ORIGINAL ARTICLE
Olmesartan Attenuates Cardiac Hypertrophy and Improves Cardiac Diastolic Function in Spontaneously Hypertensive Rats Through Inhibition of Calcineurin Pathway Mingqiang Fu, MD,* Jingmin Zhou, MD,* Jianfeng Xu, MD,* Hongmin Zhu, MD,* Jianquan Liao, MD,* Xiaotong Cui, MD,* Aijun Sun, PhD,* Michael Fu, MD,† Yunzeng Zou, PhD,*‡ Kai Hu, MD,* and Junbo Ge, MD, PhD*‡
Objective: To test whether olmesartan ameliorates cardiac diastolic
Key Words: spontaneously hypertensive rats, olmesartan, diastolic function, calcineurin pathway
dysfunction in spontaneously hypertensive rats (SHRs) through calcineurin pathway.
(J Cardiovasc Pharmacol Ô 2014;63:218–226)
Methods: Twenty-four male SHRs of 6 months were divided into saline- (n = 12) and olmesartan-treated (n = 12) groups. Agematched WKY (n = 12) rats served as controls. Saline (10 mL$kg$d) or the same volume of olmesartan liquor (2.5 mg$kg$d) was administered by gavage for 3 months. Heart rate, systolic blood pressure, cardiac structure, and function and histological studies were determined. Expression of calcineurin and downstream NFAT3 were also detected. Results: Compared with age-matched Wistar Kyoto rats, SHRs of 6 months exhibited evident cardiac hypertrophy and diastolic dysfunction as demonstrated by elevated systolic blood pressure and E/E0 , decreased E/A and E0 /A0 , while F, left ventricular ejection fraction and fractional shortening remained unimpaired. Treatment with olmesartan significantly decreased systolic blood pressure and ventricular hypertrophy, attenuated fibrosis, and improved diastolic function (all P , 0.05). Meanwhile, both calcineurin and NFAT3 expressions were downregulated in olmesartan group compared with the other 2 groups (both P , 0.05). Conclusions: These data suggest the beneficial effect of olmesartan on cardiac structure and diastolic dysfunction, and it may be mediated through calcineurin pathway. This indicates a new therapeutic target for diastolic dysfunction.
Received for publication June 18, 2013; accepted October 23, 2013. From the *Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, China; †Department of Medicine, Sahlgrenska University Hospital/Sahlgrenska, Gothenburg, Sweden; and ‡Institutes of Biomedical Sciences, Fudan University, Shanghai, China. Supported by grants from the National Basic Research Program of China (973 Program, 2012CB518605) and the National Natural Science Foundation of China (81070105) to J. Zhou and the National Natural Science Foundation of China (81100145) to J. Xu. M. Fu and J. Zhou have contributed equally. The authors report no conflicts of interest. Reprints: Junbo Ge, MD, PhD, Department of Cardiology, Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, Room 413, Building 9, 180 Fenglin Road, Shanghai 200032, China (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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INTRODUCTION Heart failure with preserved ejection fraction (HFPEF) is now well recognized and accounts for 30%–50% of heart failure patients, and it usually occurs in hypertension, diabetic mellitus, and elderly.1,2 The principle mechanism for HFPEF is considered to be left ventricular diastolic dysfunction (LVDD). LVDD is reported to be present in 30% of the general adult population without congestive heart failure and in half of patients with hypertension, and LVDD is found to be an independent risk factor for HFPEF and associated with increased cardiovascular morbidity and mortality even when asymptomatic.3,4 However, the precise mechanisms for LVDD still remain to be elucidated. LVDD is manifested as delayed relaxation and/or increased myocardial stiffness of left ventricle, with progressive left ventricular hypertrophy and fibrosis.5,6 Recently, an intracellular signaling pathway that includes the Ca2+/calmodulin activated serine/threonine phosphatase calcineurin (CaN) and downstream nuclear factor of activated T cells (NFAT3) have been shown to be a key mechanism mediating the development of cardiac hypertrophy,7 and angiotensin II is reported to be an upstream activator of calcineurin.8 However, Dupont et al have previously demonstrated that the onset of LVDD in spontaneously hypertensive rats (SHRs) is not related to hypertension or hypertrophy, and diastolic dysfunction precedes myocardial hypertrophy in the development of hypertension.9,10 Considering the aforementioned evidence, whether or not calcineurin pathway is involved in LVDD needs to be clarified. Renin-angiotensin system (RAS) is activated in HFPEF, though the usefulness of angiotensin-converting enzyme inhibitors, angiotensin II type 1 receptor blockers, and b-blockers has not been recommended in current HFPEF management guidelines, our meta-analysis and registry data showed RAS antagonists was associated with lower all-cause mortality.11,12 Therefore, the present study was designed to examine the effects of olmesartan, the newest RAS inhibitor, on cardiac hypertrophy and diastolic dysfunction in a rat model of spontaneous hypertension with a focus on calcineurin pathway. J Cardiovasc Pharmacol ä Volume 63, Number 3, March 2014
J Cardiovasc Pharmacol ä Volume 63, Number 3, March 2014
Olmesartan Attenuates Diastolic Dysfunction
METHODS
gained images and data were stored and then downloaded to a magnetooptical disk for offline analyses.
Experimental Animals Twelve male Wistar Kyoyo (WKY) rats and 24 male SHRs were purchased from Shanghai Laboratory Animal Centre, Chinese Academy Sciences, at 5 months. All rats were maintained in a specific pathogen-free room under conditions of temperature- and humidity-controlled with a 12:12-hour light–dark cycle and given standard laboratory chow and tap water ad libitum. All the animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Publication No. 85-23, revised 1996) and were approved by the Animal Care Committee in Zhongshan Hospital, Fudan University. Beginning at 6 months, the rats were divided into 3 groups: WKY + saline group, SHR + saline group, and SHR + olmesartan group (n = 12, respectively). Saline (10 mL$kg$d) or the same volume of olmesartan liquor (2.5 mg$kg$d) was administered by gastric gavage once a day for 3 months.
Histological Examinations Six rats from each group were euthanized after echocardiography completed. Then, the left ventricle was excised and weighed immediately to calculate left ventricular mass index, expressed as ratio of the left ventricular tissue weight by milligram to total BW by gram. Middle section of left ventricular short axis around papillary muscle level was then fixed in 10% formalin, embedded in paraffin, and cut into 4-mmthick sections and stained either with hematoxylin–eosin for cardiomyocyte size or with the Masson trichrome staining to evaluate the extent of fibrosis. Digital photographs were thereafter taken and analyzed using a high-resolution digital image analysis system (Qwin V3; Leica, Germany).
Western Blot Left ventricular tissues were homogenized in lysis buffer (Cell Signalling Technology, Inc.) with protease inhibitor cocktail (Sigma Chemical Co, St Louis, MO) and then electrophoresed in 10% polyacrylamide gel and transferred to PVDF membrane. The protein expressions of CaN and nuclear factor of activated T cells (NFAT3) were detected by immune blotting with antibody against CaN and NFAT3 (1:1000–1:2000, both were from Cell Signaling Technology, Inc), respectively. After 3 washes, the blots were incubated with horseradish peroxidase–conjugated secondary antibody (1:4000–1:5000, Kangchen Biotechnology, China). GAPDH (1:4000, Kangchen Biotechnology) was used as the internal control. Bands were visualized with ImageLab Software (BioRad Laboratories, Inc).
Assessment of Physiological Parameters and Echocardiography Body weight (BW) was determined by an electronic weigher, and systolic blood pressure and heart rate were measured by a tail-cuff method (BP-98A; Softron, Tokyo, Japan). Thereafter, the rats were lightly anaesthetized with ketamine hydrochloride (50 mg/kg, intraperitonealy) with a heart rate at 380 6 25 bpm. After anesthesia and precordial region being shaved, the rats were transferred to a heated platform and transthoracic echocardiographic measurements were performed in the supine position with a commercially available echocardiographic system (Vevo770 High Resolution Imaging System; VisualSonics, Toronto, Canada) using a 17.5 MHz scanhead. Two-dimensional guided M-mode echocardiography and pulsed-wave Doppler and tissue Doppler echocardiography were used to assess cardiac structure and functions. All measurements were averaged for 4 consecutive cardiac cycles and were carried out by 2 experienced technicians independently who were unaware of the experimental protocol. The
Quantitative Real-Time Polymerase Chain Reaction Total RNA was isolated from the left ventricular tissues by Trizol reagent (Invitrogen). After purification, RNA was subjected to the quantitative real-time polymerase chain reaction analysis for expressions of atrial natriuretic peptide (ANP) and skeletal a-actin (SAA) on an iCycler system (BioRad, Hercules, CA). The primers were as follows: for b-actin
TABLE 1. Routine Parameters Before and After Interventions in WKY and SHRs WKY + Saline Parameters SBP, mm Hg HR, bpm BW, g LVMI, mg/g LVEF, % FS, %
6 mo 115 364 372 2.4 83.8 53.5
6 6 6 6 6 6
9 11 17 0.2 0.7 1.2
SHR + Saline 9 mo
123 357 378 2.5 83.6 53.7
6 6 6 6 6 6
11 10 21 0.3 0.9 1.7
6 mo 185 384 352 1.8 90.8 63.1
6 6 6 6 6 6
16* 15‡ 22‡ 0.3* 0.4* 0.8*
SHR + Olmesartan 9 mo
202 388 393 2.2 90.5 63.1
6 6 6 6 6 6
14* 11‡ 19‡ 0.2* 0.7* 1.1*
6 mo 186 386 355 1.8 91.2 63.5
6 6 6 6 6 6
9 mo
17* 12‡ 18‡ 0.2* 0.6* 1.2*
174 385 382 1.6 90.4 63.0
6 6 6 6 6 6
19*† 12‡ 17‡§ 0.2*† 0.5* 1.3*
Data are means 6 SD. *P , 0.01 versus corresponding Wistar rats. †P , 0.01 versus 9-month-old SHR + saline group. ‡P , 0.05 versus corresponding Wistar rats. §P , 0.05 versus 9-month-old SHR + saline group. SBP, systolic blood pressure; HR, heart rate; BW, body weight; LVMI, left ventricular mass index; LVEF, left ventricular ejection fraction; FS, fractional shortening.
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FIGURE 1. Left ventricular morphology assessed by 2-dimensional echocardiography. LVEDD: left ventricular end-diastolic diameter; LVESD: left ventricular end-systolic diameter; A, B, and C represent 6-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively; D, E, and F represent 9-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively. *P , 0.05 versus corresponding 6-month-old rats, **P , 0.01 versus corresponding 6-month-old rats; †P , 0.05 versus 9-month-old SHR + saline group, ††P , 0.01 versus 9-month-old SHR + saline group.
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Olmesartan Attenuates Diastolic Dysfunction
FIGURE 2. Cross-sectional area of cardiac myocytes in the hearts of the 3 groups; A, B, and C represent 6-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively; D, E, and F represent 9-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively. *P , 0.05 versus corresponding 6-month-old rats, **P , 0.01 versus corresponding 6-month-old rats; †P , 0.05 versus 9-month-old SHR + saline group, ††P , 0.01 versus 9-month-old SHR + saline group.
gene, sense, 50 -CCCTAAGGCCAACCGTGAA-30 and antisense, 50 -GAGGCATACAGGGACAACACAG-30 ; for ANP gene, sense, 50 -GGTGTCCAA-CACAGATCTGA-30 and antisense, 50 -CACTAGACCACTCATCTAC-30 ; and for SAA gene, sense, 50 -AGCAGATGTGGATCACCAAG-30 and antisense, 50 -CTGCAACCA-CAGCACGATTG-30 . A comparative computed tomography method was used to determine relative quantification of messenger RNA expressions.
P , 0.05 was considered statistically significant. All analyses were carried out with SPSS16.0 statistical package for Windows (SPSS Inc, Chicago, IL).
Statistical Analysis
The baseline systolic blood pressure was markedly higher in the two 6-month-old SHR groups when compared with the WKY rats (P , 0.01). Treatment of olmesartan decreased blood pressure by 14% in SHR + olmesartan group
All values are presented as means 6 SD. Differences among groups were assessed by 1-way analysis of variance with a Student–Newman–Keuls test for post hoc analysis.
Ó 2014 Lippincott Williams & Wilkins
RESULTS Effects of Olmesartan on Blood Pressure and BW
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FIGURE 3. Relative area of fibrosis (%) in the hearts of the 3 groups; A, B, and C represent 6-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively; D, E, and F represent 9-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively. *P , 0.05 versus corresponding 6-month-old rats, **P , 0.01 versus corresponding 6-month-old rats; †P , 0.05 versus 9-month-old SHR + saline group, ††P , 0.01 versus 9-month-old SHR + saline group.
compared with SHR + saline group (P , 0.01) while no effect in heart rate was found among the 3 groups. As is illustrated in Table 1, BW and left ventricular mass index were significantly increased at the end of the study in SHR + saline group while with a lesser extent in SHR + olmesartan group.
Effect of Olmesartan on Cardiac Morphology Persistent high blood pressure induced overt left ventricular hypertrophy in SHRs of 6 months as demonstrated by M-mode echocardiographic parameters. Interventricular
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septum thickness in diastole and left ventricular posterior wall thickness all exhibited significant increases than those of agematched WKY rats (P , 0.01). Olmesartan therapy for 3 months significantly decreased left ventricular hypertrophy in SHR + olmesartan group compared with SHR + saline group (P , 0.01) (Fig. 1). As were with the echocardiographic changes of wall thickness, histological studies demonstrated that myocardial samples from the SHR groups developed obvious cardiac hypertrophy and fibrosis when compared with WKY rats Ó 2014 Lippincott Williams & Wilkins
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Olmesartan Attenuates Diastolic Dysfunction
FIGURE 4. Left ventricular diastolic function assessed by pulsed-wave Doppler imaging (upper left) and tissue Doppler imaging (lower left). E/A: ratio of maximal velocities of the E and A waves at mitral orifice level; E0 /A0 : ratio of maximal velocity of early (E0 ) and late (A0 ) diastolic waves at the level of lateral mitral annulus; E/E0 : ratio of E to E0 ; A, B, and C represent 6-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively; D, E, and F represent 9-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively. *P , 0.05 versus corresponding 6-month-old rats, **P , 0.01 versus corresponding 6-month-old rats; †P , 0.05 versus 9-month-old SHR + saline group, **††P , 0.01 versus 9-month-old SHR + saline group.
(P , 0.01) as exhibited by increased cardiomyocyte crosssectional area of left ventricle and myocardial fibrosis area. And olmesartan reduced both adverse remodeling at the end of the study between SHR + olmesartan group and SHR + saline group (Figs. 2, 3).
Effect of Olmesartan on Cardiac Functions Echocardiographic analyses of cardiac functions were determined by left ventricular ejection fraction, fractional shorting for systolic function, and ratio of maximal velocities of the E and A waves of mitral inflow (E/A) by pulsedwave Doppler imaging, ratio of maximal velocity of early to late diastolic waves (E0 /A0 ) at the level of lateral mitral annulus by tissue Doppler imaging, and E/E0 for diastolic function. As showed in Figure 4, a significant decrease in diastolic functional parameters, E/A and E0 /A0 , and an Ó 2014 Lippincott Williams & Wilkins
increase of E/E0 , were observed in 6-month-old SHRs in comparison with their respective WKY controls, indicating an abnormal diastolic function; olmesartan treatment attenuated diastolic dysfunction in SHR + olmesartan group compared with SHR + saline group (P , 0.01). There were no changes in systolic function during the time frame of the study in both SHR groups (Table 1).
Expressions of Fetal Genes and CaN/ NFAT3 Pathway Left ventricular hypertrophy and LVDD were prominently accompanied with upregulated fetal-type gene expressions (ANP and SAA) (Fig. 5) and activated CaN/NFAT3 pathway in SHRs. Olmesartan significantly ameliorated the hypertrophic responses and diastolic abnormalities by downregulating protein levels of CaN and NFAT3 (Fig. 6). www.jcvp.org |
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FIGURE 5. Relative ANP and SAA messenger RNA expressions in the hearts of the 3 groups. *P , 0.05 versus corresponding 6-month-old rats, **P , 0.01 versus corresponding 6-month-old rats; †P , 0.05 versus 9month-old SHR + saline group; ††P , 0.01 versus 9-month-old SHR + saline group.
DISCUSSION The present study demonstrated that blockade of AT1 receptor with olmesartan attenuated LVDD in parallel with
cardiac hypertrophy in a SHR model, and calcineurin/NFAT3 pathway played important roles in mediating the adverse process. These observations may assist the understanding of
FIGURE 6. Relative calcineurin and NFAT3 protein expressions in the hearts of the 3 groups. *P , 0.05 versus corresponding 6-month-old rats, **P , 0.01 versus corresponding 6-month-old rats; †P , 0.05 versus 9month-old SHR + saline group, ††P , 0.01 versus 9-month-old SHR + saline group.
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the molecular mechanisms through which LVDD occurred and progressed into HFPEF in hypertension. Hypertension is one of the most common cause to induce diastolic dysfunction, thus SHRs were used hereby to mimic the disease progression from hypertension to LVDD, for SHR is a well-established hypertension model, which resembles human essential hypertension in many aspects and a useful tool for studying disease transitions and underlying mechanisms.13,14 Our previous observational study with a broad time frame in SHRs showed that 3-month-old rats exhibited highly elevated systolic blood pressure with overt diastolic dysfunction at 6 months when systolic function was still unimpaired till 18 months.15 Previous experimental studies suggest that the renin–angiotensin–aldosterone system plays a central role in the progression of hypertension in HFPEF as demonstrated by the preventive effects of angiotensin II type I receptor blocker and angiotensin-converting enzyme inhibitor initiated before the onset of left ventricular hypertrophy, fibrosis, and diastolic dysfunction.16 However, it remains unclear whether olmesartan provides therapeutic effects in the case of administration from an established LVDD. Hence, we chose SHRs of 6 months in the present study when diastolic dysfunction explicitly existed to elucidate olmesartan on diastolic function and underlying mechanisms. Early recognition of LVDD may assist in additional risk stratification and subsequently guide the appropriate pharmacological interventions. Thus, regression of LVDD may be considered an important therapeutic target during HFPEF. As is known, Doppler parameters of LVDD may be abnormal in the absence of overt heart failure, but assessment of left ventricular diastolic properties cannot be relied on traditional Doppler parameters because such indexes as E/A, E deceleration time, and isovolumic relaxation time are mainly load dependent. In contrast, tissue Doppler imaging parameters such as E0 /A0 are significantly less preload dependent, and a combination with E/E0 , a true determinant of symptoms and prognosis, allows for better representation of left ventricular relaxation and left ventricular chamber compliance.17,18 Olmesartan is a widely used angiotensin II type I receptor blocker for the treatment of hypertension, not only in terms of lowering blood pressure with renoprotective activity but also for inducing more prominent suppression of ventricular hypertrophy and fibrosis, leading to suppression of myocardial stiffening and improvement of relaxation as is evident in the present study and in others.19,20 This study also partly explains why RAS inhibitors are useful in the management of patients with HFPEF, and especially in agreement with the recent report of Aldo-DHF in which aldosterone antagonist improve the diastolic dysfunction in patients with HFPEF.21 Zou et al22 had previously demonstrated that calcineurin was critically involved in the development of pressure overloadinduced cardiac hypertrophy. Many others also proved that calcineurin and its downstream protein NFAT3 played important roles in pathological hypertrophy and that inhibiting calcineurin could reverse myocardial hypertrophy.23,24 Meanwhile, we demonstrated that calcineurin and its pathway were involved in cardiac fibroblasts proliferation and transdifferentiation into myofibroblasts (data not published). Taking Ó 2014 Lippincott Williams & Wilkins
Olmesartan Attenuates Diastolic Dysfunction
together, olmesartan, through downregulates calcineurin/ NFAT3 pathway, reverses myocyte hypertrophy and interstitial fibrosis and thus attenuates diastolic dysfunction. In conclusion, this study suggests that olmesartan attenuates the manifestations of early-stage diastolic heart failure in SHRs. These benefits associated with the use of olmesartan are likely related to downregulation in CaN/ NFAT3 pathway thus alleviating adverse myocardial remodeling and diastolic dysfunction. This may shed light on mechanisms underlying HFPEF. However, further study still needs to elucidate the causal association between calcineurin and its pathway and LVDD.
CONCLUSIONS Diastolic dysfunction is one of the main mechanisms of diastolic heart failure, and no drugs have been proved to improve it. The present study suggested the beneficial effect of olmesartan on cardiac structure and diastolic dysfunction, and this may be partly mediated through calcineurin pathway. These findings therefore indicate a new therapeutic target for diastolic dysfunction. REFERENCES 1. Shah SJ, Heitner JF, Sweitzer NK, et al. Baseline characteristics of patients in the treatment of preserved cardiac function heart failure with an aldosterone antagonist (TOPCAT) trial. Circ Heart Fail. 2013;6:184–192. 2. Lam CS, Carson PE, Anand IS, et al. Sex differences in clinical characteristics and outcomes in elderly patients with heart failure and preserved ejection fraction: the Irbesartan in Heart Failure with Preserved Ejection Fraction (I-PRESERVE) trial. Circ Heart Fail. 2012;5:571–578. 3. Desai CS, Colangelo LA, Liu K, et al. Prevalence, prospective risk markers, and prognosis associated with the presence of left ventricular diastolic dysfunction in young adults: the coronary artery risk development in young adults study. Am J Epidemiol. 2013;177:20–32. 4. Kane GC, Karon BL, Mahoney DW, et al. Progression of left ventricular diastolic dysfunction and risk of heart failure. JAMA. 2011;306:856–863. 5. Owan TE, Redfield MM. Epidemiology of diastolic heart failure. Prog Cardiovasc Dis. 2005;47:320–332. 6. van Heerebeek L, Borbély A, Niessen HW, et al. Myocardial structure and function differ in systolic and diastolic heart failure. Circulation. 2006;113:1966–1973. 7. Molkentin JD, Lu JR, Antos CL, et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell. 1998;93:215–228. 8. Saygili E, Rana OR, Meyer C, et al. The angiotensin-calcineurin-NFAT pathway mediates stretch-induced up-regulation of matrix metalloproteinases-2/-9 in atrial myocytes. Basic Res Cardiol. 2009;104:435–448. 9. Dupont S, Maizel J, Mentaverri R, et al. The onset of left ventricular diastolic dysfunction in SHR rats is not related to hypertrophy or hypertension. Am J Physiol Heart Circ Physiol. 2012;302:H1524–H1532. 10. Aeschbacher BC, Hutter D, Fuhrer J, et al. Diastolic dysfunction precedes myocardial hypertrophy in the development of hypertension. Am J Hypertens. 2001;14:106–113. 11. Fu M, Zhou J, Sun A, et al. Efficacy of ACE inhibitors in chronic heart failure with preserved ejection fraction—a meta analysis of 7 prospective clinical studies. Int J Cardiol. 2012;155:33–38. 12. Lund LH, Benson L, Dahlström U, et al. Association between use of renin-angiotensin system antagonists and mortality in patients with heart failure and preserved ejection fraction. JAMA. 2012;308:2108–2117. 13. Boluyt MO, Bing OH, Lakatta EG. The ageing spontaneously hypertensive rat as a model of the transition from stable compensated hypertrophy to heart failure. Eur Heart J. 1995;16(suppl N):19–30. 14. Trippodo NC, Frohlich ED. Similarities of genetic (spontaneous) hypertension. Man and rat. Circ Res. 1981;48:309–319. 15. Fu M, Zhou J, Qian J, et al. Adiponectin through its biphasic serum level is a useful biomarker during transition from diastolic dysfunction to systolic dysfunction—an experimental study. Lipids Health Dis. 2012;11:106–116.
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16. Sakata Y, Masuyama T, Yamamoto K, et al. Renin angiotensin system-dependent hypertrophy as a contributor to heart failure in hypertensive rats: different characteristics from renin angiotensin system-independent hypertrophy. J Am Coll Cardiol. 2001;37: 293–299. 17. Shim CY, Kim SA, Choi D, et al. Clinical outcomes of exercise-induced pulmonary hypertension in subjects with preserved left ventricular ejection fraction: implication of an increase in left ventricular filling pressure during exercise. Heart. 2011;97:1417–1424. 18. Lee SW, Choi EY, Jung SY, et al. E/E’ ratio is more sensitive than E/A ratio for detection of left ventricular diastolic dysfunction in patients with systemic sclerosis. Clin Exp Rheumatol. 2010;28(suppl 58): S12–S17. 19. Kadowaki D, Anraku M, Tasaki Y, et al. Evaluation for antioxidant and renoprotective activity of olmesartan using nephrectomy rats. Biol Pharm Bull. 2009;32:2041–2045.
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J Cardiovasc Pharmacol ä Volume 63, Number 3, March 2014 20. Yokoyama H, Averill DB, Brosnihan KB, et al. Role of blood pressure reduction in prevention of cardiac and vascular hypertrophy. Am J Hypertens. 2005;18:922–929. 21. Edelmann F, Wachter R, Schmidt AG, et al; Aldo-DHF investigators. Effect of spironolactone on diastolic function and exercise capacity in patients with heart failure with preserved ejection fraction: the Aldo-DHF randomized controlled trial. JAMA. 2013;309:781–791. 22. Zou Y, Hiroi Y, Uozumi H, et al. Calcineurin plays a critical role in the development of pressure overload-induced cardiac hypertrophy. Circulation. 2001;104:97–101. 23. Finsen AV, Lunde IG, Sjaastad I, et al. Syndecan-4 is essential for development of concentric myocardial hypertrophy via stretch-induced activation of the calcineurin-NFAT pathway. PLoS One. 2011;6:e28302. 24. Ye J, Cardona M, Llovera M, et al. Translation of myocyte enhancer factor-2 is induced by hypertrophic stimuli in cardiomyocytes through a calcineurin-dependent pathway. J Mol Cell Cardiol. 2012;53:578–587.
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Olmesartan Attenuates Cardiac Hypertrophy and Improves Cardiac Diastolic Function in Spontaneously Hypertensive Rats Through Inhibition of Calcineurin Pathway Mingqiang Fu, MD,* Jingmin Zhou, MD,* Jianfeng Xu, MD,* Hongmin Zhu, MD,* Jianquan Liao, MD,* Xiaotong Cui, MD,* Aijun Sun, PhD,* Michael Fu, MD,† Yunzeng Zou, PhD,*‡ Kai Hu, MD,* and Junbo Ge, MD, PhD*‡
Objective: To test whether olmesartan ameliorates cardiac diastolic
Key Words: spontaneously hypertensive rats, olmesartan, diastolic function, calcineurin pathway
dysfunction in spontaneously hypertensive rats (SHRs) through calcineurin pathway.
(J Cardiovasc Pharmacol Ô 2014;63:218–226)
Methods: Twenty-four male SHRs of 6 months were divided into saline- (n = 12) and olmesartan-treated (n = 12) groups. Agematched WKY (n = 12) rats served as controls. Saline (10 mL$kg$d) or the same volume of olmesartan liquor (2.5 mg$kg$d) was administered by gavage for 3 months. Heart rate, systolic blood pressure, cardiac structure, and function and histological studies were determined. Expression of calcineurin and downstream NFAT3 were also detected. Results: Compared with age-matched Wistar Kyoto rats, SHRs of 6 months exhibited evident cardiac hypertrophy and diastolic dysfunction as demonstrated by elevated systolic blood pressure and E/E0 , decreased E/A and E0 /A0 , while F, left ventricular ejection fraction and fractional shortening remained unimpaired. Treatment with olmesartan significantly decreased systolic blood pressure and ventricular hypertrophy, attenuated fibrosis, and improved diastolic function (all P , 0.05). Meanwhile, both calcineurin and NFAT3 expressions were downregulated in olmesartan group compared with the other 2 groups (both P , 0.05). Conclusions: These data suggest the beneficial effect of olmesartan on cardiac structure and diastolic dysfunction, and it may be mediated through calcineurin pathway. This indicates a new therapeutic target for diastolic dysfunction.
Received for publication June 18, 2013; accepted October 23, 2013. From the *Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, China; †Department of Medicine, Sahlgrenska University Hospital/Sahlgrenska, Gothenburg, Sweden; and ‡Institutes of Biomedical Sciences, Fudan University, Shanghai, China. Supported by grants from the National Basic Research Program of China (973 Program, 2012CB518605) and the National Natural Science Foundation of China (81070105) to J. Zhou and the National Natural Science Foundation of China (81100145) to J. Xu. M. Fu and J. Zhou have contributed equally. The authors report no conflicts of interest. Reprints: Junbo Ge, MD, PhD, Department of Cardiology, Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, Room 413, Building 9, 180 Fenglin Road, Shanghai 200032, China (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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INTRODUCTION Heart failure with preserved ejection fraction (HFPEF) is now well recognized and accounts for 30%–50% of heart failure patients, and it usually occurs in hypertension, diabetic mellitus, and elderly.1,2 The principle mechanism for HFPEF is considered to be left ventricular diastolic dysfunction (LVDD). LVDD is reported to be present in 30% of the general adult population without congestive heart failure and in half of patients with hypertension, and LVDD is found to be an independent risk factor for HFPEF and associated with increased cardiovascular morbidity and mortality even when asymptomatic.3,4 However, the precise mechanisms for LVDD still remain to be elucidated. LVDD is manifested as delayed relaxation and/or increased myocardial stiffness of left ventricle, with progressive left ventricular hypertrophy and fibrosis.5,6 Recently, an intracellular signaling pathway that includes the Ca2+/calmodulin activated serine/threonine phosphatase calcineurin (CaN) and downstream nuclear factor of activated T cells (NFAT3) have been shown to be a key mechanism mediating the development of cardiac hypertrophy,7 and angiotensin II is reported to be an upstream activator of calcineurin.8 However, Dupont et al have previously demonstrated that the onset of LVDD in spontaneously hypertensive rats (SHRs) is not related to hypertension or hypertrophy, and diastolic dysfunction precedes myocardial hypertrophy in the development of hypertension.9,10 Considering the aforementioned evidence, whether or not calcineurin pathway is involved in LVDD needs to be clarified. Renin-angiotensin system (RAS) is activated in HFPEF, though the usefulness of angiotensin-converting enzyme inhibitors, angiotensin II type 1 receptor blockers, and b-blockers has not been recommended in current HFPEF management guidelines, our meta-analysis and registry data showed RAS antagonists was associated with lower all-cause mortality.11,12 Therefore, the present study was designed to examine the effects of olmesartan, the newest RAS inhibitor, on cardiac hypertrophy and diastolic dysfunction in a rat model of spontaneous hypertension with a focus on calcineurin pathway. J Cardiovasc Pharmacol ä Volume 63, Number 3, March 2014
J Cardiovasc Pharmacol ä Volume 63, Number 3, March 2014
Olmesartan Attenuates Diastolic Dysfunction
METHODS
gained images and data were stored and then downloaded to a magnetooptical disk for offline analyses.
Experimental Animals Twelve male Wistar Kyoyo (WKY) rats and 24 male SHRs were purchased from Shanghai Laboratory Animal Centre, Chinese Academy Sciences, at 5 months. All rats were maintained in a specific pathogen-free room under conditions of temperature- and humidity-controlled with a 12:12-hour light–dark cycle and given standard laboratory chow and tap water ad libitum. All the animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Publication No. 85-23, revised 1996) and were approved by the Animal Care Committee in Zhongshan Hospital, Fudan University. Beginning at 6 months, the rats were divided into 3 groups: WKY + saline group, SHR + saline group, and SHR + olmesartan group (n = 12, respectively). Saline (10 mL$kg$d) or the same volume of olmesartan liquor (2.5 mg$kg$d) was administered by gastric gavage once a day for 3 months.
Histological Examinations Six rats from each group were euthanized after echocardiography completed. Then, the left ventricle was excised and weighed immediately to calculate left ventricular mass index, expressed as ratio of the left ventricular tissue weight by milligram to total BW by gram. Middle section of left ventricular short axis around papillary muscle level was then fixed in 10% formalin, embedded in paraffin, and cut into 4-mmthick sections and stained either with hematoxylin–eosin for cardiomyocyte size or with the Masson trichrome staining to evaluate the extent of fibrosis. Digital photographs were thereafter taken and analyzed using a high-resolution digital image analysis system (Qwin V3; Leica, Germany).
Western Blot Left ventricular tissues were homogenized in lysis buffer (Cell Signalling Technology, Inc.) with protease inhibitor cocktail (Sigma Chemical Co, St Louis, MO) and then electrophoresed in 10% polyacrylamide gel and transferred to PVDF membrane. The protein expressions of CaN and nuclear factor of activated T cells (NFAT3) were detected by immune blotting with antibody against CaN and NFAT3 (1:1000–1:2000, both were from Cell Signaling Technology, Inc), respectively. After 3 washes, the blots were incubated with horseradish peroxidase–conjugated secondary antibody (1:4000–1:5000, Kangchen Biotechnology, China). GAPDH (1:4000, Kangchen Biotechnology) was used as the internal control. Bands were visualized with ImageLab Software (BioRad Laboratories, Inc).
Assessment of Physiological Parameters and Echocardiography Body weight (BW) was determined by an electronic weigher, and systolic blood pressure and heart rate were measured by a tail-cuff method (BP-98A; Softron, Tokyo, Japan). Thereafter, the rats were lightly anaesthetized with ketamine hydrochloride (50 mg/kg, intraperitonealy) with a heart rate at 380 6 25 bpm. After anesthesia and precordial region being shaved, the rats were transferred to a heated platform and transthoracic echocardiographic measurements were performed in the supine position with a commercially available echocardiographic system (Vevo770 High Resolution Imaging System; VisualSonics, Toronto, Canada) using a 17.5 MHz scanhead. Two-dimensional guided M-mode echocardiography and pulsed-wave Doppler and tissue Doppler echocardiography were used to assess cardiac structure and functions. All measurements were averaged for 4 consecutive cardiac cycles and were carried out by 2 experienced technicians independently who were unaware of the experimental protocol. The
Quantitative Real-Time Polymerase Chain Reaction Total RNA was isolated from the left ventricular tissues by Trizol reagent (Invitrogen). After purification, RNA was subjected to the quantitative real-time polymerase chain reaction analysis for expressions of atrial natriuretic peptide (ANP) and skeletal a-actin (SAA) on an iCycler system (BioRad, Hercules, CA). The primers were as follows: for b-actin
TABLE 1. Routine Parameters Before and After Interventions in WKY and SHRs WKY + Saline Parameters SBP, mm Hg HR, bpm BW, g LVMI, mg/g LVEF, % FS, %
6 mo 115 364 372 2.4 83.8 53.5
6 6 6 6 6 6
9 11 17 0.2 0.7 1.2
SHR + Saline 9 mo
123 357 378 2.5 83.6 53.7
6 6 6 6 6 6
11 10 21 0.3 0.9 1.7
6 mo 185 384 352 1.8 90.8 63.1
6 6 6 6 6 6
16* 15‡ 22‡ 0.3* 0.4* 0.8*
SHR + Olmesartan 9 mo
202 388 393 2.2 90.5 63.1
6 6 6 6 6 6
14* 11‡ 19‡ 0.2* 0.7* 1.1*
6 mo 186 386 355 1.8 91.2 63.5
6 6 6 6 6 6
9 mo
17* 12‡ 18‡ 0.2* 0.6* 1.2*
174 385 382 1.6 90.4 63.0
6 6 6 6 6 6
19*† 12‡ 17‡§ 0.2*† 0.5* 1.3*
Data are means 6 SD. *P , 0.01 versus corresponding Wistar rats. †P , 0.01 versus 9-month-old SHR + saline group. ‡P , 0.05 versus corresponding Wistar rats. §P , 0.05 versus 9-month-old SHR + saline group. SBP, systolic blood pressure; HR, heart rate; BW, body weight; LVMI, left ventricular mass index; LVEF, left ventricular ejection fraction; FS, fractional shortening.
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FIGURE 1. Left ventricular morphology assessed by 2-dimensional echocardiography. LVEDD: left ventricular end-diastolic diameter; LVESD: left ventricular end-systolic diameter; A, B, and C represent 6-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively; D, E, and F represent 9-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively. *P , 0.05 versus corresponding 6-month-old rats, **P , 0.01 versus corresponding 6-month-old rats; †P , 0.05 versus 9-month-old SHR + saline group, ††P , 0.01 versus 9-month-old SHR + saline group.
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FIGURE 2. Cross-sectional area of cardiac myocytes in the hearts of the 3 groups; A, B, and C represent 6-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively; D, E, and F represent 9-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively. *P , 0.05 versus corresponding 6-month-old rats, **P , 0.01 versus corresponding 6-month-old rats; †P , 0.05 versus 9-month-old SHR + saline group, ††P , 0.01 versus 9-month-old SHR + saline group.
gene, sense, 50 -CCCTAAGGCCAACCGTGAA-30 and antisense, 50 -GAGGCATACAGGGACAACACAG-30 ; for ANP gene, sense, 50 -GGTGTCCAA-CACAGATCTGA-30 and antisense, 50 -CACTAGACCACTCATCTAC-30 ; and for SAA gene, sense, 50 -AGCAGATGTGGATCACCAAG-30 and antisense, 50 -CTGCAACCA-CAGCACGATTG-30 . A comparative computed tomography method was used to determine relative quantification of messenger RNA expressions.
P , 0.05 was considered statistically significant. All analyses were carried out with SPSS16.0 statistical package for Windows (SPSS Inc, Chicago, IL).
Statistical Analysis
The baseline systolic blood pressure was markedly higher in the two 6-month-old SHR groups when compared with the WKY rats (P , 0.01). Treatment of olmesartan decreased blood pressure by 14% in SHR + olmesartan group
All values are presented as means 6 SD. Differences among groups were assessed by 1-way analysis of variance with a Student–Newman–Keuls test for post hoc analysis.
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RESULTS Effects of Olmesartan on Blood Pressure and BW
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FIGURE 3. Relative area of fibrosis (%) in the hearts of the 3 groups; A, B, and C represent 6-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively; D, E, and F represent 9-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively. *P , 0.05 versus corresponding 6-month-old rats, **P , 0.01 versus corresponding 6-month-old rats; †P , 0.05 versus 9-month-old SHR + saline group, ††P , 0.01 versus 9-month-old SHR + saline group.
compared with SHR + saline group (P , 0.01) while no effect in heart rate was found among the 3 groups. As is illustrated in Table 1, BW and left ventricular mass index were significantly increased at the end of the study in SHR + saline group while with a lesser extent in SHR + olmesartan group.
Effect of Olmesartan on Cardiac Morphology Persistent high blood pressure induced overt left ventricular hypertrophy in SHRs of 6 months as demonstrated by M-mode echocardiographic parameters. Interventricular
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septum thickness in diastole and left ventricular posterior wall thickness all exhibited significant increases than those of agematched WKY rats (P , 0.01). Olmesartan therapy for 3 months significantly decreased left ventricular hypertrophy in SHR + olmesartan group compared with SHR + saline group (P , 0.01) (Fig. 1). As were with the echocardiographic changes of wall thickness, histological studies demonstrated that myocardial samples from the SHR groups developed obvious cardiac hypertrophy and fibrosis when compared with WKY rats Ó 2014 Lippincott Williams & Wilkins
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FIGURE 4. Left ventricular diastolic function assessed by pulsed-wave Doppler imaging (upper left) and tissue Doppler imaging (lower left). E/A: ratio of maximal velocities of the E and A waves at mitral orifice level; E0 /A0 : ratio of maximal velocity of early (E0 ) and late (A0 ) diastolic waves at the level of lateral mitral annulus; E/E0 : ratio of E to E0 ; A, B, and C represent 6-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively; D, E, and F represent 9-month-old Wistar + saline group, SHR + saline group, and SHR + olmesartan group, respectively. *P , 0.05 versus corresponding 6-month-old rats, **P , 0.01 versus corresponding 6-month-old rats; †P , 0.05 versus 9-month-old SHR + saline group, **††P , 0.01 versus 9-month-old SHR + saline group.
(P , 0.01) as exhibited by increased cardiomyocyte crosssectional area of left ventricle and myocardial fibrosis area. And olmesartan reduced both adverse remodeling at the end of the study between SHR + olmesartan group and SHR + saline group (Figs. 2, 3).
Effect of Olmesartan on Cardiac Functions Echocardiographic analyses of cardiac functions were determined by left ventricular ejection fraction, fractional shorting for systolic function, and ratio of maximal velocities of the E and A waves of mitral inflow (E/A) by pulsedwave Doppler imaging, ratio of maximal velocity of early to late diastolic waves (E0 /A0 ) at the level of lateral mitral annulus by tissue Doppler imaging, and E/E0 for diastolic function. As showed in Figure 4, a significant decrease in diastolic functional parameters, E/A and E0 /A0 , and an Ó 2014 Lippincott Williams & Wilkins
increase of E/E0 , were observed in 6-month-old SHRs in comparison with their respective WKY controls, indicating an abnormal diastolic function; olmesartan treatment attenuated diastolic dysfunction in SHR + olmesartan group compared with SHR + saline group (P , 0.01). There were no changes in systolic function during the time frame of the study in both SHR groups (Table 1).
Expressions of Fetal Genes and CaN/ NFAT3 Pathway Left ventricular hypertrophy and LVDD were prominently accompanied with upregulated fetal-type gene expressions (ANP and SAA) (Fig. 5) and activated CaN/NFAT3 pathway in SHRs. Olmesartan significantly ameliorated the hypertrophic responses and diastolic abnormalities by downregulating protein levels of CaN and NFAT3 (Fig. 6). www.jcvp.org |
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FIGURE 5. Relative ANP and SAA messenger RNA expressions in the hearts of the 3 groups. *P , 0.05 versus corresponding 6-month-old rats, **P , 0.01 versus corresponding 6-month-old rats; †P , 0.05 versus 9month-old SHR + saline group; ††P , 0.01 versus 9-month-old SHR + saline group.
DISCUSSION The present study demonstrated that blockade of AT1 receptor with olmesartan attenuated LVDD in parallel with
cardiac hypertrophy in a SHR model, and calcineurin/NFAT3 pathway played important roles in mediating the adverse process. These observations may assist the understanding of
FIGURE 6. Relative calcineurin and NFAT3 protein expressions in the hearts of the 3 groups. *P , 0.05 versus corresponding 6-month-old rats, **P , 0.01 versus corresponding 6-month-old rats; †P , 0.05 versus 9month-old SHR + saline group, ††P , 0.01 versus 9-month-old SHR + saline group.
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the molecular mechanisms through which LVDD occurred and progressed into HFPEF in hypertension. Hypertension is one of the most common cause to induce diastolic dysfunction, thus SHRs were used hereby to mimic the disease progression from hypertension to LVDD, for SHR is a well-established hypertension model, which resembles human essential hypertension in many aspects and a useful tool for studying disease transitions and underlying mechanisms.13,14 Our previous observational study with a broad time frame in SHRs showed that 3-month-old rats exhibited highly elevated systolic blood pressure with overt diastolic dysfunction at 6 months when systolic function was still unimpaired till 18 months.15 Previous experimental studies suggest that the renin–angiotensin–aldosterone system plays a central role in the progression of hypertension in HFPEF as demonstrated by the preventive effects of angiotensin II type I receptor blocker and angiotensin-converting enzyme inhibitor initiated before the onset of left ventricular hypertrophy, fibrosis, and diastolic dysfunction.16 However, it remains unclear whether olmesartan provides therapeutic effects in the case of administration from an established LVDD. Hence, we chose SHRs of 6 months in the present study when diastolic dysfunction explicitly existed to elucidate olmesartan on diastolic function and underlying mechanisms. Early recognition of LVDD may assist in additional risk stratification and subsequently guide the appropriate pharmacological interventions. Thus, regression of LVDD may be considered an important therapeutic target during HFPEF. As is known, Doppler parameters of LVDD may be abnormal in the absence of overt heart failure, but assessment of left ventricular diastolic properties cannot be relied on traditional Doppler parameters because such indexes as E/A, E deceleration time, and isovolumic relaxation time are mainly load dependent. In contrast, tissue Doppler imaging parameters such as E0 /A0 are significantly less preload dependent, and a combination with E/E0 , a true determinant of symptoms and prognosis, allows for better representation of left ventricular relaxation and left ventricular chamber compliance.17,18 Olmesartan is a widely used angiotensin II type I receptor blocker for the treatment of hypertension, not only in terms of lowering blood pressure with renoprotective activity but also for inducing more prominent suppression of ventricular hypertrophy and fibrosis, leading to suppression of myocardial stiffening and improvement of relaxation as is evident in the present study and in others.19,20 This study also partly explains why RAS inhibitors are useful in the management of patients with HFPEF, and especially in agreement with the recent report of Aldo-DHF in which aldosterone antagonist improve the diastolic dysfunction in patients with HFPEF.21 Zou et al22 had previously demonstrated that calcineurin was critically involved in the development of pressure overloadinduced cardiac hypertrophy. Many others also proved that calcineurin and its downstream protein NFAT3 played important roles in pathological hypertrophy and that inhibiting calcineurin could reverse myocardial hypertrophy.23,24 Meanwhile, we demonstrated that calcineurin and its pathway were involved in cardiac fibroblasts proliferation and transdifferentiation into myofibroblasts (data not published). Taking Ó 2014 Lippincott Williams & Wilkins
Olmesartan Attenuates Diastolic Dysfunction
together, olmesartan, through downregulates calcineurin/ NFAT3 pathway, reverses myocyte hypertrophy and interstitial fibrosis and thus attenuates diastolic dysfunction. In conclusion, this study suggests that olmesartan attenuates the manifestations of early-stage diastolic heart failure in SHRs. These benefits associated with the use of olmesartan are likely related to downregulation in CaN/ NFAT3 pathway thus alleviating adverse myocardial remodeling and diastolic dysfunction. This may shed light on mechanisms underlying HFPEF. However, further study still needs to elucidate the causal association between calcineurin and its pathway and LVDD.
CONCLUSIONS Diastolic dysfunction is one of the main mechanisms of diastolic heart failure, and no drugs have been proved to improve it. The present study suggested the beneficial effect of olmesartan on cardiac structure and diastolic dysfunction, and this may be partly mediated through calcineurin pathway. These findings therefore indicate a new therapeutic target for diastolic dysfunction. REFERENCES 1. Shah SJ, Heitner JF, Sweitzer NK, et al. Baseline characteristics of patients in the treatment of preserved cardiac function heart failure with an aldosterone antagonist (TOPCAT) trial. Circ Heart Fail. 2013;6:184–192. 2. Lam CS, Carson PE, Anand IS, et al. Sex differences in clinical characteristics and outcomes in elderly patients with heart failure and preserved ejection fraction: the Irbesartan in Heart Failure with Preserved Ejection Fraction (I-PRESERVE) trial. Circ Heart Fail. 2012;5:571–578. 3. Desai CS, Colangelo LA, Liu K, et al. Prevalence, prospective risk markers, and prognosis associated with the presence of left ventricular diastolic dysfunction in young adults: the coronary artery risk development in young adults study. Am J Epidemiol. 2013;177:20–32. 4. Kane GC, Karon BL, Mahoney DW, et al. Progression of left ventricular diastolic dysfunction and risk of heart failure. JAMA. 2011;306:856–863. 5. Owan TE, Redfield MM. Epidemiology of diastolic heart failure. Prog Cardiovasc Dis. 2005;47:320–332. 6. van Heerebeek L, Borbély A, Niessen HW, et al. Myocardial structure and function differ in systolic and diastolic heart failure. Circulation. 2006;113:1966–1973. 7. Molkentin JD, Lu JR, Antos CL, et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell. 1998;93:215–228. 8. Saygili E, Rana OR, Meyer C, et al. The angiotensin-calcineurin-NFAT pathway mediates stretch-induced up-regulation of matrix metalloproteinases-2/-9 in atrial myocytes. Basic Res Cardiol. 2009;104:435–448. 9. Dupont S, Maizel J, Mentaverri R, et al. The onset of left ventricular diastolic dysfunction in SHR rats is not related to hypertrophy or hypertension. Am J Physiol Heart Circ Physiol. 2012;302:H1524–H1532. 10. Aeschbacher BC, Hutter D, Fuhrer J, et al. Diastolic dysfunction precedes myocardial hypertrophy in the development of hypertension. Am J Hypertens. 2001;14:106–113. 11. Fu M, Zhou J, Sun A, et al. Efficacy of ACE inhibitors in chronic heart failure with preserved ejection fraction—a meta analysis of 7 prospective clinical studies. Int J Cardiol. 2012;155:33–38. 12. Lund LH, Benson L, Dahlström U, et al. Association between use of renin-angiotensin system antagonists and mortality in patients with heart failure and preserved ejection fraction. JAMA. 2012;308:2108–2117. 13. Boluyt MO, Bing OH, Lakatta EG. The ageing spontaneously hypertensive rat as a model of the transition from stable compensated hypertrophy to heart failure. Eur Heart J. 1995;16(suppl N):19–30. 14. Trippodo NC, Frohlich ED. Similarities of genetic (spontaneous) hypertension. Man and rat. Circ Res. 1981;48:309–319. 15. Fu M, Zhou J, Qian J, et al. Adiponectin through its biphasic serum level is a useful biomarker during transition from diastolic dysfunction to systolic dysfunction—an experimental study. Lipids Health Dis. 2012;11:106–116.
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16. Sakata Y, Masuyama T, Yamamoto K, et al. Renin angiotensin system-dependent hypertrophy as a contributor to heart failure in hypertensive rats: different characteristics from renin angiotensin system-independent hypertrophy. J Am Coll Cardiol. 2001;37: 293–299. 17. Shim CY, Kim SA, Choi D, et al. Clinical outcomes of exercise-induced pulmonary hypertension in subjects with preserved left ventricular ejection fraction: implication of an increase in left ventricular filling pressure during exercise. Heart. 2011;97:1417–1424. 18. Lee SW, Choi EY, Jung SY, et al. E/E’ ratio is more sensitive than E/A ratio for detection of left ventricular diastolic dysfunction in patients with systemic sclerosis. Clin Exp Rheumatol. 2010;28(suppl 58): S12–S17. 19. Kadowaki D, Anraku M, Tasaki Y, et al. Evaluation for antioxidant and renoprotective activity of olmesartan using nephrectomy rats. Biol Pharm Bull. 2009;32:2041–2045.
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J Cardiovasc Pharmacol ä Volume 63, Number 3, March 2014 20. Yokoyama H, Averill DB, Brosnihan KB, et al. Role of blood pressure reduction in prevention of cardiac and vascular hypertrophy. Am J Hypertens. 2005;18:922–929. 21. Edelmann F, Wachter R, Schmidt AG, et al; Aldo-DHF investigators. Effect of spironolactone on diastolic function and exercise capacity in patients with heart failure with preserved ejection fraction: the Aldo-DHF randomized controlled trial. JAMA. 2013;309:781–791. 22. Zou Y, Hiroi Y, Uozumi H, et al. Calcineurin plays a critical role in the development of pressure overload-induced cardiac hypertrophy. Circulation. 2001;104:97–101. 23. Finsen AV, Lunde IG, Sjaastad I, et al. Syndecan-4 is essential for development of concentric myocardial hypertrophy via stretch-induced activation of the calcineurin-NFAT pathway. PLoS One. 2011;6:e28302. 24. Ye J, Cardona M, Llovera M, et al. Translation of myocyte enhancer factor-2 is induced by hypertrophic stimuli in cardiomyocytes through a calcineurin-dependent pathway. J Mol Cell Cardiol. 2012;53:578–587.
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