Muscle Myosin Heavy Chain Protein Research Articles

Skeletal muscle myosin heavy chain fragmentation as a potential marker of protein degradation in response to resistance training and disuse atrophy.

We examined how resistance exercise (RE), cycling exercise and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation. The 1boutRE study involved younger men (n=8; 5±2years of RE experience) performing a lower body RE bout with vastus lateralis (VL) biopsies being obtained prior to and acutely following exercise. With the 10weekRT study, VL biopsies were obtained in 36 younger adults before and 24h after their first/naïve RE bout. Participants also engaged in 10weeks of resistance training and donated VL biopsies before and 24h after their last RE bout. VL biopsies were also examined in an acute cycling study (n=7) and a study involving 2weeks of leg immobilization (n=20). In the 1boutRE study, fragmentation of all MyHC isoforms (MyHCTotal) increased 3h post-RE (∼200%, P=0.018) and returned to pre-exercise levels by 6h post-RE. Interestingly, a greater magnitude increase in MyHC type IIa versus I isoform fragmentation occurred 3h post-RE (8.6±6.3-fold vs. 2.1±0.7-fold, P=0.018). In 10weekRT participants, the first/naïve and last RE bouts increased MyHCTotal fragmentation 24h post-RE (+65% and +36%, P<0.001); however, the last RE bout response was attenuated compared to the first bout (P=0.045). Although cycling exercise did not alter MyHCTotal fragmentation, ∼8% VL atrophy with 2weeks of leg immobilization increased MyHCTotal fragmentation (∼108%, P<0.001). Mechanistic C2C12 myotube experiments indicated that MyHCTotal fragmentation is likely due to calpain proteases. In summary, RE and disuse atrophy increase MyHC protein fragmentation. Research into how ageing and disease-associated muscle atrophy affect these outcomes is needed. HIGHLIGHTS: What is the central question of this study? How different exercise stressors and disuse affect skeletal muscle myosin heavy chain fragmentation. What is the main finding and its importance? This investigation is the first to demonstrate that resistance exercise and disuse atrophy lead to skeletal muscle myosin heavy chain protein fragmentation in humans. Mechanistic in vitro experiments provide additional evidence that MyHC fragmentation occurs through calpain proteases.

Skeletal muscle myosin heavy chain protein fragmentation as a potential marker of protein degradation in response to resistance training and disuse atrophy.

We sought to examine how resistance exercise (RE), cycling exercise, and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation in humans. In the first study (1boutRE), younger adult men (n=8; 5±2 years of RE experience) performed a lower body RE bout with vastus lateralis (VL) biopsies obtained immediately before, 3-, and 6-hours post-exercise. In the second study (10weekRT), VL biopsies were obtained in untrained younger adults (n=36, 18 men and 18 women) before and 24 hours (24h) after their first/naïve RE bout. These participants also engaged in 10 weeks (24 sessions) of resistance training and donated VL biopsies before and 24h after their last RE bout. VL biopsies were also examined from a third acute cycling study (n=7) and a fourth study involving two weeks of leg immobilization (n=20, 15 men and 5 women) to determine how MyHC fragmentation was affected. In the 1boutRE study, the fragmentation of all MyHC isoforms (MyHCTotal) increased 3 hours post-RE (~ +200%, p=0.018) and returned to pre-exercise levels by 6 hours post-RE. Immunoprecipitation of MyHCTotal revealed ubiquitination levels remained unaffected at the 3- and 6-hour post-RE time points. Interestingly, a greater increase in magnitude for MyHC type IIa versus I isoform fragmentation occurred 3-hours post-RE (8.6±6.3-fold versus 2.1±0.7-fold, p=0.018). In all 10weekRT participants, the first/naïve and last RE bouts increased MyHCTotal fragmentation 24h post-RE (+65% and +36%, respectively; p<0.001); however, the last RE bout response was attenuated compared to the first bout (p=0.045). The first/naïve bout response was significantly elevated in females only (p<0.001), albeit females also demonstrated a last bout attenuation response (p=0.002). Although an acute cycling bout did not alter MyHCTotal fragmentation, ~8% VL atrophy with two weeks of leg immobilization led to robust MyHCTotal fragmentation (+108%, p<0.001), and no sex-based differences were observed. In summary, RE and disuse atrophy increase MyHC protein fragmentation. A dampened response with 10 weeks of resistance training, and more refined responses in well-trained men, suggest this is an adaptive process. Given the null polyubiquitination IP findings, more research is needed to determine how MyHC fragments are processed. Moreover, further research is needed to determine how aging and disease-associated muscle atrophy affect these outcomes, and whether MyHC fragmentation is a viable surrogate for muscle protein turnover rates.

Abstract 9: Smooth Muscle Cell Tgfbr2 Deletion in Mice Causes Aortic Hypercontractility and Impaired Endothelium-Dependent Relaxation

Background: Abnormal smooth muscle cell (SMC) TGF-β signaling is proposed as a critical driver in the development of thoracic aortic aneurysms and dissections (TAAD) associated with Marfan and Loeys-Dietz Syndromes as well as nonsyndromic TAAD. However, the mechanisms by which altered SMC TGF-β signaling causes TAAD are poorly understood. Others have proposed that loss of SMC TGF-β signaling causes TAAD by impairing SMC contractility, leading to aortic medial degeneration and dilation. However, mice generated in our lab with deficient SMC TGF-β signaling (due to SMC-specific deletion of the type II TGF-β receptor) have thicker aortic medias and increased mRNA encoding SMC contractile proteins. These observations predict increased contractility. Methods &amp; Results: We addressed this apparent contradiction experimentally by measuring vasomotor function (by tension myography) and contractile protein expression (by immunoblotting) in aortas of mice with normal or deficient SMC TGF-β signaling. Isolated aortic rings from mice with deficient SMC TGF-β signaling showed increased contraction to phenylephrine (Emax: 13.5 mN vs 7.8 mN in controls; p&lt;0.0001; n=19-20) and potassium chloride (Emax: 7.5 mN vs 5.7 mN in controls; p&lt;0.0001; n=19-20). Moreover, levels of smooth muscle myosin heavy chain protein were at least as high in aortas with deficient SMC TGF-β signaling as in control aortas, consistent with their capacity to generate increased contractile force. Surprisingly, aortic segments from mice with deficient SMC TGF-β signaling also had impaired endothelium-dependent relaxation to acetylcholine (Emax: 37% vs 97% in controls; p&lt;0.0001; n=19-20). Endothelium-independent relaxation to sodium nitroprusside was similar between the two groups. CD31 immunostaining of vessel segments revealed equivalent endothelial integrity in both groups. Conclusion: Physiologic SMC TGF-β signaling is an important determinant of both SMC contractility and endothelial function. Disruption of physiologic SMC TGF-β signaling may lead to TAAD through direct effects on SMC as well as through indirect effects on endothelial function. Our results also suggest an unanticipated role for SMC TGF-β signaling in regulating endothelial-mediated vasomotor function.

Architecture of the human renal inner medulla and functional implications.

The architecture of the inner stripe of the outer medulla of the human kidney has long been known to exhibit distinctive configurations; however, inner medullary architecture remains poorly defined. Using immunohistochemistry with segment-specific antibodies for membrane fluid and solute transporters and other proteins, we identified a number of distinctive functional features of human inner medulla. In the outer inner medulla, aquaporin-1 (AQP1)-positive long-loop descending thin limbs (DTLs) lie alongside descending and ascending vasa recta (DVR, AVR) within vascular bundles. These vascular bundles are continuations of outer medullary vascular bundles. Bundles containing DTLs and vasa recta lie at the margins of coalescing collecting duct (CD) clusters, thereby forming two regions, the vascular bundle region and the CD cluster region. Although AQP1 and urea transporter UT-B are abundantly expressed in long-loop DTLs and DVR, respectively, their expression declines with depth below the outer medulla. Transcellular water and urea fluxes likely decline in these segments at progressively deeper levels. Smooth muscle myosin heavy chain protein is also expressed in DVR of the inner stripe and the upper inner medulla, but is sparsely expressed at deeper inner medullary levels. In rodent inner medulla, fenestrated capillaries abut CDs along their entire length, paralleling ascending thin limbs (ATLs), forming distinct compartments (interstitial nodal spaces; INSs); however, in humans this architecture rarely occurs. Thus INSs are relatively infrequent in the human inner medulla, unlike in the rodent where they are abundant. UT-B is expressed within the papillary epithelium of the lower inner medulla, indicating a transcellular pathway for urea across this epithelium.

Resveratrol Inhibits Phenotypic Switching of Neointimal Vascular Smooth Muscle Cells After Balloon Injury Through Blockade of Notch Pathway

Phenotypic switching of vascular smooth muscle cells (VSMCs) plays an initial role in neointimal hyperplasia, the main cause of many occlusive vascular diseases. The aim of this study was to measure the effects of resveratrol (RSV) on the phenotypic transformation of VSMCs and to investigate its mechanism of action. Cultured VSMCs isolated from rat thoracic aorta were prepared with serum starvation for 72 hours followed by RSV treatment (50-200 μmol/L) and 10% serum stimulation. Male Sprague-Dawley rats, subjected to carotid arteries injury from a balloon catheter, were exposed to intraperitoneal injection of RSV (1 mg/kg) or saline and were killed after 7 or 28 days. Compared with cells in the serum-induced group, VSMCs in the RSV or N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment group exhibited significant decreases of proliferation and migration. The total and cytoplasmic Notch-1 levels were declined by RSV, accompanied by a significant increase in smooth muscle α-actin and smooth muscle myosin heavy chain protein. The expression of Notch-1, Jagged-1, Hey-1, and Hey-2 mRNA in balloon-injured arteries at 7 days was decreased by RSV treatment. Arteries from RSV-treated rats showed less neointimal hyperplasia, lower collagen content, and a lower rate of cells positive for proliferating cell nuclear antigen 28 days after injury, compared with saline controls. The results indicate that RSV can attenuate phenotypic switching of VSMCs after arterial injury through inhibition of the Notch pathway.

Sodium Ferulate Inhibits Neointimal Hyperplasia in Rat Balloon Injury Model

Background/AimNeointimal formation after vessel injury is a complex process involving multiple cellular and molecular processes. Inhibition of intimal hyperplasia plays an important role in preventing proliferative vascular diseases, such as restenosis. In this study, we intended to identify whether sodium ferulate could inhibit neointimal formation and further explore potential mechanisms involved.MethodsCultured vascular smooth muscle cells (VSMCs) isolated from rat thoracic aorta were pre-treated with 200 µmol/L sodium ferulate for 1 hour and then stimulated with 1 µmol/L angiotensin II (Ang II) for 1 hour or 10% serum for 48 hours. Male Sprague-Dawley rats subjected to balloon catheter insertion were administrated with 200 mg/kg sodium ferulate (or saline) for 7 days before sacrificed.ResultsIn presence of sodium ferulate, VSMCs exhibited decreased proliferation and migration, suppressed intracellular reactive oxidative species production and NADPH oxidase activity, increased SOD activation and down-regulated p38 phosphorylation compared to Ang II-stimulated alone. Meanwhile, VSMCs treated with sodium ferulate showed significantly increased protein expression of smooth muscle α-actin and smooth muscle myosin heavy chain protein. The components of Notch pathway, including nuclear Notch-1 protein, Jagged-1, Hey-1 and Hey-2 mRNA, as well as total β-catenin protein and Cyclin D1 mRNA of Wnt signaling, were all significantly decreased by sodium ferulate in cells under serum stimulation. The levels of serum 8-iso-PGF2α and arterial collagen formation in vessel wall were decreased, while the expression of contractile markers was increased in sodium ferulate treated rats. A decline of neointimal area, as well as lower ratio of intimal to medial area was observed in sodium ferulate group.ConclusionSodium ferulate attenuated neointimal hyperplasia through suppressing oxidative stress and phenotypic switching of VSMCs.

SMB myosin heavy chain knockout enhances tonic contraction and reduces the rate of force generation in ileum and stomach antrum

The role of SMA and SMB smooth muscle myosin heavy chain (MHC) isoforms in tonic and phasic contractions was studied in phasic (longitudinal ileum and stomach circular antrum) and tonic (stomach circular fundus) smooth muscle tissues of SMB knockout mice. Knocking out the SMB MHC gene eliminated SMB MHC protein expression and resulted in upregulation of the SMA MHC protein without altering the total MHC protein level. Switching from SMB to SMA MHC protein expression decreased the rate of the force transient and increased the sustained tonic force in SMB((-/-)) ileum and antrum with high potassium (KPSS) but not with carbachol (CCh) stimulation. The increased tonic contraction under the depolarized condition was not through changes in second messenger signaling pathways (PKC/CPI-17 or Rho/ROCK signaling pathway) or LC(20) phosphorylation. Biochemical analyses showed that the expression of contractile regulatory proteins (MLCK, MLCP, PKCδ, and CPI-17) did not change significantly in tissues tested except for PKCα protein expression being significantly decreased in the SMB((-/-)) antrum. However, specifically activating PKCα with phorbol dibutyrate (PDBu) was not significantly different in knockout and wild-type tissues, with total force being a fraction of the force generation with KPSS or CCh stimulation in SMB((-/-)) ileum and antrum. Taken together, these data show removing the SMB MHC protein expression with a compensatory increase in the SMA MHC protein results in enhanced sustained KPSS-induced tonic contraction with a reduced rate of force generation in these phasic tissues.

Nitric oxide synthase inhibition prevents activity‐induced calcineurin–NFATc1 signalling and fast‐to‐slow skeletal muscle fibre type conversions

The calcineurin–NFAT (nuclear factor of activated T-cells) signalling pathway is involved in the regulation of activity-dependent skeletal muscle myosin heavy chain (MHC) isoform type expression. Emerging evidence indicates that nitric oxide (NO) may play a critical role in this regulatory pathway. Thus, the purpose of this study was to investigate the role of NO in activity-induced calcineurin–NFATc1 signalling leading to skeletal muscle faster-to-slower fibre type transformations in vivo. Endogenous NO production was blocked by administering L-NAME (0.75 mg ml(−1)) in drinking water throughout 0, 1, 2, 5 or 10 days of chronic low-frequency stimulation (CLFS; 10 Hz, 12 h day(−1)) of rat fast-twitch muscles (L+Stim; n = 30) and outcomes were compared with control rats receiving only CLFS (Stim; n = 30). Western blot and immunofluorescence analyses revealed that CLFS induced an increase in NFATc1 dephosphorylation and nuclear localisation, sustained by glycogen synthase kinase (GSK)-3β phosphorylation in Stim, which were all abolished in L+Stim. Moreover, real-time RT-PCR revealed that CLFS induced an increased expression of MHC-I, -IIa and -IId(x) mRNAs in Stim that was abolished in L+Stim. SDS-PAGE and immunohistochemical analyses revealed that CLFS induced faster-to-slower MHC protein and fibre type transformations, respectively, within the fast fibre population of both Stim and L+Stim groups. The final fast type IIA to slow type I transformation, however, was prevented in L+Stim. It is concluded that NO regulates activity-induced MHC-based faster-to-slower fibre type transformations at the transcriptional level via inhibitory GSK-3β-induced facilitation of calcineurin–NFATc1 nuclear accumulation in vivo, whereas transformations within the fast fibre population may also involve translational control mechanisms independent of NO signalling.

Aortic dissection

Aortic dissection is an acute lesion of the aortic wall accompanied by separation of the media due to rupture or intramural hematoma. The incidence rate of aortic dissection is 5 to 30 cases per million people a year. Acute aortic dissection is a highly lethal cardiovascular emergency with an incidence of 2000 new cases per year in the United States and 3000 in Europe. The mortality rate of aortic dissection is 3.2/100 000 per year. In case of sudden death of nonhospitalized patients, aortic dissection was proved in 1.5% of necropsy cases. Most of patients die within 48 hours after admission or 1.4% per each hour. The main clinical manifestations of aortic dissection are acute myocardial infarction, stroke, pulmonary embolism, acute heart failure, acute pancreatitis, mesenteries thrombosis, which mislead the physician. The main measure, which might reduce the mortality, is early diagnosis of aortic dissection. The standard diagnosis is based on clinical symptoms and verification by instrumental (imaging) methods. An alternative mean for diagnosis of aortic dissection might be the determination of concentration of smooth muscle myosin heavy chain protein in blood serum, the peak of which is found after 3 hours after the onset of pain. Normal value of smooth muscle myosin heavy chain protein concentration is 2.5 mg/L, while in case of aortic dissection it exceeds 22.4 mg/L. This diagnostic method has not been introduced in Lithuania yet.

Xin, an actin binding protein, is expressed within muscle satellite cells and newly regenerated skeletal muscle fibers

Xin is a muscle-specific actin binding protein of which its role and regulation within skeletal muscle is not well understood. Here we demonstrate that Xin mRNA is robustly upregulated (>16-fold) within 12 h of skeletal muscle injury and is localized to the muscle satellite cell population. RT-PCR confirmed the expression pattern of Xin during regeneration, as well as within primary muscle myoblast cultures, but not other known stem cell populations. Immunohistochemical staining of single myofibers demonstrate Xin expression colocalized with the satellite cell marker Syndecan-4 further supporting the mRNA expression of Xin in satellite cells. In situ hybridization of regenerating muscle 5-7 days postinjury illustrates Xin expression within newly regenerated myofibers. Promoter-reporter assays demonstrate that known myogenic transcription factors [myocyte enhancer factor-2 (MEF2), myogenic differentiation-1 (MyoD), and myogenic factor-5 (Myf-5)] transactivate Xin promoter constructs supporting the muscle-specific expression of Xin. To determine the role of Xin within muscle precursor cells, proliferation, migration, and differentiation analysis using Xin, short hairpin RNA (shRNA) were undertaken in C2C12 myoblasts. Reducing endogenous Xin expression resulted in a 26% increase (P < 0.05) in cell proliferation and a 20% increase (P < 0.05) in myoblast migratory capacity. Skeletal muscle myosin heavy chain protein levels were increased (P < 0.05) with Xin shRNA administration; however, this was not accompanied by changes in myoglobin protein (another marker of differentiation) nor overt morphological differences relative to differentiating control cells. Taken together, the present findings support the hypothesis that Xin is expressed within muscle satellite cells during skeletal muscle regeneration and is involved in the regulation of myoblast function.

Skeletal muscle myosin heavy chain isoforms and energy metabolism after clenbuterol treatment in the rat.

Prolonged treatment with the beta(2)-adrenoceptor agonist clenbuterol (1-2 mg. kg body mass(-1). day (-1)) is known to induce the hypertrophy of fast-contracting fibers and the conversion of slow- to fast-contracting fibers. We investigated the effects of administering a lower dose of clenbuterol (250 microgram. kg body mass(-1). day (-1)) on skeletal muscle myosin heavy chain (MyHC) protein isoform content and adenine nucleotide (ATP, ADP, and AMP) concentrations. Male Wistar rats were administered clenbuterol (n = 8) or saline (n = 6) subcutaneously for 8 wk, after which the extensor digitorum longus (EDL) and soleus muscles were removed. We demonstrated an increase of type IIa MyHC protein content in the soleus from approximately 0.5% in controls to approximately 18% after clenbuterol treatment (P < 0.05), which was accompanied by an increase in the total adenine nucleotide pool (TAN; approximately 19%, P < 0.05) and energy charge [E-C = (ATP + 0.5 ADP)/(ATP + ADP + AMP); approximately 4%; P < 0.05]. In the EDL, a reduction in the content of the less prevalent type I MyHC protein from approximately 3% in controls to 0% after clenbuterol treatment (P < 0.05) occurred without any alterations in TAN and E-C. These findings demonstrate that the phenotypic changes previously observed in slow muscle after clenbuterol administration at 1-2 mg. kg body mass(-1). day(-1) are also observed at a substantially lower dose and are paralleled by concomitant changes in cellular energy metabolism.

Smooth and Striated Muscle Development in the Intrinsic Urethral Sphincter

Purpose The intrinsic urethral sphincter is composed of adjacent striated and smooth muscle. We studied the sequential expression of smooth and striated muscle proteins to gain insight into the ontogeny of intrinsic sphincter development. Materials and Methods The intrinsic urethral sphincters of timed Fischer 344 rat embryos at 14, 16 and 18 days of gestation, neonates on postnatal day 1 and adult animals were examined. Serial sections of the urethra and adjacent levator ani muscles were studied histologically with hematoxylin and eosin, anti-alpha-smooth muscle actin, anti-alpha-sarcomeric actin and antistriated muscle myosin heavy chain antibodies. Results The intrinsic urethral sphincter was identified within the periurethral mesenchyma as early as day 14 of gestation. Although striated myotubules were identified within the urethra by hematoxylin and eosin staining starting on postnatal day 1, striated muscle myosin heavy chain protein was absent in the embryonic and neonatal development of the sphincter, and it was expressed only in the mature myotubule of adults. alpha-Smooth muscle actin was expressed throughout the urethral sphincter of embryonic and neonatal animals. In adults alpha-smooth muscle actin was confined to the smooth muscle component of the urethra. Co-expression of alpha-smooth and alpha-sarcomeric muscle actin by the striated sphincter myotubule was noted only in neonates. Conclusions Development of the intrinsic urethral sphincter is characterized by sequential expression of well characterized muscle marker proteins. The co-expression of smooth and striated muscle markers by developing sphincter myotubule suggests the possibility that trans-differentiation of smooth to striated muscle occurs in the developing genitourinary tract.

Smooth and striated muscle development in the intrinsic urethral sphincter.

The intrinsic urethral sphincter is composed of adjacent striated and smooth muscle. We studied the sequential expression of smooth and striated muscle proteins to gain insight into the ontogeny of intrinsic sphincter development. The intrinsic urethral sphincters of timed Fischer 344 rat embryos at 14, 16 and 18 days of gestation, neonates on postnatal day 1 and adult animals were examined. Serial sections of the urethra and adjacent levator ani muscles were studied histologically with hematoxylin and eosin, anti-alpha-smooth muscle actin, anti-alpha-sarcomeric actin and antistriated muscle myosin heavy chain antibodies. The intrinsic urethral sphincter was identified within the periurethral mesenchyma as early as day 14 of gestation. Although striated myotubules were identified within the urethra by hematoxylin and eosin staining starting on postnatal day 1, striated muscle myosin heavy chain protein was absent in the embryonic and neonatal development of the sphincter, and it was expressed only in the mature myotubule of adults. alpha-Smooth muscle actin was expressed throughout the urethral sphincter of embryonic and neonatal animals. In adults alpha-smooth muscle actin was confined to the smooth muscle component of the urethra. Co-expression of alpha-smooth and alpha-sarcomeric muscle actin by the striated sphincter myotubule was noted only in neonates. Development of the intrinsic urethral sphincter is characterized by sequential expression of well characterized muscle marker proteins. The co-expression of smooth and striated muscle markers by developing sphincter myotubule suggests the possibility that trans-differentiation of smooth to striated muscle occurs in the developing genitourinary tract.

Interaction of thyroid hormone and functional overload on skeletal muscle isomyosin expression.

This study examined the interaction of exogenous thyroid hormone 3,5,3'-triiodothyronine (T3) and functional overload on skeletal muscle myosin heavy chain (MHC) expression, studied at both the protein and mRNA level of analysis. Animals were allocated to the following groups: 1) normal control, 2) overload control, 3) hyperthyroid control, and 4) hyperthyroid+overload. Overload of the rat plantaris was accomplished by surgical removal of its synergists (soleus and gastrocnemius), and the animals were made hyperthyroid by injections of T3 (350 micrograms/kg every other day). After overload of 8 wk, muscle enlargement occurred by 53% for both overload groups (P < 0.05). This was accompanied by a 330 and 82% increase in the relative content of type I and IIa MHC, respectively, and a corresponding decrease by 16 and 44% in type IIx and IIb MHC, respectively, in the overload control group (P < 0.05 vs. normal control). Changes in the relative and absolute content of mRNA for these MHCs paralleled the protein response. Exogenous T3 completely reversed the upregulation of type I MHC and the downregulation of type IIx associated with overload at both the protein and mRNA level (P < 0.05). However, T3 was only partially effective in blunting the downregulation of IIb MHC and the upregulation of IIa MHC (protein and mRNA) accompanying the overload.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoretic separation of rat skeletal muscle myosin heavy-chain isoforms

A new technique for the sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation of rat skeletal muscle myosin heavy-chain (MHC) isoforms is presented. This technique allows for the separation of the four identified MHC isoforms known to be present in adult rat skeletal muscle. These types of MHC are commonly called I, IIa, IIx or IId, and IIb. The procedure can be performed using minigel electrophoresis systems and does not involve preparation of gradient-separating gels or the use of special cooling devices. The procedure accommodates both silver and Coomasie Blue staining. Thus the procedure is simple to perform and highly repeatable, providing high-resolution separation of MHC protein isoforms. The percent composition of the four adult MHCs in rat soleus, medial gastrocnemius, diaphragm, and levator ani muscles by use of this procedure and Coomasie Blue staining is similar to that previously reported. This new technique provides a novel and easy-to-perform method for the separation of rat skeletal muscle MHC isoforms.

Myosin functional domains encoded by alternative exons are expressed in specific thoracic muscles of Drosophila.

The Drosophila 36B muscle myosin heavy chain (MHC) gene has five sets of alternatively spliced exons that encode functionally important domains of the MHC protein and provide a combinatorial potential for expression of as many as 480 MHC isoforms. In this study, in situ hybridization analysis has been used to examine the complexity and muscle specificity of MHC isoform expression in the fibrillar indirect flight muscle (IFM), the tubular direct flight muscles (DFM) and tubular tergal depressor of the trochanter muscle (TDT), and the visceral esophageal muscle in the adult thorax. Our results show that alternative splicing of the MHC gene transcripts is precisely regulated in these thoracic muscles, which express three MHC isoforms. Individual thoracic muscles each express transcripts of only one isoform, as detectable by in situ hybridization. An apparently novel fourth MHC isoform, with sequence homology to the rod but not to the head domain of the 36B MHC, is expressed in two direct flight muscles. These findings form a basis for transgenic experiments designed to analyze the muscle-specific functions of MHC domains encoded by alternative exons.

Alternative myosin hinge regions are utilized in a tissue-specific fashion that correlates with muscle contraction speed.

By comparing the structure of wild-type and mutant muscle myosin heavy chain (MHC) genes of Drosophila melanogaster, we have identified the defect in the homozygous-viable, flightless mutant Mhc10. The mutation is within the 3' splice acceptor of an alternative exon (exon 15a) that encodes the central region of the MHC hinge. The splice acceptor defect prevents the accumulation of mRNAs containing exon 15a, whereas transcripts with a divergent copy of this exon (exon 15b) are unaffected by the mutation. In situ hybridization and Northern blot analysis of wild-type organisms reveals that exon 15b is used in larval MHCs, whereas exons 15a and/or 15b are used in adult tissues. Because Mhc10 mutants fail to accumulate transcripts encoding MHC protein with hinge region a, analysis of their muscle-specific reduction in thick filament number serves as a sensitive assay system for determining the pattern of accumulation of MHCs with alternative hinge regions. Electron microscopic comparisons of various muscles from wild-type and Mhc10 adults reveals that those that contract rapidly or develop high levels of tension utilize only hinge region a, those that contract at moderate rates accumulate MHCs of both types, and those that are slowly contracting have MHCs with hinge region b. The presence of alternative hinge-coding exons and their highly tissue-specific usage suggests that this portion of the MHC molecule is important to the isoform-specific properties of MHC that lead to the different physiological and ultrastructural characteristics of various Drosophila muscle types. The absence of other alternative exons in the rod-coding region, aside from those shown previously to encode alternative carboxyl termini, demonstrates that the bulk of the myosin rod is not involved in the generation of isoform-specific properties of the MHC molecule.

Ultrastructural and molecular analyses of homozygous-viable Drosophila melanogaster muscle mutants indicate there is a complex pattern of myosin heavy-chain isoform distribution.

We describe the ultrastructural and initial molecular characterization of four homozygous-viable, dominant-flightless mutants of Drosophila melanogaster. Genetic mapping indicates that these mutations are inseparable from the known muscle myosin heavy-chain (MHC) allele Mhc1, and each mutation results in a muscle-specific reduction in MHC protein accumulation. The indirect flight muscles (IFMs) of each of these homozygous mutants fail to accumulate MHC, lack thick filaments, and do not display normal cylindrical myofibrils. As opposed to the null phenotype observed in the IFM, normal amounts of MHC accumulate in the leg muscles of three of these mutants, whereas the fourth mutant shows a 45% reduction in leg muscle MHC. The ultrastructure of the tergal depressor of the trochanter muscle TDT, or jump muscle) is normal in one mutant, completely lacks thick filaments in a second mutant, and displays a reduction of thick filaments in two mutants. The thick filament reduction in this latter class of mutants is limited to the four smaller anterior cells of the TDT, indicating that the TDT is a mixed fiber-type muscle. Because all isoforms of muscle MHC are encoded by alternative splicing of transcripts from a single gene, our results suggest that there is a complex pattern of MHC isoform accumulation in Drosophila. The phenotypes of the homozygous-viable mutants provide evidence for the differential localization of MHC isoforms in different muscles, within the same muscle, and even within a single muscle cell. The mutant characteristics also suggest that the use of some alternative exons is shared among the IFM, TDT, and additional muscles whereas the use of others is unique to the IFM.

Functional Domains of the Drosophila melanogaster Muscle Myosin Heavy-Chain Gene Are Encoded by Alternatively Spliced Exons

The single-copy Drosophila muscle myosin heavy-chain (MHC) gene, located at 36B(2L), has a complex exon structure that produces a diversity of larval and adult muscle MHC isoforms through regulated alternative RNA splicing. Genomic and cDNA sequence analyses revealed that this 21-kilobase MHC gene encodes these MHC isoforms in 19 exons. However, five sets of these exons, encoding portions of the S1 head and the hinge domains of the MHC protein, are tandemly repeated as two, three, four, or five divergent copies, which are individually spliced into RNA transcripts. RNA hybridization studies with exon-specific probes showed that at least 10 of the 480 possible MHC isoforms that could arise by alternative RNA splicing of these exons are expressed as MHC transcripts and that the expression of specific members of alternative exon sets is regulated, both in stage and in muscle-type specificity. This regulated expression of specific exons is of particular interest because the alternatively spliced exon sets encode discrete domains of the MHC protein that likely contribute to the specialized contractile activities of different Drosophila muscle types. The alternative exon structure of the Drosophila MHC gene and the single-copy nature of this gene in the Drosophila genome make possible transgenic experiments to test the physiological functions of specific MHC protein domains and genetic and molecular experiments to investigate the mechanisms that regulate alternative exon splicing of MHC and other muscle gene transcripts.

Functional domains of the Drosophila melanogaster muscle myosin heavy-chain gene are encoded by alternatively spliced exons.

The single-copy Drosophila muscle myosin heavy-chain (MHC) gene, located at 36B(2L), has a complex exon structure that produces a diversity of larval and adult muscle MHC isoforms through regulated alternative RNA splicing. Genomic and cDNA sequence analyses revealed that this 21-kilobase MHC gene encodes these MHC isoforms in 19 exons. However, five sets of these exons, encoding portions of the S1 head and the hinge domains of the MHC protein, are tandemly repeated as two, three, four, or five divergent copies, which are individually spliced into RNA transcripts. RNA hybridization studies with exon-specific probes showed that at least 10 of the 480 possible MHC isoforms that could arise by alternative RNA splicing of these exons are expressed as MHC transcripts and that the expression of specific members of alternative exon sets is regulated, both in stage and in muscle-type specificity. This regulated expression of specific exons is of particular interest because the alternatively spliced exon sets encode discrete domains of the MHC protein that likely contribute to the specialized contractile activities of different Drosophila muscle types. The alternative exon structure of the Drosophila MHC gene and the single-copy nature of this gene in the Drosophila genome make possible transgenic experiments to test the physiological functions of specific MHC protein domains and genetic and molecular experiments to investigate the mechanisms that regulate alternative exon splicing of MHC and other muscle gene transcripts.