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Cold exposure is associated with oxidative stress and cardiac dysfunction. The endothelin (ET) system, which plays a key role in myocardial homeostasis, may participate in cold exposure-induced cardiovascular dysfunction. This study was designed to examine the role of ET-1 in cold stress-induced cardiac geometric and contractile responses. Wild-type (WT) and ETA receptor knockout (ETAKO) mice were assigned to normal or cold exposure (4°C) environment for 2 and 5 weeks prior to evaluation of cardiac geometry, contractile, and intracellular Ca2+ properties. Levels of the temperature sensor transient receptor potential vanilloid (TRPV1), mitochondrial proteins for biogenesis and oxidative phosphorylation, including UCP2, HSP90, and PGC1α were evaluated. Cold stress triggered cardiac hypertrophy, depressed myocardial contractile capacity, including fractional shortening, peak shortening, and maximal velocity of shortening/relengthening, reduced intracellular Ca2+ release, prolonged intracellular Ca2+ decay and relengthening duration, generation of ROS and superoxide, as well as apoptosis, the effects of which were blunted by ETAKO. Western blotting revealed downregulated TRPV1 and PGC1α as well as upregulated UCP2 and activation of GSK3β, GATA4, and CREB in cold-stressed WT mouse hearts, which were obliterated by ETAKO. Levels of HSP90, an essential regulator for thermotolerance, were unchanged. The TRPV1 agonist SA13353 attenuated whereas TRPV1 antagonist capsazepine mimicked cold stress- or ET-1-induced cardiac anomalies. The GSK3β inhibitor SB216763 ablated cold stress-induced cardiac contractile (but not remodeling) changes and ET-1-induced TRPV1 downregulation. These data suggest that ETAKO protects against cold exposure-induced cardiac remodeling and dysfunction mediated through TRPV1 and mitochondrial function.

Aging is often accompanied with geometric and functional changes in the heart, although the underlying mechanisms remain unclear. Recent evidence has described a potential role of Akt and autophagy in aging-associated organ deterioration. This study was to examine the impact of cardiac-specific Akt activation on aging-induced cardiac geometric and functional changes and underlying mechanisms involved. Cardiac geometry, contractile and intracellular Ca(2+) properties were evaluated using echocardiography, edge-detection and fura-2 techniques. Level of insulin signaling and autophagy was evaluated by western blot. Our results revealed cardiac hypertrophy (enlarged chamber size, wall thickness, myocyte cross-sectional area), fibrosis, decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca(2+) release and clearance in aged (24-26 month-old) mice compared with young (3-4 month-old) mice, the effects of which were accentuated by chronic Akt activation. Aging enhanced Akt and mTOR phosphorylation while reducing that of PTEN, AMPK and ACC with a more pronounced response in Akt transgenic mice. GSK3β phosphorylation and eNOS levels were unaffected by aging or Akt overexpression. Levels of beclin-1, Atg5 and LC3-II-to-LC3-I ratio were decreased in aged hearts, the effect of which with the exception of Atg 5 was exacerbated by Akt overactivation. Levels of p62 were significantly enhanced in aged mice with a more pronounced increase in Akt mice. Neither aging nor Akt altered β-glucuronidase activity and cathepsin B although aging reduced LAMP1 level. In addition, rapamycin reduced aging-induced cardiomyocyte contractile and intracellular Ca(2+) dysfunction while Akt activation suppressed autophagy in young but not aged cardiomyocytes. In conclusion, our data suggest that Akt may accentuate aging-induced cardiac geometric and contractile defects through a loss of autophagic regulation.

Obesity is associated with oxidative stress and mitochondrial and myocardial dysfunction, although interaction among which remains elusive. This study was designed to evaluate the impact of the free radical scavenger metallothionein on high-fat diet-induced myocardial, intracellular Ca(2+), and mitochondrial dysfunction. FVB and metallothionein transgenic mice were fed a high- or low-fat diet for 5 months to induce obesity. Echocardiography revealed decreased fractional shortening, increased end-systolic diameter, and cardiac hypertrophy in high-fat-fed FVB mice. Cardiomyocytes from high-fat-fed FVB mice displayed enhanced reactive oxygen species (ROS) production, contractile and intracellular Ca(2+) defects including depressed peak shortening and maximal velocity of shortening/relengthening, prolonged duration of relengthening, and reduced intracellular Ca(2+) rise and clearance. Transmission microscopy noted overt mitochondrial damage with reduced mitochondrial density. Western blot analysis revealed enhanced phosphorylation of nuclear factor Foxo3a without changes in Foxo3a, Foxo1a, pFoxo1a, silent information regulator (Sirt), and Akt and pAkt in hearts of high-fat diet-fed FVB mice. The peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), a key regulator of mitochondrial biogenesis, was significantly depressed by high-fat diet feeding and in vitro palmitic acid treatment. RT-PCR further depicted reduced levels of the PGC-1alpha downstream nuclear respiratory factors 1 and 2, mitochondrial transcription factor A, and mitochondrial DNA copy number in hearts of high-fat-fed FVB mice. Intriguingly, the high-fat diet-induced alterations in ROS, myocardial contractile, and mitochondrial and cell signaling were negated by metallothionein, with the exception of pFoxo3a. These data suggest that metallothionein may protect against high-fat diet-induced cardiac dysfunction possibly associated with upregulation of PGC-1alpha and preservation of mitochondrial biogenesis.

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.

OBJECTIVES This study was designed to evaluate the interaction between aging and obesity on cardiac contractile and intracellular Ca2+ properties. METHODS Cardiomyocytes from young (4-mo) and aging (12- and 18-mo) male lean and the leptin deficient ob/ob obese mice were treated with leptin (0.5, 1.0 and 50 nM) for 4 hrs in vitro. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obesity models at young and older age were used for comparison. Cardiomyocyte contractile 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 (TR(90)), intracellular Ca2+ levels and decay. O2(-) levels were measured by dihydroethidium fluorescence. RESULTS Our results revealed reduced survival in ob/ob mice. Aging and obesity reduced PS, +/- dL/dt, intracellular Ca2+ rise, prolonged TR(90) and intracellular Ca2+ decay, enhanced O2(-) production and p(47phox) expression without an additive effect of the two, with the exception of intracellular Ca2+ rise. Western blot analysis exhibited reduced Ob-R expression and STAT-3 phosphorylation in both young and aging ob/ob mice, which was restored by leptin. Aging and obesity reduced phosphorylation of Akt, eNOS and p38 while promoting pJNK and pIkappaB. Low levels of leptin reconciled contractile, intracellular Ca2+ and cell signaling defects as well as O2(-) production and p(47phox) upregulation in young but not aging ob/ob mice. High level of leptin (50 nM) compromised contractile and intracellular Ca2+ response as well as O2(-) production and stress signaling in all groups. High fat diet-induced and db/db obesity displayed somewhat comparable aging-induced mechanical but not leptin response. CONCLUSIONS Taken together, our data suggest that aging and obesity compromise cardiac contractile function possibly via phosphorylation of Akt, eNOS and stress signaling-associated O2(-) release.