Pathway In Cardiomyocytes Research Articles

Midkine's Role in Cardiac Pathology.

Midkine (MDK) is a heparin-binding growth factor that is normally expressed in mid-gestational development mediating mesenchymal and epithelial interactions. As organisms age, expression of MDK diminishes; however, in adults, MDK expression is associated with acute and chronic pathologic conditions such as myocardial infarction and heart failure (HF). The role of MDK is not clear in cardiovascular disease and currently there is no consensus if it plays a beneficial or detrimental role in HF. The lack of clarity in the literature is exacerbated by differing roles that circulating and myocardial MDK play in signaling pathways in cardiomyocytes (some of which have yet to be elucidated). Of particular interest, serum MDK is elevated in adults with chronic heart failure and higher circulating MDK is associated with worse cardiac function. In addition, pediatric HF patients have higher levels of myocardial MDK. This review focuses on what is known about the effect of exogenous versus myocardial MDK in various cardiac disease models in an effort to better clarify the role of midkine in HF.

\u03b21-adrenergic receptor O-glycosylation regulates N-terminal cleavage and signaling responses in cardiomyocytes

β1-adrenergic receptors (β1ARs) mediate catecholamine actions in cardiomyocytes by coupling to both Gs/cAMP-dependent and Gs-independent/growth-regulatory pathways. Structural studies of the β1AR define ligand-binding sites in the transmembrane helices and effector docking sites at the intracellular surface of the β1AR, but the extracellular N-terminus, which is a target for post-translational modifications, typically is ignored. This study identifies β1AR N-terminal O-glycosylation at Ser37/Ser41 as a mechanism that prevents β1AR N-terminal cleavage. We used an adenoviral overexpression strategy to show that both full-length/glycosylated β1ARs and N-terminally truncated glycosylation-defective β1ARs couple to cAMP and ERK-MAPK signaling pathways in cardiomyocytes. However, a glycosylation defect that results in N-terminal truncation stabilizes β1ARs in a conformation that is biased toward the cAMP pathway. The identification of O-glycosylation and N-terminal cleavage as novel structural determinants of β1AR responsiveness in cardiomyocytes could be exploited for therapeutic advantage.

Time course of changes in oxidative stress and stress-induced proteins in cardiomyocytes exposed to doxorubicin and prevention by vitamin C.

We previously reported that Vitamin C (Vit C) protects against doxorubicin (Dox)-induced cardiotoxicity by reducing oxidative stress, p38 mitogen-activated kinase (MAPK) and p53 activation and rescuing cell death in isolated adult cardiomyocytes. The pattern of activation and the role of oxidative stress as well as down-stream mechanisms for such protection remain elusive. Therefore the present study aims to analyze time-dependant generation of reactive oxygen species (ROS) and the activation of stress induced signalling pathways in cardiomyocytes treated with Dox and Vit C. The data provides further understanding of heart pathophysiology in response to Dox at the cellular level, and may help to optimize the timing of various therapeutic approaches. Cardiomyocytes isolated from adult Sprague-Dawley rats were exposed to Dox (10 μM), Vit C (25 μM), and Dox + Vit C for different time intervals up to 24 h. p38-JNK (SB203580) and p53 (pifithrin-α) inhibitors were used to determine the role of each respective signalling protein. Dox administration to cardiomyocytes increased the levels of ROS in a time-dependent manner that followed the activation of stress-induced proteins p53, p38 and JNK MAPKs, culminating in an increase in autophagy and apoptosis markers. Dox-induced increase in ROS was alleviated by Vit C adjuvant treatment at all time-points and this was also correlated with blunting of the activation of the studied signaling pathways leading to the prevention of apoptosis and preservation of cell viability. Protective effect of Vit C against the activation of stress induced proteins, autophagy and apoptosis was mainly attributed to its antioxidant properties even though blockage of p38, JNK and p53 by pharmacological inhibitors also suppressed Dox-induced apoptosis. ROS is defined as a key inducer of cardiomyocyte damage under Dox exposure; Vit C could effectively counteract all Dox-induced changes in cardiomyocytes and may potentially be used as an antioxidant adjuvant therapy to protect against Dox-induced cardiomyopathy.

Potential phytoestrogen alternatives exert cardio-protective mechanisms via estrogen receptors.

The 17 beta-estradiol (E2) is a sex hormone that is most abundant and most active estrogen in premenopausal women. The importance of E2 in providing cardioprotection and reducing the occurrence of heart disease in women of reproductive age has been well recognized. There are three subtype of estrogen receptors (ERs), including ERα, ERβ and GPR30 have been identified and accumulating evidence reveal their roles on E2-mediated genomic and nongenomic pathway in cardiomyocytes against various cardiac insults. In this review, we focus on the estrogen and ERs mediated signaling pathways in cardiomyocytes that determines cardio-protection against various stresses and further discuss the clinical implication of ERs and phytoestrogens. Further we provide some insights on phytoeostrogens which may play as alternatives in estrogen replacement therapies.

176 Serotonin receptor 2b (5-ht2b) modulates cardiomyocyte proliferation by regulating the hippo pathway

Heart failure is one of the leading causes of death worldwide. In part, this is due to the inadequate regenerative capacity of cardiomyocytes post-injury. Modulation of the Hippo signalling pathway...

Luhong formula inhibits myocardial fibrosis in a paracrine manner by activating the gp130/JAK2/STAT3 pathway in cardiomyocytes

Ethnopharmacological relevanceLuhong formula (LHF)—a traditional Chinese medicine containing Cervus nippon Temminck, Carthamus tinctorius L., Cinnamomum cassia Presl, Codonopisis pilosula( Franch.) Nannf., Astragalus membranaceus ( Fisch.) Bge. var. mongholicus ( Bge.) Hsiao, Lepidium apetalum Willd—is used in the treatment of heart failure. Aim of the studyTo investigate the antifibrotic efficacy of LHF in a myocardial infarction-induced rat model of heart failure and to determine its mechanism of action. Material and methodsMyocardial infarction was induced in rats by coronary artery ligation, and cardiac fibroblasts were isolated. Neonatal rat cardiomyocytes (NRCMs) were isolated from 2 to 3-day-old Sprague-Dawley male rats, and cardiomyocyte hypertrophy was induced by isoprenaline. Histological examination was carried out to estimate the degree of myocardial fibrosis. Expression of gp130/JAK2/STAT3 pathway proteins was measured by western blot. The mRNA levels of downstream genes of gp130/JAK2/STAT3 pathway (i.e., CTGF, TSP-1, and TIMP1) were determined by RT-PCR; while CTGF, TSP-1, and TIMP1 protein levels were measured by ELISA. To investigate paracrine effects, cell proliferation and collagen synthesis was measured after treating cardiac fibroblasts with the conditioned media from isoprenaline-treated NRCMs. ResultsHistopathological changes showed that LHF inhibited myocardial fibrosis in heart failure rats. Treatment with LHF up-regulated gp130, JAK2, and STAT3 protein expression in heart tissue, and down-regulated CTGF, TSP-1, and TIMP1 gene expression. Isoprenaline-treated NRCMs displayed lower expression of the gp130, JAK2, and STAT3 pathway proteins and higher secretion of its downstream signaling molecules (CTGF, TSP-1, TIMP1). LHF inhibited cardiac fibroblast proliferation and collagen synthesis after treatment with the conditioned media from isoprenaline-treated NRCMs. ConclusionLHF treatment attenuates myocardial fibrosis in vivo. LHF inhibits cardiac fibroblasts proliferation and collagen synthesis in a paracrine manner by activating the gp130/JAK2/STAT3 pathway in cardiomyocytes, thereby inhibiting the secretion of downstream profibrogenic cytokines.

Effects of Trimetazidine on PDCD4/NF-κB/TNF-α Pathway in Coronary Microembolization

Background/Aims: The local inflammatory response caused by coronary microembolization (CME) is the primary cause of progressive cardiac dysfunction. The PDCD4/NF-κB/TNF-α signaling pathway plays a significant role in CME-induced myocardial Inflammation. Trimetazidine (TMZ) reduces myocardial injury, caused by percutaneous coronary intervention, through relieving the CME-induced myocardial systolic dysfunction. Therefore, the present study investigated the role of TMZ pre-treatment in the protection of myocardium after CME and PDCD4/NF-κB/TNF-α in mini pigs. Methods: 20 Bama mini pigs were randomized into sham operation (sham), microembolization (CME), TMZ, and siRNA-PDCD4 groups (n = 5). The CME model was established by injecting polyethylene microspheres via microcatheter into the left anterior descending coronary artery. The TMZ group was injected 2.5 mg/kg drug via ear vein 30 min before CME; whereas, the siRNA-PDCD4 group was transfected with PDCD4 siRNA at the left anterior descending coronary artery via microcatheter 72h before CME. Cardiac function indexes were measured using cardiac echocardiography. The mRNA expression of PDCD4 and TNF-α in the myocardium was detected by quantitative fluorescence PCR, and the protein expression of PDCD4, NF-κB (p65), and TNF-α by Western blot. Results: Echocardiographic parameters showed lower cardiac function and higher serum cTnI level in the CME group than sham, which was manifested as reduced left ventricular ejection fraction (LVEF), left ventricular fractional shortening (FS), cardiac output (CO), and increased left ventricular diastolic diameter (LVEDd). Compared to the CME group, the CME-induced cardiac function injury was reduced, and the serum cTnI level was decreased in the TMZ and siRNA-PDCD4 groups. The expressions of PDCD4, NF-κB (p65), and TNF-α were significantly increased in the CME than the sham groups (P < 0.05), and significantly decreased in the TMZ and siRNA-PDCD4 groups than the CME group (P < 0.05). Conclusion: TMZ pretreatment effectively reduced the myocardial damage caused by CME via inhibiting the PDCD4/NF-κB/ TNF-α pathway in cardiomyocytes.

Melatonin-Induced Protective Effects on Cardiomyocytes Against Reperfusion Injury Partly Through Modulation of IP3R and SERCA2a Via Activation of ERK1.

BackgroundMelatonin is a neuroendocrine hormone synthesized primarily by the pineal gland that is indicated to effectively prevent myocardial reperfusion injury. It is unclear whether melatonin protects cardiac function from reperfusion injury by modulating intracellular calcium homeostasis.ObjectiveDemonstrate that melatonin protect against myocardial reperfusion injury through modulating IP3R and SERCA2a to maintain calcium homeostasis via activation of ERK1 in cardiomyocytes.MethodsIn vitro experiments were performed using H9C2 cells undergoing simulative hypoxia/reoxygenation (H/R) induction. Expression level of ERK1, IP3R and SERCA2a were assessed by Western Blots. Cardiomyocytes apoptosis was detected by TUNEL. Phalloidin-staining was used to assess alteration of actin filament organization of cardiomyocytes. Fura-2 /AM was used to measure intracellular Ca2+ concentration. Performing in vivo experiments, myocardial expression of IP3R and SERCA2a were detected by immunofluorescence staining using myocardial ischemia/ reperfusion (I/R) model in rats.ResultsIn vitro results showed that melatonin induces ERK1 activation in cardiomyocytes against H/R which was inhibited by PD98059 (ERK1 inhibitor). The results showed melatonin inhibit apoptosis of cardiomyocytes and improve actin filament organization in cardiomyocytes against H/R, because both could be reversed by PD98059. Melatonin was showed to reduce calcium overload, further to inhibit IP3R expression and promote SERCA2a expression via ERK1 pathway in cardiomyocytes against H/R. Melatonin induced lower IP3R and higher SERCA2a expression in myocardium that were reversed by PD98059.Conclusionmelatonin-induced cardioprotection against reperfusion injury is at least partly through modulation of IP3R and SERCA2a to maintain intracellular calcium homeostasis via activation of ERK1.

Role of carvacrol in cardioprotection against myocardial ischemia/reperfusion injury in rats through activation of MAPK/ERK and Akt/eNOS signaling pathways

Carvacrol (CAR) is a compound isolated from some essential oils, many studies have demonstrated its therapeutic potential on different diseases. This study aims to evaluate the protective effect of CAR against myocardial ischemia/reperfusion (I/R) injury in rats. Male adult rats underwent ligation of the left anterior descending coronary artery (LAD) in I/R models. Rats were treated with CAR after LAD. The levels of I/R- induced infarct size, cardiomyocyte apoptosis and cardiac functional impairment were examined. Levels of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) were detected by western blotting. Cardiomyocytes induced by hypoxic reperfusion (H/R) injury were tested by Hoechst 33258. Our results revealed that CAR administration significantly protected the heart function, attenuated myocardial infarct size, increased SOD and CAT levels, reduced MDA level and especially decreased cardiomyocytes apoptosis. Western blotting showed that CAR treatment up-regulated phosphorylated ERK (p-ERK), while producing no impact onp38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK). The cardioprotection of CAR was reversed by the ERK inhibitor PD-98059, demonstrating the involvement of the MAPK/ERK pathway in the anti-apoptotic mechanisms of CAR. Besides, the results in vitro also showed the protective efficiency of CAR on cardiomyocytes H/R injury. Furthermore, pretreatment with CAR markedly increased the activation of Akt/eNOS pathway in cardiomyocytes subjected to H/R, and the protective effects of CAR were abolished in the presence of the Akt inhibitor LY294002. Therefore, the cardioprotective effects of CAR may be attributed to its antioxidant and antiapoptotic activities through activations of the MAPK/ERK and Akt/eNOS signaling pathways.

Abstract 12941: MURC/Cavin-4 Deficiency Reduces Infarct Size and Preserves Cardiac Function in a Mouse Model of Ischemia-Reperfusion Injury

Introduction: Cardiac ischemia-reperfusion (I/R) injury is considered clinically important issue in cardioprotective strategy for ischemic heart disease. Muscle-restricted coiled-coil protein (MURC)/Cavin-4 is a member of the cavin family, which is a component of caveola. MURC/Cavin-4 is involved in myofibrillar organization by regulating Rho/ROCK signaling pathway and cardiac hypertrophy through ERK1/2 activation. MURC/Cavin-4 mutations are observed in patients with dilated cardiomyopathy. However, the role of MURC/Cavin-4 in cardiac I/R injury remains unknown. Methods and Results: Cardiac I/R was performed on MURC/Cavin-4-/- mice and WT mice. Mice were subjected to 1 hour ischemia by ligating left anterior descending artery, followed by 24 hours reperfusion. MURC/Cavin-4 mRNA expression was continuously increased after reperfusion in WT mouse hearts. Compared with WT mice, MURC/Cavin-4-/- mice exhibited significantly smaller infarct size (IS), which was assessed as proportion of the area at risk (AAR) (IS/AAR[%], MURC/Cavin-4-/- vs. WT: 52.7 ± 3.1 vs. 69.8 ± 12.3, p &lt; 0.05). Echocardiography showed that MURC/Cavin-4 deficiency significantly preserved cardiac function (LVEF[%], MURC/Cavin-4-/- vs. WT: 54.2 ± 5.6 vs. 36.2 ± 2.0, p &lt; 0.01; FS[%], MURC/Cavin-4-/- vs. WT: 27.0 ± 3.7 vs. 16.9 ± 1.1, p &lt; 0.01, respectively). Hydrogen peroxide (H 2 O 2 ), the most abundant form of reactive oxygen species (ROS) produced during I/R, causes inflammation, apoptosis and subsequent tissue damages. In neonatal rat cardiomyocytes, MURC/Cavin-4 siRNA reduced cell death induced by H 2 O 2 compared with control siRNA, as determined by TUNEL staining (TUNEL positive cells[%],MURC/Cavin-4 siRNA vs. control siRNA: 45.1 ± 7.2 vs. 65.7 ± 12.1, p &lt; 0.05). Western blot analysis showed that p38 MAPK activation by H 2 O 2 was inhibited in cardiomyocytes transfected with MURC/Cavin-4 siRNA compared with control siRNA. Akt/PKB signaling was not influenced by MURC/Cavin-4 siRNA transfection. Conclusions: Our results indicate that MURC/Cavin-4 plays a crucial role in the development of cardiac I/R injury through ROS-induced apoptosis signaling pathway in cardiomyocytes. MURC/Cavin-4 might be a novel therapeutic target for cardiac I/R injury.

Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized. Murine-induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated. Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac-specific markers, GATA4, Nkx2.5 and Tnnt2. Although β-AR signalling is the foremost signalling pathway in cardiomyocytes, EPI-enhanced cardiac differentiation depended more on α-AR signalling than β-AR signalling. In addition, selective activation of α1 -AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of α2 - or β-AR signalling induced no or less differentiation, respectively. EPI- and α1 -AR-dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK-ERK1/2 pathway and regulation of miPS cell-cycle progression. These results demonstrate that activation of ARs, particularly of α1 -ARs, promoted miPSC differentiation into cardiac lineages via MEK-ERK1/2 signalling.

Abstract 299: Natural Product Derivative Bio Promotes Recovery After Myocardial Infarction via Unique Modulation of the Cardiac Microenvironment

Rationale: Myocardial infarction (MI) induces cardiac remodeling, which is regulated by the cardiac microenvironment and results in scarring and loss of cardiac function. Targeting the microenvironment represents a novel therapeutic approach. Objective: To investigate the therapeutic effect of a natural compound derivative, BIO [(2’Z,3’E)-6-Bromoindirubin-3′-oxime], on cardiac microenvironment cells and remodeling post-MI. Methods and Results: Using a series of co-culture studies, BIO was shown to induce proliferation in cardiomyocytes and, conversely, inhibit proliferation in cardiac fibroblasts. In addition, BIO produced anti-fibrotic effects in the fibroblasts, such as reduced motility and expression changes in fibrosis-related factors. In macrophages, BIO inhibited the expression of pro-inflammatory inducible nitric oxide synthase. Interestingly, BIO modulated molecular crosstalk between cardiac fibroblasts and differentiating macrophages to increase expression of anti-inflammatory M2 macrophage markers. In the optically transparent zebrafish-based model of heart failure, BIO induced cardiomyocyte proliferation to recover survival rate. BIO is a known glycogen synthase kinase-3β inhibitor, but these effects could not be recapitulated using the classical inhibitor, lithium chloride; indicating novel, potential therapeutic effects of BIO on remodeling. We characterized these novel effects of BIO as differential modulation of p27 expression and potent induction of interleukin-10 in microenvironment cells. In rat MI model, BIO reduced fibrosis and improved cardiac performance. Histological analysis revealed a greater presence of anti-inflammatory M2 macrophages in the infarction zone. BIO treatment also reduced serum levels of pro-fibrotic interleukin-6. Conclusions: BIO differentially activates signaling pathways in cardiomyocytes, cardiac fibroblasts and cardiac macrophages to reduce fibrosis, increase cardiomyocyte proliferation and promote recovery post-MI.

Mesenchymal stem cells attenuate inflammatory processes in the heart and lung via inhibition of TNF signaling.

Mesenchymal stem cells (MSC) have been used to treat different clinical conditions although the mechanisms by which pathogenetic processes are affected are still poorly understood. We have previously analyzed the homing of bone marrow-derived MSC to diseased tissues characterized by a high degree of mononuclear cell infiltration and postulated that MSC might modulate inflammatory responses. Here, we demonstrate that MSC mitigate adverse tissue remodeling, improve organ function, and extend lifespan in a mouse model of inflammatory dilative cardiomyopathy (DCM). Furthermore, MSC attenuate Lipopolysaccharide-induced acute lung injury indicating a general role in the suppression of inflammatory processes. We found that MSC released sTNF-RI, which suppressed activation of the NFκBp65 pathway in cardiomyocytes during DCM in vivo. Substitution of MSC by recombinant soluble TNF-R partially recapitulated the beneficial effects of MSC while knockdown of TNF-R prevented MSC-mediated suppression of the NFκBp65 pathway and improvement of tissue pathology. We conclude that sTNF-RI is a major part of the paracrine machinery by which MSC effect local inflammatory reactions.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-016-0573-2) contains supplementary material, which is available to authorized users.

Rationale, application and clinical qualification for NT-proBNP as a surrogate end point in pivotal clinical trials in patients with AL amyloidosis.

Amyloid light-chain (LC) amyloidosis (AL amyloidosis) is a rare and fatal disease for which there are no approved therapies. In patients with AL amyloidosis, LC aggregates progressively accumulate in organs, resulting in organ failure that is particularly lethal when the heart is involved. A significant obstacle in the development of treatments for patients with AL amyloidosis, as well as for those with any disease that is rare, severe and heterogeneous, has been satisfying traditional clinical trial end points (for example, overall survival or progression-free survival). It is for this reason that many organizations, including the United States Food and Drug Administration through its Safety and Innovation Act Accelerated Approval pathway, have recognized the need for biomarkers as surrogate end points. The international AL amyloidosis expert community is in agreement that the N-terminal fragment of the pro-brain natriuretic peptide (NT-proBNP) is analytically validated and clinically qualified as a biomarker for use as a surrogate end point for survival in patients with AL amyloidosis. Underlying this consensus is the demonstration that NT-proBNP is an indicator of cardiac response in all interventional studies in which it has been assessed, despite differences in patient population, treatment type and treatment schedule. Furthermore, NT-proBNP expression is directly modulated by amyloidogenic LC-elicited signal transduction pathways in cardiomyocytes. The use of NT-proBNP will greatly facilitate the development of targeted therapies for AL amyloidosis. Here, we review the data supporting the use of NT-proBNP, a biomarker that is analytically validated, clinically qualified, directly modulated by LC and universally accepted by AL amyloidosis specialists, as a surrogate end point for survival.

A biphasic effect of TNF-α in regulation of the Keap1/Nrf2 pathway in cardiomyocytes

Antagonizing TNF-α signaling attenuates chronic inflammatory disease, but is associated with adverse effects on the cardiovascular system. Therefore the impact of TNF-α on basal control of redox signaling events needs to be understand in more depth. This is particularly important for the Keap1/Nrf2 pathway in the heart and in the present study we hypothesized that inhibition of a low level of TNF-α signaling attenuates the TNF-α dependent activation of this cytoprotective pathway. HL-1 cardiomyocytes and TNF receptor1/2 (TNFR1/2) double knockout mice (DKO) were used as experimental models. TNF-α (2–5ng/ml, for 2h) evoked significant nuclear translocation of Nrf2 with increased DNA/promoter binding and transactivation of Nrf2 targets. Additionally, this was associated with a 1.5 fold increase in intracellular glutathione (GSH). Higher concentrations of TNF-α (>10–50ng/ml) were markedly suppressive of the Keap1/Nrf2 response and associated with cardiomyocyte death marked by an increase in cleavage of caspase-3 and PARP. In vivo experiments with TNFR1/2-DKO demonstrates that the expression of Nrf2-regulated proteins (NQO1, HO-1, G6PD) were significantly downregulated in hearts of the DKO when compared to WT mice indicating a weakened antioxidant system under basal conditions. Overall, these results indicate that TNF-α exposure has a bimodal effect on the Keap1/Nrf2 system and while an intense inflammatory activation suppresses expression of antioxidant proteins a low level appears to be protective.

Forkhead box O (FOXO) 3 modulates hypoxia-induced autophagy through AMPK signalling pathway in cardiomyocytes.

Autophagy is promoted as a response to such environmental stress conditions as ATP depletion and excessive accumulation of reactive oxygen species (ROS). Multiple signalling pathways, including AMP-activated protein kinase (AMPK), are indicated to promote autophagy in ischaemic/hypoxic (I/R) heart. However, it's far more to clarify the orchestrated cross-talk between AMPK and other signalling pathways in the autophagy. In the present study, we investigated the autophagy induction by hypoxia in Rat H9C2 cardiomyocytes with LC3-EGFP reporter, EM and Western blot analysis. Then, we examined the promotion of forkhead box O (FOXO) 3, one member of FOXO transcriptional protein family, by hypoxia in Rat H9C2 cells and determined the mediation of FOXO 3 in the hypoxia-induced autophagy in H9C2 cells. In addition, we investigated the role of AMPK signalling in the FOXO3-mediated, hypoxia-induced autophagy in H9C2 cells. It was demonstrated that hypoxia induced significant autophagy in H9C2 cells, via promoting autophagic vesicles, inducing the conversion of LC3-I to LC3-II and up-regulating autophagy-related (ATG) markers. Moreover, FOXO3 was up-regulated by the hypoxia in H9C2 cells; and the knockdown of FOXO3 significantly reduced the hypoxia-induced autophagy. In addition, AMPK signalling was significantly promoted by hypoxia in H9C2 cells, and the chemical manipulation of AMPK exerted significant influence on the hypoxia-induced autophagy and on the FOXO3 level. In conclusion, FOXO3 regulated the hypoxia-induced autophagy in cardiomyocytes, and AMPK mediated the FOXO3 promotion during the autophagy induction by hypoxia, implying the key regulatory role of FOXO3 and AMPK signalling in the hypoxia-induced autophagy in cardiomyocytes.

The Circular RNA Cdr1as Promotes Myocardial Infarction by Mediating the Regulation of miR-7a on Its Target Genes Expression

ObjectivesRecent studies have demonstrated the role of Cdr1as (or CiRS-7), one of the well-identified circular RNAs (circRNAs), as a miR-7a/b sponge or inhibitor in brain tissues or islet cells. This study aimed to investigate the presence of Cdr1as/miR-7a pathway in cardiomyocytes, and explore the mechanism underlying the function of miR-7a in protecting against myocardial infarction (MI)-induced apoptosis.MethodsMouse MI injury model was established and evaluated by infarct size determination. Real-time PCR was performed to quantify the expression of Cdr1as and miR-7a in cardiomyocytes. Cell apoptosis was determined by caspase-3 activity analysis and flow cytometry assays with Annexin V/PI staining. Transfection of Cdr1as overexpressing plasmid and miR-7a mimic were conducted for gain-of-function studies. Luciferase reporter assay and western blot analysis were performed to verity potential miR-7a targets.ResultsCdr1as and miR-7a were both upregulated in MI mice with increased cardiac infarct size, or cardiomyocytes under hypoxia treatment. Cdr1as overexpression in MCM cells promoted cell apoptosis, but was then reversed by miR-7a overexpression. The SP1 was identified as a new miR-7a target, in line with previously identified PARP, while miR-7a-induced decrease of cell apoptosis under hypoxia treatment was proven to be inhibited by PARP-SP1 overexpression. Moreover, Cdr1as overexpression in vivo increased cardiac infarct size with upregulated expression of PARP and SP1, while miR-7a overexpression reversed these changes.ConclusionsCdr1as also functioned as a powerful miR-7a sponge in myocardial cells, and showed regulation on the protective role of miR-7a in MI injury, involving the function of miR-7a targets, PARP and SP1.

Klotho inhibits angiotensin II-induced cardiomyocyte hypertrophy through suppression of the AT1R/beta catenin pathway

Myocardial hypertrophy is an independent risk factor for cardiac morbidity and mortality. The antiaging protein klotho reportedly possesses a protective role in cardiac diseases. However, the precise mechanisms underlying the cardioprotective effects of klotho remain unknown. This study was aimed to determine the effects of klotho on angiotensin II (Ang II)-induced hypertrophy in neonatal rat cardiomyocytes and the possible mechanism of actions. We found that klotho significantly inhibited Ang II-induced hypertrophic growth of neonatal cardiomyocytes, as evidenced by decreased [3H]-Leucine incorporation, cardiomyocyte surface area and β-myosin heavy chain (β-MHC) mRNA expression. Meanwhile, klotho inhibited Ang II–stimulated activation of the Wnt/β-catenin pathway in cardiomyocytes, as evidenced by decreased protein expression of active β-catenin, downregulated protein and mRNA expression of the β-catenin target genes c-myc and cyclin D1, and increased β-catenin phosphorylation. Inhibition of the Wnt/β-catenin pathway by the specific inhibitor XAV939 markedly attenuated Ang II-induced cardiomyocyte hypertrophy. The further study revealed that klotho treatment significantly downregulated protein expression of Ang II receptor type I (AT1R) but not type II (AT2R). The AT1R antagonist losartan inhibited Ang II–stimulated activation of the Wnt/β-catenin pathway and cardiomyocyte hypertrophy. Our findings suggest that klotho inhibits Ang II-induced cardiomyocyte hypertrophy through suppression of the AT1R/β-catenin signaling pathway, which may provide new insights into the mechanism underlying the protective effects of klotho in heart diseases, and raise the possibility that klotho may act as an endogenous antihypertrophic factor by inhibiting the Ang II signaling pathway.

Disrupted desmosomes drive fibrosis

![Figure][1] Compared with control cells (left), fibronectin is up-regulated in cardiomyocytes lacking PKP2 (right). [Dubash et al.][2] describe how the loss of desmosomal proteins activates a profibrotic signaling pathway in cardiomyocytes. Plakophilin-2 (PKP2) is a key structural

MiR-182 Modulates Myocardial Hypertrophic Response Induced by Angiogenesis in Heart.

Myocardial hypertrophy is an adaptive response to hemodynamic demands. Although angiogenesis is critical to support the increase in heart mass with matching blood supply, it may also promote a hypertrophic response. Previously, we showed that cardiac angiogenesis induced by placental growth factor (PlGF), promotes myocardial hypertrophy through the paracrine action of endothelium-derived NO, which triggers the degradation of regulator of G protein signaling 4 (RGS4) to activate the Akt/mTORC1 pathways in cardiomyocytes. Here, we investigated whether miRNAs contribute to the development of hypertrophic response associated with myocardial angiogenesis. We show that miR-182 is upregulated concurrently with the development of hypertrophy in PlGF mice, but not when hypertrophy was blocked by concomitant expression of PlGF and RGS4, or by PlGF expression in eNOS−/− mice. Anti-miR-182 treatment inhibits the hypertrophic response and prevents the Akt/mTORC1 activation in PlGF mice and NO-treated cardiomyocytes. miR-182 reduces the expression of Bcat2, Foxo3 and Adcy6 to regulate the hypertrophic response in PlGF mice. Particularly, depletion of Bcat2, identified as a new miR-182 target, promotes AktSer473/p70-S6KThr389 phosphorylation and cardiomyocyte hypertrophy. LV pressure overload did not upregulate miR-182. Thus, miR-182 is a novel target of endothelial-cardiomyocyte crosstalk and plays an important role in the angiogenesis induced-hypertrophic response.