Muscle Myosin Isoforms Research Articles

Selective fast fiber damage after leg press exercise leading to failure: a pole vaulter case report

The aim of this study was to investigate, in a trained pole vaulter (PV) and in an endurance-trained physical education student (PE), the effect of a leg press exercise leading to failure (LPF) on changes in serum activity of muscle enzymes and serum concentration of fast (FM) and slow (SM) myosin isoforms, while simultaneously examining mechanical output components as indicators of performance and fatigue developed throughout exercise. A case report study design based on an observational comparison of response between two dichotomous participants, PV and PE, was used. Differences between the participants’ exercise outputs were examined by unpaired t-test or Mann-Whitney test and serum levels of muscle enzymes and myosin isoforms were analyzed at baseline and 24 and 48 hours after LPF. Exercise output analyses showed that the PV’s average fatigue index was significantly higher (P = 0.004). Moreover, during the first six sets, the concentric average power exerted by the PV was significantly (P < 0.01) higher (range: 14% to 35%) than that of the PE. The PV only showed acute mild increases of serum creatine kinase (CK) and FM 24 hours after exercise. In contrast, the PE presented persistent serum rises of several muscle enzymes and SM until 48 h after exercise. The PV’s exercise output revealed an explosive (power-oriented) profile leading to selective mild damage of fast fibers. In contrast, the PE exercise output showed a fatigue-resistant profile, which induced greater muscle enzyme activity and SM serum concentration, suggesting a higher extent of slow fiber damage. Keywords: muscle damage, creatine kinase, myosin isoforms, power output.

Full‐length plasma skeletal muscle myosin isoform deficiency is associated with coagulopathy in acutely injured patients

BackgroundSkeletal muscle myosin (SkM) molecules are procoagulant both in vitro and in vivo. The association of plasma SkM isoforms with blood coagulability and hemostatic capacity has not been defined. ObjectivesWe hypothesized that coagulopathy in acutely injured patients is associated with procoagulant plasma SkM heavy chain levels. MethodsTo test this hypothesis, citrated whole blood and plasma from 104 trauma patients were collected and studied to obtain data for rapid thrombelastography, international normalized ratios, and plasma SkM levels. Coagulability parameters were dichotomized by the threshold for the hypercoagulable trauma‐induced coagulopathy. ResultsLower plasma full‐length SkM heavy chain (full‐SkM) levels were associated with higher international normalized ratio values (>1.3) (p = .03). The full‐SkM levels were also associated with a lower rate of clot propagation (thrombelastography angle <65°) (p = .004), and plasma full‐SkM levels were positively correlated with the thrombelastography angle (r2 = .32, p = .0007). The trauma patient group with the lower plasma full‐SkM levels showed an association with lower clot strength (maximum amplitude <55 mm) (p = .002), and plasma full‐SkM levels positively correlated with maximum amplitude (r2 = .27, p = .005). Hyperfibrinolysis was associated with significantly decreased full‐SkM levels (p = .03). Trauma patients who required red blood cells and fresh frozen plasma transfusions had lower plasma full‐SkM levels compared with those without transfusions (p = .04 and .02, respectively). ConclusionsIn acutely injured trauma patients, lower levels of plasma full‐SkM levels are linked to hypocoagulability in trauma‐induced coagulopathy, implying that SkM plays a role in the hemostatic capacity in trauma patients and may contribute to trauma‐induced coagulopathy.

Tissue-specific expression of myosin phosphatase subunits and isoforms in smooth muscle of mice and humans.

Alternative splicing of exon24 (E24) of myosin phosphatase targeting subunit 1 (Mypt1) by setting sensitivity to nitric oxide (NO)/cGMP-mediated relaxation is a key determinant of smooth muscle function. Here we defined expression of myosin phosphatase (MP) subunits and isoforms by creation of new genetic mouse models, assay of human and mouse tissues, and query of public databases. A Mypt1-LacZ reporter mouse revealed that Mypt1 transcription is turned on early in development during smooth muscle differentiation. Mypt1 is not as tightly restricted in its expression as smooth muscle myosin heavy chain (Myh11) and its E6 splice variant. Mypt1 is enriched in mature smooth versus nonmuscle cells. The E24 splice variant and leucine zipper minus protein isoform that it encodes is enriched in phasic versus tonic smooth muscle. In the vascular system, E24 splicing increases as vessel size decreases. In the gastrointestinal system, E24 splicing is most predominant in smooth muscle of the small intestine. Tissue-specific expression of MP subunits and Mypt1 E24 splicing is conserved in humans, whereas a splice variant of the inhibitory subunit (CPI-17) is unique to humans. A Mypt1 E24 mini-gene splicing reporter mouse generated to define patterns of E24 splicing in smooth muscle cells (SMCs) dispersed throughout the organ systems was unsuccessful. In summary, expression of Mypt1 and splicing of E24 is part of the program of smooth muscle differentiation, is further enhanced in phasic smooth muscle, and is conserved in humans. Its low-level expression in nonmuscle cells may confound its measurement in tissue samples.

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).

The Central Role of the F-Actin Surface in Myosin Force Generation.

Simple SummaryAlthough actin is a highly conserved protein, it is involved in many diverse cellular processes. Actin owes its diversity of function to its ability to bind to a host of actin-binding proteins (ABPs) that localize across its surface. Among the most studied ABPs is the molecular motor, myosin. Myosin generates force on actin filaments by pairing ATP hydrolysis, product release, and actin-binding to the conformational changes that lead to movement. Central to this process is the progression of myosin binding to the actin surface as it moves through its ATPase cycle. During binding, actin acts as a myosin ATPase activator, catalyzing essential hydrolysis release steps. Here, we use the current model of actin-myosin binding as a roadmap to describe the portions of the actin-myosin interface that are sequentially formed throughout the motor cycle. At each step, we compare the interactions of a diverse set of high-resolution actin-myosin cryo-electron microscopy structures to define what portions of the interface are conserved and which are isoform-specific.Actin is one of the most abundant and versatile proteins in eukaryotic cells. As discussed in many contributions to this Special Issue, its transition from a monomeric G-actin to a filamentous F-actin form plays a critical role in a variety of cellular processes, including control of cell shape and cell motility. Once polymerized from G-actin, F-actin forms the central core of muscle-thin filaments and acts as molecular tracks for myosin-based motor activity. The ATP-dependent cross-bridge cycle of myosin attachment and detachment drives the sliding of myosin thick filaments past thin filaments in muscle and the translocation of cargo in somatic cells. The variation in actin function is dependent on the variation in muscle and non-muscle myosin isoform behavior as well as interactions with a plethora of additional actin-binding proteins. Extensive work has been devoted to defining the kinetics of actin-based force generation powered by the ATPase activity of myosin. In addition, over the past decade, cryo-electron microscopy has revealed the atomic-evel details of the binding of myosin isoforms on the F-actin surface. Most accounts of the structural interactions between myosin and actin are described from the perspective of the myosin molecule. Here, we discuss myosin-binding to actin as viewed from the actin surface. We then describe conserved structural features of actin required for the binding of all or most myosin isoforms while also noting specific interactions unique to myosin isoforms.

Skeletal Muscle Myosin Is Procoagulant By Binding Factor XI Via Its A3 Domain and Enhancing Factor XI Activation By Thrombin

Blood coagulation mechanisms play key roles in health and disease. Pilot studies using selected human plasmas showed the potential associations of plasma skeletal muscle myosin (SkM) isoforms and phenotypes with pulmonary embolism and thrombin generation, suggesting SkM may contribute to blood coagulation reactions in plasma. Here we report that ex vivo studies of the coagulability of fresh flowing human blood over SkM-coated surfaces showed that an anti-factor XI (FXI) mAb, but not an anti-tissue factor mAb, inhibited clot formation, indicating that FXI is an essential contributor for the normal observed procoagulant response of blood during its exposure to immobilized SkM. This raised the question of whether procoagulant SkM's requirement for FXI involves direct or indirect effects on FXI.To assess direct interactions between SkM and FXI, Bio-Layer Interferometry (BLI) (Octet Red system) was used to record kinetics for binding of soluble FXI to immobilized SkM. BLI data showed that FXI bound to SkM with a Kd of 0.2 nM (k on= 2.92x10 6 M -1s -1 and k off=9.25x10 -3 s -1) (Fig. 1A). In contrast, prekallikrein (PK) did not bind to the SkM (Fig.1A), indicating the specificity of SkM for binding FXI. The anti-FXI mAb1A6, which recognizes the Apple (A)3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying SkM binds the FXI A3 domain.Studies using purified clotting factors were made to identify which FXI-related activities might be affected by SkM. When FXI activation by thrombin was evaluated under conditions where polyphosphate (PolyP) 100-mer and 700-mer enhance FXI activation, SkM concentration-dependently enhanced FXI activation by thrombin (Fig. 1B). Whereas alkaline phosphatase destroyed PolyP's ability to stimulate FXI activation by thrombin, it did not cause a reduction of SkM's ability to enhance FXI activation, indicating SkM's activity is independent of PolyP-like sequences in SkM. Small unilamellar phospholipid vesicles (20% phosphatidylserine (PS) / 80% phosphatidylcholine) did not affect FXI activation by thrombin; furthermore, reagents that neutralize procoagulant PS, i.e., lactadherin, annexin V, and phospholipase A2, did not affect SkM's enhancement of FXI activation by thrombin, indicating that this activity is not due to anionic phospholipids linked to SkM.The effects of SkM on FXI autoactivation and FXI activation by FXIIa were evaluated. As is well known, PolyP and some other anionic reagents, e.g., nucleic acid polymers, enhance not only FXI activation by thrombin but also FXI autoactivation and FXI activation by FXIIa. However, SkM did not significantly affect FXI autoactivation or FXI activation by factor XIIa, further emphasizing that SkM's enhancement of FXI activation by thrombin is not due to any PolyP-like compounds and that it is a unique property of procoagulant SkM. This also suggests that SkM has a unique mechanism for its procoagulant activity on FXI activation which is limited to the thrombin positive feedback loop.To evaluate further the basis for interactions between FXI and SkM, we employed FXI- PK chimeras because BLI binding studies showed that, in contrast to FXI, PK did not bind to SkM. Recombinant FXI proteins in which each of the four A domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding A domain from PK and were used to identify the site of factor XI to interact with SkM for FXI activation by thrombin. The FXI chimera with the substitution of the PKA1 domain was not activated by thrombin, which is consistent with the fact that the FXI A1 domain is an interactive site for thrombin. Thrombin activation of the two FXI chimeras (FXI/PKA3 and FXI/PKA4) with substitutions of either the A3 or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain (FXI/PKA2) did not reduce the enhancement of activation by thrombin compared to wild type FXI. Furthermore, mAb1A6, which recognizes the A3 domain and which inhibited the prothrombotic activity of fresh blood flowing over a SkM-coated surface, potently inhibited FXI binding to SkM in BLI studies. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains of FXI.In summary, we found that SkM's ex vivo procoagulant activity requires FXI, that SkM enhances FXI activation by thrombin and this requires FXI's A3 and A4 domains, and that SkM's high affinity binding of FXI requires the FXI A3 domain (Fig. 1C). [Display omitted] DisclosuresGruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment. Gailani: Anthos Therapeutics: Consultancy; Aronora: Membership on an entity's Board of Directors or advisory committees; Bayer Pharma: Consultancy; Bristol Myer Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain

We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal β-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D ) in the absence of actin is completely reversed for each chimera compared with the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared with wild-type (WT) IFI, whereas for EMB-3b these values are increased compared with wild-type (WT) EMB. In the presence of actin, ADP affinity (KAD ) is unchanged for IFI-3a, compared with IFI, but ADP affinity for EMB-3b is increased, compared with EMB, and shifted toward IFI values. ATP-induced dissociation of acto-S1 (K1k+2 ) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicates that the exon 3-encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating actomyosin crossbridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.

Characterization of the Functional Diversity of the Synthetic Nanomachine Powered by Different Muscle Myosin Isoforms

The functional diversity of two ortholog isoforms of the fast-skeletal muscle myosin (amphibian and mammalian) can be precisely defined using a synthetic nanomachine and a simulation model able to interface the output of the nanomachine to that of the muscle of origin. The nanomachine is made by an ensemble of HMM fragments, carried on a piezoelectric nanopositioner and brought to interact with an actin filament attached with the correct polarity to a bead trapped into the focus of a Dual Laser (Pertici et al., Nat Commun 9:3532, 2018). In the present version, the bead-actin filament assembly is prepared with the Ca2+-insensitive gelsolin fragment TL40, to make [Ca2+] an independent parameter and allow future studies on the Ca2+-dependence of the sarcomeric proteins action. In this way, even in the simple actin-myosin assay, we reveal a class-specific Ca2+-sensitivity of the amphibian myosin performance. In 2 mM ATP, following the attainment of the isometric plateau force (F0), up to five different isotonic force-velocity points for each interaction are recorded by switching the control from position- to force-feedback. The different sensitivity to temperature of mammalian and frog fast-skeletal muscle performance is tested comparing the force-velocity relations of the two isoforms at the same temperature (∼24°C). Just like for the muscle of origin, the unloaded shortening velocity of the nanomachine powered by mammalian isoform is ∼1/2 that of the amphibian. Model simulation with a unique actin-myosin kinetic scheme allows interfacing in situ and in vivo performances, demonstrating the ability of the nanomachine to recapitulate the emergent properties of the half-sarcomere of the muscle of origin and identify, at the level of a specific protein, the functional diversity of muscles in vivo. Supported by IIT-SEED, Genova and Fondazione CR Firenze, Italy.

In Situ Characterization of the Working Stroke of the Slow and Fast Isoforms of Muscle Myosin

Muscle performance depends on the myosin heavy chain (MHC) isoform expressed in the myosin II motor. Stiffness measurements at sarcomere level in skinned fibres from either soleus or psoas muscles of rabbit have recently indicated that the stiffness and the force of the slow myosin isoform (soleus), are three times smaller than those of the fast isoform (psoas) (Percario et al. J Physiol 596:1243, 2018). Here the investigation on the mechano-kinetic features of the two isoforms in situ is extended to the comparison of the size and speed of the working stroke, measured on the isotonic velocity transient elicited by stepwise drops in force of different sizes superimposed on the plateau force (T0) of maximally Ca2+ activated skinned fibres from either muscle (sarcomere length 2.4 μm, temperature 12°C, 4% dextran T-500). The analysis of the early rapid shortening, due to the synchronisation of the working stroke in the attached motors shows that at high load (0.8 T0) the size of the working stroke is smaller in the slow isoform (2 nm) than in the fast isoform (5 nm), while it is similar at low load (7-8 nm at 0.2 T0). The rate of the working stroke increases by ∼6 times with the decrease in load from 0.8 to 0.2 T0, but, at any load, is one order of magnitude slower in the slow isoform than in the fast isoform. A reduced size of the working stroke of the slow isoform, together with the smaller force per motor, opens the question on the mechanism that can explain the high efficiency of the slow muscle with respect to the fast muscle. Supported by MIUR-PRIN 2010R8JK2X and Telethon GGP12282 (Italy).

Impact of anti-S2 Peptides on myosin S2 Isoforms and HCM Mutants

Myosin subfragment-2 (S2) is an unstable coiled coil that influences the binding of myosin heads to actin. Instability in the myosin S2 could underlie one of the causes for hyper-contractility in Hypertrophic Cardiomyopathy (HCM). Supporting this hypothesis, HCM mutations E924K and E930del in myosin S2 reduced myosin S2 coiled coil stability compared to wild type as measured by Forester's Resonance Energy Transfer (FRET) and Gravitational Force Spectroscopy (GFS). To modulate the stability of the S2, anti-S2 peptides called stabilizer and destabilizer peptides because they are designed to increase and decrease the stability of the MYH7 S2 coiled coil, were tested on a range of human S2 peptide sequences. The anti-S2 peptides were tested on myosin S2 peptides across MYH11 (smooth), MYH7 (cardiac), and MYH2 (skeletal) with GFS and FRET. The results demonstrated that the mechanical stability was increased by stabilizer and decreased by destabilizer across the cardiac and skeletal myosin S2 peptides but not for smooth muscle isoform. Dose-response curves for FRET assays indicated that both peptides bound at nanomolar concentrations to wild type cardiac and skeletal, but not smooth muscle isoforms. The wild type cardiac S2 peptide required more force to unzip than skeletal, smooth, or mutant peptides in the absence of stabilizer peptide. Effectively, the stabilizer peptide improved the stability of both HCM mutants (E924K and E930del). The stabilizer peptide enhanced dimer formation in mutant cardiac, and skeletal myosin S2 peptides, and destabilizer increased flexibility of cardiac and skeletal myosin S2 wild type peptide. This study demonstrates that the structural stability of myosin S2 can be altered by designed small peptides as well as mutations. These anti-S2 peptides share similar specificities and high affinities for the tested striated muscle isoforms but not the smooth muscle myosin isoform.

Functional Comparison of Homologous Mutations in Human Beta, Perinatal, and Embryonic Muscle Myosin Isoforms

Functional Comparison of Homologous Mutations in Human Beta, Perinatal, and Embryonic Muscle Myosin Isoforms

Expressing a Z-disk nebulin fragment in nebulin-deficient mouse muscle: effects on muscle structure and function

BackgroundNebulin is a critical thin filament-binding protein that spans from the Z-disk of the skeletal muscle sarcomere to near the pointed end of the thin filament. Its massive size and actin-binding property allows it to provide the thin filaments with structural and regulatory support. When this protein is lost, nemaline myopathy occurs. Nemaline myopathy causes severe muscle weakness as well as structural defects on a sarcomeric level. There is no known cure for this disease.MethodsWe studied whether sarcomeric structure and function can be improved by introducing nebulin’s Z-disk region into a nebulin-deficient mouse model (Neb cKO) through adeno-associated viral (AAV) vector therapy. Following this treatment, the structural and functional characteristics of both vehicle-treated and AAV-treated Neb cKO and control muscles were studied.ResultsIntramuscular injection of this AAV construct resulted in a successful expression of the Z-disk fragment within the target muscles. This expression was significantly higher in Neb cKO mice than control mice. Analysis of protein expression revealed that the nebulin fragment was localized exclusively to the Z-disks and that Neb cKO expressed the nebulin fragment at levels comparable to the level of full-length nebulin in control mice. Additionally, the Z-disk fragment displaced full-length nebulin in control mice, resulting in nemaline rod body formation and a worsening of muscle function. Neb cKO mice experienced a slight functional benefit from the AAV treatment, with a small increase in force and fatigue resistance. Disease progression was also slowed as indicated by improved muscle structure and myosin isoform expression.ConclusionsThis study reveals that nebulin fragments are well-received by nebulin-deficient mouse muscles and that limited functional benefits are achievable.

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.

Blebbistatin modulates prostatic cell growth and contrapctility through myosin II signaling.

To investigate the effect of blebbistatin (BLEB, a selective myosin inhibitor) on regulating contractility and growth of prostate cells and to provide insight into possible mechanisms associated with these actions. BLEB was incubated with cell lines of BPH-1 and WPMY-1, and intraprostatically injected into rats. Cell growth was determined by flow cytometry, and in vitro organ bath studies were performed to explore muscle contractility. Smooth muscle (SM) myosin isoform (SM1/2, SM-A/B, and LC17a/b) expression was determined via competitive reverse transcriptase PCR. SM myosin heavy chain (MHC), non-muscle (NM) MHC isoforms (NMMHC-A and NMMHC-B), and proteins related to cell apoptosis were further analyzed via Western blotting. Masson's trichrome staining was applied to tissue sections. BLEB could dose-dependently trigger apoptosis and retard the growth of BPH-1 and WPMY-1. Consistent with in vitro effect, administration of BLEB to the prostate could decrease rat prostatic epithelial and SM cells via increased apoptosis. Western blotting confirmed the effects of BLEB on inducing apoptosis through a mechanism involving MLC20 dephosphorylation with down-regulation of Bcl-2 and up-regulation of BAX and cleaved caspase 3. Meanwhile, NMMHC-A and NMMHC-B, the downstream proteins of MLC20, were found significantly attenuated in BPH-1 and WPMY-1 cells, as well as rat prostate tissues. Additionally, BLEB decreased SM cell number and SM MHC expression, along with attenuated phenylephrine-induced contraction and altered prostate SMM isoform composition with up-regulation of SM-B and down-regulation of LC17a, favoring a faster contraction. Our novel data demonstrate BLEB regulated myosin expression and functional activity. The mechanism involved MLC20 dephosphorylation and altered SMM isoform composition.

Skeletal muscle in aged mice reveals extensive transformation of muscle gene expression

BackgroundAging leads to decreased skeletal muscle function in mammals and is associated with a progressive loss of muscle mass, quality and strength. Age-related muscle loss (sarcopenia) is an important health problem associated with the aged population.ResultsWe investigated the alteration of genome-wide transcription in mouse skeletal muscle tissue (rectus femoris muscle) during aging using a high-throughput sequencing technique. Analysis revealed significant transcriptional changes between skeletal muscles of mice at 3 (young group) and 24 (old group) months of age. Specifically, genes associated with energy metabolism, cell proliferation, muscle myosin isoforms, as well as immune functions were found to be altered. We observed several interesting gene expression changes in the elderly, many of which have not been reported before.ConclusionsThose data expand our understanding of the various compensatory mechanisms that can occur with age, and further will assist in the development of methods to prevent and attenuate adverse outcomes of aging.

Troponin Subunits are Expressed in the Rat Afferent Arteriole

Vascular smooth muscle cells of the renal afferent arteriole are unusual: they must be able to contract very rapidly in response to a sudden increase in systemic blood pressure, thereby protecting 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 M et al (2003) FASEB J 17: 2284–2286). Since 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 skeletal muscle and slow skeletal muscle/cardiac TnC isoforms and slow skeletal muscle and cardiac TnI isoforms were detected by RT‐PCR and confirmed by cDNA sequencing. Furthermore, cardiac and slow skeletal muscle TnI isoforms, but not fast skeletal muscle TnI were detected in isolated afferent arterioles at the protein level by proximity ligation assay. We conclude that, in addition to Ca2+‐mediated phosphorylation of myosin 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.Support or Funding InformationSupported by the Canadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Postinjury Exercise and Platelet-Rich Plasma Therapies Improve Skeletal Muscle Healing in Rats But Are Not Synergistic When Combined

Background: Skeletal muscle injuries are the most common sports-related injury and a major concern in sports medicine. The effect of platelet-rich plasma (PRP) injections on muscle healing is still poorly understood, and current data are inconclusive. Purpose: To evaluate the effects of an ultrasound-guided intramuscular PRP injection, administered 24 hours after injury, and/or posttraumatic daily exercise training for 2 weeks on skeletal muscle healing in a recently established rat model of skeletal muscle injury that highly mimics the muscle trauma seen in human athletes. Study Design: Controlled laboratory study. Methods: A total of 40 rats were assigned to 5 groups. Injured rats (medial gastrocnemius injury) received a single PRP injection (PRP group), daily exercise training (Exer group), or a combination of a single PRP injection and daily exercise training (PRP-Exer group). Untreated and intramuscular saline–injected animals were used as controls. Muscle force was determined 2 weeks after muscle injury, and muscles were harvested and evaluated by means of histological assessment and immunofluorescence microscopy. Results: Both PRP (exhibiting 4.8-fold higher platelet concentration than whole blood) and exercise training improved muscle strength (maximum tetanus force, TetF) in approximately 18%, 20%, and 30% of rats in the PRP, PRP-Exer, and Exer groups, respectively. Specific markers of muscle regeneration (developmental myosin heavy chain, dMHC) and scar formation (collagen I) demonstrated the beneficial effect of the tested therapies in accelerating the muscle healing process in rats. PRP and exercise treatments stimulated the growth of newly formed regenerating muscle fibers (1.5-, 2-, and 2.5-fold increase in myofiber cross-sectional area in PRP, PRP-Exer, and Exer groups, respectively) and reduced scar formation in injured skeletal muscle (20%, 34%, and 41% of reduction in PRP, PRP-Exer, and Exer groups, respectively). Exercise-treated muscles (PRP-Exer and Exer groups) had significantly reduced percentage of dMHC-positive regenerating fibers (35% and 47% decrease in dMHC expression, respectively), indicating that exercise therapies accelerated the muscle healing process witnessed by the more rapid replacement of the embryonic-developmental myosin isoform by mature muscle myosin isoforms. Conclusion: Intramuscular PRP injection and, especially, treadmill exercise improve histological outcome and force recovery of the injured skeletal muscle in a rat injury model that imitates sports-related muscle injuries in athletes. However, there was not a synergistic effect when both treatments were combined, suggesting that PRP does not add any beneficial effect to exercise-based therapy in the treatment of injured skeletal muscle. Clinical Relevance: This study demonstrates the efficacy of an early active rehabilitation protocol or single intramuscular PRP injection on muscle recovery. The data also reveal that the outcome of the early active rehabilitation is adversely affected by the PRP injection when the two therapies are combined, and this could explain why PRP therapies have failed in randomized clinical trials where the athletes have adhered to postinjection rehabilitation protocols based on the principle of early, active mobilization.

X-Ray Crystallography Structures of Drosophila Striated Muscle Myosin II

Skeletal muscle myosin class II proteins are molecular machines that convert the chemical energy derived from the hydrolysis of ATP into mechanical work used to power muscle contraction. Drosophila melanogaster contains one gene encoding all striated muscle myosin II isoforms. The objective of this study is to gain insight into how alternative exon selection imparts myosin protein isoform biochemical and biophysical specificity. To this end, two His-tagged recombinant proteins encoding an indirect flight muscle myosin isoform (IFI) and an embryonic body wall myosin isoform (EMB) were expressed in and purified from the indirect flight muscles (IFM) of engineered fly lines lacking endogenous IFM myosin (Caldwell et al., Methods, 2012). The purified His-tagged myosins retain ATPase activity similar to their corresponding untagged isoforms. The myosin subfragment-1 (S1) contains the myosin heavy chain motor domain and the essential light chain. We determined the three-dimensional structure of IFI S1 at 2.5 A resolution and EMB S1 at 2.2 A resolution. They are the first insect myosin protein structures determined by X-ray crystallography (EMB is PDB 4QBD). Both structures are in the post rigor enzymatic state, as determined by comparisons with known myosin structures. For the EMB structure, two copies of the myosin molecule with slight conformational differences were resolved in the asymmetric unit. The electron density revealed a citrate molecule (contained in the crystallization solution) bound in the nucleotide-binding pocket. For the IFI structure, there is one myosin molecule in the asymmetric unit. The nucleotide-binding pocket contains an ADP molecule. Differences in conformation within regions of the proteins encoded by alternative exons suggest a source of the observed physiological differences in the embryonic and adult flight muscle fibers. (Funded by NIH R01GM32443 to SIB).

Analysis of Methionine Oxidation in Myosin Isoforms in Porcine Skeletal Muscle by LC-MS/MS Analysis.

The purpose of this study was to analyze oxidized methionines in the myosin isoforms of porcine longissimus thoracis, psoas major, and semimembranosus muscles by liquid chromatography (LC) and mass spectrometry (MS). A total of 836 queries matched to four myosin isoforms (myosin-1, -2, -4, and -7) were analyzed and each myosin isoform was identified by its unique peptides (7.3-13.3). Forty-four peptides were observed from all three muscles. Seventeen peptides were unique to the myosin isoform and the others were common peptides expressed in two or more myosin isoforms. Five were identified as oxidized peptides with one or two methionine sulfoxides with 16 amu of mass modification. Methionines on residues 215 (215), 438 (438), 853 (851), 856 (854), 1071 (1069), and 1106 (1104) of myosin-1 (myosin-4) were oxidized by the addition of oxygen. Myosin-2 had two oxidized methionines on residues 215 and 438. No queries matched to myosin-7 were observed as oxidized peptides. LC-MS/MS allows analysis of the oxidation of specific amino acids on specific residue sites, as well as in specific proteins in the food system.

Tuning of shortening speed in coleoid cephalopod muscle: no evidence for tissue‐specific muscle myosin heavy chain isoforms

The contractile protein myosin II is ubiquitous in muscle. It is widely accepted that animals express tissue-specific myosin isoforms that differ in amino acid sequence and ATPase activity in order to tune muscle contractile velocities. Recent studies, however, suggested that the squid Doryteuthis pealeii might be an exception; members of this species do not express muscle-specific myosin isoforms, but instead alter sarcomeric ultrastructure to adjust contractile velocities. We investigated whether this alternative mechanism of tuning muscle contractile velocity is found in other coleoid cephalopods. We analyzed myosin heavy chain transcript sequences and expression profiles from muscular tissues of a cuttlefish, Sepia officinalis, and an octopus, Octopus bimaculoides, in order to determine if these cephalopods express tissue-specific myosin heavy chain isoforms. We identified transcripts of four and six different myosin heavy chain isoforms in S. officinalis and O. bimaculoides muscular tissues, respectively. Transcripts of all isoforms were expressed in all muscular tissues studied, and thus S. officinalis and O. bimaculoides do not appear to express tissue-specific muscle myosin isoforms. We also examined the sarcomeric ultrastructure in the transverse muscle fibers of the arms of O. bimaculoides and the arms and tentacles of S. officinalis using transmission electron microscopy and found that the fast contracting fibers of the prey capture tentacles of S. officinalis have shorter thick filaments than those found in the slower transverse muscle fibers of the arms of both species. It thus appears that coleoid cephalopods, including the cuttlefish and octopus, may use ultrastructural modifications rather than tissue-specific myosin isoforms to adjust contractile velocities.