Rescue of cardiomyocyte dysfunction by phospholamban ablation does not prevent ventricular failure in genetic hypertrophy.

Cardiac hypertrophy, either compensated or decompensated, is associated with cardiomyocyte contractile dysfunction from depressed sarcoplasmic reticulum (SR) Ca(2+) cycling. Normalization of Ca(2+) cycling by ablation or inhibition of the SR inhibitor phospholamban (PLN) has prevented cardiac failure in experimental dilated cardiomyopathy and is a promising therapeutic approach for human heart failure. However, the potential benefits of restoring SR function on primary cardiac hypertrophy, a common antecedent of human heart failure, are unknown. We therefore tested the efficacy of PLN ablation to correct hypertrophy and contractile dysfunction in two well-characterized and highly relevant genetic mouse models of hypertrophy and cardiac failure, Galphaq overexpression and human familial hypertrophic cardiomyopathy mutant myosin binding protein C (MyBP-C(MUT)) expression. In both models, PLN ablation normalized the characteristically prolonged cardiomyocyte Ca(2+) transients and enhanced unloaded fractional shortening with no change in SR Ca(2+) pump content. However, there was no parallel improvement in in vivo cardiac function or hypertrophy in either model. Likewise, the activation of JNK and calcineurin associated with Galphaq overexpression was not affected. Thus, PLN ablation normalized contractility in isolated myocytes, but failed to rescue the cardiomyopathic phenotype elicited by activation of the Galphaq pathway or MyBP-C mutations.

Evaluation of left ventricular diastolic function from spectral and color M-mode Doppler in genetically altered mice.

Doppler indices of transmitral flow are commonly used to assess noninvasively left ventricular (LV) diastolic function in species larger than mice. The objective of our study was to characterize patterns of LV diastolic function in 2 genetically altered mouse models using Doppler- and color M-mode echocardiography. Phospholamban (PLB) reversibly inhibits the sarcoplasmic reticulum Ca(2+) ATPase (SERCA) and is a key regulator of myocardial relaxation. Twelve-week-old PLB knockout mice (PLB/KO) were examined in parallel with age-matched transgenic mice expressing a mutant form of PLB (PLB/N27A) that exhibited superinhibition of SERCA. Transmitral Doppler flow indexes, including isovolumic relaxation time, the ratio of peak early-to-late filling velocities, and deceleration time of peak early transmitral velocity indicate impaired diastolic filling in the PLB/N27A mutants, but improved LV diastolic function in the PLB/KO mice. In addition, a relatively load-independent parameter of LV relaxation measured by color M-mode Doppler, the propagation velocity of early flow into the LV cavity confirmed the observed differences. We conclude that transmitral filling patterns and color M-mode flow propagation velocity reflect changes in myocardial relaxation in mice with genetically altered levels of PLB and may be useful tools to characterize LV diastolic function in other mouse models of disease.

Structural and functional implications of the phospholamban hinge domain: impaired SR Ca2+ uptake as a primary cause of heart failure.

OBJECTIVE: The role of sarcoplasmic reticulum (SR) in the onset and progression of heart failure is controversial. We tested the hypothesis that impairment of SR Ca2+ sequestration may be a primary cause for progressive left ventricular (LV) dysfunction and the phospholamban hinge domain may be critical in this process. METHODS: A phospholamban hinge domain mutant (PLB/N27A) was introduced in the cardiac compartment of the phospholamban null mouse. An integrative approach was used to characterize the resulting cardiac phenotype at a structural, cellular, whole organ and intact animal level. RESULTS: NMR analysis revealed a defined alteration in the alpha-helical configuration between residues Q22 to F35 in mutant phospholamban. Transgenic lines expressing similar levels of mutant compared to wild-type phospholamban exhibited super-inhibition of the SR Ca2+ ATPase affinity for Ca2+ (EC50 0.52 microM) in oxalate-supported Ca2+ uptake measurements, which translated into impaired relaxation and attenuated responses to beta-adrenergic stimulation. Importantly, a blunted force-frequency relation was observed in mutant hearts preceding left ventricular dilation. Upon aging to 10 months, the predominantly diastolic dysfunction progressed to congestive heart failure, characterized by induction of a fetal gene program, cardiac remodeling, lung congestion, depressed systolic function and early mortality. CONCLUSION: Increased inhibition of Ca2+ sequestration may be a causative factor in the development of left ventricular dysfunction and myocyte remodeling leading to heart failure. Furthermore, the hinge domain may play an important role in transmitting PLB's regulatory effects on SERCA.