Muscle Isoform Research Articles

Biochemical and structural investigation of the dynamic regulation mechanism of pyruvate kinase muscle isoform 2 using amino acids

Biochemical and structural investigation of the dynamic regulation mechanism of pyruvate kinase muscle isoform 2 using amino acids

The loss of slow skeletal muscle isoform of troponin T in spindle intrafusal fibres explains the pathophysiology of Amish nemaline myopathy.

The pathogenic mechanism and the neuromuscular reflex-related phenotype (e.g. tremors accompanied by clonus) of Amish nemaline myopathy, as well as of other recessively inherited TNNT1 myopathies, remain to be clarified. The truncated slow skeletal muscle isoform of troponin T (ssTnT) encoded by the mutant TNNT1 gene is unable to incorporate into myofilaments and is degraded in muscle cells. By contrast to extrafusal muscle fibres, spindle intrafusal fibres of normal mice contain a significant level of cardiac TnT and a low molecular weight splice form of ssTnT. Intrafusal fibres of ssTnT-knockout mice have significantly increased cardiac TnT. Rotarod and balance beam tests have revealed abnormal neuromuscular co-ordination in ssTnT-knockout mice and a blunted response to a spindle sensitizer, succinylcholine. The loss of ssTnT and a compensatory increase of cardiac TnT in intrafusal nuclear bag fibres may increase myofilament Ca2+ -sensitivity and tension, impairing spindle function, thus identifying a novel mechanism for the development of targeted treatment. A nonsense mutation at codon Glu180 of TNNT1 gene causes Amish nemaline myopathy (ANM), a recessively inherited disease with infantile lethality. TNNT1 encodes the slow skeletal muscle isoform of troponin T (ssTnT). The truncated ssTnT is unable to incorporate into myofilament and is degraded in muscle cells. The symptoms of ANM include muscle weakness, atrophy, contracture and tremors accompanied by clonus. An ssTnT-knockout (KO) mouse model recapitulates key features of ANM such as atrophy of extrafusal slow muscle fibres and increased fatigability. However, the neuromuscular reflex-related symptoms of ANM have not been explained. By isolating muscle spindles from ssTnT-KO and control mice aiming to examine the composition of myofilament proteins, we found that, in contrast to extrafusal fibres, intrafusal fibres contain a significant level of cardiac TnT and the low molecular weight splice form of ssTnT. Intrafusal fibres from ssTnT-KO mice have significantly increased cardiac TnT. Rotarod and balance beam tests revealed impaired neuromuscular co-ordination in ssTnT-KO mice, indicating abnormality in spindle functions. Unlike the wild-type control, the beam running ability of ssTnT-KO mice had a blunted response to a spindle sensitizer, succinylcholine. Immunohistochemistry detected ssTnT and cardiac TnT in nuclear bag fibres, whereas fast skeletal muscle TnT was detected in nuclear chain fibres, and cardiac α-myosin was present in one of the two nuclear bag fibres. The loss of ssTnT and a compensatory increase of cardiac TnT in nuclear bag fibres would increase myofilament Ca2+ -sensitivity and tension, thus affecting spindle activities. This mechanism provides an explanation for the pathophysiology of ANM, as well as a novel target for treatment.

Thermal unfolding of various human non-muscle isoforms of tropomyosin

Tropomyosin (Tpm) is an α-helical coiled-coil protein dimer, which forms a continuous head-to-tail polymer along the actin filament. In striated muscles, Tpm plays an important role in the Ca2+-dependent regulation of muscle contraction. However, little is known about functional and especially structural properties of the numerous non-muscle Tpm isoforms. In the present work, we have applied circular dichroism (CD) and differential scanning calorimetry (DSC) to investigate thermal unfolding and domain structure of various non-muscle human Tpm isoforms. These isoforms, the products of two different genes, TPM1 and TPM3, also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results clearly demonstrate that structural properties of various non-muscle Tpm isoforms can be quite different depending on the presence of different alternatively spliced exons in their genes. These data show for the first time a significant difference in the thermal unfolding between muscle and non-muscle Tpm isoforms and indicate that replacement of alternatively spliced exons alters the stability of certain domains in the Tpm molecule.

Heterozygous variants in MYBPC1 are associated with an expanded neuromuscular phenotype beyond arthrogryposis.

Encoding the slow skeletal muscle isoform of myosin binding protein-C, MYBPC1 is associated with autosomal dominant and recessive forms of arthrogryposis. The authors describe a novel association for MYBPC1 in four patients from three independent families with skeletal muscle weakness, myogenic tremors, and hypotonia with gradual clinical improvement. The patients carried one of two de novo heterozygous variants in MYBPC1, with the p.Leu263Arg variant seen in three individuals and the p.Leu259Pro variant in one individual. Both variants are absent from controls, well conserved across vertebrate species, predicted to be damaging, and located in the M-motif. Protein modeling studies suggested that the p.Leu263Arg variant affects the stability of the M-motif, whereas the p.Leu259Pro variant alters its structure. In vitro biochemical and kinetic studies demonstrated that the p.Leu263Arg variant results in decreased binding of the M-motif to myosin, which likely impairs the formation of actomyosin cross-bridges during muscle contraction. Collectively, our data substantiate that damaging variants in MYBPC1 are associated with a new form of an early-onset myopathy with tremor, which is a defining and consistent characteristic in all affected individuals, with no contractures. Recognition of this expanded myopathic phenotype can enable identification of individuals with MYBPC1 variants without arthrogryposis.

Expression of troponin subunits in the rat renal afferent arteriole.

Vascular smooth muscle cells of the renal afferent arteriole are unusual in that they must be able to contract very rapidly in response to a sudden increase in systemic blood pressure in order to protect the downstream glomerular capillaries from catastrophic damage. We showed that this could be accounted for, in part, by exclusive expression, at the protein level, of the "fast" (B) isoforms of smooth muscle myosin II heavy chains in the afferent arteriole, in contrast to other vascular smooth muscle cells such as the rat aorta and efferent arteriole which express exclusively the "slow" (A) isoforms (Shiraishi et al. (2003) FASEB. J. 17, 2284-2286). As contraction of the more rapidly contracting striated (skeletal and cardiac) muscles is regulated by the thin filament-associated troponin (Tn) system, we hypothesized that Tn or a Tn-like system may exist in afferent arteriolar cells and contribute to the unusually rapid contraction of this tissue in response to increased intraluminal pressure. We examined the expression of TnC (Ca2+ -binding subunit), TnI (inhibitory subunit), and TnT (tropomyosin-binding subunit) in vascular smooth muscle cells of the rat renal afferent arteriole at the mRNA level. Fast-twitch skeletal muscle and slow-twitch skeletal muscle/cardiac TnC isoforms and slow-twitch skeletal muscle and cardiac TnI isoforms were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by cDNA sequencing. Furthermore, cardiac and slow-twitch skeletal muscle TnI isoforms, but not fast-twitch skeletal muscle TnI, were detected in isolated afferent arterioles at the protein level by proximity ligation assay. Finally, striated muscle myosin II heavy chain expression was identified in isolated rat afferent arterioles by RT-PCR. We conclude that, in addition to Ca2+ -mediated phosphorylation of myosin II regulatory light chains, contraction of the afferent arteriole may be regulated by a mechanism normally associated with the much more rapidly contracting cardiac and skeletal muscles, which involves Ca2+ binding to TnC, leading to alleviation of inhibition of the actomyosin MgATPase by TnI and tropomyosin and rapid contraction of the vessel.

Arsenic trioxide inhibits EMT in hepatocellular carcinoma by promoting lncRNA MEG3 via PKM2

Hepatocellular carcinoma (HCC) presents a great burden for patients worldwide, and metastasis of HCC remains problematic. Arsenic trioxide is a traditional drug that has shown excellent efficacy when applied as cancer therapy. Our study explored the antimetastatic mechanism of arsenic trioxide in HCC. We investigated changes in pyruvate kinase muscle isoform 2 (PKM2) and maternal expression gene 3 (MEG3) following treatment with arsenic trioxide in HCC cells. Consequently, arsenic trioxide negatively regulated PKM2 and positively regulated MEG3. We explored migration ability and the expression of the epithelial to mesenchymal transition (EMT)-related biomarkers E-cadherin, N-cadherin and Vimentin by silencing MEG3 under arsenic trioxide treatment. The wound healing assay showed that arsenic trioxide inhibited the migration of HCC, but silencing MEG3 partially reversed this effect. On the other hand, the EMT-related biomarkers are alleviated under the treatment of arsenic trioxide, but this effect deteriorated when MEG3 is silenced. In conclusion, our study demonstrates a novel mechanism by which arsenic trioxide inhibits EMT in hepatocellular carcinoma by promoting lncRNA MEG3 and PKM2 negatively regulating MEG3.

SAT-013 Dihydrotestosterone Does Not Modulate Key Lipogenesis Regulator in Skeletal Muscle from Female Mice

Polycystic ovary syndrome (PCOS) affects 10% of women and is hallmarked by hyperandrogenemia (HA), which is also a component of non-alcoholic fatty liver disease (NAFLD). In this project, we aimed to investigate how androgen and androgen receptor (AR) impact a key player of lipogenesis, sterol regulatory element-binding protein 1 (SREBP1). To this end, skeletal muscle from lean, female wild type mice and mice administered dihydrotestosterone (DHT) for one month were extracted and analyzed to determine the effect of low dose DHT on lipogenic cytosolic protein and gene expression. We performed Western blot and real-time quantitative polymerase chain reaction (qRT-PCR) analysis of lipogenic intermediates in homogenates of an energy storage tissue. Skeletal muscle was used as a control. Low-dose DHT lowered the active form of cytosolic SREBP-2 in skeletal muscle. However, FAS and p-ACC protein and mRNA expression levels were unchanged in skeletal muscle. The results indicate that SREBP-2 is the primary SREBP isoform in skeletal muscle but that it is not playing a role in DHT-induced lipogenic gene expression or NAFLD. Thank you to HUCM, JHUSOM, and Georgetown University Medical Center (GUMC) for supporting this work.

CPI-17-mediated contraction of vascular smooth muscle is essential for the development of hypertension in obese mice

Several factors have been implicated in obesity-related hypertension, but the genesis of the hypertension is largely unknown. In this study, we found a significantly upregulated expression of CPI-17 (C-kinase-potentiated protein phosphatase 1 inhibitor of 17 kDa) and protein kinase C (PKC) isoforms in the vascular smooth muscles of high-fat diet (HFD)-fed obese mice. The obese wild-type mice showed a significant elevation of blood pressure and enhanced calcium-sensitized contraction of vascular smooth muscles. However, the obese CPI-17-deficient mice showed a normotensive blood pressure, and the calcium-sensitized contraction was consistently reduced. In addition, the mutant muscle displayed an abolished responsive force to a PKC activator and a 30%–50% reduction in both the initial peak force and sustained force in response to various G protein-coupled receptor (GPCR) agonists. Our observations showed that CPI-17-mediated calcium sensitization is mediated through a GPCR/PKC/CPI-17/MLCP/RLC signaling pathway. We therefore propose that the upregulation of CPI-17-mediated calcium-sensitized vasocontraction by obesity contributes to the development of obesity-related hypertension.

Overcoming transarterial chemoembolization resistance through metabolic reprogramming in hepatocellular carcinoma

Background: In the United States, death from hepatocellular carcinoma (HCC) has risen 43% in the last 16 years. For patients not amenable to curative therapy, transarterial chemoembolization (TACE) is the locoregional therapy of choice. Despite a survival advantage to best supportive care, 40% of patients’ tumors will have no response to TACE. We hypothesize that TACE resistance is related to preferential oxygen independent glycolysis through the activation of pyruvate kinase muscle isoform 2 (PKM2), the rate limiting enzyme of glycolysis. Additionally, inhibition of PKM2 will induce cellular metabolic reprogramming and in resistant tumors induce the conversion to TACE responder. Methods: Through transcriptomic analysis of multiple patient cohorts a 15 gene signature, TACE Navigator, was identified which can reliably predict an individual's TACE response. Transcriptomic analyses of TACE responder vs. non-responders identified differentially expressed genes associated with both hypoxia and glycolysis pathways. Further bioinformatics analyses revealed PKM2 as the potential driver for TACE resistance. HCC cell lines were characterized as responder and non-responder based on both transcriptomic analyses and in vitro TACE modeling. Functional studies were performed in vitro using immunoblotting, quantitative polymerase chain reaction, glycolysis and hypoxia assays. Results: PKM2 expression was elevated in patients who did not respond to TACE compared to responders (p < 0.0001). Multivariable Cox regression demonstrated high PKM2 expression to be associated with worse overall survival (HR 3.02, CI 1.51–6.04, p < 0.01) and Kaplan-Meier analysis demonstrated decreased median overall survival (27.5 months to undefined, p < 0.0001). Relative mRNA expression of PKM2 was found to be 2.76 times higher in the non-responder (p < 0.01) and this difference increased to 8.03-fold higher in hypoxia as compared to the responder line (p < 0.0001). Additionally, baseline levels of glycolysis were 2.5 times higher in the non-responder (p < 0.01). In vitro TACE modeling demonstrated no change in cell proliferation in the non-responder line when exposed to doxorubicin in hypoxia compared to a 4.47-fold decrease in the responder. After PKM2 knockdown the non-responder resulted in a 20-fold decreased (p < 0.001) in proliferation with lentiviral shRNA and 6.77-fold decrease (p < 0.05) with pharmacologic inhibitor. Conclusion: Elevated PKM2 is associated with treatment resistance and worse overall survival in patients who receive TACE. In vitro data suggest that elevated PKM2 is associated with increased utilization of the glycolysis pathway, resulting in oxygen independent cell metabolism. Through PKM2 knockdown, non-responder cells can be reprogrammed to act as responders. By reprogramming the therapy resistant phenotype, TACE efficacy could be improved in patients.

Epidermal growth factor receptor controls glycogen phosphorylase in T cells through small GTPases of the RAS family

We recently uncovered a regulatory pathway of the muscle isoform of glycogen phosphorylase (PYGM) that plays an important role in regulating immune function in T cells. Here, using various enzymatic, pulldown, and immunoprecipitation assays, we describe signaling cross-talk between the small GTPases RAS and RAP1A, member of RAS oncogene family (RAP1) in human Kit 225 lymphoid cells, which, in turn, is regulated by the epidermal growth factor receptor (EGFR). We found that this communication bridge is essential for glycogen phosphorylase (PYG) activation through the canonical pathway because this enzyme is inactive in the absence of adenylyl cyclase type 6 (ADCY6). PYG activation required stimulation of both exchange protein directly activated by cAMP 2 (EPAC2) and RAP1 via RAS and ADCY6 phosphorylation, with the latter being mediated by Raf-1 proto-oncogene, Ser/Thr kinase (RAF1). Consistent with this model, PYG activation was EGFR-dependent and may be initiated by the constitutively active form of RAS. Consequently, PYG activation in Kit 225 T cells could be blocked with specific inhibitors of RAS, EPAC, RAP1, RAF1, ADCY6, and cAMP-dependent protein kinase. Our results establish a new paradigm for the mechanism of PYG activation, which depends on the type of receptor involved.

Structures of the heart specific SERCA2a Ca2+-ATPase.

The sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) performs active reuptake of cytoplasmic Ca2+ and is a major regulator of cardiac muscle contractility. Dysfunction or dysregulation of SERCA2a is associated with heart failure, while restoring its function is considered as a therapeutic strategy to restore cardiac performance. However, its structure has not yet been determined. Based on native, active protein purified from pig ventricular muscle, we present the first crystal structures of SERCA2a, determined in the CPA-stabilized E2-AlF4- form (3.3Å) and the Ca2+-occluded [Ca2]E1-AMPPCP form (4.0Å). The structures are similar to the skeletal muscle isoform SERCA1a pointing to a conserved mechanism. We seek to explain the kinetic differences between SERCA1a and SERCA2a. We find that several isoform-specific residues are acceptor sites for post-translational modifications. In addition, molecular dynamics simulations predict that isoform-specific residues support distinct intramolecular interactions in SERCA2a and SERCA1a. Our experimental observations further indicate that isoform-specific intramolecular interactions are functionally relevant, and may explain the kinetic differences between SERCA2a and SERCA1a.

Loss of the Slow Skeletal Muscle Isoform of Troponin T Impairs Motor Coordination in Mice

The loss of the slow skeletal muscle isoform of troponin T (ssTnT) encoded by TNNT1 causes lethal myopathies with symptoms including abnormal neuromuscular reflexes. It has been a challenging question that how the loss of one isoform of TnT results in extremely severe phenotypes while the fast isoform of TnT is present in the patient muscles. We investigated ssTnT knockout (KO) mice for the role of ssTnT in muscle spindles that are a mechanosensors critical to normal neuromuscular function. Immunohistological and spindle protein content studies indicated that unlike extrafusal muscle fibers, adult ssTnT-KO mouse spindle intrafusal fibers express high levels of cardiac TnT, which is significantly increased in intrafusal fibers of ssTnT-KO mice. Impaired motor coordination was observed in ssTnT-KO mice with slower walking during a balance beam performance test as compared with age/sex-matched wildtype (WT) control (p<0.05). An interesting observation from the balance beam performance test is that subcutaneous injection of succinylcholine (SCh), a muscle relaxant believed to alter spindle activities, significantly increased the time for WT mice to walk across the balance beam (p<0.01) whereas SCh treatment produced a trend for decreased time across the beam in ssTnT-KO mice (p=0.10). Taken together, our data suggest that the loss of ssTnT in the KO mice impairs spindle intrafusal fiber contractility, identifying a novel therapeutic target. The attenuated effect of SCh treatment on motor coordination in the ssTnT-KO mice may be due to the potentially adaptive increased expression of cardiac TnT in spindle intrafusal fibers. Further testing is currently underway to assessing the hypothesized adaptation of spindle intrafusal fiber TnT isoform expression and function in ssTnT-KO mice during postnatal development.

Biventricular Increases in Mitochondrial Fission Mediator (MiD51) and Proglycolytic Pyruvate Kinase (PKM2) Isoform in Experimental Group 2 Pulmonary Hypertension-Novel Mitochondrial Abnormalities.

Introduction: Group 2 pulmonary hypertension (PH), defined as a mean pulmonary arterial pressure ≥25 mmHg with elevated pulmonary capillary wedge pressure >15 mmHg, has no approved therapy and patients often die from right ventricular failure (RVF). Alterations in mitochondrial metabolism, notably impaired glucose oxidation, and increased mitochondrial fission, contribute to right ventricle (RV) dysfunction in PH. We hypothesized that the impairment of RV and left ventricular (LV) function in group 2 PH results in part from a proglycolytic isoform switch from pyruvate kinase muscle (PKM) isoform 1 to 2 and from increased mitochondrial fission, due either to upregulation of expression of dynamin-related protein 1 (Drp1) or its binding partners, mitochondrial dynamics protein of 49 or 51 kDa (MiD49 or 51).Methods and Results: Group 2 PH was induced by supra-coronary aortic banding (SAB) in 5-week old male Sprague Dawley rats. Four weeks post SAB, echocardiography showed marked reduction of tricuspid annular plane systolic excursion (2.9 ± 0.1 vs. 4.0 ± 0.1 mm) and pulmonary artery acceleration time (24.3 ± 0.9 vs. 35.4 ± 1.8 ms) in SAB vs. sham rats. Nine weeks post SAB, left and right heart catheterization showed significant biventricular increases in end systolic and diastolic pressure in SAB vs. sham rats (LV: 226 ± 15 vs. 103 ± 5 mmHg, 34 ± 5 vs. 7 ± 1 mmHg; RV: 40 ± 4 vs. 22 ± 1 mmHg, and 4.7 ± 1.5 vs. 0.9 ± 0.5 mmHg, respectively). Picrosirius red staining showed marked biventricular fibrosis in SAB rats. There was increased muscularization of small pulmonary arteries in SAB rats. Confocal microscopy showed biventricular mitochondrial depolarization and fragmentation in SAB vs. sham cardiomyocytes. Transmission electron microscopy confirmed a marked biventricular reduction in mitochondria size in SAB hearts. Immunoblot showed marked biventricular increase in PKM2/PKM1 and MiD51 expression. Mitofusin 2 and mitochondrial pyruvate carrier 1 were increased in SAB LVs.Conclusions: SAB caused group 2 PH. Impaired RV function and RV fibrosis were associated with increases in mitochondrial fission and expression of MiD51 and PKM2. While these changes would be expected to promote increased production of reactive oxygen species and a glycolytic shift in metabolism, further study is required to determine the functional consequences of these newly described mitochondrial abnormalities.

Potent antiproliferative effect of fatty-acid derivative AIC-47 on leukemic mice harboring BCR-ABL mutation.

Therapy based on targeted inhibition of BCR‐ABL tyrosine kinase has greatly improved the prognosis for patients with Philadelphia chromosome (Ph)‐positive leukemia and tyrosine kinase inhibitors (TKI) have become standard therapy. However, some patients acquire resistance to TKI that is frequently associated with point mutations in BCR‐ABL. We previously reported that a medium‐chain fatty‐acid derivative AIC‐47 induced transcriptional suppression of BCR‐ABL and perturbation of the Warburg effect, leading to growth inhibition in Ph‐positive leukemia cells. Herein, we showed that AIC‐47 had anti‐leukemic effects in either wild type (WT)‐ or mutated‐BCR‐ABL‐harboring cells. AIC‐47 suppressed transcription of BCR‐ABL gene regardless of the mutation through downregulation of transcriptional activator, c‐Myc. Reprogramming of the metabolic pathway has been reported to be associated with resistance to anti‐cancer drugs; however, we found that a point mutation of BCR‐ABL was independent of the profile of pyruvate kinase muscle (PKM) isoform expression. Even in T315I‐mutated cells, AIC‐47 induced switching of the expression profile of PKM isoforms from PKM2 to PKM1, suggesting that AIC‐47 disrupted the Warburg effect. In a leukemic mouse model, AIC‐47 greatly suppressed the increase in BCR‐ABL mRNA level and improved hepatosplenomegaly regardless of the BCR‐ABL mutation. Notably, the improvement of splenomegaly by AIC‐47 was remarkable and might be equal to or greater than that of TKI. These findings suggest that AIC‐47 might be a promising agent for overcoming the resistance of Ph‐positive leukemia to therapy.

Adaptation of Skeletal Muscle to Prolonged Activity: Role of Myosin

The aim of this short review is to describe the role of myosin isoforms during the adaptation of skeletal muscle to prolonged physical activity (for example endurance exercise) and to show the coordination between changes in muscle oxidative capacity and myofibrillar apparatus in slow-twitch and fast-twitch muscles. Adaptational changes in myosin isoforms during long lasting muscle activity (decrease of MyHC IIb isoforms relative content and increase of that MyHC IIa and decrease of MyLC 1 fast isoforms in fast-twitch muscles) are in good coordination with changes of muscle oxidative capacity. These changes show that during regular endurance exercise fast-twitch muscle fibers (type IIA) are also recruited and create the potential source of increase in endurance capacity during the process of adaptation to the prolonged physical activity.

Quantitative proteomics analysis to identify biomarkers of chronic myofascial pain and therapeutic targets of dry needling in a rat model of myofascial trigger points.

BackgroundProteomics analysis may provide important information regarding the pathogenesis of chronic myofascial pain and the mechanisms underlying the treatment effects of dry needling.Materials and methodsThis study used a rat model of myofascial trigger points (MTrPs) to perform a proteomics analysis. Three biological replicate experiments were used to compare the proteomes of healthy control rats, a rat model of MTrP, MTrP model rats following dry needling of MTrPs, and MTrP model rats following dry needling of non-MTrPs. Tandem mass tag (TMT) labeling technology based on nanoscale liquid chromatography-tandem mass spectrometry was used. Hierarchical clustering, gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and protein–protein interaction network analysis were performed to characterize the proteins. To validate the TMT results, three candidate biomarker proteins were verified using parallel reaction monitoring and Western blot analysis.ResultsA total of 2,635 proteins were identified. GO and KEGG enrichment analyses showed that the glycolysis/gluconeogenesis pathways played dominant roles in the pathogenesis of chronic myofascial pain. The three candidate biomarker proteins were the pyruvate kinase muscle isozyme (encoded by the PKM gene), the muscle isoform of glycogen phosphorylase (encoded by the PYGM gene), and myozenin 2 (encoded by the MYOZ2 gene). The validation results were consistent with the TMT results.ConclusionThis is the first proteomics study that has investigated the pathogenesis of chronic myofascial pain and the mechanisms underlying the treatment effects of dry needling in an in vivo rat model of MTrPs, which might promote our understanding of the molecular mechanisms underlying chronic myofascial pain.

Acylcarnitine profiles in serum and muscle of dairy cows receiving conjugated linoleic acids or a control fat supplement during early lactation

Acylcarnitines (ACC) are formed when fatty acid (FA)-coenzyme A enters the mitochondria for β-oxidation and the tricarboxylic acid cycle through the carnitine shuttle. Concentrations of ACC may vary depending on the metabolic conditions, but can accumulate when rates of β-oxidation exceed those of tricarboxylic acid. This study aimed to characterize muscle and blood serum acylcarnitine profiles, to determine the mRNA abundance of muscle carnitine acyltransferases, and to test whether dietary supplementation (from d 1 in milk) with conjugated linoleic acids (CLA; 100 g/d; each 12% of trans-10,cis-12 and cis-9,trans-11 CLA; n = 11) altered these compared with control fat-supplemented cows (CTR; n = 10). Blood samples and biopsies from the semitendinosus musclewere collected on d -21, 1, 21, and 70 relative to parturition. Serum and muscle ACC profiles were quantified using a targeted metabolomics approach. The CLA supplement did not affect the variables examined. The serum concentration of free carnitine decreased with the onset of lactation. The concentrations of acetylcarnitine, hydroxybutyrylcarnitine, and the sum of short-chain ACC in serum were greater from d -21 to 21 than thereafter. The serum concentrations of long-chain ACC tetradecenoylcarnitine (C14:1) and octadecenoylcarnitine (C18:1) concentrations were greater on d 1 and 21 compared with d -21. Muscle carnitine remained unchanged, whereas short- and medium-chain ACC, including propenoylcarnitine (C3:1), hydroxybutyrylcarnitine, hydroxyhexanoylcarnitine, hexenoylcarnitine (C6:1), and pimelylcarnitine were increased on d 21 compared with d -21 and decreased thereafter. In muscle, the concentrations of long-chain ACC (from C14 to C18) were elevated on d 1. The mRNA abundance of carnitine palmitoyltransferase 1, muscle isoform (CPT1B) increased 2.8-fold from d -21 to 1, followed by a decline to nearly prepartum values by d 70, whereas that of CPT2 did not change over time. The majority of serum and muscle short- and long-chain ACC were positively correlated with the FA concentrations in serum, whereas serum carnitine and C5 were negatively correlated with FA. Time-related changes in the serum and muscle ACC profiles were demonstrated that were not affected by the CLA supplement at the dosage used in the present study. The elevated concentrations of long-chain ACC species in muscle and of serum acetylcarnitine around parturition point to incomplete FA oxidation were likely due to insufficient metabolic adaptation in response to the load of FA around parturition.

Discovery of a Novel Nav1.7 Inhibitor From Cyriopagopus albostriatus Venom With Potent Analgesic Efficacy

Spider venoms contain a vast array of bioactive peptides targeting ion channels. A large number of peptides have high potency and selectivity toward sodium channels. Nav1.7 contributes to action potential generation and propagation and participates in pain signaling pathway. In this study, we describe the identification of μ-TRTX-Ca2a (Ca2a), a novel 35-residue peptide from the venom of Vietnam spider Cyriopagopus albostriatus (C. albostriatus) that potently inhibits Nav1.7 (IC50 = 98.1 ± 3.3 nM) with high selectivity against skeletal muscle isoform Nav1.4 (IC50 > 10 μM) and cardiac muscle isoform Nav1.5 (IC50 > 10 μM). Ca2a did not significantly alter the voltage-dependent activation or fast inactivation of Nav1.7, but it hyperpolarized the slow inactivation. Site-directed mutagenesis analysis indicated that Ca2a bound with Nav1.7 at the extracellular S3–S4 linker of domain II. Meanwhile, Ca2a dose-dependently attenuated pain behaviors in rodent models of formalin-induced paw licking, hot plate test, and acetic acid-induced writhing. This study indicates that Ca2a is a potential lead molecule for drug development of novel analgesics.

Generation of Functional Myocytes from Equine Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) have revolutionized human biomedicine through their use in disease modeling and therapy. In comparison, little progress has been made toward the application of iPSCs in veterinary species. In that regard, skeletal myocytes from iPSCs would have great potential for understanding muscle function and disease in the equine athlete. In this study, we generated skeletal myotubes by transducing equine iPSC-derived mesenchymal derivatives with an inducible lentiviral vector coding for the human sequence of the myogenic factor, MyoD. Myosin heavy chain-positive myotubes generated from two different iPSC lines were compared to myotubes from adult equine skeletal muscle progenitor cells (MPCs). iPSC myotubes had a smaller mean area than MPC myotubes (≤2-fold). In addition, quantitative polymerase chain reaction analyses showed that iPSC myotubes expressed MYH2 and MYH3 isoforms (at similar or lower levels than MPC myotubes), but they did not express the mature muscle isoform, MYH1. Compared to MPC myotubes, iPSC myotubes expressed reduced levels of the myogenic factors, MYOD1 and MYF6, but did not express MYF5. Finally, iPSC myotubes responded to KCl-induced membrane depolarization by releasing calcium and did so in a manner similar to MPC myotubes. In conclusion, this is the first study to report the generation of functional myocytes from equine iPSCs.

Abstract 435: Post-transcriptional Regulation of Tropomyosin Isoforms Altered in Human Heart Diseases

Background: Tropomyosin 1 (TPM1) is a coiled coil protein that forms polymers along with the actin filament, and it is required for myofibril organization, myocardial contraction, and cardiac development. TPM1 has cardiac muscle and non-muscle specific protein isoforms that dictate its function in cardiac muscle contraction or in cytoskeleton. TPM1 mutations in exon 2b, 5, 6b or 8 are associated with cardiovascular diseases including familial hypertrophic cardiomyopathy, dilated cardiomyopathy and left ventricular non-compaction. Thus, it is of great importance to investigate the mechanisms that control tropomyosin isoforms in order to identify novel therapeutic targets for cardiovascular diseases in which TPM1 is mutated. Method: We used nanopore sequencing technology using MinION and poly(A)click seq (PAC-seq) to identify different TPM1 transcripts generated in rat heart derived H9c2 cells. RT-PCR and western blotting were conducted to measure mRNA and protein levels of TPM1 respectively. Results: We identified 10 different isoforms of TPM1 using nanopore sequencing and determined several exons that are extensively spliced and favored in cardiac cells. We further identified an RNA binding protein that controls cardiac specific isoforms of TPM1 via alternative splicing and polyadenylation. Importantly, we found that this RNA binding protein regulates alternative splicing of exon 6b of TPM1, which is mutated in patients with cardiomyopathies. Conclusions: We identified an RNA binding protein, which controls expression of cardiac specific TPM1 isoforms by alternative polyadenylation and alternative splicing.