Environmental stresses converge on the mitochondria that can trigger or inhibit cell death. Excitable, post-mitotic cells (such as cardiac myocytes in heart, and neurons in brain), in response to sub-lethal noxious stress engage mechanisms affording protection from subsequent insults. These protection mechanisms involve activation of endogenous signaling which can confer significant resistance to oxidant and other stresses associated with hypoxia/reoxygenation (i.e., during a heart attack or stroke), which promotes the enhanced capacity for cell survival. However, the upstream signaling mechanisms have remained an area of active debate, and the end effector(s) have remained unsolved. We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species- (ROS-) threshold for the mitochondrial permeability transition (mPTP) in cardiac myocytes, and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria. We demonstrate that a wide variety of cardio/neuroprotective agents acting via distinct upstream mechanisms all promote cell survival by limiting mPTP induction. We found that protection can be triggered in 2 general ways dependent and independent of regulatory mitochondrial swelling which converge via inhibition of GSK-3b on the end effector, the permeability transition pore complex, preventing the mPTP. Cell protection exhibiting a memory (i.e., preconditioning) results from triggered mitochondrial swelling (due to enhanced K+ accumulation via influx and/or retention) causing enhanced substrate oxidation and ROS production, leading to redox activation of PKC which in turn inhibits GSK-3b (via phosphorylation of ser-9). We concluded that GSK-3b (and specifically its inactivation) is a major, required integration point for a multitude of upstream signals acting on an end-effector responsible for cardioprotection (the mitochondrial permeability transition pore). When cell protection signaling pathways are activated, we found that the Bcl-2 family members relay the signal from GSK-3b onto a target at or in close proximity to the pore. Thus, the effect of the convergence of these signaling pathways via inhibition of GSK-3b, relayed through Bcl-2 proteins, on the end effector, the permeability transition pore complex, to limit mPTP induction, is the general mechanism of cardiomyocyte protection. We propose that clinical treatment strategies designed to inhibit the master switch kinase, GSK-3b, to protect the permeability transition pore complex from mPTP induction, would be effective to reduce the size of infarction during episodes of heart attack or stroke by preventing the death of cardiac myocytes and neurons (respectively). Signaling defects underlying the age-assocciated loss of the capacity for ischemic preconditioning are being examined which could lead to testable clinical therapies relevant to the preservation of healthy aging. Experiments are ongoing examining the hypothesis that the sensitivity of mPTP to ROS can serve as a biomarker of mitochondrial fitness during aging. The mitochondrial fitness of muscle stem cells may in part underlie the development of age-related sarcopenia. A healthy and nutritionally well-adjusted lifestyle should favor balanced energy supply and demand in concert with mitochondrial fission-fusion and mitophagy-biogenesis processes leading to proper organelle turnover and energy-redox function of the mitochondrial population. Perturbed fusion-fission dynamics and defective mitophagy result in impaired mitochondrial quality and consequently in spread mitochondrial damage as reflected by energetic-redox impairment. In this scenario damaged mitochondria cannot be removed and accumulate due to failure of the fission process interrupting normal mitophagy. Degradation of mitochondrial quality results in the overall average energetic-redox impairment of the mitochondrial network, as shown by mitochondrial populations isolated from organs affected by metabolic disease or by ablation of fission proteins. Autophagy/mitophagy, and mitochondrial function relate directly to our main new hypothetical idea that citrate/AcCoA cycling controlled by ATP citrate lyase regulates key acetylation/deacetylation processes in the mitochondrial, cytoplasmic and nuclear compartments and thereby has a potentially central and unifying role in the control of aging and longevity. Our results suggest that diverse interventions that have been proven to prolong lifespan in multiple animal experimental models all decrease global acetylation in cardiomyocytes and neurons and positively affect the autophagy outcome. Within the hypothetical framework that the health status of the mitochondrial network affects/governs the health span of cells and the organism, thus influencing longevity (1), we are studying mitochondrial function and metabolic remodeling as a function of age in high and low running capacity (HCR and LCR) rats, obtained by inbreeding from an original NIH outbred colony. HCR and LCR rats exhibit a very significant difference in lifespan that parallels the innate differences in running capacity. The HCR exhibit a 40% longer lifespan than LCR rats, a trait associated with a higher capacity to use lipids than glucose as substrate, under exercising conditions. We hypothesized that the respiratory reserve capacity of the cardiac cell in the longer-living HCR will be higher than in LCR, along with relatively enhanced turnover of mitochondria thus determining improved mitochondrial health. Faster aging due to deficit in mitochondrial energetics and turnover will result in a progressively damaged mitochondrial population. The mitochondrial respiratory reserve (quantified as the difference in respiratory flux under maximal uncoupling minus baseline conditions) of isolated cardiac myocytes from HCR-LCR rats at 5, 17 and 24 months of age, was analyzed under different substrate combinations (glucose, palmitate, or glucose+palmitate) with high throughput Seahorse technology. In the same samples, parallel studies of mitochondrial turnover (autophagy/mitophagy) using laser-scanning fluorescence confocal imaging and electron microscopy were performed. Main results show that lipid (palmitate)-based mitochondrial respiratory reserve is higher in HCR than LCR cardiomyocytes but decreases from 5 to 17 months of age in both groups. However, and unexpectedly, respiratory reserve increased thereafter, always more in HCR than LCR and with palmitate alone or in combination with glucose. Our experimental protocol enabled us to dissect the effect of glucose on respiratory reserve which remained much lower exhibiting a slight decrease over the age range analyzed in both groups. In agreement with our hypothesis, the turnover as well as steady state level of autophagy/mitophagy in cardiomyocytes was higher in the longer-living HCRs. The acetyl-CoA levels were always in the lower, likely permissive, concentration range (< 1mM) for HCR, in agreement with the imaging and electron microscopy studies. Interestingly, mitochondrial energetics in HCR-LCR rats showed a similar pattern of substrate consumption as in cardiomyocytes, further supporting our findings with respiration measurements and analyses.

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National Institute on Aging (NIA)
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Aon, Miguel A; Cortassa, Sonia; Juhaszova, Magdalena et al. (2016) Mitochondrial health, the epigenome and healthspan. Clin Sci (Lond) 130:1285-305
Gonzalez-Freire, Marta; de Cabo, Rafael; Bernier, Michel et al. (2015) Reconsidering the Role of Mitochondria in Aging. J Gerontol A Biol Sci Med Sci 70:1334-42
Cortassa, Sonia; Caceres, Viviane; Bell, Lauren N et al. (2015) From metabolomics to fluxomics: a computational procedure to translate metabolite profiles into metabolic fluxes. Biophys J 108:163-72
Zorov, Dmitry B; Juhaszova, Magdalena; Sollott, Steven J (2014) Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev 94:909-50
Yaniv, Yael; Juhaszova, Magdalena; Sollott, Steven J (2013) Age-related changes of myocardial ATP supply and demand mechanisms. Trends Endocrinol Metab 24:495-505
Ahmet, Ismayil; Tae, Hyun-Jin; Juhaszova, Magdalena et al. (2011) A small nonerythropoietic helix B surface peptide based upon erythropoietin structure is cardioprotective against ischemic myocardial damage. Mol Med 17:194-200
Yaniv, Yael; Juhaszova, Magdalena; Wang, Su et al. (2011) Analysis of mitochondrial 3D-deformation in cardiomyocytes during active contraction reveals passive structural anisotropy of orthogonal short axes. PLoS One 6:e21985
Juhaszova, Magdalena; Zorov, Dmitry B; Yaniv, Yael et al. (2009) Role of glycogen synthase kinase-3beta in cardioprotection. Circ Res 104:1240-52
Zorov, Dmitry B; Juhaszova, Magdalena; Yaniv, Yael et al. (2009) Regulation and pharmacology of the mitochondrial permeability transition pore. Cardiovasc Res 83:213-25
Ahmet, Ismayil; Spangler, Edward; Shukitt-Hale, Barbara et al. (2009) Blueberry-enriched diet protects rat heart from ischemic damage. PLoS One 4:e5954

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