Slow Skeletal Muscle Research Articles

Changes in the Mechanical Properties of Fast and Slow Skeletal Muscle after 7 and 21 Days of Restricted Activity in Rats.

Disuse muscle atrophy is usually accompanied by changes in skeletal muscle structure, signaling, and contractile potential. Different models of muscle unloading can provide valuable information, but the protocols of experiments with complete immobilization are not physiologically representative of a sedentary lifestyle, which is highly prevalent among humans now. In the current study, we investigated the potential effects of restricted activity on the mechanical characteristics of rat postural (soleus) and locomotor (extensor digitorum longus, EDL) muscles. The restricted-activity rats were kept in small Plexiglas cages (17.0 × 9.6 × 13.0 cm) for 7 and 21 days. After this, soleus and EDL muscles were collected for ex vivo mechanical measurements and biochemical analysis. We demonstrated that while a 21-day movement restriction affected the weight of both muscles, in soleus muscle we observed a greater decrease. The maximum isometric force and passive tension in both muscles also significantly changed after 21 days of movement restriction, along with a decrease in the level of collagen 1 and 3 mRNA expression. Furthermore, the collagen content itself changed only in soleus after 7 and 21 days of movement restriction. With regard to cytoskeletal proteins, in our experiment we observed a significant decrease in telethonin in soleus, and a similar decrease in desmin and telethonin in EDL. We also observed a shift towards fast-type myosin heavy chain expression in soleus, but not in EDL. In summary, in this study we showed that movement restriction leads to profound specific changes in the mechanical properties of fast and slow skeletal muscles. Future studies may include evaluation of signaling mechanisms regulating the synthesis, degradation, and mRNA expression of the extracellular matrix and scaffold proteins of myofibers.

Conditionally dominant negative impacts of clinically recessive TNNT1 myopathy mutations.

TNNT1 nonsense mutation causing premature termination of the translation of slow skeletal muscle troponin T (ssTnT) has been reported with recessive inheritance and loss-of-function phenotypes. We studied the phenotypes of mouse models of Tnnt1 myopathies for pathological impacts besides the loss of slow fiber functions. Mice with a knock-in stop codon at Glu180 mimicking the Amish nemaline myopathy (ANM-KI) show similar recessive phenotypes to that of Tnnt1-knockout (ssTnT-KO) mice (Wei et al. J. Physiol. 2014) with loss of slow fibers, low fatigue-resistance, active regeneration, inflammatory immune cell infiltration and compensatory hypertrophy of fast fibers in slow fiber-rich muscles. ANM-KI mice showed significantly more loss of slow fibers, more active regeneration, worse post-fatigue recovery and more hypertrophy of fast fibers than that of ssTnT-KO mice at young age and advancing with age. A feature of TNNT1 premature termination mutations is decreased tropomyosin-binding affinity and loss of thin filament incorporation. Therefore, the more severe than ssTnT-KO myopathic phenotypes of ANM-KI mice suggest negative impacts of non-myofilament incorporated TnT fragment on causing degeneration of slow and mix fiber type muscles. Since hypertrophy of fast fibers was not seen in ANM-KI mice before 7 days of age when soleus muscle is not utilized due to premature neuromuscular development, work- and/or load-induced slow and mix fiber muscle injury may worsen the negative impacts by increasing the total level of non-myofilament incorporated TnT and fragments from the release of fast skeletal muscle TnT in mix type fibers. Since non-myofilament-incorporated TnT is rapidly degraded in normal muscle cells, the cytotoxic effect of non-myofilament-incorporated TnT and fragment may have high potency (Jeong et al. AJP, 2009) and this pathogenic mechanism presents a molecular target for the treatment of TNNT1 myopathies and for improving muscle health of carriers.

Effects of ATP on Time Parameters of Contractility of Rats’ Slow and Fast Skeletal Muscles in Normal and Hypothermic Conditions

We have previously shown that hypothermia leads to an increase in the synaptic modulating effects of ATP but not of adenosine in several different animal skeletal muscles. In this paper, we studied the effect of ATP on the amplitude–time parameters of single and tetanic contractions of rats’ isolated fast (1) and slow (2) muscles at different temperatures. We found that when muscles were stimulated by the electrical field (0.1 Hz, 0.5 ms, 10 V), with a decrease in the bath temperature from 37 °C to 14 °C (3), there was an increase in the half-relaxation time of the slow muscle (m. soleus), but not of the fast muscle (m. EDL). Similar effects were observed using a carbachol-induced contraction technique, which suggests the postsynaptic (4) nature of the expansion of the contractile response of the slow muscle induced by ATP (5). To confirm the postsynaptic nature of the observed phenomenon, experiments were performed at a high calcium level (7.2 mM), in which the presynaptic effects of ATP were shown to be offset. We found that the hypercalcium condition did not significantly change the effects of ATP on the measured parameters in both muscles. To record muscle tetanic contractions, we gradually increased the frequency of electrical impulses with the increment of 2.5 Hz to achieve the fusion frequencies of 12.5 Hz for m. soleus and 17.5 Hz for m. EDL at normal temperatures. ATP (100 μM) did not change the fusion frequency for both muscles at a normal temperature but decreased this parameter for the slow muscle to 5 Hz at 14 °C without affecting that for the fast muscle. We conclude that ATP potentiates a hypothermia-induced increase in the half-relaxation time of the contraction of rats’ slow, but not fast, skeletal muscles by acting on postsynaptic P2 receptors (6).

Exon skipping caused by splicing mutation in TNNT1 nemaline myopathy.

The TNNT1 gene encoding the slow skeletal muscle TnT has been identified as a causative gene for nemaline myopathy. TNNT1 nemaline myopathy is mainly characterized by neonatal-onset muscle weakness, pectus carinatum and respiratory insufficiency. Herein, we report on a Chinese girl with TNNT1 nemaline myopathy with mild clinical phenotypes without thoracic deformities or decreased respiratory function. Muscle biopsy showed moderate to marked type 1 fiber atrophy and nemaline rods. Next-generation sequencing identified the compound heterozygous c. 587dupA (p. D196Efs*41) and c. 387+5G>A mutations in the TNNT1 gene according to the transcript NM_003283.4. RNA sequencing revealed complete exon 9 skipping caused by the c. 387+5G>A mutation. Through quantitative PCR, we found that both the truncation c. 587dupA (p. D196Efs*41) and the splicing c. 387+5G>A mutations triggered nonsense-mediated mRNA decay (NMD). Western blotting showed the residual amount of the truncated TNNT1 protein by deletion of exon 9, which may ameliorate the disease to some extent.

Fast and slow skeletal myosin binding protein-C and aging

Aging is associated with skeletal muscle strength decline and cardiac diastolic dysfunction. The structural arrangements of the sarcomeric proteins, such as myosin binding protein-C (MyBP-C) are shown to be pivotal in the pathogenesis of diastolic dysfunction. Yet, the role of fast (fMyBP-C) and slow (sMyBP-C) skeletal muscle MyBP-C remains to be elucidated. Herein, we aimed to characterize MyBP-C and its paralogs in the fast tibialis anterior (TA) muscle from adult and old mice. Immunoreactivity preparations showed that the relative abundance of the fMyBP-C paralog was greater in the TA of both adult and old, but no differences were noted between groups. We further found that the expression level of cardiac myosin binding protein-C (cMyBP-C), an important modulator of cardiac output, was lowered by age. Standard SDS-PAGE along with Pro-Q Diamond phosphoprotein staining did not identify age-related changes in phosphorylated MyBP-C proteins from TA and cardiac muscles; however, it revealed that MyBP-C paralogs in fast skeletal and cardiac muscle were highly phosphorylated. Mass spectrometry further identified glycogen phosphorylase, desmin, actin, troponin T, and myosin regulatory light chain 2 as phosphorylated myofilament proteins in both ages. MyBP-C protein-bound carbonyls were determined using anti-DNP immunostaining and found the carbonyl level of fMyBP-C, sMyBP-C, and cMyBP-C to be similar between old and adult animals. In summary, our data showed some differences regarding the MyBP-C paralog expression and identified an age-related reduction of cMyBP-C expression. Future studies are needed to elucidate which are the age-driven post-translational modifications in the MyBP-C paralogs.

Early Divergence of the C-Terminal Variable Region of Troponin T Via a Pair of Mutually Exclusive Alternatively Spliced Exons Followed by a Selective Fixation in Vertebrate Heart.

Troponin T (TnT) is the thin filament anchoring subunit of troponin complex and plays an organizer role in the Ca2+-regulation of striated muscle contraction. From an ancestral gene emerged ~ 700 million years ago in Bilateria, three homologous genes have evolved in vertebrates to encode muscle type-specific isoforms of TnT. Alternative splicing variants of TnT are present in vertebrate and invertebrate muscles to add functional diversity. While the C-terminal region of TnT is largely conserved, it contains an alternatively spliced segment emerged early in C. elegans, which has evolved into a pair of mutually exclusive exons in arthropods (10A and 10B of Drosophila TpnT gene) and vertebrates (16 and 17 of fast skeletal muscle Tnnt3 gene). The C-terminal alternatively spliced segment of TnT interfaces with the other two subunits of troponin with functional significance. The vertebrate cardiac TnT gene that emerged from duplication of the fast TnT gene has eliminated this alternative splicing by the fixation of an exon 17-like constitutive exon, indicating a functional value in slower and rhythmic contractions. The vertebrate slow skeletal muscle TnT gene that emerged from duplication of the cardiac TnT gene has the exon 17-like structure conserved, indicating its further function in sustained and fatigue resistant contractions. This functionality-based evolution is consistent with the finding that exon 10B-encoded segment of Drosophila TnT homologous to the exon 17-encoded segment of vertebrate fast TnT is selectively expressed in insect heart and leg muscles. The evolution of the C-terminal variable region of TnT demonstrates a submolecular mechanism in modifying striated muscle contractility and for the treatment of muscle and heart diseases.

Abstract P1026: Creg1 Inhibits Cardiomyocyte Proliferation And Promotes Myocardial Differentiation And Compaction During Postnatal Cardiac Development

Introduction: Overexpression of cellular repressor of E1A-stimulated genes 1 (CREG1) promotes the formation of compact, contracting myocardium-like structures during cardiomyogenesis from embryonic stem cells. Cell biology studies suggest that CREG1 promotes differentiation and inhibits proliferation of developing cardiomyocytes. This study aims to understand the role of CREG1 during myocardial development in vivo . Methods: Cardiac tissues were harvested from wild-type (WT) and constitutive Creg1 knockout (KO) postnatal day 1.5 and 17 through timed mating of heterozygous Creg1 mice. Creg1 KO mice were compared with Creg1 WT littermates using immunoblotting, immunofluorescence, and methylene blue staining. Results: Postnatal day 1.5 Creg1 KO hearts showed lower levels of expression of slow skeletal muscle troponin I (TnI) compared with WT counterparts. No significant difference in expression was seen between WT and KO Creg1 hearts for myosin light chain 3 (MLC3), myosin heavy chain (MHC), and cardiac troponin I. Immunofluorescence staining revealed increased numbers of PCNA (Proliferating Cell Nuclear Antigen) positive nuclei in postnatal day 1.5 Creg1 KO hearts, suggesting enhanced cardiomyocyte proliferation after loss of CREG1. Methylene blue staining in postnatal day 17 hearts displayed decreased myocardial compaction in Creg1 KO mice. Conclusion: CREG1 may promote cardiomyocyte differentiation and inhibit proliferation during postnatal myocardial development. Ablation of CREG1 results in cardiac compaction defects. Our findings suggest an important role for CREG1 in cardiac development.

Small Molecule RPI-194 Stabilizes Activated Troponin to Increase the Calcium Sensitivity of Striated Muscle Contraction.

Small molecule cardiac troponin activators could potentially enhance cardiac muscle contraction in the treatment of systolic heart failure. We designed a small molecule, RPI-194, to bind cardiac/slow skeletal muscle troponin (Cardiac muscle and slow skeletal muscle share a common isoform of the troponin C subunit.) Using solution NMR and stopped flow fluorescence spectroscopy, we determined that RPI-194 binds to cardiac troponin with a dissociation constant KD of 6–24 μM, stabilizing the activated complex between troponin C and the switch region of troponin I. The interaction between RPI-194 and troponin C is weak (KD 311 μM) in the absence of the switch region. RPI-194 acts as a calcium sensitizer, shifting the pCa50 of isometric contraction from 6.28 to 6.99 in mouse slow skeletal muscle fibers and from 5.68 to 5.96 in skinned cardiac trabeculae at 100 μM concentration. There is also some cross-reactivity with fast skeletal muscle fibers (pCa50 increases from 6.27 to 6.52). In the slack test performed on the same skinned skeletal muscle fibers, RPI-194 slowed the velocity of unloaded shortening at saturating calcium concentrations, suggesting that it slows the rate of actin-myosin cross-bridge cycling under these conditions. However, RPI-194 had no effect on the ATPase activity of purified actin-myosin. In isolated unloaded mouse cardiomyocytes, RPI-194 markedly decreased the velocity and amplitude of contractions. In contrast, cardiac function was preserved in mouse isolated perfused working hearts. In summary, the novel troponin activator RPI-194 acts as a calcium sensitizer in all striated muscle types. Surprisingly, it also slows the velocity of unloaded contraction, but the cause and significance of this is uncertain at this time. RPI-194 represents a new class of non-specific troponin activator that could potentially be used either to enhance cardiac muscle contractility in the setting of systolic heart failure or to enhance skeletal muscle contraction in neuromuscular disorders.

A.3 A novel recessive TNNT1 congenital core-rod myopathy in French Canadians

Background: Mutations in the slow skeletal muscle troponin T (TNNT1) gene cause a congenital nemaline myopathy resulting in death from respiratory insufficiency in early infancy. We report on four French Canadians with a novel congenital TNNT1 myopathy. Methods: Patients underwent lower extremity and paraspinal MRI, quadriceps biopsy and genetic testing. TNNT1 expression in muscle was assessed by quantitative PCR and immunoblotting. Wild type or mutated TNNT1 mRNAs were co-injected with morpholinos in a zebrafish knockdown model to assess for rescue of the morphant phenotype. Results: Four patients shared a novel missense homozygous mutation in TNNT1. They developed from childhood slowly progressive limb-girdle weakness with spinal rigidity and contractures. They suffered from restrictive lung disease and recurrent episodes of rhabdomyolysis. Older patients remained ambulatory into their sixties. Lower extremity MRI showed symmetrical myopathic changes. Paraspinal MRI showed diffuse fibro-fatty involution. Biopsies showed multi-minicores. Nemaline rods were seen in half the patients. TNNT1 mRNA expression was similar in controls and patients, while levels of TNNT1 protein were reduced in patients. Wild type TNNT1 mRNA rescued the zebrafish morphants but mutant transcripts failed to do so. Conclusions: This study expands the spectrum of TNNT1-related myopathy to include a milder clinical phenotype caused by a functionally-confirmed novel mutation.

RPI‐194 is a Novel Troponin Activator that Increases the Calcium Sensitivity of Striated Muscle Contraction

IntroductionSystolic heart failure is a cardiac disease in which the contractile function of heart muscle becomes weakened and unable to pump blood with adequate force. As of yet, no existing positive inotrope is able to lower the mortality rate in systolic heart failure. Cardiac troponin complex (cTn) is a trimeric complex consisting of troponin C (cTnC), troponin I (cTnI) and troponin T (cTnT). cTnC regulates blood supply to the heart by turning muscle contraction on and off in a calcium‐dependent manner. More precisely, the regulatory N‐domain of cTnC (cNTnC) binds the switch region of cTnI to activate contraction. Drug‐like molecules need to bind to this cNTnC‐cTnI switch region interface and modulate the pumping activity of the heart. Our objective was to identify a small molecule that has the ability to stabilize the activated conformation of cTn by binding to this interface and thereby enhance cardiac muscle contraction.HypothesisA cardiac troponin activator could potentially compensate the impaired contractile function of the heart in the treatment of systolic heart failureMethodsA total of 47 new compounds were designed using existing knowledge of calcium sensitizers. The compounds were screened against a recombinantly purified cNTnC‐cTnI chimeric construct, named gChimera, using nuclear magnetic resonance and fluorescence spectroscopy. We identified a novel small molecule cardiac troponin activator, RPI‐194, and measured its binding affinity to recombinantly purified cNTnC and gChimera. We also investigated the activity of RPI‐194 in rat skinned cardiac muscle trabeculae, isolated mouse cardiomyocytes, and isolated working mouse hearts. RPI‐194 activity was also tested in skinned skeletal muscle fibers from rats as both cardiac and slow skeletal muscle share the same isoform of cTnC.ResultsWe demonstrated that a small molecule troponin activator RPI‐194 binds to gChimera with a KD of 12‐24 µM and is able to stabilize activated cTn. It also slowed down the rate of calcium release from reconstituted cTn. RPI‐194 showed increasing calcium sensitivity of isometric contraction in skinned cardiac muscle trabeculae, as well as in slow and fast skeletal muscle fibers, suggesting it to be a calcium sensitizer, with cross‐reactivity within striated muscle. Contrarily, RPI‐194 was unable to slow down calcium release from isolated cNTnC. It also reduced the velocity of unloaded shortening in skeletal muscle fibers, suggesting that it slows the rate of actin‐myosin cross‐bridging, although no effect on myosin ATPase activity was found. RPI‐194 decreased the velocity and amplitude of contractions in isolated cardiomyocytes but contractility was preserved in isolated working hearts.ConclusionsRPI‐194 is a small molecule troponin activator that acts as a calcium sensitizer in striated muscle. Because of the isoform sharing of cTnC in cardiac and slow skeletal muscle, it is nearly impossible to develop a cardiac‐specific troponin activator. Whole animal model studies are needed to determine the impact of RPI‐194 on the different striated muscle types and whether this would be acceptable for therapeutic intervention.

Apocynin Attenuates Diabetes-Induced Skeletal Muscle Dysfunction by Mitigating ROS Generation and Boosting Antioxidant Defenses in Fast-Twitch and Slow-Twitch Muscles.

In response to diabetes mellitus, skeletal muscle is negatively affected, as is evident by reduced contractile force production, increased muscle fatigability, and increased levels of oxidative stress biomarkers. Apocynin is a widely used NADPH oxidase inhibitor, with antioxidant and anti-inflammatory potential. It has been effective for amelioration of a variety of disorders, including diabetic complications. Therefore, the present study was conducted to evaluate the effects and action mechanisms of apocynin in slow- and fast-twitch diabetic rat muscles. Male Wistar rats were rendered diabetic by applying intraperitoneally a single dose of streptozotocin (45 mg/kg). Apocynin treatment (3 mg/kg/day) was administered over 8 weeks. Fasting blood glucose (FBG), insulin tolerance and body weight gain were measured. Both slow (soleus) and fast (extensor digitorum longus, EDL) skeletal muscles were used for muscle function evaluation, oxidative stress markers, and evaluating gene expression using qRT-PCR. Treatment with apocynin significantly reduced FBG levels and enhanced insulin tolerance. Apocynin also prevented muscle contractile dysfunction in EDL muscle but had no significant effect on this parameter in soleus muscles. However, in both types of muscles, apocynin mitigated the oxidative stress by decreasing ROS levels and increasing total glutathione levels and redox state. Concomitantly, apocynin also statistically enhanced Nrf-2 and GLU4 mRNA expression and downregulated NOX2, NOX4, and NF-κB mRNA. Collectively, apocynin exhibits properties myoprotective in diabetic animals. These findings indicate that apocynin predominantly acts as an antioxidant in fast-twitch and slow-twitch muscles but has differential impact on contractile function.

Expression profiles of protein markers regulating the mitochondrial lifecycle in skeletal muscle of acute spinal cord transected rats

Spinal cord transection (ST) inactivates the neuromuscular complex and triggers progressive muscular atrophy of the affected skeletal muscles. Disruption in neural input also leads to a reduction in mitochondrial volume. The mitochondria lifecycle can be generally divided into processes of biogenesis, expression of OXPHOS proteins, fusion, fission, and mito/autophagy; specifically, the balance between biogenesis vs. mitophagy dictates the rate of formation and destruction, respectively. Here, we examined the expression profiles of the markers associated with the mitochondrial lifecycle up to one month following ST in phenotypically slow (soleus; SOL) and mixed (plantaris; PLT) skeletal muscle. We hypothesized that ST would induce a reduction in mitochondrial DNA (mtDNA) copy number, decrease expression in markers associated with biogenesis, and increased proteins that regulate mito/autophagy. Adult female Sprague Dawley rats were randomly divided into control (CON; n=6), 1 day (1dST; n=5), 8 day (8dST; n=8), and 28 day post‐ST (28dST; n=8). Compared to CON relative SOL and PLT muscle masses (absolute mass / body mass) were 98, 65, and 62% and 85, 60, and 73% at 1, 8 and 28d post‐ST, respectively (p<0.05). Compared to CON, no significant changes were observed in mtDNA copy number in ST muscles following amplification using standard end‐point PCR protocols for mtDNA (ND1, COX1, or ATP6) and nuclear (β2M) genes (p>0.05). Total protein was isolated from SOL and PLT, separated using standard SDS‐PAGE protocol, and probed for markers associated with various stages of the mitochondrial lifecycle using standard western blot protocols. Upstream markers known to influence mitochondrial lifecycle signaling (Rev‐Erbα, AMPK) showed a varied temporal and muscle‐specific response: in 1dST SOL muscles, Rev‐Erbα increased 74% from CON then decreased 43% at 28dST, whereas 8dST PLT muscle exhibited a 44% increase (p<0.05). In 8dST SOL and PLT muscles, the pAMPK:AMPK ratio was elevated 366 and 85% from CON, respectively (p<0.05). Markers for mitochondrial biogenesis (PGC‐1α, NRF1, NRF2) in SOL and PLT muscles were largely unchanged, although a ~50% decrease in Tfam protein expression was observed at each ST time point compared to CON (p<0.05). Mitofusion2, a marker for mitochondrial fusion, was unchanged (p>0.05). However, for markers detecting mitochondrial fission (MFF, Drp1, Fis1), we observed a ~60% increase in MFF expression in 1dST SOL and PLT muscles (p<0.05). As expected markers for mitophagy (Parkin, PINK, p62, LC3 I/II) were significantly elevated: Parkin increased ~70% at 28dST (p<0.05), whereas LC3 I/II increased 65% and 112% at 1dST in in SOL and PLT muscles, respectively (p<0.05), and remained significantly elevated in PLT muscles at 8dST (27%) and 28d (37%) (p<0.05). Taken together, these data suggest that mitochondrial volume and expression of markers regulating mitochondrial biogenesis and fusion are not influenced by acute ST despite a decrease in muscle mass. However, selective markers for fission and mitophagy are significantly elevated in both SOL and PLT muscles, suggesting that mitochondrial destruction mechanism are activated acutely following ST injury. Lastly, selective proteins of the OXPHOS protein complex significantly increased in 1dST SOL and PLT muscles suggesting possible activity‐dependent regulation of mitochondrial gene expression.

Localized TPC1-mediated Ca2+ release from endolysosomes contributes to myoseptal junction development in zebrafish.

In the trunk of developing zebrafish embryos, adjacent myotome blocks transmit contractile force via myoseptal junctions (MJs), which are dynamic structures that connect the actin cytoskeleton of skeletal muscle cells to extracellular matrix components via transmembrane protein complexes in the sarcolemma. Here, we report that the endolysosomal ion channel, two-pore channel type 1 (TPC1, encoded by tpcn1), generates highly localized non-propagating Ca2+ transients that play a distinct and required role in the capture and attachment of superficial slow skeletal muscle cells at MJs. Use of antisense morpholinos or CRISPR/Cas9 gene editing to disrupt tpcn1 gene expression resulted in abnormal MJ phenotypes, including slow skeletal muscle cells detaching from or crossing the myosepta. We also report that TPC1-decorated endolysosomes are dynamically associated with MJs in a microtubule-dependent manner, and that attenuating tpcn1 expression or TPC1 function disrupted endolysosomal trafficking and resulted in an abnormal distribution of β-dystroglycan (encoded by dag1; a key transmembrane component of the dystrophin-associated protein complex). Taken together, our data suggest that localized TPC1-generated Ca2+ signals facilitate essential endolysosomal trafficking and membrane contact events, which help form and maintain MJs following the onset of slow skeletal muscle cell contractile activity. This article has an associated First Person interview with the first author of the paper.

UCHL1 Regulates Lipid and Perilipin 2 Level in Skeletal Muscle.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that was originally found in neurons. We found that UCHL1 is highly expressed in slow oxidative skeletal muscles, but its functions remain to be fully understood. In this study, we observed that UCHL1 protein levels in skeletal muscle and C2C12 myotubes were downregulated by fasting or glucose starvation respectively. Skeletal muscle selective knockout (smKO) of UCHL1 resulted in a significant reduction of lipid content in skeletal muscle and improved glucose tolerance. UCHL1 smKO did not significantly change the levels of key proteins involved in oxidative metabolism such as SDHA, Akt, or PDH. Interestingly, while the levels of the major lipases and lipid transporters were unchanged, perilipin 2 was significantly downregulated in UCHL1 smKO muscle. Consistently, in C2C12 myotubes, UCHL1 siRNA knockdown also reduced perilipin 2 protein level. This data suggests that UCHL1 may stabilize perilipin 2 and thus lipid storage in skeletal muscle.

The Effect of High and Low-Intensity Interval Training on TRF1 and TRF2 Gene Expression in Slow and Fast-Twitch Skeletal Muscles of C57BL/6 Mice: An Experimental Study

The Effect of High and Low-Intensity Interval Training on TRF1 and TRF2 Gene Expression in Slow and Fast-Twitch Skeletal Muscles of C57BL/6 Mice: An Experimental Study

Mechanisms of the modulation of actin-myosin interactions by A1-type myosin light chains

BackgroundThe interaction of N-terminal extension of the myosin A1 essential light chain (A1 ELC) with actin is receiving increasing attention as a target in utilizing synthetic A1 ELC N-terminal-derived peptides in cardiac dysfunction therapy. MethodsTo elucidate the mechanism by which these peptides regulate actin-myosin interaction, here we have investigated their effects on the myosin subfragment 1 (S1)-induced polymerization of G-actin. ResultsThe MLCFpep and MLCSpep peptides spanning the 3–12 of A1 ELC sequences from fast and slow skeletal muscle, respectively, increased the rate of actin polymerization not only by S1(A2) but also the rate of S1(A1)-induced actin polymerization, suggesting that they did not interfere with the direct binding of A1 ELC with actin. The efficiency of actin polymerization in the presence of the N-terminal ELC peptides depended on their sequence. Substitution of aspartic acid for neutral asparagine at position 5 of MLCFpep dramatically enhanced its ability to stimulate S1-induced polymerization and enabled it to initiate polymerization of G-actin in the absence of S1. ConclusionsThese and other results presented in this work suggest that the modulation of myosin motor activity by N-terminal ELC peptides is exerted through a change in actin filament conformation rather than through blocking the A1 ELC-actin interaction. General significanceThe results imply the possibility of enhancing therapeutic effects of these peptides by modifications of their sequence.

Transcriptomic Analysis for Pork Color—The Ham Halo Effect in Biceps Femoris

Pork color is a major indicator of product quality that guides consumer purchasing decisions. Recently, industryhas received an increase in consumer complaints about the lightness and nonuniformity of ham color, primarily lighter colorin the periphery termed “ham halo” that is not caused by manufacturing procedures. This effect is seen in fresh and processed hams and the outer lighter muscle is associated with lower myoglobin concentration, pH, and type I fibers. The objective of this study was to identify differences in gene expression profiles between light and normal-colored portions of the biceps femoris muscle from pork hams. RNA-sequencing (RNA-seq) was performed for paired light and normal-colored muscle samples from 10 animals showing the ham halo effect. Over 50 million paired-end reads (2×75 bp) per library were obtained. An average of 99.74% of trimmed high-quality reads was mapped to the Sscrofa 11.1 genome assembly. Differentially expressed genes (DEGs) were identified using both the DESeq2 and GFOLD software packages. A total of 14,049 genes were expressed in the biceps femoris; 13,907 were expressed in both light and normal muscle, while 56 and 86 genes were only expressed in light and normal muscle, respectively. Analysis with DESeq2 identified 392 DEGs with 359 genes being more highly expressed in normal-colored muscle. A total of 61 DEGs were identified in the DESeq2 analysis and identified in at least 7 of the 10 individual animal analyses. All 61 of these DEGs were up-regulated in normal-colored muscle. Gene Ontology enrichment analysis of DEGs identified the transition between fast and slow fibers and skeletal muscle adaptation and contraction as the most significant biological process terms. The evaluation of gene expression by RNA-seq identified DEGs between regions of the biceps femoris with the ham halo effect that are associated with variation in pork color.

Definition of the mechanokinetic parameters accounting for the functional diversity of slow and fast isoforms of skeletal muscle myosin with a synthetic nanomachine

The mechanokinetic parameters characterising the force and power produced by fast (psoas) and slow (soleus) skeletal rabbit muscle are defined by feeding to a model simulation the mechanical output of a synthetic nanomachine (Pertici et al., Nat Commun 9:3532, 2018) powered by 8 heavy-mero-myosin fragments (HMM) of myosin II purified from either muscle. Implementation of a length clamp control for the minimization of the trap compliance allows to integrate the measurements of all the relevant steady state parameters (the isometric force (F0), the force-velocity relation and the maximum power (Pmax)) with transient kinetics parameters (rate of isometric force development (r) and force fluctuation due to individual motor attachment-detachment).

Troponin C mutations alter twitch kinetics and force-frequency relationship of fast and slow twitch skeletal muscle

Troponin C (TnC) is critically involved in the contraction of striated muscle. The role of TnC on the mechanical process of skeletal muscle has been thoroughly investigated. However, the impact of TnC mutations on the force-frequency relationship (FFR) of skeletal muscle is yet to be investigated. The objective of this study is to determine the effect of TnC mutations on the force-frequency relationship and contractile kinetics in fast and slow-twitch skeletal muscle. To investigate this key question, we measured the extensor digitorum longus (EDL) and soleus force-frequency response in a murine model with a TnC mutation (expressed after TnCL48QAAV or TnCD73NAAV injection) that causes increased calcium sensitivity (L48Q) or a decreased calcium sensitivity (D73N) and compared the FFR to the wildtype counterparts.

Conditionally myopathic effects of exon 8 deleting mutation of slow skeletal muscle troponin T in compound heterozygous transgenic mouse model

Cases of inherited myopathy with in-frame deletion of the segment encoded by exons 8 of TNNT1 gene encoding slow skeletal muscle troponin T (ssTnT-ΔE8) were reported in compound heterozygotes with loss of function TNNT1 mutations such as exon 14 deletion. We constructed a transgenic mouse line expressing ssTnT-ΔE8 driven by a cloned Tnnt1 promoter. High level over-expressions produced barely detectable ssTnT-ΔE8 integration in the myofilaments corresponding to marginal pathophysiological effects, consistent with the absence of clinical phenotype of heterozygous carriers.