Huoxue Wentong Formula ameliorates myocardial infarction in rats through inhibiting CaMKII oxidation and phosphorylation.
ABSTRACT: Background:The Chinese medicine Huoxue Wentong Formula (HXWTF) was used to treat thoracic obstruction and angina pectoris in clinic, which has not been investigated in myocardial ischemia-induced apoptosis and angiogenic function. Here we aimed to investigate the roles of HXWTF in rats with myocardial ischemia-induced apoptosis and angiogenesis disorders, as well as to reveal the potential mechanisms. Methods:Male SD rats were subjected to coronary artery ligation followed by HXWTF (420, 840 and 1680 mg/kg/day, p.o.) or isosorbide mononitrate (6.3 mg/kg/day, p.o.) treatment for 4 weeks. Electrocardiogram (ECG) and Echocardiography (ECHO) were used to measure cardiac function. Hematoxylin and eosin (H&E) staining and CD34/?-SMA immunohistochemical staining were performed to observe the ischemic heart sections pathological changes and angiogenesis. Then, the effects on cardiomyocyte apoptosis of H9c2 and tube formation of HCMECs were observed, as well as the changes in the levels of total calmodulin dependent protein kinase II (t-CaMKII), phosphorylated CaMKII (p-CaMKII), oxidized CaMKII (ox-CaMKII), CD34, and Bcl-2/Bax ratio were detected. Results:Rats with coronary artery ligation exhibited abnormal cardiac function, enlarged myocardial space, disorderly arranged myocardial fibers, inflammatory cells infiltrated, and aggravated myocardial cell apoptosis, along with angiogenesis dysfunction. The expressions of CD34, p-CaMKII, and ox-CaMKII were elevated and Bcl-2/Bax ratio was diminished in ischemic hearts and H/SD-treated H9c2 or HCMECs, while HXWTF treatment completely rescued angiogenic dysfunction, inhibited cardiomyocyte apoptosis, and down-regulated cardiac CaMKII oxidation and phosphorylation activities. Conclusion:Our study demonstrates that HXWTF improves myocardial infarction possibly through inhibiting CaMKII oxidation and phosphorylation levels, facilitating angiogenic function and alleviating cardiomyocyte apoptosis. Thus, therapeutics targeting CaMKII activities may be a promising strategy for rescuing ischemic cardiomyopathy.
Project description:Acute myocardial infarction (AMI) is an ischemic heart disease with high mortality worldwide. AMI triggers a hypoxic microenvironment and induces extensive myocardial injury, including autophagy and apoptosis. MiRNAs, which are a class of posttranscriptional regulators, have been shown to be involved in the development of ischemic heart diseases. We have previously reported that hypoxia significantly alters the miRNA transcriptome in rat cardiomyoblast cells (H9c2), including miR-27a-5p. In the present study, we further investigated the potential function of miR-27a-5p in the cardiomyocyte response to hypoxia, and showed that miR-27a-5p expression was downregulated in the H9c2 cells at different hypoxia-exposed timepoints and the myocardium of a rat AMI model. Follow-up experiments revealed that miR-27a-5p attenuated hypoxia-induced cardiomyocyte injury by regulating autophagy and apoptosis via <i>Atg7</i>, which partly elucidated the anti-hypoxic injury effects of miR-27a-5p. Taken together, this study shows that miR-27a-5p has a cardioprotective effect on hypoxia-induced H9c2 cell injury, suggesting it may be a novel target for the treatment of hypoxia-related heart diseases.
Project description:miRNAs have been implicated in processing of cardiac hypoxia/reoxygenation (H/R)-induced injury. Recent studies demonstrated that miR-19a might provide a potential cardioprotective effect on myocardial disease. However, the effect of miR-19a in regulating myocardial ischemic injury has not been previously addressed. The present study was to investigate the effect of miR-19a on myocardial ischemic injury and identified the potential molecular mechanisms involved. Using the H/R model of rat cardiomyocytes H9C2 in vitro, we found that miR-19a was in low expression in H9C2 cells after H/R treatment and H/R dramatically decreased cardiomyocyte viability, and increased lactate dehydrogenase (LDH) release and cardiomyocyte apoptosis, which were attenuated by co-transfection with miR-19a mimic. Dual-luciferase reporter assay and Western blotting assay revealed that PTEN was a direct target gene of miR-19a, and miR-19a suppressed the expression of PTEN via binding to its 3'-UTR. We further identified that overexpression of miR-19a inhibited the expression of PTEN at the mRNA and protein levels. Moreover, PTEN was highly expressed in H/R H9C2 cells and the apoptosis induced by H/R was associated with the increase in PTEN expression. Importantly, miR-19a mimic significantly increased p-Akt levels under H/R. In conclusion, our findings indicate that miR-19a could protect against H/R-induced cardiomyocyte apoptosis by inhibiting PTEN /PI3K/p-Akt signaling pathway.
Project description:Myocardial ischemia is a condition with insufficient oxygen supporting the heart tissues, which may result from myocardial infarction or trauma-induced hemorrhagic shock. In order to develop better preventive and therapeutic strategies for myocardial ischemic damage, it is important that we understand the mechanisms underlying this type of injury. Mitochondrial-derived vesicles (MDVs) have been proposed as a novel player in maintaining mitochondrial quality control. This study aimed to investigate the role and possible mechanisms of MDVs in ischemia/hypoxia-induced myocardial apoptosis. H9C2 cardiomyocytes were used for the cellular experiments. A 40% fixed blood volume hemorrhagic shock rat model was used to construct the acute general ischemic models. MDVs were detected using immunofluorescence staining with PDH and TOM20. Exogenous MDVs were reconstituted in vitro from isolated mitochondria under different hypoxic conditions. The results demonstrate that MDV production was negatively correlated with cardiomyocyte apoptosis under hypoxic conditions; exogenous MDVs inhibited hypoxia-induced cardiomyocyte apoptosis; and MDV-mediated protection against hypoxia-induced cardiomyocyte apoptosis was accomplished via Bcl-2 interactions in the mitochondrial pathway. This study provides evidence that MDVs protect cardiomyocytes against hypoxic damage by inhibiting mitochondrial apoptosis. Our study used a novel approach that expands our understanding of MDVs and highlights that MDVs may be part of the endogenous response to hypoxia designed to mitigate damage. Strategies that stimulate cardiomyocytes to produce cargo-specific MDVs, including Bcl-2 containing MDVs, could theoretically be helpful in treating ischemic/hypoxic myocardial injury.
Project description:RATIONALE:Ca2+/calmodulin-dependent protein kinase (CaMK)II is a multifunctional kinase involved in vital cellular processes such as Ca(2+) handling and cell fate regulation. In mammalian heart, 2 primary CaMKII isoforms, deltaB and deltaC, localize in nuclear and cytosolic compartments, respectively. Although previous studies have established an essential role of CaMKII-deltaC in cardiomyocyte apoptosis, the functional role of the more abundant isoform, CaMKII-deltaB, remains elusive. OBJECTIVE:Here, we determined the potential role of CaMKII-deltaB in regulating cardiomyocyte viability and explored the underlying mechanism. METHODS AND RESULTS:In cultured neonatal rat cardiomyocytes, the expression of CaMKII-deltaB and CaMKII-deltaC was inversely regulated in response to H2O2-induced oxidative stress with a profound reduction of the former and an increase of the later. Similarly, in vivo ischemia/reperfusion (IR) led to an opposite regulation of these CaMKII isoforms in a rat myocardial IR model. Notably, overexpression of CaMKII-deltaB protected cardiomyocytes against oxidative stress-, hypoxia-, and angiotensin II-induced apoptosis, whereas overexpression of its cytosolic counterpart promoted apoptosis. Using cDNA microarray, real-time PCR and Western blotting, we demonstrated that overexpression of CaMKII-deltaB but not CaMKII-deltaC elevated expression of heat shock protein (HSP)70 family members, including inducible (i)HSP70 and its homolog (Hst70). Moreover, overexpression of CaMKII-deltaB led to phosphorylation and activation of heat shock factor (HSF)1, the primary transcription factor responsible for HSP70 gene regulation. Importantly, gene silencing of iHSP70, but not Hst70, abolished CaMKII-deltaB-mediated protective effect, indicating that only iHSP70 was required for CaMKII-deltaB elicited antiapoptotic signaling. CONCLUSIONS:We conclude that cardiac CaMKII-deltaB and CaMKII-deltaC were inversely regulated in response to oxidative stress and IR injury, and that in contrast to CaMKII-deltaC, CaMKII-deltaB serves as a potent suppressor of cardiomyocyte apoptosis triggered by multiple death-inducing stimuli via phosphorylation of HSF1 and subsequent induction of iHSP70, marking both CaMKII-delta isoforms as promising therapeutic targets for the treatment of ischemic heart disease.
Project description:: Although adipose-derived stem cells (ASCs) hold the promise of effective therapy for myocardial infarction, low cardiac retention of implanted ASCs has hindered their therapeutic efficiency. We investigated whether an externally applied static magnetic field (SMF) enhances cardiac localization of "magnetic" cells and promotes heart function recovery when ASCs are preloaded with superparamagnetic iron oxide (SPIO) nanoparticles. The influence of SMF (0.1 Tesla) on the biological activities of SPIO-labeled ASCs (SPIOASCs) was investigated first. Fifty-six female rats with myocardial infarction underwent intramyocardial injection of cell culture medium (CCM) or male SPIOASCs with or without the subcutaneous implantable magnet (CCM-magnet or SPIOASC-magnet). Four weeks later, endothelial differentiation, angiogenic cytokine secretion, angiogenesis, cardiomyocyte apoptosis, cell retention, and cardiac performance were examined. The 0.1-Tsela SMF did not adversely affect the viability, proliferation, angiogenic cytokine secretion, and DNA integrity of SPIOASCs. The implanted SPIOASCs could differentiate into endothelial cell, incorporate into newly formed vessels, and secrete multiple angiogenic cytokines. Four weeks after cell transplantation, the number of cardiac SPIOASCs was significantly increased, vascular density was markedly enlarged, fewer apoptotic cardiomyocytes were present, and heart contractile function was substantially improved in the SPIOASC-magnet treated rats in comparison with the SPIOASC-treated rats. The SPIOASCs could differentiate into endothelial cells, incorporate into vessels, promote angiogenesis, and inhibit ischemic cardiomyocyte apoptosis. An externally applied SMF offered a secure environment for biological properties of SPIOASCs, increased the cardiac retention of implanted magnetic SPIOASCs, and further enhanced heart function recovery after myocardial infarction.This pilot proof-of-concept study suggests that a 0.1-Tesla static magnetic field does not adversely affect the viability, proliferation, angiogenic cytokine secretion, or DNA integrity of the superparamagnetic iron oxide-labeled adipose-derived stem cells (SPIOASCs). Implantation of adipose-derived stem cells promotes myocardial neovascularization and inhibits ischemic cardiomyocyte apoptosis through endothelial differentiation, incorporation into vessels, and paracrine factor secretion. An externally applied static magnetic field enhanced myocardial retention of intramyocardially injected "magnetic" SPIOASCs and promoted cardiac function recovery after myocardial infarction. With further preclinical optimization, this approach may improve the outcome of current stem cell therapy for ischemic myocardial infarction.
Project description:Pigment epithelium-derived factor (PEDF) is a pleiotropic gene with anti-inflammatory, antioxidant and anti-angiogenic properties. However, recent reports about the effects of PEDF on cardiomyocytes are controversial, and it is not known whether and how PEDF acts to inhibit hypoxic or ischemic endothelial injury in the heart. In the present study, adult Sprague-Dawley rat models of acute myocardial infarction (AMI) were surgically established. PEDF-small interfering RNA (siRNA)-lentivirus (PEDF-RNAi-LV) or PEDF-LV was delivered into the myocardium along the infarct border to knockdown or overexpress PEDF, respectively. Vascular permeability, cardiomyocyte apoptosis, myocardial infarct size and animal cardiac function were analyzed. We also evaluated PEDF's effect on the suppression of the endothelial permeability and cardiomyocyte apoptosis under hypoxia in vitro. The results indicated that PEDF significantly suppressed the vascular permeability and inhibited hypoxia-induced endothelial permeability through PPAR?-dependent tight junction (TJ) production. PEDF protected cardiomyocytes against ischemia or hypoxia-induced cell apoptosis both in vivo and in vitro via preventing the activation of caspase-3. We also found that PEDF significantly reduced myocardial infarct size and enhanced cardiac function in rats with AMI. These data suggest that PEDF could protect cardiac function from ischemic injury, at least by means of reducing vascular permeability, cardiomyocyte apoptosis and myocardial infarct size.
Project description:Tilianin is a naturally occurring phenolic compound with a cardioprotective effect against myocardial ischemia/reperfusion injury (MIRI). The aim of our study was to determine the potential targets and mechanism of action of tilianin against cardiac injury induced by MIRI. An in silico docking model was used in this study for binding mode analysis between tilianin and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Oxygen-glucose deprivation/reperfusion- (OGD/R-) injured H9c2 cardiomyocytes and ischemia/reperfusion- (I/R-) injured isolated rat hearts were developed as in vitro and ex vivo models, respectively, which were both treated with tilianin in the absence or presence of a specific CaMKII inhibitor KN93 for target verification and mechanistic exploration. Results demonstrated the ability of tilianin to facilitater the recovery of OGD/R-induced cardiomyocyte injury and the maintenance of cardiac function in I/R-injured hearts. Tilianin interacted with CaMKII? with an efficient binding performance, a favorable binding score, and restraining p-CaMKII and ox-CaMKII expression in cardiomyocytes injured by MIRI. Importantly, inhibition of CaMKII abolished tilianin-mediated recovery of OGD/R-induced cardiomyocyte injury and maintenance of cardiac function in I/R-injured hearts, accompanied by the disability to protect mitochondrial function. Furthermore, the protective effects of tilianin towards mitochondrion-associated proapoptotic and antiapoptotic protein counterbalance and c-Jun N-terminal kinase (JNK)/nuclear factor- (NF-) ?B-related inflammation suppression were both abolished after pharmacological inhibition of CaMKII. Our investigation indicated that the inhibition of CaMKII-mediated mitochondrial apoptosis and JNK/NF-?B inflammation might be considered as a pivotal mechanism used by tilianin to exert its protective effects on MIRI cardiac damage.
Project description:Recent studies have shown that several upstream signaling elements of apoptosis and necroptosis are closely associated with acute injury in the heart. In our study, we observed that miR-105 was notably dysregulated in rat hearts with myocardial infarction (MI). Thus, the purpose of this study was to test the hypothesis that miR-105 participates in the regulation of RIP3/p-MLKL- and BNIP3-dependent necroptosis/apoptosis in H9c2 cells and MI rat hearts. Our results show that the RIP3/p-MLKL necroptotic pathway and BNIP3-dependent apoptosis signaling are enhanced in H9c2 cells under hypoxic conditions, whereas, compared with these pathways in the controls, those in miR-105-treated H9c2 cells are suppressed. Mechanistically, we identified miR-105 as the miRNA directly suppressing the expression of RIP3 and BNIP3, two important mediators involved in cell necroptosis and apoptosis. Furthermore, MI rat hearts injected with miR-105 had decreased infarct sizes, indicating that miR-105 is among three miRNAs that function simultaneously to suppress necroptotic/apoptotic cell death pathways and to inhibit MI-induced cardiomyocyte cell death at multiple levels. Taken together, miR-105 may constitute a new therapeutic strategy for cardioprotection in ischemic heart disease.
Project description:Ischemic cardiovascular disease represents one of the largest epidemics currently facing the aging population. Current literature has illustrated the efficacy of autologous, stem cell therapies as novel strategies for treating these disorders. The CD34+ hematopoetic stem cell has shown significant promise in addressing myocardial ischemia by promoting angiogenesis that helps preserve the functionality of ischemic myocardium. Unfortunately, both viability and angiogenic quality of autologous CD34+ cells decline with advanced age and diminished cardiovascular health.To offset age- and health-related angiogenic declines in CD34+ cells, we explored whether the therapeutic efficacy of human CD34+ cells could be enhanced by augmenting their secretion of the known angiogenic factor, sonic hedgehog (Shh).When injected into the border zone of mice after acute myocardial infarction, Shh-modified CD34+ cells (CD34(Shh)) protected against ventricular dilation and cardiac functional declines associated with acute myocardial infarction. Treatment with CD34(Shh) also reduced infarct size and increased border zone capillary density compared with unmodified CD34 cells or cells transfected with the empty vector. CD34(Shh) primarily store and secrete Shh protein in exosomes and this storage process appears to be cell-type specific. In vitro analysis of exosomes derived from CD34(Shh) revealed that (1) exosomes transfer Shh protein to other cell types, and (2) exosomal transfer of functional Shh elicits induction of the canonical Shh signaling pathway in recipient cells.Exosome-mediated delivery of Shh to ischemic myocardium represents a major mechanism explaining the observed preservation of cardiac function in mice treated with CD34(Shh) cells.
Project description:Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA-/- mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.