Rationale: We previously showed that transgenic mice expressing Ca²⁺/calmodulin-dependent protein kinase II δ_C (CaMKII-TG) develop dilated cardiomyopathy associated with increased ryanodine receptors (RyR2) phosphorylation, enhanced sarcoplasmic reticulum (SR) Ca²⁺ leak and lowering of SR Ca²⁺ load. We hypothesized that phospholamban (PLN) ablation would restore SR Ca²⁺ load and prevent the decreased ventricular contractility, dilation and mortality seen in CaMKII-TG.

Objective: Our objectives were to generate CaMKII-TG mice lacking PLN, determine whether the maladaptive effects of cardiac CaMKIIδ_C expression were corrected, and establish the mechanistic basis for these changes.

Methods and Results: CaMKII-TG were crossed with PLN knockout (PLN-KO) mice to generate KO/TG mice. Myocytes from wild type (WT), CaMKII-TG, PLN-KO and KO/TG were compared. The decreased SR Ca²⁺ load and twitch Ca²⁺ transients seen in CaMKII-TG were normalized in KO/TG. Surprisingly the heart failure phenotype was exacerbated, as indicated by increased left ventricular dilation, decreased ventricular function, increased apoptosis and greater mortality. In KO/TG myocytes SR Ca²⁺ sparks and leak were significantly increased, presumably because of the combined effects of restored SR Ca²⁺ load and RyR2 phosphorylation. Mitochondrial Ca²⁺ loading was increased in cardiomyocytes from KO/TG versus WT or CaMKII-TG mice and this was dependent on elevated SR Ca²⁺ sparks. Cardiomyocytes from KO/TG showed poor viability, improved by inhibiting SR Ca²⁺ release and mitochondrial Ca²⁺ loading.

Conclusions: Normalizing cardiomyocyte SR Ca²⁺ loading in the face of elevated CaMKII and RyR2 phosphorylation leads to enhanced SR Ca²⁺ leak and mitochondrial Ca²⁺ elevation, associated with exacerbated cell death, heart failure and mortality.