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). Both the diazoxide-activated mitochondrial ATP dependent K+ channel (mitoKATP, which we have identified and are now chararcterizing), and the Ca2+-activated K+ channel, for example, can serve as mitochondrial K+ influx mechanisms (that can mediate mitochondrial regulatory swelling-dependent protection, etc). The delta-opioid peptide, DADLE, and the NHE-inhibitors, HOE 642 (cariporide) and HOE 694, each produce mitochondrial regulatory swelling-dependent protection independently of the mitoKATP. Alternatively, receptor tyrosine kinase or certain G-protein coupled receptor activation elicits cell protection (without mitochondrial swelling or durable memory) by inhibiting GSK-3b, via either PKB/Akt and mTOR/p70s6k, PKC, or PKA pathways. Examples of this latter class include insulin (via Akt and mTOR/p70s6k) and the direct GSK-3 inhibitor, Li+. We found that siRNA knock-down of GSK-3b, but not GSK-3a, induced the protection state, and that transgenic mice expressing a cardiac-restricted, constitutively active, non-inhibitable form of GSK-3b (GSK-3b S9A) were resistant to a whole battery of upstream signals that were effective to induce the protected state in WT mice. 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. As a key glycolytic enzyme, hexokinase (HK) provides a functional link between mitochondria and cytosol by governing the preferential utilization of mitochondrial ATP for glucose phosphorylation. In the late 1970s and early 1980s it was demonstrated that HKI &II are VDAC-binding proteins, and since then this glycolytic enzyme has been recognized as the main VDAC modulator. There had been considerable debate over the past several years regarding the role of HKII (one of the mitochondrial-bound isoforms) in mPTP regulation, and we sought to definitively answer the question. Glucose and ATP act both as catalytic substrates and modulators of HK-VDAC binding. In addition, different drugs may facilitate the release of HK from mitochondria, thereby mimicking the natural effect of glucose-6-phosphate. Antifungal drugs such as clotrimazol and bifonazol are effective hexokinase-releasing agents. Indeed, we found that clotrimazol does reduce the mPTP ROS-threshold in cardiomyocytes, although this drug could have other non-specific actions. Since it is known that the N-terminal sequence of the mitochondrial HKI and II has high affinity to VDAC, a peptide corresponding to the N-terminal 29 AA residue domain common for both the HKI and HKII (HKII-VDB;VDAC-binding domain) was constructed and found to effectively release the mitochondrial-bound HK pool. We found that this peptide substantially decreased the mPTP ROS-threshold in cardiomyocytes (IC50 400 nM), while a control peptide had no such effect (proving the specificity of the active peptide). It was also found that forced release of HKII by this peptide invokes a cell death signal that engages mPTP opening. Surprisingly, this peptide was also fully effective in VDAC1/3 -/- fibroblasts (where the VDAC2 isoform was also found not to be bound to HKII) proving the complete dispensability of VDACs for the induction of apoptosis in these cells. Thus, HKII detachment from another site might be the signal to initiate apoptosis by relay of a conformational change from OMM to IMM and to components/modulators of the mPTP, independently of VDACs presence.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000823-20
Application #
8736632
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
2013
Total Cost
$750,557
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
Zip Code
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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