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Reconstruction of Functional Insect Flight Muscle Fibers with Rabbit Skeletal Muscle Actin
118a
Sunday, February 12, 2017
584-Pos Board B349 A Simplified Flexible Chain Model of Calcium Regulated Myosin-Actin Interaction Leonard P. Heinz, Rainer H.A. Fink. Medical Biophysics Unit, Institute for Physiology und Pathophysiology, Heidelberg University, Heidelberg, Germany. While the process of muscle contraction is relatively well understood at the molecular level, the collective behavior of many molecular motors has become an interesting topic for computer simulations in recent years. Duke’s model has proven to provide a good description of the myosin-actin interaction and the Flexible Chain Model can be used to describe the regulatory behavior of the troponin-tropomyosin complex. Based on these models, we developed and implemented a simple computational motility assay that covers calcium-regulated muscle activation and allows to study the cooperative effects of many motormolecules working in parallel as well as the relationships between contraction speed and isometric force versus calcium concentration. Like in Duke’s model, myosin heads are modeled using a simple 3-state system corresponding to detached, attached and strongly attached to actin. The behavior of the troponin-tropomyosin complex, located at every 7th actin binding site, is treated using a simple 2-state model, allowing troponin to be attached or detached to actin. Both troponin and myosin control a continuous energy landscape that models the steric blocking of actin binding sites by tropomyosin. This troponin-potential is computed using radial basis interpolation. It is locally reduced, if a myosin head binds to actin and increased, if a binding site is occupied by troponin, causing the system to act cooperatively. 585-Pos Board B350 Reconstruction of Functional Insect Flight Muscle Fibers with Rabbit Skeletal Muscle Actin Hiroyuki Iwamoto. Res. & Util. Div., SPring-8, JASRI, Sayo-gun, Hyogo, Japan. Stretch activation (SA, delayed rise of force after stretch) is a mechanism essential for the fast wing-beat of insects with asynchronous flight muscles. To determine which constituent protein(s) is (are) indispensable for SA, we have been conducting experiments to replace the endogenous actin of bumblebee flight muscle with rabbit skeletal muscle actin, and we have demonstrated that actin filaments can be regenerated as judged from X-ray diffraction patterns (2016 annual meeting). In this experiment, the endogenous actin is removed by gelsolin, and after this, rabbit G-actin is polymerized in situ. To prevent spontaneous contraction, a myosin inhibitor must be added to the solutions. For vertebrate skeletal and cardiac muscles, the preferred inhibitor is BDM (butanedione monoxime, Fujita et al., 1996). However, BDM is not an effective inhibitor for insect flight muscle. For this reason, we used blebbistatin instead, and the actin filaments were successfully restored. The problem is that the inhibition by blebbistatin is irreversible, and it is not reversed by extensive washout or irradiation with blue light. Here we used a high concentration of BDM (100 mM) and repeated the experiments. The actin filaments were restored, and unlike in the case of blebbistatin, actin-based layer line reflections were intensified after washout of BDM and ATP. This indicates that the ability of the endogenous myosin to form rigor complexes is not compromised by the use of BDM. We are currently trying to activate the flight muscle fibers prepared in this way. 586-Pos Board B351 The Skeletal Muscle Molecular Clock Regulates Titin Isoform Expression Lance A. Riley1,2, Xiping Zhang1,2, Karyn A. Esser1,2. 1 Physiology and Functional Genomics, University of Florida, Gainesville, FL, USA, 2Myology Institute, University of Florida, Gainesville, FL, USA. The circadian clock transcription factors, BMAL1 and CLOCK, are fundamental transcriptional regulators of cell time keeping and critical cell specific genes important for homeostasis. To determine the specific role of the molecular clock in adult skeletal muscle, our lab developed the inducible, skeletal muscle specific Bmal1 knockout (iMSBmal1-/-) mouse. Notably, skeletal muscles from these mice exhibit decreased specific tension and reduced unstimulated tension developed during a fatigability test. To begin to discern the molecular mechanisms that link the changes in the molecular clock with changes in muscle function, we tested whether iMSBmal1-/- muscle would also exhibit changes in sarcomeric protein expression. We determined that the tibialis anterior muscle of the iMSBmal1-/- mice exhibits a significant increase (38% in iMSBmal1-/- mice vs. 19% in iMSBmal1þ/þ mice) in expression of a longer isoform of titin. It is established that titin, the giant filamentous protein that maintains sarcomeric structure, underlies passive
tension, and contributes to active tension development. To determine if this increased heterogeneity of titin isoforms within the TA had an effect on sarcomere length, we performed immunohistochemistry. An antibody to a-actinin was used to demarcate Z-lines in longitudinal sections of the tibialis anterior muscle and sarcomere length was measured as the distance between Z-lines. While average sarcomere length was not different between iMSBmal1þ/þ and iMSBmal1-/- muscle, variation in sarcomere length was increased following Bmal1 knockout in skeletal muscle. Further studies with our model are currently ongoing and will lead to increased knowledge of the importance of titin isoform maintenance in a variety of pathological conditions. 587-Pos Board B352 Cacium-Induced SR Calcium Leak in Dysferlin-Null Murine Muscle Fibers Valeriy Lukyanenko, Joaquin Muriel, Robert J. Bloch. Physiology, University of Maryland, Baltimore, MD, USA. Osmotic shock injury (OSI) decreases the amplitude of voltage-induced Ca2þtransients (VICTs) in dysferlin-null (A/J) but not control (A/WySnJ) myofibers and also markedly increases sarcoplasmic [Ca2þ] (Kerr et al., Proc. Natl. Acad. Sci. USA, 2013). We compared the effects of drugs that target L-type Ca2þchannels (LTCC: diltiazem, nifedipine, verapamil) and ryanodine receptors (RyR1: dantrolene, tetracaine, S107) on A/WySnJ and A/J FDB myofibers in culture to assess their effect on VICTs following OSI. We also examined A/J fibers transfected to express N-terminal Venus chimaeras of dysferlin (V-Dysf). All Ca2þ antagonists inhibited VICTs in A/J and A/WySnJ fibers at high concentrations, but 1-10 mM diltiazem specifically increased VICT amplitudes by ~15% in A/J fibers, restoring them to values close to controls. All the inhibitors at low concentrations improved recovery of VICTs in A/J fibers after OSI. The fact that inhibitors of the LTCC and of the RyR1 protect A/J fibers from OSI-induced loss of VICTs strongly suggests that the damage caused to the VICTs by OSI is mediated by Ca2þ-induced SR Ca2þ leak through the RyR1. Consistent with this, injured A/J fibers produced Ca2þ waves, indicative of Ca2þ-induced Ca2þ release (CICR). Treatment of A/J fibers with 10 mM S107 (stabilizer of RyR1-FKBP coupling that reduces Ca2þ leak) or expression of V-Dysf both protected A/J fibers against the loss in amplitude of VICTs following OSI and prevented OSI-induced Ca2þ waves. Our data suggest that, in the absence of dysferlin, OSI causes increased leak of SR Ca2þ through the RyR1, leading to CICR. We conclude that dysferlin stabilizes the coupling of the LTCC and RyR1 to reduce Ca2þ leak when fibers are mechanically stressed. Supported by the Jain Foundation, MDA and NIH (RO1 AR064268). 588-Pos Board B353 MG29 Interacts with Bin1 for Maintaining T-Tubule Structure in Skeletal Muscle Physiology and Regeneration Xinyu Zhou1, Kristyn Gumpper1, Xinxin Wang1, Junwei Wu1, Tao Tan1, Miyuki Nishi2, Hiroshi Takeshima2, Jianjie Ma1, Hua Zhu1. 1 Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH, USA, 2Department of Biological Chemistry, Kyoto University, Kyoto, Japan. Mitsugumin 29 (MG29), a member of the synaptophysin-like family proteins, is a transmembrane protein mainly expressed in the t-tubule membranes of skeletal muscle. Electron microscopy analysis of skeletal muscle derived from mg29-/- mice revealed morphological defects of t-tubules, indicating importance of MG29 in maintenance of muscle structure and function. To determine the function of MG29 in muscle physiology and regeneration, we performed co-immunoprecipitation and found that MG29 can bind to Bin1, another t-tubular protein. Two-color STORM super-resolution imaging analysis confirmed co-localization of MG29 and Bin1 on t-tubules of skeletal muscle. Interestingly, organized distribution Bin1 is severely disrupted in mg29-/- muscle. For testing the role of MG29 in muscle regeneration, we injured gastrocnemius muscle with cardiotoxin (CTX) and tracked muscle repair and regeneration. Western blot showed that following CTX injury, MG29 protein levels were transiently reduced from day 1 to day 3, followed by recovery associated with muscle regeneration. Protein level of Bin1 in wild type muscle remained unchanged during the CTX-injury and recover process. Compared with wild type muscle, the mg29-/- muscle displayed delayed regeneration with significantly reduced levels of Bin1 following CTX-induced injury. Together our data suggest that functional interaction between MG29 and Bin1 contribute to maintenance of t-tubule network and its remodeling process associated with muscle injury and regeneration. Targeting the MG29/Bin1 complex might provide a potential effective approach for treatment of muscle diseases.
Sunday, February 12, 2017
584-Pos Board B349 A Simplified Flexible Chain Model of Calcium Regulated Myosin-Actin Interaction Leonard P. Heinz, Rainer H.A. Fink. Medical Biophysics Unit, Institute for Physiology und Pathophysiology, Heidelberg University, Heidelberg, Germany. While the process of muscle contraction is relatively well understood at the molecular level, the collective behavior of many molecular motors has become an interesting topic for computer simulations in recent years. Duke’s model has proven to provide a good description of the myosin-actin interaction and the Flexible Chain Model can be used to describe the regulatory behavior of the troponin-tropomyosin complex. Based on these models, we developed and implemented a simple computational motility assay that covers calcium-regulated muscle activation and allows to study the cooperative effects of many motormolecules working in parallel as well as the relationships between contraction speed and isometric force versus calcium concentration. Like in Duke’s model, myosin heads are modeled using a simple 3-state system corresponding to detached, attached and strongly attached to actin. The behavior of the troponin-tropomyosin complex, located at every 7th actin binding site, is treated using a simple 2-state model, allowing troponin to be attached or detached to actin. Both troponin and myosin control a continuous energy landscape that models the steric blocking of actin binding sites by tropomyosin. This troponin-potential is computed using radial basis interpolation. It is locally reduced, if a myosin head binds to actin and increased, if a binding site is occupied by troponin, causing the system to act cooperatively. 585-Pos Board B350 Reconstruction of Functional Insect Flight Muscle Fibers with Rabbit Skeletal Muscle Actin Hiroyuki Iwamoto. Res. & Util. Div., SPring-8, JASRI, Sayo-gun, Hyogo, Japan. Stretch activation (SA, delayed rise of force after stretch) is a mechanism essential for the fast wing-beat of insects with asynchronous flight muscles. To determine which constituent protein(s) is (are) indispensable for SA, we have been conducting experiments to replace the endogenous actin of bumblebee flight muscle with rabbit skeletal muscle actin, and we have demonstrated that actin filaments can be regenerated as judged from X-ray diffraction patterns (2016 annual meeting). In this experiment, the endogenous actin is removed by gelsolin, and after this, rabbit G-actin is polymerized in situ. To prevent spontaneous contraction, a myosin inhibitor must be added to the solutions. For vertebrate skeletal and cardiac muscles, the preferred inhibitor is BDM (butanedione monoxime, Fujita et al., 1996). However, BDM is not an effective inhibitor for insect flight muscle. For this reason, we used blebbistatin instead, and the actin filaments were successfully restored. The problem is that the inhibition by blebbistatin is irreversible, and it is not reversed by extensive washout or irradiation with blue light. Here we used a high concentration of BDM (100 mM) and repeated the experiments. The actin filaments were restored, and unlike in the case of blebbistatin, actin-based layer line reflections were intensified after washout of BDM and ATP. This indicates that the ability of the endogenous myosin to form rigor complexes is not compromised by the use of BDM. We are currently trying to activate the flight muscle fibers prepared in this way. 586-Pos Board B351 The Skeletal Muscle Molecular Clock Regulates Titin Isoform Expression Lance A. Riley1,2, Xiping Zhang1,2, Karyn A. Esser1,2. 1 Physiology and Functional Genomics, University of Florida, Gainesville, FL, USA, 2Myology Institute, University of Florida, Gainesville, FL, USA. The circadian clock transcription factors, BMAL1 and CLOCK, are fundamental transcriptional regulators of cell time keeping and critical cell specific genes important for homeostasis. To determine the specific role of the molecular clock in adult skeletal muscle, our lab developed the inducible, skeletal muscle specific Bmal1 knockout (iMSBmal1-/-) mouse. Notably, skeletal muscles from these mice exhibit decreased specific tension and reduced unstimulated tension developed during a fatigability test. To begin to discern the molecular mechanisms that link the changes in the molecular clock with changes in muscle function, we tested whether iMSBmal1-/- muscle would also exhibit changes in sarcomeric protein expression. We determined that the tibialis anterior muscle of the iMSBmal1-/- mice exhibits a significant increase (38% in iMSBmal1-/- mice vs. 19% in iMSBmal1þ/þ mice) in expression of a longer isoform of titin. It is established that titin, the giant filamentous protein that maintains sarcomeric structure, underlies passive
tension, and contributes to active tension development. To determine if this increased heterogeneity of titin isoforms within the TA had an effect on sarcomere length, we performed immunohistochemistry. An antibody to a-actinin was used to demarcate Z-lines in longitudinal sections of the tibialis anterior muscle and sarcomere length was measured as the distance between Z-lines. While average sarcomere length was not different between iMSBmal1þ/þ and iMSBmal1-/- muscle, variation in sarcomere length was increased following Bmal1 knockout in skeletal muscle. Further studies with our model are currently ongoing and will lead to increased knowledge of the importance of titin isoform maintenance in a variety of pathological conditions. 587-Pos Board B352 Cacium-Induced SR Calcium Leak in Dysferlin-Null Murine Muscle Fibers Valeriy Lukyanenko, Joaquin Muriel, Robert J. Bloch. Physiology, University of Maryland, Baltimore, MD, USA. Osmotic shock injury (OSI) decreases the amplitude of voltage-induced Ca2þtransients (VICTs) in dysferlin-null (A/J) but not control (A/WySnJ) myofibers and also markedly increases sarcoplasmic [Ca2þ] (Kerr et al., Proc. Natl. Acad. Sci. USA, 2013). We compared the effects of drugs that target L-type Ca2þchannels (LTCC: diltiazem, nifedipine, verapamil) and ryanodine receptors (RyR1: dantrolene, tetracaine, S107) on A/WySnJ and A/J FDB myofibers in culture to assess their effect on VICTs following OSI. We also examined A/J fibers transfected to express N-terminal Venus chimaeras of dysferlin (V-Dysf). All Ca2þ antagonists inhibited VICTs in A/J and A/WySnJ fibers at high concentrations, but 1-10 mM diltiazem specifically increased VICT amplitudes by ~15% in A/J fibers, restoring them to values close to controls. All the inhibitors at low concentrations improved recovery of VICTs in A/J fibers after OSI. The fact that inhibitors of the LTCC and of the RyR1 protect A/J fibers from OSI-induced loss of VICTs strongly suggests that the damage caused to the VICTs by OSI is mediated by Ca2þ-induced SR Ca2þ leak through the RyR1. Consistent with this, injured A/J fibers produced Ca2þ waves, indicative of Ca2þ-induced Ca2þ release (CICR). Treatment of A/J fibers with 10 mM S107 (stabilizer of RyR1-FKBP coupling that reduces Ca2þ leak) or expression of V-Dysf both protected A/J fibers against the loss in amplitude of VICTs following OSI and prevented OSI-induced Ca2þ waves. Our data suggest that, in the absence of dysferlin, OSI causes increased leak of SR Ca2þ through the RyR1, leading to CICR. We conclude that dysferlin stabilizes the coupling of the LTCC and RyR1 to reduce Ca2þ leak when fibers are mechanically stressed. Supported by the Jain Foundation, MDA and NIH (RO1 AR064268). 588-Pos Board B353 MG29 Interacts with Bin1 for Maintaining T-Tubule Structure in Skeletal Muscle Physiology and Regeneration Xinyu Zhou1, Kristyn Gumpper1, Xinxin Wang1, Junwei Wu1, Tao Tan1, Miyuki Nishi2, Hiroshi Takeshima2, Jianjie Ma1, Hua Zhu1. 1 Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH, USA, 2Department of Biological Chemistry, Kyoto University, Kyoto, Japan. Mitsugumin 29 (MG29), a member of the synaptophysin-like family proteins, is a transmembrane protein mainly expressed in the t-tubule membranes of skeletal muscle. Electron microscopy analysis of skeletal muscle derived from mg29-/- mice revealed morphological defects of t-tubules, indicating importance of MG29 in maintenance of muscle structure and function. To determine the function of MG29 in muscle physiology and regeneration, we performed co-immunoprecipitation and found that MG29 can bind to Bin1, another t-tubular protein. Two-color STORM super-resolution imaging analysis confirmed co-localization of MG29 and Bin1 on t-tubules of skeletal muscle. Interestingly, organized distribution Bin1 is severely disrupted in mg29-/- muscle. For testing the role of MG29 in muscle regeneration, we injured gastrocnemius muscle with cardiotoxin (CTX) and tracked muscle repair and regeneration. Western blot showed that following CTX injury, MG29 protein levels were transiently reduced from day 1 to day 3, followed by recovery associated with muscle regeneration. Protein level of Bin1 in wild type muscle remained unchanged during the CTX-injury and recover process. Compared with wild type muscle, the mg29-/- muscle displayed delayed regeneration with significantly reduced levels of Bin1 following CTX-induced injury. Together our data suggest that functional interaction between MG29 and Bin1 contribute to maintenance of t-tubule network and its remodeling process associated with muscle injury and regeneration. Targeting the MG29/Bin1 complex might provide a potential effective approach for treatment of muscle diseases.