The failing human heart expresses tumor necrosis factor-alpha (TNF-alpha). However, its pathophysiological significance is not clear. We previously reported that robust overexpression of TNF-alpha in the murine heart causes lethal myocarditis. In this study, we modified the transgene to reduce the production of TNF-alpha by preserving the destabilizing sequence in TNF-alpha cDNA. Expression was driven by the murine alpha-myosin heavy chain promoter. Use of this modified construct allowed to the establish a mutine transgenic line (TG). TG offspring were examined at 6, 12, and 24 weeks. All showed a significantly higher heart weight-to-body weight ratio. Northern blot analysis confirmed the expression of transgene in the heart, and enzyme-linked immunosorbent assay demonstrated the presence of TNF-alpha protein. The TG heart demonstrated a mild, diffuse, lymphohistiocytic interstitial inflammatory infiltrate. Cardiomyocyte necrosis and apoptosis were present but not abundant. Magnetic resonance imaging showed that the TG heart was significantly dilated with reduced ejection fraction. Although the left ventricular dP/dtmax was not different at baseline, its responsiveness to isoproterenol was significantly blunted in TG. Atrial natriuretic factor was expressed in the TG ventricle. A group of TG died spontaneously, and subsequent autopsies revealed exceptional dilation of the heart, increased lung weight, and pleural effusion, suggesting that they died of congestive heart failure. The cumulative mortality rate at 6 months was 23%. In conclusion, the mouse overexpressing TNF-alpha recapitulated the phenotype of congestive heart failure. This provides a novel model to elucidate the role of this cytokine in the development of congestive heart failure.

Paleogenetics is an emerging field that resurrects ancestral proteins from now-extinct organisms to test, in the laboratory, models of protein function based on natural history and Darwinian evolution. Here, we resurrect digestive alcohol dehydrogenases (ADH4) from our primate ancestors to explore the history of primate-ethanol interactions. The evolving catalytic properties of these resurrected enzymes show that our ape ancestors gained a digestive dehydrogenase enzyme capable of metabolizing ethanol near the time that they began using the forest floor, about 10 million y ago. The ADH4 enzyme in our more ancient and arboreal ancestors did not efficiently oxidize ethanol. This change suggests that exposure to dietary sources of ethanol increased in hominids during the early stages of our adaptation to a terrestrial lifestyle. Because fruit collected from the forest floor is expected to contain higher concentrations of fermenting yeast and ethanol than similar fruits hanging on trees, this transition may also be the first time our ancestors were exposed to (and adapted to) substantial amounts of dietary ethanol.experimental paleogenetics | alcohol dehydrogenase | ethanol | primates | evolution

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Background-Tumor necrosis factor (TNF)-␣ plays a pathophysiological role in heart failure. Although both TNF receptor 1 (TNFR1) and 2 (TNFR2) are present in the heart, comparatively little is known about the role of TNFR2. Methods and Results-We bred TNFR1-knockout (KO) or TNFR2KO mice to transgenic (TG) mice with cardiac-specific overexpression of TNF-␣ and analyzed resultant progeny. Six groups of male and female mice were studied: wild type (WT) with wild receptors (WT/W), TG with wild receptors (TG/W), TG with heterozygous receptor KO (TG/R1 ϩ/Ϫ or TG/R2 ϩ/Ϫ ), and TG with homozygous receptor KO (TG/R1 Ϫ/Ϫ or TG/R2 Ϫ/Ϫ ). Both male and female TG mice displayed cardiac hypertrophy, dilation, and reduced cardiac function. Male TG mice were more severely affected than genotypically matched females and died of heart failure at a younger age. Survival, cardiac function, and remodeling of TG/R1 ϩ/Ϫ and TG/R1 Ϫ/Ϫ mice were improved relative to TG/W mice in both males and females. However, the survival of female TG/R2 ϩ/Ϫ and TG/R2 Ϫ/Ϫ mice was worse than that of TG/W mice, with increased left ventricular dimension and left ventricular weight/body weight ratios. The cardiac TNF-␣ protein level was upregulated in TG/R1

IntroductionEpidemiologic studies have observed important differences in survival between men and women with heart failure (1-5). For example, the population-based Framingham Heart Study found that after the onset of symptomatic heart failure, the prognosis of women was significantly better than men (3,4). Similar observations were reported in patients with advanced heart failure, and these sex-related differences could not be attributed to a high incidence of coronary disease in men (1, 2). Furthermore, hormone-replacement therapy appeared to effect improved survival in postmenopausal women with nonischemic heart failure (6).Sex-related differences in the physiologic response to stress have also been identified in spontaneously hypertensive rats (7), transgenic mice overexpressing the cardiac Na + /Ca 2+ exchanger (8), and rats with pressure overload-induced hypertrophy (9). However, studies in these animal models of heart failure have not assessed sexrelated survival nor have they elucidated underlying mechanisms that could account for these differences.Recently, we developed transgenic mice (TNF1.6) with cardiac-specific overexpression of TNF-α and have characterized their progression to congestive heart failure (10, 11). These transgenic mice developed ventricular hypertrophy, cardiac dilatation, interstitial infiltrates and fibrosis, attenuation of adrenergic responsiveness, and re-expression of atrial natriuretic factor in the ventricle. These mice had a 6-month mortality of nearly 25%. Furthermore, the mice that died spontaneously demonstrated exceptional dilatation of the heart, organized atrial thrombus, and massive pleural effusion, suggesting that they died of congestive heart failure (10). These characterizations have been confirmed by a separate group studying a similar transgenic model (12,13). In this study we report the presence of sex-related differences in survival of TNF1.6 mice and have begun to elucidate cellular pathways that might be of etiologic importance in affecting sexrelated differences. Methods TNF-α transgenic mice.Studies used previously characterized transgenic mice (TNF1.6) (10). For RNA and protein analysis, animals were sacrificed, and excised ventricle or liver tissues were snap-frozen in liquid nitrogen. Blood samples were collected, and peripheral white blood cells were isolated by lysing erythrocytes with 1.7% NH 4 Cl. The mice were handled according to protocols approved by the Institutional Animal Care and Use Committee, University of Pittsburgh.Experimental design. Survival analysis was used to assess differences between male and female transgenic mice. Echocardiography was performed to compare the cardiac structure and function between the sexes in vivo. To assess whether phenotypic differences arose from differ- Epidemiological evidence suggests that the prognosis of heart failure in women is better than in men. In our murine model of dilated cardiomyopathy arising from cardiac-specific overexpression of TNF-α, the 6-month survival rate was significantly better in fem...