Rosuvastatin-attenuated heart failure in aged spontaneously hypertensive rats via PKCα/β2 signal pathway

There are controversies concerning the capacity of Rosuvastatin to attenuate heart failure in end-stage hypertension. The aim of the study was to show whether the Rosuvastatin might be effective or not for the heart failure treatment. Twenty-one spontaneously hypertensive rats (SHRs) aged 52 weeks with heart failure were randomly divided into three groups: two receiving Rosuvastatin at 20 and 40 mg/kg/day, respectively, and the third, placebo for comparison with seven Wistar-Kyoto rats (WKYs) as controls. After an 8-week treatment, the systolic blood pressure (SBP) and echocardiographic features were evaluated; mRNA level of B-type natriuretic peptide (BNP) and plasma NT-proBNP concentration were measured; the heart tissues were observed under electron microscope (EM); myocardial sarcoplasmic reticulum Ca2+ pump (SERCA-2) activity and mitochondria cytochrome C oxidase (CCO) activity were measured; the expressions of SERCA-2a, phospholamban (PLB), ryanodine receptor2 (RyR2), sodium–calcium exchanger 1 (NCX1), Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase inhibitor-1 (PPI-1) were detected by Western blot and RT-qPCR; and the total and phosphorylation of protein kinase Cα/β (PKCα/β) were measured. Aged SHRs with heart failure was characterized by significantly decreased left ventricular ejection fraction and left ventricular fraction shortening, enhanced left ventricular end-diastolic diameter and LV Volume, accompanied by increased plasma NT-proBNP and elevated BNP gene expression. Damaged myofibrils, vacuolated mitochondria and swollen sarcoplasmic reticulum were observed by EM. Myocardium mitochondria CCO and SERCA-2 activity decreased. The expressions of PLB and NCX1 increased significantly with up-regulation of PPI-1 and down-regulation of CaMKII, whereas that of RyR2 decreased. Rosuvastatin was found to ameliorate the heart failure in aged SHRs and to improve changes in SERCA-2a, PLB, RyR2, NCX1, CaMKII and PPI-1; PKCα/β2 signal pathway to be suppressed; the protective effect of Rosuvastatin to be dose dependent. In conclusion, the heart failure of aged SHRs that was developed during the end stage of hypertension could be ameliorated by Rosuvastatin.


Introduction
Hypertension has become the most familiar 'epidemic disease' in the world. Because of its harmful complications in the cardiovascular system, it has been identified as the mayor risk factor for high cardiac mortality. Although plenty of new anti-hypertensive medications have been developed [1,2], ventricular hypertrophy and the subsequent congestive heart failure are still the common occurrences in most aged hypertensives [3]. Therefore, an effective, safe and widely used agent is urgently needed to attenuate the progress of heart failure.
The HMG-CoA-reductase inhibitors (statins) exert a protective effect on the cardiovascular system [4,5], in addition to their lipidlowering action [6][7][8], as indicated by the heart function restoration in the hypertensive patients with or without coronary diseases [9][10][11]. Furthermore, the JUPITER trial suggested that Rosuvastatin could effectively decrease C-reactive protein (CRP) level, thereby reducing cardiovascular event rates [12,13]. Braunwald [14] doubted that this effect might be based on reducing inflammation per se, without an effect on LDL-C. However, the exact role and underlying mechanism of these protective effects are not completely clear.
The purpose of this study was to observe the impairment of cardiac systolic function in aged spontaneously hypertensive rats (SHRs). We reported here that Rosuvastatin could ameliorate the impairment that occurs at the end stage of hypertension in the aged SHRs; and provide evidence indicating signalling pathways are involved.

Material and methods Animals
Twenty-one male SHRs, aged 52 weeks and weighing approximately 400 g, and seven age-matched normotensive male Wistar-Kyoto rats (WKY) as controls (Animal Administration Center of Fudan University, Shanghai, China) were bred ad libitum. SHRs were randomly divided into three groups, the first two receiving Rosuvastatin at a dose of 20 mg/kg/day (SHR+LD, n = 7) and 40 mg/kg/day (SHR+HD, n = 7) respectively; the third, placebo (SHR, n = 7) for comparison with seven WKYs as controls. Rosuvastatin and vehicle were daily administered through an intra-gastric tube for 8 weeks. All animal experimental procedures were approved by the Animal Care and Use Committee of Fudan University and performed in accordance with the guide for the care and use of laboratory animals (NIH publication no. 85-23, National Academy Press, Washington, DC, USA, revised 1996).

Measurement of systolic blood pressure
All the rats were trained to adapt themselves to the restraining cages and tail-cuff apparatus for the standard non-invasive tail-cuff method before SBP measurement [15], which was performed at the beginning without exception, and then every 4 weeks under their conscious conditions. Each measurement was repeated thrice.

Determination of plasma NT-proBNP concentration
The animals were killed so that their blood samples were collected from the abdominal aorta, the blood serum separated and NT-proBNP concentration determined by ELISA according to the manufacturer's instructions (R & D Systems, Minneapolis, MN, USA).

Measurement of cardiac function
Trans-thoracic echocardiographic analysis was performed with an animal-specific instrument (VisualSonics ® Vevo770 ® ; VisualSonicsInc, Toronto, Canada), as previously described [16]. The animals were anaesthetized with isoflurane before M-mode images of the left ven-tricle were recorded when the heart rate was near 400 bpm. To evaluate the function of their hearts, the following parameters were measured: the left ventricular end-diastolic diameter (LVIDd), left ventricular volume (LV Volume), left ventricular ejection fraction (LVEF), left ventricular short-axis fractional shortening (LVFS), left ventricular stroke volume (SV).

Transmission electron microscopy
From each group the cardiac tissues were minced into small pieces ( 1 mm 3 ) and fixed in 2.5% glutaraldehyde in 0.1 mol/l sodium cacodylate buffer (pH 7.3) for 2 hrs. The specimens were rinsed in buffer, postfixed in cacodylate-buffered 2% OsO 4 , stained en bloc in uranyl acetate, dehydrated gradient in ethanol and embedded in epoxy resin. Finally, 50-70 nm super thin slices were prepared, stained with uranyl acetate and lead citrate and examined under an electron microscope (Philips TECNA10; Philips, Amsterdam, The Netherlands).

Measurement of mitochondria cytochrome c oxidase activity
Mitochondria were isolated from left ventricle as described previously [17]. The final crude mitochondrial pellet was resuspended in sucrosehistidine-EDTA buffer, and the protein concentrations were determined via bicinchoninic acid method. The cytochrome c oxidase (CCO) activity was measured as described by Subbuswamy [18].

Measurement of SERCA-2 activity
The protein extraction of the cardiac tissues was prepared, as previously described [19]; the activity of SERCA-2 was measured according to the operating instructions of Ca 2+ -ATPase kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Western blot
The ventricle tissues were removed rapidly from the rats to be stored at À80°C. The expressions of phosphorylated PKC and calcium-handling proteins were measured via Western blot and normalized to the protein level of b-actin or GAPDH. From the frozen ventricle tissues were extracted the total proteins, whose concentration was determined with a BCA protein assay. SERCA2a, PLB, PPI-1, NCX1 (1:1000; ABCAM, Cambridge, UK), CaMKII, RyR, PKCa, PKCb1, PKCb2, phospho-PKCa, phospho-PKCb1 and phospho-PKCb2 (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were examined by Western blot as previously described [15], and the optic densities were analysed using ImagePro 5.0 (Media Cybernetics, Inc., Silver Spring, MD, USA).

Statistical analysis
The results were presented as mean ± S.E.M. and analysed using oneway ANOVA followed by Fisher's LSD test for multiple comparisons using the SPSS software package, version 16.0 (SPSS Inc., Chicago, IL, USA), P < 0.05 considered as statistically significant.

Lowering effect of Rosuvastatin on blood pressure
The average SBP was found to be higher in SHR controls than in WKYs by 59.4% (P < 0.01). However, no significant difference was observed among SHR+LD, SHR+HD and SHR controls after 4 and 8 weeks, respectively (P > 0.05; Table 1).

Effect of Rosuvastatin on cardiac structure and function of SHRs
Echocardiographic measurements were conducted in vitro to prove whether myocardium of SHR underwent heart malfunction (Fig. 1A). It was found that LVEF and LVFS were lower in SHR controls than in WKYs by 37.5% and 45.5% respectively (P < 0.05). In SHR+HD, LVEF and LVFS increased by 52.9% and 66.3%, respectively, when compared with SHR controls (P < 0.05). However, SV showed no significant difference among these four groups (P > 0.05) ( Fig. 1B and C).
The results also indicated an increase in LVIDd and LV volume in SHR controls by 34.8% and 80%, respectively, when compared with WKYs (P < 0.05). In SHR+HD, significant reductions developed in LVIDd and LV volume by 20.1% and 33.9%, respectively (P < 0.05; Fig. 1D and E).
According to the general changes, the results of the transmission electron microscopy showed the losses and damages of myofibrils in SHR controls when compared with WKYs ( Fig. 2A). Swollen, fragmented and vacuolated mitochondria were observed to be evident in SHRs, and the cristae in the mitochondria appeared distorted and in some cases were completely lysed. Furthermore, there appeared dilated and swollen sarcoplasmic reticulum in SHRs (Fig. 2B). Treated with Rosuvastatin, SHRs showed improved cardiac structure as indicated by the reversed losses and damages of myofibrils, and increased volume of myofibrils. Moreover, the swollen, fragmented and vacuolated mitochondria were alleviated, whereas the sarcoplasmic reticulum was not as dilated and swollen as that of SHR controls. The improvement of the cardiac structure was more apparent in SHR +HD than in SHR+LD ( Fig. 2C and D).
In addition to the morphologic alterations, plasma NT-proBNP levels were examined to be significantly increased in SHR controls in comparison with WKYs, and to be decreased in SHR+LD and SHR +HD by 12.1% and 19.5% respectively (P < 0.01; Table 3). Meanwhile, the mRNA level of BNP examined by real-time RT-qPCR showed a 5.35-fold increase in SHR controls, and a decrease in SHR+LD and SHR+HD by 47.4% and 72.3% respectively (P < 0.01; Fig. 3).

Regulation of the mRNA and protein expressions of Ca 2+ -cycling protein in SHRs
To investigate the mechanism of Rosuvastatin in the attenuated cardiac function of SHRs, the expressions of myocardium Ca 2+ -handling proteins were evaluated.  PPI-1, dephosphorylation of the PLB and thus incapable of activating SERCA2a, was significantly up-regulated by 85.9% in SHR controls (P < 0.01), and significantly down-regulated in SHR+LD and SHR+HD by 56.4% and 77.3% respectively (P < 0.01). Meanwhile, the expression of CaMKII up-regulated significantly by 40.9% in SHR +HD (P < 0.01) but not SHR+LD (P > 0.05), when compared with SHR controls (Fig. 4A).
The expression of NCX1 was significantly up-regulated in SHR controls, but reversed in SHR+HD. Meanwhile, the protein expressions of SERCA2a, PLB and RyR2 were highly consistent with those of RT-qPCR, as indicated by Western blot (Fig. 4B and D).

Decreased expressions and phosphorylation of PKCa and PKCb2 in SHRs
PKC signalling pathway has been identified as a given pathway in the Ca 2+ -regulating process. It was found that Rosuvastatin down-regulated significantly the mRNA expression of PKCa and PKCb by 64.5% and 67.7% in SHR+LD, respectively, and by 7.29% and 66.4% in SHR+HD respectively (P < 0.01). Furthermore, the ratios of phosphorylation to the total PKCa and PKCb2 were significantly inhibited, but it was not true of PKCb1 in SHR+LD and SHR+HD when compared with SHR controls (Fig. 5).

Discussion
Most of the hypertensives tend to develop cardiac hypertrophy and even heart failure, although they receive the administration of antihypertensive medications. Rosuvastatin has been reported to be controversial in attenuating cardiac function [9], improving survival rate in those with diastolic heart failure [10] and decreasing all-cause mortality following a diagnosis of heart failure [11]. However, the mechanisms of these protective effects remain unknown.
In the present study, we used 52-week-old SHR as the model of the end-stage hypertensive heart disease, finding that the aged SHRs developed systolic heart failure characterized by the abnormal parameters of echocardiography and the increased NT-proBNP. We  also found that the mitochondria were swollen, fragmented and vacuolated and the sarcoplasmic reticulum (SR) were dilated and swollen in SHRs. The mitochondria CCO and SERCA2 activity were down-regulated. In addition, the decreased expressions of SER-CA2a, RyR2 and CaMKII were accompanied by an increase in PLB, NCX1 and PPI-1, suggesting a possible mechanism that the failure of SR can induce the impairments of Ca 2+ -handling proteins, thus causing heart failure. Rosuvastatin treatment can ameliorate heart failure of aged SHRs independent of blood pressure lowering, via dephosphorylation of PKCa/b2, followed by dephosphorylation of PPI-1 and increased expression of CaMKII, thus promoting phosphorylation of PLB and up-regulating SERCA2 activity.
It is well recognized that high blood pressure increases ventricular wall tension, stimulates cardiomyocytes to express cytokines [20], causes ventricular remodelling and consequently develops heart failure, the pathological changes of which can be attenuated with a reduction in SBP, thereby improving cardiac function. Statins have been reported on lowering blood pressure in both SHR and WKY/L-NAME rats by reducing total peripheral vascular resistance [21]. However, there have been controversies on their anti-hypertensive effects. Loch and Saka reported that statin treatment failed to alter blood pressure in Dahl salt-sensitive rats [22,23].
In the present study, no significant changes in SBP were observed among the three SHR groups. The main reason respon-sible for this could be that 52-week-old SHRs had developed severe endothelial compromise, perivascular fibrosis and arterial stiffness. In this case, Rosuvastatin with weak anti-hypertensive effect could not alter significantly the peripheral resistance and the resultant SBP. This result was consistent with the report by Prandin MG that the confounding factors, such as age and baseline SBP, might influence the blood pressure of each individual [24].
Previous studies have shown that statins can ameliorate BNP upregulation [25] and restore heart function in hypertensives [9] in addition to their lipid-lowering effects. This is also the case for the results from this study that a high concentration of Rosuvastatin could reverse the expressions of SERCA-2a, RyR2, PLB, NCX1, PPI-1 and CaMKII, thus ameliorating the cardiac malfunction at the end stage of hypertension.
However, there have been controversies around the therapeutic effect of statins on cardiac malfunction. In John Kjekshus' trial, even Rosuvastatin could reduce the total number of hospitalizations for heart failure, but produce no effect on heart failure mortality [26]. The UNIVERSE trials also failed to demonstrate a therapeutic effect of statin on LV remodelling in the patients with chronic heart failure [27]. It could be that those enrolled had been well treated for heart failure with a standard therapy before Rosuvastatin, which reduced significantly the population with potentially fatal heart failure enrolled in the trial. This could also be because the patients with the heart failure of ischaemic or non-ischaemic aetiology were not differentially enrolled. Thus, the different inclusion and exclusion criteria could cause different pathophysiological and aetiological changes in heart failure, confusing the consistence of the results in the cohort.
The previous literature has shown that PKCa/b function as fundamental regulators of cardiac contractility and Ca 2+ -handling proteins in cardiomyocyte [28][29][30][31], and PKCb2 inhibition attenuates myocardial infarction and hypertension-induced heart failure [32,33]. Moreover, the enhancement in cardiac contractility associated with PKCa gene deletion protected the myocardium against pressure overload-induced heart failure and dilated cardiomyopathy [34].
Our previous study [15] indicated that Rosuvastatin could decrease the expression of angiotensin II type 1 receptor (AT 1 R), one type of G-protein coupling receptor, in the ventricle, and resultantly inhibit mitogen-activated protein kinase (MAPK) signal pathway, thus ameliorate cardiac hypertrophy in hypertensive heart disease. To investigate whether PKC is involved in the inhibitory effect of AT 1 R by Rosuvastatin, we examined the mRNA and protein expression of PKCa/b in the myocardium, and found down-regulation of PKCa/b as well as decreased ratios of phosphorylation to total PKCa and PKCb2 after Rosuvastatin   It was reported that the decreased expression of PKC resulted in inactivation of PPI-1, activating the phosphorylation of protein phosphatase-1, which in turn regulated PLB phosphorylation [38,39], thereby promoting the function of SERCA-2a responsible for Ca 2+ loading and the magnitude of the Ca 2+ transients [40,41]. In this study, therefore, a decrease in SERCA2a expression along with an increase in PLB and NCX1 protein level in SHRs could lead to a decreased SR function causing a decreased SR Ca 2+ store and rate of Ca 2+ releasing from the SR. These changes were found to be capable of affecting the contractile function of the heart, which were reversed following the administration of Rosuvastatin, suggesting that Rosuvastatin could ameliorate the changes in Ca 2+ -handling protein via PKC signalling pathway.
It can be concluded from this study that Rosuvastatin therapy was proved to exert a protective effect on the end stage of hypertension with dephosphorylation of PKCa/b2, causing the consequent dephosphorylation of PPI-1 and increased expression of CaMKII, which could result in the decreased expression of PLB and increased SERCA2a, RyR2 in the aged SHRs.
Beyond lipid lowering, statins have many pleiotropic effects, such as stimulating stem cells [42,43], activating immune system [44], activating heme oxygenase-1 at the stress response elements [45] and having synergistic effects with telmisartan on endothelial progenitor cells [46]. Those we discovered above can be one of the statins' pleiotropic effects, which can make statins potentially beneficial to patients with heart failure, for whom hypertension is the main risk factor.