Effect Of S100A8/A9 On Myocardial Hypertrophic Preconditioning

Background and Objective:Heart failure (HF) is the end point of numerous cardiovascular diseases, and is also the main cause of disability and death. Cardiac hypertrophy is the main pathological basis of the progression of heart failure. Hypertrophic growth of ventricular myocytes is a hallmark feature of numerous forms of cardiovascular disease. Myocardial hypertrophy at the cellular level is characterized by an increase of cardiomyocyte protein synthesis and cell volume, it is crucial for the transition from adaptive to maladaptive cardiac function with progression to irreversible changes. Another hallmark feature of pathological hypertrophic remodeling is accumulation and deposition of excessive extracellular matrix. Cardiac hypertrophy and remodeling plays an important role to promote the irreversible change of the heart and shift the heart function to the overt heart failure stage. Clinical and experimental studies have shown that withdrawal of pressure overload, such as aortic debanding in animals and aortic valve replacement in patients, makes the regression of myocardial hypertrophy and various beneficial molecular changes. During this process, antihypertrophic factors were induced to play a key role in antihpertrophy. It has been reported that intermittent overload promotes the improvement of myocardial performance in adult animals, producing both a mild hypertrophic response and a favorable gene expression profile. It was also reported that in an animal model short-term antihypertensive therapy can prolong the antihypertrophic function of myocardium to protect the heart. However, whether the removal of short-term or long-term pressure overload renders the heart resistant to subsequent prolonged stimulation did not have been reported.Murry et al first reported the seminal discovery by of ischemic preconditioning in 1986, a phenomenon in which several episodes of brief ischemia followed by reperfusion protected cardiac muscle cells from a subsequent prolonged ischemic insult. Since then, a similar protective effect of ischemic preconditioning has been observed in many organs, including brain, liver, and kidney, supporting the notion that ischemic preconditioning is a fundamental property shared by different cell types. Different forms of nonischemic preconditioning have been demonstrated in the heart, including mechanical stretch, heat stress, metabolic challenge, and pharmacological agents. The cardioprotective effects have also been extended beyond myocyte viability to pathological hypertrophy and remodeling. However, the vast majority of studies investigating preconditioning have focused on the protective effects of ischemic preconditioning. In contrast, other nonischemic types of preconditioning have received relatively little attention, and whether the principles of preconditioning can be applied to other pathological cardiac states remains unexplored. It has been reported that the prevalence of myocardial hypertrophy is less than 50% in patients with essential hypertension, suggesting that some factors which resist prohypertrophic stimulation exist in many patients. Experimental studies have demonstrated that some factors can prevent cardiac hypertrophy independent of an antihypertensive effect, but how to induce such antihypertrophic factors for antihypertrophic purposes remains unclear. An earlier report demonstrating that exercise-induced preconditioning protected against the development of pressure overload-induced hypertrophy. Based on the above points mentioned here, we first propose bold hypothesis that transient hypertrophic stimulation to the heart would make the heart resistant to subsequent hypertrophic stress and slow the progression to heart failure, which termed "myocardial hypertrophic preconditioning". In preliminary experiments we have demonstrated that precondioning by prohypertrophic factors renders the heart resistant to subsequent hypertrophic stress and delays the progression from myocardial hypertrophy to heart failure, as well as improve cardiac function and inhibit cardiac remodeling, indicting the presence of "myocardial hypertrophic preconditioning". Myocardial hypertrophy preconditioning against myocardial hypertrophy might be related with the increase expression of S100A8/A9 factors after release of hypertrophic stimulation. But its mechanism of myocardium preconditioning is not clear, S100A8 and S100A9 are members of the S100 calcium-binding family of proteins, S100A8 and S100A9 are likely to exert their intracellular regulatory activities by interacting with specific targets in a Ca2+-dependent manner. Ca2+is essential for transcriptional activation during cardiac hypertrophy. Among the Ca2+-dependent signaling pathways implicated in cardiac hypertrophy, the activation of calcineurin and subsequent nuclear translocation of NFAT are important. We hypothesized that S100A8/A9 might be involved in cardiac hypertrophy preconditioning by regulating the calcineurin-NFAT signaling pathway. In this study, we attempted to further study the expression of S100A8/A9 in myocardial hypertrophy preconditioning and investigated the function and involved mechanisms of recombinant S100A8/A9 and recombinant lentiviral knockdown of S100A8/A9 to further study on the myocardial hypertrophic preconditioning.MethodsTransverse aortic constriction (TAC) induced myocardial hypertrophy preconditioning and mechanisms involved in vivo1.1 Creation of Transverse aortic constriction (TAC)-induced myocardial hypertrophy model and Experimental ProtocolsC57BL/6 male mice (8-10 weeks,18-25 g) were subjected to TAC or debanding or sham operation as described elsewhere. The mice were anaesthetized with a mixture of xylazine (5 mg/kg, ip) and ketamine (100 mg/kg, ip). After anesthesia artificially ventilated with animal ventilator. Briefly, after a thoracotomy in the 2nd intercostal space, Then Blunt isolated aortic arch to exposed it,7-0 silk ligature was tied around the transverse aorta and a 27 G blunted needle which was subsequently removed. In sham-operated animals, the ligature was tied loosely around the aorta arch. At the indicated time, in preconditioning group a debanding operation was performed by carefully removing the ligature.To contruct the model of TAC-induced hypertrophic preconditioning,6-7 mice were randomly divided into each group, Four groups were included:Sham group and TAC group, observation for 6 weeks; Prel+TAC group:debanding the aorta after 3 days of TAC, and banded again 4 days later followed by observation for 6 weeks; Pre2+TAC group:debanding the aorta after 1 week of TAC, and banded again 1 week later followed by observation for 6 weeks. These mice and sham-operated ones were sacrificed by overdose anesthesia (pentobarbital sodium 150mg/kg, ip) at 1-8 weeks after the operation.1.2 Echocardiography and Left ventricular hemodynamicsCardiac function and remodeling were dynamically evaluated with echocardiography at 7days、21 days and 35 days after TAC. Two-dimensional parasternal short-axis images of the left ventricle (LV) were obtained at the level of the papillary muscles. From M-mode tracings, the LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), LV diastolic posterior wall thickness (PWd), LV posterior wall systolic thickness (PWs) and LV fractional shortening (LVFS) were measured. LVFS= (LVEDd-LVESd)/LVEDd x 100. LV hemodynamic was evaluated using a Millar catheter.1.3 The expression level of S100A8/A9 was detected by Western BlotThe expression levels after remove the stimulated were divided into 4 groups:1) Sham group; 2) TAC 1 w group:TAC for one week; 3) T3dDld group:debanding the aorta for 1 day after 3 days of TAC; 4) T1wD1d group:debanding the aorta for lday after 1 week of TAC.The expression and and its duration of S100A8/A9 after hypertrophy preconditioning:there were 5 groups:1) Sham group; 2) TAC 1 w group:TAC for one week; 4) Tlw/Dlw/Tlw 组:debanding the aorta after 1 week of TAC, and banded again 1 week later followed by observation; 5) T1w/D1w/T6w group: debanding the aorta after 1 week of TAC, and banded again 6 week later followed by observation.Western blot were performed to evaluate expression levels of S100A8 and S100 A9 protein.The effect and mechanism of upregulation of S100A8/A9 induced by myocardial hypertrophy preconditioning2.1 Neonatal rat cardiomyocytes (NRCs) were stimulated by Norepinephrine(NE)The isolation and culturing of neonatal rat cardiomyocytes (NRCs) were performed。Three groups were designed:(1) NE group:1μmol/L norepinephrine (NE, dissolved in Dulbecco’s Modification of Eagle’s Medium (DMEM)) for 48 h, (2) Pre+NE group:after stimulation for 12 h, NE was removed for another 12 h, and then NE was added again to stimulate for 48 h. (3) Control group:DMEM treatment for 48 h. Cardiomyocytes were harvested and western blot were performed to evaluate expression levels of S100A8 and S100A9 protein.2.2 Effects of recombinant murine S100A8/S100A9 on NRVCs and fibroblastsEight groups were designed as follows:(1) NE group:1 μmol/L NE treatment for 48 hours; (2) NE+S100A8 group:treatment with 1 μmol/L NE and S100A8 (1 μg/mL) for 48 hours; (3) NE+S100A9 group:treatment with 1 μmol/L NE and S100A9 (1 μg/mL) for 48 hours; (4) NE+S100A8/A9 group:treatment with 1 μmol/L NE and S100A8/A9 (1 μg/mL) for 48 hours; (5) control group:treatment with DMEM for 48 hours; (6) S100A8 group:S100A8 (1 μg/mL) for 48 hours without NE stimulated; (7) S100A9 group:S100A9 (1 μg/mL) for 48 hours without NE stimulated; (8)S100A8/A9 group:treatment with S100A8/A9 (1 μg/mL) for 48 hours without NE stimulated. Cell surface area and expression of ANP, β-MHC, calcineurin and nuclear factor of activated T cells (NFAT) in cardiomyocytes, procollagen I and procollagen Ⅲ mRNA in fibroblasts were analyzed.Effects of recombinant Lentivirus Carrying Overexpressed or Short Hairpin RNA for S100A8 or A9 on NRVCs and fibroblasts3.1 Construction and identification of recombinant lentivirusThe shRNA target sequences of S100A8 and A9 genes and the full coding sequence of S100A8-or A9 were cloned into the Lentiviral vector which contains the ZsGreenl marker to construct recombinant lentivirus and empty vector. The overexpression or knockdown of S100A8 or A9 was achieved by transfecting cultured neonatal rat cardiomyocytes with the recombinant lentivirus (multiplicity of infection (MOI)=5). After transfection for 24hr, the virus-containing transduction medium was replaced with fresh growth medium. Further incubating the cells for 72 hours to allow the recombinant lentivirus to achieve the maximum effect. Infection efficiency and silencing/overexpression effect were evaluated using a fluorescence microscopy, real-time-PCR and western blot.3.2 Cell viability assayNeonatal rat cardiomyocytes were infected with lentivirus carrying S100A8/A9 (MOI= 5/10/15) for 72 hours or recombinant S100A8/A9 (1 and 10 μg/mL) for 48 hours. Cell viability was measured using methyl thiazolyl tetrazolium (MTT) assay.3.3 Effect of silence of S100A8 or A9 on NRVCs and fibroblasts:Cultured neonatal rat cardiomyocytes and fibroblasts were stimulated with lentivirus carrying shRNA target sequences of S100A8/A9 (Lv-ShRNA-S100A8 and Lv-ShRNA-S100A9) for 72 hours. Then cardiomyocytes surface area and expression of ANP, β-MHC, calcineurin and nuclear factor of activated T cells (NFAT) in cardiomyocytes, procollagen I and procollagen III mRNA in fibroblasts were analyzed.Results:Antihypertrophic effect of hypertrophic preconditioning in vivo and upregulate the expression of S100A8/A91.1 Hypertrophic preconditioning improves the pathophysiology of HFIn TAC mice, At 7days、21 day and 35 days serial echocardiography showed a time-dependent increase of diastolic and systolic left ventricular wall thickness (Pwd/Pws), as well as LVEDD and LVESD, while LVFS decreased over time. In contrast, hypertrophic preconditioning significantly slowed the increase of LV wall thickness, the enlargement of LV dimensions, and the decline of LVFS. No significant differences were noted between the 2 preconditioning groups. Six weeks after TAC, Echocardiographic LV dimensions were smaller, LV fractional shortening was larger, LV end-diastolic pressure was lower, and LV contractility was higher in the preconditioning groups than in TAC alone group (all P<0.05). No significant differences were noted between them on LV posterior wall thickness (LV cavity enlargement in TAC group would decrease wall thickness).1.2 Hypertrophic preconditioning unregulate the expression of S100A8/A9These findings indicated that hypertrophic preconditioning has an inhibitory effect on cardiac hypertrophy and slow the progression of HF. we examined the expression of S100A8 and S100A9 after the preconditioning of stimulation. Western Blot showed that myocardial protein expression of S100A8 and S100A9 was significantly increased in mice 1 day after debanding that had been preceded by 3 days or 1 week of TAC. We further checked how long the upregulation of S100A8/A9 would persist in the two preconditioning groups. The results demonstrated that S100A8 or A9 was significantly increased in response to preconditioning, which was continued until 1 week and 6 weeks after reimposition of hypertrophic stimuli.Hypertrophic preconditioning upregulates the expression of S100A8/A9 and recombinant protein S100A8/A9 attenuates hypertrophy and fibrosis in vitro2.1 Hypertrophic preconditioning upregulates the expression of S100A8/A9In the cultured cardiomyocytes, Western blot found that expression of S100A8 and S100A9 proteins in cultured NRVCs was similar between control cells and NE-stimulated cells, but was markedly upregulated at 12 hours after the withdrawal of NE.2.2 Recombinant S100A8/A9 attenuates hypertrophy and fibrosis in vitroWe investigated whether recombinant S100A8 and S100A9 proteins had antihypertrophic effects in cultured NRVCs and fibroblasts. The treatment with either S100A8 or S100A9 (or both proteins) significantly suppressed the NE-induced increase in the surface area of cardiomyocytes. In comparison with control cells, exposure to NE for 48 hours increased the expression of ANP and β-MHC mRNA in NRVCs, and the expression of procollagen I and III mRNA in fibroblasts, as well, whereas treatment with S100A8, S100A9, or both of these proteins prevented such changes. These findings suggested that S100A8 and S100A9 could attenuate NE-induced hypertrophy and fibrosis in cultured cardiac cells. The exposure of NRVCs to NE resulted in increased expression of calcineurin, but this was abrogated by the treatment with either S100A8 or S100A9, or both of these proteins. When the subcellular localization of NFATc3 was assessed by Western blotting, it was primarily localized in the cytoplasm of control cells and underwent translocation to the nucleus in response to NE stimulation, whereas treatment with S100A8 or S100A9, or both of these proteins, inhibited NE-induced nuclear translocation of NFATc3.Silencing of S100A8/A9 attenuates the antihypertrophic Effects of hypertrophic preconditioningThe aforementioned results suggest the important role of S100A8/A9 in myocardial hypertrophy. We then used approaches for overexpression and silence of function to further address this issue.3.1 Identification of recombinant lentivirusImmunofluorescence staining showed that the recombinant virus overexpression/knockdown of S100A8/A9 could effectively infect cardiac muscle cells, and when MOI=5 could infect more than 70%of the cells. Infection of cardiomyocytes with lentivirus-S100A8 or A9 upregulated S100A8 or A9 by >200-fold, which was much higher than the upregulation amplitude in response to debanding (about6-fold). RT-PCR and Western Blot demonstrated that the expression of S100A8 and S100A9 (Lv-ShRNA-S100A8 andLv-ShRNA-S100A9) was significantly decreased compared with the control group.3.2 Cardiomyocyte viability in response to endogenous and extraneous S100A8 or S100A9The cell viability test (MTT) showed that high-dose overexpression of endogenous S100A8 or A9 and a high dose of exogenous S100A8 or A9 increased cardiomyocyte death, suggesting that the role of S100A8/A9 is dose dependent. Accordingly, we chose the S100A8/A9-silencing approach for further mechanism research.3.3 Silencing of S100A8/A9 Attenuates the Antihypertrophic Effects of Hypertrophic PreconditioningCompared with the preconditioning group, both Lv-Sh-S100A8 and A9 significantly reduced the hypertrophic preconditioning effects manifested by an increase of cardiomyocyte cell surface area, the upregulation of ANP and P-MHC in cardiomyocytes, increase of calcineurin protein levels, and nuclear translocation of NFAT3and the upregulation of procollagens in fibroblasts.Conclusions:1. Myocardial hypertrophy preconditioning upregulate the expression of S100A8/A9 and which was continued until 1 week and 6 weeks after reimposition of hypertrophic stimuli. Recombinant protein of S100A8/A9 exerts antihypertrophic effect through inhibite the calcineurin/NFAT Pro-hypertrophic signaling pathway in vitro.2. Silencing of S100A8/A9 Attenuates the Antihypertrophic Effects of Hypertrophic Preconditioning through inhibited NE-induced the activation of calcineurin/NFAT signaling pathway. Both Lv-Sh-S100A8 and A9 significantly reduced the hypertrophic preconditioning effects manifested by an increase of cardiomyocyte cell surface area, the upregulation of ANP and β-MHC in cardiomyocytes, and the upregulation of procollagens in fibroblasts increase of calcineurin protein levels and nuclear translocation of NFAT3...