Hongzhu Li

Role of dopamine D2 receptors in ischemia/reperfusion induced apoptosis of cultured neonatal rat cardiomyocytes

BackgroundMyocardial ischemia/reperfusion injury is the major cause of morbidity and mortality for cardiovascular diseases. Dopamine D2 receptors are expressed in cardiac tissues. However, the roles of dopamine D2 receptors in myocardial ischemia/reperfusion injury and cardiomyocyte apoptosis are unclear. Here we investigated the effects of both dopamine D2 receptors agonist (bromocriptine) and antagonist (haloperidol) on apoptosis of cultured neonatal rat ventricular myocytes induced by ischemia/reperfusion injury.MethodsMyocardial ischemia/reperfusion injury was simulated by incubating primarily cultured neonatal rat cardiomyocytes in ischemic (hypoxic) buffer solution for 2 h. Thereafter, these cells were incubated for 24 h in normal culture medium.ResultsTreatment of the cardiomyocytes with 10 μM bromocriptine significantly decreased lactate dehydrogenase activity, increased superoxide dismutase activity, and decreased malondialdehyde content in the culture medium. Bromocriptine significantly inhibited the release of cytochrome c, accumulation of [Ca2+]i, and apoptosis induced by ischemia/reperfusion injury. Bromocriptine also down-regulated the expression of caspase-3 and -9, Fas and Fas ligand, and up-regulated Bcl-2 expression. In contrast, haloperidol (10 μM) had no significant effects on the apoptosis of cultured cardiomyocytes under the aforementioned conditions.ConclusionsThese data suggest that activation of dopamine D2 receptors can inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion damage through various pathways.

Involvement of calcium-sensing receptor in cardiac hypertrophy-induced by angiotensinII through calcineurin pathway in cultured neonatal rat cardiomyocytes

Cardiac hypertrophy is a common pathological change accompanying cardiovascular disease. Recently, some evidence indicated that calcium-sensing receptor (CaSR) expressed in the cardiovascular tissue. However, the functional involvement of CaSR in cardiac hypertrophy remains unclear. Previous studies have shown that CaSR caused accumulation of inositol phosphate to increase the release of intracellular calcium. Moreover, Ca(2+)-dependent phosphatase calcineurin (CaN) played a vital role in the development of cardiac hypertrophy. Therefore, we investigated the expression of CaSR in cardiac hypertrophy-induced by angiotensin II (AngII) and the effects of CaSR activated by GdCl(3) on the related signaling transduction pathways. The results showed that AngII induced cardiac hypertrophy and up-regulated the expression of CaSR, meanwhile increased the intracellular calcium concentration ([Ca(2+)](i)) and activated CaN hypertrophic signaling pathway. Compared with AngII alone, the above changes were further obvious when adding GdCl(3). But the effects of GdCl(3) on the cardiac hypertrophy were attenuated by CsA, a specific inhibitor of CaN. In conclusion, these results suggest that CaSR is involved in cardiac hypertrophy-induced by AngII through CaN pathway in cultured neonatal rat cardiomyocytes.

L-arginine inhibits isoproterenol-induced cardiac hypertrophy through nitric oxide and polyamine pathways.

Polyamines (putrescine, spermidine and spermine) are essential for cell growth and differentiation. Nitric oxide exhibits antihypertrophic functions and inhibits cardiac remodelling. However, the metabolism of polyamines and the potential interactions with nitric oxide in cardiac hypertrophy remain unclear. We randomly divided Wistar rats into four treatment groups: controls, isoproterenol (ISO), ISO and L-arginine, and L-arginine. Isoproterenol (5 mg/kg/day, subcutaneously) and/or L-arginine (800 mg/kg/day, intraperitoneally) was administered once daily for 7 days. The expression of atrial natriuretic peptide mRNA was determined by reverse transcription-polymerase chain reaction, and fibrogenesis of heart was assessed by Van Gieson staining. Polyamines were measured with high-performance liquid chromatography, and plasma nitric oxide content and lactate dehydrogenase (LDH) activity were determined with a spectrophotometer. The expression levels of ornithine decarboxylase, spermidine/spermine N1-acetyltransferase (SSAT), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) were analysed by Western blot. Heart-to-body weight ratio, left ventricle-to-body weight ratio, atrial natriuretic peptide mRNA expression, collagen fibres and LDH activity were elevated, both ornithine decarboxylase and SSAT proteins were up-regulated, and total polyamines were increased in the group treated with ISO. Additionally, the expression of iNOS was up-regulated, eNOS was down-regulated, and nitric oxide levels were low. Notably, cotreatment with L-arginine reversed most of these changes except for SSAT expression,which was further up-regulated. We propose that increased polyamines and decreased nitric oxide are involved in cardiac hypertrophy induced by ISO and suggest that L-arginine pre-treatment can attenuate cardiac hypertrophy through the regulation of key enzymes of the polyamine and nitric oxide pathways.

Calcium-sensing receptors induce apoptosis in cultured neonatal rat ventricular cardiomyocytes during simulated ischemia/reperfusion.

Calcium‐sensing receptors (CaSRs) are G‐protein coupled receptors which regulate systemic calcium homeostasis and also participate in cell proliferation, differentiation and apoptosis. We have previously shown that CaSR can induce apoptosis in isolated rat adult hearts and in normal rat neonatal cardiomyocytes. However, no knowledge exists concerning the role of CaSR in apoptosis induced by ischemia and reperfusion in neonatal cardiac myocytes. Therefore, in the present study, we incubated primary neonatal rat ventricular cardiomyocytes in ischemia‐mimetic solution for 2 h, then re‐incubated them in a normal culture medium for 24 h to establish a model of simulated ischemia/reperfusion (I/R). We assayed the apoptotic ratio of the cardiomyocytes by flow cytometry; observed morphological alterations by transmission electron microscope; analyzed the expression of caspase‐3, Bcl‐2, CaSR, extracellular signal‐regulated protein kinase (ERK), and Fas/Fas ligand (FasL) by Western blotting; and measured the concentration of intracellular calcium by Laser Confocal Scanning Microscopy. The results showed that simulated I/R increased the expression of CaSR and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSR, further enhanced CaSR expression, along with increases in intracellular calcium and apoptosis in cardiomyocytes during I/R. Activation of CaSR down‐regulated Bcl‐2 expression, up‐regulated caspase‐3 and Fas/FasL expression and stimulated ERK1/2 phosphorylation. In summary, CaSR is involved in I/R injury and apoptosis of neonatal rat ventricular cardiomyocytes by inhibiting Bcl‐2, inducing calcium overload and activating the Fas/FasL death receptor pathway.

Mediation of exogenous hydrogen sulfide in recovery of ischemic post-conditioning-induced cardioprotection via down-regulating oxidative stress and up-regulating PI3K/Akt/GSK-3β pathway in isolated aging rat hearts

The physiological and pathological roles of hydrogen sulfide (H2S) in the regulation of cardiovascular functions have been recognized. Cystathionine gamma-lyase (CSE) is a major H2S-producing enzyme in cardiovascular system. Ischemic post-conditioning (PC) provides cadioprotection in young hearts but lost in the aging hearts. The involvement of H2S in the recovery of PC-induced cardioprotection in the aging hearts is unclear. In the present study, we demonstrated that ischemia/reperfusion (I/R) decreased H2S production rate and CSE expression, aggravated cardiomyocytes damage, apoptosis and myocardial infarct size, reduced cardiac function, increased the levels of Bcl-2, caspase-3 and caspase-9 mRNA, enhanced oxidative stress in isolated young and aging rat hearts. I/R also increased the release of cytochrome c and down-regulated the phosphorylation of PI3K, Akt and GSK-3β in the aging rat hearts. We further found that PC increased H2S production rate and CSE expressions, and protected young hearts from I/R-induced cardiomyocytes damage, all of which were disappeared in the aging hearts. Supply of NaHS not only increased PC-induced cardioprotection in the young hearts, but also lightened I/R induced-myocardial damage and significantly recovered the cardioprotective role of PC against I/R induced myocardial damage in the aging hearts. LY294002 (a PI3K inhibitor) abolished but N-acetyl-cysteine (NAC, an inhibitor of reactive oxygen species, ROS) further enhanced the protective role of H2S against I/R induced myocardial damage in the aging hearts. In conclusion, these results demonstrate that exogenous H2S recovers PC-induced cardioprotection via inhibition of oxidative stress and up-regulation of PI3K-Akt-GSK-3β pathway in the aging rat hearts. These findings suggested that H2S might be a novel target for the treatment of aging cardiovascular diseases.

Increased expression of calcium-sensing receptors in atherosclerosis confers hypersensitivity to acute myocardial infarction in rats

Acute myocardial infarction (AMI) is a leading cause of death worldwide. Most cases of AMI result from coronary atherosclerosis (AS). The pathogenic mechanisms underlying AS lesions and AMI are incompletely understood. Calcium-sensing receptors (CaSR) belong to a family of G-protein-coupled receptors. We previously discovered that CaSR was expressed in the heart tissue of adult rats. CaSR may contribute to AMI in AS. We initially established a rat model of AS by injection of vitamin D3 and feeding with a high-fat diet. Isoproterenol (ISO) was used to induce AMI. The MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH), cardiac troponin T (cTnT), tetrazolium chloride staining, and cardiac function parameters were selected as indicators of myocardial damage or necrosis. Cardiac apoptosis was analyzed by transferase dUTP nick-end labeling (TUNEL) assay. Expression of CaSR, Bcl-2, Bax, caspase-3, p-ERK1/2, p-JNK, and p-p38 were determined by Western blot analysis. Compared with the control group, levels of cTnT, CK-MB, and LDH; number of TUNEL-positive cells; and expression of CaSR, Bax, caspase-3, p-ERK1/2, p-JNK and p-p38, were significantly increased, whereas cardiac function and expression of Bcl-2 were decreased markedly in isoproterenol (ISO)-treated group (C/ISO) and AS groups. These changes were significant in the AS/ISO group than in the C/ISO group or AS group. The upregulation of CaSR during AS formation renders hypersensitivity to AMI. Activation of the pro-apoptotic mitochondria pathway and JNK-p38 MAPK pathway triggered by increased expression of CaSR may be one of molecular mechanisms underlying AMI in AS.

Effect of Dopamine Receptor 1 on Apoptosis of Cultured Neonatal Rat Cardiomyocytes in Simulated Ischaemia/Reperfusion

Dopamine receptors exist in many tissues, including rat cardiac tissue. However, the physiological importance of dopamine receptors in the homeostatic regulation of cardiac function is unclear. In this study, a model of ischaemia/reperfusion was established by culturing primary neonatal rat cardiomyocytes in ischaemia-mimetic solution for 2 hr, followed by incubation in normal culture medium for 24 hr. Lactate dehydrogenase activity, superoxide dismutase activity and malondialdehyde content were determined colorimetrically with a spectrophotometer. Apoptotic cell death was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling staining and flow cytometry, and morphological alterations were observed with transmission electron microscopy. The intracellular free calcium concentration ([Ca2+]i) was measured by confocal laser scanning microscopy. Finally, the expression of dopamine receptor 1 (DR1), caspase-3, -8 and -9, Fas, Fas ligand and Bcl-2 and the release of cytochrome c were analysed by Western blot. The results showed that DR1 expression was increased markedly during ischaemia/reperfusion. Treatment with 10 microM SKF-38393 (DR1 agonist) significantly increased lactate dehydrogenase activity, decreased superoxide dismutase activity and increased malondialdehyde content in the culture medium. The DR1 agonist promoted the release of cytochrome c, accumulation of [Ca2+]i, and apoptosis induced by ischaemia/reperfusion. Furthermore, SKF-38393 up-regulated the expression of caspase-3, -8 and -9, Fas and Fas ligand, and down-regulated Bcl-2 expression. In contrast, 10 microM SCH-23390 (DR1 antagonist) had no significant effects on the above indicators. In conclusion, DR1 activation is involved in the apoptosis of cultured neonatal rat cardiomyocytes in simulated ischaemia/reperfusion through the mitochondrial and death receptor pathways.

Effects of polyamines on apoptosis induced by simulated ischemia/reperfusion injury in cultured neonatal rat cardiomyocytes

We incubated neonatal Sprague—Dawley rat cardiomyocytes in primary culture in a medium simulating ischemia (consisting of hypoxia plus serum deprivation) for 2 h, then re‐incubated them for 24 h in normal culture medium to establish a model of simulated ischemia/reperfusion (I/R) injury. Apoptotic cell death was measured by MTT assay, TUNEL staining and flow cytometry. Morphological alterations were assessed by transmission electron microscopy, the expression of caspases‐3 and −9 and Bcl‐2 and the release of cytochrome c by Western blotting, and the intracellular free‐calcium concentration ([Ca2+]i) by laser confocal scanning microscopy. The results showed that pretreatment with 10 μmol/l spermine or spermidine significantly inhibited apoptosis in the I/R cells, suppressed the expression of caspases‐3 and −9 and cytochrome c release, up‐regulated Bcl‐2 expression and decreased [Ca2+]i. However, pretreatment with 10 μmol/l putrescine had the opposite effects. Evidence for this “double‐edged” effect of polyamines on apoptosis in I/R‐injured cardiomyocytes is presented for the first time. It may suggest a novel pharmacological target for preventing and treating cardiac ischemia/reperfusion injury.

The Calcium‐Sensing Receptor Mediates Hypoxia‐Induced Proliferation of Rat Pulmonary Artery Smooth Muscle Cells Through MEK1/ERK1,2 and PI3K Pathways

Activation of the calcium-sensing receptor (CaSR) leads to an increase of intracellular calcium concentration and alteration of cellular activities. High level of intracellular calcium is involved in hypoxia-induced proliferation of pulmonary arterial smooth muscle cells (PASMCs). However, whether the CaSR is expressed in PAMSCs and is related to the hypoxia-induced proliferation of PASMCs is unclear. In this study, the expression and distribution of CaSRs were detected by RT-PCR, western blotting and immunofluorescence; the intracellular concentration of free calcium ([Ca(2+) ](i) ) was determined by confocal laser scanning microscopy; cell proliferation was tested using an MTT and BrdU incorporation assay; cell cycle analysis was carried out using a flow cytometric assay; and the expression of proliferating cell nuclear antigen (PCNA), extracellular signal-regulated protein kinase 1,2 (ERK1,2) and AKT were analysed by western blotting. We observed that both CaSR mRNA and protein were expressed in rat PASMCs. Lowering of oxygen from 21% to 2.5% led to increased [Ca(2+) ](i) and CaSR expression. This condition of hypoxia also stimulated PASMCs proliferation accompanying with increased phosphorylation of ERK1,2 and AKT. GdCl(3) (an agonist of CaSR) or NPS2390 (an antagonist of CaSR) amplified or weakened the effect of hypoxia, respectively. PD98059 (a MEK1 inhibitor) or LY294002 (a PI3K inhibitors) decreased the up-regulation of PCNA expression and the increase of the cell proliferation index induced by hypoxia and GdCl(3) in PASMCs. Our results suggest that CaSR is expressed in rat PASMCs, and that CaSR activation through MEK1/ERK1,2 and PI3 kinase pathways is involved in hypoxia-induced proliferation of PASMCs.

Involvement of exogenous H2S in recovery of cardioprotection from ischemic post-conditioning via increase of autophagy in the aged hearts.

BACKGROUND Hydrogen sulfide (H2S), which is a member of the gasotransmitter family, plays an important physiological and pathological role in cardiovascular system. Ischemic post-conditioning (PC) provides myocardial protective effect in the young hearts but not in the aged hearts. Exogenous H2S restores PC-induced cardioprotection by inhibition of mitochondrial permeability transition pore (mPTP) in the aged hearts. However, whether H2S contributes to the recovery of PC-induced cardioprotection via up-regulation of autophagy in the aged hearts is unclear. METHODS The isolated aged rat hearts (24-months-old, 450-500g) and aged cardiomyocytes-induced by d-galactose were exposed to an ischemia/reperfusion (I/R) and PC protocol. RESULTS We found PC lost cardioprotection in the aged hearts and cardiomyocytes. NaHS (a H2S donor) significantly restored cardioprotection of PC through decreasing myocardial damage, infarct size, and apoptosis, improving cardiac function, increasing cell viability and autophagy in the aged hearts and cardiomyocytes. 3-MA (an autophagy inhibitor) abolished beneficial effect of NaHS in the aged hearts. In addition, in the aged cardiomyocytes, NaHS up-regulated AMPK/mTOR pathway, and the effect of NaHS on PC was similar to the overexpression of Atg 5, treatment of AICAR (an AMPK activator) or Rapamycin (a mTOR inhibitor, an autophagy activator), respectively. CONCLUSIONS These results suggest that exogenous H2S restores cardioprotection from PC by up-regulation of autophagy via activation of AMPK/mTOR pathway in the aged hearts and cardiomyocytes.