Heavy Metal Scavenger Metallothionein Mitigate Deep Hypothermia-Induced Myocardial Contractile Anomalies:Role Of Autophagy

Low ambient temperature environment exposure increased the risk of cardiovascular morbidity and mortality although the underlying mechanism remains unclear. This study was designed to examine the impact of cardiac overexpression of metallothionein, a cysteine-rich heavy metal scavenger, on low temperature (4℃)-induced changes in myocardial function and underlying mechanism involved with a focus on autophagy. Cold exposure (4℃for3weeks) promoted oxidative stress, protein damage, increased left ventricular end systolic and diastolic diameter, suppressed fractional shortening and whole heart contractility, the effects of which were significantly attenuated or ablated by metallothionein. Levels of the autophagy markers LC3B-Ⅱ, Beclin-1and Atg7were significantly upregulated with unchanged autophagy adaptor protein p62. Fluorescent immunohistochemistry revealed abundant LC3B puncta in cold temperature-exposed mouse hearts. Co-immunoprecipitation revealed increased dissociation between Bcl2and Beclin-1. Cold exposure reduced phosphorylation of the autophagy inhibitory signaling molecules Akt and mTOR, increased ULK1phosphorylation along with dampened eNOS phosphorylation (without changes in their total protein expression). These cold exposure-induced changes in myocardial function, autophagy and autophagy signaling cascades were significantly alleviated or mitigated by metallothionein. Inhibition of autophagy using3-methyladenine in vivo reversed cold exposure-induced cardiomyocyte contractile defects. Cold exposure-induced cardiomyocyte dysfunction was attenuated by antioxidant N-acetylcysteine and lysosomal inhibitor bafilomycin Al. Collectively, these findings suggested that metallothionein protects against cold exposure-induced cardiac anomalies possibly through attenuation of cardiac autophagy. PART1Heavy Metal Scavenger Metallothionein Mitigates Cold Exposure-Induced Myocardial Contractile AnomaliesObjective:To elucidate whether heavy metal scavenger metallothionein could mitigate cold exposure-induced myocardial contractile anomalies.Methods:Five to six month-old male metallothionein(dark brown)mice and their wild-type FVB littermates(white) were housed at room temperature or low ambient temperature in a cold room(4℃) for3weeks. Cardiac geometry and function were evaluated in anesthetized(ketamine80mg/kg and xylazine12mg/kg, i.p.) mice using the2-D guided M-mode echocardiography (Sonos5500) equipped with a15-6MHz linear transducer at room or cold (4℃) temperature based on the mouse group assignment. The Langendorff perfused heart function was assessed using the ADInstruments PowerLab(?) system.Results:Neither cold exposure nor metallothionein, or both, affected body weight, heart rate, LV wall thickness, LV mass,(absolute or normalized value) and cardiac output. Cold exposure significantly increased LVESD, LVEDD and reduced fractional shortening, the effects of which were significantly attenuated or abrogated by metallothionein. Cardiac performance was also evaluated using the Langendorff perfused heart system. Mechanical indices including left ventricular developed pressure (LVDP), maximal rate of pressure development (+dP/dt) and decline (-dP/dt) were similar between FVB and metallothionein mice under normal temperature. Cold exposure significantly depressed LVDP,+dP/dt and-dP/dt in FVB mice, the effects of which were significantly attenuated or obliterated by metallothionein.There is no difference between FVB-NT group and MT-NT group.Conclusion:Data from our current study revealed that metallothionein overexpression rescues against cold exposure-induced myocardial contractile dysfunction. PART2Heavy Metal Scavenger Metallothionein Protects Aginst Cold Exposure-Induced Myocardial Contractile Anomalies Possibly through Attenaution of Cardiac autophagyObjective:To investigate whether heavy metal scavenger metallothionein protecting aginst cold exposure-induced myocardial contractile anomalies is through attenaution of cardiac autophagy.Methods:Considering that autophagy, a regulated cellular process through which mammalian cells degrade and recycle macromolecules, organelles and nutrients is positively associated with cold exposure, essential protein markers of autophagy [LC3B, Atg7, Beclin-1and unc-51-like kinase (ULK-1)], as well as the autophagosome cargo protein p62were monitored in myocardium from FVB and metallothionein mice with or without low ambient temperature exposure. Fluorescent immunohistochemistry was used as an alternative approach for autophagy assessment. Immunoprecipitation technique was employed to evaluate the dissociation of Beclin-1from Bcl2, a process favoring autophagy induction. To explore the possible signaling mechanisms involved in autophagy regulation, both autophagy stimulatory and inhibitory signaling molecules including AMP-dependent protein kinase (AMPK), Akt and mammalian target of rapamycin (mTOR) were scrutinized. Levels of endothelial nitric oxide synthase (eNOS) and p70S6kinase (p70S6K), pivotal cell signaling molecules governing cardiac homeostasis downstream of Akt were evaluated. To evaluate the role of autophagosome formation and the late-stage autophagolysosome fusion (autophagy flux) in cold exposure-induced cardiac contractile anomalies, the effects of autophagosome inhibitor3-methyl adenine (3-MA, in vivo and in vitro) and the lysosomal inhibitor bafilomycin Al (in vitro) on cold stress-induced changes in autophagy and cardiac contractile function were tested.Results:Our results depicted that cold exposure significantly upregulated the levels of LC3B-Ⅱ, LC3B-Ⅱ-to-LC3B-Ⅰ ratio, Beclin-1, Atg7and ULK1Ser777phosphorylation (absolute or normalized value) without affecting levels of total ULK1and the autophagy adaptor protein p62. While metallothionein transgene itself did not affect the expression of these autophagy markers, it significantly ablated cold-exposure-induced elevation in LC3B-Ⅱ, LC3B-Ⅱ-to-LC3B-I ratio, Beclin-1, Atg7and ULK1phosphorylation without affecting other autophagy markers tested. Similar to Western blot finding, metallothionein and autophagy inhibition using the autophagy inhibitor3-MA (10mg/kg/wk, i.p., for3weeks) significantly alleviated cold exposure-induced rise in LC3+puncta without eliciting any overt effect by themselves. Cold exposure reduced phosphorylation (absolute or normalized value) of the autophagy inhibitory signaling molecules including Akt, mTOR and eNOS (without affecting that of AMPK and p70s6k). Total protein expression of Akt, mTOR, eNOS, AMPK and p70s6k was unaffected by cold exposure. Metallothionein significantly attenuated cold exposure-induced changes in the phosphorylation of Akt, mTOR and eNOS without affecting the responses of AMPK and p70s6k. Our result indicated that the amount of Bcl2that immunoprecipitated with Beclin-1was significantly decreased in FVB mice exposed to low temperature. While metallothionein transgene itself did not affect the ratio between Bcl2and Beclin-1, it removed cold exposure-induced dissociation between Bcl2and Beclin-1. Level of Bcl2was unaffected by cold exposure or metallothionein. Our data also revealed that cold exposure significantly depressed peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt) and prolonged time-to-90%relengthening (TR90) without affecting resting cell length and time-to-peak shortening (TPS) in murine cardiomyocytes. Similar to the effect of3-MA, Bafilomycin Al attenuated or ablated cold exposure-induced cardiomyocyte dysfunction including depressed PS,±dL/dt and prolonged TR90. There is no difference between FVB-NT group and MT-NT group.Conclusion:Our data favor the notion that autophagy induction may play an essential role in cold stress-and metallothionein-elicited responses in cardiac mechanical function. Our findings further revealed a likely role of Akt, mTOR and eNOS signaling in cold stress-and metallothionein-induced regulation of myocardial autophagy and contractile function. PART3Heavy Metal Scavenger Metallothionein Protects Aginst Cold Exposure-Induced Myocardial Contractile Anomalies Possibly through Depressing of Oxidative StressObjective:To investigate whether heavy metal scavenger metallothionein protecting aginst cold exposure-induced myocardial contractile anomalies is through depressing of oxidative stress.Methods:To examine the role of oxidative stress in cold exposure-induced cardiomyocyte abnormalities, cardiomyocytes from FVB mice maintained at room temperature or cold environment (4℃) for3weeks were incubated with or without the antioxidant N-acetylcysteine (NAC,500(μM) or3-MA (10mM, used as a positive control) for4hours in vitro prior to evaluation of autophagy and cardiomyocyte contractile function. Reduced and oxidized glutathione [GSH and glutathione disulfide (GSSG)], Intracellular reactive oxygen species (ROS) measurement,and Protein carbonyl assay were also used to investigate the role of oxidative stress in cold exposure-induced cardiomyocyte abnormalities.Results:Sustained cold exposure promoted ROS production and carbonyl formation, as well as decreased levels of glutathione and the GSH/GSSG ratio without affecting the levels of oxidized glutathione (GSSG), the effect of which was significantly attenuated by metallothionein. NAC and3-MA significantly attenuated or mitigated cold exposure-induced elevation in LC3B-Ⅱ and LC3B-Ⅱ-to-LC3B-Ⅰ ratio without affecting p62accumulation. Neither NAC nor3-MA affected autophagosome formation (LC3B-Ⅱ and LC3B-Ⅱ-to-LC3B-Ⅰ ratio) or autophagolysosome fusion (p62) in cardiomyocytes from mice maintained under normal temperature. Both NAC and3-MA significantly attenuated or ablated cold exposure-induced cardiomyocyte contractile dysfunction including depressed PS,±dL/dt and prolonged TR90. Neither NAC nor3-MA overtly affected cardiomyocyte contractile properties in mice maintained under normal temperature. There is no difference between FVB-NT group and MT-NT group.Conclusion:These findings favored an essential role of oxidative stress in cold exposure-and metallothionein-induced changes in myocardial contractile function.