Bacterial endotoxin lipopolysaccharide (LPS) is responsible for the multiorgan dysfunction that characterizes septic shock and is causal in the myocardial depression that is a common feature of endotoxemia in patients. In this setting the myocardial dysfunction appears to be due, in part, to the production of proinflammatory cytokines. A line of evidence also indicates that LPS stimulates autophagy in cardiomyocytes. However, the signal transduction pathway leading to autophagy and its role in the heart are incompletely characterized. In this work, we wished to determine the effect of LPS on autophagy and the physiological significance of the autophagic response. Autophagy was monitored morphologically and biochemically in HL-1 cardiomyocytes, neonatal rat cardiomyocytes, and transgenic mouse hearts after the administration of bacterial LPS or TNF-alpha. We observed that autophagy was increased after exposure to LPS or TNF-alpha, which is induced by LPS. The inhibition of TNF-alpha production by AG126 significantly reduced the accumulation of autophagosomes both in cell culture and in vivo. The inhibition of p38 MAPK or nitric oxide synthase by pharmacological inhibitors also reduced autophagy. Nitric oxide or H(2)O(2) induced autophagy in cardiomyocytes, whereas N-acetyl-cysteine, a potent antioxidant, suppressed autophagy. LPS resulted in increased reactive oxygen species (ROS) production and decreased total glutathione. To test the hypothesis that autophagy might serve as a damage control mechanism to limit further ROS production, we induced autophagy with rapamycin before LPS exposure. The activation of autophagy by rapamycin suppressed LPS-mediated ROS production and protected cells against LPS toxicity. These findings support the notion that autophagy is a cytoprotective response to LPS-induced cardiomyocyte injury; additional studies are needed to determine the therapeutic implications.

Our understanding of autophagy has expanded greatly in recent years, largely due to the identification of the many genes involved in the process and to the development of better methods to monitor the process, such as green fluorescent protein-LC3 to visualize autophagosomes in vivo. A number of groups have demonstrated a tight connection between autophagy and mitochondrial turnover. Mitochondrial quality control is the process whereby mitochondria undergo successive rounds of fusion and fission with a dynamic exchange of components to segregate functional and damaged elements. Removal of the mitochondrion that contains damaged components is accomplished via autophagy (mitophagy). Mitophagy also serves to eliminate the subset of mitochondria producing the most reactive oxygen species, and episodic removal of mitochondria will reduce the oxidative burden, thus linking the mitochondrial free radical theory of aging with longevity achieved through caloric restriction. Mitophagy must be balanced by biogenesis to meet tissue energy needs, but the system is tunable and highly dynamic. This process is of greatest importance in long-lived cells such as cardiomyocytes, neurons, and memory T cells. Autophagy is known to decrease with age, and the failure to maintain mitochondrial quality control through mitophagy may explain why the heart, brain, and components of the immune system are most vulnerable to dysfunction as organisms age.

Autophagy is a highly regulated intracellular degradation process by which cells remove cytosolic long-lived proteins and damaged organelles. The mitochondrial permeability transition (MPT) results in mitochondrial depolarization and increased reactive oxygen species production, which can trigger autophagy. Therefore, we hypothesized that the MPT may have a role in signaling autophagy in cardiac cells. Mitochondrial membrane potential was lower in HL-1 cells subjected to starvation compared to cells maintained in full medium. Mitochondrial membrane potential was preserved in starved cells treated with cyclosporin A (CsA), suggesting the MPT pore is associated with starvation-induced depolarization. Starvation-induced autophagy in HL-1 cells, neonatal rat cardiomyocytes and adult mouse cardiomyocytes was inhibited by CsA. Starvation failed to induce autophagy in CypD-deficient murine cardiomyocytes, whereas in myocytes from mice overexpressing CypD the levels of autophagy were enhanced even under fed conditions. Collectively, these results demonstrate a role for CypD and the MPT in the initiation of autophagy. We also analyzed the role of the MPT in the degradation of mitochondria by biochemical analysis and electron microscopy. HL-1 cells subjected to starvation in the presence of CsA had higher levels of mitochondrial proteins (by Western blot), more mitochondria and less autophagosomes (by electron microscopy) then cells starved in the absence of CsA. Our results suggest a physiologic function for CypD and the MPT in the regulation of starvation-induced autophagy. Starvation-induced autophagy regulated by CypD and the MPT may represent a homeostatic mechanism for cellular and mitochondrial quality control.

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