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ER-mitochondria remodeling and mitochondrial complex I dysfunction in Dystrophin-deficient mice
Archives of Cardiovascular Disease Supplements (2019) 11, 232—235
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Topic 15 - Heart failure/cardiomyopathy—D Tuesday, April 2, 2019 340
Intracardiac extracellular vesicle release in post-infarction diabetic hearts S.M.I. Mazlan ∗ , V. Duval , C. Devue , M. Robillard , C.M. Boulanger , J.S. Silvestre , X. Loyer Paris Cardiovascular Research Centre, Inserm, Paris, France ∗ Corresponding author. E-mail address: [email protected] (S.M.I. Mazlan) Introduction Cardiovascular disease (CVD) is the main cause of death in non-communicable diseases. In response to myocardial infarction (MI), extracellular vesicles (EVs) including large (lEVs) and small (sEVs), are released within and from the heart to facilitate intercellular communication and maintaining cardiac homeostasis. Objective As diabetes increases the risk of CVD, the purpose of the study was to investigate how diabetes influences the release of intracardiac EVs after MI. Method C57BL/6 J male mice were fed normal chow diet or highfat diet (HFD) for 3 months. HFD fed mice were glucose intolerant as attested by the measure of GTT above 200 mg/mL. Mice were subjected to MI by permanent ligation of the left anterior descending artery and sham animals underwent similar surgical procedure without ligation. Left ventricles from sham or MI mice were then harvested at either 15, 24, 48 or 72 hours after surgery (N = 5 per group at each time point) and processed for EV extraction by differential centrifugation. EVs were quantified and analyzed via Tunable Resistive Pulse Sensing Technology (TRPS), flow cytometry and Western blot. Results In chow diet fed mice, release of both lEVs and sEVs was increased at 24 h post-MI when compared to shams. These findings were in agreement with previous data obtained in younger control animals. In diabetic mice, lEVs peaked at 24 h post-MI and this increase was slightly greater than that observed in chow diet fed mice. However, there were no differences in sEV release between sham and MI diabetic mice. TRPS analysis revealed that diabetes does not change EV size and population. Furthermore, both control and diabetic derived EVs harboured cardiomyocyte marker (Troponin T) as revealed by Western blot.
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Conclusion Our results show that diabetes modulates the release of both intracardiac sEVs and lEVs after MI. Further work will be needed to fully investigate the functional impact of cardiac EVs in the diabetic heart after MI. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2019.02.112 488
Metformin reverses cardiac SR/ER-mitochondria remodeling and mitochondrial complex I dysfunction in Dystrophin-deficient mice C. Angebault-Prouteau ∗ , M. Lacote , A. Lacampagne , J. Fauconnier PhyMedExp, Université de Montpellier, Inserm, CNRS, Montpellier, France ∗ Corresponding author. E-mail address: [email protected] (C. Angebault-Prouteau) Introduction Besides skeletal and diaphragmatic muscle dysfunction, Duchenne muscular dystrophy (DMD) exhibit a progressive cardiomyopathy with an altered Ca2 + handling and a defect in mitochondrial respiration capacity. Aim The aim of the present study was to determine if the sarco-endoplasmic reticulum (SR/ER)-mitochondria interaction and mitochondrial Ca2 + homeostasis contribute to the progression of the cardiomyopathy. Methods and results Ventricular myocytes were isolated from 3-month old Dystrophin-deficient mice (mdx mice). Using proximity ligation assay, we observed an increase interaction between the SR/ER Ca2 + release channels, IP3R1, and the porine of the mitochondria, VDAC1, associated with an increase expression levels of IP3R1 and SIGMA1R. Similarly, the mitochondrial Ca2 + uniporter (MCU) and its regulated subunit, MICU1, expressions levels were enhanced in mdx heart. Furthermore the mitochondrial Ca2 + uptake kinetics and the mitochondrial Ca2 + content were significantly increased. Using oxygraphy experiment, we observed a severe decrease in respiration driven by the complex I with a stimulation in anion superoxide production measured with Mitosox red
Topic 15 - Heart failure/cardiomyopathy—D fluorescent dye. Finally, mdx mice were treated with the complex I modulator metformin at 200 mg/kg/day during one month. Metformin normalize the SR/ER-mitochondria interaction, decreases MICU1 expression and mitochondrial Ca2 + content and tends to restore complex I alteration in cardiomyocytes. Conclusions In summary, our data demonstrate for the first time that in the DMD heart the mitochondrial dysfunction is linked to an excessive SR/ER-mitochondria coupling with an increase in mitochondrial Ca2 + uptake and complex I dysfunction. Such remodeling could be reversed by metformin providing a novel therapeutic perspective in DMD. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2019.02.113
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Altered reticulum-mitochondria interactions contribute to mitochondrial Ca2 + signaling dysfunction in the diabetic mice heart M. Dia 1,2,∗ , J. Rieusset 3 , E. Tubbs 3 , N. Bendridi 3 , L. Gomez 1 , M. Ovize 1 , M. Kurdi 2 , M. Paillard 1 1 Laboratoire CarMeN-équipe 5 Cardioprotection, Inserm, INRA, Université Claude-Bernard Lyon-1, INSA-Lyon, Univ-Lyon, 69500 Bron, France 2 Laboratory of Experimental and clinical Pharmacology, Faculty of Sciences, Lebanese University, Beyrouth, Liban 3 Laboratoire CarMeN-équipe 3, Inserm, INRA, Université Claude-Bernard Lyon-1, INSA-Lyon, Univ-Lyon, 69921 Oullins, France ∗ Corresponding author. E-mail address: [email protected] (M. Dia) Introduction Diabetic cardiomyopathy has been linked to Ca2 + signaling alterations, notably a decreased mitochondrial Ca2 + uptake. Uncovering the changes occurring at Ca2 + microdomains between reticulum and mitochondria in the heart has launched a new area of investigation for cardiometabolic diseases. Objective We here aimed to study if the impairment of mitochondrial Ca2 + handling could be due to a dysregulation of the reticulum-mitochondria interactions or of the mitochondrial Ca2 + uniporter in the diabetic mice heart. Methods Mice were either fed with a standard diet (SD: 16.9% proteins, 4.3% lipids) or a high-fat high sucrose diet (HFHSD: 20% proteins, 36% lipids) for 16 weeks. Cardiac mitochondria associated membranes (MAM) composition was analyzed by proteomics and immunoblotting. Proximity ligation assay, calcium imaging and hypoxia/reoxygenation were performed on isolated cardiomyocytes. Results Our HFHSD mice displayed a cardiac insulin resistance and hypertrophy. Decreased MAM/pure mitochondria content in the HFHSD vs SD heart was observed, with an elevated level of proteins involved in lipid metabolism and a decrease in tethering proteins. Decreased IP3R-VDAC proximity upon HFHSD was concomitant to a reduced IP3R-stimulated Ca2 + transfer to mitochondria, with no changes in mitochondrial calcium uniporter protein expression and function. Additionally, decreased amplitude of cytosolic Ca2 + transients was seen in the HFHSD cardiomyocytes, with no significant changes in the reticular Ca2 + release level. Besides, increased cell death susceptibility was measured after both in vitro
233 hypoxia/reoxygenation and in vivo ischemia/reperfusion under HFHSD. Conclusion Our data, therefore, indicate that decreased reticulum-mitochondria interactions trigger an impaired mitochondrial Ca2 + handling in the diabetic mice heart, with no alteration of the mitochondrial Ca2 + uniporter, while the enhanced susceptibility to cell death could be referred to lipid toxicity. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2019.02.114 411
Molecular basis of high glucose-mediated cardiac calcium mishandling M. Samia el Hayek 1,∗ , P. Gerbaud 1 , C. Valdivia 2 , S. Gomez 1 , F. Lefebvre 1 , J. Chen 3 , H. Valdivia 2 , J.P. Benitah 1 , A.M. Gomez 1 , L. Pereira 1 1 University of Paris-Sud, Chatenay Malabry, France 2 Department of Medicine, University of Wisconsin, Madison 3 Department of Medicine, University of California, San Diego, CA, États-Unis ∗ Corresponding author. E-mail address: [email protected] (M. Samia el Hayek) Introduction Epidemiological and preclinical studies have pointed out a correlation between hyperglycemia and increasing risk of heart failure and cardiac death. In cardiomyocytes, hyperglycemia has been shown to alter Ca2 + signalling via CaMKII. Yet, the underlying mechanisms are still unclear. Objective To determine the molecular basis of high glucosemediated Ca2 + mishandling. Methods Ventricular cardiomyocytes were isolated from control and Epac2-KO mice. Parallel experiments were repeated in human cardiomyocytes differentiated from induced pluripotent stem cells (h-iPSC-CM). Ca2 + signaling was studied in cells loaded with a fluorescent Ca2 + dye and treated with high glucose (HG) ± ESI-05 (Epac2 inhibitor). We assessed ryanodine receptor (RyR2) phosphorylation state by western blot and measured single RyR2 channel activity incorporated into bilayers. Results HG-mediated SR Ca2 + leak and pro-arrhythmic events were prevented with Epac2 blocker (ESI-05) in normal mice cells and with Epac2 deletion (Epac2-KO) (Ca2 + sparks frequency N/100 m/s: 0.23 ± 0.15 for HG + ESI-05, 0.09 ± 0.04 for Epac2KO + HG vs. 0.16 ± 0.06 for normal glucose, P = N.S.). HG treatment increased RyR2 phosphorylation at the CaMKII site (S2814, ∼ 29% increase, P < 0.05) leading to a dramatic increase in RyR2 open probability (P0 = 0.76 ± 0.07 in HG vs. 0.22 ± 0.07 in NG, P < 0.01) fully prevented with ESI-05 (P0 = 0.15 ± 0.03, P = N.S.). Similarly, in h-iPSC-CM, chronic HG (1 week) increased the percentage of cells presenting pro-arrhythmic events (85% of cells). Electrically evoked [Ca2 + ]i transients were decreased in h-IPSC-CM (F/F0 = 2.10 ± 0.07 vs. 2.94 ± 0.17, P < 0.01) and associated to higher RyR-S2814 phosphorylation. Those effects were blunted by ESI-05. Conclusion Our data suggests that HG alters Ca2 + signalling via Epac2—mediated pro-arrhythmic events due a CaMKII-dependent increase of RyR2 activity. Overtime, this newly described mechanism lowers systolic Ca2 + release as seen in diabetic-associated cardiomyopathy. Disclosure of interest The authors declare that they have no competing interest.
Available online at
ScienceDirect www.sciencedirect.com
Topic 15 - Heart failure/cardiomyopathy—D Tuesday, April 2, 2019 340
Intracardiac extracellular vesicle release in post-infarction diabetic hearts S.M.I. Mazlan ∗ , V. Duval , C. Devue , M. Robillard , C.M. Boulanger , J.S. Silvestre , X. Loyer Paris Cardiovascular Research Centre, Inserm, Paris, France ∗ Corresponding author. E-mail address: [email protected] (S.M.I. Mazlan) Introduction Cardiovascular disease (CVD) is the main cause of death in non-communicable diseases. In response to myocardial infarction (MI), extracellular vesicles (EVs) including large (lEVs) and small (sEVs), are released within and from the heart to facilitate intercellular communication and maintaining cardiac homeostasis. Objective As diabetes increases the risk of CVD, the purpose of the study was to investigate how diabetes influences the release of intracardiac EVs after MI. Method C57BL/6 J male mice were fed normal chow diet or highfat diet (HFD) for 3 months. HFD fed mice were glucose intolerant as attested by the measure of GTT above 200 mg/mL. Mice were subjected to MI by permanent ligation of the left anterior descending artery and sham animals underwent similar surgical procedure without ligation. Left ventricles from sham or MI mice were then harvested at either 15, 24, 48 or 72 hours after surgery (N = 5 per group at each time point) and processed for EV extraction by differential centrifugation. EVs were quantified and analyzed via Tunable Resistive Pulse Sensing Technology (TRPS), flow cytometry and Western blot. Results In chow diet fed mice, release of both lEVs and sEVs was increased at 24 h post-MI when compared to shams. These findings were in agreement with previous data obtained in younger control animals. In diabetic mice, lEVs peaked at 24 h post-MI and this increase was slightly greater than that observed in chow diet fed mice. However, there were no differences in sEV release between sham and MI diabetic mice. TRPS analysis revealed that diabetes does not change EV size and population. Furthermore, both control and diabetic derived EVs harboured cardiomyocyte marker (Troponin T) as revealed by Western blot.
1878-6480/
Conclusion Our results show that diabetes modulates the release of both intracardiac sEVs and lEVs after MI. Further work will be needed to fully investigate the functional impact of cardiac EVs in the diabetic heart after MI. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2019.02.112 488
Metformin reverses cardiac SR/ER-mitochondria remodeling and mitochondrial complex I dysfunction in Dystrophin-deficient mice C. Angebault-Prouteau ∗ , M. Lacote , A. Lacampagne , J. Fauconnier PhyMedExp, Université de Montpellier, Inserm, CNRS, Montpellier, France ∗ Corresponding author. E-mail address: [email protected] (C. Angebault-Prouteau) Introduction Besides skeletal and diaphragmatic muscle dysfunction, Duchenne muscular dystrophy (DMD) exhibit a progressive cardiomyopathy with an altered Ca2 + handling and a defect in mitochondrial respiration capacity. Aim The aim of the present study was to determine if the sarco-endoplasmic reticulum (SR/ER)-mitochondria interaction and mitochondrial Ca2 + homeostasis contribute to the progression of the cardiomyopathy. Methods and results Ventricular myocytes were isolated from 3-month old Dystrophin-deficient mice (mdx mice). Using proximity ligation assay, we observed an increase interaction between the SR/ER Ca2 + release channels, IP3R1, and the porine of the mitochondria, VDAC1, associated with an increase expression levels of IP3R1 and SIGMA1R. Similarly, the mitochondrial Ca2 + uniporter (MCU) and its regulated subunit, MICU1, expressions levels were enhanced in mdx heart. Furthermore the mitochondrial Ca2 + uptake kinetics and the mitochondrial Ca2 + content were significantly increased. Using oxygraphy experiment, we observed a severe decrease in respiration driven by the complex I with a stimulation in anion superoxide production measured with Mitosox red
Topic 15 - Heart failure/cardiomyopathy—D fluorescent dye. Finally, mdx mice were treated with the complex I modulator metformin at 200 mg/kg/day during one month. Metformin normalize the SR/ER-mitochondria interaction, decreases MICU1 expression and mitochondrial Ca2 + content and tends to restore complex I alteration in cardiomyocytes. Conclusions In summary, our data demonstrate for the first time that in the DMD heart the mitochondrial dysfunction is linked to an excessive SR/ER-mitochondria coupling with an increase in mitochondrial Ca2 + uptake and complex I dysfunction. Such remodeling could be reversed by metformin providing a novel therapeutic perspective in DMD. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2019.02.113
052
Altered reticulum-mitochondria interactions contribute to mitochondrial Ca2 + signaling dysfunction in the diabetic mice heart M. Dia 1,2,∗ , J. Rieusset 3 , E. Tubbs 3 , N. Bendridi 3 , L. Gomez 1 , M. Ovize 1 , M. Kurdi 2 , M. Paillard 1 1 Laboratoire CarMeN-équipe 5 Cardioprotection, Inserm, INRA, Université Claude-Bernard Lyon-1, INSA-Lyon, Univ-Lyon, 69500 Bron, France 2 Laboratory of Experimental and clinical Pharmacology, Faculty of Sciences, Lebanese University, Beyrouth, Liban 3 Laboratoire CarMeN-équipe 3, Inserm, INRA, Université Claude-Bernard Lyon-1, INSA-Lyon, Univ-Lyon, 69921 Oullins, France ∗ Corresponding author. E-mail address: [email protected] (M. Dia) Introduction Diabetic cardiomyopathy has been linked to Ca2 + signaling alterations, notably a decreased mitochondrial Ca2 + uptake. Uncovering the changes occurring at Ca2 + microdomains between reticulum and mitochondria in the heart has launched a new area of investigation for cardiometabolic diseases. Objective We here aimed to study if the impairment of mitochondrial Ca2 + handling could be due to a dysregulation of the reticulum-mitochondria interactions or of the mitochondrial Ca2 + uniporter in the diabetic mice heart. Methods Mice were either fed with a standard diet (SD: 16.9% proteins, 4.3% lipids) or a high-fat high sucrose diet (HFHSD: 20% proteins, 36% lipids) for 16 weeks. Cardiac mitochondria associated membranes (MAM) composition was analyzed by proteomics and immunoblotting. Proximity ligation assay, calcium imaging and hypoxia/reoxygenation were performed on isolated cardiomyocytes. Results Our HFHSD mice displayed a cardiac insulin resistance and hypertrophy. Decreased MAM/pure mitochondria content in the HFHSD vs SD heart was observed, with an elevated level of proteins involved in lipid metabolism and a decrease in tethering proteins. Decreased IP3R-VDAC proximity upon HFHSD was concomitant to a reduced IP3R-stimulated Ca2 + transfer to mitochondria, with no changes in mitochondrial calcium uniporter protein expression and function. Additionally, decreased amplitude of cytosolic Ca2 + transients was seen in the HFHSD cardiomyocytes, with no significant changes in the reticular Ca2 + release level. Besides, increased cell death susceptibility was measured after both in vitro
233 hypoxia/reoxygenation and in vivo ischemia/reperfusion under HFHSD. Conclusion Our data, therefore, indicate that decreased reticulum-mitochondria interactions trigger an impaired mitochondrial Ca2 + handling in the diabetic mice heart, with no alteration of the mitochondrial Ca2 + uniporter, while the enhanced susceptibility to cell death could be referred to lipid toxicity. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2019.02.114 411
Molecular basis of high glucose-mediated cardiac calcium mishandling M. Samia el Hayek 1,∗ , P. Gerbaud 1 , C. Valdivia 2 , S. Gomez 1 , F. Lefebvre 1 , J. Chen 3 , H. Valdivia 2 , J.P. Benitah 1 , A.M. Gomez 1 , L. Pereira 1 1 University of Paris-Sud, Chatenay Malabry, France 2 Department of Medicine, University of Wisconsin, Madison 3 Department of Medicine, University of California, San Diego, CA, États-Unis ∗ Corresponding author. E-mail address: [email protected] (M. Samia el Hayek) Introduction Epidemiological and preclinical studies have pointed out a correlation between hyperglycemia and increasing risk of heart failure and cardiac death. In cardiomyocytes, hyperglycemia has been shown to alter Ca2 + signalling via CaMKII. Yet, the underlying mechanisms are still unclear. Objective To determine the molecular basis of high glucosemediated Ca2 + mishandling. Methods Ventricular cardiomyocytes were isolated from control and Epac2-KO mice. Parallel experiments were repeated in human cardiomyocytes differentiated from induced pluripotent stem cells (h-iPSC-CM). Ca2 + signaling was studied in cells loaded with a fluorescent Ca2 + dye and treated with high glucose (HG) ± ESI-05 (Epac2 inhibitor). We assessed ryanodine receptor (RyR2) phosphorylation state by western blot and measured single RyR2 channel activity incorporated into bilayers. Results HG-mediated SR Ca2 + leak and pro-arrhythmic events were prevented with Epac2 blocker (ESI-05) in normal mice cells and with Epac2 deletion (Epac2-KO) (Ca2 + sparks frequency N/100 m/s: 0.23 ± 0.15 for HG + ESI-05, 0.09 ± 0.04 for Epac2KO + HG vs. 0.16 ± 0.06 for normal glucose, P = N.S.). HG treatment increased RyR2 phosphorylation at the CaMKII site (S2814, ∼ 29% increase, P < 0.05) leading to a dramatic increase in RyR2 open probability (P0 = 0.76 ± 0.07 in HG vs. 0.22 ± 0.07 in NG, P < 0.01) fully prevented with ESI-05 (P0 = 0.15 ± 0.03, P = N.S.). Similarly, in h-iPSC-CM, chronic HG (1 week) increased the percentage of cells presenting pro-arrhythmic events (85% of cells). Electrically evoked [Ca2 + ]i transients were decreased in h-IPSC-CM (F/F0 = 2.10 ± 0.07 vs. 2.94 ± 0.17, P < 0.01) and associated to higher RyR-S2814 phosphorylation. Those effects were blunted by ESI-05. Conclusion Our data suggests that HG alters Ca2 + signalling via Epac2—mediated pro-arrhythmic events due a CaMKII-dependent increase of RyR2 activity. Overtime, this newly described mechanism lowers systolic Ca2 + release as seen in diabetic-associated cardiomyopathy. Disclosure of interest The authors declare that they have no competing interest.