Role And Mechanisms Of Calcineurin In Left Ventricular Diastolic Dysfunction Induced By Pressure Overload

Diastolic heart failure (also known as heart failure with preserved ejection fraction) is currently the focus of basic and clinical research, since this syndrome is not the same as that of systolic heart failure in the pathogenesis and clinical features. Hypertension, diabetes millitus as well as elderly women are always conditions accompanied with diastolic heart failure. It is believed left ventricular diastolic dysfunction is the earliest manifestation of diastolic heart failure, which is characterized by delayed relaxation and/or increased myocardial stiffness. However, there is not much research on how a normal heart progressed to diastolic dysfunction and diastolic heart failure under pathological stimuli. On one hand, there is no well accepted animal model of diastolic dysfunction; on the other hand, the concept of diastolic dysfunction and diastolic heart failure is appreciated in recent years. Different from the treatment for systolic heart failure which can effectively alleviate symptoms and improve prognosis by aggressively reversing negative myocardial remodeling, treatment for diastolic heart failure is mainly targeted on the primary diseases which may induce diastolic dysfunction as well as diastolic heart failure (ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure2012). Angiotensin converting enzyme inhibitors or angiotensin receptor blockers are proved to significantly reduce heart failure hospitalization and mortality in systolic heart failure patients, whereas these benefits still need to be confirmed in diastolic heart failure patients.Hypertension is the most common cause of diastolic dysfunction and diastolic heart failure, and it is previously demonstrated that calcineurin, a calcium/calmodulin dependent serine/threonine protein phosphatase, together with its downstream signal nucleus factor of activated T cell (NFAT3) plays important roles in adverse myocardial remodeling in hypertension. However, the effects and precise mechanisms of calcineurin and its pathway in the main constitutes of the heart, that is, myocytes and cardiac fibroblasts, in pressure overload induced diastolic dysfunction remain to be elucidated.We previously conducted a meta analysis which concluded that in patients with heart failure with preserved ejection fraction, ACE inhibitors reduced the risk of all cause mortality without affecting cardiovascular mortality and the incidence of any cause rehospitalization, though there was a beneficial trend for heart failure related readmission in subgroup patients. Therefore, further mechanism investigation and early identification for diastolic dysfunction may contribute to early intervention and improve the prognosis in diastolic heart failure patients. The present study aims:1) to select and assess a spontaneously hypertensive rat model on diastolic function;2) to clarify the role of calcineurin pathway in pressure overload induced diastolic dysfunction through mechanical stretch of myocytes and cardiac fibroblasts at molecular level;3) to test whether olmesartan ameliorates cardiac diastolic dysfunction in spontaneously hypertensive rats (SHR) through calcineurin (CaN) pathway. In brief, we hope to provide some theoretical basis and clinical intervention target for diastolic dysfunction and diastolic heart failure through this study.Part One Evaluation of left ventricular diastolic function in spontaneously hypertensive rat model by high-resolution echocardiographyObjective To evaluatie left ventricular diastolic function in spontaneously hypertensive rats (SHR) by high-resolution echocardiography.Methods Three-month-old male SHR (n=15) were used and followed until9months old at intervals of3months (n=5for each time point), Wistar rats of the same age (n=15) served as controls. Comprehensive echocardiographic parameters of cardiac structure and function were obtained through M-mode, Pulsed-wave Doppler and Tissue Doppler imaging with a17.5MHz probe. And serum N-terminal pro-B type natriuretic peptide (NT-proNBP) was measured by enzyme linked immunosorbent assay (ELISA), with histological examinations also determined to observe changes of heart tissue.Results Compared with Wistar rats, three-month-old SHR demonstrated significant elevated systolic blood pressure (168.0±4.5mmHg vs105.8±8.4mmHg, P<0.01) as well as NT-proBNP (284.38±6.54fmol/ml vs190.77±15.47fmol/ml, P<0.01), and increased interventricular septal thickness (IVS,1.58±0.08mm vs1.16±0.13mm, P<0.01), left ventricular posterior wall thickness (LVPW,1.65±0.08mm vs1.39±0.18mm, P<0.01), E/E’(26.28±1.76vs18.85±3.69, P<0.01) while SHR at six months and 9months exhibited substantial decreased E/A (2.05±0.23vs2.78±0.14for6months, P<0.05;1.92±0.13vs2.67±0.31for9months, P<0.05) and E’/A’(0.57±0.06vs1.46±0.10for6months, P<0.05;0.61±0.07vs1.44±0.11, P<0.05) together with increased serum NT-proBNP (333.40±13.42fmol/ml vs191.42±8.25fmol/ml for6months,354.31±9.39fmol/ml vs190.36±9.13fmol/ml for9months; both P<0.01) and E/E’(30.85±1.35vs21.08±1.64for6months,31.77±5.22vs21.54±2.11for9months, both P<0.01). There were no statistical differences pertaining to deceleration time (DT) and isovolumic relaxation time (IVRT) between SHR and Wistar rats among each age group. And E/A, E’/A’ decreased while E/E’ increased for SHR of6months and9months when compared to the values of3-months-old SHR. No difference of diastolic parameters was found in Wistar rats. Systolic function assessed by left ventricular ejection fraction (LVEF) and fractional shortening (FS) both remained unchanged for SHR and Wistar rats. HE staining demonstrated cardiomyocyte hypertrophy in SHR with advent of diastolic dysfunction as well as interstitial/perivascular fibrosis visualized by Masson trichrome staining.Conclusions SHR of6months exhibited explicit diastolic dysfunction, characterized by cardiomyocyte hypertrophy and interstitial/perivascular fibrosis, echocardio-graphic E/A, E’/A’ and E/E’ are the most valuable indices for assessing cardiac structure and diastolic function. SHR is a qualified model for the studying in mechanisms and intervention of diastolic dysfunction and diastolic heart failure. Part Two Role of calcineurin in cardiacmyocytes hypertrophy and apoptosis induced by mechanical stretchObjective To investigate if calcineurin and its pathway is involved in cardiacmyocyte hypertrophy and apoptosis induced by mechanical stretch.Methods Cardiomyocyte cultures were prepared by dissociation of1-3day old neonatal rat hearts and were differentially plated to remove fibroblasts. Mechanical stretch was applied to induce the pressure overload response. The cultured cardiomyocytes were randomly divided into control group, mechanical stretch group (MS group), calcineurin siRNA+mechanical stretch group (CaN-siRNA+MS group), FK506+mechanical stretch group (FK506+MS group), calcineurin overexpression+mechanical stretch group (CaN-overex+MS group). After the desired time of incubation, total proteins or RNA were extracted from cultured cardiomyocytes to detect changes of hypertrophic signals (ANP, SAA) and apoptotic proteins (Bax/Bcl-2).Results In vitro experiments showed that calcineurin RNAi and overexpression lentiviruses were successfully conducted. Compared with control group, MS group and CaN-overex+MS group induced profound hypertrophic and apoptotic responses, accompanied with overexpression of calcineurin/NFAT3, while CaN-siRNA+MS group and FK506+MS group demonstrated a downregulation of calcineurin and NFAT3, together with lower level of ANP, SAA, Bax and increased expression of Bcl-2.Conclusions Calcineurin pathway was essential for mechanical stretch induced cardiomyocytes adverse remodeling, interfere with or abolish calcineurin/NFAT3signal transduction could reverse hypertrophic response and attenuate cell apoptosis. Part Three Role of calcineurin in cardiac fibroblasts proliferation, transdifferentiation and apoptosis induced by mechanical stretchObjective To investigate if calcineurin pathway is involved in cardiac fibroblasts proliferation, transdifferentiation and apoptosis induced by mechanical stretch.Methods Primary cardiac fibroblasts from1-to2-day-old Sprague-Dawley rats were isolated by trypsinization and purified by differential anchoring velocity technology. The cultured cells of passage2-3were divided into:control group, mechanical stretch group (MS group), calcineurin siRNA+mechanical stretch group (CaN-siRNA+MS group), FK506+mechanical stretch group (FK506+MS group), calcineurin overexpression+mechanical stretch group (CaN-overex+MS group). Calcineurin and downstream NFAT3expressions were determined by western blot. Cardiac fibroblasts proliferation was determined by cell counting kit-8and detection of proliferating cell nuclear antigen (PCNA), cell transdifferentiation was measured by alpha-smooth muscle actin (a-SMA) through western blot and apoptosis by Bax/Bcl-2. With incubation of TGF-β1, we investigated the mechanism of calcineurin pathway in cardiac fibroblasts proliferation and transdifferentiation.Results Compared with control group, mechanical stretch promoted cardiac fibroblasts proliferation, transdifferentiation as well as apoptosis, overexpression of calcineurin could induce more cardiac fibroblasts adverse remodeling alike mechanical stretch. Pretreatment with calcineurin siRNA significantly attenuated cardiac fibroblasts proliferation, transdifferentiation and apoptosis when later stimulated by mechanical stretch, whereas pretreatment with FK506, a calcineurin inhibitor, only moderately inhibited cardiac fibroblasts proliferation and apoptosis with no obvious effect on cell transdifferentiation. Meanwhile, TGF-β1sitimulated cardiac fibroblasts proliferation and transdifferentiation was accompanied by upregulation of calcineurin and NFAT3proteins.Conclusion This study demonstrates that mechanical stretch promoted cardiac fibroblasts proliferation, transdifferentiation and apoptosis via activating calcineurin/NFAT3pathway, thus the calcineurin pathway might be a potential target of reversing myocardial fibrosis. Part Four Olmesartan attenuates hypertrophy and cardiac diastolic dysfunction in spontaneously hypertensive rats through inhibition of calcineurin pathwayObjective The present study aimed to test whether olmesartan ameliorates cardiac diastolic dysfunction in spontaneously hypertensive rats (SHR) through calcineurin (CaN) pathway.Methods Twenty-four male SHR of6months old were divided into saline-(n=12) and olmesartan-treated (n=12) group. Age matched Wistar-Kyoto (WKY, n=12) rats were served as controls. Saline (10ml·kg·d) or the same volume of olmesartan liquor (2.5mg·kg·d) was administered by gavage for3months. Heart rate (HR), systolic blood pressure (SBP), cardiac structure and function as well as histological studies were determined. Expression of CaN and downstream NFAT3were also detected.Results SHR of6months old exhibited evident cardiac hypertrophy and diastolic dysfunction as demonstrated by elevated SBP and E/E’, decreased E/A and E’/A’ while left ventricular ejection fraction (LVEF) and fractional shortening (FS) remained unimpaired, compared with age matched WKY rats. Treatment with olmesartan significantly decreased systolic blood pressure and ventricular hypertrophy, attenuated fibrosis and improved diastolic function (all p<0.05). Meanwhile, CaN/NFAT3expressions were both down-regulated in olmesartan group as compared with the other two groups (both p<0.05).Conclusions These data suggest the beneficial effect of olmesartan on cardiac structure and diastolic dysfunction, and CaN pathway was involved, at least in part, as one of the underlying mechanisms. This indicates a new therapeutic mechanism for olmesartan in diastolic dysfunction.