The long-term goals of this project are to 1) understand the role of mitochondria in ischemia-reperfusion injury and cardioprotection; 2) to understand the role of altered ion homeostasis and altered metabolism in ischemia-reperfusion and cardioprotection and 3) to understand changes in cytosolic and mitochondrial signaling involved in cardioprotection and cell death. It is proposed that ischemic preconditioning (PC) initiates signaling that converges on mitochondria and results in cardioprotection. PC is known to involve nitric oxide signaling. We tested the hypothesis that caveolea might serve as a signaling module to transmit signals from G-protein coupled receptors on the plasma membrane to the mitochondria. Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important roles in ischemic preconditioning (IPC)-induced cardioprotection. Mitochondria are key regulators of preconditioning and most proteins showing an increase in SNO with IPC are mitochondrial. However, it is not clear how IPC transduces NO/SNO signaling to mitochondria. In this study using Langendorff perfused mouse hearts, we found that IPC-induced cardioprotection was blocked by treatment with either N-nitro-L-arginine methyl ester (L-NAME, a constitutive NO synthase inhibitor), ascorbic acid (a reducing agent to decompose SNO), or methyl-b-cyclodextrin (MbCD, a cholesterol sequestering agent to disrupt caveolae). IPC not only activated AKT/eNOS signaling but also led to translocation of eNOS to mitochondria. MCD treatment disrupted caveolae structure, leading to dissociation of eNOS from caveolin-3 and blockade of IPC-induced activation of the AKT/eNOS signaling pathway. A significant increase in mitochondrial SNO was found in IPC hearts compared to perfusion control, and the disruption of caveolae by MCD treatment not only abolished IPC-induced cardioprotection, but also blocked IPC-induced increase in SNO. In conclusion, these results suggest that caveolae transduce IPC-induced eNOS/NO/SNO acute cardioprotective signaling in the heart. We also test the role of a novel G-protein coupled receptor the extracellular Ca2+-sensing receptor (CaSR) in cardioprotection. The CaSR responds to changes not only in extracellular Ca2+ but also to many other ligands. CaSR has been found to be expressed in the hearts and cardiovascular system. In this study, we confirmed that CaSR is expressed in mouse cardiomyocytes, and showed that it is predominantly localized in caveolae. We investigated whether CaSR plays a cardioprotective role in ischemic preconditioning (IPC). Hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following treatments: (1) control perfusion;(2) perfusion with a specific CaSR antagonist, NPS2143;(3) IPC (four cycles of 5 min of global ischemia and 5 min of reperfusion);or (4) perfusion with NPS2143 prior to and during IPC. Following these treatments hearts were subjected to 20 min of no-flow global ischemia and 120 min of reperfusion. Compared with control, IPC significantly improved post-ischemic left ventricular functional recovery and reduced infarct size. Although NPS2143 perfusion alone did not change the hemodynamic function and did not change the extent of post-ischemic injury, NPS2143 treatment abolished cardioprotection of IPC. Through immunoblot analysis, it was demonstrated that IPC significantly increased the levels of phosphorylated ERK1/2, AKT, and GSK3β, which were also prevented by NPS2143 treatment. Taken together, the distribution of CaSR in caveolae along with NPS2143-blockable IPC-induced cardioprotective signaling suggest that the activation of CaSR during IPC is cardioprotective, a process involving caveolae. Another project involves examining the role of cyclophilin D in cell physiology and pathology. Following ischemia and reperfusion a mitochondrial pore, known as the mitochondrial transition pore (mPTP) opens and leads to cell death. Cyclophilin is the only identified component of mPTP. Mitochondrial permeability transition pore (mPTP) opening plays a critical role in mediating cell death during ischemia/ reper-fusion (I/R) injury. Our previous studies have shown that protein S-nitrosylation (SNO) plays a protective role in I/R injury and that the SNO of cyclophilin D (CypD), a critical mPTP mediator, may be a functional target in orchestrating cytoprotection. To investigate whether SNO of CypD might attenuate mPTP activation, we mutated cysteine 203 of CypD, the SNO site, to a serine residue (C203S) and determined its effects on mPTP opening. Treatment of wildtype (WT) mouse embryonic fibroblasts (MEFs) with H2O2 resulted in an ≈50% loss of the mitochondrial calcein fluorescence, suggesting substantial activation of the mPTP. Consistent with the reported role of CypD in mPTP activation, CypD null (CypD-/-) MEFs exhibited significantly less mPTP opening. Addition of a nitric oxide donor, GSNO, to WT but not CypD-/- MEFs prior to H2O2 attenuated mPTP opening. To test whether C203 is required for this protection, we infected CypD-/- MEFs with a C203S-CypD vector. Surprisingly, C203S-CypD re-constituted MEFs were resistant to mPTP opening in the presence or absence of GSNO, suggesting a crucial role for C203 in mPTP activation. To determine whether mutation of C203S-CypD would alter mPTP in vivo, we injected a recombinant adenovirus encoding C203S-CypD or WT CypD into CypD-/- mice via tail-vein. Mitochondria isolated from livers of CypD-/- mice or mice expressing C203S-CypD were resistant to Ca2+-induced swelling as compared to WT CypD reconstituted mice. Our results indicate that the cysteine 203 residue of CypD is necessary for redox stress-induced activation of mPTP. We were also interested in examining the physiological role of cyclophilin D. Isolated mitochondria from mice deficient in cyclophilin D (CypD-/-) are less sensitive to Ca2+-induced opening of the mitochondrial permeability transition (MPT) in vitro. Thus, the lack of CypD enables heart mitochondria to take up more Ca2+ before undergoing the MPT. We hypothesize that the MPT serves as a Ca2+-safety valve that can open to release excess Ca2+, but not necessarily result in death. If the MPT is blocked in CypD-/- mice, we hypothesize that matrix Ca2+ (Ca2+m) would be higher in CypD-/- mice compared to WT and this would activate Ca2+-sensitive NADH dehydrogenases (e.g., pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH)), which would in turn, alter oxidative metabolism and increase oxygen consumption. Consistent with this, we found altered expression levels of PDH E1 subunit and the alpha-KGDH E2 subunit in CypD-/- hearts using 2D DIGE proteomics. To evaluate differences in metabolism, we perfused hearts with 13C-glucose and 13C-palmitate and looked at their contribution to the acetyl-CoA pool by measuring label incorporation into the C4 of glutamate. The 13C-labeled glucose or palmitate enters the Krebs cycle and labels the alpha-KG pool that is in equilibrium with glutamate, which is usually present at higher levels. The ratio of glucose to palmitate metabolism in CypD-/- hearts was 1.5-fold higher than in WT, which would suggest increased PDH activity. 13C-labeling into succinate compared to glutamate was also increased significantly in CypD-/- hearts, and this result would be consistent with increased activity of alpha-KGDH relative to other competing reactions. We measured alpha-KGDH activity to evaluate whether Krebs cycle flux upstream of succinate was elevated in CypD-/- hearts and found a 1.4 fold increase in alpha-KGDH activity. Therefore, these results demonstrate that the loss of a MPT component, CypD, results in physiological flux changes in the Krebs cycle and oxidative metabolism that are consistent with increased Ca2+m.

Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2011
Total Cost
$527,890
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
Zip Code
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