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. It has been proposed that cells die following ischemia and reperfusion because of a rise in mitochondrial calcium which leads to activation of the mitochondrial permeability transition pore (MPT). The increase in mitochondrial calcium is proposed to occur via calcium uptake by the mitochondrial calcium uniporter. The composition of the MPT is unknown, but cyclophilin D has been shown to be a regulator of the MPT. We examined the role of calcium uptake by mitochondrial calcium uniporter (MCU) in regulating cell death and metabolism. In collaboration with Dr. Toren Finkel, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU(-/-) mice have no apparent capacity to rapidly uptake calcium. Whereas basal metabolism seems unaffected, the skeletal muscle of MCU(-/-) mice exhibited alterations in the phosphorylation and activity of pyruvate dehydrogenase. In addition, MCU(-/-) mice exhibited marked impairment in their ability to perform strenuous work. We further show that mitochondria from MCU(-/-) mice lacked evidence for calcium-induced permeability transition pore (PTP) opening. Surprisingly, the lack of PTP opening does not seem to protect MCU(-/-) cells and tissues from cell death. Following global I/R in a Langendorff perfused heart model infarct size was indistinguishable between WT hearts and hearts from MCU-/- mice. To further complicate the picture, cyclosporine A, an inhibitor of the mPTP, reduced infarct size in WT hearts, but not in MCU-/- hearts. We have been testing whether loss of MCU leads to alterations in other cell death pathways. We also examined the response of the mouse to addition of isoproterenol. We have also performed studies to examine the role of MCU regulatory proteins in regulating mitochondrial calcium homeostasis. Knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) abrogates rapid mitochondrial Ca2+ uptake and permeability transition pore (PTP) opening. However, hearts from global MCU-KO mice were not protected from ischemic injury. Furthermore, MCU-KO hearts were resistant to protection afforded by cyclosporin A (CsA), a pore desensitizer that inhibits binding of cyclophilin D (CypD) to the PTP. This study investigates the hypothesis that the lack of protection in MCU-KO may be explained by alterations in PTP opening due to compensatory changes in CypD signaling. To investigate whether pore opening can occurs in MCU-KO, Ca2+ uptake and swelling were measured in isolated mitochondria in the presence of the Ca2+ ionophore ETH129 to permit Ca2+ entry into the matrix. With ETH129, MCU-KO mitochondria were able to take up Ca2+ and underwent pore opening similar to WT. To investigate the Ca2+ sensitivity of PTP in MCU-KO, basal Ca2+ was set to the same level in mitochondria from KO and WT prior to a Ca2+ uptake assay. MCU-KO underwent PTP opening before WT, suggesting that PTP Ca2+ sensitivity is altered in the absence of MCU. To determine whether CypD-mediated regulation of PTP opening may be different following global MCU deletion, experiments were performed to examine the interaction between CypD and the proposed PTP component ATP synthase. Mitochondria isolated from WT and MCU KO hearts were incubated with an immunocapture antibody to pulldown ATP synthase. Interestingly, preliminary results suggest that there was more CypD associated with ATP synthase in MCU KO in comparison to WT (n=5, P=0.088). As phosphorylation of CypD has been proposed to enhance PTP opening, immunoprecipitation experiments were performed using an antibody for phosphorylated proteins. MCU KO mitochondria had an increase in the amount of phosphorylated CypD (n=7, P=0.058). These results suggest that absence of MCU may alter PTP opening such that less Ca2+ is required to trigger PTP, which may be due to compensatory changes in CypD-mediated pore regulation. Our previous study in mouse embryonic fibroblasts showed that cysteine 202 of cyclophilin D (CyPD) is necessary for redox stress-induced activation of the mitochondrial permeability transition pore (mPTP). To further investigate the essential function of this cysteine residue in situ, we used CRISPR to develop a knock-in mouse model (C57BL/6N stain), where CyPD cysteine 202 was mutated to a serine (C202S-KI). The amount of total CyPD expressed in the CyPD C202S-KI did not differ compared to the wild-type (WT). However, the CyPD C202S-KI mouse hearts elicit a significant cardioprotective effect against ischemia-reperfusion (I/R) injury in the Langendorff perfused heart model. After 20 min of global ischemia followed by 90 min of reperfusion, the post-ischemic recovery of rate pressure product (RPP= heart rate x LVDP) was 45.04.2% in CyPD WT and 59.64.0% in CyPD C202S-KI mice. Myocardial infarct size was decreased in CyPD C202S-KI mouse hearts versus CyPD WT mice (24.54.7% vs 49.82.7%). Isolated heart mitochondria from CyPD C202-KI mice had a higher calcium retention capacity compared to CyPD WT mice (140.020.82 vs 213.316.67 umol Ca+2/g protein). However, in contrast to CyPD knockout mice which exhibit more pronounced cardiac hypertrophy in response to pressure overload stimulation than control mice, CyPD C202S-KI mice developed a comparable level of hypertrophy to their WT littermate in an angiotensin II-induced hypertrophy model delivered by implanted osmotic minipumps. In conclusion, these results show that mutated CyPD C202S affords cardioprotection against I/R injury, suggesting that the redox-modification of cysteine 202 might play an important role in the regulation of CyPD and its downstream targets such as mPTP.
| Parks, Randi J; Murphy, Elizabeth; Liu, Julia C (2018) Mitochondrial Permeability Transition Pore and Calcium Handling. Methods Mol Biol 1782:187-196 |
| Lindsey, Merry L; Bolli, Roberto; Canty Jr, John M et al. (2018) Guidelines for experimental models of myocardial ischemia and infarction. Am J Physiol Heart Circ Physiol 314:H812-H838 |
| Murphy, Elizabeth; Glancy, Brian; Steenbergen, Charles (2018) What You Eat Affects Your Shape. Circ Res 122:8-10 |
| Ulmer, Bärbel M; Stoehr, Andrea; Schulze, Mirja L et al. (2018) Contractile Work Contributes to Maturation of Energy Metabolism in hiPSC-Derived Cardiomyocytes. Stem Cell Reports 10:834-847 |
| Parks, Randi J; Menazza, Sara; Holmström, Kira M et al. (2018) Cyclophilin D-mediated regulation of the permeability transition pore is altered in mice lacking the mitochondrial calcium uniporter. Cardiovasc Res : |
| Han, Kim; Hassanzadeh, Shahin; Singh, Komudi et al. (2017) Parkin regulation of CHOP modulates susceptibility to cardiac endoplasmic reticulum stress. Sci Rep 7:2093 |
| Menazza, Sara; Sun, Junhui; Appachi, Swathi et al. (2017) Non-nuclear estrogen receptor alpha activation in endothelium reduces cardiac ischemia-reperfusion injury in mice. J Mol Cell Cardiol 107:41-51 |
| Liu, Julia C; Parks, Randi J; Liu, Jie et al. (2017) The In Vivo Biology of the Mitochondrial Calcium Uniporter. Adv Exp Med Biol 982:49-63 |
| Glancy, Brian; Hartnell, Lisa M; Combs, Christian A et al. (2017) Power Grid Protection of the Muscle Mitochondrial Reticulum. Cell Rep 19:487-496 |
| Maack, Christoph; Murphy, Elizabeth (2017) Metabolic cardiomyopathies - fighting the next epidemic. Cardiovasc Res : |
Showing the most recent 10 out of 65 publications
