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. We also examined the role of succinylation in regulating cardiac function and response to ischemia and reperfusion. Succinylation refers to modification of lysine residues with succinyl groups donated by succinyl-CoA. Sirtuin5 (Sirt5) is a mitochondrial NAD+-dependent deacylase that catalyzes the removal of succinyl groups from proteins. Sirt5 and protein succinylation are conserved across species, suggesting functional importance of the modification. Sirt5 loss impacts liver metabolism but the role of succinylation in the heart has not been explored. We combined affinity enrichment with proteomics and mass spectrometry to analyze total succinylated lysine content of mitochondria isolated from WT and Sirt5-/- mouse hearts. We identified 887 succinylated lysine residues in 184 proteins. 44 peptides (5 proteins) occurred uniquely in WT samples, 289 (46 proteins) in Sirt5-/- samples, and 554 (133 proteins) were common to both groups. The 46 unique proteins in Sirt5-/- heart participate in metabolic processes such as fatty acid β-oxidation (Eci2) and branched chain amino acid catabolism, and include respiratory chain proteins (Ndufa7, 12, 13, Dhsa). We performed label-free analysis of the peptides common to WT and Sirt5-/- hearts. 16 peptides from 9 proteins were significantly increased in Sirt5-/- by at least 30%. The adenine nucleotide transporter 1 showed the highest increase in succinylation in Sirt5-/- (108.4 fold). The data indicates that succinylation is widespread in the heart and enriched in metabolic pathways. We examined whether the loss of Sirt5 would impact ischemia-reperfusion (I/R) injury and we found an increase in infarct size in Sirt5-/- hearts compared to WT littermates (68.5+/-1.1% Sirt5-/- vs 39.6+/- 6.8% WT) following 20 minutes of ischemia and 90 minutes reperfusion. We further demonstrate that the degree of I/R injury in Sirt5-/- heart is restored to WT levels by pretreatment with dimethyl malonate, a competitive inhibitor of succinate dehydrogenase (SDH), implicating alteration in SDH activity as causative of the injury. We provided the first large scale description of the cardiac prolyl hydroxylome and demonstrated that prolyl hydroxylation alters protein stability, translation and splicing in human cardiomyocytes (Stoehr et al110). We used human iPSC-CM in combination with pulse-chase, stable isotope labeling by amino acids in cell culture (SILAC) and proteomics to analyze the effects of a prolyl hydroxylase inhibitor on protein degradation and synthesis. Cells were cultured in light medium (12C and 14N) and then switched to heavy medium (13C and 15N) and simultaneously treated with vehicle or a prolyl hydroxylase inhibitor, dimethyl-oxalylglycine (DMOG). The decay of the light amino acid provides a measure of protein degradation. We identified 167 proteins that exhibit differences in degradation with DMOG; 164 of the 167 were stabilized. Regulation of protein turnover is increasingly recognized as an important regulator of cardiac function and disease.111-113 Proteins involved in RNA splicing, such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ), were stabilized with DMOG. Analyzing incorporation of the heavy amino acids provides a measure of protein synthesis. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as -cardiac actin. To identify novel prolyl hydroxylated proteins, we searched the mass spectrometry data for prolyl hydroxylation and identified 78 prolyl hydroxylated proteins. Gene ontology (GO) terms enriched in the data set are summarized in Fig. 7. We identified SRSF2, SFPQ, -cardiac actin and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels; this finding is consistent with studies in hibernating animals that show a change in protein synthesis in the absence of a change in mRNA. Because RNA splicing factors were prolyl hydroxylated we investigated splicing with and without inhibition of prolyl hydroxylation and detected 369 alternative splicing events that were significantly different, with a preponderance of exon skipping. This study provides the first extensive characterization of the cardiac prolyl hydroxylome and demonstrates that inhibition of oxygen and alpha-ketoglutarate hydroxylases alters protein stability, translation and splicing.
| 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 : |
| 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 : |
| 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 |
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