreased the endogenous PLB level by 54%, which was associated with a 35% increase in Ca2+ affinity of SERCA2 a nd 21% faster diastolic Ca2+ decline. However, in adult cardiac myocytes, A dv/asPLB failed to significantly alter the endogenous PLB level, the SERCA2 activity, or most of the contractile parameters. Conclusions-K3E/R14E is a dominant negative mutant of PLB that disrupts the structural integrity and function of the endogenous PLB and consequently e nhances SERCA2 activity and myocyte contractility. In neonatal myocytes, th e decrease in steady-state abundance of PLB by asPLB also leads to increase d SERCA2 activity.