Study Of The Mechanism Of Heavy Metal Scavenger Metallothionein In Myocardial Protection

Cardiovascular disease(CVD) has become the leading threat to human health. Themortality and morbidity exceeded malignant tumors and it becomes the No.1cause ofdeath globally. There are3millions people who died of cardiovascular diseases,accounting for40%of all mortality causes. The mechanism of cardiovascular disease isnot clear, therefore, studies on cardiovascular disease prevention are particularlyimportant.The main function of endoplasmic reticulum is synthesis, folding and transportationof protein. About a third of the cellular protein synthesis is carried out in endoplasmicreticulum. Therefore, maintaining homeostasis of the endoplasmic reticulum is requiredto ensure the normal functions of the network. But many factors can cause imbalance inthe steady state and lead to endoplasmic reticulum stress: for example, ischemia-reperfusion injury, oxidative stress, detrimental effects of physical or chemical factors.Over the past10years, studies found that endoplasmic reticulum stress played animportant role in the pathogenesis of many cardiovascular diseases. In recent years, withthe increasing pressure of work and life, the incidence of hypertension has increased overyears. Increasing literature reported that endoplasmic reticulum stress was involved in the pathophysiology of hypertension. However, the underlying mechanism has yet beenentirely clear. Recent studies confirmed that, as a rich source of cysteine, and a heavymetal scavenger with strong metal binding capability, metallothionein (MT) had goodcardioprotective function, particularly in aging, obesity, insulin resistance, diabetes, andheart disease. By clearing excessive reactive oxygen species (ROS) and reactive nitrogenradicals (RNS), MT restored the oxidation-reduction balance and thus achieved effectivecardiac protection. Recently, it was reported that by reducing oxidative stress, MTeffectively protected mitochondrial function and remarkably reduced endoplasmicreticulum stress. Therefore, we hypothesize that MT can effectively reduce endoplasmicreticulum stress-induced myocardial injury. The aim of this study is to provide a newperspective and theoretical foundation for the study of the mechanism of myocardialinjury.This study was based on the literature review and our preliminary work. Firstly, westudied whether MT has protective effect on endoplasmic reticulum stress-inducedmyocardial injury and determined the role of autophagy and apoptosis in this process.Secondly, we investigated the role of MT to hypertensive myocardial injury protection.Finally, we studied the mechanism of myocardial injury at the mitochondria level.Part I. Heavy Metal Scavenger Metallothionein Attenuates ERStress-Induced Myocardial Contractile AnomaliesExperiment I: Role of AutophagyAims: Endoplasmic reticulum (ER) stress increases the risk of cardiovascular morbidityand mortality. However, the underlying mechanism remains elusive. This study wasdesigned to examine the Effects of cardiac over-expression of metallothionein, acysteine-rich heavy metal scavenger, on ER stress-induced changes in myocardialfunction and investigate the underlying mechanism involved with a focus on autophagy. Methods: Wild-type friendly virus B (FVB) and transgenic mice with cardiac-specificover-expression of metallothionein were challenged with the ER stress inducertunicamycin (1mg/kg, ip for48h). After48hours several indexes were evaluated,including ultrasonic cardiogram, cardiac systolic function, cardiac diastolic function andcalcium function. DCF staining was used to show the amount of released ROS. Westernblot was used to detect ER stress related proteins GRP78, GADD153, p-eIF2α, p-IRE1ɑand the expression of autophagy related proteins p-ULK1(Ser777), Atg5, Atg7, LC3B andp62. Then, the myocardial cells were treated with autophagic inhibitor3-MA orN-acetylcysteine (NAC), an antioxidant. The myocardial systolic and diastolic functionswere evaluated.Results: Our results showed that ER stress led to compromised echocardiographic andcardiomyocyte contractile function, and intracellular Ca2+mishandling. Tunicamycininduced ER stress and oxidative stress, increased left ventricular end systolic anddiastolic diameter, as well as suppressed fractional shortening and whole heartcontractility, however, the effects of which were significantly attenuated or ablated bymetallothionein. Levels of the autophagy markers such as phosphorylated ULK1, Atg5,Atg7, LC3B and the autophagy adaptor p62were significantly upregulated bymetallothionein. The ER stress-induced changes in myocardial function, autophagy andautophagy signaling were distinctly mitigated or alleviated by metallothionein. Inhibitionof autophagy by3-methyladenine in vitro reversed ER stress-induced cardiomyocytecontractile deficiency. Meanwhile, ER stress-induced dysfunction of cardiomyocytes wasattenuated by the antioxidant.Experiment II: Role of ApoptosisAims: Endoplasmic reticulum (ER) stress elicits myocardial oxidative stress, apoptosisand myocardial dysfunction. This study was designed to evaluate the impact of cardiacoverexpression of metallothionein, a cysteine-rich free radical scavenger, on ERstress-induced myocardial dysfunction and elucidate the underlying mechanism with afocus on apoptosis. Methods: Wild-type friendly virus B (FVB) and transgenic mice with cardiac-specificoverexpression of metallothionein were challenged with the ER stress inducertunicamycin (1mg/kg, ip for48h). After48hours several indexes were evaluated,including ultrasonic cardiogram, cardiac systolic function, cardiac diastolic function andcalcium function. JC-1was used to measure the membrane potential of mitochondrial.Western blot was used to detect ER stress related proteins GRP78, GADD153, p-eIF2α,p-IRE1ɑ and determine the expression of apoptosis related proteins BAX, Bcl-2,Caspase-8, Caspase-9and Caspase-12.Results: Our data showed that tunicamycin treatment elicited cardiac remodeling(increasing ventricular end systolic and diastolic diameters without affecting ventricularwall thickness), suppressed fractional shortening, isolated cardiomyocyte contractilefunction, and compromised intracellular Ca2+handling, the effects of which wereobliterated by metallothionein. Treatment with tunicamycin caused mitochondrialdysfunction of cardiomyocyte, as evidenced by decreased mitochondrial membranepotential (m, JC-1staining), which was restored by metallothionein. In addition,tunicamycin treatment dramatically activated myocardial intrinsic apoptosis pathway, asevidenced by increased protein expression of Bax, Caspase-9, Caspase-12, as well asCaspase-3activity. Interestingly, overexpression of metallothionein significantlyalleviated tunicamycin-induced myocardial apoptosis.Part II. Heavy Metal Scavenger Metallothionein AttenuatesHypertension-Induced Myocardial Contractile AnomaliesExperiment I: Role of AutophagyAims: To study whether heavy metal scavenger metallothionein (MT) can improve thehypertensive heart disease induced by L-NAME, and to explore the roles ofautophagy and mammalian target of rapamycin (mTOR) signaling in this process.Methods: Wild type FVB mice (male,5-6months) and cardiac specific MToverexpression mice were randomly divided into four groups: FVB, FVB+L-NAME, MTand MT+L-NAME. In FVB+L-NAME or MT+L-NAME groups, L-NAME (NO synthase inhibitor,0.1g/L) was added in the drinking water.14days later, several indexeswere evaluated, including blood pressure, ultrasonic cardiogram, cardiac systolicfunction, cardiac diastolic function and calcium function. WGA and Masson staining wasused to determine the size of cardiomyocyte and the deGRPe of myocardial fibrosis.Western blot was used to detect mTOR related proteins p-AMPKα (Thr172), p-TSC2(Thr1462), p-mTOR (Ser2448), p-p70S6K (Ser424) and determine the expression ofautophagy related proteins p-ULK1(Ser777) and LC3B. Then, the mice were treated withautophagy inhibitor3-MA or agonist rapamycin (RAPA), and the myocardial systolic anddiastolic functions were evaluated.Results:14days later, the blood pressure in FVB+L-NAME or MT+L-NAME groupwas higher than corresponding control group (p<0.05). The results of ultrasoniccardiogram showed that LVESD and LVEDD in the FVB+L-NAME were increased andFS was decreased (compared with FVB group, p<0.05). These indexes were improved inMT+L-NAME group (compared with FVB+L-NAME group, p<0.05). Studies incardiomyocyte model showed that L-NAME significantly decreased the cellsystolic peak,-dL/dt,+dL/dt and ΔFFI, and increased TR90and Ca2+decay rate(compared with control group, p<0.05). MT treatment reversed the damage induced byL-NAME (compared with L-NAME group, p<0.05). The results of Masson stainingshowed that cardiac fibrosis was increased significantly in FVB+L-NAME group(compared with FVB group, p<0.05). The results of WGA staining showed thatL-NAME treatment significantly increased the cardiomyocyte size (compared withcontrol group, p<0.05). MT treatment reversed the fibrosis and cell size induced byL-NAME in vivo or in vitro (p<0.05). The Western bolt showed that L-NAME treatmentsignificantly decreased the expression of p-AMPKα (Thr172), p-TSC2(Thr1462),p-ULK1(Ser777) and LC3B, and increased the expression of p-mTOR (Ser2448) andp-p70S6K (Ser424)(compared with FVB group, p<0.05). MT overexpression reversed theeffects of L-NAME on these proteins (compared with FVB+L-NAME group, p<0.05). Invivo, the cell systolic peak,-dL/dt,+dL/dt and TR90were significantly decreased inFVB+L-NAME+3MA (compared with FVB+L-NAME group, p<0.05) andMT+L-NAME+3MA (compared with MT+L-NAME group, p<0.05). RAPA treatmentimproved the systolic function impaired by L-NAME (p<0.05) Experiment II: Role of ApoptosisAims: This study was designed to investigate to whether heavy metal scavengermetallothionein (MT) can improve the hypertensive heart disease induced by L-NAME,and to explore the roles of mitochondrial function, membrane potential and apoptosissignaling pathways in this process.Methods: Wild type FVB mice (male,5-6months) and cardiac specific MToverexpression mice were randomly divided into four groups: FVB, FVB+L-NAME, MTand MT+L-NAME. In FVB+L-NAME or MT+L-NAME groups, L-NAME(NO synthaseinhibitor,0.1g/L) was added in the drinking water. Fourteen days later, blood pressure,ultrasonic cardiogram, cardiac systolic function, cardiac diastolic function and calciumfunction were evaluated. JC-1and TUNEL staining were used to measure the membranepotential of mitochondrial and detect myocardial apoptosis. Western blot was used todetect the expression of apoptosis related proteins BAX, p-BAD, BAD and Bcl-2. Theexpression level of Caspase-9and Caspase-12were also detected.Results:14days later, the blood pressure in FVB+L-NAME or MT+L-NAME groupwas higher than control group (p<0.05). The results of ultrasonic cardiogram showedthat LVESD and LVEDD in the FVB+L-NAME were increased and FS was decreased(compared with FVB group, p<0.05). These indexes were improved in MT+L-NAMEgroup (compared with FVB+L-NAME group, p<0.05). Studies in cardiomyocyte modelshowed that L-NAME significantly decreased the cell systolic peak,-dL/dt,+dL/dt andΔFFI, and increased TR90andCa2+decay rate (compared with control group, p<0.05).MT treatment reversed the damage induced by L-NAME (compared with L-NAMEgroup, p<0.05). The results of Masson staining showed that cardiac fibrosis wasincreased significantly in FVB+L-NAME group (compared with FVB group, p<0.05).The results of JC-1staining showed that L-NAME significantly decreased themitochondrial membrane potential level (compared with control group, p<0.05). Theresults of TUNEL staining showed that L-NAME significantly increased the number ofapoptotic cardiomyocytes (compared with control group, p<0.05). MT treatment reversed the decrease of the mitochondrial membrane potential level and the increase of apoptoticcardiomyocytes (p<0.05). The Western bolt detection showed that L-NAME treatmentsignificantly decreased the expression of p-BAD and Bcl-2, increased the expression ofCaspase-9and Caspase-12(compared with FVB group, p<0.05). MT overexpressionreversed the effects of L-NAME on the expression of these proteins (comparedwith FVB+L-NAME group, p<0.05).SummaryThrough these studies, we found that metallothionein (MT) can effectively reduceendoplasmic reticulum stress-induced myocardial injury. MT, through its antioxidantfunction, reduced endoplasmic reticulum stress of the endoplasmic reticulum, reducedthe level of cardiac autophagy, restored mitochondrial membrane potential and function,and finally reduced myocardial apoptosis. Meanwhile, in the L-NAME inducedhypertension model, MT restored the level of cardiac autophagy by inhibiting mTOR,restored mitochondrial membrane potential and function, reduced ER stress and the levelof apoptosis and improved hypertensive myocardial injury induced by L-NAME.