Christopher L. Antos

Environmental stresses converge on the mitochondria that can trigger or inhibit cell death.Excitable, postmitotic cells, in response to sublethal noxious stress, engage mechanisms that afford protection from subsequent insults.We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species (ROS) threshold for the mitochondrial permeability transition (MPT) in cardiomyocytes and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria.Cell protection that exhibits a memory (preconditioning) results from triggered mitochondrial swelling that causes enhanced substrate oxidation and ROS production, leading to redox activation of PKC, which inhibits glycogen synthase kinase-3 (GSK-3).Alternatively, receptor tyrosine kinase or certain G protein-coupled receptor activation elicits cell protection (without mitochondrial swelling or durable memory) by inhibiting GSK-3, via protein kinase B/Akt and mTOR/p70 s6k pathways, PKC pathways, or protein kinase A pathways.The convergence of these pathways via inhibition of GSK-3 on the end effector, the permeability transition pore complex, to limit MPT induction is the general mechanism of cardiomyocyte protection.Nonstandard abbreviations used: bisindolylmaleimide I (BIS); 2-chloro-N6-cyclopentyladenosine (CCPA); diazoxide (Dz); 2,7-dichlorodihydrofluorescein diacetate (DCF); glucagon-like peptide-a (GLP-1); glycogen synthase kinase-3 (GSK-3); 5-hydroxydecanoate (5HD); indanyloxyacetic acid 94 (IAA94); mitochondrial ATPdependent K + channel (mitoKATP); mitochondrial permeability transition (MPT); N-acetyl-L-cysteine (NAC); partial fatty acid oxidation (PFAO); Na/H exchange (NHE); preconditioning (PC); protein kinase A (PKA); protein kinase B (PKB); reactive oxygen species (ROS); receptor for activated C kinase (RACK); Sanglifehrin A (SFA); short interfering RNA (siRNA); S-nitroso-N-acetyl-penicillamine (SNAP); tetramethylrhodamine methyl ester (TMRM); transgenic (TG); transmembrane potential (); trimetazidine (TMZ); Tyr-D-Ala-Gly-Phe-D-Leu (DADLE).

The adult myocardium responds to a variety of pathologic stimuli by hypertrophic growth that frequently progresses to heart failure. The calcium/calmodulin-dependent protein phosphatase calcineurin is a potent transducer of hypertrophic stimuli. Calcineurin dephosphorylates members of the nuclear factor of activated T cell (NFAT) family of transcription factors, which results in their translocation to the nucleus and activation of calcium-dependent genes. Glycogen synthase kinase-3 (GSK-3) phosphorylates NFAT proteins and antagonizes the actions of calcineurin by stimulating NFAT nuclear export. To determine whether activated GSK-3 can act as an antagonist of hypertrophic signaling in the adult heart in vivo, we generated transgenic mice that express a constitutively active form of GSK-3 beta under control of a cardiac-specific promoter. These mice were physiologically normal under nonstressed conditions, but their ability to mount a hypertrophic response to calcineurin activation was severely impaired. Similarly, cardiac-specific expression of activated GSK-3 beta diminished hypertrophy in response to chronic beta-adrenergic stimulation and pressure overload. These findings reveal a role for GSK-3 beta as an inhibitor of hypertrophic signaling in the intact myocardium and suggest that elevation of cardiac GSK-3 beta activity may provide clinical benefit in the treatment of pathologic hypertrophy and heart failure.

We have produced null mutant mouse embryonic stem cells for the cell adhesion molecule E-cadherin. Such E-cadherin-/- ES cells are defective in cell aggregation; this defect can be corrected by transfection with cDNA for either E-cadherin or N-cadherin driven by a constitutive promoter. The presence (or absence) of E-cadherin regulates the expression of the transcription factor T-brachyury, indicating that cadherins play a role in linking cell surface receptors and gene expression. Comparative analysis of the parental and the genetically altered ES cell lines was performed to examine cell differentiation and the capability to form organized tissues. While differentiating E-cadherin-/- ES cells are still able to express various early and late differentiation markers, they show a clear-cut deficiency in forming organized structures. This phenotype can be rescued by constitutive expression of E-cadherin, which results exclusively in formation of epithelia. In contrast, rescue transfectants expressing N-cadherin show no epithelial structures, instead forming neuroepithelium and cartilage. These results provide the first evidence that specific cadherins directly stimulate differentiation into certain types of tissues.