Cardiomyocyte Autophagy Research Articles

Long non-coding RNA nuclear-enriched abundant transcript 1 inhibition blunts myocardial ischemia reperfusion injury via autophagic flux arrest and apoptosis in streptozotocin-induced diabetic rats

Background and aimsThis study aimed to investigate the effects of long non-coding RNA (lncRNA)-nuclear-enriched abundant transcript (Neat1) on myocardial ischemia reperfusion injury in diabetic rats ex vivo and in vivo. MethodsScreening for LncRNA Neat1 expression was performed in rat myocardial tissues using microarray analysis and verified by qRT-PCR. Cell viability of rat cardiomyocytes was analyzed by MTT assay. Levels of autophagy-related proteins Atg7, Atg5, LC3-II/LC3-I and p62 were determined by Western blot assay. Left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular ejection fraction (LVEF) and fractioning shortening were obtained by transthoracic echocardiography. Left ventricular end systolic pressure (LVESP), left ventricular end diastolic pressure (LVEDP), maximum rate of increase or decrease of left ventricular pressure (±dp/dtmax) and heart rate were obtained by computer algorithms and an interactive videographics programme. Myocardial infarct size was determined by Evans blue and triphenyltetrazolium chloride (TTC) staining. Myocardial apoptotic index was analyzed by TUNEL assay and immunohistochemical staining. Autophagic flux was examined by evaluating fluorescent LC3 puncta. ResultsNeat1 was highly expressed in ischemia reperfusion-treated diabetic rat myocardial tissues. Overexpression of Neat1 promoted the production of lactate dehydrogenase, inhibited superoxide dismutase content and cardiomyocyte viability. Neat1 overexpression also promoted the production of serum myocardial enzymes, including creatine kinase and creatine kinase-MB, and increased infarct size. By promoting myocardial apoptosis and autophagy, Neat1 aggravated myocardial ischemia reperfusion (I/R) injury in diabetic rats. Neat1 promoted cardiomyocyte autophagy by up-regulating Foxo1 expression to increase hypoxia-reoxygenation injury. ConclusionsI/R treatment caused more injuries in diabetic rats compared with normal rats. Elevated Neat1 expression aggravates myocardial ischemia reperfusion injury via activation of apoptosis and autophagy in diabetic rats. Foxo1 is one of the molecular mechanisms underlying Neat1-induced autophagy.

Exosomal Mst1 transfer from cardiac microvascular endothelial cells to cardiomyocytes deteriorates diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) is characterized by cardiac microvascular endothelial cells (CMECs) injury and cardiomyocyte (CM) dysfunction. Exosomes mediated cellular communication between CMECs and CM has emerging roles in the pathogenesis of DCM, but the underlining mechanisms are unclear. Mammalian sterile 20-like kinase 1 (Mst1), a key component in Hippo pathway which participates in regulating organ size, apoptosis and autophagy, is involved in the development of DCM. We generated the endothelial-specific Mst1 transgenic mice (Tg-Mst1EC) and constructed diabetic model with streptozotocin (STZ). Interestingly, Tg-Mst1EC mice suffered from worse cardiac function and aggravated insulin resistance compared with non-transgenic (NTg) diabetic mice. The content of Mst1 protein was increased, while Mst1 mRNA had no significant change in CM isolated from diabetic Tg-Mst1EC mice. In vitro, CMECs-derived exosomes were taken up by CM and increased Mst1 protein content which inhibited autophagy, as well as enhanced apoptosis in high glucose (HG) cultured CM as evidenced by immunofluorescence and western blot analysis. In addition, Mst1 inhibited glucose uptake under diabetic condition by disrupting the glucose transporter type 4 (GLUT4) membrane translocation through decreasing the interaction between Daxx and GLUT4, as well as enhancing the association of Mst1 and Daxx. Our study exemplifies pleiotropic effects of Mst1-enriched exosomes released from CMECs on inhibiting autophagy, promoting apoptosis and suppressing the glucose metabolism in CM.

Abstract 485: Enhanced Chaperone Mediated Autophagy in Cardiomyocytes Ameliorates Pathology Induced by Hypoxic and Proteotoxic Stresses

Rationale: Protein degradation pathways play a critical role in maintaining cardiac homeostasis. Chaperone mediated autophagy (CMA) is a protein degradation pathway unique to mammalian cells, but little is known about CMA in cardiac disease. Preliminary studies in our lab showed increased CMA activity in mouse hearts subjected to MI (1.81 fold) and TAC surgery (3.5 fold), as well as, in hearts from mutant CryAB R120G transgenic mice (3 fold). Thus, we hypothesized that CMA plays a critical role in hypoxia- and proteotoxicity-induced cardiac stresses. CMA selectively targets substrate proteins for lysosomal degradation using CMA-specific lysosome membrane protein type 2a (LAMP2a) receptor. LAMP2a is both necessary and sufficient for CMA activity. Methods: An adenovirus and a siRNA were created to overexpress and silence LAMP2a levels to study the gain and loss of CMA in neonatal rat ventricular cardiomyocytes (NRVMs). Two models of cardiomyocyte stress were employed: 1) a hypoxic stress model that exposed NRVMs to CoCl 2 , a hypoxia mimetic agent; and 2) a proteotoxic model where mutant CryAB R120G , a missense mutation of αB-crystallin, was expressed causing the accumulation of insoluble protein aggregates. A more specific measure of CMA activity was also determined in lysosomal fractions of NRVMs. Results: The LAMP2a adenovirus and siRNA showed successful increase and knockdown of LAMP2a levels in NRVMs, respectively. CoCl 2 -induced hypoxia significantly increased CMA activity in NRVMs (26%). LAMP2a overexpression further augmented CoCl 2 -induced CMA activity (55%) and increased the percent of CMA-active lysosomes. LAMP2a siRNA blunted the CoCl 2 -induction of LAMP2a protein levels (65%) and silenced CMA activity in intact lysosomes. Further, CoCl 2 increased apoptosis which was rescued by enhanced CMA activity as shown by TUNEL staining. Thus, CMA is regulated by hypoxic stress and modulates cardiac cell survival. In a proteotoxic model, CryAB R120G and LAMP2a co-infection decreased CryAB R120G protein levels and insoluble aggregate pathology. Our data suggest that CMA may be cardioprotective against hypoxic and proteotoxic stresses. Impact: Defining the role of CMA in the heart could open new therapeutic pathways for the treatment of heart failure.

Knockdown of lncRNA AK139328 alleviates myocardial ischaemia/reperfusion injury in diabetic mice via modulating miR-204-3p and inhibiting autophagy.

This study was aimed at investigating the effects of lncRNA AK139328 on myocardial ischaemia/reperfusion injury (MIRI) in diabetic mice. Ischaemia/reperfusion (I/R) model was constructed in normal mice (NM) and diabetic mice (DM). Microarray analysis was utilized to identify lncRNA AK139328 overexpressed in DM after myocardial ischaemia/reperfusion (MI/R). RT‐qPCR assay was utilized to investigate the expressions of lncRNA AK139328 and miR‐204‐3p in cardiomyocyte and tissues. Left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular ejection fraction (LVEF) and fractioning shortening (FS) were obtained by transthoracic echocardiography. Haematoxylin‐eosin (HE) staining and Masson staining were utilized to detect the damage of myocardial tissues degradation of myocardial fibres and integrity of myocardial collagen fibres. Evans Blue/TTC staining was used to determine the myocardial infarct size. TUNEL staining was utilized to investigate cardiomyocyte apoptosis. The targeted relationship between lncRNA AK139328 and miR‐204‐3p was confirmed by dual‐luciferase reporter gene assay. MTT assay was used for analysis of cardiomyocyte proliferation. Western blot was utilized to investigate the expression of alpha smooth muscle actin (α‐SMA), Atg7, Atg5, LC3‐II/LC3‐I and p62 marking autophagy. Knockdown of lncRNA AK139328 relieved myocardial ischaemia/reperfusion injury in DM and inhibited cardiomyocyte autophagy as well as apoptosis of DM. LncRNA AK139328 modulated miR‐204‐3p directly. MiR‐204‐3p and knockdown of lncRNA AK139328 relieved hypoxia/reoxygenation injury via inhibiting cardiomyocyte autophagy. Silencing lncRNA AK139328 significantly increased miR‐204‐3p expression and inhibited cardiomyocyte autophagy, thereby attenuating MIRI in DM.

Ginsenoside Rb1 Ameliorates Autophagy of Hypoxia Cardiomyocytes from Neonatal Rats via AMP-Activated Protein Kinase Pathway.

To investigate whether ginsenoside-Rb1 (Gs-Rb1) improves the CoCl-induced autophagy of cardiomyocytes via upregulation of adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway. Ventricles from 1- to 3-day-old Wistar rats were sequentially digested, separated and incubated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 3 days followed by synchronization. Neonatal rat cardiomyocytes were randomly divided into 7 groups: control group (normal level oxygen), hypoxia group (500 μmol/L CoCl2), Gs-Rb1 group (200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), Ara A group (500 μmol/L Ara A + 500 μmol/L CoCl2), Ara A+ Gs-Rb1 group (500 μmol/L Ara A + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), AICAR group [1 mmol/L 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) + 500 μmol/L CoCl2], and AICAR+Gs-Rb1 group (1 mmol/L AICAR + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2). Cells were treated for 12 h and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cardiac troponin I (cTnI) levels were detected by enzyme-linked immunosorbent assay (ELISA). AMPK activity was assessed by 2',7'-dichlorofluorescein diacetate (DCFH-DA) ELISA assay. The protein expressions of Atg4B, Atg5, Atg6, Atg7, microtubule-associated protein 1A/1B-light chain 3 (LC3), P62, and active-cathepsin B were measured by Western blot. Gs-Rb1 significantly improved the cell viability of hypoxia cardiomyocytes (P<0.01). However, the viability of hypoxia-treated cardiomyocytes was significantly inhibited by Ara A (P<0.01). Gs-Rb1 increased the AMPK activity of hypoxia-treated cardiomyocytes. The AMPK activity of hypoxia-treated cadiomyocytes was inhibited by Ara A (P<0.01) and was not affected by AICAR =0.983). Gs-Rb1 up-regulated Atg4B, Atg5, Beclin-1, Atg7, LC3B II, the LC3B II/I ratio and cathepsin B activity of hypoxia cardiomyocytes (P<0.05), each of these protein levels was significantly enhanced by Ara A (all P<0.01), but was not affected by AICAR (all P>0.05). Gs-Rb1 significantly down-regulated P62 levels of hypoxic cardiomyocytes (P<0.05). The P62 levels of hypoxic cardiomyocytes were inhibited by Ara A (P<0.05) and were not affected by AICAR (P=0.871). Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the upregulation of AMPK pathway.

Diabetes mellitus and heart failure: insights from a toxic relationship

Diabetes mellitus and heart failure: insights from a toxic relationship

Beyond the Cardiomyocyte: Consideration of HIPPO Pathway Cell-Type Specificity.

Increasing interest in the HIPPO signaling pathway has stemmed largely from its ability to modulate cardioprotection and heart regeneration after injury, responses originating from manipulation within the cardiomyocyte. This viewpoint discusses the need for definitive understanding of HIPPO pathway function in cardiac nonmyocytes and highlights the necessity of considering cell-type specificity for effective therapeutic targeting of the HIPPO pathway in cardiovascular disease. The HIPPO pathway is a highly conserved kinase cascade that determines organ size by regulating cell survival, proliferation, and differentiation. On activation, the core components of the pathway, MST (mammalian sterile 20-like kinase) 1/2, SAV1 (salvador 1), LATS (large tumor suppressor kinase) 1/2, and MOB1 (Mps one binder kinase activator-like 1), serve to phosphorylate and repress activity of the transcriptional cofactors YAP (Yes-associated protein) and TAZ (transcriptional coactivator with a PDZ-binding domain). When HIPPO is inhibited, YAP/TAZ cytosolic retention is relinquished, allowing for nuclear localization and association with multiple transcription factors, the most established being the TEAD (TEA domain) family (Figure 1). Investigation of the HIPPO pathway in the heart has garnered increased attention during the past decade and justifiably so. HIPPO is fundamental in maintaining adult heart homeostasis and modulating responses to injury. To date, the majority of studies investigating HIPPO signaling in the heart have focused on the cardiomyocyte. In response to myocardial infarction or pressure overload stress, the core HIPPO kinase MST1 is activated in cardiomyocytes. MST1 signals through canonical (to restrain YAP) and noncanonical (to antagonize BCL-XL [B-cell lymphoma-extra large] and Beclin1) mechanisms resulting in the inhibition of cardiomyocyte proliferation and autophagy and the enhancement of cell death.1–3 Conversely, HIPPO pathway inhibition limits injury and promotes tissue regeneration through YAP-dependent increases in cardiomyocyte proliferation and survival.4 As a result, interventions that activate cardiomyocyte …

Autophagy-A key pathway for cardiac health and longevity.

As average life expectancy continues to rise in the developed world, age-associated pathologies are increasing in prevalence. The hallmarks of cardiac ageing include cardiomyocyte loss, fibrosis and hypertrophy, all of which contribute to an increased incidence of cardiac disease. At the molecular level, cellular ageing is characterized by increased ROS production, mitochondrial dysfunction and the accumulation of damaged proteins and organelles. Cardiomyocytes and other senescent cell types rely upon autophagy, a lysosome-mediated degradation pathway, to remove potentially toxic protein aggregates and damaged organelles from the cellular milieu. However, increasing lines of evidence point to an age-associated decrease in cardiomyocyte autophagy, with predictably negative consequences for cardiac function and health. Conversely, stimulation of autophagy has been shown to improve cellular health and cardiac function and to increase lifespan in numerous model organisms. Clearly, autophagy represents a critical pathway for cellular vitality, as well as a promising therapeutic target for the treatment of age-related cardiac pathologies. In this review, we will discuss the mechanism of autophagy and its regulation in the cell, the role of autophagy in the ageing heart, and how the autophagy pathway might be targeted to improve cardiac health.

Sphingosine 1 phosphate receptor-1 (S1PR1) signaling protects cardiac function by inhibiting cardiomyocyte autophagy.

ObjectiveTo investigate the role of sphingosine-1-phosphate (S1P) and its receptors in cardiomyocyte autophagy, cardiomyocyte hypertrophy and cardiac function.MethodsCardiomyocytes were isolated from neonatal Vista rats. Autophagy and hypertrophy of cardiomyocytes were induced via starvation culture and phenylephrine (PE), respectively, and S1P was used to treat the cardiomyocytes. The effect of S1P on cardiomyocyte autophagy was evaluated by the number of autophagosomes, the expression of autophagy-related proteins and autophagic marker genes in cardiomyocytes. The effect of S1P on cardiomyocyte hypertrophy was evaluated by examining the surface area of cardiomyocytes and the expression of hypertrophic genes. Subsequently, different small interfering RNAs (siRNAs) were used to knockdown the expression of the three types of S1P receptors on cardiomyocytes and to analyze the type of receptor that mediates S1P signaling in cardiomyocytes. Finally, sphingosine 1 phosphate receptor-1 (S1PR1) was knockout in the mouse cardiomyocytes using the Cas9 technique. The effect of S1PR1 on cardiac autophagy and cardiac hypertrophy was examined by assessing cardiomyocyte autophagy, cardiomyocyte hypertrophy and cardiac function.ResultsStarvation-induced cardiomyocyte autophagy and PE-induced cardiomyocyte hypertrophy were significantly attenuated by S1P. The results showed that the formation of autophagosomes was decreased, the autophagy-associated protein LC3II/I and the expression of autophagic marker genes Atg5, Atg12, Beclin1 and LC3B decreased after S1P treatment. The surface area of the cardiomyocytes was decreased, and the expression of hypertrophic genes, including atrial natriuretic factor (ANF), skeletal muscle and cardiac actin (SKA), myosin heavy chain (β-MHC) and brain natriuretic peptide (BNP) were all decreased after S1P treatment. The autophagy and hypertrophy of cardiomyocytes in the S1PR1 knocked-down group were significantly increased compared to those in the control group, the S1PR2 and the S1PR3 knocked-down groups. In vivo, the knockout of S1PR1 in cardiomyocytes exacerbated stress-induced cardiac autophagy, cardiac hypertrophy and the impairment of cardiac function.ConclusionS1P could inhibit cardiomyocyte autophagy, thereby inhibiting cardiomyocyte hypertrophy and protecting cardiac function by activating S1PR1 in pressure-overloaded cardiomyocytes in mice.

HMGB1 contributes to adriamycin-induced cardiotoxicity via up-regulating autophagy

Adriamycin (ADR) is one of the most widely used and effective anthracycline antitumor agents in the treatment of certain hematological malignancies and solid tumors. However, the severe cardiotoxicity of ADR limits its clinical application. So far, the mechanism of the cardiotoxicity of ADR has not been completely clarified. In our research, cardiomyocyte autophagy and cardiac damage were observed in accompany ADR treatment, and autophagy appeared earlier than cardiac damage. Inhibition of autophagy by silencing ATG7 improved the survival rate of cardiomyocytes treated with ADR. The release of HMGB1 increased after ADR treatment, and silencing HMGB1 could reverse cardiomyocyte damage by attenuating autophagy. In addition, the expression of YAP was decreased, and overexpressing YAP down-regulated HMGB1 and alleviated cardiomyocyte damage. These results indicated that autophagy was the leading cause of ADR-induced cardiotoxicity, and HMGB1 played a vital role in the process of up-regulating autophagy.

Ablation of toll-like receptor 4 attenuates aging-induced myocardial remodeling and contractile dysfunction through NCoRI-HDAC1-mediated regulation of autophagy

Aging is usually accompanied with overt structural and functional changes as well as suppressed autophagy in the heart although the precise regulatory mechanisms are somewhat unknown. Here we evaluated the role of the innate proinflammatory mediator toll-like receptor 4 (TLR4) in cardiac aging and the underlying mechanism with a focus on autophagy. Cardiac geometry and function were monitored in young or old wild-type (WT) and TLR4 knockout (TLR4−/−) mice using echocardiography, IonOptix® edge-detection and fura-2 techniques. Levels of autophagy and mitophagy, nuclear receptor corepressor 1 (NCoR1) and histone deacetylase I (HDAC1) were examined using western blot. Transmission electronic microscopy (TEM) was employed to monitor myocardial ultrastructure. Our results revealed that TLR4 ablation alleviated advanced aging (24 months)-induced changes in myocardial remodeling (increased heart weight, chamber size, cardiomyocyte cross-sectional area), contractile function and intracellular Ca2+ handling as well as autophagy and mitophagy [Beclin-1, Atg5, LC3B, PTEN-induced putative kinase 1 (PINK1), Parkin and p62]. Aging downregulated levels of NCoR1 and HDAC1 as well as their interaction, the effects were significantly attenuated or negated by TLR4 ablation. Advanced aging disturbed myocardial ultrastructure as evidenced by loss of myofilament alignment and swollen mitochondria, which was obliterated by TLR4 ablation. Moreover, aging suppressed autophagy (GFP-LC3B puncta) in neonatal mouse cardiomyocytes, the effect of which was negated by the TLR4 inhibitor CLI-095. Inhibition of HDCA1 using apicidin cancelled off CLI095-induced beneficial response of GFP-LC3B puncta against aging. Our data collectively indicate a role for TLR4-mediated autophagy in cardiac remodeling and contractile dysfunction in aging through a HDAC1-NCoR1-dependent mechanism.

Hydrogen Sulfide Regulating Myocardial Structure and Function by Targeting Cardiomyocyte Autophagy.

Objective:Hydrogen sulfide (H2S), a gaseous signal molecule, plays a crucial role in many pathophysiologic processes in the cardiovascular system. Autophagy has been shown to participate in the occurrence of many cardiac diseases. Increasing evidences indicated that H2S regulates myocardial structure and function in association with the altered autophagy and plays a “switcher” role in the autophagy of myocardial diseases. The aim of this review was to summarize these insights and provide the experimental evidence that H2S targets cardiomyocyte autophagy to regulate cardiovascular function.Data Sources:This review was based on data in articles published in the PubMed databases up to October 30, 2017, with the following keywords: “hydrogen sulfide,” “autophagy,” and “cardiovascular diseases.”Study Selection:Original articles and critical reviews on H2S and autophagy were selected for this review.Results:When autophagy plays an adaptive role in the pathogenesis of diseases, H2S restores autophagy; otherwise, when autophagy plays a detrimental role, H2S downregulates autophagy to exert a cardioprotective function. For example, H2S has beneficial effects by regulating autophagy in myocardial ischemia/reperfusion and plays a protective role by inhibiting autophagy during the operation of cardioplegia and cardiopulmonary bypass. H2S postpones cardiac aging associated with the upregulation of autophagy but improves the left ventricular function of smoking rats by lowering autophagy.Conclusions:H2S exerts cardiovascular protection by regulating autophagy. Cardiovascular autophagy would likely become a potential target of H2S therapy for cardiovascular diseases.

Metformin inhibits autophagy and mitophagy in cardiomyocytes

Metformin, a first‐line medication for the treatment of type 2 diabetes mellitus, has been suggested by several clinical studies to be cardioprotective. However, the mechanisms underlying metformin's cardioprotection remain debated. One current hypothesis proposes that metformin protects the heart by increasing autophagy in cardiomyocytes, leading to increased removal of protein aggregates and damaged organelles including mitochondria. In this study, we tested this hypothesis using both in vitro and in vivo models. Rat H9C2 cardiac myoblasts were treated with 3 mM metformin for 24 hours and autophagy/mitophagy were determined by Western blot analysis of LC3‐II, a well‐established marker of autophagic vesicles. Surprisingly, metformin significantly reduced LC3‐II levels in either total cell lysates or mitochondrial fractions; and lysosomal inhibitors increased LC3‐II in metformin‐treated cells to a less degree as compared with vehicle‐treated cells. These results suggest that metformin inhibited rather than stimulated autophagy and mitophagy, contrary to the current belief. This was confirmed by a novel dual fluorescent mitophagy reporter showing reduced mitochondrial fragments being degraded in the lysosomes in the presence of metformin. The reduced mitophagy was also found in the heart from the mitophagy reporter mice that received 200 mg/kg of metformin through gavage. Collectively, these results indicate that the cardioprotective benefit of metformin observed in many different models may be through mechanisms other than increased autophagy and mitophagy.Support or Funding InformationThis work was supported by the National Institutes of Health National Center for Research Resources.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P21‐Activated Kinase 1 is Necessary for Autophagy and Mitophagy in Cardiomyocytes

P21‐Activated Kinase 1 (PAK1) is a serine‐threonine kinase that regulates multiple signal transduction pathways in cardiomyocytes. Despite the large body of evidence indicating a key role for PAK1 in cardiac protective signalling, the mechanisms underlying the cardioprotective effects of PAK1 remain elusive. Autophagy is a cellular process in which unwanted organelles and proteins are degraded and recycled to maintain cellular homeostasis. Mitophagy is a form of selective autophagy that eliminates damaged mitochondria from the cell via the lysosome system to control mitochondrial quality. Autophagy and mitophagy are essential for cell survival in response to starvation, but may become detrimental under other conditions. How autophagy and mitophagy are regulated for adaptation in different contexts is under intense investigation. In this study, we explored whether PAK1 regulates autophagy and mitophagy in cardiomyocytes. PAK1 was knocked down by using siRNA in H9c2 cardiac myoblasts and then the expression levels of the autophagosome marker LC3‐II were analysed by Western blot. Interestingly, downregulation of PAK1 reduced LC3‐II levels in the total cell lysates, which was not significantly increased by the lysosomal protease inhibitors pepstatin A (pepA) and E64d, suggesting that autophagy flux is reduced by downregulation of PAK1. Similarly, PAK1 knockdown reduced LC3‐II expression levels in mitochondria either in the presence or absence of pepA and E64d, indicating limited mitochondrial degradation or mitophagy in the absence of PAK1. These results were reproduced in mouse hearts lacking PAK1. Together, our results suggest that PAK1 is essential to maintain the activities of autophagy and mitophagy in cardiomyocytes in vitro and in vivo. Current studies in our lab are investigating the functional significance of autophagy and mitophagy in PAK1‐induced myocardial protection.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of autophagy in cardiomyocytes by calmodulin antagonists

Macroautophagy (hereafter referred to as autophagy) is a catabolic process that regulates important aspects of myocardial metabolism in which cytoplasmic contents including proteins, other macromolecules, and organelles are sequestered in double membrane vesicles and delivered to lysosomes for destruction. While numerous studies have indicated involvement of signaling via Ca2+ in autophagic flux, the precise mechanisms remain unknown. Calmodulin (CaM) is the ubiquitous transducer of intracellular Ca2+ signals. CaM binds to an estimated network of over 300 cellular proteins, yet is expressed insufficiently for its targets. We have begun to examine the role of CaM in autophagic flux in cardiomyocytes. In H9C2 cardiomyocytes, amino acid starvation for 2 hrs triggers autophagic flux, as assessed by the formation of LC3‐II and the decrease in p62 protein levels. Chloroquine dose‐dependently reduces starvation‐induced autophagic flux. Competitive binding assays using purified CaM and FRET‐based CaM biosensors demonstrate direct CaM binding by chloroquine. Consistently, W‐7 and calmidazolium, two other structurally distinct CaM antagonists, dose‐dependently inhibit starvation‐induced formation of autophagic flux. These data demonstrate a role for CaM in autophagic flux in cardiomyocytes.Support or Funding InformationAHA 15SDG25090279 Grant (to E Wauson) and NIH HL112184 grant (to Q‐K Tran)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA‐17‐3p inhibits excessive post‐hypoxic autophagy and attenuates H9C2 cardiomyocytes reoxygenation injury via PTEN‐Akt‐mTOR signaling

BackgroundThe microRNA (miR)‐17~92 is one of the best‐characterized polycistronic miRNA clusters, which encodes six individual miRNAs and contributes to various cellular and biological processes. In particular, miR‐17‐3p (a passenger miRNA of miR‐17) has been shown to indirectly inhibit Phosphatase and tensin homolog (PTEN) and attenuate myocardial ischemia/reperfusion (I/R) injury, but the underneath mechanism is unclear. Inhibition of PTEN can upregulate Akt/mTOR signaling and therefore suppresses the excessive autophagy, which may be a mechanism that underlies myocardial I/R injury. We, thus, hypothesized that miR‐17‐3p may protect against hypoxia/reoxygenation (H/R) induced cell injury by inhibiting post‐hypoxic excessive autophagy in H9C2 cardiomyocytes via PTEN‐Akt‐mTOR signaling pathway.Methods and resultsIn rat H9C2 cardiomyocytes, H/R (6 hours hypoxia followed by 6 hours reoxygenation) significantly enhanced the expression of miR‐17‐3p (P &lt;0.05 vs. Control), which was concomitant by increased lactic acid dehydrogenase (LDH) leakage (cell injury marker, P&lt;0.05 vs. Control) and the levels of p62 and the ratio of LC3II/I (autophagy markers, P&lt;0.05 vs. Control), suggesting that miR‐17‐3p may be involved in the pathogenesis of H/R induced autophagic cell death. To explore the potential role of miR‐17‐3p in H/R‐induced cell injury, H9C2 cells were transfected with the miR‐17‐3p agomir or its negative control. The overexpression of miR‐17‐3p can significantly attenuate H/R‐induced cell injury (reduced LDH level, P &lt;0.05 vs. H/R) and inhibit H/R‐induced excessive autophagy (decreased levels of p62 and LC3II/I, P &lt;0.05 vs. H/R). Given the important role of PTEN‐Akt‐mTOR signaling in autophagy, the effects of miR‐17‐3p on the key events in the PTEN‐Akt‐mTOR signaling cascade during H/R were examined. As anticipated, miR‐17‐3p overexpression significantly down‐regulated PTEN expression and up‐regulated the levels of phosphorylated Akt1 (Thr308) and mTOR (P&lt;0.05 vs. H/R), indicating that the miR‐17‐3p may protect against excessive post‐hypoxic autophagic cell death in H9C2 cardiomyocytes via PTEN‐Akt‐mTOR signaling pathway.ConclusionUpregulating PTEN‐Akt‐mTOR axis and the subsequent inhibition of excessive autophagy may represent the major mechanism whereby miR‐17‐3p attenuates H/R injury in H9C2 cardiomyocytes.Support or Funding InformationStudied supported by RGC/GRF grants (17158616M, 17117217 M)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lactone Component From Ligusticum chuanxiong Alleviates Myocardial Ischemia Injury Through Inhibiting Autophagy.

The dysregulation of autophagy is associated with a series of cardiovascular diseases, such as myocardial ischemia injury. Lactone component from Ligusticum chuanxiong (LLC) is the major constituent of the traditional Chinese herb L. chuanxiong Hort., which has been reported to hold potential cardioprotective effects. In this study, to determine whether LLC protects the heart through regulation of autophagy, we explored the effects of LLC on cardioprotection and autophagy in myocardial ischemia injured rats and H9c2 cardiomyocytes. Our results showed that LLC significantly reduced infarct size and serum levels of lactate dehydrogenase, creatine kinase, and cardiac troponin and ameliorated histological features in a dose-dependent manner. Similar protections were observed in cardiomyocytes subjected to oxygen-glucose deprivation (OGD). Meanwhile, LLC inhibited autophagy induced by myocardial ischemia injury, characterized by increased autophagic vacuoles, LC3-II/LC3-I ratio and the expression of Beclin 1, whereas decreased the expression of p62. Additionally, LLC combined with a lysosomal inhibitor chloroquine (CQ) reduced LC3-II/LC3-I ratio in cardiomyocytes compared with CQ alone. Furthermore, LLC-afforded cardioprotection was abolished by a specific PI3K inhibitor LY294002. Collectively, these findings demonstrated that cardioprotective effects of LLC were related to restoration of autophagic flux through the activation of PI3K/Akt/mTOR signaling pathway.

Ginsenoside Rb1 inhibits autophagy through regulation of Rho/ROCK and PI3K/mTOR pathways in a pressure-overload heart failure rat model.

This study was designed to explore the relationship between ginsenoside Rb1 (Grb1) and high-load heart failure (HF) in rats. The parameters of cardiac systolic function (left ventricular posterior wall thickness (LVPWT), left ventricular internal diastolic diameter (LVID), fraction shortening (FS) and mitral valves (MVs)) of rat hearts in each group were inspected by echocardiogram. The expressions of rat myocardial contractile proteins, autophagy-related proteins and the activation of Rho/ROCK and PI3K/mTOR pathways were detected by Western blot. LVPWT, FS, MVs and the expression of myocardial contractile proteins α-MHC, apoptosis-related proteins Bcl-2 and signalling pathway involved proteins pAkt and mTOR were significantly reduced in the HF, HF+5mg/kg Grb1 (HF+Grb1-5) and HF+Grb1+arachidonic acid (AA) groups with LVID, β-MHC, cell apoptosis, cell autophagy and Rho/ROCK significantly increased compared with the control group, of which the tendency was contrary to the HF+20mg/kg Grb1 (HF+Grb1-20) group compared with the HF group (P<0.05). In the HF+Grb1+AA group, there was no significant change in the above indexes compared with the HF group. The results indicated that Grb1 can exert anti-HF function by inhibiting cardiomyocyte autophagy of rats through regulation of Rho/ROCK and PI3K/mTOR pathways.

Melatonin Balance the Autophagy and Apoptosis by Regulating UCP2 in the LPS-Induced Cardiomyopathy.

To explore the mechanism of mitochondrial uncoupling protein 2 (UCP2) mediating the protective of melatonin when septic cardiomyopathy. UCP2 knocked out mice and cardiomyocytes were used to study the effect of melatonin in response to LPS. Indicators of myocardial and mitochondria injury including mitochondrial membrane potential, mitochondrial permeability transition pore, calcium loading, ROS, and ATP detection were assessed. In addition cell viability and apoptosis as well as autophagy-associated proteins were evaluated. Melatonin was able to protect heart function from LPS, which weakened in the UCP2-knockout mice. Consistently, genipin, a pharmacologic inhibitor of UCP2, augmented LPS-induced damage of AC16 cells. In contrast, melatonin upregulated UCP2 expression and protected the cells from the changes in morphology, mitochondrial membrane potential loss, mitochondrial Ca2+ overload, the opening of mitochondrial permeability transition pore, and subsequent increased ROS generation as well as ATP reduction. Mitophagy proteins (Beclin-1 and LC-3β) were increased while apoptosis-associated proteins (cytochrome C and caspase-3) were decreased when UCP2 was up-regulated. In conclusion, UCP2 may play a protecting role against LPS by regulating the balance between autophagy and apoptosis of cardiomyocytes, and by which mechanisms, it may contribute to homeostasis of cardiac function and cardiomyocytes activity. Melatonin may protect cardiomyocytes through modulating UCP2.

Polycystin-2-dependent control of cardiomyocyte autophagy

AimsConsiderable evidence points to critical roles of intracellular Ca2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca2+ homeostasis and autophagy. Methods and resultsActivation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca2+ chelation using BAPTA-AM, whereas removal of extracellular Ca2+ had no effect, pointing to a role of intracellular Ca2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca2+-channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca2+. Furthermore, PC2 ablation was associated with impaired Ca2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca2+ stores. Finally, we provide evidence that Ca2+-mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types. ConclusionTogether, this study unveils PC2 as a novel regulator of autophagy acting through control of intracellular Ca2+ homeostasis.