This study provides several new findings showing that resveratrol prevents the transition from cardiac hypertrophy to HF induced by PO. First, we demonstrated resveratrol reversed LV hypertrophy induced by PO with time limitation. Second, we showed that resveratrol strongly and continuously prevented the cardiac structural and functional exacerbation due to PO. Third, we showed that resveratrol exerted multiple actions that may contribute to the beneficial effects including (1) inhibition of cardiac fibrosis, (2) prevention of myocardium ultrastructural alterations, (3) modulation of activation and expression of Ca2+ handling proteins.
Cardioprotective mechanisms of resveratrol
The development of HF is associated with marked myocardial fibrosis which is characterized by excessive extracellular matrix (ECM) deposition and myocardial stiffness [
13]. Limiting pathological myocardial fibrosis represents a potential therapeutic target to prevent HF. Previous studies have demonstrated that resveratrol has beneficial effects to reduce cardiac fibrosis in a variety of pathological models [
14],[
15]. For instance, resveratrol alleviated cardiac fibrosis in spontaneously hypertensive rats [
16] and decreased left ventricular interstitial and perivascular fibrosis in DOCA-salt rats [
17]. We demonstrated that resveratrol inhibited interstitial and perivascular fibrosis due to PO, indicative of the improvement of myocardial compliance and diastolic function. Indeed, increased LV pressure half-time, an index of diastolic heart function, was detected in banded rats, but prevented by resveratrol (see Additional file
3). Meanwhile, inhibition of cardiac fibrosis by resveratrol could explain the findings that LVm/BW in treated rats was remarkably lower than in banded rats at 12 weeks postsurgery while no difference in the size of cardiomyocytes was detected by histological analysis between two groups.
Second, we found that resveratrol protected against myocardial ultrastructural abnormalities induced by PO. The most obvious detriment to myocardial ultrastructure induced by PO was decrease of myofibrils as a result of rupture and degradation of myofilaments. Notably, resveratrol treatment normalized myocardial ultrastructure as evidenced by intact myofibrils and organized sarcomeres, which may be attributed to multiple mechanisms beneficial to cell survival including prevention of cardiomyocyte apoptosis, regulation of autophagy, and reduction of oxidative stress [
14],[
18]-[
20]. The other striking abnormality caused by PO was swollen and disordered mitochondria revealed by TEM. Resveratrol only partially prevented mitochondrial lesions. However, other studies demonstrated that resveratrol significantly attenuated the abnormality of mitochondrial ultrastructure in sepsis-induced myocardial depression with a large dose (30 mg/kg or 60 mg/kg) [
21], and preserved mitochondrial function and biogenesis contributing to improve cardiac energy metabolism and reduce oxidative stress in other models [
5],[
20],[
22],[
23]. This difference could be explained by the diversity of resveratrol dosage, the period of treatment, the stage of HF and animal models.
Ca
2+ cycling which refers to the release and reuptake of intracellular Ca
2+ is highly regulated in cardiomyocytes and determines the process of cardiac muscle contraction and relaxation. Defects in the regulation of Ca
2+ handling proteins contribute to HF. To our knowledge, the effects of resveratrol on Ca
2+ handling proteins in HF remain unclear. Impaired SERCA
2 function and enhanced NCX activity have been proposed as causes of reduced SR Ca
2+ load in HF. Moreover, PLB, a regulator of the affinity of SERCA
2 for Ca
2+, balances SR Ca
2+ uptake through inhibiting the affinity of SERCA
2 for Ca
2+ by unphosphorylated PLB and relieving this inhibition by phosphorylation. As a negative regulator of SERCA
2, hypophosphorylated PLB is an important cause of deficient SR Ca
2+ uptake in failing hearts. Accordingly, both SERCA
2 overexpression and PLB inhibition have been designed as therapeutic strategies for HF [
6]. In this study, PO caused substantially decreased SERCA
2 expression and ATPase activity, elevated NCX
1, increased PLB and decreased p-PLB, indicating that SR Ca
2+ load in this animal model is deficient, consequently leading to impaired contractile function. Resveratrol effectively upregulated SERCA
2 levels, ATPase activity and the ratio of phosphorylated to unphosphorylated PLB, accompanied by a significant downregulation of NCX
1, indicative of improved SR Ca
2+ load in hearts. Consistent with our results, a previous study showed that increased SERCA
2a expression by resveratrol improved contractile function in chronic type 1 diabetes [
12].
Additionally, we detected a significant increase in expression and phosphorylation of CaMKII due to PO. It has been reported that CaMKII hyperphosphorylation of RyR
2 accounts for excessive diastolic SR Ca
2+ leak in non-ischemic (aortic banding) cardiomyopathy, but not in ischemia (post-MI) in which PKA phosphorylation of RyR
2 is involved [
24]. This leads to increased RyR
2 open probability and a diastolic SR Ca
2+ leak because of a higher sensitivity to Ca
2+-induced Ca
2+ release at low cytoplasmic Ca
2+ concentrations. In our experiment, resveratrol attenuated the increase in CaMKII level and completely inhibited hyperphosphorylation of CaMKII. It has been demonstrated that RyR
2 leak was inhibited through inhibition of CaMKII phosphorylation. Knock-in mice with an inactivated CaMKII phosphorylation site on RyR
2 had lower SR Ca
2+ leak and improved SR Ca
2+ load, and were relatively protected from HF development after transverse aortic constriction [
25]. In patients with HF, CaMKII- but not PKA-dependent RyR
2 phosphorylation was significantly increased, accompanied by increased SR Ca
2+ leak, reduced systolic Ca
2+ transients, depletion of SR Ca
2+ storage and elevated diastolic Ca
2+ levels. Moreover, CaMKII inhibition, but not inhibition of PKA yielded a reduction of the SR Ca
2+ leak [
25]. Therefore, we speculate that CaMKII downregulation by resveratrol plays a major role in preventing the development of cardiac dysfunction in aortic banded animals via inhibition of RyR
2 leak.
Although the activity of RyR
2 channel in HF was investigated in many studies, very little is known about expression of RyR
2 and its significance in failing heart. We found that RyR
2 protein expression was significantly decreased in myocardium of HF and resveratrol effectively enhanced the expression of RyR
2. We speculate that downregulation of RyR
2 may decrease systolic Ca
2+ release and impair cardiac contractility. Similarly, Kubalova et al. found that RyR
2 content in failing hearts was decreased to approximately half of control values, but the levels of other proteins of the Ca
2+ release channel complex such as triadin and junctin were not changed. The altered stoichiometry of triadin and junctin to RyR
2 may increase activity of RyR
2, thus leading to the abnormal Ca
2+ handling in HF [
26].
A prevailing theory is that one of the major mechanisms proposed to underlie resveratrol mediated cardioprotection is reduction of oxidative stress [
4],[
27],[
28]. Polydatin, a resveratrol glucoside, has been shown to prevent enhanced Ca
2+ spark-mediated SR leak by reducing oxidative stress in RyR
2 in burn-traumatized heart, leading to protection of cardiac function against burn injury [
29]. Further studies are needed to understand whether reduction of oxidative stress by resveratrol could regulate RyR
2 and other Ca
2+ handling proteins in aortic banded rats.