Myocardial Hypertrophic Preconditioning Attenuates Cardiomyocyte Hypertrophy And Slows Progression To Heart Failure Through Up-regulation Of S100A8/A9

Background and Objective:Characterized with increased cardiomyocyte protein synthesis and increased cell volume, myocardial hypertrophy (MH) is crucial for the transition of cardiac function from adaptive to maladaptive status and progression to irreversible change. Although some degree of cardiac hypertrophy serves to reduce wall stress and helps compensate for increased load on the myocardium, sustained prohypertrophic signaling within cardiomyocytes is detrimental and a major factor contributing to the progression to heart failure (HF). Clinical and experimental investigations showed that withdrawal of pressure overload such as aortic debanding in animals and aortic valve replacement for patients with aortic stenosis leads to regression of myocardial hypertrophy, and during which beneficial molecular alterations occur. It was reported that intermittent systolic overload promoted better myocardial performance in adult animals, and displayed a mild hypertrophic response and a favorable fetal gene expression profile. However, it is completely unknown whether removal of short or long term pressureoverload would render the heart resistant to subsequent prolonged prohypertrophic stimuli.The phenomenon of ischemic preconditioning, whereby brief episodes of ischemia and reperfusion render the heart resistant to subsequent prolonged ischemia, has received much attention since its first report by Murry et al. in1986. In addition to ischemia, pretreatment with hypoxia, hyperbaric oxygen or some drugs can also induce protective effect of preconditioning. Likewise, is it possible that short term of hypertrophic stimulation render the heart resistant to subsequent hypertrophic stress? Coincidently, it was reported that in an animal study short-term antihypertensive therapy can prolong the antihypertrophic effect of myocardium to protect the heart. In addition, removal of cardiac pressure overload was reported to significantly change the gene expression profile, and some of which are antihypertrophic genes. It appears that anti-hypertension or removal of prohypertrophic stimuli would produce an antihypertrophic memory, but it is unclear how long such memory would persist.It is well known that similar intensity of pressure overload (e.g. hypertension) may lead to different degree of MH, thus the prevalence of MH in patients with essential hypertension is less than50%, which suggest that factors resistant to prohypertrophic stimulation presents in some patients. Experimental studies demonstrate that some factors could prevent cardiac hypertrophy independent on the antihypertensive effects. But it remains unclear how to induce such antihypertrophic factors and make use of them for therapeutic purpose. Based on the clues mentioned above, here we propose a new concept termed "myocardial hypertrophic preconditioning"(MHP). Our hypothesis is that short term hypertrophic stimulation would render the heart resistant to subsequent hypertrophic stress and slow the progression of heart failure (HF). In this study, we attempted to experimentally prove the phenomenon of MHP and investigate the mechanism involved.Methods:1. Cell cultureThe isolation and culturing of neonatal rat ventricular cardiomyocytes (NRVCs) was performed. Cells were divided into three groups:(1) NE group:1μmol/L norepinephrine (NE, dissolved in Dulbecco’s Modification of Eagle’s Medium (DMEM)) for48h;(2) NE+PRE group:after stimulation for12h, NE was removed for another12h, and then NE was added again and stimulation for48h;(3) Control group:DMEM treatment for48h. Cardiomyocytes were harvested and analyzed for cell surface and expression of atrial natriuretic peptide (ANP) and beta-myosin heavy chains (β-MHC).2. Drug-induced myocardial hypertrophyC57BL/6male mice (8wk,20-25g) were intraperitoneally anesthetized with pentobarbital (50mg/kg). After anesthesia, the mice were treated with phenylephrine (PE,30mg/kg/day) or vehicle by osmotic minipump (Alzet) to induce cardiac hypertrophy. After4days of drug-induced stimulation, the animals were sacrificed to obtain heart and calculate heart weighe/body weight ratio. The mice were divided into3groups, with6-7mice in each group, as follows:(1) PE group:PE infusion for4d;(2) PE+PRE group:PE for1d, infusion stop for1d, PE for2d, infusion stop for2d, and than PE for4d;(3) Control group:ascorbic acid infusion for4d.3. Transverse aortic constriction (TAC) induced myocardial hypertrophyC57BL/6male mice (8wk,20-25g) were anesthetized with pentobarbital sodium (50mg/kg). The chest cavity was entered in the second intercostals space at the left upper sternal border through a small incision, and the thymus was then gently deflected out of the field of view to expose the aortic arch. After the transverse aorta was isolated between the carotid arteries, it was constricted by a7-0silk suture ligature tied firmly against a27-gauge needle. The gauge needle was promptly removed and chest was closed. Myoardial hypertrophy and subsequent heart failure were induced.4. M-mode echocardiographyCardiac function and remodeling were evaluated with echocardiography at35 days after TAC. From M-mode tracing, LV end-diastolic diameter (LVEDd), LV end-systolic diameter (LVESd), diastolic and systolic LV wall thickness (Pwd, Pws) and left ventricular shortening (LVFS) were measured. LVFS=(LVEDd-LVESd)/LVEDd×100.5. Histological examinationsFor histological examinations hearts were fixed in4%paraformaldehyde and embedded in paraffin4-6μm sections which were then stained with HE or Masson. Cross section area of cardiomyocytes and myocardial fibrosis were calculated using Image J software.6. Experimental protocols for TAC miceExperiments were divided into two protocols,6-7mice in each group.Protocol1:Early window protective effect of preconditioning in pressure overload mice. Three groups were included:Sham group and TAC group, observation for7days; TAC+PRE group:TAC for3days, debanding for4days, then second TAC, and finally observation for7days.Protocol2:Late window protective effect of preconditioning in pressure overloaded mice. Four groups were designed:Sham group and TAC group, observation for6weeks; TAC+PRE1group:TAC for3days, debanding for4days, then second TAC, and finally observation for6weeks; TAC+PRE2group:TAC for1week, debanding for1week, then second TAC, and finally observation for6weeks.7. The expression of S100A8/A9Real-time PCR and Western blot were performed to evaluate expression levels of S100A8and S100A9mRNA and S100A9protein in the heart of mice and cultured cardiomyocytes.8. Experimental protocol for investigating the role of S100A8/A9in cardiomyocyte hypertrophy and fibrosis. The isolation and culturing of neonatal rat ventricular cardiomyocytes and fibroblasts were performed. Experimental groups were designed as follows:(1) NE group:1μmol/L NE treatment for48h;(2) NE+S100A8group:treatment with1μmol/L NE and S100A8(1μg/mL) for48h;(3) NE+S100A9group:treatment with1μmol/L NE and S100A9(1μg/mL) for48h;(4) NE+S100A8/A9group:treatment with1μmol/L NE and S100A8/A9(1μg/mL) for48h;(5) Control group:treatment with DMEM for48h. We analyzed the cell surface area and expression of ANP, P-MHC, calcineurin, nuclear factor of activated T cells (NFAT) in cardiomyocytes, procollagen Ⅰ and procollagen Ⅲ mRNA in fibroblasts.Results:1. Antihypertrophic Effect of Hypertrophic Preconditioning (HP) in vitroIn comparison with Control group, the expression levels of hyperophic mark genes ANP (ANP/β-actin:1.00±0.06vs.2.53±0.05, P=0.00) and β-MHC (β-MHC/β-actin:0.98±0.02vs.2.67±0.15, P=0.000) were significantly up-regulated in NE group, while hypertrophic preconditioning significantly inhibited the NE-induced upregulation of ANP and β-MHC, P<0.05-0.001). Compared with NE group, the cardiomyocyte area in NE+PRE group decreased by about29%(2.29±0.02vs.1.62±0.02folds of control, P=0.000)2. Antihypertrophic Effect of HP in vivoIn the mice using PE infusion to induce myocardial hypertrophy, the HW/BW in PE+PRE group was significantly smaller than in PE group (4.02±0.04vs.4.18±0.06mg/g, P=0.044). Similarly, one week after TAC, the HW/BW of mice was smaller in TAC+PRE group than in TAC group (5.35±0.17vs.5.99±0.22mg/g, P=0.014). Six weeks after TAC, the HW/BW also smaller in the two TAC+PRE groups than in TAC group (5.32±0.14in TAC+PRE1, and5.43±0.11in TAC+PRE2,7.16±0.33in TAC mg/g, P<0.01), while the cardiomyocyte surface area was significantly smaller in TAC+PRE1and TAC+PRE2groups than in TAC group, and the increase of fetal genes ANP and P-MHC in the two preconditioning groups was significantly attenuated.3. Hypertrophic preconditioning slows the progression of cardiac remodelingM-mode echocardiographic examination in TAC mice showed an increase in diastolic and systolic left ventricular wall thickness, LVEDD and LVESD,(P≤0.001) at35days after TAC. In contrast, HP significantly slowed the progression of LV wall thickness increase and LV enlargement (all P<0.05). In addition, histological analysis showed that myocardial fibrosis was significantly attenuated in both preconditioning groups than in TAC group.4. Hypertrophic preconditioning improves heart failureTAC-induced congestive heart failure was manifested by increases in lung weight to body weight ratio (LW/BW). Six weeks after TAC, the LW/BW was markedly decreased in the TAC+PRE1and TAC+PRE2groups (5.98±0.12in TAC+PRE1group, P=0.008and6.15±0.11in TAC+PRE2group,9.88±1.00mg/g in TAC group mg/g, P=0.046). In addition, LVFS at35days after TAC in TAC group was significantly lower compared with that in Sham group (P<0.001), while LVFS in TAC+PRE1group and TAC+PRE2group were significantly higher than that in the TAC group (all P<0.05).The above findings indicate that HP attenuates cardiomyocyte hypertrophy and slows progression to heart failure. Next we tried to search the potential mechanism.5. Up-regulation of S100A8/A9in response to prohypertrophic stimuli removal.It has been reported that S100A9was one of the genes specifically induced during the regression of cardiac hypertrophy. We than examined the expression of S100A8and S100A9in response to prohypertrophic stimuli removal. We noted that expression levels of S100A8and S100A9mRNA and S100A9protein in cultured NRVCs were similar between control group and NE group, but they were markedly up-regulated at12hours after NE removal. Consistently, myocardial expression of S100A9and/or S100A8was also significantly increased in mice at1day after debanding preceded by3days or1week of TAC.6. S100A8/A9Attenuates Hypertrophy and Fibrosis in vitro.We further investigated the effects of S100A8and S100A9protein on cultured cardiomyocytes and fibroblasts. Compared with the control groups,48h of exposure to NE increased the mRNA expression of ANP (ANP/β-actin:1.04±0.17vs.5.52±1.32, P=0.00) and β-MHC(β-MHC/β-actin:1.03±0.16vs.4.22±1.36, P=0.004) in NVRCs, procollagen Ⅰ and procollagen Ⅲ in fibroblasts, while treatment with S100A8, S100A9or both of them prevented these changes (all P<0.05). In addition, treatment with S100A8or S100A9or both of them significantly suppressed the NE-induced increase in cell surface area in NRVCs.7. S100A8/A9suppresses the prohypertrophic calcineurin/NFAT pathway.Compared with the control group, exposure of NRVCs to NE resulted in an increase in the expression of calcineurin (calcineurin/β-actin:0.18±0.02vs.0.55±0.02, P=0.000), which was abrogated by treatment with S100A8or S100A9or both of them (all P<0.05). Subcellular localization of NFATc3was assessed by Western blot. In the control groups, NFATc3was primarily localized in the cytoplasm, which was translocated into the nucleus in response to NE stimulation. Treatment with S100A8or S100A9or both of them inhibited NE-induced NFATc3nuclear translocation.Conclusions:1. Precondioning by prohypertrophic factors renders the heart resistant to subsequent hypertrophic stress and delays the progression from myocardial hypertrophy to heart failure, indicting the presence of "myocardial hypertrophic preconditioning".2. Myocardial hypertrophy preconditioning exerts antihypertrophic effect, inhibits cardiac remodeling and improves heart failure.3. S100A8/A9are up-regulated in response to hypertrophic stimuli removal and exerts antihypertrophic effect.4. S100A8/A9stimulation suppresses the prohypertrophic calcineurin/NFAT pathway.