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Emerging Role of the Mitochondrial Outer Membrane Translocator Protein (TSPO) in Heart Failure and Mitochondrai Quality Control
Tuesday, February 14, 2017 CL transfer function of NM23-H4. Our current model suggests that NM23H4/OPA1 complexes exist in healthy mitochondria at the inner membrane to maintain OPA1 functions in membrane fusion and dynamics. Upon OPA1 cleavage, an early step during mitophagy, NM23-H4 may be released from these complexes, allowing simultaneous interaction of the hexameric NM23-H4 complex with inner and outer mitochondrial membrane and CL transfer. (1) Schlattner et al. (2015) Naunyn Schmiedebergs Arch. Pharmacol. 388, 271-8. (2) Kagan et al. (2016) Cell Death Diff. 23, 1140-51. (3) Boissan et al. (2014) Science 344, 1510-5. 1594-Plat Emerging Role of the Mitochondrial Outer Membrane Translocator Protein (TSPO) in Heart Failure and Mitochondrai Quality Control Phung N. Thai1, Daniel Daugherty1, Bert J. Frederich2, Samuel Galice1, Wenbin Deng1, Donald M. Bers1, Saul Schaefer1, Elena N. Dedkova1. 1 University of California, Davis, CA, USA, 2University of California, San Francisco, CA, USA. Introduction: Mitochondrial dysfunction is a hallmark of heart failure (HF), resulting in decreased energy production and impaired mitochondrial quality control (mitophagy) with resultant contractile dysfunction and cell death. The 18-kDa mitochondrial translocator protein (TSPO) has been considered a part of the mitochondrial permeability transition pore (mPTP) for a long time; however, its role in mPTP regulation is now debatable and the precise role of the TSPO in cardiac physiology and HF remains poorly understood. Objective: Determine the role of TSPO in a murine pressure-overload model of HF. Methods and Results: Conditional, cardiac-specific TSPO knockout (KO) mice were generated using the Cre-loxP system. Pressure overload by transverse aortic constriction (TAC) for 8 weeks significantly increased TSPO expression in wild type (WT) mice, but not in KO mice. While WT TAC mice showed a marked reduction in systolic function, KO TAC mice did not have a significant reduction in ejection fraction, and also exhibited fewer clinical HF signs (less cardiac dilation and fibrosis). TAC reduced mitochondrial calcium uptake and enhanced mPTP opening in permeabilized myocytes from WT animals. Calcium uptake was restored in TAC KO mice without significant effect on mPTP activity. The restoration of the mitochondrial calcium uptake coincided with reduced oxidative stress, improved oxygen consumption rate, and enhanced ATP generation and mitochondrial reserve capacity. In WT TAC, elevated mitochondrial accumulation of mCherry-Parkin1 was not coupled to the increase in LC3-autophagosomes formation indicating impaired mitophagy. In contrast, mitophagy was normalized in TSPO KO. Conclusions: These data suggest a novel mechanism to prevent HF at the cellular level via normalization of TSPO levels which leads to preservation of mitochondrial calcium uptake, oxidative phosphorylation, and mitochondrial mt-QC resulting in preservation of mitochondrial integrity and cell survival. 1595-Plat Dysregulation of Mitochondrial Permeability Transition Pore (MPTP) is Associated with Enhanced ROS Production in Skeletal Muscle of an ALS Mouse Model Yajuan Xiao1,2, Jianxun Yi1, Chehade Karam2, Xuejun Li1, Kamal Dhakal1, Dosuk Yoon1, Jingsong Zhou1,2. 1 Kansas City University of Medicine and Biosciences, Kansas City, MO, USA, 2Molecular Biophysics and Physiology, Rush University, Chicago, IL, USA. Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease. Oxidative stress is implicated in ALS pathophysiology. Using ROS BriteÔ 570, a cytosolic ROS detector, we found a significant increase in ROS production
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of muscle fibers derived from the ALS G93A mice. Mitochondria are the major source of ROS production. In order to detect early changes in ROS-related mitochondrial metabolic function, we generated double transgenic mice (G93A/cpYFP) that carry human ALS mutation SOD1G93A and mt-cpYFP transgenes, in which mt-cpYFP monitors dynamic changes of ROS-related mitoflash events at the single mitochondrion level. Remarkably, G93A muscle cells show early and disease stage-dependent changes in mitoflash events. The mitoflash activity at the age of 2 months (before ALS symptom onset) is marked by an increased flashing area in G93A muscle fibers, while their kinetics properties remain unchanged. After ALS onset (3-month old), there are drastic changes in the kinetics of mitoflash signal with prolonged FDHM (full duration at half maximum) of the mitoflash signal. Thus, mitoflash provides a sensitive and quantitative biomarker for evaluating ROSrelated mitochondrial dysfunction. It is known that uncontrolled opening of mitochondrial permeability transition pore (mPTP) is a key step to promote mitochondrial ROS production. While the molecular composition of mPTP is still incompletely understood, studies have shown that cyclophilin D (CypD) promotes the opening of mPTP and phosphorylated form of GSK3b participates in maintaining mPTP in the closed state. We used subcellular fractionation to isolate mitochondria from skeletal muscle of G93A mice, and found an increased CypD expression level in muscle mitochondria, while the phosphorylated form of GSK-3b was significantly reduced in mitochondria of G93A muscle. Thus, both may act as triggering factors for mPTP opening in G93A muscle. 1596-Plat An Alternative Splice Variant of Chloride Intracellular Channel 5 Protein, (CLIC5B) Regulates Cardiac Mitochondrial Localization and Function of CLIC5 Devasena Ponnalagu, Ahmed Tafsirul Hussain, Shubha Gururaja Rao, Harpreet Singh. Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA, USA. Mitochondrial bioenergetics is critical for the cell survival. Mitochondrial ion channels like BKCa and KATP play a significant role in cardioprotection from ischemia-reperfusion (IR) injury possibly by modulating the mitochondrial physiology. Interestingly, inner mitochondrial membrane (IMM) channels lack canonical mitochondrial targeting sequence (MTS) but localize to mitochondria. In case of mitoBKCa, a c-terminal splice variant, DEC, (BKCa-DEC) was reported to target BKCa to mitochondria of adult cardiomyocytes. Recently, we reported chloride intracellular channel (CLIC) proteins, specifically, CLIC4 and CLIC5 are enriched in the outer and inner membrane of rat cardiac mitochondria, respectively, in spite of lacking any of the classical MTS. Surprisingly, two distinct bands of CLIC5 at ~30 kDa (CLIC5A) and ~50 kDa, were observed in the rat cardiac mitochondrial lysates which could be attributed to the presence of another isoform (~50 kDa, CLIC5B). Our recent findings further demonstrate that variants CLIC5A and CLIC5B are present in the heart, but lungs and kidneys lack CLIC5B. Also, CLIC5 localized to the Percoll-purified mitochondria isolated from rat heart (69.13 5 12.2%, n=3) and rat cardiomyocyte cells, H9C2 (55.12 5 7.1%) but not in mouse lung epithelial (11 5 2.1 %) cells which lacked CLIC5B. Thus, suggesting that the splice variant of CLIC5, promotes its localization to the IMM. Also, we observed that cardiac mitochondria isolated from clic5-/- mice exhibited increased reactive oxygen species (ROS, p< 0.05, n=3) production whereas the clic5-/- lung mitochondria did not show any change, supporting the role of CLIC5B in regulating the mitochondrial function. Our study further establishes splice variation as a conserved mechanism for targeting ion channels to mitochondria. As CLICs are cardioprotective, understanding mitochondrial localization of CLIC5 will further aid in development of novel therapeutics.
325a
of muscle fibers derived from the ALS G93A mice. Mitochondria are the major source of ROS production. In order to detect early changes in ROS-related mitochondrial metabolic function, we generated double transgenic mice (G93A/cpYFP) that carry human ALS mutation SOD1G93A and mt-cpYFP transgenes, in which mt-cpYFP monitors dynamic changes of ROS-related mitoflash events at the single mitochondrion level. Remarkably, G93A muscle cells show early and disease stage-dependent changes in mitoflash events. The mitoflash activity at the age of 2 months (before ALS symptom onset) is marked by an increased flashing area in G93A muscle fibers, while their kinetics properties remain unchanged. After ALS onset (3-month old), there are drastic changes in the kinetics of mitoflash signal with prolonged FDHM (full duration at half maximum) of the mitoflash signal. Thus, mitoflash provides a sensitive and quantitative biomarker for evaluating ROSrelated mitochondrial dysfunction. It is known that uncontrolled opening of mitochondrial permeability transition pore (mPTP) is a key step to promote mitochondrial ROS production. While the molecular composition of mPTP is still incompletely understood, studies have shown that cyclophilin D (CypD) promotes the opening of mPTP and phosphorylated form of GSK3b participates in maintaining mPTP in the closed state. We used subcellular fractionation to isolate mitochondria from skeletal muscle of G93A mice, and found an increased CypD expression level in muscle mitochondria, while the phosphorylated form of GSK-3b was significantly reduced in mitochondria of G93A muscle. Thus, both may act as triggering factors for mPTP opening in G93A muscle. 1596-Plat An Alternative Splice Variant of Chloride Intracellular Channel 5 Protein, (CLIC5B) Regulates Cardiac Mitochondrial Localization and Function of CLIC5 Devasena Ponnalagu, Ahmed Tafsirul Hussain, Shubha Gururaja Rao, Harpreet Singh. Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA, USA. Mitochondrial bioenergetics is critical for the cell survival. Mitochondrial ion channels like BKCa and KATP play a significant role in cardioprotection from ischemia-reperfusion (IR) injury possibly by modulating the mitochondrial physiology. Interestingly, inner mitochondrial membrane (IMM) channels lack canonical mitochondrial targeting sequence (MTS) but localize to mitochondria. In case of mitoBKCa, a c-terminal splice variant, DEC, (BKCa-DEC) was reported to target BKCa to mitochondria of adult cardiomyocytes. Recently, we reported chloride intracellular channel (CLIC) proteins, specifically, CLIC4 and CLIC5 are enriched in the outer and inner membrane of rat cardiac mitochondria, respectively, in spite of lacking any of the classical MTS. Surprisingly, two distinct bands of CLIC5 at ~30 kDa (CLIC5A) and ~50 kDa, were observed in the rat cardiac mitochondrial lysates which could be attributed to the presence of another isoform (~50 kDa, CLIC5B). Our recent findings further demonstrate that variants CLIC5A and CLIC5B are present in the heart, but lungs and kidneys lack CLIC5B. Also, CLIC5 localized to the Percoll-purified mitochondria isolated from rat heart (69.13 5 12.2%, n=3) and rat cardiomyocyte cells, H9C2 (55.12 5 7.1%) but not in mouse lung epithelial (11 5 2.1 %) cells which lacked CLIC5B. Thus, suggesting that the splice variant of CLIC5, promotes its localization to the IMM. Also, we observed that cardiac mitochondria isolated from clic5-/- mice exhibited increased reactive oxygen species (ROS, p< 0.05, n=3) production whereas the clic5-/- lung mitochondria did not show any change, supporting the role of CLIC5B in regulating the mitochondrial function. Our study further establishes splice variation as a conserved mechanism for targeting ion channels to mitochondria. As CLICs are cardioprotective, understanding mitochondrial localization of CLIC5 will further aid in development of novel therapeutics.