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Skeletal Muscle Deficiencies in Homozygous Fast-Skeletal Myosin Binding Protein-C Mutant Mice
14a
Sunday, February 28, 2016
81-Plat Skeletal Muscle Deficiencies in Homozygous Fast-Skeletal Myosin Binding Protein-C Mutant Mice Brian L. Lin, Suresh Govindan, Sakthivel Sadayappan. Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA. Myosin Binding Protein-C (MyBP-C) consists of three isoforms: slowskeletal, fast-skeletal, and cardiac. In the present study, our objective is to elucidate the role of fast-skeletal Myosin Binding Protein-C (fsMyBP-C). Using recombinant MyBP-C N-termini, we have previously demonstrated that the fast-skeletal isoform regulates muscle contraction more similarly to cardiac vs. slow-skeletal MyBP-C. However, the physiological implications of such regulation in skeletal muscle remains unclear. Therefore, we generated homozygous knock-out mice that do not express fsMyBP-C in either alleles, as well as heterozygous mice that express fsMyBP-C in only one allele. Currently, no myopathies are associated with mutations in fsMyBP-C, fsMyBP-C expression is dysregulated in skeletal muscle diseases, such as Duchenne Muscular Dystrophy. Both homozygous and heterozygous mice are viable and do not currently exhibit differences in longevity. However, expression of fsMyBPC are drastically different: homozygous mice do not express fsMyBP-C, but heterozygous mice exhibit the same pattern of expression in skeletal muscle as wild-type (WT) mice. In WT mice, fsMyBP-C is expressed at levels comparable to slow-skeletal MyBP-C in the extensor digitalis longus (EDL) and tibialis anterior (TA) (fast-type and mixed type skeletal muscles, respectively). Interestingly, small amounts of fsMyBP-C are also expressed in the soleus, a slow-type muscle. To analyze functional changes in EDL and soleus muscles, we used Force-ATPase experiments to analyze steady-state myofilament properties, such as force generation, Ca2þ-sensitivity, and tension cost. No significant functional changes were observed in heterozygous mice, nor were they observed in the soleus muscle. However, functional deficits became apparent in EDL muscles of homozygous mice, but could be recovered by the addition of recombinant fsMyBP-C N-termini. We propose that homozygous mice may be a useful model for isolating the effects of fsMyBP-C in health and disease. 82-Plat Mechanochemical Evolution of the Giant Muscle Protein Titin as Inferred from Ancient Proteins Raul Perez-Jimenez, Aitor Manteca, David De Sancho, Elı´as Herrero-Gala´n, Jorge Alegre Cebollada. CIC nanoGUNE, San Sebastian, Spain. We use phylogenetic analysis and single-molecule atomic force spectroscopy (AFS) to investigate the nanomechanical evolution of titin, a giant protein responsible for the elasticity, integrity and signal transduction of muscle filaments. We bring back to life an eight-domain fragment of titin (I65-I72) from different extinct species ranging from 179 to 356 Mya that have been compared with their modern descendants. Using AFS techniques we have observed that the mechanical stability of titin domains and the presence of disulfide bonds are key elements in the evolution of titin. Our experiments together with structure prediction analysis and biochemical assays confirm that ancient titin domains were richer in disulfide bonds and have higher mechanical stability. While titin domains from birds and reptiles have kept similar mechanochemical features than those of the ancient ones, titin from mammals show lower mechanical stability and lower number of disulfide bonds. Our findings show a paleomechanical trend that suggests that disulfide bonds are mechanical regulators in titin that have disappeared over the course of evolution in mammals generating more flexible and extensible titin domains. We hypothesize that this substitution derived in physiological changes that allowed the muscular development and morphological diversity of modern vertebrates. A comparative analysis between animal physiological properties and titin mechanochemical properties supports our hypothesis and also allows us inferring some physiological properties of ancient species. 83-Plat The Muscle Mechanical Basis of Freeman-Sheldon Syndrome Kaylyn M. Bell1, William A. Kronert2, Yiming Guo2, Deepti Rao2, Alice Huang1, Sanford I. Bernstein2, Douglas M. Swank1. 1 Biology, Rensselaer Polytechnic Institute, Troy, NY, USA, 2Biology, San Diego State University, San Diego, CA, USA. We have generated the first animal models for Distal Arthrogryposis type 2A, commonly known as Freeman-Sheldon Syndrome (FSS). FSS is characterized by congenital contractures resulting from point mutations in skeletal muscle contractile proteins, yet how these mutations alter muscle contractile properties is not known. We created two myosin heavy chain transgenes containing point
mutations Y583S and T178I, each known to cause dominant FSS. We expressed them in Drosophila muscles, isolated the indirect flight muscles from heterozygous mutant flies (mutation/þ), and performed skinned muscle fiber mechanics. The most striking mechanical alteration was a dramatic increase in muscle stiffness. Y583S and T178I fibers’ elastic moduli increased 40% and 30%, respectively, under relaxed conditions and 70% and 46% when activated. The increased stiffness contributed to decreased work and power generation. Maximum power decreased by 45% and 62% because elevated stiffness increased work absorbed values during the lengthening portion of work-loop power measurements. A contributing factor to both decreased power and increased active stiffness was a reduction in rate for at least one stronglybound cross-bridge cycle transition. This was inferred from 17% and 31% decreases in optimal frequency for power generation, and 27% and 39% decreases in apparent muscle rate constant 2pb. Another possible contributor to the stiffness changes might be muscle ultrastructure alterations. While we performed mechanics experiments on fibers from 2-hour-old flies, which showed normal muscle ultrastructure, disruption in myofibril hexagonal packing and morphology appeared at 2-days and increased with age. Impaired muscle function caused 29% and 63% decreases in flight ability for Y583S and T178I heterozygotes, respectively. We conclude that Freeman-Sheldon Syndrome likely results from highly elevated muscle fiber stiffness and decreased ability of the muscles to power limb movement. Supported by NIH grants R01 AR064274 to D.M.S. and R01 GM32443 to S.I.B. 84-Plat Structural and Functional Impacts of Novel Mutations in Slow Skeletal Muscle Troponin T Found in Non-Amish TNNT1 Nemaline Myopathies Chinthaka K. Amarasinghe, Jian-Ping Jin. Physiology, Wayne State University School of Medicine, Detroit, MI, USA. Troponin T (TnT) is the thin filament anchoring subunit of the troponin complex and has two tropomyosin-binding sites for the incorporation of troponin into the sarcomeric structure. A nonsense mutation in exon 11 of the slow skeletal muscle troponin T (ssTnT) gene (TNNT1) truncating the polypeptide chain at Glu180 was found to cause nemaline myopathy (NM) in the Amish, an autosomal recessive disease with severe lethal phenotype. More NM TNNT1 mutations have recently been reported with similar recessive phenotypes. Here we engineered protein constructs representing the mutant ssTnT to investigate their impact on tropomyosin-binding and integration into the thin filament regulatory system. Like the Glu180X mutation, two novel nonsense mutations in exon 9 and exon 11 truncate the ssTnT polypeptide chain at Ser108 and Leu203, respectively, to delete the C-terminal region tropomyosin-binding site 2. A splicing site mutation causes a deletion of a 39 amino acid segment from the middle region tropomyosin-binding site 1. To understand the molecular mechanisms underlying these TNNT1 mutations, we expressed and purified the mutant ssTnT proteins and analyzed their tropomyosin-binding affinity using solid-phase protein binding assays. The results demonstrate that both the Ser108X and exon 8 deletion mutations have similarly decreased tropomyosin-binding as that of Glu180X. We recently showed that the affinity of tropomyosin-binding site 1 is modulated by the isoform-specific N-terminal variable region, with ssTnT having the weakest tropomyosin-binding affinity. Therefore, the N-terminal variable region-based conformational and functional modulation may be a therapeutic target for TNNT1 myopathies. While Leu203X has both tropomyosin-binding sites intact, its recessive phenotype suggests that the incorporation into troponin complex may be required for high affinity binding of TnT to tropomyosin, which may be a protective mechanism against the Leu203X mutation. 85-Plat Molecular Mechanism of Novel Deletions in TPM3 that cause a Hypercontractile Phenotype with Congenital Muscle Stiffness Steven Marston1, Maria Papadaki1, Massimiliano Memo1, Andrew Messer1, Sandra Donkervoort2, Carsten Bonneman2, Kristen Nowak3, Royston Ong3, Elyshia McNamara3. 1 Imperial College London, London, United Kingdom, 2Neuromuscular and Neurogenetic Disorders of Childhood Section, National Institutes of Health, Bethesda, MD, USA, 3Harry Perkins Institute of Medical Research, University of Western Australia, Perth, Australia. Patients with ‘stiff child’ syndrome usually have mutations at the interface of actin and tropomyosin that could affect the equilibrium of the Ca2þ-dependent switch of muscle. ACTA1 K326N was previously reported and we now report two de novo TPM3 (Tpm3.12 protein) mutations, DE218 and DE224, resulting in a significant hypercontractile phenotype with marked congenital muscle stiffness associated with ventilatory failure in one case.
Sunday, February 28, 2016
81-Plat Skeletal Muscle Deficiencies in Homozygous Fast-Skeletal Myosin Binding Protein-C Mutant Mice Brian L. Lin, Suresh Govindan, Sakthivel Sadayappan. Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA. Myosin Binding Protein-C (MyBP-C) consists of three isoforms: slowskeletal, fast-skeletal, and cardiac. In the present study, our objective is to elucidate the role of fast-skeletal Myosin Binding Protein-C (fsMyBP-C). Using recombinant MyBP-C N-termini, we have previously demonstrated that the fast-skeletal isoform regulates muscle contraction more similarly to cardiac vs. slow-skeletal MyBP-C. However, the physiological implications of such regulation in skeletal muscle remains unclear. Therefore, we generated homozygous knock-out mice that do not express fsMyBP-C in either alleles, as well as heterozygous mice that express fsMyBP-C in only one allele. Currently, no myopathies are associated with mutations in fsMyBP-C, fsMyBP-C expression is dysregulated in skeletal muscle diseases, such as Duchenne Muscular Dystrophy. Both homozygous and heterozygous mice are viable and do not currently exhibit differences in longevity. However, expression of fsMyBPC are drastically different: homozygous mice do not express fsMyBP-C, but heterozygous mice exhibit the same pattern of expression in skeletal muscle as wild-type (WT) mice. In WT mice, fsMyBP-C is expressed at levels comparable to slow-skeletal MyBP-C in the extensor digitalis longus (EDL) and tibialis anterior (TA) (fast-type and mixed type skeletal muscles, respectively). Interestingly, small amounts of fsMyBP-C are also expressed in the soleus, a slow-type muscle. To analyze functional changes in EDL and soleus muscles, we used Force-ATPase experiments to analyze steady-state myofilament properties, such as force generation, Ca2þ-sensitivity, and tension cost. No significant functional changes were observed in heterozygous mice, nor were they observed in the soleus muscle. However, functional deficits became apparent in EDL muscles of homozygous mice, but could be recovered by the addition of recombinant fsMyBP-C N-termini. We propose that homozygous mice may be a useful model for isolating the effects of fsMyBP-C in health and disease. 82-Plat Mechanochemical Evolution of the Giant Muscle Protein Titin as Inferred from Ancient Proteins Raul Perez-Jimenez, Aitor Manteca, David De Sancho, Elı´as Herrero-Gala´n, Jorge Alegre Cebollada. CIC nanoGUNE, San Sebastian, Spain. We use phylogenetic analysis and single-molecule atomic force spectroscopy (AFS) to investigate the nanomechanical evolution of titin, a giant protein responsible for the elasticity, integrity and signal transduction of muscle filaments. We bring back to life an eight-domain fragment of titin (I65-I72) from different extinct species ranging from 179 to 356 Mya that have been compared with their modern descendants. Using AFS techniques we have observed that the mechanical stability of titin domains and the presence of disulfide bonds are key elements in the evolution of titin. Our experiments together with structure prediction analysis and biochemical assays confirm that ancient titin domains were richer in disulfide bonds and have higher mechanical stability. While titin domains from birds and reptiles have kept similar mechanochemical features than those of the ancient ones, titin from mammals show lower mechanical stability and lower number of disulfide bonds. Our findings show a paleomechanical trend that suggests that disulfide bonds are mechanical regulators in titin that have disappeared over the course of evolution in mammals generating more flexible and extensible titin domains. We hypothesize that this substitution derived in physiological changes that allowed the muscular development and morphological diversity of modern vertebrates. A comparative analysis between animal physiological properties and titin mechanochemical properties supports our hypothesis and also allows us inferring some physiological properties of ancient species. 83-Plat The Muscle Mechanical Basis of Freeman-Sheldon Syndrome Kaylyn M. Bell1, William A. Kronert2, Yiming Guo2, Deepti Rao2, Alice Huang1, Sanford I. Bernstein2, Douglas M. Swank1. 1 Biology, Rensselaer Polytechnic Institute, Troy, NY, USA, 2Biology, San Diego State University, San Diego, CA, USA. We have generated the first animal models for Distal Arthrogryposis type 2A, commonly known as Freeman-Sheldon Syndrome (FSS). FSS is characterized by congenital contractures resulting from point mutations in skeletal muscle contractile proteins, yet how these mutations alter muscle contractile properties is not known. We created two myosin heavy chain transgenes containing point
mutations Y583S and T178I, each known to cause dominant FSS. We expressed them in Drosophila muscles, isolated the indirect flight muscles from heterozygous mutant flies (mutation/þ), and performed skinned muscle fiber mechanics. The most striking mechanical alteration was a dramatic increase in muscle stiffness. Y583S and T178I fibers’ elastic moduli increased 40% and 30%, respectively, under relaxed conditions and 70% and 46% when activated. The increased stiffness contributed to decreased work and power generation. Maximum power decreased by 45% and 62% because elevated stiffness increased work absorbed values during the lengthening portion of work-loop power measurements. A contributing factor to both decreased power and increased active stiffness was a reduction in rate for at least one stronglybound cross-bridge cycle transition. This was inferred from 17% and 31% decreases in optimal frequency for power generation, and 27% and 39% decreases in apparent muscle rate constant 2pb. Another possible contributor to the stiffness changes might be muscle ultrastructure alterations. While we performed mechanics experiments on fibers from 2-hour-old flies, which showed normal muscle ultrastructure, disruption in myofibril hexagonal packing and morphology appeared at 2-days and increased with age. Impaired muscle function caused 29% and 63% decreases in flight ability for Y583S and T178I heterozygotes, respectively. We conclude that Freeman-Sheldon Syndrome likely results from highly elevated muscle fiber stiffness and decreased ability of the muscles to power limb movement. Supported by NIH grants R01 AR064274 to D.M.S. and R01 GM32443 to S.I.B. 84-Plat Structural and Functional Impacts of Novel Mutations in Slow Skeletal Muscle Troponin T Found in Non-Amish TNNT1 Nemaline Myopathies Chinthaka K. Amarasinghe, Jian-Ping Jin. Physiology, Wayne State University School of Medicine, Detroit, MI, USA. Troponin T (TnT) is the thin filament anchoring subunit of the troponin complex and has two tropomyosin-binding sites for the incorporation of troponin into the sarcomeric structure. A nonsense mutation in exon 11 of the slow skeletal muscle troponin T (ssTnT) gene (TNNT1) truncating the polypeptide chain at Glu180 was found to cause nemaline myopathy (NM) in the Amish, an autosomal recessive disease with severe lethal phenotype. More NM TNNT1 mutations have recently been reported with similar recessive phenotypes. Here we engineered protein constructs representing the mutant ssTnT to investigate their impact on tropomyosin-binding and integration into the thin filament regulatory system. Like the Glu180X mutation, two novel nonsense mutations in exon 9 and exon 11 truncate the ssTnT polypeptide chain at Ser108 and Leu203, respectively, to delete the C-terminal region tropomyosin-binding site 2. A splicing site mutation causes a deletion of a 39 amino acid segment from the middle region tropomyosin-binding site 1. To understand the molecular mechanisms underlying these TNNT1 mutations, we expressed and purified the mutant ssTnT proteins and analyzed their tropomyosin-binding affinity using solid-phase protein binding assays. The results demonstrate that both the Ser108X and exon 8 deletion mutations have similarly decreased tropomyosin-binding as that of Glu180X. We recently showed that the affinity of tropomyosin-binding site 1 is modulated by the isoform-specific N-terminal variable region, with ssTnT having the weakest tropomyosin-binding affinity. Therefore, the N-terminal variable region-based conformational and functional modulation may be a therapeutic target for TNNT1 myopathies. While Leu203X has both tropomyosin-binding sites intact, its recessive phenotype suggests that the incorporation into troponin complex may be required for high affinity binding of TnT to tropomyosin, which may be a protective mechanism against the Leu203X mutation. 85-Plat Molecular Mechanism of Novel Deletions in TPM3 that cause a Hypercontractile Phenotype with Congenital Muscle Stiffness Steven Marston1, Maria Papadaki1, Massimiliano Memo1, Andrew Messer1, Sandra Donkervoort2, Carsten Bonneman2, Kristen Nowak3, Royston Ong3, Elyshia McNamara3. 1 Imperial College London, London, United Kingdom, 2Neuromuscular and Neurogenetic Disorders of Childhood Section, National Institutes of Health, Bethesda, MD, USA, 3Harry Perkins Institute of Medical Research, University of Western Australia, Perth, Australia. Patients with ‘stiff child’ syndrome usually have mutations at the interface of actin and tropomyosin that could affect the equilibrium of the Ca2þ-dependent switch of muscle. ACTA1 K326N was previously reported and we now report two de novo TPM3 (Tpm3.12 protein) mutations, DE218 and DE224, resulting in a significant hypercontractile phenotype with marked congenital muscle stiffness associated with ventilatory failure in one case.