Shi-Yan Li

Cardiac contractile dysfunction in Lep/Lep obesity is accompanied by NADPH oxidase activation, oxidative modification of sarco(endo)plasmic reticulum Ca2+-ATPase and myosin heavy chain isozyme switch.

Aims/hypothesisObesity is an independent risk factor for heart diseases but the underlying mechanism is not clear. This study examined cardiac contraction, oxidative stress, oxidative modification of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and the myosin heavy chain (MHC) isoform switch in obese mice.MethodsMechanical properties were evaluated in ventricular myocytes from C57BL/6J lean and Lep/Lep obese mice (formerly known as ob/ob mice), including peak shortening (PS), time to 50 or 90% PS, time to 50 or 90% relengthening (TR50, TR90), maximal velocity of shortening/relengthening (±dL/dt), intracellular Ca2+ and its decay (τ). Oxidative stress, lipid peroxidation, protein damage and SERCA activity were assessed by glutathione/glutathione disulfide, malondialdehyde, protein carbonyl and 45Ca2+ uptake, respectively. NADPH oxidase was determined by immunoblotting.ResultsMyocytes from Lep/Lep mice displayed depressed PS and ± dL/dt, prolonged TR50, TR90, elevated resting [Ca2+]i, prolonged τ, reduced contractile capacity at high stimulus frequencies and diminished responsiveness to extracellular Ca2+ compared with lean controls. Cardiac glutathione/glutathione disulfide was decreased whereas malondialdehyde, protein carbonyl, membrane p47phox and membrane gp91phox were increased in the Lep/Lep group. SERCA isoenzyme 2a was markedly modified by oxidation in Lep/Lep hearts and associated with decreased 45Ca2+ uptake. The MHC isozyme displayed a shift from the α to the β isoform in Lep/Lep hearts. Short-term incubation of angiotensin II with myocytes mimicked the mechanical defects, SERCA oxidation and 45Ca2+ uptake seen in Lep/Lep myocytes. Incubation of the NADPH oxidase inhibitor apocynin with Lep/Lep myocytes alleviated contractile defects without reversing SERCA oxidation or activity.Conclusions/interpretationThese data indicate that obesity-related cardiac defects may be related to NADPH oxidase activation, oxidative damage to SERCA and the MHC isozyme switch.

Aging induces cardiac diastolic dysfunction, oxidative stress, accumulation of advanced glycation endproducts and protein modification

Evidence suggests that aging, per se, is a major risk factor for cardiac dysfunction. Oxidative modification of cardiac proteins by non‐enzymatic glycation, i.e. advanced glycation endproducts (AGEs), has been implicated as a causal factor in the aging process. This study was designed to examine the role of aging on cardiomyocyte contractile function, cardiac protein oxidation and oxidative modification. Mechanical properties were evaluated in ventricular myocytes from young (2‐month) and aged (24–26‐month) mice using a MyoCam® system. The mechanical indices evaluated were peak shortening (PS), time‐to‐PS (TPS), time‐to‐90% relengthening (TR90) and maximal velocity of shortening/relengthening (± dL/dt). Oxidative stress and protein damage were evaluated by glutathione and glutathione disulfide (GSH/GSSG) ratio and protein carbonyl content, respectively. Activation of NAD(P)H oxidase was determined by immunoblotting. Aged myocytes displayed a larger cell cross‐sectional area, prolonged TR90, and normal PS, ± dL/dt and TPS compared with young myocytes. Aged myocytes were less tolerant of high stimulus frequency (from 0.1 to 5 Hz) compared with young myocytes. Oxidative stress and protein oxidative damage were both elevated in the aging group associated with significantly enhanced p47phox but not gp91phox expression. In addition, level of cardiac AGEs was ∼2.5‐fold higher in aged hearts than young ones determined by AGEs‐ELISA. A group of proteins with a molecular range between 50 and 75 kDa with pI of 4–7 was distinctively modified in aged heart using one‐ or two‐dimension SDS gel electrophoresis analysis. These data demonstrate cardiac diastolic dysfunction and reduced stress tolerance in aged cardiac myocytes, which may be associated with enhanced cardiac oxidative damage, level of AGEs and protein modification by AGEs.

Cardiac overexpression of alcohol dehydrogenase exacerbates chronic ethanol ingestion-induced myocardial dysfunction and hypertrophy: Role of insulin signaling and ER stress

Chronic alcohol intake leads to alcoholic cardiomyopathy characterized by cardiac hypertrophy and contractile dysfunction possibly related to the toxicity of the ethanol metabolite acetaldehyde. This study examined the impact of augmented acetaldehyde exposure on myocardial function, geometry, and insulin signaling via cardiac-specific overexpression of alcohol dehydrogenase (ADH). ADH transgenic and wild-type FVB mice were placed on a 4% alcohol diet for 12 weeks. Echocardiographic, glucose tolerance, glucose uptake, insulin signaling, and ER stress indices were evaluated. Mice consuming alcohol exhibited glucose intolerance, dampened cardiac glucose uptake, cardiac hypertrophy and contractile dysfunction, all of which with the exception of whole body glucose tolerance were exaggerated by the ADH transgene. Cardiomyocytes from ethanol-fed mice exhibited depressed insulin-stimulated phosphorylation insulin receptor (tyr1146) and IRS-1 (tyrosine) as well as enhanced serine phosphorylation of IRS-1. ADH-augmented alcohol-induced effect of IRS-1 phosphorylation (tyrosine/serine). Neither alcohol nor adh affected expression of insulin receptor and IRS-1. Alcohol reduced phosphorylation of Akt and GSK-3beta as well as GSK-3beta expression and the effect was exaggerated by ADH. The transcriptional factors GATA4, c-jun and c-jun phosphorylation were upregulated by alcohol, which was amplified by ADH. The ratios of phospho-c-Jun/c-Jun and phospho-GATA4/GATA4 remained unchanged. Chronic alcohol intake upregulated expression of the endoplasmic reticulum stress markers eIF2alpha, IRE-1alpha, GRP78 and gadd153, the effect of which was exaggerated by ADH. These data suggest that elevated cardiac acetaldehyde exposure via ADH may exacerbate alcohol-induced myocardial dysfunction, hypertrophy, insulin insensitivity and ER stress, indicating a key role of ADH gene in alcohol-induced cardiac dysfunction and insulin resistance.

High-fat diet enhances visceral advanced glycation end products, nuclear O-Glc-Nac modification, p38 mitogen-activated protein kinase activation and apoptosis.

High‐fat diet intake often leads to obesity, insulin resistance and hypertension, which present a common and detrimental health problem. However, precise mechanism underlying tissue damage due to high‐fat diet‐induced obesity has not been carefully elucidated. The present study was designed to examine the effect of high‐fat diet intake on visceral advanced glycation end products (AGEs) formation, nuclear O‐Glc‐NAc modification and apoptosis in heart, liver and kidney. Adult male Sprague‐Dawley weight‐matched rats were fed for 12 weeks with a high‐fat diet (45% kcal from fat) or an isocaloric low‐fat diet (10% kcal from fat). High‐fat diet feeding significantly elevated body weight. Blood pressure and heart rate were comparable between the two rat groups. Competitive enzyme‐linked immunosorbent assay showed significantly elevated serum AGE levels, visceral AGE formation, caspase‐3 activation and cytoplasmic DNA fragmentation in heart and liver but not kidney samples of high‐fat diet fed rats compared with those from low‐fat diet fed group. Western blot analysis further revealed that high‐fat diet feeding induced overt nuclear O‐Glc‐NAc modification and p38 mitogen‐activated protein kinase activation in heart and liver although not in kidney samples of the high‐fat diet‐fed rats. Collectively, our results indicated that high‐fat diet intake is associated with obesity accompanied by elevated serum and visceral AGEs, visceral post‐translational nuclear O‐Glc‐NAcylated modification and apoptosis, which may contribute to high‐fat diet‐induced tissue damage.

Cardiac overexpression of antioxidant catalase attenuates aging-induced cardiomyocyte relaxation dysfunction.

Catalase, an enzyme which detoxifies H2O2, may interfere with cardiac aging. To test this hypothesis, contractile and intracellular Ca2+ properties were evaluated in cardiomyocytes from young (3-4 months) and old (26-28 months) FVB and transgenic mice with cardiac overexpression of catalase. Contractile indices analyzed included peak shortening (PS), time-to-90% PS (TPS90), time-to-90% relengthening (TR90), half-width duration (HWD), maximal velocity of shortening/relengthening (+/-dL/dt) and intracellular Ca2+ levels or decay rate. Levels of advanced glycation endproduct (AGE), Na+/Ca2+ exchanger (NCX), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), phospholamban (PLB), myosin heavy chain (MHC), membrane Ca2+ and K+ channels were measured by western blot. Catalase transgene prolonged survival while did not alter myocyte function by itself. Aging depressed+/-dL/dt, prolonged HWD, TR90 and intracellular Ca2+ decay without affecting other indices in FVB myocytes. Aged FVB myocytes exhibited a stepper decline in PS in response to elevated stimulus or a dampened rise in PS in response to elevated extracellular Ca2+ levels. Interestingly, aging-induced defects were nullified or significantly attenuated by catalase. AGE level was elevated by 5-fold in aged FVB compared with young FVB mice, which was reduced by catalase. Expression of SERCA2a, NCX and Kv1.2 K+ channel was significantly reduced although levels of PLB, L-type Ca2+ channel dihydropyridine receptor and beta-MHC isozyme remained unchanged in aged FVB hearts. Catalase restored NCX and Kv1.2 K+ channel but not SERCA2a level in aged mice. In summary, our data suggested that catalase protects cardiomyocytes from aging-induced contractile defect possibly via improved intracellular Ca2+ handling.

CARDIAC‐SPECIFIC OVEREXPRESSION OF CATALASE PROLONGS LIFESPAN AND ATTENUATES AGEING‐INDUCED CARDIOMYOCYTE CONTRACTILE DYSFUNCTION AND PROTEIN DAMAGE

1 Oxidative stress plays a role in senescence‐associated organ deterioration. This is supported by the beneficial effects of anti‐oxidants against ageing‐related organ damage, although their role in cardiac ageing has not been elucidated. 2 The aim of the present study was to examine the impact of cardiac‐specific overexpression of catalase, an enzyme for H2O2 detoxification, on cardiac contractile function and protein damage in young (3–4 months) and old (26–28 months) male mice. Lifespan was analysed using the Kaplan–Meier survival curve. Cardiomyocyte contractile indices at various stimulus frequencies (0.1–5.0 Hz) were analysed, including peak shortening (PS), time to 90% PS, time to 90% relengthening (TR90) and maximal velocity of shortening/relengthening (±dL/dt). Protein damage was assessed using protein carbonyl formation. Catalase transgenic mice showed longer lifespan than wild‐type FVB mice. The catalase transgene itself did not alter bodyweight or organ weight, or myocyte function. Ageing depressed ±dL/dt and prolonged TR90, but had no effect on other indices in FVB mice. Increased frequency triggered decreases in PS amplitude were exaggerated in aged FVB myocytes. Interestingly, ageing‐induced mechanical defects were significantly attenuated in myocytes from catalase mice. Protein carbonyl formation was elevated in aged FVB compared with young FVB mice, which was significantly diminished in catalase mice. The proteomes of the myocardium of young or old FVB and catalase mice were compared using two‐dimensional gel electrophoresis and mass spectrometry. Six proteins with differential expression between yound and old FVB groups were tentatively identified, some of which were reversed by catalase. 3 In summary, the present data suggest that catalase protects cardiomyocytes from ageing‐induced contractile defects and protein damage.

Possible involvement of NADPH oxidase and JNK in homocysteine-induced oxidative stress and apoptosis in human umbilical vein endothelial cells

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases, although the mechanism leading to vascular dysfunction is not clear. The aim of this study was to examine the effect of homocysteine (Hcy) on oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were challenged for 24 h with Hcy (10 μM-3 mM) in the presence of various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin (100 μM), the p38 mitogen-activated protein kinase inhibitor SB203580 (2.5 μM), the extracellular signal-regulated kinase inhibitor U0126 (2.5 μM), the stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) inhibitor JNK inhibitor II (10 μM), and antioxidants α-tocopherol (5 μg/mL) and N-acetyl cysteine (NAC, 2 mM). Reactive oxygen species (ROS) were detected using 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4′,6′-diamidino-2′-phenylindoladihydrochloride staining, annexin-V phosphatidyl-serine/propidium iodide, and caspase-3 assay. NADPH oxidase and SAPK/JNK signal were evaluated with immunoblotting. Hcy significantly enhanced ROS generation and apoptosis after 24-h incubation. Apocynin prevented Hcy-induced ROS generation but only partially restored Hcy-induced apoptosis. JNK inhibitor II, α-tocopherol, and NAC partially reduced Hcy-induced apoptosis, although SB203580 and U0126 had no effect. Immunoblotting analysis confirmed upregulation of NADPH oxidase and SAPK/JNK signaling. Collectively, our results suggested that Hcy may induce oxidative stress and apoptosis through an NADPH oxidase and/or JNK-dependent mechanisms(s).

Malondialdehyde inhibits cardiac contractile function in ventricular myocytes via a p38 mitogen-activated protein kinase-dependent mechanism.

Increased oxidative stress plays a significant role in the etiology of cardiovascular disease. Lipid peroxidation, initiated in the presence of hydroxy radicals resulting in the production of malondialdehyde, directly produces oxidative stress. This study was designed to examine the direct impact of malondialdehyde on ventricular contractile function at the single cardiac myocyte level. Ventricular myocytes from adult rat hearts were stimulated to contract at 0.5 Hz, and mechanical and intracellular Ca2+ properties were evaluated using an IonOptix Myocam® system. Contractile properties analyzed included peak shortening amplitude (PS), time‐to‐PS (TPS), time‐to‐90% relengthening (TR90), maximal velocity of shortening/relengthening (±dLdt), and Ca2+‐induced intracellular Ca2+ fluorescence release (CICR) and intracellular Ca2+ decay (τ). p38 mitogen‐activated protein (MAP) kinase phosphorylation was assessed with Western blot. Our results indicated that malondialdehyde directly depressed PS, ±dLdt and CICR in a concentration‐dependent manner and shortened TPS without affecting TR90 and τ. Interestingly, the malondialdehyde‐induced cardiac mechanical effect was abolished by both the p38 MAP kinase inhibitor SB203580 (1 and 10 μM) and the antioxidant vitamin C (100 μM). Western blot analysis confirmed direct phosphorylation of p38 MAP kinase by malondialdehyde. These findings revealed a novel role of malondialdehyde and p38 MAP kinase in lipid peroxidation and oxidative stress‐associated cardiac dysfunction.

Benefit and risk of exercise on myocardial function in diabetes.

Regular physical activity promotes cardiorespiratory fitness and has been considered a cornerstone for non-pharmacological treatment of more than 17 million Americans with diabetes mellitus. Physical exercise has been shown to positively affect certain cardiovascular risk factors such as insulin resistance, glucose metabolism, blood pressure and body fat composition, which are closely associated with diabetes and heart disease. With the increasingly sedentary life style in our society, routine daily exercise of moderate intensity is highly recommended to reduce cardiovascular risk, the leading cause of death in diabetic patients. Exercise produces many beneficial effects to the heart function such as reduced incidence of coronary heart disease, attenuated severity of diabetic cardiomyopathy, improved cardiac performance, cardiac reserve and autonomic regulation. Nevertheless, many diabetic patients do not appear to gain much benefit from exercise or may even be at risk of performing physical exercise. This review summarizes the benefit and risk of exercise on diabetic heart function, with a special emphasis on myocardial and autonomic function.

Aldehyde dehydrogenase-2 transgene ameliorates chronic alcohol ingestion-induced apoptosis in cerebral cortex.

Chronic intake of alcohol results in multiple organ damage including brain. This study was designed to examine the impact of facilitated acetaldehyde breakdown via transgenic overexpression of mitochondrial aldehyde dehydrogenase-2 (ALDH2) on alcohol-induced cerebral cortical injury. ALDH2 transgenic mice were produced using the chicken beta-actin promoter. Wild-type FVB and ALDH2 mice were placed on a 4% alcohol or control diet for 12 weeks. Protein damage and apoptosis were evaluated with carbonyl formation, caspase and TUNEL assays. Western blot was performed to examine expression (or its activation) of ALDH2, the pro- and anti-apoptotic proteins caspase-8, Bax, Bcl-2, Omi/HtrA2, apoptosis repressor with caspase recruitment domain (ARC), FLICE-like inhibitory protein (FLIP), X-linked inhibitor of apoptosis protein (XIAP), Akt, glycogen synthase kinase-3beta (GSK-3beta), p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Chronic alcohol intake led to elevated apoptosis in the absence of overt protein damage, the effect of which was ablated by the overexpression of ALDH2 transgene. Consistently, ALDH2 transgene significantly attenuated alcohol-induced upregulation of Bax, Omi/HtrA2 and XIAP as well as downregulation of Bcl-2 and ARC without affecting alcohol-induced increase of FLIP in cerebral cortex. Phosphorylation of Akt and GSK-3beta was dampened while total/phosphorylated JNK and p38 phosphorylation were elevated following chronic alcohol intake, the effects of which were abrogated by ALDH2 transgene. Expression of total Akt, GSK-3beta, p38 and ERK (total or phosphorylated) was not affected by either chronic alcohol intake or ALDH2 transgene. Our results suggested that transgenic overexpression of ALDH2 rescues chronic alcoholism-elicited cerebral injury possibly via a mechanism associated with Akt, GSK-3beta, p38 and JNK signaling.