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Mitochondrial membrane protein Sigmar1 regulates mitochondrial dynamics and function
Abstracts
(ROS) signalling and promotes reductive stress, which is leading hypertrophic cardiomyopathy.
doi:10.1016/j.yjmcc.2017.07.061
050 Mitochondrial membrane protein Sigmar1 regulates mitochondrial dynamics and function Shafiul Alama, Chowdhury S. Abdullaha, Richa Aishwaryab, Jonette M. Greena, A. Wayne Orra, Sumitra Miriyalac, Manikandan Panchatcharamc, Hanna Osinskad, John N. Lorenze, Jeffrey Robbinsd, Shenuarin Bhuiyana a
Department of Pathology and Translational Pathobiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, USA b Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, USA c Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, USA d Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Centre, Cincinnati, OH, USA e Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Rationale: Sigma 1 receptor (Sigmar1) is a molecular chaperone protein known to reside at the interface between the mitochondria and endoplasmic reticulum (ER) in different cellular systems. We recently reported that Sigmar1 is highly expressed in cardiomyocytes but its molecular function still remains unknown. Objective: We investigated the subcellular localization and molecular function of Sigmar1 in regulating mitochondrial function in the heart. Methods and Results: Subcellular fractionation and biochemical experiments confirmed Sigmar1 expression in the mitochondrial- and mitochondrial-associated ER membrane fraction. We isolated mitochondria from the mouse and human heart cell lysates and found Sigmar1 as an integral mitochondrial outer membrane protein by biochemical experiments. To define the molecular function of Sigmar1, we used the Sigmar1 knockout mice. Sigmar1 knockout mice showed normal morphometry but exhibited significantly altered cardiac hemodynamics and ultrastructure. Ultrastructural analysis by transmission electron microscopy revealed accumulation of irregular shaped highly fused mitochondria and sarcomere disorganization. Examining the mitochondrial dynamics regulatory proteins, we found that Sigmar1 KO hearts showed decreased level of the fission protein, Drp-1 and increased expression of fusion protein, OPA1. Analysis of mitochondrial bioenergetics showed significantly reduced basal respiration and ATP turnover in the mitochondria isolated from Sigmar1 KO hearts. We also found decreased mitochondrial DNA copy number and ATP content in the Sigmar1 KO hearts. Conclusions: Our findings suggested that Sigmar1 is an integral mitochondrial outer membrane protein necessary to maintain normal mitochondrial morphology and function in the heart.
doi:10.1016/j.yjmcc.2017.07.062
051 miR-181c Regulates Mitochondrial Calcium Influx by targeting Cytochrome C Oxidase subunit 1 Soroosh Solhjooa, Sangeetha Kannana,b, Deepthi Ashoka, Brian O'Rourkea, Charles Steenbergena, Samarjit Dasa
a
151
Johns Hopkins University, Baltimore, MD, USA B.S.Abdur Rahman University, Chennai, Tamil Nadu, India
b
We have identified a microRNA, miR-181c, which can translocate into the mitochondria of cardiomyocytes, and regulate the mitochondrial gene cytochrome c oxidase subunit 1 (mt-COX1). Recently, we have also demonstrated that miR-181c/d-/- mice are protected against ischemia/reperfusion (I/R) injury by attenuating oxidative stress in the heart. Previous data also suggest that overexpression of miR-181c in the heart can activate Ca2 + entry into mitochondria. Here, we investigate the mechanism by which miR-181c regulates Ca2 + influx into the mitochondrial matrix. We overexpressed miR181c in H9c2 cells cultured under 1 mM N-acetyl cysteine. After 48 hr of transfection, we have identified a significant increase of PDH activity, suggesting miR-181c regulates mitochondrial Ca2 + influx, which in turn stimulate ROS production. Using miR-181c/d-/- mice, we found both Mitochondrial Calcium Uptake 1 (MICU1) and mitochondrial respiratory complex IV (COX IV) expression are markedly higher in the heart. Immunoprecipitated with MICU1, and then immunoblot for different sub-units of COX IV confirmed a protein-protein interaction between MICU1 and COX IV. We have also found significantly less Pyruvate Dehydrogenase (PDH) activity in neonatal mouse ventricular myocytes (NMVMs) isolated from miR-181c/d-/- mouse compared to C57BL6 (WT), suggesting significantly lower mitochondrial Ca2 +-concentrations in the miR-181c/ d-/- group. Utilizing a coverslip induced I/R-model, we observe that siRNAs against MICU1 (si-MICU1) during the ischemic phase significantly increase Ca2 +-entry into the mitochondria of the NMVMs. Lowering MICU1 also significantly increases Ca2 +-entry into the mitochondria after 30 min of ischemia in miR-181c/d-/- NMVMs. Furthermore, 30 min ischemia followed by 30 min reperfusion in NMVM monolayers led to significantly less oscillatory instability in mitochondrial inner membrane potential (ΔΨm) in miR-181c/d-/NMVMs compared with WT NMVMs. However, using si-MICU1 in the miR-181c/d-/- NMVM group attenuated mitochondrial protection against I/R-injury. MICU1 is directly associated with complex IV. Thus, miR-181c can regulate mitochondrial Ca2 +-entry by targeting mt-COX1 during I/R injury. doi:10.1016/j.yjmcc.2017.07.063
052 Dual optical mapping of the innervated Langendorff-perfused heart reveals novel insights into acute electrophysiological responses to sympathetic stimulation Lianguo Wanga, Srinivas Tapaa, Samantha Stuarta, Rachel Mylesb, Kieran Brackc, Andre Ngc, Donald Bersa, Crystal Ripplingera a
University of California Davis, Davis, USA University of Glasgow, Glasgow, UK c University of Leicester, Leicester, UK b
Background: Sympathetic activation of the cardiac tissue has been recognized as one of the key adaptive mechanisms in the mammalian heart. However, few studies have looked at the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (AP) and intracellular Ca2 + transients (CaT). Methods and Results: The heart and posterior thoracic cavity from rabbits (NZW, N=9) and mice (C57Bl6, N =8) were dissected and perfused through the descending aorta for dual optical mapping of transmembrane potential (Vm) and intracellular Ca2 + to study the effects of direct sympathetic nerve stimulation (SNS). SNS was achieved through spinal cord stimulation at T1-T3. Our results show that acute SNS (60 sec, 5-10Hz) increased heart rate (HR) and CaT
(ROS) signalling and promotes reductive stress, which is leading hypertrophic cardiomyopathy.
doi:10.1016/j.yjmcc.2017.07.061
050 Mitochondrial membrane protein Sigmar1 regulates mitochondrial dynamics and function Shafiul Alama, Chowdhury S. Abdullaha, Richa Aishwaryab, Jonette M. Greena, A. Wayne Orra, Sumitra Miriyalac, Manikandan Panchatcharamc, Hanna Osinskad, John N. Lorenze, Jeffrey Robbinsd, Shenuarin Bhuiyana a
Department of Pathology and Translational Pathobiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, USA b Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, USA c Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, USA d Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Centre, Cincinnati, OH, USA e Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Rationale: Sigma 1 receptor (Sigmar1) is a molecular chaperone protein known to reside at the interface between the mitochondria and endoplasmic reticulum (ER) in different cellular systems. We recently reported that Sigmar1 is highly expressed in cardiomyocytes but its molecular function still remains unknown. Objective: We investigated the subcellular localization and molecular function of Sigmar1 in regulating mitochondrial function in the heart. Methods and Results: Subcellular fractionation and biochemical experiments confirmed Sigmar1 expression in the mitochondrial- and mitochondrial-associated ER membrane fraction. We isolated mitochondria from the mouse and human heart cell lysates and found Sigmar1 as an integral mitochondrial outer membrane protein by biochemical experiments. To define the molecular function of Sigmar1, we used the Sigmar1 knockout mice. Sigmar1 knockout mice showed normal morphometry but exhibited significantly altered cardiac hemodynamics and ultrastructure. Ultrastructural analysis by transmission electron microscopy revealed accumulation of irregular shaped highly fused mitochondria and sarcomere disorganization. Examining the mitochondrial dynamics regulatory proteins, we found that Sigmar1 KO hearts showed decreased level of the fission protein, Drp-1 and increased expression of fusion protein, OPA1. Analysis of mitochondrial bioenergetics showed significantly reduced basal respiration and ATP turnover in the mitochondria isolated from Sigmar1 KO hearts. We also found decreased mitochondrial DNA copy number and ATP content in the Sigmar1 KO hearts. Conclusions: Our findings suggested that Sigmar1 is an integral mitochondrial outer membrane protein necessary to maintain normal mitochondrial morphology and function in the heart.
doi:10.1016/j.yjmcc.2017.07.062
051 miR-181c Regulates Mitochondrial Calcium Influx by targeting Cytochrome C Oxidase subunit 1 Soroosh Solhjooa, Sangeetha Kannana,b, Deepthi Ashoka, Brian O'Rourkea, Charles Steenbergena, Samarjit Dasa
a
151
Johns Hopkins University, Baltimore, MD, USA B.S.Abdur Rahman University, Chennai, Tamil Nadu, India
b
We have identified a microRNA, miR-181c, which can translocate into the mitochondria of cardiomyocytes, and regulate the mitochondrial gene cytochrome c oxidase subunit 1 (mt-COX1). Recently, we have also demonstrated that miR-181c/d-/- mice are protected against ischemia/reperfusion (I/R) injury by attenuating oxidative stress in the heart. Previous data also suggest that overexpression of miR-181c in the heart can activate Ca2 + entry into mitochondria. Here, we investigate the mechanism by which miR-181c regulates Ca2 + influx into the mitochondrial matrix. We overexpressed miR181c in H9c2 cells cultured under 1 mM N-acetyl cysteine. After 48 hr of transfection, we have identified a significant increase of PDH activity, suggesting miR-181c regulates mitochondrial Ca2 + influx, which in turn stimulate ROS production. Using miR-181c/d-/- mice, we found both Mitochondrial Calcium Uptake 1 (MICU1) and mitochondrial respiratory complex IV (COX IV) expression are markedly higher in the heart. Immunoprecipitated with MICU1, and then immunoblot for different sub-units of COX IV confirmed a protein-protein interaction between MICU1 and COX IV. We have also found significantly less Pyruvate Dehydrogenase (PDH) activity in neonatal mouse ventricular myocytes (NMVMs) isolated from miR-181c/d-/- mouse compared to C57BL6 (WT), suggesting significantly lower mitochondrial Ca2 +-concentrations in the miR-181c/ d-/- group. Utilizing a coverslip induced I/R-model, we observe that siRNAs against MICU1 (si-MICU1) during the ischemic phase significantly increase Ca2 +-entry into the mitochondria of the NMVMs. Lowering MICU1 also significantly increases Ca2 +-entry into the mitochondria after 30 min of ischemia in miR-181c/d-/- NMVMs. Furthermore, 30 min ischemia followed by 30 min reperfusion in NMVM monolayers led to significantly less oscillatory instability in mitochondrial inner membrane potential (ΔΨm) in miR-181c/d-/NMVMs compared with WT NMVMs. However, using si-MICU1 in the miR-181c/d-/- NMVM group attenuated mitochondrial protection against I/R-injury. MICU1 is directly associated with complex IV. Thus, miR-181c can regulate mitochondrial Ca2 +-entry by targeting mt-COX1 during I/R injury. doi:10.1016/j.yjmcc.2017.07.063
052 Dual optical mapping of the innervated Langendorff-perfused heart reveals novel insights into acute electrophysiological responses to sympathetic stimulation Lianguo Wanga, Srinivas Tapaa, Samantha Stuarta, Rachel Mylesb, Kieran Brackc, Andre Ngc, Donald Bersa, Crystal Ripplingera a
University of California Davis, Davis, USA University of Glasgow, Glasgow, UK c University of Leicester, Leicester, UK b
Background: Sympathetic activation of the cardiac tissue has been recognized as one of the key adaptive mechanisms in the mammalian heart. However, few studies have looked at the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (AP) and intracellular Ca2 + transients (CaT). Methods and Results: The heart and posterior thoracic cavity from rabbits (NZW, N=9) and mice (C57Bl6, N =8) were dissected and perfused through the descending aorta for dual optical mapping of transmembrane potential (Vm) and intracellular Ca2 + to study the effects of direct sympathetic nerve stimulation (SNS). SNS was achieved through spinal cord stimulation at T1-T3. Our results show that acute SNS (60 sec, 5-10Hz) increased heart rate (HR) and CaT