| The growth hormone–releasing peptide ghrelin is a polypeptide of28amino acidresidues discovered by Kojima et al. in1999; it is an endogenous ligand of the growthhormone secretagogue receptor (GHS-R) and has several biological functions, includingstimulation of growth hormone (GH) secretion and promotion of food intaken, which hasbeen linked to obesity. In addition, as a cardiovascular hormone, ghrelin has a variety offunctions that potentially benefits cardiovascular system, including increasing cardiaccontractility, protecting endothelial cells, regulating myocardial energy metabolism ofatherosclerosis, and protecting from ischemia/reperfusion injury as well as improving theprognosis of myocardial infarction and heart failure. However, the specific cellular andmolecular mechanisms underlying these effects are not yet fully understood.In this study, we detected the levels of ghrelin and angiotensin (Ang) II in patients andrats with heart failure to explore the effects of ghrelin on cardiac function. We then furtherinvestigated whether the ghrelin affected cardiac myocyte apoptosis, and thus affectedcardiac function. Moreover, we used ghrelin to intervene in apoptosis of Ang II-inducedprimary neonatal rat cardiomyocytes and investigated the effects and mechanisms of ghrelinon the apoptosis of Ang II-induced cardiomyocyte.Methods:1. We enrolled patients with congestive heart failure (CHF) and healthy control subjectsrandomly. Blood samples were collected and the serum levels of ghrelin and Ang II weredetected.2. The rat model of heart failure was established by ligation of the left anteriordescending (LAD) of female Wistar rats. Then, the rats were divided into sham operation(SO) group, model (myocardial infarction, MI) group and MI-Ghrelin group. Then, thefollowing indexes, LV end-diastolic pressure (LVEDP), left ventricular systolic pressure(LVSP),±dp/dtmax(mmHg/s), the pathological changes of the heart, the ratio of leftventricular mass to body weight (LVM/BW) and the levels of brain natriuretic peptide (BNP), were observed and detected to determine whether the rat model of heart failure wassuccessful and the effects of ghrelin on cardiac function.After successful establishment of the rat model of heart failure, the follwing indexeswere detected:2.1Serum levels of ghrelin and Ang II in rats were examined by ELISA.2.2Myocardial apoptosis was evaluated by TUNEL staining.2.3The expressions of apoptosis-related molecules (Bcl-2, Bax and of caspase-3) wereexamined by real-time quantitative PCR and immunohistochemical staining.2.4The expressions of endoplasmic reticulum stress-related molecules (GRP78,caspase-12and CHOP) mRNA were examined by real-time quantitative PCR.2.5The expressions of Ang II type1(AT1) receptors were examined by real-timequantitative PCR and immunohistochemical staining.3. Last, the primary neonatal rat cardiomyocytes were cultured and identified byimmunohistochemical staining in vitro. Cardiamyocyte apoptosis was induced by AngⅡandghrelin was added to interfer in the role of AngⅡ.The cells were divided into the followinggroups: Ghrelin (0.1μM), Ang II (0.1μM), Ghrelin+Ang II (0.1μM ghrelin+0.1μM AngII), and the control respectively, then they were cultured dishes for24h. Finally, the follwingindexes were detected:3.1Cell viabilities were analyzed by MTT assay and the number of apoptoticcardiomyocytes was evaluated by DNA ladder, TUNEL staining and flow cytometry.3.2The expressions of caspase-3were examined by real-time quantitative PCR,western blot and immunohistochemical staining.3.3The expressions of Bcl-2and Bax were examined by real-time quantitative PCR andimmunohistochemical staining.3.4The expressions of endoplasmic reticulum stress-related molecules (GRP78,caspase-12and CHOP) were examined by real-time quantitative PCR and western blot.3.5The expressions of Ang II type1(AT1) receptors were examined by real-timequantitative PCR, western blot and immunohistochemical staining. The expressions of AngII type2(AT2) receptors and ghrelin receptors (GHS-R1a, GHS-R1b) mRNA wereexamined by real-time quantitative PCR.Results:1. The results of clinical study: The results of clinical trials showed that the serum levels of Ang II and ghrelin wereboth significantly increased in patients with heart failure, and the levels of ghrelin wereassociated with the increases in Ang II.2. The results of animal tests:2.1The results of the rat heart failure model study showed that compared with SOgroup, in MI group, LVEDP was significantly increased, LVSP and±dp/dtmax(mmHg/s)were significantly decreased; the ratio of left ventricular mass to body weight (LVM/BW)was significantly increased; fibrosis and scar tissues formed in infracted area and the serumlevels of BNP were significantly elevated. However, compared with MI group, inMI-Ghrelin group, LVEDP was significantly decreased, LVSP and±dp/dtmax(mmHg/s) weresignificantly increased; the ratio of left ventricular mass to body weight (LVM/BW) wassignificantly decreased and the serum levels of BNP were significantly decreased. The aboveresults showed that the model of heart failure was successfully constructed and ghrelin couldimprove the cardiac function in CHF rats.2.2The serum levels of Ang II and ghrelin were both significantly increased in rats withheart failure, and the results were consistent with the results of clinical studies.2.3Compared with SO group, the number of apoptotic myocardial cells wassignificantly increased in MI group. Howere, Compared with MI group, the number ofapoptotic myocardial cells was significantly decreased in MI-Ghrelin group. The aboveresults showed that ghrelin could improve the cardiac function through inhibition of theapoptosis of the myocardial cells.2.4An imbalance between Bcl-2and Bax expression was closely correlated withapoptosis, and caspase-3was a key molecule in the apoptosis pathway. This study alsodemonstrated that compared with SO group, the expressions of Bax and caspase-3were bothsignificantly increased while the expression of Bcl-2was significantly decreased in MIgroup. However, compared with MI group, the expressions of Bax and caspase-3were bothsignificantly decreased, while the expression of Bcl-2was significantly increased inMI-Ghrelin group.2.5The endoplasmic reticulum stress (ERS) was an important pathway for cellapoptosis. To further explore the role of ERS, this study examined the expressions of theERS-related molecules GRP78, CHOP and caspase-12and found that compared with SOgroup, the expressions of GRP78, CHOP and caspase-12were all significantly increased inMI group. Howere, compared with MI group, the expressions of GRP78, CHOP and caspase-12were all significantly decreased in MI-Ghrelin group. The above results showedthat endoplasmic reticulum stress participated in the pathological process of heart failure.2.6The effect of Ang II on cardiomyocytes was related to the activation of specificreceptors. We determined the AT1R expression and found that AT1R was significantlyincreased in MI group. However, compared with MI group, the expressions of AT1R weresignificantly increased in MI-Ghrelin group. The above results showed that the AT1R wasup-regulated in myocardial of HF, high levels of Ang II might play biological roles throughupregulating the AT1R expression while ghrelin inhibited the roles of Ang II via reducingAT1R expression.3. The results of in vitro experiments:3.1The viability of Ang II-treated cardiomyocytes was significantly decreased,compared with that in control group. The administration of ghrelin to Ang II-treated cellsprevented Ang II-induced cell death.3.2The apoptotic cells were examined by DNA ladder, TUNEL staining and flowcytometry. The results showed that the number of apoptotic cardiomyocytes wassignificantly increased in Ang II, while the number of apoptotic cells in Ang II+Ghrelingroup was significantly decreased compared with that in Ang II group.3.3This study also demonstrated that in Ang Ⅱ group, the expressions of Bax andcaspase-3were both significantly increased while the expression of Bcl-2was significantlydecreased. We also found that compared to the AngⅡgroup, in Ghrelin+AngⅡgroup, theexpressions of Bax and caspase-3were both significantly decreased, while the expression ofBcl-2was significantly increased.3.4The expressions of GRP78, CHOP and caspase-12were all significantly increasedin Ang II group. However, compared with Ang II group, the expressions of GRP78, CHOPand caspase-12were all significantly decreased in Ang II+Ghrelin group. These datasuggested that the Ang II-induced apoptosis of cardiomyocytes was at least partiallymediated by the ER stress pathway and might be inhibited by ghrelin.3.5Our data showed that both AT1and AT2receptors expressions significantlyincreased in the Ang II group. However, Ang II+Ghrelin treatment only down-regulated theAT1receptor, did not alter the expression of the AT2receptor. Additionally, AT1receptorexpression was significantly reduced in ghrelin group compared to control group but AT2receptor expression in the ghrelin group was not significantly different from that of thecontrol group. These data indicated that Ang II might bind mainly to AT1receptor and induce cardiomyocyte apoptosis and ghrelin inhibits cardiomyocyte apoptosis via reducingAng II-upregulated AT1receptor.3.6Moreover, to further explore the effects and mechanisms of ghrelin on cardiacmyocyte apoptosis, we deteced the expressions of ghrelin receptors GHS-R1a and GHS-R1bin cardiomyocytes and found that ghrelin receptors GHS-R1a and GHS-R1b were expressedin cardiomyocytes, and were significantly increased after co-incubation of ghrelin with AngII. It was suggested that ghrelin might exert a cardio-protective function against AngII-induced cardiomyocyte apoptosis through upregulating the expressions of GHS-R1a andGHS-R1b.Conclusions:1. The serum levels of Ang II and ghrelin are both significantly increased in patientswith heart failure and in rats with heart failure, and the levels of ghrelin are associated withthe increases in Ang II.2. Ghrelin can downregulate the expressions of GRP78, caspase-12, CHOP, Bax andcaspase-3, and upregulate the expressions of Bcl-2, thereby inhibit the apoptosis ofmyocardial cells and improve the cardiac function.3. The mechanisms of ghrelin anti-heart failure may be up-regulating the expression ofghrelin receptors (GHS-R1a, GHS-R1b), down-regulating the expression of AT1R andinhibiting the Ang II-induced cardiomyocyte apoptosis.
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