CTRP9 protein protects against myocardial injury following ischemia-reperfusion through AMP-activated protein kinase (AMPK)-dependent mechanism

Ischemic heart disease is the major cause of death in Western countries. CTRP9 (C1q/TNF-related protein 9) is a fat-derived plasma protein that has salutary effects on glucose metabolism and vascular function. However, the functional role of CTRP9 in ischemic heart disease has not been clarified. Here, we examined the regulation of CTRP9 in response to acute cardiac injury and investigated whether CTRP9 modulates cardiac damage after ischemia and reperfusion. Myocardial ischemia-reperfusion injury resulted in reduced plasma CTRP9 levels and increased plasma free fatty acid levels, which were accompanied by a decrease in CTRP9 expression and an increase in NADPH oxidase component expression in fat tissue. Treatment of cultured adipocytes with palmitic acid or hydrogen peroxide reduced CTRP9 expression. Systemic administration of CTRP9 to wild-type mice, before the induction of ischemia or at the time of reperfusion, led to a reduction in myocardial infarct size following ischemia-reperfusion. Administration of CTRP9 also attenuated myocyte apoptosis in ischemic heart, which was accompanied by increased phosphorylation of AMP-activated protein kinase (AMPK). Treatment of cardiac myocytes with CTRP9 protein reduced apoptosis in response to hypoxia/reoxygenation and stimulated AMPK phosphorylation. Blockade of AMPK activity reversed the suppressive actions of CTRP9 on cardiomyocyte apoptosis. Knockdown of adiponectin receptor 1 diminished CTRP9-induced increases in AMPK phosphorylation and survival of cardiac myocytes. Our data suggest that CTRP9 protects against acute cardiac injury following ischemia-reperfusion via an AMPK-dependent mechanism.     

GH-releasing peptides improve cardiac dysfunction and cachexia and suppress stress-related hormones and cardiomyocyte apoptosis in rats with heart failure

Growth hormone (GH)-releasing peptides (GHRP), a class of synthetic peptidyl GH secretagogues, have been reported to exert a cardioprotective effect on cardiac ischemia. However, whether GHRP have a beneficial effect on chronic heart failure (CHF) is unclear, and the present work aims to clarify this issue. At 9 wk after pressure-overload CHF was created by abdominal aortic banding in rats, one of four variants of GHRP (GHRP-1, -2, and -6 and hexarelin, 100 mug/kg) or saline was injected subcutaneously twice a day for 3 wk. Echocardiography and cardiac catheterization were performed to monitor cardiac function and obtain blood samples for hormone assay. GHRP treatment significantly improved left ventricular (LV) function and remodeling in CHF rats, as indicated by increased LV ejection fraction, LV end-systolic pressure, and diastolic posterior wall thickness and decreased LV end-diastolic pressure and LV end-diastolic dimension. GHRP also significantly alleviated development of cardiac cachexia, as shown by increases in body weight and tibial length in CHF rats. Plasma CA, renin, ANG II, aldosterone, endothelin-1, and atrial natriuretic peptide were significantly elevated in CHF rats but were significantly decreased in GHRP-treated CHF rats. GHRP suppressed cardiomyocyte apoptosis and increased cardiac GH secretagogue receptor mRNA expression in CHF rats. GHRP also decreased myocardial creatine kinase release in hypophysectomized rats subjected to acute myocardial ischemia. We conclude that chronic administration of GHRP alleviates LV dysfunction, pathological remodeling, and cardiac cachexia in CHF rats, at least in part by suppressing stress-induced neurohormonal activations and cardiomyocyte apoptosis.     

Tissue kallikrein infusion prevents cardiomyocyte apoptosis, inflammation and ventricular remodeling after myocardial infarction

We investigated the effect of tissue kallikrein infusion on cardiac protection at acute and sub-acute phases after myocardial infarction (MI). Immediately after MI, rats were infused with purified tissue kallikrein, with or without icatibant (a kinin B2 receptor antagonist). Intramyocardial injection of kallikrein reduced myocardial infarct size and inhibited cardiomyocyte apoptosis at 1 day after MI associated with increased nitric oxide levels, Akt and glycogen synthase kinase-3beta phosphorylation and decreased caspase-3 activation. Kallikrein infusion for 7 days improved cardiac function, normalized left ventricular wall thickness and decreased monocyte/macrophage infiltration in the infarct heart. Kallikrein treatment reduced NADH oxidase expression and activity, superoxide formation and malondialdehyde levels, and reduced MAPK and Ikappa-Balpha phosphorylation, NF-kappaB activation and MCP-1 and VCAM-1 expression. Kallikrein's effects were all blocked by icatibant. These results indicate that kallikrein through kinin B2 receptor activation prevents apoptosis, inflammation and ventricular remodeling by increased nitric oxide formation and suppression of oxidative stress-mediated signaling pathways.     

Antioxidant metallothionein alleviates endoplasmic reticulum stress-induced myocardial apoptosis and contractile dysfunction

Abstract

Aims. Endoplasmic reticulum (ER) stress exerts myocardial oxidative stress, apoptosis, and contractile anomalies, although the precise interplay between ER stress and apoptosis remains elusive. This study was designed to examine the impact of the cysteine-rich free radical scavenger metallothionein on ER stress-induced myocardial contractile defect and underlying mechanisms. Methods and results. Wild-type friendly virus B and transgenic mice with cardiac-specific overexpression of metallothionein were challenged with the ER stress inducer tunicamycin (1 mg/kg, intraperitoneal, 48 h) prior to the assessment of myocardial function, oxidative stress, and apoptosis. Our results revealed that tunicamycin promoted cardiac remodeling (enlarged left ventricular end systolic/diastolic diameters with little changes in left ventricular wall thickness), suppressed fractional shortening and cardiomyocyte contractile function, elevated resting Ca²⁺, decreased stimulated Ca²⁺ release, prolonged intracellular Ca²⁺ clearance, and downregulated sarco(endo)plasmic reticulum Ca²⁺-ATPase levels, the effects of which were negated by metallothionein. Treatment with tunicamycin caused cardiomyocyte mitochondrial injury, as evidenced by decreased mitochondrial membrane potential (??m, assessed by JC-1 staining), the effect of which was negated by the antioxidant. Moreover, tunicamycin challenge dramatically facilitated myocardial apoptosis as manifested by increased Bax, caspase 9, and caspase 12 protein levels, as well as elevated caspase 3 activity. Interestingly, metallothionein transgene significantly alleviated tunicamycin-induced myocardial apoptosis. Conclusion. Taken together, our data favor a beneficial effect of metallothionein against ER stress-induced cardiac dysfunction possibly associated with attenuation of myocardial apoptosis.     

Interfering with long chain noncoding RNA ANRIL expression reduces heart failure in rats with diabetes by inhibiting myocardial oxidative stress

This study is performed to elucidate whether long-chain noncoding RNA ANRIL has an effect on diabetes, and further explore the mechanism of ANRIL in diabetes. The rat model of diabetes was established via intraperitoneal injection of streptozotocin. The modeled rats were grouped into normal, diabetes, siRNA-NC, and ANRIL siRNA groups. Besides, the expression of ANRIL, cardiac function, inflammatory factor levels, cardiomyocyte apoptosis, and levels of oxidative stress index were all determined. Upregulated ANRIL was found in myocardial tissue of diabetic rats. Downregulated ANRIL improved cardiac function index and the expression of inflammatory factors, improved the pathological state of myocardial tissue and myocardial remodeling, decreased myocardial collagen deposition area and cardiomyocyte apoptosis and reduced the oxidative level of myocardial tissue in diabetic rats. This present study suggests that upregulated ANRIL is found in myocardial tissue of diabetic rats. Additionally, silencing of ANRIL reduces myocardial injury in diabetes by inhibiting myocardial oxidative stress.     

Adiponectin ameliorates hyperglycemia-induced cardiac hypertrophy and dysfunction by concomitantly activating Nrf2 and Brg1

Hyperglycemia-induced oxidative stress is implicated in the development of cardiomyopathy in diabetes that is associated with reduced adiponectin (APN) and heme oxygenase-1 (HO-1). Brahma-related gene 1 (Brg1) assists nuclear factor-erythroid-2-related factor-2 (Nrf2) to activate HO-1 to increase myocardial antioxidant capacity in response to oxidative stress. We hypothesized that reduced adiponectin (APN) impairs HO-1 induction which contributes to the development of diabetic cardiomyopathy, and that supplementation of APN may ameliorate diabetic cardiomyopathy by activating HO-1 through Nrf2 and Brg1 in diabetes. Control (C) and streptozotocin-induced diabetic (D) rats were untreated or treated with APN adenovirus (1×10⁹ pfu) 3 weeks after diabetes induction and examined and terminated 1 week afterward. Rat left ventricular functions were assessed by a pressure–volume conductance system, before the rat hearts were removed to perform histological and biochemical assays. Four weeks after diabetes induction, D rats developed cardiac hypertrophy evidenced as increased ratio of heart weight to body weight, elevated myocardial collagen I content, and larger cardiomyocyte cross-sectional area (all P<0.05 vs C). Diabetes elevated cardiac oxidative stress (increased 15-F_2t-isoprostane, 4-hydroxynonenal generation, 8-hydroxy-2′-deoxyguanosine, and superoxide anion generation), increased myocardial apoptosis, and impaired cardiac function (all P<0.05 vs C). In D rats, myocardial HO-1 mRNA and protein expression were reduced which was associated with reduced Brg1 and nuclear Nrf2 protein expression. All these changes were either attenuated or prevented by APN. In primarily cultured cardiomyocytes (CMs) isolated from D rats or in the embryonic rat cardiomyocytes cell line H9C2 cells incubated with high glucose (HG, 25 mM), supplementation of recombined globular APN (gAd, 2 μg/mL) reversed HG-induced reductions of HO-1, Brg1, and nuclear Nrf2 protein expression and attenuated cellular oxidative stress, myocyte size, and apoptotic cells. Inhibition of HO-1 by ZnPP (10 μM) or small interfering RNA (siRNA) canceled all the above gAd beneficial effects. Moreover, inhibition of Nrf2 (either by the Nrf2 inhibitor luteolin or siRNA) or Brg1 (by siRNA) canceled gAd-induced HO-1 induction and cellular protection in CMs and in H9C2 cells incubated with HG. In summary, our present study demonstrated that APN reduced cardiac oxidative stress, ameliorated cardiomyocyte hypertrophy, and prevented left ventricular dysfunction in diabetes by concomitantly activating Nrf2 and Brg1 to facilitate HO-1 induction.     

Luteolin limits infarct size and improves cardiac function after myocardium ischemia/reperfusion injury in diabetic rats

Background

The present study was to investigate the effects and mechanism of Luteolin on myocardial infarct size, cardiac function and cardiomyocyte apoptosis in diabetic rats with myocardial ischemia/reperfusion (I/R) injury.

Methodology/Principal Findings

Diabetic rats underwent 30 minutes of ischemia followed by 3 h of reperfusion. Animals were pretreated with or without Luteolin before coronary artery ligation. The severity of myocardial I/R induced LDH release, arrhythmia, infarct size, cardiac function impairment, cardiomyocyte apoptosis were compared. Western blot analysis was performed to elucidate the target proteins of Luteolin. The inflammatory cytokine production were also examined in ischemic myocardium underwent I/R injury. Our results revealed that Luteolin administration significantly reduced LDH release, decreased the incidence of arrhythmia, attenuated myocardial infarct size, enhanced left ventricular ejection fraction and decreased myocardial apoptotic death compared with I/R group. Western blot analysis showed that Luteolin treatment up-regulated anti-apoptotic proteins FGFR2 and LIF expression, increased BAD phosphorylation while decreased the ratio of Bax to Bcl-2. Luteolin treatment also inhibited MPO expression and inflammatory cytokine production including IL-6, IL-1a and TNF-a. Moreover, co-administration of wortmannin and Luteolin abolished the beneficial effects of Luteolin.

Conclusions/Significance

This study indicates that Luteolin preserves cardiac function, reduces infarct size and cardiomyocyte apoptotic rate after I/R injury in diabetic rats. Luteolin exerts its action by up-regulating of anti-apoptotic proteins FGFR2 and LIF expression, activating PI3K/Akt pathway while increasing BAD phosphorylation and decreasing ratio of Bax to Bcl-2.     

Deficiency in Beclin1 attenuates alcohol-induced cardiac dysfunction via inhibition of ferroptosis

BackgroundBinge drinking leads to compromised mitochondrial integrity and contractile function in the heart although little effective remedy is readily available. Given the possible derangement of autophagy in ethanol-induced cardiac anomalies, this study was designed to examine involvement of Beclin1 in acute ethanol-induced cardiac contractile dysfunction, in any, and the impact of Beclin1 haploinsufficiency on ethanol cardiotoxicity with a focus on autophagy-related ferroptosis.MethodsWT and Beclin1 haploinsufficiency (BECN^+/?) mice were challenged with ethanol for one week (2 g/kg, i.p. on day 1, 3 and 7) prior to assessment of cardiac injury markers (LDH, CK-MB), cardiac geometry, contractile and mitochondrial integrity, oxidative stress, lipid peroxidation, apoptosis and ferroptosis.ResultsEthanol exposure compromised cardiac geometry and contractile function accompanied with upregulated Beclin1 and autophagy, mitochondrial injury, oxidative stress, lipid peroxidation and apoptosis, and ferroptosis (GPx4, SLC7A11, NCOA4). Although Beclin1 deficiency did not affect cardiac function in the absence of ethanol challenge, it alleviated ethanol-induced changes in cardiac injury biomarkers, cardiomyocyte area, interstitial fibrosis, echocardiographic and cardiomyocyte mechanical properties along with mitochondrial integrity, oxidative stress, lipid peroxidation, apoptosis and ferroptosis. Ethanol challenge evoked pronounced ferroptosis (downregulated GPx4, SLC7A11 and elevated NCOA4, lipid peroxidation), the effect was alleviated by Beclin1 haploinsufficiency. Inhibition of ferroptosis using LIP-1 rescued ethanol-induced cardiac mechanical anomalies. In vitro study noted that ferroptosis induction using erastin abrogated Beclin1 haploinsufficiency-induced response against ethanol.ConclusionsIn sum, our data suggest that Beclin1 haploinsufficiency benefits acute ethanol challenge-induced myocardial remodeling and contractile dysfunction through ferroptosis-mediated manner.     

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX’s cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury.     

The Cardioprotective Effect of Vitamin E (Alpha-Tocopherol) Is Strongly Related to Age and Gender in Mice

Vitamin E (VitE) only prevented cardiovascular diseases in some patients and the mechanisms remain unknown. VitE levels can be affected by aging and gender. We hypothesize that age and gender can influence VitE’s cardioprotective effect. Mice were divided into 4 groups according to age and gender, and each group of mice were divided into a control group and a VitE group. The mice were administered water or VitE for 21 days; Afterward, the cardiac function and myocardial infarct size and cardiomyocyte apoptosis were measured after myocardial ischemia reperfusion(MI/R). VitE may significantly improved cardiac function in young male mice and aged female mice by enhancing ERK1/2 activity and reducing JNK activity. Enhanced expression of HSP90 and Bcl-2 were also seen in young male mice. No changes in cardiac function and cardiac proteins were detected in aged male mice and VitE was even liked to exert a reverse effect in cardiac function in young mice by enhancing JNK activity and reducing Bcl-2 expression. Those effects were in accordance with the changes of myocardial infarction size and cardiomyocyte apoptosis in each group of mice. VitE may reduce MI/R injury by inhibiting cardiomyocyte apoptosis in young male mice and aged female mice but not in aged male mice. VitE was possibly harmful for young female mice, shown as increased cardiomyocyte apoptosis after MI/R. Thus, we speculated that the efficacy of VitE in cardiac protection was associated with age and gender.     

Downregulation of p300/CBP-associated factor inhibits cardiomyocyte apoptosis via suppression of NF-κB pathway in ischaemia/reperfusion injury rats

Cardiomyocyte apoptosis is the main reason of cardiac injury after myocardial ischaemia-reperfusion (I/R) injury (MIRI), but the role of p300/CBP-associated factor (PCAF) on myocardial apoptosis in MIRI is unknown. The aim of this study was to investigate the main mechanism of PCAF modulating cardiomyocyte apoptosis in MIRI. The MIRI model was constructed by ligation of the rat left anterior descending coronary vessel for 30 min and reperfusion for 24 h in vivo. H9c2 cells were harvested after induced by hypoxia for 6 h and then reoxygenation for 24 h (H/R) in vitro. The RNA interference PCAF expression adenovirus was transfected into rat myocardium and H9c2 cells. The area of myocardial infarction, cardiac function, myocardial injury marker levels, apoptosis, inflammation and oxidative stress were detected respectively. Both I/R and H/R remarkably upregulated the expression of PCAF, and downregulation of PCAF significantly attenuated myocardial apoptosis, inflammation and oxidative stress caused by I/R and H/R. In addition, downregulation of PCAF inhibited the activation of NF-κB signalling pathway in cardiomyocytes undergoing H/R. Pretreatment of lipopolysaccharide, a NF-κB pathway activator, could blunt these protective effects of PCAF downregulation on myocardial apoptosis in MIRI. These results highlight that downregulation of PCAF could reduce cardiomyocyte apoptosis by inhibiting the NF-κB pathway, thereby providing protection for MIRI. Therefore, PCAF might be a promising target for protecting against cardiac dysfunction induced by MIRI.     

The β subunit of soluble guanylyl cyclase GUCY1B3 exerts cardioprotective effects against ischemic injury via the PKCε/Akt pathway

The soluble form of guanylyl cyclase (sGC) is the main receptor for the signaling agent nitric oxide (NO), which regulates cardiomyocyte contractile function and attenuates cardiomyocyte hypertrophy. sGC catalyzes the formation of cyclic guanosine monophosphate (cGMP), a regulator of vascular tone, and cardiac NO-sGC-cGMP signaling modulates cardiac stress responses, including ischemia and reperfusion (IR) injury. Here, we investigated the role of GUCY1B3 (the β subunit of sGC) in cardiomyocyte IR injury and myocardial infarction (MI) in vitro and in vivo. GUCY1B3 was upregulated in neonatal rat ventricular myocytes in response to IR injury, and GUCY1B3 overexpression restored IR-induced cell death and apoptosis. Treatment with specific inhibitors of PKCδ, PKCε, and Akt suggested that the protective effects of GUCY1B3 were mediated by PKCε/Akt signaling. In a mouse model of coronary artery ligation-induced MI, GUCY1B3 silencing aggravated MI-induced cardiac dysfunction and increased infarct size and exacerbated cardiomyocyte apoptosis in association with the inactivation of PKCε and Akt. Our results suggest that GUCY1B3 exerts cardioprotective effects through the modulation of the PKCε/Akt activity and identify a potential mechanism involved in NO-sGC-cGMP signaling in the heart.     

Activation of catalase by apelin prevents oxidative stress-linked cardiac hypertrophy

Adipose tissue secretes a variety of bioactive factors, which can regulate cardiomyocyte hypertrophy via reactive oxygen species (ROS). In the present study we investigated whether apelin affects ROS-dependent cardiac hypertrophy. In cardiomyocytes apelin inhibited the hypertrophic response to 5-HT and oxidative stress induced by 5-HT- or H₂O₂ in a dose-dependent manner. These effects were concomitant to the increase in mRNA expression and activity of catalase. Chronic treatment of mice with apelin attenuated pressure-overload-induced left ventricular hypertrophy. The prevention of hypertrophy by apelin was associated with increased myocardial catalase activity and decreased plasma lipid hydroperoxide, as an index of oxidative stress. These results show that apelin behaves as a catalase activator and prevents cardiac ROS-dependent hypertrophy.     

Differential role of kinin B1 and B2 receptors in ischemia-induced apoptosis and ventricular remodeling

We investigated the role of kinin receptors in cardiac remodeling after ischemia/reperfusion (I/R). Bradykinin injection improved cardiac contractility, diastolic function, reduced infarct size and prevented left ventricular thinning after I/R, whereas des-Arg(9)-BK injection had no protective effects. Bradykinin, but not des-Arg(9)-BK, reduced cardiomyocyte apoptosis and increased Akt and GSK-3beta phosphorylation. Furthermore, myocardial infarct size was similar between wild type and B2 knockout mice after I/R, but significantly reduced in kinin B1 receptor knockout mice. These results indicate that the kinin B2 receptor, but not the B1 receptor, protects against I/R-induced cardiac dysfunction by inhibiting apoptosis and limiting ventricular remodeling.     

Early moderate exercise benefits myocardial infarction healing via improvement of inflammation and ventricular remodelling in rats

Thus far, the cellular and molecular mechanisms related to early (especially within 24 hours after acute myocardial infarct (MI)) exercise‐mediated beneficial effects on MI have not yet been thoroughly established. In the present study, we demonstrated that acute MI rats that underwent early moderate exercise training beginning one day after MI showed no increase in mortality and displayed significant improvements in MI healing and ventricular remodelling, including an improvement in cardiac function, a decrease in infarct size, cardiomyocyte apoptosis, cardiac fibrosis and cardiomyocyte hypertrophy, and an increase in myocardial angiogenesis, left ventricular wall thickness and the number of cardiac telocytes in the border zone. Integrated miRNA‐mRNA profiling analysis performed by the ingenuity pathway analysis system revealed that the inhibition of the TGFB1 regulatory network, activation of leucocytes and migration of leucocytes into the infarct zone comprise the molecular mechanism underlying early moderate exercise‐mediated improvements in cardiac fibrosis and the pathological inflammatory response. The findings of the present study demonstrate that early moderate exercise training beginning one day after MI is safe and leads to significantly enhanced MI healing and ventricular remodelling. Understanding the mechanism behind the positive effects of this early training protocol will help us to further tailor suitable cardiac rehabilitation programmes for humans.