Pitavastatin-attenuated cardiac dysfunction in mice with dilated cardiomyopathy via regulation of myocardial calcium handling proteins

C57BL/6 mice with dilated cardiomyopathy (DCM) were randomly divided to receive placebo or pitavastatin at a dose of 1 or 3 mg kg-1d-1. After 8 weeks treatment, mice with dilated cardiomyopathy developed serious cardiac dysfunction characterized by significantly enhanced left ventricular end-diastolic diameter (LVIDd), decreased left ventricular ejection fraction (LVEF) as well as left ventricular short axis fractional shortening (LVFS), accompanied with enlarged cardiomyocytes, and increased plasma levels of N-terminal pro-B type natriuretic peptide (NT-proBNP) and plasma angiotensin II (AngII) concentration. Moreover, myocardium sarcoplasmic reticulum Ca2+ pump (SERCA-2) activity was decreased. The ratio of phosphorylated phospholamban (PLB) to total PLB decreased significantly with the down-regulation of SERCA- -2a and ryanodine receptor (RyR2) expression. Pitavastatin was found to ameliorate the cardiac dysfunction in mice with dilated cardiomyopathy by reversing the changes in the ratios of phosphorylated PLB to total PLB, SERCA-2a and RyR2 via reducing the plasma AngII concentration and the expressions of myocardium angiotensin II type 1 receptor (AT1R) and protein kinase C (PKC)b2. The possible underlying mechanism might be the regulation of myocardial AT1R-PKCb2-Ca2+ handling proteins.

Abstract 087: Reversal of Aging-Induced Contrast Changes of Cardiac SERCA 2a and Inducible Nitric Oxide Synthase in Mice Deficient in Beta3-Adrenoreceptor: Insights into the Molecular Mechanism of Cardiac Aging

Background: We have shown previously that aging-induced cardiac dysfunction and β-adrenergic desensitization were prevented in β 3 -adrenergic receptor (AR) knockout (β 3 KO) aged mice. However, the molecular mechanism is unclear. We hypothesize that reversal of aging-induced alterations of cardiomyocyte SR Ca 2+ -ATPase (SERCA 2a) and inducible nitric oxide (NO) synthase by β 3 -AR deficiency may play a key role for the protective effect. Methods: We compared SERCA 2a, iNOS, β 1 - and β 3 -AR protein expression, myocyte contractile, and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to isoproterenol (ISO, 10 -8 M) in cardiomyocytes obtained from 2 young (Y) (~6 mo) and 2 aged (A) (~26-30 mo) groups (5/group) of wild-type (WT) and β 3 KO mice, respectively. Results: Compared with YWT, AWT myocytes had significantly decreased protein levels of SERCA 2a (AWT: 0.22 vs YWT: 0.61) and β 1 -AR (0.34 vs 0.56), but increased iNOS (0.49 vs 0.24) and β 3 -AR (0.29 vs 0.14). These changes were associated with reduced basal cell contraction (dL/dt max ) (84.3 vs 124.8 μm/s), relaxation (dR/dt max ) (-66.1 vs -98.8 μm/s), and [Ca 2+ ] iT (0.19 vs 0.23). This was accompanied by diminished ISO-stimulated inotropic response. In AWT myocytes, ISO caused significantly less increases in dL/dt max (34% vs 82%), dR/dt max (22% vs 60%), and [Ca 2+ ] iT (15% vs 35%). Compared with YWT, Yβ 3 KO did not alter basal myocyte contraction and relaxation and response to ISO stimulation, but had significantly increased protein levels of SERCA 2a (Yβ 3 KO: 1.3 vs YWT: 0.61) and reduced iNOS (0.17 vs 0.24) with relatively unchanged β 1 -AR (0.63 vs 0.60). Aβ 3 KO mice had similar alterations. Importantly, in contrast to AWT, in Aβ 3 KO myocytes, the increased SERCA 2a (1.1) and reduced iNOS (0.19) correlated with normal basal cell contraction and relaxation with preserved ISO-stimulated inotropic response. ISO caused similar increases in dL/dt max (82% vs 84%) and [Ca 2+ ] iT (31% vs 33%) compared to Yβ 3 KO mice. Conclusions: β 3 -AR deficiency prevents aging-caused downregulation of cardiac β 1 -ARs and reverses increased iNOS and decreased SERCA 2a, leading to the preservation of myocyte function, [Ca 2+ ] iT , and β-adrenergic reserve in aged hearts. Thus, blocking β 3 -AR may provide a new strategy for myocardial aging.

Age-Associated Changes in Ca2+-ATPase and Oxidative Damage in Sarcoplasmic Reticulum of Rat Heart

Altered Ca2+ handling may be responsible for the development of cardiac contractile dysfunctions with advanced age. In the present study, we investigated the roles of oxidative damage to sarcoplasmic reticulum (SR) and expression of Ca2+-ATPase (SERCA 2a) and phospholamban in age-associated dysfunction of cardiac SR. SR vesicles were prepared from hearts of 2-, 6-, 15-, and 26-month-old Wistar rats. Although activity of Ca2+-ATPase decreased with advancing age, no differences in relative amounts of SERCA 2a and phospholamban protein were observed. On the other hand, significant accumulation of protein oxidative damage occurred with aging. The results of this study suggest that age-related alteration in Ca2+-ATPase activity in the rat heart is not a consequence of decreased protein levels of SERCA 2a and phospholamban, but could arise from oxidative modifications of SR proteins. Cellular oxidative damage caused by reactive oxygen species could contribute to age-related alternations in myocardial relaxation.

The onset of left ventricular diastolic dysfunction in SHR rats is not related to hypertrophy or hypertension

Left ventricular (LV) diastolic dysfunction, particularly relaxation abnormalities, are known to be associated with the development of LV hypertrophy (LVH). Preliminary human and animal studies suggested that early LV diastolic dysfunction may be revealed independently of LVH. However, whether LV diastolic dysfunction is compromised before the onset of hypertension and LVH remains unknown. We therefore evaluated LV diastolic function in spontaneously hypertensive rats (SHR) at different ages and tested whether LV diastolic dysfunction is associated with abnormal intracellular calcium homeostasis. LV systolic and diastolic functions were evaluated by invasive and echocardiographic methods in 3-week-old (without hypertension) and 5-week-old (with hypertension) SHR and Wistar-Kyoto control rats. Basal intracytoplasmic calcium and sarcoplasmic reticulum (SR) Ca2+ contents were measured in cardiomyocytes using fura-2 AM. Sarco(endo)plasmic Ca2+-ATPase isoform 2a (SERCA 2a) and phospholamban (PLB) expressions were quantified by Western blot and quantitative RT-PCR techniques. LV relaxation dysfunction was observed in 3-week-old SHR rats before onset of hypertension and LVH. An increase in basal intracytoplasmic Ca2+ and a decrease in SR Ca2+ release were demonstrated in SHR. Decreased expression of SERCA 2a and Ser16 PLB (p16-PLB) protein levels was also observed in SHR rats, whereas mRNA expression was not decreased. For the first time, we have shown that LV myocardial dysfunction precedes hypertension in 3-week-old SHR rats. This LV myocardial dysfunction was associated with high diastolic [Ca2+]i possibly due to decreased SERCA 2a and p16-PLB protein levels. Diastolic dysfunction may be a potential predictive marker of arterial hypertension in genetic hypertension syndromes.