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Mechano-Chemo-Transduction in Rabbit Cardiomyocytes Mediated by no Signaling
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Wednesday, March 2, 2016
characterized (Coppini et al. ABS Biophysical Journal 2015) the changes in sarcomere function and E-C coupling that occur in ventricular myocardium of two HCM mouse models carrying different mutations in cTnT (R92Q and E163R). Both models exhibited diastolic dysfunction that was, however, related to different mechanisms i.e. E-C coupling abnormalities in R92Q and sarcomere changes in E163R. Here we employ these mouse models to study whether atrial remodeling is a consequence of diastolic dysfunction or is also influenced by the specific underlying mutation. Echocardiographic measurements of left atrial (LA) dimensions showed that LA area was severely increased in R92Q hearts while it was only mildly increased in E163R (in mm2 : 6.7350.5 in R92Q, 4.8250.16 in E163R vs 3.9750.26 in WT). Left atrial trabeculae were dissected and mounted isometrically to record twitch tension. We studied the steady-state force-frequency relationship and the response to positive inotropic stimuli such as Isoproterenol 10-7 mM (ISO) and 8 mM extracellular [Ca2þ]. Compared to WT, R92Q atrial trabeculae showed: (i) slower kinetics of both force development and relaxation (e.g. at 1 Hz, 50% relaxation was prolonged by 35%), (ii) impaired twitch amplitude at high pacing rates (50% reduction), (iii) depressed rested-state contractions and (iv) blunted increase of twitch tension in ISO and high [Ca2þ]. None of these changes were observed in intact E163R atrial trabeculae. These findings suggest that atrial remodeling in R92Q is more pronounced compared to E163R, and related to E-C coupling alterations. Supported by the Italian Ministry of Health (WFR GR-2011-02350583). 2961-Pos Board B338 Mechano-Chemo-Transduction in Rabbit Cardiomyocytes Mediated by no Signaling Rafael Shimkunas1,2, Zhong Jian1, Bence Hegyi1, John Shaw3, Nipavan Chiamvimonvat4, Kenneth Ginsburg1, Julie Bossuyt1, Donald M. Bers1, Kit S. Lam5, Leighton T. Izu1, Ye Chen-Izu1. 1 Pharmacology, UC Davis, Davis, CA, USA, 2Biomedical Engineering, UC Davis, Davis, CA, USA, 3Aerospace Engineering, University of Michigan, Ann Arbor, MI, USA, 4Internal Medicine/Cardiology, UC Davis, Davis, CA, USA, 5 Biochemistry and Molecular Medicine, UC Davis, Sacramento, CA, USA. Background: Increased mechanical stress under pathological conditions such as hypertension, infarction and fibrosis can cause arrhythmias and heart failure. However, little is known about Mechano-Chemo-Transduction (MCT) mechanisms that underlie heart disease development. We developed a novel Cell-in-Gel system to control mechanical loading on cardiomyocytes during excitation-contraction coupling. Here we investigate mechanical load effects on modulating Ca2þ signaling and contraction dynamics to test the hypothesis NO signaling mediates MCT. Methods: Freshly isolated adult rabbit ventricular myocytes were embedded in polyvinyl alcohol (10%) and crosslinked with tetravalent boronate-PEG (7.5%) to form a 3D viscoelastic hydrogel. The Cell-in-Gel system was continuously perfused with Tyrode’s solution and electrically paced at 0.5 Hz. Cardiomyocyte contraction and Ca2þ transients were measured in-gel and compared with load-free cells in solution. Results: Cardiomyocytes contracting in-gel under mechanical load showed augmented systolic Ca2þ transients (Fura-2 fluorescence ratio 1.47 5 0.07 ingel vs 0.85 5 0.03 load-free), revealing MCT transduces mechanical stress to increase intracellular Ca2þ release. NOS inhibition using 1 mM L-NAME reduced the in-gel Ca2þ transient by 33% (n=15), suggesting NO signaling mediates MCT. Cells in-gel showed 8% decrease in contraction (fractional shortening 13.5 5 0.4% in-gel vs 14.7 5 0.3% load-free). Cells in-gel treated with L-NAME demonstrated a further reduction in contraction (11.6 5 0.8%) as a result of decreased MCT by NOS inhibition. Importantly, 12% of cells in-gel showed mechanical load-induced alternans, which was abolished with L-NAME treatment, suggesting NO signaling contributes to load-induced Ca2þ dysregulation. Conclusion: Our findings demonstrate MCT increases the systolic Ca2þ transient to enhance contractility in response to increased afterload, serving a compensatory mechanism to autoregulate contractility. MCT is attenuated by inhibiting NOS. These results suggest NOS mediates increased Ca2þ signaling in cardiomyocytes under mechanical load, providing a mechanistic basis for the Anrep effect. 2962-Pos Board B339 The Role of ROS and Calcium for the Prolonged Force Depression after Eccentric Contractions Ha˚kan Westerblad1,2, Niklas Ivarsson1, Abram Katz1,3, Sigitas Kamandulis2, Maja Schlittler1,2, Marius Brazaitis2, Albertas Skurvydas2. 1 Department of Physiology & Pharmacology, Karolinska Institutet, Stockholm, Sweden, 2Lithuanian Sports University, Kaunas, Lithuania, 3 Ariel University, Ariel, Israel.
We recently showed that metabolically demanding high-intensity interval training results in a prolonged force depression due to increased production of reactive oxygen species (ROS) leading to defective cellular Ca2þ handling. A prolonged force depression can also be induced with mechanically demanding exercises, and we hypothesized that this deficiency also involves changes in ROS and Ca2þ. To test this hypothesis, human subjects performed mechanically demanding drop jumps, which induced a prolonged force depression especially at low stimulation frequencies: 24 hours after exercise force was decreased by ~40% at 20 Hz and ~20% at 100 Hz. Contrary to our hypothesis, we observed no signs of ROS-induced protein modifications after the drop jumps, as judged from no significant changes in the extent of malondialdehyde or carbonyl binding to protein. There was no significant change in the expression of the two key proteins in the control of sarcoplasmic reticulum Ca2þ release (the ryanodine receptor 1 (RyR1) and the dihydropyridine receptor), but there were minor changes in the amount of FKBP12 bound to RyR1 (an early increase followed by a decrease). In conclusion, the long-lasting force depressions after mechanically demanding drop jumps is not accompanied by any obvious signs of ROS-induced damage or Ca2þ dysregulation, which is in contrast to the situation after metabolically demanding exercise. 2963-Pos Board B340 The Loss of the Transmembrane Protein MG23 Affects the Fast-Twitch Feature of EDl Muscle Myuki Nishi1, Takahisa Gouda1, Nagomi Kurebayashi2, Yu Takahashi1, Shinji Komazaki3, Hua Zhu4, Hiroshi Takeshima1. 1 Biological Chemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Kyoto, Japan, 2Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine, Tokyo, Japan, 3Anatomy, Saitama Medical School, Moroyama, Japan, 4Surgery, Ohio State University, Clumbus, OH, USA. We previously identified the 23-kDa transmembrane protein MG23 in skeletal muscle. MG23 is abundantly expressed in skeletal muscle, and weakly detected in various tissues. Immunochemical and electronmicroscopic analyses suggested that MG23 forms a bowl-shaped homooligomer, and is distributed throughout the outer nuclear membrane and sarco/endoplasmic reticulum. In planar lipid bilayer membranes, purified recombinant MG23 behaved as voltage-sensitive cation-conducting channels which are equally permeable to Ca2þ and Kþ. Based on the observations, MG23 may take part in rapid cationic flux across intracellular membrane systems, while the physiological role of MG23 remains to be investigated. To survey MG23 function in vivo, we generated MG23-knockout (KO) mice. MG23-KO mice were healthy and apparently normal in growth and reproduction. In MG23-KO mice, fast-twitch EDL muscle exhibited reduced wet weight and slow relaxation after contraction. Although MG23 is unlikely to contribute directly to transcriptional machinery, gene-chip analysis detected aberrant gene expression in MG23-KO EDL muscle; for example, the expression levels of Ryr1, Cacna1s and Jph1 were obviously reduced, while Sln expression was >10-fold facilitated. On the other hand, slow-twitch soleus muscle from MG23-KO mice was apparently normal in contraction and gene expression. Our observations may imply that MG23 is a critical membranous component for the development and/or maintenance of fast-twitch properties in muscle cells. 2964-Pos Board B341 Roles of Mitsugumin53 in Skeletal Muscle Mi Kyoung Ahn1, Keon Jin Lee1, Mei Huang1, Jianjie Ma2, Eun Hui Lee1. 1 Dept. of Physiology, College of Medicine, The Catholic Univ. of Korea, Seoul, Korea, Republic of, 2Dept. of Internal Medicine, College of Medicine, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH, USA. Mitsugumin 53 (MG53), a tripartite-motif (TRIM) family protein, participates in cell membrane repair in multiple cell types. MG53 protects myocardial infarction associated with ischemia-reperfusion injury. Subcutaneous injection of purified MG53 to a mouse model of Duchenne muscular dystrophy alleviates the muscle pathology by promoting membrane repair. We previously reported the role of MG53 in mouse skeletal myotubes - MG53 binds to sarcoplasmic/ endoplasmic reticulum Ca2þ-ATPase 1a (SERCA1a) via its TRIM and PRY domains, and attenuates SERCA1a activity. In the present study, we tried to find additional roles of MG53 in skeletal muscle using single skeletal myotube Ca2þ imaging experiment, and found that MG53 regulated Ca2þ homeostasis in skeletal myotubes.
Wednesday, March 2, 2016
characterized (Coppini et al. ABS Biophysical Journal 2015) the changes in sarcomere function and E-C coupling that occur in ventricular myocardium of two HCM mouse models carrying different mutations in cTnT (R92Q and E163R). Both models exhibited diastolic dysfunction that was, however, related to different mechanisms i.e. E-C coupling abnormalities in R92Q and sarcomere changes in E163R. Here we employ these mouse models to study whether atrial remodeling is a consequence of diastolic dysfunction or is also influenced by the specific underlying mutation. Echocardiographic measurements of left atrial (LA) dimensions showed that LA area was severely increased in R92Q hearts while it was only mildly increased in E163R (in mm2 : 6.7350.5 in R92Q, 4.8250.16 in E163R vs 3.9750.26 in WT). Left atrial trabeculae were dissected and mounted isometrically to record twitch tension. We studied the steady-state force-frequency relationship and the response to positive inotropic stimuli such as Isoproterenol 10-7 mM (ISO) and 8 mM extracellular [Ca2þ]. Compared to WT, R92Q atrial trabeculae showed: (i) slower kinetics of both force development and relaxation (e.g. at 1 Hz, 50% relaxation was prolonged by 35%), (ii) impaired twitch amplitude at high pacing rates (50% reduction), (iii) depressed rested-state contractions and (iv) blunted increase of twitch tension in ISO and high [Ca2þ]. None of these changes were observed in intact E163R atrial trabeculae. These findings suggest that atrial remodeling in R92Q is more pronounced compared to E163R, and related to E-C coupling alterations. Supported by the Italian Ministry of Health (WFR GR-2011-02350583). 2961-Pos Board B338 Mechano-Chemo-Transduction in Rabbit Cardiomyocytes Mediated by no Signaling Rafael Shimkunas1,2, Zhong Jian1, Bence Hegyi1, John Shaw3, Nipavan Chiamvimonvat4, Kenneth Ginsburg1, Julie Bossuyt1, Donald M. Bers1, Kit S. Lam5, Leighton T. Izu1, Ye Chen-Izu1. 1 Pharmacology, UC Davis, Davis, CA, USA, 2Biomedical Engineering, UC Davis, Davis, CA, USA, 3Aerospace Engineering, University of Michigan, Ann Arbor, MI, USA, 4Internal Medicine/Cardiology, UC Davis, Davis, CA, USA, 5 Biochemistry and Molecular Medicine, UC Davis, Sacramento, CA, USA. Background: Increased mechanical stress under pathological conditions such as hypertension, infarction and fibrosis can cause arrhythmias and heart failure. However, little is known about Mechano-Chemo-Transduction (MCT) mechanisms that underlie heart disease development. We developed a novel Cell-in-Gel system to control mechanical loading on cardiomyocytes during excitation-contraction coupling. Here we investigate mechanical load effects on modulating Ca2þ signaling and contraction dynamics to test the hypothesis NO signaling mediates MCT. Methods: Freshly isolated adult rabbit ventricular myocytes were embedded in polyvinyl alcohol (10%) and crosslinked with tetravalent boronate-PEG (7.5%) to form a 3D viscoelastic hydrogel. The Cell-in-Gel system was continuously perfused with Tyrode’s solution and electrically paced at 0.5 Hz. Cardiomyocyte contraction and Ca2þ transients were measured in-gel and compared with load-free cells in solution. Results: Cardiomyocytes contracting in-gel under mechanical load showed augmented systolic Ca2þ transients (Fura-2 fluorescence ratio 1.47 5 0.07 ingel vs 0.85 5 0.03 load-free), revealing MCT transduces mechanical stress to increase intracellular Ca2þ release. NOS inhibition using 1 mM L-NAME reduced the in-gel Ca2þ transient by 33% (n=15), suggesting NO signaling mediates MCT. Cells in-gel showed 8% decrease in contraction (fractional shortening 13.5 5 0.4% in-gel vs 14.7 5 0.3% load-free). Cells in-gel treated with L-NAME demonstrated a further reduction in contraction (11.6 5 0.8%) as a result of decreased MCT by NOS inhibition. Importantly, 12% of cells in-gel showed mechanical load-induced alternans, which was abolished with L-NAME treatment, suggesting NO signaling contributes to load-induced Ca2þ dysregulation. Conclusion: Our findings demonstrate MCT increases the systolic Ca2þ transient to enhance contractility in response to increased afterload, serving a compensatory mechanism to autoregulate contractility. MCT is attenuated by inhibiting NOS. These results suggest NOS mediates increased Ca2þ signaling in cardiomyocytes under mechanical load, providing a mechanistic basis for the Anrep effect. 2962-Pos Board B339 The Role of ROS and Calcium for the Prolonged Force Depression after Eccentric Contractions Ha˚kan Westerblad1,2, Niklas Ivarsson1, Abram Katz1,3, Sigitas Kamandulis2, Maja Schlittler1,2, Marius Brazaitis2, Albertas Skurvydas2. 1 Department of Physiology & Pharmacology, Karolinska Institutet, Stockholm, Sweden, 2Lithuanian Sports University, Kaunas, Lithuania, 3 Ariel University, Ariel, Israel.
We recently showed that metabolically demanding high-intensity interval training results in a prolonged force depression due to increased production of reactive oxygen species (ROS) leading to defective cellular Ca2þ handling. A prolonged force depression can also be induced with mechanically demanding exercises, and we hypothesized that this deficiency also involves changes in ROS and Ca2þ. To test this hypothesis, human subjects performed mechanically demanding drop jumps, which induced a prolonged force depression especially at low stimulation frequencies: 24 hours after exercise force was decreased by ~40% at 20 Hz and ~20% at 100 Hz. Contrary to our hypothesis, we observed no signs of ROS-induced protein modifications after the drop jumps, as judged from no significant changes in the extent of malondialdehyde or carbonyl binding to protein. There was no significant change in the expression of the two key proteins in the control of sarcoplasmic reticulum Ca2þ release (the ryanodine receptor 1 (RyR1) and the dihydropyridine receptor), but there were minor changes in the amount of FKBP12 bound to RyR1 (an early increase followed by a decrease). In conclusion, the long-lasting force depressions after mechanically demanding drop jumps is not accompanied by any obvious signs of ROS-induced damage or Ca2þ dysregulation, which is in contrast to the situation after metabolically demanding exercise. 2963-Pos Board B340 The Loss of the Transmembrane Protein MG23 Affects the Fast-Twitch Feature of EDl Muscle Myuki Nishi1, Takahisa Gouda1, Nagomi Kurebayashi2, Yu Takahashi1, Shinji Komazaki3, Hua Zhu4, Hiroshi Takeshima1. 1 Biological Chemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Kyoto, Japan, 2Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine, Tokyo, Japan, 3Anatomy, Saitama Medical School, Moroyama, Japan, 4Surgery, Ohio State University, Clumbus, OH, USA. We previously identified the 23-kDa transmembrane protein MG23 in skeletal muscle. MG23 is abundantly expressed in skeletal muscle, and weakly detected in various tissues. Immunochemical and electronmicroscopic analyses suggested that MG23 forms a bowl-shaped homooligomer, and is distributed throughout the outer nuclear membrane and sarco/endoplasmic reticulum. In planar lipid bilayer membranes, purified recombinant MG23 behaved as voltage-sensitive cation-conducting channels which are equally permeable to Ca2þ and Kþ. Based on the observations, MG23 may take part in rapid cationic flux across intracellular membrane systems, while the physiological role of MG23 remains to be investigated. To survey MG23 function in vivo, we generated MG23-knockout (KO) mice. MG23-KO mice were healthy and apparently normal in growth and reproduction. In MG23-KO mice, fast-twitch EDL muscle exhibited reduced wet weight and slow relaxation after contraction. Although MG23 is unlikely to contribute directly to transcriptional machinery, gene-chip analysis detected aberrant gene expression in MG23-KO EDL muscle; for example, the expression levels of Ryr1, Cacna1s and Jph1 were obviously reduced, while Sln expression was >10-fold facilitated. On the other hand, slow-twitch soleus muscle from MG23-KO mice was apparently normal in contraction and gene expression. Our observations may imply that MG23 is a critical membranous component for the development and/or maintenance of fast-twitch properties in muscle cells. 2964-Pos Board B341 Roles of Mitsugumin53 in Skeletal Muscle Mi Kyoung Ahn1, Keon Jin Lee1, Mei Huang1, Jianjie Ma2, Eun Hui Lee1. 1 Dept. of Physiology, College of Medicine, The Catholic Univ. of Korea, Seoul, Korea, Republic of, 2Dept. of Internal Medicine, College of Medicine, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH, USA. Mitsugumin 53 (MG53), a tripartite-motif (TRIM) family protein, participates in cell membrane repair in multiple cell types. MG53 protects myocardial infarction associated with ischemia-reperfusion injury. Subcutaneous injection of purified MG53 to a mouse model of Duchenne muscular dystrophy alleviates the muscle pathology by promoting membrane repair. We previously reported the role of MG53 in mouse skeletal myotubes - MG53 binds to sarcoplasmic/ endoplasmic reticulum Ca2þ-ATPase 1a (SERCA1a) via its TRIM and PRY domains, and attenuates SERCA1a activity. In the present study, we tried to find additional roles of MG53 in skeletal muscle using single skeletal myotube Ca2þ imaging experiment, and found that MG53 regulated Ca2þ homeostasis in skeletal myotubes.