Hydrogen sulfide improves endothelial barrier function by modulating the ubiquitination degradation of KLF4 through TRAF7 S-sulfhydration in diabetic aorta.

Anomalous vascular endothelium significantly contributes to various cardiovascular diseases. VE-cadherin plays a vital role in governing the endothelial barrier. Krüppel-like factor 4(KLF4), as a transcription factor, which binds the VE-cadherin promoter and enhances its transcription. Tumor necrosis factor receptor-associated factor 7 (TRAF7) is an E3 ubiquitin ligase that has been shown to modulate the degradation of KLF4. H2S can covalently modify cysteine residues on proteins through S-sulfhydration, thereby influencing the structure and functionality of the target protein. However, the role of S-sulfhydration on endothelial barrier integrity remains to be comprehensively elucidated. This study aims to investigate whether protein S-sulfhydration in the endothelium regulates endothelial integrity and its underlying mechanism. In this study, we observed that protein S-sulfhydration was reduced in the endothelium during diabetes and TRAF7 was the main target. Overexpression of TRAF7-Cys327 mutant could mitigate the endothelial barrier damage by weakening TRAF7 interaction with KLF4 and reducing ubiquitination degradation of KLF4. In conclusion, our research demonstrates that H2S plays a pivotal role in regulating S-sulfhydration of TRAF7 at Cys327. This regulation effectively inhibits the ubiquitin-mediated degradation of KLF4, resulting in an upregulation of VE-cadherin levels. This molecular mechanism contributes to the prevention of endothelial barrier damage.

PDZK1 improves ventricular remodeling in hypertensive rats by regulating the stability of the Mas receptor.

Ventricular remodeling is one of the main causes of mortality from heart failure due to hypertension. Exploring its mechanism and finding therapeutic targets have become urgent scientific problems to be solved. A number of studies have shown that Mas, as an Ang-(1-7) specific receptor, was significantly reduced in myocardial tissue of rats undergoing hypertensive ventricular remodeling. It has been reported that Mas receptor levels are significantly downregulated in myocardium undergoing ventricular remodeling, but studies focused on intracellular and post-translational modifications of Mas are lacking. The results of this research are as follows: (1) PDZK1 interacts with the carboxyl terminus of Mas through its PDZ1 domain; (2) the expression of PDZK1 and Mas is decreased in rats undergoing hypertensive ventricular remodeling, and PDZK1 upregulation can ameliorate hypertensive myocardial fibrosis and myocardial hypertrophy; (3) PDZK1 enhances the stability of Mas protein through the proteasome pathway, and the proteasome inhibitor MG132 promotes hypertensive ventricular remodeling. PDZK1 improves ventricular remodeling in hypertensive rats by regulating Mas receptor stability. This study provides a scientific basis for the prevention and treatment of ventricular remodeling.

The Influence of baseline glycemic status on the effects of intensive blood pressure lowering: Results from the STEP randomized trial.

BACKGROUND: Intensive systolic blood pressure (SBP) lowering showed cardiovascular benefits in the Strategy of Blood Pressure Intervention in the Elderly Hypertensive Patients (STEP) trial. We investigated whether baseline glycemic status influences the effects of intensive SBP lowering on cardiovascular outcomes. METHODS: In this post hoc analysis of the STEP trial, participants were randomly assigned to receive intensive (110 to <130 mmHg) or standard SBP treatment (130 to <150 mmHg) and categorized by baseline glycemic status into three subgroups: normoglycemia, prediabetes, and diabetes. The primary outcome was a composite of stroke, acute coronary syndrome, acute decompensated heart failure, coronary revascularization, atrial fibrillation, or death from cardiovascular causes. A competing risk proportional hazards regression model was used in the analysis. RESULTS: Of the 8,318 participants, 3,275, 2,769, and 2,274 had normoglycemia, prediabetes, and diabetes, respectively. Over a median follow-up of 3.33 years, intensive SBP lowering significantly reduced the risk of the primary outcome (adjusted hazard ratio 0.73, 95% confidence interval [CI] 0.59-0.91). The adjusted hazard ratios for the primary outcome in the normoglycemia, prediabetes, and diabetes subgroups were 0.72 (95% CI 0.49-1.04), 0.69 (95% CI 0.46-1.02), and 0.80 (95% CI 0.56-1.15), respectively. The intensive SBP lowering strategy resulted in similar effects among participants in the three subgroups (all interaction P >0.05). The sensitivity analyses showed consistent results with the main analysis. CONCLUSION: The effects of intensive SBP lowering on cardiovascular outcomes were consistent among participants with normoglycemia, prediabetes, and diabetes.

Hydrogen sulfide regulates SERCA2a SUMOylation by S-Sulfhydration of SENP1 to ameliorate cardiac systole-diastole function in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is a serious complication of diabetes mellitus that eventually progresses to heart failure. The sarco(endo)plasmic reticulum calcium ATPase 2a (SERCA2a), an important calcium pump in cardiomyocytes, is closely related to myocardial systolic-diastolic function. In mammalian cells, hydrogen sulfide (H2S), as a second messenger, antioxidant, and sulfurizing agent, is involved in diverse biological processes. Despite the importance of H2S for protection against DCM, the mechanisms remain poorly understood. The aim of the present study was to determine whether H2S regulates intracellular calcium homeostasis by acting on SERCA2a to reduce cardiomyocyte apoptosis during DCM. Db/db mice were injected with NaHS for 18 weeks. Neonatal rat cardiomyocytes (NRCMs) were treated with high glucose, palmitate, oleate, and NaHS for 48 h. Compared to the NaHS-treated groups, in vivo and in vitro type 2 diabetic models both showed reduced intracellular H2S content, reduced cystathionine γ-lyase (CSE) expression, impaired cardiac function, decreased SERCA2a expression and decreased SERCA2a activity, reduced SUMOylation of SERCA2a, increased sentrin-specific protease 1 (SENP1) expression, and disruption of calcium homeostasis leading to activation of the mitochondrial apoptosis pathway. Compared to the NaHS-treated type 2 diabetes cellular model, overexpression of SENP1 C683A reduced the S-sulfhydration of SENP1, reduced the SUMOylation of SERCA2a, reduced the increased expression and activity of SERCA2a, and induced mitochondrial apoptosis in cardiomyocytes. These results suggested that exogenous H2S elevates SENP1 S-sulfhydration to increase SERCA2a SUMOylation, improve myocardial systolic-diastolic function, and decrease cardiomyocyte apoptosis in DCM.

Hydrogen Sulfide Regulates SERCA2a Ubiquitylation via Muscle RING Finger-1 S-Sulfhydration to Affect Cardiac Contractility in db/db Mice.

Hydrogen sulfide (H2S), as a gasotransmitter, is involved in various pathophysiological processes. Diabetic cardiomyopathy (DCM) is a major complication of diabetes mellitus (DM), which leads to structural and functional abnormalities of the myocardium and eventually causes heart failure (HF). Systolic and diastolic dysfunction are fundamental features of heart failure. SERCA2a, as a key enzyme for calcium transport in the endoplasmic reticulum (ER), affects the process of myocardial relaxation and contraction. H2S can protect the cardiac function against diabetic hearts, however, its mechanisms are unclear. This study found that exogenous H2S affects cellular calcium transport by regulating the H2S/MuRF1/SERCA2a/cardiac contractile pathway. Our results showed that, compared with the db/db mice, exogenous H2S restored the protein expression levels of CSE and SERCA2a, and the activity of SERCA2a, while reducing cytosolic calcium concentrations and MuRF1 expression. We demonstrated that MuRF1 could interact with SERCA2a via co-immunoprecipitation. Using LC-MS/MS protein ubiquitylation analysis, we identified 147 proteins with increased ubiquitination levels, including SERCA2a, in the cardiac tissues of the db/db mice compared with NaHS-treated db/db mice. Our studies further revealed that NaHS administration modified MuRF1 S-sulfhydration and enhanced the activity and expression of SERCA2a. Under hyperglycemia and hyperlipidemia, overexpression of the MuRF1-Cys44 mutant plasmid reduced the S-sulfhydration level of MuRF1 and decreased the ubiquitination level of SERCA2a and the intracellular Ca2+ concentration. These findings suggested that H2S modulates SERCA2a ubiquitination through MuRF1 S-sulfhydration of Cys44 to prevent decreased myocardial contractility due to increased cytosolic calcium.

Hydrogen sulphide ameliorating skeletal muscle atrophy in db/db mice via Muscle RING finger 1 S-sulfhydration.

Muscle atrophy occurs in many pathological states, including cancer, diabetes and sepsis, whose results primarily from accelerated protein degradation and activation of the ubiquitin-proteasome pathway. Expression of Muscle RING finger 1 (MuRF1), an E3 ubiquitin ligase, was increased to induce the loss of muscle mass in diabetic condition. However, hydrogen sulphide (H2 S) plays a crucial role in the variety of physiological functions, including antihypertension, antiproliferation and antioxidant. In this study, db/db mice and C2C12 myoblasts treated by high glucose and palmitate and oleate were chose as animal and cellular models. We explored how exogenous H2 S attenuated the degradation of skeletal muscle via the modification of MuRF1 S-sulfhydration in db/db mice. Our results show cystathionine-r-lyase expression, and H2 S level in skeletal muscle of db/db mice was reduced. Simultaneously, exogenous H2 S could alleviate ROS production and reverse expression of ER stress protein markers. Exogenous H2 S could decrease the ubiquitination level of MYOM1 and MYH4 in db/db mice. In addition, exogenous H2 S reduced the interaction between MuRF1 with MYOM1 and MYH4 via MuRF1 S-sulfhydration. Based on these results, we establish that H2 S prevented the degradation of skeletal muscle via MuRF1 S-sulfhydration at the site of Cys44 in db/db mice.

Exogenous H2S Promoted USP8 Sulfhydration to Regulate Mitophagy in the Hearts of db/db Mice.

Hydrogen sulfide (H2S), an important gasotransmitter, regulates cardiovascular functions. Mitochondrial damage induced by the overproduction of reactive oxygen species (ROS) results in myocardial injury with a diabetic state. The purpose of this study was to investigate the effects of exogenous H2S on mitophagy formation in diabetic cardiomyopathy. In this study, we found that exogenous H2S could improve cardiac functions, reduce mitochondrial fragments and ROS levels, enhance mitochondrial respiration chain activities and inhibit mitochondrial apoptosis in the hearts of db/db mice. Our results showed that exogenous H2S facilitated parkin translocation into mitochondria and promoted mitophagy formation in the hearts of db/db mice. Our studies further revealed that the ubiquitination level of cytosolic parkin was increased and the expression of USP8, a deubiquitinating enzyme, was decreased in db/db cardiac tissues. S-sulfhydration is a novel posttranslational modification of specific cysteine residues on target proteins by H2S. Our results showed that the S-sulfhydration level of USP8 was obviously decreased in vivo and in vitro under hyperglycemia and hyperlipidemia, however, exogenous H2S could reverse this effect and promote USP8/parkin interaction. Dithiothreitol, a reducing agent that reverses sulfhydration-mediated covalent modification, increased the ubiquitylation level of parkin, abolished the effects of exogenous H2S on USP8 deubiquitylation and suppressed the interaction of USP8 with parkin in neonatal rat cardiomyocytes treated with high glucose, oleate and palmitate. Our findings suggested that H2S promoted mitophagy formation by increasing S-sulfhydration of USP8, which enhanced deubiquitination of parkin through the recruitment of parkin in mitochondria.

Exogenous H2S Induces Hrd1 S-sulfhydration and Prevents CD36 Translocation via VAMP3 Ubiquitylation in Diabetic Hearts.

Hydrogen sulfide (H2S) plays physiological roles in vascular tone regulation, cytoprotection, and ATP synthesis. HMG-CoA reductase degradation protein (Hrd1), an E3 ubiquitin ligase, is involved in protein trafficking. H2S may play a role in controlling fatty acid uptake in diabetic cardiomyopathy (DCM) in a manner correlated with modulation of Hrd1 S-sulfhydration; however, this role remains to be elucidated. The aim of the present study was to examine whether H2S can attenuate lipid accumulation and to explain the possible mechanisms involved in the regulation of the H2S-Hrd1/VAMP3 pathway. Db/db mice and neonatal rat cardiomyocytes treated with high glucose, palmitate and oleate were used as animal and cellular models of type 2 diabetes, respectively. The expression of cystathionine-γ-lyase (CSE), Hrd1, CD36 and VAMP3 was detected by Western blot analysis. In addition, Hrd1 was mutated at Cys115, and Hrd1 S-sulfhydration was examined using an S-sulfhydration assay. VAMP3 ubiquitylation was investigated by immunoprecipitation. Lipid droplet formation was tested by TEM, BODIPY 493/503 staining and oil red O staining. The expression of CSE and Hrd1 was decreased in db/db mice compared to control mice, whereas CD36 and VAMP3 expression was increased. NaHS administration reduced droplet formation, and exogenous H2S restored Hrd1 expression, modified S-sulfhydration, and decreased VAMP3 expression in the plasma membrane. Using LC-MS/MS analysis, we identified 85 proteins with decreased ubiquitylation, including 3 vesicle-associated membrane proteins, in the cardiac tissues of model db/db mice compared with NaHS-treated db/db mice. Overexpression of Hrd1 mutated at Cys115 diminished VAMP3 ubiquitylation, whereas it increased CD36 and VAMP3 expression and droplet formation. siRNA-mediated Hrd1 deletion increased the expression of CD36 in the cell membrane. These findings suggested that H2S regulates VAMP3 ubiquitylation via Hrd1 S-sulfhydration at Cys115 to prevent CD36 translocation in diabetes.

Hydrogen sulfide regulates muscle RING finger-1 protein S-sulfhydration at Cys44 to prevent cardiac structural damage in diabetic cardiomyopathy.

BACKGROUND AND PURPOSE: Hydrogen sulfide (H2 S) plays important roles as a gasotransmitter in pathologies. Increased expression of the E3 ubiquitin ligase, muscle RING finger-1 (MuRF1), may be involved in diabetic cardiomyopathy. Here we have investigated whether and how exogenous H2 S alleviates cardiac muscle degradation through modifications of MuRF1 S-sulfhydration in db/db mice. EXPERIMENTAL APPROACH: Neonatal rat cardiomyocytes were treated with high glucose (40 mM), oleate (100 µM), palmitate (400 µM), and NaHS (100 µM) for 72 hr. MuRF1 was silenced with siRNA technology and mutation at Cys44 . Endoplasmic reticulum stress markers, MuRF1 expression, and ubiquitination level were measured. db/db mice were injected with NaHS (39 µmol·kg-1 ) for 20 weeks. Echocardiography, cardiac ultrastructure, cystathionine-γ-lyase, cardiac structure proteins expression, and S-sulfhydration production were measured. KEY RESULTS: H2 S levels and cystathionine-γ-lyase protein expression in myocardium were decreased in db/db mice. Exogenous H2 S reversed endoplasmic reticulum stress, including impairment of the function of cardiomyocytes and structural damage in db/db mice. Exogenous H2 S could suppress the levels of myosin heavy chain 6 and myosin light chain 2 ubiquitination in cardiac tissues of db/db mice, and MuRF1 was modified by S-sulfhydration, following treatment with exogenous H2 S, to reduce the interaction between MuRF1 and myosin heavy chain 6 and myosin light chain 2. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that H2 S regulates MuRF1 S-sulfhydration at Cys44 to prevent myocardial degradation in the cardiac tissues of db/db mice. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

MiR-146a down-regulates inflammatory response by targeting TLR3 and TRAF6 in Coxsackievirus B infection.

Coxsackievirus B (CVB) is the major cause of human myocarditis and dilated cardiomyopathy. Toll-like receptor 3 (TLR3) is an intracellular sensor to detect pathogen's dsRNA. TLR3, along with TRAF6, triggers an inflammatory response through NF-κB signaling pathway. In the cells infected with CVB type 3 (CVB3), the abundance of miR-146a was significantly increased. The role of miR-146a in CVB infection is unclear. In this study, TLR3 and TRAF6 were identified as the targets of miR-146a. The elevated miR-146a inhibited NF-κB translocation and subsequently down-regulated proinflammatory cytokine expression in the CVB3-infected cells. Therefore, the NF-κB pathway can be doubly blocked by miR-146a through targeting of TLR3 and TRAF6. MiR-146a may be a negative regulator on inflammatory response and an intrinsic protective factor in CVB infection.

Exogenous H2S reduces the acetylation levels of mitochondrial respiratory enzymes via regulating the NAD+-SIRT3 pathway in cardiac tissues of db/db mice.

Hydrogen sulfide (H2S), a gaseous molecule, is involved in modulating multiple physiological functions, such as antioxidant, antihypertension, and the production of polysulfide cysteine. H2S may inhibit reactive oxygen species generation and ATP production through modulating respiratory chain enzyme activities; however, the mechanism of this effect remains unclear. In this study, db/db mice, neonatal rat cardiomyocytes, and H9c2 cells treated with high glucose, oleate, and palmitate were used as animal and cellular models of type 2 diabetes. The mitochondrial respiratory rate, respiratory chain complex activities, and ATP production were decreased in db/db mice compared with those in db/db mice treated with exogenous H2S. Liquid chromatography with tandem mass spectrometry analysis showed that the acetylation level of proteins involved in the mitochondrial respiratory chain were increased in the db/db mice hearts compared with those with sodium hydrosulfide (NaHS) treatment. Exogenous H2S restored the ratio of NAD+/NADH, enhanced the expression and activity of sirtuin 3 (SIRT3) and decreased mitochondrial acetylation level in cardiomyocytes under hyperglycemia and hyperlipidemia. As a result of SIRT3 activation, acetylation of the respiratory complexe enzymes NADH dehydrogenase 1 (ND1), ubiquinol cytochrome c reductase core protein 1, and ATP synthase mitochondrial F1 complex assembly factor 1 was reduced, which enhanced the activities of the mitochondrial respiratory chain activity and ATP production. We conclude that exogenous H2S plays a critical role in improving cardiac mitochondrial function in diabetes by upregulating SIRT3.

Nox4-dependent ROS production is involved in CVB3-induced myocardial apoptosis.

Viral myocarditis is a cardiovascular disease that seriously affects human health. Its mechanism is not clear. Coxsackievirus B3 (CVB3) is a member of the picornavirus family and is the leading cause of viral myocarditis. Our group tested the genes in a mouse model of CVB3 virus infection and confirmed that the NADPH oxidase gene had a high expression trend in the acute phase of infection. Whether Nox4, the homologue of NADPH oxidase, participates in the process of viral myocarditis has not been reported. In this study, we found increased expression of Nox4 in viral myocarditis in vivo and in vitro. DPI is a non-specific inhibitor of Nox4 that improved CVB3-induced myocarditis after injection in vivo. DPI also inhibited intracellular ROS release and apoptosis in vitro. Our data indicated that Nox4-dependent ROS production was involved in CVB3-induced myocardial apoptosis.

Exogenous H2S switches cardiac energy substrate metabolism by regulating SIRT3 expression in db/db mice.

Hydrogen sulfide (H2S) is involved in diverse physiological functions, such as anti-hypertension, anti-proliferation, regulating ATP synthesis, and reactive oxygen species production. Sirtuin 3 (SIRT3) is a NAD + -dependent deacetylase that regulates mitochondrial energy metabolism. The role of H2S in energy metabolism in diabetic cardiomyopathy (DCM) may be related to regulate SIRT3 expression; however, this role remains to be elucidated. We hypothesized that exogenous H2S could switch cardiac energy metabolic substrate preference by lysine acetylation through promoting the expression of SIRT3 in cardiac tissue of db/db mice. Db/db mice, neonatal rat cardiomyocytes, and H9c2 cell line with the treatment of high glucose, oleate, and palmitate were used as animal and cellular models of type 2 diabetes. Using LC-MS/MS, we identified 76 proteins that increased acetylation, including 8 enzymes related to fatty acid ß-oxidation and 7 enzymes of the tricarboxylic acid (TCA) cycle in the db/db mice hearts compared to those with the treatment of NaHS. Exogenous H2S restored the expression of NAMPT and the ratio of NAD+/NADH enhanced the expression and activity of SIRT3. As a result of activation of SIRT3, the acetylation level and activity of fatty acid ß-oxidation enzyme LCAD and the acetylation of glucose oxidation enzymes PDH, IDH2, and CS were reduced which resulted in activation of PDH, IDH2, and CS. Our finding suggested that H2S induced a switch in cardiac energy substrate utilization from fatty acid ß-oxidation to glucose oxidation in DCM through regulating SIRT3 pathway. KEY MESSAGES: H2S regulated the acetylation level and activities of enzymes in fatty acid oxidation and glucose oxidation in cardiac tissues of db/db mice. Exogenous H2S decreased mitochondrial acetylation level through upregulating the expression and activity of SIRT3 in vivo and in vitro. H2S induced a switch in cardiac energy substrate utilization from fatty acid oxidation to glucose.

Knockdown of dishevelled-1 attenuates cyclosporine A-induced apoptosis in H9c2 cardiomyoblast cells.

Cyclosporine (CsA) has become a mainstay for immune suppression of organ transplants. It is known that patients receiving CsA manifest increased growth of aggressive cardiotoxicity. We have demonstrated that CsA induces myocardium cell apoptosis in vivo and vitro. Recently, dishevelled-1 (Dvl-1) protein, which is a cytoplasmic mediator of Wnt/ß-catenin signaling, was explored in cardiac diseases. However, whether Dvl-1 is involved in CsA-induced apoptosis remains to be determined. The aim of this study was to explore the role of Dvl-1 in CsA-induced apoptosis in H9c2 cardiomyoblast cells and to investigate the role of the Wnt/ß-catenin signaling cascade in this progress. H9c2 cells were treated with CsA in dose and time-dependent manners. We found that the appropriate concentrations and time-points of CsA-induced the expression of Dvl-1 and subsequent up-regulation of ß-catenin and c-Myc, which is consistent with previously demonstrated concentrations and time-points when H9c2 cells apoptosis occurred. Then, cells were transfected with small interfering RNA (siRNA) against Dvl-1 and stimulated with previously demonstrated concentration of CsA. Dvl-1 down-regulation decreased the apoptotic rate, caspase-3 activity, and the Bax/Bcl-2 ratio in H9c2 cells treated with CsA. Furthermore, knocking down the expression of Dvl-1 partially suppressed the activity of the Wnt/ß-catenin pathway. Moreover, we further deleted the downstream member ß-catenin by specific siRNA, and found that CsA-induced the Bax/Bcl-2 ratio and the expression of c-Myc, which were attenuated. Our results are the first to unveil this novel aspect of Dvl-1 signaling. In addition, these data provide insight into the pathogenesis and the therapeutic strategies of CsA-induced myocardial injury.

[Detection and application of PIS genetic deficiency gene in dairy goat].

The purpose of this study was to develop a molecular method for detecting polled intersex syndrome (PIS) genetic deficiency gene in dairy goat. Three pairs of primers, PIS-, PIS+, and NEI were designed based on PIS gene sequence (AF404302) to identify the PIS genetic deficiency genotype. For the normal phenotype, the fragments of 141 and 300 bp were obtained for the genotype PIS-PIS-, and 141, 449, and 300 bp for the genotype PIS-PIS+. For the PIS goat with the genotype PIS+PIS+, 449 and 300 bp were obtained. Two hundred and twenty-four dairy goats in one population were tested based on this method. The results showed that there were 150 PIS-PIS+, 70 PIS -PIS-, and 4 PIS+PIS+. The genotype frequency of PIS-PIS+ was 66.9%, and the gene frequency of PIS+ was 35.3% in the population. Therefore, the frequency of PIS offspring was over 12%. This study developed a method to detect PIS genetic deficiency dairy goat. The method could identify buck genotype accurately to avoid the occurrence of PIS genetic deficiency. The ease and accuracy show a strong potential of the method for use in marker assisted selection of dairy goats and healthy development of dairy goat industry.

[A method for screening microRNA target genes in goat skin by microRNA seed sequence mediated PCR].

The purpose of the present study was to establish a new microRNA seed mediated controllable genetic operation, namely MicroRNA Targets Finger Print (MTFP), for screening microRNA targets and detecting target gene expression profiles. Based on combining the complementary sequence of seed sequence, upstream and downstream anchor sequence including special adaptor, microRNA targets were amplified by means of the reverse transcription and special two step PCR. The polyacrylamide gel electrophoresis was used to analyze the sizes of amplified microRNA targets and their abundance of expression, which were used to screen specifically expressed genes in different physiological or experimental conditions. Specific target genes were obtained through isolation of DNA fragments and sequencing methods. As an example, by screening the targets of miR-203 in goat skin, five genes were amplified and sequenced with the sizes of 718 bp (JN709494), 349 bp (JN709495), 243 bp (JN709496), 159 bp (JN709497), and 97 bp (JN709498) from goat skin collections. The novel universal MTFP method could be applied for finding microRNA regulation targets or assessing target gene expression profile.

Cyclosporin A induces apoptosis in H9c2 cardiomyoblast cells through calcium-sensing receptor-mediated activation of the ERK MAPK and p38 MAPK pathways.

The cardiotoxicity of cyclosporine A (CsA) limits its clinical application in extensive and long-term therapies. Our group has shown that CsA induces myocardium cell apoptosis in vivo and increases calcium-sensing receptor (CaSR) expression. However, its molecular mechanism remains unknown. The purpose of this study was to determine whether CaSR plays an essential role in CsA-induced apoptosis in H9c2 cells and to investigate the role of the mitogen-activated protein kinase (MAPK) signaling cascade in this process. H9c2 cells were treated with CsA in a dose-dependent manner, and decreased Bcl-2 expression, increased Bax expression, and caspase-3 activation were observed. In a time-dependent manner, CsA increased CaSR expression, activated the extracellularly regulated kinase (ERK) and p38 MAPK pathways, and inactivated the c-Jun N-terminal kinase (JNK) MAPK signaling pathway. When H9c2 cardiomyoblast cells pretreated with gadolinium chloride (GdCl(3)), a CaSR activator, were treated with CsA, decreased phosphorylation of ERK1/2, increased phosphorylation of p38, decreased Bcl-2 expression, increased Bax expression, and activated caspase-3 were observed. Cells pretreated with the CaSR inhibitor NPS2390 inhibited this process. Furthermore, the MEK1/2 inhibitor U0126 and the p38 MAPK inhibitor SB203580 markedly blocked the effect of CsA on cell apoptosis, apoptotic-related protein expression, and caspase-3 activation. These findings showed that CsA induced apoptosis in H9c2 cells in vitro, and CaSR mediated the degradation of ERK MAPK and the upregulation of the p38 MAPK pathway involved in CsA-induced H9c2 cardiomyoblast cell apoptosis.