MTOR Dependent Autophagy Plays A Dual Role In Regulation Of Cardiomyocyte Dysfunction In Different Models

BackgroundNormal cardiac function is essential for a healthy and good quality life. Cardiomyocytes are non-regenerative cell. Therefore, the maintenance of myocardial homeostasis is extremely important. However, it is really a challenge to keep the heart healthy throughout the whole life.First, different genes, different hearts. As we all know, mutation of genes that are related to heart differentiation can lead to severe cardiac dysfunction. But in normal population, cardiac function may also be different because of genetic polymorphism, especially when the heart is challenged by stresses. In this paper, we will discuss the effect of ADH (alcohol dehydrogenase) overexpression and MIF (Macrophagy migration inhibitory factor) deficiency on cardiac function.ADH is responsible for the first step of alcohol metabolism, which catalyzes alcohol to acetaldehyde. ADH1 is the most enzymatic functional isoform of ADH. The gene which codes the ADH1 substructure-ADH1B has genetic polymorphism. The mutant ADH IB protein has superactive enzymatic function. In East Asian population, the mutation rate of ADH IB is 40-90%, which is much higher than that in African or in European population. ADH overexpression model was mostly used in researches about alcoholic diseases, while the effect of ADH itself on cardiac function is still unknown.MIF, as a cytokine secreted by T cells, has been identified as a cardioprotective factor in 2008, the story of which was published on Nature.It was reported that, in ischemic condition, MIF improved glucose uptake by activating AMPK pathway, and resulting in cardiac protection. However, the mutation on a promoter of MIF gene can lead to decrease of MIF expression, which suppressed the AMPK activation in ischemic condition. MIF plays a critical role in cardiac homeostasis.Then, the external stresses, diseases and aging are all big challenges for the heart. In this paper, we will talk about 2 models, one is the ER (endoplasmic reticulum) stress model and the other is the aging model, to discuss the acquired burdens for heart.Nowadays, pollution, unhealthy lifestyle and pressure can lead to induction of ER stress. ER stress plays important roles in different kinds of diseases, like obesity, diabetes, heart failure and myocardial ischemia-reperfusion injury. ER is a double membrane organelle, the main function of which is processing newly synthesized proteins, and correctly folding them, finally transports the correctly folded protein to Golgi. Meanwhile, ER is also an important organelle for calcium storage. In normal condition, there are about 30% proteins not correctly folded, which are then degraded by related mechanism in ER. The processing and folding procedures are easily disturbed by external stimulations. Especially under stresses like hypoxia, inflammation or oxidative stress, the ER will trigger unfolded protein response (UPR) to initiate protective mechanism, maintain the ER homeostasis. However, if the stresses remain, the UPR would be over-triggered, causing ER stress. Tunicamycin is an effective inducer of ER stress, which is already widely used in establishment of ER stress model.Aging is a big problem for human beings since ancient times. The function of organs, the metabolic level of cells and the function of organelles will be obviously reduced in aging people, as well as changes in the molecular level.Aging hearts have significantly decreased systolic and diastolic function, declined energetic metabolism level and reduced ability of calcium handling. The possible mechanisms involved in aging are oxidative stress and mitochondrial dysfunction, as many reports said. But there is still a huge gap needs to be filled in the field of aging.Autophagy is a procedure that degrades aged or damaged proteins and organelles to accomplish cell renewal and material recycle. The cell keeps a certain level of autophagy in normal condition. Too low or too high levels of autophagy are both vital. Low autophagy level is related to low renewal level, which leads to slow metabolic rate and accumulation of harmful material, finally resulting in myocardial dysfunction and even cell death. Excessive autophagy can lead to degradation of normal-functioned proteins or organelles, which will also causing myocardial dysfunction and cell death. Autophagy is closely associated with many heart diseases, such as myocardial ischemia-reperfusion injury, chronic heart failure, coronary artery disease and septic heart failure。mTOR (Mammalian target of rapamycin), a serine/threonine kinase, is an important protein that takes responsibility for metabolism and growth regulation, which is also involved in classic autophagy pathway as a negative regulator of autophagy. Phosphorylation of mTOR activates mTOR activity, which can be regulated by many proteins, like Akt (promote mTOR phosphorylation) and AMPK (suppress mTOR phosphorylation).In this paper, we will discuss the effect of ADH or MIF on cardiac function under different pathological conditions, including ER stress and aging, as well as the underlying mechanisms.PurposeThe aim of this study is to explore the effect of different genotypes of ADH or MIF on cardiac function under different pathological conditions (ER stress and aging) and the underlying mechanisms, which focus on the role of mTOR dependent autophagy and the related upstream pathway. To achieve this aim, we studied (1) The effect of cardiac specific overexpresion of ADH on ER stress-induced cardiac function and the role of mTOR dependent pathway; (2) The effect of MIF deficiency on aging-induced cardiac function and the role of mTOR dependent pathway.MethodsPart 1 Alcohol Dehydrogenase Protects against Endoplasmic Reticulum Stress-Induced Myocardial Contractile Dysfunction via Attenuation of Oxidative Stress and Autophagy: Role of PTEN-Akt-mTOR Signaling.1. Mouse ModelWide-type (FVB) or ADH cardiac overexpression mice were given a single dose of tunicamycin (i.p. lmg/kg) or equivalent of PBS respectively. Experiments were conducted 48 hours later after injection.2. Cardiac function, cardiomyocyte contractile function and calcium handlingCardiac function was detected by echocardiography. Cardiomyocytes were first isolated by using Langendorff system and digested by Librase enzyme, the contractile function of which was then detected by SoftEdge Myocam software, and the calcium handling properties was detected by a dual-excitation fluorescence photomultiplier system, using fura-2/AM fluorescence.3. Oxidative stressOxidative stress was detected in isolated cardiomyocytes by using H2DCFDA and DHE staining.4. Protein expressionProteins’expression including ER stress markers, autophagy markers and PTEN-Akt-mTOR pathway related proteins were detected by Western Blot.5. In vitro studyIsolated cardiomyocytes from WT or ADH mice were treated with tunicamycin or (and) autophagy inhibitor 3-MA, Akt inhibitor AktI, or mTOR inhibitor rapamycin. Then the myocardial contractile function was recorded using SoftEdge Myocam software.Part 2 Macrophagy Migration Inhibitory Factor (MIF) Deficiency Exacerbates Aging-Induced Cardiac Remodeling and Dysfunction Despite Improved Inflammation:Role of Autophagy Regualtion.1. Experimental models(1) Aging mouse model.3-4 months normal-feeded wild-type (C57BL/6) or MIF knockout mice were used as young mice.24 months old normal-feeded WT or MIF knockout mice were used as old mice. (2) Rapamycin treated mice model. Treatment of rapamycin followed i.p.2mg/kg/d for 8 weeks. (3) Premature senescence model and MIF depleted model. H9C2 cells were treated with doxorubicin (0.1 u M) for 24 hours to set the premature senescence model, while the H9C2 cells were treated with MIF SiRNA to set MIF depleted model.2. Cardiac function, cardiomyocyte contractile function and calcium handlingCardiac function was detected by echocardiography. Cardiomyocytes were first isolated by using Langendorff system and digested by Librase enzyme, the contractile function of which was then detected by SoftEdge Myocam software, and the calcium handling properties was detected by a dual-excitation fluorescence photomultiplier system, using fura-2/AM fluorescence.3. Myocardial morphology and heart fibrosisUsing Lectin staining examined the myocardial morphology and using Masson Trichorme staining studied the heart fibrosis.4. Glucose handling capability of mice and ATP level in heart tissueThe IPGTT experiment was used to detect the glucose handling capability of mice. ATP level in frozen heart tissue was detected by chloroform-methanol method, using a spectrofluorimeter with an excitation wavelength of 350nm and emission wavelength of 485nm.5. Protein expressionProtein expression including aging markers, autophagy markers and AMPK-mTOR pathway related proteins were detected by Western Blot.ResultsPart 1 Alcohol Dehydrogenase Protects against Endoplasmic Reticulum Stress-Induced Myocardial Contractile Dysfunction via Attenuation of Oxidative Stress and Autophagy: Role of PTEN-Akt-mTOR Signaling.1. ADH ameliorated ER stress-induced decreased cardiac function. After tunicamycin treatment, the cardiac function of WT mice was significantly decreased, which was obviously alleviated by ADH overexpression. Similarly, after tunicamycin treatment, calcium handling capability of WT mice was significantly disturbed, which was also mitigated by ADH overexpression.2. ADH reduced ER stress-induced oxidative stress level.3. ADH ameliorated tunicamycin-induced high level of ER stress and autophagy, improved tunicamyicn-induced decrease of PTEN-Akt-mTOR signaling. Treatment of tunicamycin leaded to elevated expression of ER stress markers BIP and GADD153 and autophagy markers LC3B Ⅱ, Atg5, Atg7 and P62 in WT mice, which were all significantly reversed by ADH. Tunicamycin also leaded to decreased PTEN, Akt and mTOR level, which was improved by ADH.4. ADH suppressed autophagy level through Akt-mTOR dependent pathway, which protected cardiac function against ER stress. From the result of in vitro study, the contractile function of isolated cardiomyocytes was significantly decreased after tunicamycin treatment, which was alleviated in ADH overexpression cardiomyocytes or in WT cardiomyocytes treatment with autophagy inhibitor 3-MA. However, when the ADH overexpression cardiomyocytes was treated with Akt inhibitor AktI or mTOR inhibitor rapamycin, the cardiac protective role of ADH against tunicamyin no longer existed.Part 2 Macrophagy Migration Inhibitory Factor (MIF) Deficiency Exacerbates Aging-Induced Cardiac Remodeling and Dysfunction Despite Improved Inflammation:Role of Autophagy Regualtion.1. MIF deficiency attenuated aging-induced declined glucose handling capability and cardiac dysfunction. In IPGTT experiment, at 15min and 60 min time points after glucose challenge, the blood glucose levels of old mice were overtly lower than yound mice, which was attenuated in MIF knockout old mice. The cardiac function and caridomyocyte contractile function was significantly decreased in old mice, which was even worse in MIF knockout old mice.2. MIF deficiency exacerbated aging-induced cardiomyocyte hypertrophy and myocardial fiborosis.3. AMPK-mTOR pathway and autophagy was suppressed in aging heart, which was attenuated by MIF knockout. Aging heart exhibited decreased LC3BⅡ level and increased P62 level, indicating low level of autophagy and accumulation of cell garbage. Aging heart also showed declined phosphorylated AMPK level and increased phosphorylated mTOR level, indicating the AMPK-mTOR pathway was suppressed. These changes were further emphasized by MIF deficiency.4. Doxorubicin treatment caused premature senescence in H9C2 cells, as evidenced by increased level of senescence marker SA-β-Gal. Depletion of MIF with MIF siRNA accentuated senescence of H9C2 cells, while restoration of MIF expression obviously attenuated senescence of H9C2 cells.5. Treatment of autophagy inducer ameliorated the effect of MIF deficiency on cardiac dysfunction in aging mice.6. MIF deficiency alleviated aging-induced inflammation, while the autophagy inducer rapamycin had no obvious effect on it.Conclusion.We firstly reported the cardioprotective effect of ADH itself again ER stress-induced cardiac dysfunction, and indicated the underlying mechanisms, which are alleviating the oxidative stress level trigged by ER stress, and attenuating PTEN-Akt-mTOR pathway dependent autophagy level. We also firstly showed that MIF deficiency exacerbated aging-induced cardiac dysfunction. In aging heart, the autophagy level is decreased, which leads to blockage of normal myocardial energetic metabolism as well as material renewal and finally disturbs the cardiac function. MIF can mediate activation of AMPK-mTOR dependent autophagy. Therefore, MIF deficiency resulted in further decreased autophagy level and cardiac function.In these two studies, mTOR dependent autophagy showed dual role. In the ADH-ER stress study, ER stress induced excessive mTOR dependent autophagy activation and resulted in cell death and cardiac dysfunction; while in the MIF-aging model, aging suppressed mTOR dependent autophagy level, causing decrease of myocardial metabolism rate and shortage of energy supply. In all, to achieve the goal of cardioprotection, the autophagy level should be maintained at an appropriated level. mTOR is a crucial target for autophagy regulation. Drugs targeting mTOR has great potential for clinical application.