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Our novel proposal is that TNFα exerts a direct effect on mitochondrial respiratory function in the heart, independently of its cell surface receptors. TNFα-induced cardioprotection is known to involve reactive oxygen species (ROS) and sphingolipids. We therefore further propose that this direct mitochondrial effect is mediated via ROS and sphingolipids. The protective concentration of TNFα (0.5 ng/ml) was added to isolated heart mitochondria from black 6 × 129 mice (WT) and double TNF receptor knockout mice (TNFR1&2(-/-)). Respiratory parameters and inner mitochondrial membrane potential were analyzed in the presence/absence of two antioxidants, N-acetyl-L: -cysteine or N-tert-butyl-α-(2-sulfophenyl)nitrone or two antagonists of the sphingolipid pathway, N-oleoylethanolamine (NOE) or imipramine. In WT, TNFα reduced State 3 respiration from 279.3 ± 3 to 119.3 ± 2 (nmol O₂/mg protein/min), increased proton leak from 15.7 ± 0.6% (control) to 36.6 ± 4.4%, and decreased membrane potential by 20.5 ± 3.1% compared to control groups. In TNFR1&2(-/-) mice, TNFα reduced State 3 respiration from 205.2 ± 4 to 75.7 ± 1 (p < 0.05 vs. respective control). In WT mice, both antioxidants added with TNFα restored State 3 respiration to 269.2 ± 2 and 257.6 ± 2, respectively. Imipramine and NOE also restored State 3 respiration to 248.4 ± 2 and 249.0 ± 2, respectively (p < 0.01 vs. TNFα alone). Similarly, both antioxidant and inhibitors of the sphingolipid pathway restored the proton leak to pre-TNF values. TNFα-treated mitochondria or isolated cardiac muscle fibers showed an increase in respiration after anoxia-reoxygenation, but this effect was lost in the presence of an antioxidant or NOE. Similar data were obtained in TNFR1&2(-/-) mice. TNFα exerts a protective effect on respiratory function in isolated mitochondria subjected to an anoxia-reoxygenation insult. This effect appears to be independent of its cell surface receptors, but is likely to be mediated by ROS and sphingolipids.

Objective: To evaluate the significance of the JAK-STAT pathway in insulin-induced cardioprotection from reperfusion injury., Methods: In isolated perfused rat hearts subjected to insulin therapy (0.3 mU/ml) +/- AG490 (5 microM, JAK-STAT inhibitor), the phosphorylation state of STAT3 and Akt was determined after 15 min of reperfusion. Infarct size was measured after 120 min of reperfusion. Isolated cardiac myocytes from wild type (WT) and cardiac specific STAT3 deficient mice were treated with insulin at reoxygenation following simulated ischemia (SI, 26 h). Cell viability was measured after 120 min of reoxygenation following SI, whereas phosphorylation state of Akt was measured after 15 min of reoxygenation following SI., Results: Insulin given at reperfusion led to phosphorylation of STAT3 and Akt both of which were inhibited by AG490. AG490 also blocked the insulin-dependent decrease in infarct size, supporting a role for JAK-STAT in cardioprotection. In addition, insulin protection from SI was blocked in myocytes from the STAT3 deficient mice, or in WT mice treated with AG490. Furthermore, insulin failed to phosphorylate Akt in the STAT3 deficient cardiomyocytes., Conclusion: Insulin-induced cardioprotection at reperfusion occurs through activation of STAT3. Inhibiting STAT3 by AG490, or STAT3 depletion in cardiac myocytes affects activation of Akt, suggesting close interaction between STAT3 and Akt in the cardioprotective signalling pathway activated by insulin treatment at reperfusion.