Cindy X. Fang

Metallothionein prolongs survival and antagonizes senescence-associated cardiomyocyte diastolic dysfunction: role of oxidative stress

Senescence is accompanied by oxidative stress and cardiac dysfunction, although the link between the two remains unclear. This study examined the role of antioxidant metallothionein on cardiomyocyte function, superoxide generation, the oxidative stress biomarker aconitase activity, cytochrome c release, and expression of oxidative stress‐related proteins, such as the GTPase RhoA and NADPH oxidase protein p47phox in young (5–6 mo) and aged (26–28 mo) FVB wild‐type (WT) and cardiac‐specific metallothionein transgenic mice. Metallothionein mice showed a longer life span (by ∼4 mo) than FVB mice evaluated by the Kaplan‐Meier survival curve. Compared with young cardiomyocytes, aged myocytes displayed prolonged TR90, reduced tolerance to high stimulus frequency, and slowed intracellular Ca2+ decay, all of which were nullified by metallothionein. Aging increased superoxide generation, active RhoA abundance, cytochrome c release, and p47phox expression and suppressed aconitase activity without affecting protein nitrotyrosine formation in the hearts. These aging‐induced changes in oxidative stress and related protein biomarkers were attenuated by metallothionein. Aged metallothionein mouse myocytes were more resistant to the superoxide donor pyrogallol‐induced superoxide generation and apoptosis. In addition, aging‐associated prolongation in TR90 was blunted by the Rho kinase inhibitor Y‐27632. Collectively, our data demonstrated that metallothionein may alleviate aging‐induced cardiac contractile defects and oxidative stress, which may contribute to prolonged life span in metallothionein transgenic mice.—Yang, X., Doser, T. A., Fang, C. X., Nunn, J. M., Janardhanan, R., Zhu, M., Sreejayan, N., Quinn, M. T., Ren, J. Metallothionein prolongs survival and antagonizes senescence‐associated cardiomyocyte diastolic dysfunction: role of oxidative stress FASEB J. 20, E260–E270 (2006)

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.

Hypertrophic cardiomyopathy in high-fat diet-induced obesity: role of suppression of forkhead transcription factor and atrophy gene transcription

Cellular hypertrophy is regulated by coordinated pro- and antigrowth machineries. Foxo transcription factors initiate an atrophy-related gene program to counter hypertrophic growth. This study was designed to evaluate the role of Akt, the forkhead transcription factor Foxo3a, and atrophy genes muscle-specific RING finger (MuRF)-1 and atrogin-1 in cardiac hypertrophy and contractile dysfunction associated with high-fat diet-induced obesity. Mice were fed a low- or high-fat diet for 6 mo along with a food-restricted high-fat weight control group. Echocardiography revealed decreased fractional shortening and increased end-systolic diameter and cardiac hypertrophy in high-fat obese but not in weight control mice. Cardiomyocytes from high-fat obese but not from weight control mice displayed contractile and intracellular Ca2+ defects including depressed maximal velocity of shortening/relengthening, prolonged duration of shortening/relengthening, and reduced intracellular Ca2+ rise and clearance. Caspase activities were greater in high-fat obese but not in weight control mouse hearts. Western blot analysis revealed enhanced basal Akt and Foxo3a phosphorylation and reduced insulin-stimulated phosphorylation of Akt and Foxo3a without changes in total protein expression of Akt and Foxo3a in high-fat obese hearts. RT-PCR and immunoblotting results displayed reduced levels of the atrogens atrogin-1 and MuRF-1, the upregulated hypertrophic markers GATA4 and ciliary neurotrophic factor receptor-alpha, as well as the unchanged calcineurin and proteasome ubiquitin in high-fat obese mouse hearts. Transfection of H9C2 myoblast cells with dominant-negative Foxo3a adenovirus mimicked palmitic acid (0.8 mM for 24 h)-induced GATA4 upregulation without an additive effect. Dominant-negative Foxo3a-induced upregulation of pAkt and repression of phosphatase and tensin homologue were abrogated by palmitic acid. These results suggest a cardiac hypertrophic response in high-fat diet-associated obesity at least in part through inactivation of Foxo3a by the Akt pathway.

A newly synthetic chromium complex - chromium(phenylalanine)3 improves insulin responsiveness and reduces whole body glucose tolerance

Low‐molecular‐weight organic chromium complexes such as chromium picolinate are often used as dietary supplements to improve insulin sensitivity and to correct dyslipidemia. However, toxicity associated with such chromium compounds has compromised their therapeutic value. The aim of this study was to evaluate the impact of a newly synthesized complex of chromium with phenylalanine, Cr(pa)3 on insulin‐signaling and glucose tolerance. Cr(pa)3 was synthesized by chelating chromium(III) with d‐phenylalanine ligand in aqueous solution. In mouse 3T3‐adipocytes, Cr(pa)3 augmented insulin‐stimulated glucose‐uptake as assessed by a radioactive‐glucose uptake assay. At the molecular level, Cr(pa)3 enhanced insulin‐stimulated phosphorylation of Akt in a time‐ and concentration‐dependent manner without altering the phosphorylation of insulin receptor. Oral treatment with Cr(pa)3 (150 μg/kg/d, for six weeks) in ob/ob(+/+) obese mice significantly alleviated glucose tolerance compared with untreated obese mice. Unlike chromium picolinate, Cr(pa)3 does not cleave DNA under physiological reducing conditions. Collectively, these data suggest that Cr(pa)3 may represent a novel, less‐toxic chromium supplement with potential therapeutic value to improve insulin sensitivity and glycemic control in type II diabetes.

Metallothionein antagonizes aging-induced cardiac contractile dysfunction: role of PTP1B, insulin receptor tyrosine phosphorylation and Akt.

Aging is often accompanied by reduced insulin sensitivity and cardiac dysfunction. However, the causal relationship between the two remains poorly understood. This study was designed to determine the impact of cardiac‐specific overexpression of antioxidant metallothionein (MT) on aging‐associated cardiac dysfunction and impaired insulin signaling. Contractile and intracellular Ca2+ properties were evaluated in left ventricular myocytes including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time‐to‐PS (TPS), time‐to‐90% relengthening (TR90), fura‐2 fluorescence intensity change (ΔFFI) and intracellular Ca2+ decay rate. Expression of insulin receptor, protein‐tyrosine phosphatase 1B (PTP1B), phosphorylation of insulin receptor (Tyr1146) and Akt were evaluated by Western blot analysis. Aged wild‐type FVB and MT transgenic mice (26–28 months old) displayed glucose intolerance and hyperinsulinemia. Cardiomyocytes from aged FVB mice exhibited prolonged TR90 and intracellular Ca2+ decay associated with normal PS, ± dL/dt, TPS and ΔFFI compared with those from young (2–3 months old) mice. Western blot analysis revealed reduced Akt expression and insulin (5 mU g−1)‐stimulated Akt phosphorylation, elevated PTP1B expression and diminished basal insulin receptor tyrosine phosphorylation associated with comparable insulin receptor expression in aged FVB mouse hearts. All of these aging‐related defects in cardiac contractile function and insulin signaling (although not hyperinsulinemia and glucose intolerance) were significantly attenuated or ablated by MT transgene. These data indicate that enhanced antioxidant defense is beneficial for aging‐induced cardiac contractile dysfunction and alteration in insulin signaling.

Small guanine nucleotide-binding protein Rho and myocardial function

AbstractRhoA and Rho-kinase (ROCK) participate in a wide variety of cell signal functions such as cell growth, smooth and cardiac muscle contraction, cytoskeleton rearrangement, cell migration and proliferation. In vascular smooth muscle cells, RhoA and ROCK play an important role in Ca2+ sensitization and regulate vascular smooth muscle tone. In the heart, RhoA and ROCK mediate hypertrophic response leading to cardiac hypertrophy. Recent cellular and molecular biology studies using ROCK inhibitors such as Y-27632 and fasudil have indicated a pivotal role of the RhoA-ROCK cascade in many aspects of cardiovascular function such as cardiac hypertrophy and ventricular remodeling following myocardial infarction. Inhibition of the RhoA-ROCK signaling pathway may be a suitable target for a number of cardiovascular diseases including hypertension, atherosclerosis, diabetes and hypertrophic heart failure. This review focuses on the current understanding of the RhoA-ROCK signal pathway in heart diseases and discusses the use of ROCK inhibitors as therapeutic agents for heart diseases ranging from hypertensive cardiomyopathy to heart failure.

Enhanced pulmonary inflammation following experimental intracerebral hemorrhage.

The association between brain damage and respiratory dysfunction has been recognized although mechanistic link between the two is still poorly defined. Intracerebral hemorrhage is accompanied by brain injury, stroke, and parenchymal hematoma formation with surrounding inflammation. Increase intracranial pressure as a result of intracerebral hemorrhage may promote localized activation of cytokines and coagulation system including tissue factor release. However, whether intracerebral hemorrhage triggers inflammation in noncerebral organs has not been elucidated. The aim of the present study was to examine the impact of intracerebral hemorrhage on lung inflammatory response. Intracerebral hemorrhage was induced by stereotaxic intrastriatal administration of bacterial collagenase. Expression of intracellular adhesion molecule-1 (ICAM-1), IKB-alpha, tissue factor, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) was evaluated by Western blot analysis. Our results revealed that intracerebral hemorrhage upregulated expression of ICAM-1 and tissue factor in both brain and lung, whereas it enhanced TNF-alpha and IL-1beta mainly in brain within 6 and 24 h of the brain injury. Levels of IKB-alpha remained unchanged in brain and lung tissues. Appearance of inflammatory markers in the lung was accompanied by morphological pulmonary damage. These data suggest that intracerebral hemorrhage may trigger acute inflammatory response in both brain and lung.

Phytoestrogen α-zearalanol antagonizes homocysteine-induced imbalance of nitric oxide/endothelin-1 and apoptosis in human umbilical vein endothelial cells

Although the issue of estrogen replacement therapy on cardiovascular health is debatable, it has presumable benefits for endothelial function in postmenopausal women. However, the fear of breast cancer has intimidated women contemplating estrogen treatment and limited its long-term application. An effective alternative remedy not associated with breast carcinoma is in serious demand. This study was designed to examine the effect of phytoestrogen α-zearalanol (α-ZAL) and 17β-estradiol (E2) on nitric oxide (NO) and endothelin (ET)-1 levels, apoptosis, and apoptotic enzymes in human umbilical vein endothelial cells (HUVEC). HUVEC cells were challenged for 24 h with homocysteine (10−3M), an independent risk factor for a variety of vascular diseases, in the presence of α-ZAL or E2 (10−9 to 10−6M). Release of NO and ET-1 were measured with enzyme immunoassay. Apoptosis was evaluated by fluorescence-activated cell sorter analysis. Expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), Bax, and Bcl-2 were determined using Western blot. NOS activity was evaluated with 3H-arginine to 3H-citrulline conversion. Our results indicated that Hcy significantly reduced NO production, NOS activity, enhanced ET-1/NO ratio and apoptosis, upregulated iNOS, Bax, and downregulated eNOS, Bcl-2 expression. These effects were significantly attenuated by α-ZAL and E2. ZAL displayed a similar potency compared with E2 in antagonizing Hcy-induced effects. In summary, these results suggested that α-ZAL may effectively preserve Hcy-induced decrease in NO, increase in ET-1/NO ratio and apoptosis, which contributes to protective effects of phytoestrogens on endothelial function.

Acetaldehyde promotes rapamycin-dependent activation of p70S6K and glucose uptake despite inhibition of Akt and mTOR in dopaminergic SH-SY5Y human neuroblastoma cells

Alcohol intake is one of the important lifestyle factors for the risk of insulin resistance and type 2 diabetes. Acetaldehyde, the major ethanol metabolite which is far more reactive than ethanol, has been postulated to participate in alcohol-induced tissue injury although its direct impact on insulin signaling is unclear. This study was designed to examine the effect of acetaldehyde on glucose uptake and insulin signaling in human dopaminergic SH-SY5Y cells. Akt, mammalian target of rapamycin (mTOR), ribosomal-S6 kinase (p70(S6K)), the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and insulin receptor substrate (IRS)-2 were evaluated by Western blot analysis. Glucose uptake and apoptosis were measured using [(3)H]-2-deoxyglucose uptake and caspase-3 assay, respectively. Short-term exposure (12 h) of acetaldehyde (150 muM) facilitated glucose uptake in a rapamycin-dependent manner without affecting apoptosis, IRS-2 expression and insulin-stimulated glucose uptake in SH-SY5Y cells. Acetaldehyde suppressed basal and insulin-stimulated Akt phosphorylation without affecting total Akt expression. Acetaldehyde inhibited mTOR phosphorylation without affecting total mTOR and insulin-elicited response on mTOR phosphorylation. Rapamycin, which inhibits mTOR leading to inactivation of p70(S6K), did not affect acetaldehyde-induced inhibition on phosphorylation of Akt and mTOR. Interestingly, acetaldehyde enhanced p70(S6K) activation and depressed 4E-BP1 phosphorylation, the effect of which was blunted and exaggerated, respectively, by rapamycin. Collectively, these data suggested that acetaldehyde did not adversely affect glucose uptake despite inhibition of insulin signaling cascade at the levels of Akt and mTOR, possibly due to presence of certain mechanism(s) responsible for enhanced p70(S6K) phosphorylation.

Intracerebral Hemorrhage Elicits Aberration in Cardiomyocyte Contractile Function and Intracellular Ca2+ Transients

Background and Purpose— The sequelae of intracerebral hemorrhage involve multiple organ damage including electrocardiographic alteration, although the mechanism(s) behind myocardial dysfunction is unknown. The aim of this study was to examine the impact of intracerebral hemorrhage on cardiomyocyte contractile function, intracellular Ca2+ handling, Ca2+ cycling proteins, I kappa B beta protein (I&kgr;B) phosphorylation, hypoxia-inducible factor 1&agr; (HIF-1&agr;), and nitrosative damage within 48 hours of injury. Methods— Mechanical and intracellular Ca2+ properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), fura-2 fluorescence intensity (FFI), and intracellular Ca2+ decay. Results— Myocytes from intracerebral hemorrhage rats exhibited depressed PS, ±dL/dt, prolonged TPS and TR90, as well as declined baseline FFI and slowed intracellular Ca2+ decay between 12 and 24 hours after injury. Most of these aberrations returned to normal levels 48 hours after hemorrhage with the exception of −dL/dt and TR90. Myocytes from 24-hour posthemorrhage rats exhibited a stepper negative staircase in PS with increased stimulus frequency. Cardiac expression of sarco(endo)plasmic reticulum Ca2+-ATPase 2a and phospholamban was enhanced, whereas that of Na+-Ca2+ exchanger and voltage-dependent K+ channel was decreased. I&kgr;B phosphorylation, HIF-1α, inducible NO synthase, and 3-nitrotyrosine were enhanced 12 hours after injury. Conclusions— These data demonstrated that intracerebral hemorrhage initiates cardiomyocyte contractile and intracellular Ca2+ dysregulation possibly related to altered expression of Ca2+ cycling proteins, nitrosative damage, and myocardial phosphorylation of I&kgr;B.