DR1 Activation Inhibits the Proliferation of Vascular Smooth Muscle Cells through Increasing Endogenous H2S in Diabetes.

Tissue ischemia and hypoxia caused by the abnormal proliferation of smooth muscle cells (SMCs) in the diabetic state is an important pathological basis for diabetic microangiopathy. Studies in recent years have shown that the chronic complications of diabetes are related to the decrease of endogenous hydrogen sulfide (H2S) in diabetic patients, and it has been proven that H2S can inhibit the proliferation of vascular SMCs (VSMCs). Our study showed that the endogenous H2S content and the expression of cystathionine gamma-lyase (CSE), which is the key enzyme of H2S production, were decreased in arterial SMCs of diabetic mice. The expression of PCNA and Cyclin D1 was increased, and the expression of p21 was decreased in the diabetic state. After administration of dopamine 1-like receptors (DR1) agonist SKF38393 and exogenous H2S donor NaHS, the expression of CSE was increased and the change in proliferation-related proteins caused by diabetes was reversed. It was further verified by cell experiments that SKF38393 activated calmodulin (CaM) by increasing the intracellular calcium ([Ca2+]i) concentration, which activated the CSE/H2S pathway, enhancing the H2S content in vivo. We also found that SKF38393 and NaHS inhibited insulin-like growth factor-1 (IGF-1)/IGF-1R and heparin-binding EGF-like growth factor (HB-EGF)/EGFR, as well as their downstream PI3K/Akt, JAK2/STAT3 and ERK1/2 pathways. Taken together, our results suggest that DR1 activation up-regulates the CSE/H2S system by increasing Ca2+-CaM binding, which inhibits the IGF-1/IGF-1R and HB-EGF/EGFR pathways, thereby decreasing their downstream PI3K/Akt, JAK2/STAT3 and ERK1/2 pathways to achieve the effect of inhibiting HG-induced VSMCs proliferation.

SKF38393 prevents high glucose (HG)-induced endothelial dysfunction by inhibiting the effects of HG on cystathionine γ-lyase/hydrogen sulfide activity and via a RhoA/ROCK1 pathway.

BACKGROUND: Endothelial dysfunction plays a crucial role in diabetic vascular complications. A decrease in hydrogen sulfide (H2S) levels is increasingly becoming a vital factor contributing to high glucose (HG)-induced endothelial dysfunction. Dopamine D1-like receptors (DR1) activation has important physiological functions in the cardiovascular system. H2S decreases the dysfunction of vascular endothelial cells. However, no studies have reported whether DR1 protects the function of vascular endothelial cells by regulating H2S levels. AIM: The present study aimed to determine whether DR1 regulates the levels of endogenous H2S, which exerts protective effects against HG-induced injury of human umbilical vein endothelial cells (HUVECs) via Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing kinase 1 (ROCK1) signalling. METHODS: HUVECs were exposed to HG (30 mM) or normal glucose (5.5 mM) after different treatments. Cell viability, proliferation and migration were measured by Cell Counting Kit-8, EdU cell proliferation assay, transwell assay and wound healing assay, respectively. H2S probe (7-Azido-4-Methylcoumarin) was used to detect levels of H2S. The intracellular calcium concentration ([Ca2+]i) were measured using Fluo-4 AM. The protein expressions were quantified by Western blot. RESULTS: We found that HG decreased the expression of DR1 and cystathionine γ-lyase (CSE) and H2S production. The DR1 agonist SKF38393 significantly increased DR1 and CSE expression and H2S production, whereas NaHS (a H2S donor) only increased CSE expression and H2S production but had no effect on DR1 expression. Meanwhile, SKF38393 further increased the [Ca2+]i induced by HG. In addition, HG reduced cell viability and the expression of Cyclin D1 and proliferating cell nuclear antigen and increased the expression of p21C⁢i⁢p/W⁢A⁢F-1, collagen I, collagen III, matrix metalloproteinase 9, osteopontin and α-smooth muscle actin and the activity of phosphorylated RhoA and ROCK1. SKF38393 and NaHS reversed these effects of HG. PPG (a CSE inhibitor) abolished the beneficial effect of SKF38393. These effects of SKF38393 were similar to those of Y-27632 (a ROCK inhibitor). CONCLUSION: Taken together, our results suggest that DR1 activation upregulates the CSE/H2S pathway by increasing the [Ca2+]i, which protects endothelial cells from HG-induced injury by inhibiting the RhoA/ROCK1 pathway.

DR1 activation promotes vascular smooth muscle cell apoptosis via up-regulation of CSE/H2 S pathway in diabetic mice.

The important role of hydrogen sulfide (H2 S) as a novel gasotransmitter in inhibiting proliferation and promoting apoptosis of vascular smooth muscle cells (VSMCs) has been widely recognized. The dopamine D1 receptor (DR1), a G protein coupled receptor, inhibits atherosclerosis by suppressing VSMC proliferation. However, whether DR1 contributes to VSMC apoptosis via the induction of endogenous H2 S in diabetic mice is unclear. Here, we found that hyperglycemia decreased the expressions of DR1 and cystathionine-γ-lyase (CSE, a key enzyme for endogenous H2 S production) and reduced endogenous H2 S generation in mouse arteries and cultured VSMCs. DR1 agonist SKF38393 increased DR1 and CSE expressions and stimulated endogenous H2 S generation. Sodium hydrosulfide (NaHS, a H2 S donor) increased CSE expressions and H2 S generation but had no effect on DR1 expression. In addition, high glucose (HG) increased VSMC apoptosis, up-regulated IGF-1-IGF-1R and HB-EGF-EGFR, and stimulated ERK1/2 and PI3K-Akt pathways. Overexpression of DR1, the addition of SKF38393 or supply of NaHS further promoted VSMC apoptosis and down-regulated the above pathways. Knock out of CSE or the addition of the CSE inhibitor poly propylene glycol diminished the effect of SKF38393. Moreover, calmodulin (CaM) interacted with CSE in VSMCs; HG increased intracellular Ca2+ concentration and induced CaM expression, further strengthened the interaction of CaM with CSE in VSMCs, which were further enhanced by SKF38393. CaM inhibitor W-7, inositol 1,4,5-trisphosphate (IP3 ) inhibitor 2-APB, or ryanodine receptor inhibitor tetracaine abolished the stimulatory effect of SKF38393 on CaM expression and intracellular Ca2+ concentration. Taken together, these results suggest that DR1 up-regulates CSE/H2 S signaling by inducing the Ca2+ -CaM pathway followed by down-regulations of IGF-1-IGF-1R and HB-EGF-EGFR and their downstream ERK1/2 and PI3K-Akt, finally promoting the apoptosis of VSMCs in diabetic mice.

The DR1­CSE/H2S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice.

Glomerular mesangial cell (MC) proliferation and extracellular matrix deposition are the main pathological changes in diabetic nephropathy. Hydrogen sulfide (H2S) inhibits the proliferation of MCs. Dopamine 1 receptors (DR1) are expressed in MCs and serve important physiological roles. However, it is unclear whether DR1 activation inhibits MC proliferation by increasing endogenous H2S. The present study found that the production of H2S and the expression of DR1 and cystathionine­Î³­lyase (CSE) were decreased in the renal tissues of diabetic mice and high glucose (HG)­induced MCs. SKF38393 (a DR1 agonist) increased the production of H2S and the expression of DR1 and CSE and NaHS (an exogenous H2S donor) only increased H2S production and CSE expression but not DR1 expression. HG increased the thickness of the glomerular basement membrane, cell viability and proliferation, the expression of cyclin D1, PCNA, collagen 1 and α­smooth muscle actin and the activity of phosphorylated ERK1/2 and decreased the expression of P21 and MMP9. SKF38393 and NaHS reversed the effects of HG. PPG (a CSE inhibitor) abolished the beneficial effects of SKF38393. The beneficial effects of SKF38393 were similar to those of PD98059 (an ERK1/2 inhibitor). Taken together, the findings suggested that the DR1­CSE/H2S pathway activation attenuated diabetic MC proliferation and extracellular matrix deposition by downregulating the ERK1/2 signaling pathway.

NPS2390, a Selective Calcium-sensing Receptor Antagonist Controls the Phenotypic Modulation of Hypoxic Human Pulmonary Arterial Smooth Muscle Cells by Regulating Autophagy.

BACKGROUND AND OBJECTIVES: Calcium-sensing receptor (CaSR) is known to regulate hypoxia-induced pulmonary hypertension (HPH) and vascular remodeling via the phenotypic modulation of pulmonary arterial smooth muscle cells (PASMCs) in small pulmonary arteries. Moreover, autophagy is an essential modulator of VSMC phenotype. But it is not clear whether CaSR can regulate autophagy involving the phenotypic modulation under hypoxia. METHODS: The viability of human PASMCs was detected by cell cycle and BrdU. The expressions of proliferation protein, phenotypic marker protein, and autophagy protein in human PASMCs were determined by western blot. RESULTS: Our results showed that hypoxia-induced autophagy was considerable at 24 h. The addition of NPS2390 decreased the expression of autophagy protein and synthetic phenotype marker protein osteopontin and increased the expression of contractile phenotype marker protein SMA-ɑ and calponin via suppressing downstream PI3K/Akt/mTOR signal pathways. CONCLUSIONS: Our study demonstrates that treatment of NPS2390 was conducive to inhibit the proliferation and reverse phenotypic modulation of PASMCs by regulating autophagy levels.

[Changes of myocardial function in diabetic rats and its mechanism].

OBJECTIVE: To investigate the role of calcium-sensing receptor (CaSR) in the decrease of cardiac function in type 2 diabetic rats. METHODS: Wistar rats were randomly divided into 3 groups including control, diabetic-4 week and diabetic-8 week groups. Rats in the diabetes group were fed with high-glucose and high-fat diet, and intraperitoneal injection of streptozocin (STZ,30 mg/kg) was conducted 4 weeks later to establish a type 2 diabetes model. Cardiac morphological changes were observed by HE staining, cardiac function was detected by echocardiography, and CaSR and PKC-αprotein expressions in cardiac tissue were detected by Western blot. RESULTS: Compared with the control group, the myocardium of diabetic rats showed irregular contraction zone, decreased expression of CaSR protein, increased expression of PKC-α protein, decreased systolic and diastolic functions, and gradually worsened with the prolongation of the course of the disease. CONCLUSION: Hyperglycemia inhibits the expression of CaSR protein in myocardium of diabetic rats by activating PKC-α, which can cause intracellular calcium disorder and lead to decreased cardiac function.

Calcium-sensing receptor-mediated mitogen-activated protein kinase pathway improves the status of transplanted mouse embryonic stem cells in rats with acute myocardial infarction.

Several studies have identified the critical role of calcium-sensing receptors (CaSRs) in cardiac ischaemia/reperfusion injury and cardiac hypertrophy and have demonstrated that CaSRs induce myocardial apoptosis by activating MAPKs. Using acute myocardial infarction rat models, we found that a combination therapy of CaSR inhibition and embryonic stem cell (ESC) transplantation after acute myocardial infarction (AMI) leads to a dramatic reduction in the infarct size; a significant increase in the maximum rising and falling rate (+dp/dtmax and -dp/dtmax, respectively) of left ventricular pressure; a significant decrease in left ventricular end-diastolic pressure; a significant decrease in the mRNA expression level of CaSR, Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, p-ERK, p-JNK and p-P38 protein together with apoptosis indexes in the C and E groups; and a significant decrease in cTnT levels as well as LDH and CK activity. These findings indicate that cardiac function could be enhanced significantly by combination therapy with CaSR inhibition and ESC transplantation; the effect was better than ESC transplantation alone, and the mechanism might be associated with a reduction in cell apoptosis via the inhibition of the MAPK pathway. Apoptosis could be reduced through CaSR, which regulates the MAPK pathway and apoptosis-related protein. Our study indicated that CaSR inhibitors have a pivotal role in the treatment of AMI.

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.

Exogenous spermine inhibits hypoxia/ischemia-induced myocardial apoptosis via regulation of mitochondrial permeability transition pore and associated pathways.

Myocardial infarction (MI) is associated with a high mortality rate, which is attributed to the effects of myocyte loss that occurs as a result of ischemia-induced cell death. Very few therapies can effectively prevent or delay the effects of ischemia. Polyamines (PAs) are polycations required for cell growth and division, and their use may prevent cell loss. The aim of this study was to investigate the relationship between hypoxia/ischemia (H/I)-induced cell apoptosis and PA metabolism and to investigate the ability of spermine to limit H/I injury in cardiomyocytes by blocking the mitochondrial apoptotic pathway. Neonatal rat cardiomyocytes were placed under hypoxic conditions for 24 h after being subjected to 5 µM of spermine as a pretreatment therapy. H/I induced PA catabolism, which was indicated by a 1.3-fold up-regulation of spermidine/spermine N(1)-acetyltransferase expression. Exogenous spermine significantly reduced H/I-induced cell death rate (60 ± 2 to 36 ± 2%) and apoptosis rate (42 ± 2 to 21 ± 2%); it also attenuated lactate dehyodrogenase and creatine kinase leakage (440 ± 13 and 336 ± 16 U/L to 275 ± 15 and 235 ± 13 U/L). Furthermore, it decreases calcium overload (3.8 ± 0.2 to 2.2 ± 0.1 a.u.). Moreover, spermine pretreatment remarkably decreased cytochrome c release from the mitochondria to the cytosol, lowering the expression of cleaved caspase-3 and -9. With spermine pretreatment, there was an increase in Bcl-2 levels and phosphorylation of ERK1/2, phosphoinositide 3-kinase, Akt, and GSK-3ß, preserving mitochondrial membrane potential and inducing an mitochondrial permeability transition pore opening. In conclusion, H/I decreased endogenous spermine concentrations in cardiomyocytes, which ultimately induced apoptosis. The addition of exogenous spermine effectively prevented myocyte cell death.

Exogenous H2S contributes to recovery of ischemic post-conditioning-induced cardioprotection by decrease of ROS level via down-regulation of NF-κB and JAK2-STAT3 pathways in the aging cardiomyocytes.

BACKGROUND: Hydrogen sulfide (H2S), a third member of gasotransmitter family along with nitric oxide and carbon monoxide, generated from mainly catalyzed by cystathionine-lyase, possesses important functions in the cardiovascular system. Ischemic post-conditioning (PC) strongly protects against the hypoxia/reoxygenation (H/R)-induced injury and apoptosis of cardiomyocytes. However, PC protection is ineffective in the aging cardiomyocytes. Whether H2S restores PC-induced cardioprotection by decrease of reactive oxygen species (ROS) level in the aging cardiomyocytes is unknown. METHODS: The aging cardiomyocytes were induced by treatment of primary cultures of neonatal cardiomyocytes using d-galactose and were exposed to H/R and PC protocols. Cell viability was observed by CCK-8 kit. Apoptosis was detected by Hoechst 33342 staining and flow cytometry. ROS level was analyzed using spectrofluorimeter. Related protein expressions were detected through Western blot. RESULTS: Treatment of NaHS (a H2S donor) protected against H/R-induced apoptosis, cell damage, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c). The supplementation of NaHS also decreased the activity of LDH and CK, MDA contents, ROS levels and the phosphorylation of IκBα, NF-κB, JNK2 and STAT3, and increased cell viability, the expression of Bcl-2, the activity of SOD, CAT and GSH-PX. PC alone did not provide cardioprotection in H/R-treated aging cardiomyocytes, which was significantly restored by the addition of NaHS. The beneficial role of NaHS was similar to the supply of N-acetyl-cysteine (NAC, an inhibitor of ROS), Ammonium pyrrolidinedithiocarbamate (PDTC, an inhibitor of NF-κB) and AG 490 (an inhibitor of JNK2), respectively, during PC. CONCLUSION: Our results suggest that exogenous H2S contributes to recovery of PC-induced cardioprotection by decrease of ROS level via down-regulation of NF-κB and JAK2/STAT3 pathways in the aging cardiomyocytes.

Exogenous spermine inhibits the proliferation of human pulmonary artery smooth muscle cells caused by chemically-induced hypoxia via the suppression of the ERK1/2- and PI3K/AKT-associated pathways.

Pulmonary vascular remodeling is a significant pathological feature of hypoxia-induced pulmonary hypertension (HPH), while pulmonary artery smooth muscle cell (PASMC) proliferation plays a leading role in pulmonary vascular remodeling. Spermine (Sp), a polyamine, plays a critical role in periodic cell proliferation and apoptosis. The present study was conducted to observe the association between hypoxia-induced PASMC proliferation and polyamine metabolism, and to explore the effects of exogenous Sp on PASMC poliferation and the related mechanisms. In the present study, PASMCs were cultured with cobalt chloride (CoCl2) to establish a hypoxia model, and Sp at various final concentrations (0.1, 1, 10 and 100 µM) was added to the medium of PASMCs 40 min prior to the induction of hypoxia. Cell proliferation was measured by 3-(4,5-dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) assay, cell counting kit-8 assay and 5-bromo­2'­deoxyuridine (BrdU) incorporation assay. Cell cycle progression was determined by flow cytometry, and the protein expression levels of spermidine/spermine N1-acetyltransferase (SSAT; the key enzyme in the terminal degradation of polyamine), ornithine decarboxylase (ODC; the key enzyme of polyamine biosynthesis), cyclin D1 and p27 were measured by western blot analysis. The results revealed that the proliferation of the PASMCs cultured with CoCl2 at 50 µM for 24 h markedly increased. The expression of ODC was decreased and the expression of SSAT was increased in the cells under hypoxic conditions. Exogenous Sp at concentrations of 1 and 10 µM significantly inhibited hypoxia-induced PASMC proliferation, leading to cell cycle arrest at the G1/G0 phase. In addition, Sp decreased cyclin D1 expression, increased p27 expression, and suppressed the phosphorylation of extracellular signal­regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT); however, the above-metioned parameters were not markedly affected by Sp at concentrations of 0.1 or 100 µM. These results suggest that hypoxia disrupts polyamine metabolism, and Sp at concentrations of 1 and 10 µM inhibits the increase in human PASMC proliferation caused by chemically-induced hypoxia via the suppression of the ERK1/2- and PI3K/AKT-associated pathways. This study thus offer new insight into the prevention and treatment of HPH.

Exogenous hydrogen sulfide restores cardioprotection of ischemic post-conditioning via inhibition of mPTP opening in the aging cardiomyocytes.

The physiological and pathological roles of hydrogen sulfide (H2S) in the regulation of cardiovascular functions have been recognized. H2S protects against the hypoxia/reoxygenation (H/R)-induced injury and apoptosis of cardiomyocytes, and ischemic post-conditioning (PC) plays an important role in cardioprotection from H/R injury in neonatal cardiomyocytes but not in aging cardiomyocytes. Whether H2S is involved in the recovery of PC-induced cardioprotection in aging cardiomyocytes is unclear. In the present study, we found that both H/R and PC decreased cystathionine-γ-lyase (CSE) expression and the production rate of H2S. Supplementation of NaHS protected against H/R-induced apoptosis, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c), and mPTP opening. The addition of NaHS also counteracted the reduction of cell viability caused by H/R and increased the phosphorylation of ERK1/2, PI3K, Akt, GSK-3ß and mitochondrial membrane potential. Additionally, NaHS increased Bcl-2 expression, promoted PKC-ε translocation to the cell membrane, and activated mitochondrial ATP-sensitive K channels (mitoKATP). PC alone did not provide cardioprotection in H/R-treated aging cardiomyocytes, which was significantly restored by the supplementation of NaHS. In conclusion, our results suggest that exogenous H2S restores PC-induced cardioprotection via the inhibition of mPTP opening by the activation of the ERK1/2-GSK-3ß, PI3K-Akt-GSK-3ß and PKC-ε-mitoKATP pathways in aging cardiomyocytes. These findings provide a novel target for the treatment of aging ischemic cardiomyopathy.

[Decreased expression of calcium-sensing receptor involved in the progression of diabetic cardiomyopathy].

OBJECTIVE: To observe the dynamic expression of calcium-sensing receptor(CaSR) in myocardium of diabetic rats. METHODS: Thirty male Wistar rats were randomly divided into 3 groups including control, diabetic-4 week and diabetic-8 week groups(n = 10). The type 2 diabetes mellitus models were established by intraperitoneal injection of streptozotocin (STZ, 30 mg/kg) after high-fat and high-sugar diet for one month. The cardiac morphology was observed by electron microscope. Western blot analyzed the expression of CaSR, phospholamban (PLN), a calcium handling regulator, and Ca+-ATPase(SERCA) in cardiac tissues. RESULTS: Compared with control group, the expressions of CaSR and SERCA were decreased, while the expression of PLN was significantly increased in a time-dependent manner in diabetic groups. Meanwhile diabetic rats displayed abnormal cardiac structure. CONCLUSION: These results indicate that the CaSR expression of myocardium is reduced in the progression of DCM, and its potential mechanism may be related to the imnaired intracellular calcium homeostasis.

Mediation of exogenous hydrogen sulfide in recovery of ischemic post-conditioning-induced cardioprotection via down-regulating oxidative stress and up-regulating PI3K/Akt/GSK-3ß 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-3ß 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-3ß pathway in the aging rat hearts. These findings suggested that H2S might be a novel target for the treatment of aging cardiovascular diseases.

Exercise training preserves ischemic preconditioning in aged rat hearts by restoring the myocardial polyamine pool.

BACKGROUND: Ischemic preconditioning (IPC) strongly protects against myocardial ischemia reperfusion (IR) injury. However, IPC protection is ineffective in aged hearts. Exercise training reduces the incidence of age-related cardiovascular disease and upregulates the ornithine decarboxylase (ODC)/polyamine pathway. The aim of this study was to investigate whether exercise can reestablish IPC protection in aged hearts and whether IPC protection is linked to restoration of the cardiac polyamine pool. METHODS: Rats aging 3 or 18 months perform treadmill exercises with or without gradient respectively for 6 weeks. Isolated hearts and isolated cardiomyocytes were exposed to an IR and IPC protocol. RESULTS: IPC induced an increase in myocardial polyamines by regulating ODC and spermidine/spermine acetyltransferase (SSAT) in young rat hearts, but IPC did not affect polyamine metabolism in aged hearts. Exercise training inhibited the loss of preconditioning protection and restored the polyamine pool by activating ODC and inhibiting SSAT in aged hearts. An ODC inhibitor, α-difluoromethylornithine, abolished the recovery of preconditioning protection mediated by exercise. Moreover, polyamines improved age-associated mitochondrial dysfunction in vitro. CONCLUSION: Exercise appears to restore preconditioning protection in aged rat hearts, possibly due to an increase in intracellular polyamines and an improvement in mitochondrial function in response to a preconditioning stimulus.

Involvement of calcium-sensing receptors in hypoxia-induced vascular remodeling and pulmonary hypertension by promoting phenotypic modulation of small pulmonary arteries.

Phenotype modulation of pulmonary artery smooth muscle cells (PASMCs) plays an important role during hypoxia-induced vascular remodeling and pulmonary hypertension (PAH). We had previously shown that calcium-sensing receptor (CaSR) is expressed in rat PASMCs. However, little is known about the role of CaSR in phenotypic modulation of PASMCs in hypoxia-induced PAH as well as the underlying mechanisms. In this study, we investigated whether CaSR induces the proliferation of PASMCs in small pulmonary arteries from both rats and human with PAH. PAH was induced by exposing rats to hypoxia for 7-21 days. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVI), the percentage of medial wall thickness to the external diameter (WT %), and cross-sectional total vessel wall area to the total area (WA %) of small pulmonary arteries were determined by hematoxylin and eosin (HE), masson trichrome and Weigert's staining. The protein expressions of matrix metalloproteinase (MMP)-2 and MMP-9, the tissue inhibitors of metalloproteinase (TIMP)-3, CaSR, proliferating cell nuclear antigen (PCNA), phosphorylated extracellular signal-regulated kinase (p-ERK), and smooth muscle cell (SMC) phenotype marker proteins in rat small pulmonary arteries, including calponin, SMα-actin (SMAα), and osteopontin (OPN), were analyzed by immunohistochemistry and Western blotting, respectively. In addition, immunohistochemistry was applied to paraffin-embedded human tissues from lungs of normal human and PAH patients with chronic heart failure (PAH/CHF). Compared with the control group, mPAP, RVI, WT % and WA % in PAH rats were gradually increased with the prolonged hypoxia. At the same time, the expressions of CaSR, MMP-2, MMP-9, TIMP-3, PCNA, OPN, and p-ERK were markedly increased, while the expressions of SMAα and calponin were significantly reduced in lung tissues or small pulmonary arteries of PAH rats. Neomycin (an agonist of CaSR) enhanced but NPS2390 (an antagonist of CaSR) weakened these hypoxic effects. We further found that the expression change of CaSR, PCNA, and SMC phenotypic marker proteins in PAH/CHF lungs was similar to those in PAH rats. Our data suggest that CaSR is involved in the pulmonary vascular remodeling and PAH by promoting phenotypic modulation of small pulmonary arteries.

Mediation of dopamine D2 receptors activation in post-conditioning-attenuated cardiomyocyte apoptosis.

The physiological and pathological roles of dopamine D2 receptors (DR2) in the regulation of cardiovacular functions have been recognized. DR2 activation protects hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and apoptosis, and ischemic post-conditioning (PC) plays a critical role in cardioprotection as well; however the involvement of the DR2 activation in the PC-induced cardioprotection is unknown. In the present study, we found that the H/R increased the expressions of DR2 mRNA and protein in cardiomyocytes, which were significantly enhanced by PC. Bromocriptine (Bro, a DR2 agonist) further increased DR2 expression, but Haloperidol (Hal, a DR2 antagonist) reversed the Bro-induced DR2 expressions. PC protected against H/R-induced apoptosis, the rise of [Ca(2+)]i, the expressions of cleaved caspase-3 and -9, release of cytochrome c, and mPTP opening. In addition, PC counteracted the reduction of cell viability caused by H/R, increased the phosphorylation of ERK1/2, PI3K, Akt, GSK-3ß and mitochondrial membrane potential. PC further increased Bcl-2 expression, promoted PKC-ε translocation to cell membrane, and activated the mitochondrial ATP-sensitive K channels (mKATP). Bro further enhanced the cardioprotective roles of PC, but Hal reversed these effects of Bro. Meanwhile, we found that DR2 was expressed in cell membrane and interacted with PKC-ε in PC. In conclusion, these results suggest that PC attenuates cardiomyocyte apoptosis via inhibition of mPTP opening by DR2-mediated activation of ERK1/2, PI3K-Akt-GSK-3ß and PKC-ε-mKATP. These findings provide a novel target for the treatment of ischemic cardiomyopathy.

Akt and Erk1/2 activate the ornithine decarboxylase/polyamine system in cardioprotective ischemic preconditioning in rats: the role of mitochondrial permeability transition pores.

Ornithine decarboxylase (ODC) is the first rate-limiting enzyme in polyamine biosynthesis, which is essential for cell survival. We hypothesized that the ODC/polyamine system is involved in ischemic preconditioning (IPC)-mediated cardioprotection through the activation of Erk1/2 and Akt and through the inhibition of the mitochondrial permeability transition (mPT). Isolated rat hearts were subjected to 40 min of ischemia either with or without IPC (3 cycles of 5-min global ischemia), and ODC protein expression, polyamine content, and Akt and Erk1/2 phosphorylation were evaluated after 30 min of reperfusion. IPC significantly upregulated the ODC/polyamine pathway, promoted Erk1/2 and Akt phosphorylation, and reduced the infarct size and heart dysfunction after reperfusion. An inhibitor of ODC, α-difluoromethylornithine (DFMO), abolished the IPC-induced cardioprotection. Moreover, the inhibition of the IPC-induced activation of Erk1/2 and Akt using PD98059 or wortmannin downregulated the ODC/polyamine system. In separate studies, the Ca(2+) load required to open the mPT pore was significantly lower in DFMO-treated cardiac mitochondria than in mitochondria from IPC hearts. Furthermore, spermine or spermidine significantly inhibited the mPT induced by CaCl2. These results suggest that IPC upregulates the ODC/polyamine system and mediates preconditioning cardioprotection, which may depend on the phosphorylation/activation of Erk1/2 and Akt and on the inhibition of the mPT during reperfusion.

[The protective effect of DR2 activation on hypoxia/reperfusion injury in the neonatal rat cardiomyocytes and related mechanism].

OBJECTIVE: To observe the effect of dopamine receptor (DR2) activation on hypoxia/reperfusion injury (HRI) in the neonatal rat cardiomyocytes, and to explore its mechanism. METHODS: The hypoxia/reperfusion (H/R) injury model was established in primarily cultured neonatal rat cardiomyocytes, and randomly assigned: control, H/R, bromocriptine (Bro) and haloperidol (Hal) groups. The cell apoptosis was detected using inverted microscope, transmission electron microscope and flow cytometry (FCM). The lactate dehydrogenase(LDH) and superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in cell medium were analyzed. The expression of mRNA and protein of caspase-3, caspase-8, caspase-9, Fas, Fas-L, Cyt C and Bcl-2 were detected by RT-PCR and Western blot, respectively. RESULTS: Compared with the control group, apoptosis rate, LDH activity, MDA content and the expression of pro-apoptotic factors and anti-apoptotic factors were increased, but SOD activity was decreased in H/R group. Compared with the H/R group, all index above-mentioned were down-regulated or reversed in Bro-group, and had no obvious differences in Hal-group. CONCLUSION: The neonatal rat cardiomyocytes injury and apoptosis caused by hypoxia/reperfusion can be inhibited with DR2 activation, which mechanism is related to scavenging oxygen radical.

Involvement of dopamine D2 receptors activation in ischemic post-conditioning-induced cardioprotection through promoting PKC-ε particulate translocation in isolated rat hearts.

Dopamine D2 receptors (DR2) are important regulators in many organs, including cardiac system. Protein kinase C (PKC) activation and translocation is associated with cardioprotection against ischemic post-conditioning (PC); however, the regulatory role of DR2 during this process has been unknown. This study hypothesized that the prevention of cardiomyocyte damage by DR2 activation is associated with PKC translocation to the cell membrane. In the present study, we found that the ischemia/reperfusion (I/R) increased the expressions of DR2 mRNA and protein, which were further enhanced by PC. Bromocriptine (DR2 agonist) up-regulated the PC-induced DR2 expressions, and Haloperidol (DR2 antagonist) reversed the increase of DR2 expressions by Bromocriptine. PC reduced I/R-induced cardiomyocytes damage, apoptosis and myocardial infarct size, and improved cardiac function. Compared with PC, Bromocriptine further enhanced the cardioprotective roles of PC, but Haloperidol canceled the protection effect of Bromocriptine. PC up-regulated PKC-ε translocation in the particulate fraction, which was further strengthened by Bromocriptine but canceled by Haloperidol. In the cytosolic fraction, the changes of the PKC-ε translocation were opposite to the particulate fraction. These findings suggest that DR2 activation provides cardioprotection via promoting PC-induced translocation of PKC-ε.