P313Embryonic cardiomyocytes can orchestrate various cell protective mechanisms to survive metabolic stress

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Abstract

Whereas adult cardiomyocytes are highly susceptible to stress, cardiomyocytes in the prenatal heart appear to be rather resistant. To investigate how embryonic cardiomyocytes survive metabolic stress in vivo, we utilized tissue mosaicism for mitochondrial dysfunction in 13.5 dpc mouse hearts. The latter is based on inactivation of the X-linked gene encoding Holocytochrome c synthase (Hccs), which is essential for mitochondrial respiration. In heterozygous heart conditional Hccs knockout females (cHccs+/-) random X chromosomal inactivation results in a mosaic of healthy and Hccs deficient cells in the myocardium. Microarray RNA expression analyses identified genes involved in unfolded protein response (UPR) and programmed cell death as differentially expressed in cHccs+/- versus control embryonic hearts. Activation of the UPR localized to Hccs deficient cardiomyocytes but does not involve ER stress pathways, suggesting that it is caused by defective mitochondria. Consistently, various mitochondrial chaperones, such as HSP10 and HSP60, but not ER chaperones (e.g. GRP78) are induced in defective cells. Mitochondrial dysfunction often results in oxidative stress, and indeed we detected induction of SOD2 and reduced glutathione levels in cHccs+/- hearts. Interestingly, no increase in protein oxidation was observed, suggesting that antioxidative mechanisms in Hccs deficient cardiomyocytes prevent oxidative damage. Mitochondrial dysfunction and unrestricted UPR can induce cell death, and we indeed detected activation of upstream components of both intrinsic as well as extrinsic apoptosis in cHccs+/- hearts. Cell death is not executed, however, suggesting the activation of antiapoptotic mechanisms. Whereas most apoptosis inhibitors (i.e. Bcl-2, Bcl-x, c-FLIP, XIAP, survivin) were either unchanged or downregulated in Hccs deficient cardiomyocytes, ARC (apoptosis repressor with caspase recruitment domain) was induced. Given that ARC can inhibit both apoptotic pathways as well as necrosis and attenuates UPR, we generated cHccs+/- embryos on an ARC knockout background (cHccs+/-,ARC-/-). Surprisingly, the absence of ARC does not induce cell death in embryonic Hccs deficient cardiomyocytes, although no compensatory upregulation of other apoptosis inhibitors was observed. Also, induction of mitochondrial chaperones, antioxidative defense and UPR was not compromised in cHccs+/-,ARC-/- hearts. Taken together, our data demonstrate an impressive plasticity of embryonic cardiomyocytes to respond to metabolic stress, the loss of which might be involved in the high susceptibility of postnatal cardiomyocytes to cell death.

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