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PubMed2005

Remote control of A-band cardiac thin filaments by the I-Z-I protein network of cardiac sarcomeres.

A conventional view of the role of sarcomeric thin filaments in cardiac function is that they react with cross-bridges that translate them toward the center of the sarcomere in a reaction triggered by Ca(2+) and powered by ATP. However, thin filaments also engage in a complex network of protein-protein interactions in the Z-disc. Thus, in the modern context, understanding of what thin filaments do in the heart must take into account not only A-band regions that react with cross-bridges, but also I-Z-I regions that rarely, if ever, react with cross-bridges and dwell near and within the Z-disc. To highlight these multiplex functions of the thin filament, I discuss the hypothesis that physical and chemical reactions at the interface of the thin filaments with Z-disc proteins control the docking and activity of kinases and phosphatases that control the levels of phosphorylation of thin filament regulatory proteins. Testing this hypothesis has taken on new significance with the identification of multisite phosphorylation of thin filament proteins as a critical element in the control of cardiac contraction and relaxation reserve and in maladaptive mechanisms in heart failure. Moreover, multiple mutations in Z-disc proteins that link to prevalent cardiomyopathies are likely to alter this remote control of A-band thin filament function.

Actin Cytoskeleton↗
PubMed2006

The sarcomeric Z-disc: a nodal point in signalling and disease.

The perception of the Z-disc in striated muscle has undergone significant changes in the past decade. Traditionally, the Z-disc has been viewed as a passive constituent of the sarcomere, which is important only for the cross-linking of thin filaments and transmission of force generated by the myofilaments. The recent discovery of multiple novel molecular components, however, has shed light on an emerging role for the Z-disc in signal transduction in both cardiac and skeletal muscles. Strikingly, mutations in several Z-disc proteins have been shown to cause cardiomyopathies and/or muscular dystrophies. In addition, the elusive cardiac stretch receptor appears to localize to the Z-disc. Various signalling molecules have been shown to interact with Z-disc proteins, several of which shuttle between the Z-disc and other cellular compartments such as the nucleus, underlining the dynamic nature of Z-disc-dependent signalling. In this review, we provide a systematic view on the currently known Z-disc components and the functional significance of the Z-disc as an interface between biomechanical sensing and signalling in cardiac and skeletal muscle functions and diseases.

Actin Cytoskeleton↗
PubMed1992

Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle, in vivo and in vitro.

We studied the development of transverse (T)-tubules and sarcoplasmic reticulum (SR) in relationship to myofibrillogenesis in normal and dysgenic (mdg/mdg) mouse skeletal muscle by immunofluorescent labeling of specific membrane and myofibrillar proteins. At E16 the development of the myofibrils and membranes in dysgenic and normal diaphragm was indistinguishable, including well developed myofibrils, a delicate network of T-tubules, and a prominent SR which was not yet cross-striated. In diaphragms of E18 dysgenic mice, both the number and size of muscle fibers and myofibrillar organization were deficient in comparison to normal diaphragms, as previously reported. T-tubule labeling was abnormal, showing only scattered tubules and fragments. However, many muscle fibers displayed cross striation of sarcomeric proteins and SR comparable to normal muscle. In cultured myotubes, cross-striated organization of sarcomeric proteins proceeded essentially in two stages: first around the Z-line and later in the A-band. Sarcomeric organization of the SR coincided with the first stage, while the appearance of T-tubules in the mature transverse orientation occurred infrequently, only after A-band maturation. In culture, myofibrillar and membrane organization was equivalent in normal and dysgenic muscle at the earlier stage of development, but half as many dysgenic myotubes reached the later stage as compared to normal. We conclude that the mdg mutation has little effect on the initial stage of membrane and myofibril development and that the deficiencies often seen at later stages result indirectly from the previously described absence of dihydropyridine receptor function in the mutant.

Animals↗
PubMed1999

A nemaline myopathy mutation in alpha-tropomyosin causes defective regulation of striated muscle force production.

Nemaline myopathy (NM) is a rare autosomal dominant skeletal muscle myopathy characterized by severe muscle weakness and the subsequent appearance of nemaline rods within the muscle fibers. Recently, a missense mutation inTPM3, which encodes the slow skeletal alpha-tropomyosin (alphaTm), was linked to NM in a large kindred with an autosomal-dominant, childhood-onset form of the disease. We used adenoviral gene transfer to fully differentiated rat adult myocytes in vitro to determine the effects of NM mutant human alphaTm expression on striated muscle sarcomeric structure and contractile function. The mutant alphaTm was expressed and incorporated correctly into sarcomeres of adult muscle cells. The primary defect caused by expression of the mutant alphaTm was a decrease in the sensitivity of contraction to activating Ca(2+), which could help explain the hypotonia seen in NM. Interestingly, NM mutant alphaTm expression did not directly result in nemaline rod formation, which suggests that rod formation is secondary to contractile dysfunction and that load-dependent processes are likely involved in nemaline rod formation in vivo.

Adenoviridae↗
PubMed2001

Decoy oligonucleotide characterization of GATA-4 transcription factor in hypertrophic agonist induced responses of cardiac myocytes.

GATA-4 transcription factor is required for normal cardiac development. However, it is unknown whether GATA-4 is an essential mediator of hypertrophic responses in the heart. Rat B-type natriuretic peptide (BNP) gene promoter contains a region of two adjacent GATA binding sites (between -68 and -97) with high affinity for GATA-4. In order to block GATA-4 dependent signaling in cultured neonatal rat ventricular myocytes we administered a synthetic 30-bp phosphorothioated double-stranded DNA complementary to the rat BNP promoter region (between -68 and -97) as a "decoy" cis-element to bind GATA-4. GATA decoy oligodeoxynucleotide treatment of cardiomyocytes blocked GATA-4 DNA binding activity in electrophoretic mobility shift analysis and decreased baseline expression of cardiac natriuretic peptides and GATA-dependent promoter activity. In contrast, blocked GATA-4 DNA binding did not prevent endothelin-1 or phenylephrine induced expression of cardiac natriuretic peptides. Mutation of GATA binding sites at -80 and -91 rat BNP promoter downregulated baseline but did not affect endothelin-1 or angiotensin II induced promoter activity. Additively, GATA decoy oligodeoxynucleotide treatment was insufficient to block endothelin-1 induced activation of protein synthesis or sarcomeric protein assembly. In conclusion, a targeted disruption of GATA-4 DNA binding activity is insufficient to prevent hypertrophic agonist induced responses of ventricular myocytes.

Angiotensin II↗
PubMed2003

Familial hypertrophic cardiomyopathy-linked alterations in Ca2+ binding of human cardiac myosin regulatory light chain affect cardiac muscle contraction.

The ventricular isoform of human cardiac regulatory light chain (HCRLC) has been shown to be one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular and/or septal hypertrophy, myofibrillar disarray, and sudden cardiac death. Our recent studies have demonstrated that the properties of isolated HCRLC could be significantly altered by the FHC mutations and that their detrimental effects depend upon the specific position of the missense mutation. This report reveals that the Ca(2+) sensitivity of myofibrillar ATPase activity and steady-state force development are also likely to change with the location of the specific FHC HCRLC mutation. The largest effect was seen for the two FHC mutations, N47K and R58Q, located directly in or near the single Ca(2+)-Mg(2+) binding site of HCRLC, which demonstrated no Ca(2+) binding compared with wild-type and other FHC mutants (A13T, F18L, E22K, P95A). These two mutants when reconstituted in porcine cardiac muscle preparations increased Ca(2+) sensitivity of myofibrillar ATPase activity and force development. These results suggest the importance of the intact Ca(2+) binding site of HCRLC in the regulation of cardiac muscle contraction and imply its possible role in the regulatory light chain-linked pathogenesis of FHC.

Adenosine Triphosphatases↗
PubMed2005

The Z-disc proteins myotilin and FATZ-1 interact with each other and are connected to the sarcolemma via muscle-specific filamins.

Myotilin and the calsarcin family member FATZ-1 (also called calsarcin-2 or myozenin-1) are recently discovered sarcomeric proteins implicated in the assembly and stabilization of the Z-discs in skeletal muscle. The essential role of myotilin in skeletal muscle is attested by the observation that certain forms of myofibrillar myopathy and limb girdle muscular dystrophy are caused by mutations in the human myotilin gene. Here we show by transfection, biochemical and/or yeast two-hybrid assay that: (1) myotilin is able to interact with the C-terminal region of FATZ-1 and that the N- or C-terminal truncations of myotilin abrogate binding; (2) myotilin can also interact with another calsarcin member, FATZ-2 (calsarcin-1, myozenin-2); (3) myotilin and FATZ-1 bind not only to the C-terminal region of filamin-C containing the Ig repeats 19-24, but also to the other two filamins, filamin-A and filamin-B, as well as the newly identified filamin-Bvar-1variant; (4) the binding of myotilin to filamin-C involves binding sites in its N-terminal region, whereas FATZ-1 associates with filamin-C via sequences within either its N- or C-terminal region; and finally, (5) the C-terminal region of filamin-C like filamin-B and filamin-Bvar-1, shows binding activity with the beta1A integrin subunit. Our findings further dissect the molecular interactions within the Z-disc that are essential for its organization, and provide evidence for a novel connection between Z-disc proteins and the sarcolemma via filamins and beta1 integrins. These data shed new light on the complex organization of the Z-disc that is highly relevant to understanding muscular dystrophies.

Animals↗
PubMed2003

Regulatory light chains of striated muscle myosin. Structure, function and malfunction.

Striated (skeletal and cardiac) muscle is activated by the binding of Ca(2+) to troponin C and is regulated by the thin filament proteins, tropomyosin and troponin. Unlike in molluscan or smooth muscles, the myosin regulatory light chains (RLC) of striated muscles do not play a major regulatory role and their function is still not well understood. The N-terminal domain of RLC contains a 'Ca(2+)-Mg(2+)'-binding site and, analogous to that of smooth muscle myosin, also contains a phosphorylation site. During muscle contraction, the increase in Ca(2+) concentration activates the Ca(2+)/calmodulin-dependent myosin light chain kinase and leads to phosphorylation of the RLC. In agreement with other laboratories we have demonstrated that phosphorylation and Ca(2+) binding to the RLC play an important modulatory role in striated muscle contraction. Furthermore, the ventricular isoform of human cardiac RLC has been shown to be one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray and sudden cardiac death. Our recent studies have demonstrated that phosphorylation and Ca(2+) binding to human ventricular RLC are significantly altered by the FHC mutations and that their detrimental effects depend upon the specific position of the missense mutation, whether located in the proximity of the RLC 'Ca(2+)-Mg(2+)'-binding site or the phosphorylation site (Serine 15). We have also shown that there is a functional coupling between Ca(2+) and/or Mg(2+) binding to the RLC and phosphorylation and that the FHC mutations can affect this relationship. Further in vivo studies are necessary to investigate the mechanisms involved in the pathogenesis of RLC-linked FHC.

Amino Acid Sequence↗
PubMed1989

Developmental regulation of intermediate filament and actin mRNAs during myogenesis is disrupted by oncogenic ras genes.

Terminal differentiation of skeletal myoblasts is accompanied by down-regulation of vimentin and beta-, gamma-actins and up-regulation of desmin and sarcomeric alpha-actin(s). To investigate whether the normal decline in expression of vimentin and beta-, gamma-actins was coupled to withdrawal of proliferating myoblasts from the cell cycle or was a direct consequence of terminal differentiation, expression of the mRNAs encoding the actin microfilaments and intermediate filaments was examined in differentiation-defective C2 myoblasts bearing oncogenic ras genes. When transferred to mitogen-deficient medium, myoblasts transfected with the valine-12 allele of the human Harvey (H)-ras gene ceased dividing but failed to appropriately down-regulate vimentin and beta-, gamma-actin mRNAs. On the contrary, the level of vimentin mRNA expression was increased about 2.5-fold. Conversely, alpha-sarcomeric actin and desmin mRNAs continued to be expressed at basal levels in ras-transfected myoblasts that had withdrawn from the cell cycle. The ability of the ras oncogene to interfere with developmental regulation of actin and intermediate filament mRNAs was dependent upon mutational activation and was not observed in myoblasts transfected with proto-oncogenic ras alleles. These results demonstrate that, in addition to preventing up-regulation of muscle-specific gene products during myogenesis, the oncogenic forms of ras interfere with the mechanism(s) responsible for down-regulation of genes whose expression declines during myoblast fusion.

Actins↗
PubMed1996

Ablation of the murine alpha myosin heavy chain gene leads to dosage effects and functional deficits in the heart.

The alpha-myosin heavy chain (alpha-MyHC) is the major contractile protein expressed in the myocardium of adult mice. We have produced mice carrying a null mutation of alpha-MyHC by homologous recombination in murine ES cells. Homozygous null animals die between 11 and 12 d in utero of gross heart defects, while alpha-MyHC+/- heterozygotes survive and appear externally normal. The presence of a single functional alpha-MyHC+ allele in heterozygous animals results in reduced levels of the transcript and protein as well as fibrosis and alterations in sarcomeric structure. Examination of heart function using a working heart preparation revealed severe impairment of both contractility and relaxation in a subset of the alpha-MyHC+/- animals. Thus, two alpha-MyHC+ alleles are necessary for normal cardiac development, and hemizygosity for the normal allele can result in altered cardiac function.

Alleles↗
PubMed2005

Calcineurin function is required for myofilament formation and troponin I isoform transition in Drosophila indirect flight muscle.

Mutations in the Drosophila calcineurin B2 gene cause the collapse of indirect flight muscles during mid stages of pupal development. Examination of cell fate-specific markers indicates that unlike mutations in genes such as vestigial, calcineurin B2 does not cause a shift in cell fate from indirect flight muscle to direct flight muscle. Genetic and molecular analyses indicate a severe reduction of myosin heavy chain gene expression in calcineurin B2 mutants, which accounts at least in part for the muscle collapse. Myofibrils in calcineurin B2 mutants display a variety of phenotypes, ranging from normal to a lack of sarcomeric structure. Calcineurin B2 also plays a role in the transition to an adult-specific isoform of troponin I during the late pupal stages, although the incompleteness of this transition in calcineurin B2 mutants does not contribute to the phenotype of muscle collapse. Together, these findings suggest a molecular basis for the indirect flight muscle hypercontractility phenotype observed in flies mutant for Drosophila calcineurin B2.

Actin Cytoskeleton↗
PubMed2006

Cardiac myosin binding protein C phosphorylation is cardioprotective.

Cardiac myosin binding protein C (cMyBP-C) has three phosphorylatable serines at its N terminus (Ser-273, Ser-282, and Ser-302), and the residues' phosphorylation states may alter thick filament structure and function. To examine the effects of cMyBP-C phosphorylation, we generated transgenic mice with cardiac-specific expression of a cMyBP-C in which the three phosphorylation sites were mutated to aspartic acid, mimicking constitutive phosphorylation (cMyBP-C(AllP+)). The allele was bred into a cMyBP-C null background (cMyBP-C((t/t))) to ensure the absence of endogenous dephosphorylated cMyBP-C. cMyBP-C(AllP+) was incorporated normally into the cardiac sarcomere and restored normal cardiac function in the null background. However, subtle changes in sarcomere ultrastructure, characterized by increased distances between the thick filaments, indicated that phosphomimetic cMyBP-C affects thick-thin filament relationships, and yeast two-hybrid data and pull-down studies both showed that charged residues in these positions effectively prevented interaction with the myosin heavy chain. Confirming the physiological relevance of these data, the cMyBP-C(AllP+:(t/t)) hearts were resistant to ischemia-reperfusion injury. These data demonstrate that cMyBP-C phosphorylation functions in maintaining thick filament spacing and structure and can help protect the myocardium from ischemic injury.

Amino Acid Sequence↗
PubMed2004

Multi-minicore disease revisited.

Multi-minicore disease (MmD) is an infrequent congenital myopathy, defined by structural changes in optic and electron microscopy, namely, multiple small areas lacking oxidative enzyme activity and focal disorganization of contractile proteins involving at most a few sarcomeres. The classical form of the disease manifests as more or less severe hypotonia and generalized weakness with predominance in axial and proximal limb muscles. Clinical variants also exist. Usually MmD is inherited as an autosomal recessive trait. Genetic heterogeneity is recognized and up to now mutations in the genes of RYR1 and SEPN1 have been detected. We record three unrelated cases of MmD. Case 1, with the classical benign form, was followed-up for 15 years. Case 2, presenting pharyngolaryngeal involvement and severe delay of head control, improved gradually, until independent gait was acquired at age of six years. A moderate restriction of daily life activities remains. Case 3, of antenatal-onset, was expressed by arthrogryposis of hands, predominance of scapular girdle deficit and a stable course after ten years on physiotherapy. All cases were selected by the characteristic morphological abnormalities in biceps brachii samples, including electron microscopy. Emphasis is given to case 2 due to type 1 fiber uniformity and mild endomysial fibrosis, posing a difficult differential diagnosis with congenital muscular dystrophy were it not for the significant number of multi-minicores.

Adolescent↗
PubMed1988

A common factor regulates skeletal and cardiac alpha-actin gene transcription in muscle.

The skeletal and cardiac alpha-actin genes are coexpressed in muscle development but exhibit distinctive tissue-specific patterns of expression. We used an in vivo competition assay and an in vitro electrophoretic mobility shift assay to demonstrate that both genes interact with a common trans-acting factor(s). However, there was at least one gene-specific cis-acting sequence in the skeletal alpha-actin gene that interacted with a trans-acting factor which was not rate limiting in the expression of the cardiac alpha-actin gene. The common factor(s) interacted with several cis-acting regions that corresponded to sequences that are required for the transcriptional modulation of these sarcomeric alpha-actin genes in muscle cells. These regulatory regions contained the sequence motif CC(A + T-rich)6GG, which is known as a CArG box. Results of in vivo competition assays demonstrated that the factor(s) bound by the skeletal alpha-actin gene is also essential for the maximal activity of the cardiac alpha-actin, simian virus 40 (SV40), alpha 2(I)-collagen, and the beta-actin promoters in muscle cells. In contrast, fibroblastic cells contained functionally distinct transcription factor(s) that were used by the SV40 enhancer but that did not interact with the sarcomeric alpha-actin cis-acting sequences. The existence of functionally different factors in these cell types may explain the myogenic specificity of these sarcomeric alpha-actin genes. Results of in vitro studies suggested that both the sarcomeric alpha-actin genes interact with the CArG box-binding factor CBF and that the skeletal alpha-actin promoter contains multiple CBF-binding sites. In contrast, CBF did not interact in vitro with a classical CAAT box, the SV40 enhancer, or a linker scanner mutation of an alpha-actin CArG box. Furthermore, methylation interference and DNase I footprinting assays demonstrated the precise sites of interaction of CBF with three CArG motifs at positions -98, -179, and -225 in the human skeletal alpha-actin gene.

Actins↗
PubMed2005

Increase in tension-dependent ATP consumption induced by cardiac troponin T mutation.

How different mutations in cardiac troponin T (cTnT) lead to distinct secondary downstream cellular remodeling in familial hypertrophic cardiomyopathy (FHC) remains elusive. To explore the molecular basis for the distinct impact of different mutations in cTnT on cardiac myocytes, we studied mechanical activity of detergent-skinned muscle fiber bundles from different lines of transgenic (TG) mouse hearts that express wild-type cTnT (WTTG), R92W cTnT, R92L cTnT, and Delta-160 cTnT (deletion of amino acid 160). The amount of mutant cTnT is approximately 50% of the total myocellular cTnT in both R92W and R92L TG mouse hearts and approximately 35% in Delta-160 TG mouse hearts. Myofilament Ca2+ sensitivity was enhanced in all mutant cTnT TG cardiac muscle fibers. Compared with the WTTG fibers, Ca2+ sensitivity increased significantly at short sarcomere length (SL) of 1.9 microm (P < 0.001) in R92W TG fibers by 2.2-fold, in R92L by 2.0-fold, and in Delta-160 by 1.3-fold. At long SL of 2.3 microm, Ca2+ sensitivity increased significantly (P < 0.01) in a similar manner (R92W, 2.5-fold; R92L, 1.9-fold; Delta-160, 1.3-fold). Ca2+-activated maximal tension remained unaltered in all TG muscle fibers. However, tension-dependent ATP consumption increased significantly in Delta-160 TG muscle fibers at both short SL (23%, P < 0.005) and long SL (37%, P < 0.0001), suggesting a mutation-induced change in cross-bridge detachment rate constant. Chronic stresses on relative cellular ATP level in cardiac myocytes may cause a strain on energy-dependent Ca2+ homeostatic mechanisms. This may result in pathological remodeling that we observed in Delta-160 TG cardiac myocytes where the ratio of sarco(endo)plasmic reticulum Ca2+-ATPase 2/phospholamban decreased significantly. Our results suggest that different types of stresses imposed on cardiac myocytes would trigger distinct cellular signaling, which leads to remodeling that may be unique to some mutants.

Adenosine Triphosphate↗
PubMed2026

Functional Validation of a Novel Homozygous TTN Splice-Site Variant Reveals Aberrant Splicing in Hypertrophic Cardiomyopathy.

The TTN gene encodes a crucial structural protein within cardiac sarcomeres, and its variants may contribute to hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy; however, phenotype and genotype are different. Whole-exome sequencing (WES) was conducted on a Chinese proband diagnosed with HCM. In silico splicing prediction tools and minigene assays were employed to investigate the impact of the identified variant on mRNA splicing. A literature review was performed to retrieve and analyze previously reported splicing variants in the TTN gene associated with HCM. A 42-year-old male proband presented with nonobstructive HCM and paroxysmal atrial arrhythmias. A novel homozygous TTN variant was found, predicted to cause a 14-base pair deletion at a splice acceptor site. Two asymptomatic offspring were found to carry the heterozygous variant. Based on variant interpretation guidelines, the variant met the PM2_supporting criterion and was classified as a variant of uncertain significance (VUS). The predicted aberrant splicing effect was subsequently confirmed by the minigene splicing assay, demonstrating altered pre-mRNA splicing leading to an in-frame insertion/deletion (p.Arg32498_Glu32504delinsGln). Functional verification confirmed that this mutation conforms to the PM4 criterion, suggesting that it can be reclassified as a tepid VUS (scoring 3 points). The functional result might provide pathogenic evidence. We also reviewed 28 previously reported splicing variants in TTN associated with HCM. Of these, 57.1% (16/28) localized to the I-band region, whereas 21.4% (6/28) were situated in the A-band domain of titin. Notably, 21.4% (6/28) co-occurred with pathogenic variants in other sarcomeric genes (MYH7 or MYBPC3), correlating with more severe clinical phenotypes. Reclassification and reinterpretation of the variants revealed that none met the level of likely pathogenic or higher. The present case contributes a homozygous splice-site variant with experimentally confirmed aberrant splicing and an in-frame protein alteration in titin. The focus of this report is the Mendelian genetic basis of the proband's cardiomyopathy phenotype, and our data provide additional case-level and functional evidence for a possible role of specific TTN splicing defects in HCM.

Humans↗
PubMed2004

Complete genomic structure of the human nebulin gene and identification of alternatively spliced transcripts.

The giant nebulin protein is a fundamental structural component of the thin filaments of the striated muscle sarcomere. Nebulin binds to actin and the size of nebulin correlates with actin filament length, suggesting that nebulin may determine the length of the thin filaments during myofibrillogenesis. We have previously described the genomic organization of the 3' end of the nebulin gene (NEB), and identified 18 different NEB mutations in patients with autosomal recessive nemaline myopathy. Here we present the genomic organization of the entire nebulin gene, and the identification of numerous alternatively spliced mRNAs. The gene comprises 183 exons spanning 249 kb of the genomic sequence. The translation initiation codon is in exon 3, and the stop codon and the 3' UTR are in exon 183. There are four regions with alternatively spliced exons, that is, exons 63-66, 82-105, 143-144 and 166-177, giving rise to a number of different transcripts. The alternatively spliced exons 143-144 give rise to two different transcripts varying between muscle types and between muscles of different developmental stages. The alternatively spliced exons 166-177 express at least 20 different transcripts in adult human tibialis anterior muscle alone. Preliminary results show several transcripts in both of the two remaining alternatively spliced regions. Extensive alternative splicing of NEB may explain why nemaline myopathy patients with homozygous truncating mutations show expression of the carboxy-terminus of the nebulin protein contrary to expectations. The use of alternative transcripts might also explain why severe phenotypes are rare among patients with two truncating mutations.

Alternative Splicing↗
PubMed1992

Myosin light chain-2 mutation affects flight, wing beat frequency, and indirect flight muscle contraction kinetics in Drosophila.

We have used a combination of classical genetic, molecular genetic, histological, biochemical, and biophysical techniques to identify and characterize a null mutation of the myosin light chain-2 (MLC-2) locus of Drosophila melanogaster. Mlc2E38 is a null mutation of the MLC-2 gene resulting from a nonsense mutation at the tenth codon position. Mlc2E38 confers dominant flightless behavior that is associated with reduced wing beat frequency. Mlc2E38 heterozygotes exhibit a 50% reduction of MLC-2 mRNA concentration in adult thoracic musculature, which results in a commensurate reduction of MLC-2 protein in the indirect flight muscles. Indirect flight muscle myofibrils from Mlc2E38 heterozygotes are aberrant, exhibiting myofilaments in disarray at the periphery. Calcium-activated Triton X-100-treated single fiber segments exhibit slower contraction kinetics than wild type. Introduction of a transformed copy of the wild type MLC-2 gene rescues the dominant flightless behavior of Mlc2E38 heterozygotes. Wing beat frequency and single fiber contraction kinetics of a representative rescued line are not significantly different from those of wild type. Together, these results indicate that wild type MLC-2 stoichiometry is required for normal indirect flight muscle assembly and function. Furthermore, these results suggest that the reduced wing beat frequency and possibly the flightless behavior conferred by Mlc2E38 is due in part to slower contraction kinetics of sarcomeric regions devoid or partly deficient in MLC-2.

Allosteric Regulation↗