Cardiac-specific overexpression of a superinhibitory pentameric phospholamban mutant enhances inhibition of cardiac function in vivo

Phospholamban is a regulator of the Ca2+ affinity of the cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2d) and of cardiac contractility, In vitro expression studies have shown that several mutant phospholamban monomers are superinhibitory, suggesting that monomeric phospholamban is the active species. However, a phospholamban Asn(27) --> Ala (N27A) mutant, which maintained a normal pentamer to monomer ratio, was shown to act as a superinhibitor of SERCA2a Ca2+ affinity, To determine whether the pentameric N27A mutant is superinhibitory in vivo, transgenic mice with cardiac-specific overexpression of mutant phospholamban mere generated. Quantitative immunoblotaing revealed a 61 +/- 6% increase in total phospholamban in mutant hearts, with 90% of the overexpressed protein being pentameric, The EC,, value for Ca2+ dependence of Ca2+ uptake was 0.69 +/- 0.07 mu m in mutant hearts, compared with 0.29 +/- 0.02 mu M in wild-type hearts or 0.43 +/- 0.03 mu M in hearts overexpressing wild-type PLB by 2-fold. Myocytes from phospholamban N27A mutant hearts also exhibited more depressed contractile parameters than wild-type phospholamban overexpressing cells. The shortening fraction was 52%, rates of shortening and relengthening were 46% and 38% respectively, and time for 80% decay of the Ca2+ signal was 146%, compared with wild-types (100%). Langendorff-perfuse mutant hearts also demonstrated depressed contractile parameters, Furthermore, in vivo echocardiography showed a depression in the ratio of early to late diastolic transmitral velocity and a 79% prolongation of the isovolumic relaxation time. Isoproterenol stimulation did not fully relieve the depressed contractile parameters at the cellular, organ, and intact animal levels. Thus, pentameric phospholamban N27A mutant can act as a superinhibitor of the affinity of SERCA2a for Ca2+ and of cardiac contractility in vivo.