Mitochondrial aldehyde dehydrogenase 2 accentuates aging-induced cardiac remodeling and contractile dysfunction: role of AMPK, Sirt1, and mitochondrial function.

Cardiac aging is associated with compromised myocardial function and morphology although the underlying mechanism remains elusive. Aldehyde dehydrogenase 2 (ALDH2), an essential mitochondrial enzyme governing cardiac function, displays polymorphism in humans. This study was designed to examine the role of ALDH2 in aging-induced myocardial anomalies. Myocardial mechanical and intracellular Ca(2+) properties were examined in young (4-5 months) and old (26-28 months) wild-type and ALDH2 transgenic mice. Cardiac histology, mitochondrial integrity, O2(-) generation, apoptosis, and signaling cascades, including AMPK activation and Sirt1 level were evaluated. Myocardial function and intracellular Ca(2+) handling were compromised with advanced aging; the effects were accentuated by ALDH2. Hematoxylin and eosin and Masson trichrome staining revealed cardiac hypertrophy and interstitial fibrosis associated with greater left-ventricular mass and wall thickness in aged mice. ALDH2 accentuated aging-induced cardiac hypertrophy but not fibrosis. Aging promoted O2(-) release, apoptosis, and mitochondrial injury (mitochondrial membrane potential, levels of UCP-2 and PGC-1α), and the effects were also exacerbated by ALDH2. Aging dampened AMPK phosphorylation and Sirt1, the effects of which were exaggerated by ALDH2. Treatment with the ALDH2 activator Alda-1 accentuated aging-induced O2(-) generation and mechanical dysfunction in cardiomyocytes, the effects of which were mitigated by cotreatment with activators of AMPK and Sirt1, AICAR, resveratrol, and SRT1720. Examination of human longevity revealed a positive correlation between life span and ALDH2 gene mutation. Taken together, our data revealed that ALDH2 enzyme may accentuate myocardial remodeling and contractile dysfunction in aging, possibly through AMPK/Sirt1-mediated mitochondrial injury.

Cell proliferation and interleukin-6-type cytokine signaling are implicated by gene expression responses in early optic nerve head injury in rat glaucoma.

PURPOSE: In glaucoma, the optic nerve head (ONH) is the principal site of initial axonal injury, and elevated intraocular pressure (IOP) is the predominant risk factor. However, the initial responses of the ONH to elevated IOP are unknown. Here the authors use a rat glaucoma model to characterize ONH gene expression changes associated with early optic nerve injury. METHODS: Unilateral IOP elevation was produced in rats by episcleral vein injection of hypertonic saline. ONH mRNA was extracted, and retrobulbar optic nerve cross-sections were graded for axonal degeneration. Gene expression was determined by microarray and quantitative PCR (QPCR) analysis. Significantly altered gene expression was determined by multiclass analysis and ANOVA. DAVID gene ontology determined the functional categories of significantly affected genes. RESULTS: The Early Injury group consisted of ONH from eyes with <15% axon degeneration. By array analysis, 877 genes were significantly regulated in this group. The most significant upregulated gene categories were cell cycle, cytoskeleton, and immune system process, whereas the downregulated categories included glucose and lipid metabolism. QPCR confirmed the upregulation of cell cycle-associated genes and leukemia inhibitory factor (Lif) and revealed alterations in expression of other IL-6-type cytokines and Jak-Stat signaling pathway components, including increased expression of IL-6 (1553%). In contrast, astrocytic glial fibrillary acidic protein (Gfap) message levels were unaltered, and other astrocytic markers were significantly downregulated. Microglial activation and vascular-associated gene responses were identified. CONCLUSIONS: Cell proliferation and IL-6-type cytokine gene expression, rather than astrocyte hypertrophy, characterize early pressure-induced ONH injury.

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.

Transgenic overexpression of aldehyde dehydrogenase-2 rescues chronic alcohol intake-induced myocardial hypertrophy and contractile dysfunction.

BACKGROUND: Chronic alcoholism leads to the onset and progression of alcoholic cardiomyopathy through toxic mechanisms of ethanol and its metabolite, acetaldehyde. This study examined the impact of altered acetaldehyde metabolism through systemic transgenic overexpression of aldehyde dehydrogenase-2 (ALDH2) on chronic alcohol ingestion-induced myocardial damage. METHODS AND RESULTS: ALDH2 transgenic mice were produced with the chicken beta-actin promoter. Wild-type FVB and ALDH2 mice were placed on a 4% alcohol diet or a control diet for 14 weeks. Myocardial and cardiomyocyte contraction, intracellular Ca(2+) handling, histology (hematoxylin and eosin, Masson trichrome), protein damage, and apoptosis were determined. Western blot was used to monitor the expression of NADPH oxidase, calcineurin, apoptosis-stimulated kinase (ASK-1), glycogen synthase kinase-3beta (GSK-3beta), GATA4, and cAMP-response element binding (CREB) protein. ALDH2 reduced the chronic alcohol ingestion-induced elevation in plasma and tissue acetaldehyde levels. Chronic alcohol consumption led to cardiac hypertrophy, reduced fractional shortening, cell shortening, and impaired intracellular Ca(2+) homeostasis, the effect of which was alleviated by ALDH2. In addition, the ALDH2 transgene significantly attenuated chronic alcohol intake-induced myocardial fibrosis, protein carbonyl formation, apoptosis, enhanced NADPH oxidase p47(phox) and calcineurin expression, as well as phosphorylation of ASK-1, GSK-3beta, GATA4, and CREB. CONCLUSIONS: The present results suggest that transgenic overexpression of ALDH2 effectively antagonizes chronic alcohol intake-elicited myocardial hypertrophy and contractile defect through a mechanism that is associated, at least in part, with phosphorylation of ASK-1, GSK-3beta, GATA4, and CREB. These data strongly support the notion that acetaldehyde may be an essential contributor to the chronic development of alcoholic cardiomyopathy.

Global changes in optic nerve head gene expression after exposure to elevated intraocular pressure in a rat glaucoma model.

PURPOSE: In glaucoma, the optic nerve head (ONH) is the likely site of initial injury and elevated intraocular pressure (IOP) is the best-known risk factor. This study determines global gene expression changes in the pressure-injured ONH. METHODS: Unilateral sustained IOP elevation (glaucoma, n = 46) or optic nerve transection (n = 10) was produced in rats. ONHs were removed, and the retrobulbar optic nerves were graded for degeneration. Gene expression in the glaucomatous ONH with extensive injury was compared with that in the fellow ONH (n = 6/group), by using cDNA microarrays. Data from 12 arrays were normalized, significant differences in gene expression determined, and significantly affected gene classes identified. For the remaining ONH, grouped by experimental condition and degree of injury, quantitative reverse transcriptase-PCR (qPCR) and ANOVA were used to compare selected message levels. RESULTS: Microarray analysis identified more than 2000 significantly regulated genes. For 225 of these genes, the changes were greater than twofold. The most significantly affected gene classes were cell proliferation, immune response, lysosome, cytoskeleton, extracellular matrix, and ribosome. A 2.7-fold increase in ONH cellularity confirmed glaucoma model cell proliferation. By qPCR, increases in levels of periostin, collagen VI, and transforming growth factor beta1 were linearly correlated to the degree of IOP-induced injury. For cyclinD1, fibulin 2, tenascin C, TIMP1, and aquaporin-4, correlations were significantly nonlinear, displaying maximum change with focal injury. CONCLUSIONS: In the ONH, pressure-induced injury results in cell proliferation and dramatically altered gene expression. For specific genes, expression levels were most altered by focal injury, suggesting that further array studies may identify initial, and potentially injurious, altered processes.

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 Ca(2+) 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 (TR(90)), fura-2 fluorescence intensity change (DeltaFFI) and intracellular Ca(2+) 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 TR(90) and intracellular Ca(2+) decay associated with normal PS, +/- dL/dt, TPS and DeltaFFI 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.

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 approximately 4 mo) than FVB mice evaluated by the Kaplan-Meier survival curve. Compared with young cardiomyocytes, aged myocytes displayed prolonged TR(90), 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.