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Optogenetic Modulation of Cardiomyocyte Excitability
424a
Tuesday, February 14, 2017
2083-Pos Board B403 The Effect of Ribonucleotide Reductase Overexpression on Cardiomyocyte Metabolism Jason D. Murray1, Farid Moussavi-Harami2, Michael Regnier3. 1 Physiology and Biophysics, University of Washington, Seattle, WA, USA, 2 Cardiology, University of Washington, Seattle, WA, USA, 3Bioengineering, University of Washington, Seattle, WA, USA. Our group has repeatedly reported that of 2-deoxy-ATP (dATP) can be used by myosin and significantly increases contraction of cardiac muscle at all levels of calcium activation. We have also demonstrated that overexpressing the enzyme ribonucleotide reductase (RNR) in cardiomyocytes using viral vectors increases the rate and magnitude of contraction and increases left ventricular contraction in normal and infarcted hearts. This increase in work likely results in a concomitant increase in energy utilization. Thus an important question in developing this approach as a therapy is to determine whether and how elevated dATP affects cardiac metabolism, which is the goal of this current study. Preliminary data suggest that upregulating RNR results in an increase in the relative contribution of succinate dehydrogenase to mitochondrial respiration in the heart, possibly indicating an enhanced utilization of fatty acid oxidation despite no change in overall respiratory capacity. While mitochondria have a low affinity for dADP, creatine kinase phosphorylates dADP at a similar rate as ADP, allowing for rapid regeneration of dATP. The overexpression of RNR in cardiomyocytes normally results in a tenfold increase in cytosolic concentration of 2-deoxy-ATP (dATP), such that it becomes ~1% of the total ATP pool. We now have a viral vector for expressing a constitutively active form of the Rrm1 subunit (D57N mutation) that may increase cytosolic [dATP] as high as 10% of the ATP pool in cardiomyocytes and in vitro experiments indicate this level does not reduce mitochondrial respiratory capacity. Ongoing and planned experiments will compare the effects of overexpression of RNR vs. D57N containing RNR on contractile and metabolic properties of cardiomyocytes. 2084-Pos Board B404 Cardiac Fatty Acid Binding Protein (FABP3) Depletes SR Calcium Load in Ventricular Myocytes Wenjie Li, Shaoran Zhang, Siwei Zhou, Lingling Jiang, Wei Wang. Biochemistry and Molecular Biology, Hebei Medical University, Shijiazhuang, China. Rationale: Cardiac fatty acid-binding protein (FABP3) is a cardiac-specific member of lipid-binding protein family and its expression level is often reduced in patients with diabetic heart diseases. Recent evidence suggests that FABP3 suppresses calcium transient and shortening of isolated rat cardiomyocytes. However, the underlying mechanisms are largely elusive. Objective: To determine the role of FABP3 in regulating SR calcium release. Results: In STZinduced type I diabetes mouse model (DM), protein expression level of FABP3 was elevated, cardiac function was reduced and negatively correlated with FABP3 expression level. Amplitudes of cell shortening and calcium transient are both impaired in DM cardiomyocytes, which can be mimicked by applying FABP3 at pathological concentration to control cardiomyocytes. FABP3 reduces calcium transient amplitude of cardiomyocytes in a dose dependent manner (EC50 = 0.052 nmol/L). SR calcium content is reduced in DM cardiomyocytes and FABP3 depletes SR calcium content in cardiomyocytes. FABP3 colocalizes with SERCA, inhibits SERCA activity with a greater EC50 (0.49 nmol/L). Co-immunoprecipitation study suggests elevated FABP3 promotes binding between SERCA and phospholamban. FABP3 also colocalizes with RyR2, binds to RyR2 and promotes RyR2-mediated SR calcium leak. Conclusion: Increased FABP3 expression level in DM mice compromises cardiac function by reducing SR calcium load via two independent approaches: (A) to reduce SERCA activity by promoting PLB-SERCA interaction and (B) to enhance RyR2-mediated SR calcium leak. 2085-Pos Board B405 Optogenetics Design of Mechanistically-Based Stimulation Patterns for Cardiac Defibrillation Claudia Crocini1, Cecilia Ferrantini2, Raffaele Coppini2, Marina Scardigli1, Ping Yan3, Leslie M. Loew3, Godfrey L. Smith4, Elisabetta Cerbai2, Corrado Poggesi2, Francesco S. Pavone1, Leonardo Sacconi1. 1 LENS, Sesto Fiorentino, Italy, 2University of Florence, Florence, Italy, 3 University of Connecticut Health Center, Farmington, CT, USA, 4University of Glasgow, Glasgow, United Kingdom. Current rescue therapies for life-threatening arrhythmias disregard the pathological electro-anatomical substrate and base their efficacy on a generalized electrical discharge. Here, we developed an all-optical platform to examine less invasive defibrillation strategies. An ultrafast wide-field macroscope operating at 2 KHz (100 x 100 pixel) was developed to optically map action
potential propagation with a red-shifted voltage sensitive dye (di-4ANBDQPQ) in whole mouse hearts. Control of the electrical activity was achieved by employing transgenic mouse hearts expressing Channel Rhodopsin-2 (ChR2). In order to draw arbitrarily-chosen ChR2 stimulation patterns with sub-millisecond temporal resolution, the macroscope was implemented with a random-access scanning head based on acousto-optic deflectors (AODs). AODs rapidly scan the laser beam across the whole field of view exciting different volume with a commutation time of few ms. At the end of one cycle the AODs return to the initial position and repeat the stimulation cycle. Alternatively, a simpler optical solution based on digital micromirror device (DMD) in combination with a high power LED was used to manipulate light positioning in a real simultaneous manner. We employed the macroscope to study the mechanistic features of ventricular tachycardia and we designed mechanistically-based cardioversion/defibrillation patterns exploiting the transient refractoriness of myocardium produced by the ChR2 stimulation. Multiple regions of conduction block revealed to efficiently defibrillate arrhythmic hearts but with lower energy requirements as compared to whole ventricle interventions. To confirm that the cardioversion efficiency is rigorously dependent on the mechanistic-based design, we positioned multiple regions of conduction block regardless of the re-entry arrhythmic wavefront obtaining a dramatically reduced cardioversion rate. In conclusion, this work demonstrates that defibrillation energies can be substantially reduced by applying discrete stimulation patterns and promotes the investigation of new anti-arrhythmic strategies. 2086-Pos Board B406 Optogenetic Modulation of Cardiomyocyte Excitability Ramona Kopton1, Eva Rog-Zielinska2, Urszula Siedlecka2, Jonas Wietek3, Peter Hegemann3, Peter Kohl1, Franziska Schneider1. 1 Institute for Experimental Cardiovascular Medicine, University Heart Centre Freiburg - Bad Krozingen, Freiburg, Germany, 2National Heart and Lung Institute, Imperial College London, London, United Kingdom, 3 Experimental Biophysics, Institute for Biology, Humboldt-University Berlin, Berlin, Germany. Optogenetics is a fast-developing technology, first applied to neuroscience research. A number of studies have begun to transfer the optogenetic approach to cardiovascular research, with a focus on electrical regulation of specific cell types in the heart. There is demand to develop tools to selectively modulate electrical activity in cardiomyocytes (CM) and to interfere with membrane currents in non-myocytes (NM) to advance our understanding of heterocellular-electrotonic coupling in vitro and in vivo. We here report on cell-type specific activation of co-cultured CM and NM using the light-gated cation channel ChR2. Furthermore, we tested three anionselective channelrhodopsins (ACR) for their potential to inhibit action potential initiation and propagation. Neonatal hearts of the lines WT1-VSFPþ;þ, aMHC-VSFPþ;þ and WT1-ChR2H134Rþ;þ were isolated and digested. Cultured cells were transfected with cDNA of ACRs coupled to fluorescent marker proteins. In order to analyse heterocellular coupling we performed whole-cell patchclamp recordings on CM cocultured with NM from the WT1-ChR2H134Rþ;þ line. In a first set of experiments, pulsed ChR2 activation in NM evoked action potentials in patched CM, indicating direct electrical coupling. Next, we tested three different ACR for their potential to hyperpolarise cardiac cells (GtACR1-eGFP, iCþþ mCherry, Phobos-mCherry). Transfected cells were patch-clamped in the current-clamp mode to follow light-induced changes in membrane voltage. Notably, ACR activation lead to hyperpolarisation in isolated cardiac NM and to depolarisation in CM. We would like to inhibit cardiac electrical activity by optogenetic hyperpolarisation of NM. As we have shown ACR photocurrents depolarise CM and evoke action potentials, different to the inhibitory effect reported in neurons. We expect that NM, which are connected to CM, have more negative potentials, resulting in depolarising ACR currents also in NM. Therefore, we are looking for a stronger tool to inhibit cardiac activity and plan to generate a light-activated Kþ-channel. 2087-Pos Board B407 Antihypertrophic Effects of Diazoxide Involves Changes in MIR-132 Expression in Adult Rat Cardiomycytes Gayathri Narasimhan, Elba Carrillo, Ascencion Herna´ndez, Maria C. Garcı´a, Jorge A. Sanchez. Pharmacology, Cinvestav, Mexico, D.F., Mexico. Diazoxide (DZX), a mitochondrial KATP channel opener has anti-hypertrophic effects in cardiac hypertrophy models but the mechanism involved is unclear. Here we report that DZX prevents cardiac hypertrophy induced by long-term beta-adrenergic stimulation by regulating miR-132 expression.
Tuesday, February 14, 2017
2083-Pos Board B403 The Effect of Ribonucleotide Reductase Overexpression on Cardiomyocyte Metabolism Jason D. Murray1, Farid Moussavi-Harami2, Michael Regnier3. 1 Physiology and Biophysics, University of Washington, Seattle, WA, USA, 2 Cardiology, University of Washington, Seattle, WA, USA, 3Bioengineering, University of Washington, Seattle, WA, USA. Our group has repeatedly reported that of 2-deoxy-ATP (dATP) can be used by myosin and significantly increases contraction of cardiac muscle at all levels of calcium activation. We have also demonstrated that overexpressing the enzyme ribonucleotide reductase (RNR) in cardiomyocytes using viral vectors increases the rate and magnitude of contraction and increases left ventricular contraction in normal and infarcted hearts. This increase in work likely results in a concomitant increase in energy utilization. Thus an important question in developing this approach as a therapy is to determine whether and how elevated dATP affects cardiac metabolism, which is the goal of this current study. Preliminary data suggest that upregulating RNR results in an increase in the relative contribution of succinate dehydrogenase to mitochondrial respiration in the heart, possibly indicating an enhanced utilization of fatty acid oxidation despite no change in overall respiratory capacity. While mitochondria have a low affinity for dADP, creatine kinase phosphorylates dADP at a similar rate as ADP, allowing for rapid regeneration of dATP. The overexpression of RNR in cardiomyocytes normally results in a tenfold increase in cytosolic concentration of 2-deoxy-ATP (dATP), such that it becomes ~1% of the total ATP pool. We now have a viral vector for expressing a constitutively active form of the Rrm1 subunit (D57N mutation) that may increase cytosolic [dATP] as high as 10% of the ATP pool in cardiomyocytes and in vitro experiments indicate this level does not reduce mitochondrial respiratory capacity. Ongoing and planned experiments will compare the effects of overexpression of RNR vs. D57N containing RNR on contractile and metabolic properties of cardiomyocytes. 2084-Pos Board B404 Cardiac Fatty Acid Binding Protein (FABP3) Depletes SR Calcium Load in Ventricular Myocytes Wenjie Li, Shaoran Zhang, Siwei Zhou, Lingling Jiang, Wei Wang. Biochemistry and Molecular Biology, Hebei Medical University, Shijiazhuang, China. Rationale: Cardiac fatty acid-binding protein (FABP3) is a cardiac-specific member of lipid-binding protein family and its expression level is often reduced in patients with diabetic heart diseases. Recent evidence suggests that FABP3 suppresses calcium transient and shortening of isolated rat cardiomyocytes. However, the underlying mechanisms are largely elusive. Objective: To determine the role of FABP3 in regulating SR calcium release. Results: In STZinduced type I diabetes mouse model (DM), protein expression level of FABP3 was elevated, cardiac function was reduced and negatively correlated with FABP3 expression level. Amplitudes of cell shortening and calcium transient are both impaired in DM cardiomyocytes, which can be mimicked by applying FABP3 at pathological concentration to control cardiomyocytes. FABP3 reduces calcium transient amplitude of cardiomyocytes in a dose dependent manner (EC50 = 0.052 nmol/L). SR calcium content is reduced in DM cardiomyocytes and FABP3 depletes SR calcium content in cardiomyocytes. FABP3 colocalizes with SERCA, inhibits SERCA activity with a greater EC50 (0.49 nmol/L). Co-immunoprecipitation study suggests elevated FABP3 promotes binding between SERCA and phospholamban. FABP3 also colocalizes with RyR2, binds to RyR2 and promotes RyR2-mediated SR calcium leak. Conclusion: Increased FABP3 expression level in DM mice compromises cardiac function by reducing SR calcium load via two independent approaches: (A) to reduce SERCA activity by promoting PLB-SERCA interaction and (B) to enhance RyR2-mediated SR calcium leak. 2085-Pos Board B405 Optogenetics Design of Mechanistically-Based Stimulation Patterns for Cardiac Defibrillation Claudia Crocini1, Cecilia Ferrantini2, Raffaele Coppini2, Marina Scardigli1, Ping Yan3, Leslie M. Loew3, Godfrey L. Smith4, Elisabetta Cerbai2, Corrado Poggesi2, Francesco S. Pavone1, Leonardo Sacconi1. 1 LENS, Sesto Fiorentino, Italy, 2University of Florence, Florence, Italy, 3 University of Connecticut Health Center, Farmington, CT, USA, 4University of Glasgow, Glasgow, United Kingdom. Current rescue therapies for life-threatening arrhythmias disregard the pathological electro-anatomical substrate and base their efficacy on a generalized electrical discharge. Here, we developed an all-optical platform to examine less invasive defibrillation strategies. An ultrafast wide-field macroscope operating at 2 KHz (100 x 100 pixel) was developed to optically map action
potential propagation with a red-shifted voltage sensitive dye (di-4ANBDQPQ) in whole mouse hearts. Control of the electrical activity was achieved by employing transgenic mouse hearts expressing Channel Rhodopsin-2 (ChR2). In order to draw arbitrarily-chosen ChR2 stimulation patterns with sub-millisecond temporal resolution, the macroscope was implemented with a random-access scanning head based on acousto-optic deflectors (AODs). AODs rapidly scan the laser beam across the whole field of view exciting different volume with a commutation time of few ms. At the end of one cycle the AODs return to the initial position and repeat the stimulation cycle. Alternatively, a simpler optical solution based on digital micromirror device (DMD) in combination with a high power LED was used to manipulate light positioning in a real simultaneous manner. We employed the macroscope to study the mechanistic features of ventricular tachycardia and we designed mechanistically-based cardioversion/defibrillation patterns exploiting the transient refractoriness of myocardium produced by the ChR2 stimulation. Multiple regions of conduction block revealed to efficiently defibrillate arrhythmic hearts but with lower energy requirements as compared to whole ventricle interventions. To confirm that the cardioversion efficiency is rigorously dependent on the mechanistic-based design, we positioned multiple regions of conduction block regardless of the re-entry arrhythmic wavefront obtaining a dramatically reduced cardioversion rate. In conclusion, this work demonstrates that defibrillation energies can be substantially reduced by applying discrete stimulation patterns and promotes the investigation of new anti-arrhythmic strategies. 2086-Pos Board B406 Optogenetic Modulation of Cardiomyocyte Excitability Ramona Kopton1, Eva Rog-Zielinska2, Urszula Siedlecka2, Jonas Wietek3, Peter Hegemann3, Peter Kohl1, Franziska Schneider1. 1 Institute for Experimental Cardiovascular Medicine, University Heart Centre Freiburg - Bad Krozingen, Freiburg, Germany, 2National Heart and Lung Institute, Imperial College London, London, United Kingdom, 3 Experimental Biophysics, Institute for Biology, Humboldt-University Berlin, Berlin, Germany. Optogenetics is a fast-developing technology, first applied to neuroscience research. A number of studies have begun to transfer the optogenetic approach to cardiovascular research, with a focus on electrical regulation of specific cell types in the heart. There is demand to develop tools to selectively modulate electrical activity in cardiomyocytes (CM) and to interfere with membrane currents in non-myocytes (NM) to advance our understanding of heterocellular-electrotonic coupling in vitro and in vivo. We here report on cell-type specific activation of co-cultured CM and NM using the light-gated cation channel ChR2. Furthermore, we tested three anionselective channelrhodopsins (ACR) for their potential to inhibit action potential initiation and propagation. Neonatal hearts of the lines WT1-VSFPþ;þ, aMHC-VSFPþ;þ and WT1-ChR2H134Rþ;þ were isolated and digested. Cultured cells were transfected with cDNA of ACRs coupled to fluorescent marker proteins. In order to analyse heterocellular coupling we performed whole-cell patchclamp recordings on CM cocultured with NM from the WT1-ChR2H134Rþ;þ line. In a first set of experiments, pulsed ChR2 activation in NM evoked action potentials in patched CM, indicating direct electrical coupling. Next, we tested three different ACR for their potential to hyperpolarise cardiac cells (GtACR1-eGFP, iCþþ mCherry, Phobos-mCherry). Transfected cells were patch-clamped in the current-clamp mode to follow light-induced changes in membrane voltage. Notably, ACR activation lead to hyperpolarisation in isolated cardiac NM and to depolarisation in CM. We would like to inhibit cardiac electrical activity by optogenetic hyperpolarisation of NM. As we have shown ACR photocurrents depolarise CM and evoke action potentials, different to the inhibitory effect reported in neurons. We expect that NM, which are connected to CM, have more negative potentials, resulting in depolarising ACR currents also in NM. Therefore, we are looking for a stronger tool to inhibit cardiac activity and plan to generate a light-activated Kþ-channel. 2087-Pos Board B407 Antihypertrophic Effects of Diazoxide Involves Changes in MIR-132 Expression in Adult Rat Cardiomycytes Gayathri Narasimhan, Elba Carrillo, Ascencion Herna´ndez, Maria C. Garcı´a, Jorge A. Sanchez. Pharmacology, Cinvestav, Mexico, D.F., Mexico. Diazoxide (DZX), a mitochondrial KATP channel opener has anti-hypertrophic effects in cardiac hypertrophy models but the mechanism involved is unclear. Here we report that DZX prevents cardiac hypertrophy induced by long-term beta-adrenergic stimulation by regulating miR-132 expression.