Muscle abnormalities in Drosophila melanogaster heldup mutants are caused by missing or aberrant troponin-I isoforms

We have investigated the molecular bases of muscle abnormalities in four Drosophila melanogaster heldup mutants. We find that the heldup gene encodes troponin-I, one of the principal regulatory proteins associated with skeletal muscle thin filaments. heldup3, heldup4, and heldup5 mutants, all of which have grossly abnormal flight muscle myofibrils, lack mRNAs encoding one or more troponin-I isoforms. In contrast, heldup2, an especially interesting mutant wherein flight muscles are atrophic, synthesizes the complete mRNA complement. By sequencing mutant troponin-I cDNAs we demonstrate that the molecular basis for muscle degeneration in heldup2 is conversion of an invariant alanine residue to valine. We finally show that degeneration of heldup2 thin filament/Z-disc networks can be prevented by eliminating thick filaments from flight muscles using a null allele of the sarcomeric myosin heavy chain gene. This latter observation suggests that actomyosin interactions exacerbate the structural or functional defect resulting from the troponin-I mutation.     

A nonsense mutation within the act88F actin gene disrupts myofibril formation in Drosophila indirect flight muscles

We have investigated the molecular basis of muscle abnormalities in the flightless Drosophila mutant lfm(3)7. This EMS-induced, semi-dominant allele was isolated by Mogami and Hotta (1981) and was shown to disrupt the organization of myofibrils in indirect flight muscles. Here we demonstrate that lfm(3)7 contains a nonsense mutation within codon 355 of the act88F actin gene. A single G greater than A transition converts a tryptophan (TGG) codon to an opal (TGA) terminator, thus deleting the carboxy-terminal 20 amino acids of an actin isoform that accumulates only in thoracic flight muscles. The truncated actin polypeptide is stable, and retains antigenicity to at least two anti-Drosophila actin monoclonal antibodies. We suggest that abnormalities in lfm(3)7 flight muscles result from incorporation of the mutant actin isoform into assembling myofibrils.     

Rediscovery and further characterization of the aeroplane (ae) wing posture mutation in Drosophila melanogaster.

In 1931, Theodore Quelprud characterized a novel spontaneous mutation in Drosophila melanogaster, which was named aeroplane (ae) based on its abnormal wing posture. Although the characterization of the original ae locus was minimal, it is very likely that another allele of this extinct mutation has now been identified. aeroplane-like (ae-l) was isolated as a by-product of a transformation experiment. The apparent wing paralysis is not caused by any obvious abnormalities in the thorax, wing, indirect flight muscles or direct flight muscles. Classical genetic complementation analyses of ae-l with other genes in the region suggest that it represents an allele of a novel locus. Unexpectedly, a molecular examination revealed that the physical lesion identified in the ae-l mutant is exceptionally close to the homeotic gene teashirt (tsh) and, indeed, may represent an unusual allele of teashirt.     

A direct screen identifies new flight muscle mutants on the Drosophila second chromosome.

An ethyl methanesulfonate mutagenesis of Drosophila melanogaster was undertaken, and >3000 mutagenized second chromosomes were generated. More than 800 homozygous viable lines were established, and adults were screened directly under polarized light for muscle defects. A total of 16 mutant strains in which the indirect flight muscles were reduced in volume or disorganized or were otherwise abnormal were identified. These fell into seven recessive and one semidominant complementation groups. Five of these eight complementation groups, including the semidominant mutation, have been mapped using chromosomal deficiencies and meiotic recombination. Two complementation groups mapped close to the Myosin heavy chain gene, but they are shown to be in different loci. Developmental analysis of three mutations showed that two of these are involved in the early stages of adult myogenesis while the other showed late defects. This is the first report of results from a systematic and direct screen for recessive flight muscle defects. This mutant screen identifies genes affecting the flight muscles, which are distinct from those identified when screening for flightlessness.     

Dense cataract and microphthalmia--new spontaneous mutation in BALB/c mice

We describe a new spontaneous mutation in BALB/c mice that causes abnormal phenotype, such as congenital cataract and microphthalmia. This abnormality was found to be inheritable because offspring with the same abnormality were produced by backcrossing the abnormal male to its normal female parent. Results of various crosses made to determine the mode of inheritance indicated that this abnormality is attributable to mutation of an autosomal recessive gene. Slit lamp examination of the mutant eyes revealed total lenticular opacity, disturbed typical iris pattern, and abnormal pupillary muscle development. Histologic changes in mutant eyes between gestation day 13 and postnatal day 1 indicated various eye and lens abnormalities, including microphthalmia; underdeveloped iris, optic stalk, cornea, and retina; degenerated lens fibers with lost fibrillar structure; and vacuoles of various sizes at the posterior border of the lens. Mild opacity of the lens was found to progress with age and became denser, resembling mature cataract, and occupying the lens completely at the age of six to eight weeks. We, therefore, temporarily designated this abnormality as dense cataract and microphthalmia, with the gene symbol dcm.     

Defective Mitochondrial Dynamics Is an Early Event in Skeletal Muscle of an Amyotrophic Lateral Sclerosis Mouse Model

Mitochondria are dynamic organelles that constantly undergo fusion and fission to maintain their normal functionality. Impairment of mitochondrial dynamics is implicated in various neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS) is an adult-onset neuromuscular degenerative disorder characterized by motor neuron death and muscle atrophy. ALS onset and progression clearly involve motor neuron degeneration but accumulating evidence suggests primary muscle pathology may also be involved. Here, we examined mitochondrial dynamics in live skeletal muscle of an ALS mouse model (G93A) harboring a superoxide dismutase mutation (SOD1^G93A). Using confocal microscopy combined with overexpression of mitochondria-targeted photoactivatable fluorescent proteins, we discovered abnormal mitochondrial dynamics in skeletal muscle of young G93A mice before disease onset. We further demonstrated that similar abnormalities in mitochondrial dynamics were induced by overexpression of mutant SOD1^G93A in skeletal muscle of normal mice, indicating the SOD1 mutation drives ALS-like muscle pathology in the absence of motor neuron degeneration. Mutant SOD1^G93A forms aggregates inside muscle mitochondria and leads to fragmentation of the mitochondrial network as well as mitochondrial depolarization. Partial depolarization of mitochondrial membrane potential in normal muscle by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused abnormalities in mitochondrial dynamics similar to that in the SOD1^G93A model muscle. A specific mitochondrial fission inhibitor (Mdivi-1) reversed the SOD1^G93A action on mitochondrial dynamics, indicating SOD1^G93A likely promotes mitochondrial fission process. Our results suggest that accumulation of mutant SOD1^G93A inside mitochondria, depolarization of mitochondrial membrane potential and abnormal mitochondrial dynamics are causally linked and cause intrinsic muscle pathology, which occurs early in the course of ALS and may actively promote ALS progression.     

A single nucleotide deletion in the skeletal muscle-specific calcium channel transcript of muscular dysgenesis (mdg) mice.

The skeletal muscle-specific dihydropyridine-sensitive calcium channel is a critical component of excitation-contraction coupling in skeletal muscle. A recessive mutation in mice, muscular dysgenesis (mdg), has previously been described as resulting in defective excitation-contraction coupling. Although the channel-forming subunit (alpha 1) of the skeletal calcium channel is not detectable immunologically, specific mRNA of normal size is present in dysgenic muscle. cDNA for this calcium channel alpha 1 subunit has now been cloned from dysgenic muscle and sequenced in its entirety. A single nucleotide deletion occurs at nucleotide 4010 of the cDNA, resulting in a shift of the translational reading frame. The mutation has been confirmed by direct sequencing of PCR products from homozygous mutant and normal muscle. The mutant polypeptide is predicted to contain the first three repeating domains, five of the normal six transmembrane helices of the last repeating domain, and an altered and truncated C terminus. The mature mRNA encoding the dysgenic alpha 1 subunit appears to be labile. It is possible that premature termination of translation renders the mutant mRNA subject to degradation by nucleases. This work resolves a long-standing controversy on the nature of the primary genetic defect in muscular dysgenesis.     

Drosophila melanogaster troponin-T mutations engender three distinct syndromes of myofibrillar abnormalities

In vertebrates troponin complexes interact co-operatively with tropomyosin dimers to modulate skeletal muscle contraction. In order further to investigate troponin assembly and function in vivo, we are developing molecular genetic approaches. Here we report characterization of the gene that encodes Drosophila tropinin-T and analyses of muscle defects engendered by several mutant alleles. We found that the Drosophila troponin-T locus specifies at least three proteins having sequences similar to vertebrate troponin-T. All are significantly larger than any avian or mammalian isoforms, however, due to a highly acidic carboxy-terminal extension. Comparisons of the chromosomal arrangements of vertebrate and Drosophila troponin-T genes revealed that the location of one intron-exon boundary is conserved. This observation and the similarity of vertebrate and Drosophila troponin-T primary sequences suggest that the respective proteins are homologous, and that troponin-T pre-dates the divergence of vertebrate and invertebrate organisms. In situ hybridization of the Drosophila troponin-T gene to polytene chromosomes demonstrated that it resides within subdivision 12A of the X chromosome, precisely where upheld and indented thorax flight muscle mutations have been mapped previously. We determined the nucleotide sequences of troponin-T genes in five extant mutants. All have deleterious alterations, directly establishing that upheld and indented thorax muscle abnormalities are due to defective troponin-T. Two of the alleles, upheld2 and upheld3, apparently disrupt RNA splicing and eliminate most or all troponin-T from flight and jump muscles, while the remaining three alleles change the identities of single amino acids of troponin-T. Electron microscopy of mutant muscles revealed that the two null alleles eliminate thin filaments, except where they are bound by electron-dense material presumed to be Z-disc proteins. Two of the point mutations, upheld101 and indented thorax3, do not perturb assembly of myofibrils, but cause their degeneration within days after muscles begin to be utilized. The final mutation, upheldwhu, reduces the diameter of the myofibril lattice by approximately one-half. We propose hypotheses to explain how each troponin-T mutation engenders the observed myofibrillar defects.     

Recovery of Dominant, Autosomal Flightless Mutants of Drosophila Melanogaster and Identification of a New Gene Required for Normal Muscle Structure and Function

To identify further mutations affecting muscle function and development in Drosophila melanogaster we recovered 22 autosomal dominant flightless mutations. From these we have isolated eight viable and lethal alleles of the muscle myosin heavy chain gene, and seven viable alleles of the indirect flight muscle (IFM)-specific Act88F actin gene. The Mhc mutations display a variety of phenotypic effects, ranging from reductions in myosin heavy chain content in the indirect flight muscles only, to reductions in the levels of this protein in other muscles. The Act88F mutations range from those which produce no stable actin and have severely abnormal myofibrillar structure, to those which accumulate apparently normal levels of actin in the flight muscles but which still have abnormal myofibrils and fly very poorly. We also recovered two recessive flightless mutants on the third chromosome. The remaining five dominant flightless mutations are all lethal alleles of a gene named lethal(3)Laker. The Laker alleles have been characterized and the gene located in polytene bands 62A10,B1-62B2,4. Laker is a previously unidentified locus which is haplo-insufficient for flight. In addition, adult wild-type heterozygotes and the lethal larval trans-heterozygotes show abnormalities of muscle structure indicating that the Laker gene product is an important component of muscle.     

Expression of the inclusion body myopathy 3 mutation in Drosophila depresses myosin function and stability and recapitulates muscle inclusions and weakness

Hereditary myosin myopathies are characterized by variable clinical features. Inclusion body myopathy 3 (IBM-3) is an autosomal dominant disease associated with a missense mutation (E706K) in the myosin heavy chain IIa gene. Adult patients experience progressive muscle weakness. Biopsies reveal dystrophic changes, rimmed vacuoles with cytoplasmic inclusions, and focal disorganization of myofilaments. We constructed a transgene encoding E706K myosin and expressed it in Drosophila (E701K) indirect flight and jump muscles to establish a novel homozygous organism with homogeneous populations of fast IBM-3 myosin and muscle fibers. Flight and jump abilities were severely reduced in homozygotes. ATPase and actin sliding velocity of the mutant myosin were depressed >80% compared with wild-type myosin. Light scattering experiments and electron microscopy revealed that mutant myosin heads bear a dramatic propensity to collapse and aggregate. Thus E706K (E701K) myosin appears far more labile than wild-type myosin. Furthermore, mutant fly fibers exhibit ultrastructural hallmarks seen in patients, including cytoplasmic inclusions containing aberrant proteinaceous structures and disorganized muscle filaments. Our Drosophila model reveals the unambiguous consequences of the IBM-3 lesion on fast muscle myosin and fibers. The abnormalities observed in myosin function and muscle ultrastructure likely contribute to muscle weakness observed in our flies and patients.     

Functional recovery of troponin I in a Drosophila heldup mutant after a second site mutation.

To identify proteins that interact in vivo with muscle components we have used a genetic approach based on the isolation of suppressors of mutant alleles of known muscle components. We have applied this system to the case of troponin I (TnI) in Drosophila and its mutant allele heldup2 (hdp2). This mutation causes an alanine to valine substitution at position 116 after a single nucleotide change in a constitutive exon. Among the isolated suppressors, one of them results from a second site mutation at the TnI gene itself. Muscles endowed with TnI mutated at both sites support nearly normal myofibrillar structure, perform notably well in wing beating and flight tests, and isolated muscle fibers produce active force. We show that the structural and functional recovery in this suppressor does not result from a change in the stoichiometric ratio of TnI isoforms. The second site suppression is due to a leucine to phenylalanine change within a heptameric leucine string motif adjacent to the actin binding domain of TnI. These data evidence a structural and functional role for the heptameric leucine string that is most noticeable, if not specific, in the indirect flight muscle.     

Circling mouse: possible animal model for deafness

Mutant mice with abnormalities are potentially useful as models for studying human defects. Here we report a group of mice with abnormal behavioral patterns. A new spontaneous mutant mouse exhibited hyperactive behavior at about seven days of age, followed by tight circling behavior. Breeding studies suggest that this mutation is caused by a single gene defect inherited in an autosomal recessive manner. Consequently, this mutation is referred to as a circling (cir) mouse mutation with the gene symbol cir. Auditory test results identified clearly the hearing loss of the cir, compared with wild-type mice. Pathologic studies confirmed developmental defects in cochlea and spiral ganglions that were correlated to the abnormal behavior observed in the cir mice. Thus, cir mice may be useful as a model for studying inner ear abnormalities and deafness in humans.     

Protein synthesis during flight muscle development in normal and wupB indirect flight muscles of Drosophila melanogaster

We have looked at protein synthesis in Drosophila pupae during normal and abnormal development of indirect flight muscle. Abnormal development was followed in the dominant flightless mutant wupB isolated by Hotta and Benzer (Genetic Mechanisms of Development, pp. 129–167. Academic Press, New York, 1972). The mutant muscles in adult wupB flies have abnormal morphology and disorganized myofibrils. Protein synthesis in developing muscle was followed on SDS-polyacrylamide gels. During early stages of development (55–60 h) protein synthesis patterns are similar in the mutant and the wild-type. However, at 61 h, the mutant shows a transient increase in synthesis of the 68 and 70 kDa heat shock proteins. This is followed at about 70 h by a divergence of the patterns of synthesis of other proteins seen in the mutant and wild type. These results suggest that induction of heat shock protein synthesis is an early event in abnormal morphogenesis in this mutant.     

A mutation in serca underlies motility dysfunction in accordion zebrafish

Zebrafish acquire the ability for fast swimming early in development. The motility mutant accordion (acc) undergoes exaggerated and prolonged contractions on both sides of the body, interfering with the acquisition of patterned swimming responses. Our whole cell recordings from muscle indicate that the defect is not manifested in neuromuscular transmission. However, imaging of skeletal muscle of larval acc reveals greatly prolonged calcium transients and associated contractions in response to depolarization. Positional cloning of acc identified a serca mutation as the cause of the acc phenotype. SERCA is a sarcoplasmic reticulum transmembrane protein in skeletal muscle that mediates calcium re-uptake from the myoplasm. The mutation in SERCA, a serine to phenylalanine substitution, is likely to result in compromised protein function that accounts for the observed phenotype. Indeed, direct evidence that mutant SERCA causes the motility dysfunction was provided by the finding that wild type fish injected with an antisense morpholino directed against serca, exhibited accordion-like contractions and impaired swimming. We conclude that the motility dysfunction in embryonic and larval accordion zebrafish stems directly from defective calcium transport in skeletal muscle rather than defective CNS drive.     

The Calsequestrin Mutation CASQ2D307H Does Not Affect Protein Stability and Targeting to the Junctional Sarcoplasmic Reticulum but Compromises Its Dynamic Regulation of Calcium Buffering

Mutations in cardiac ryanodine receptor (RYR2) and cardiac calsequestrin (CASQ2) genes are linked to catecholaminergic polymorphic ventricular tachycardia, a life-threatening genetic disease. They predispose young individuals to cardiac arrhythmia in the absence of structural abnormalities. One such mutation that changes an aspartic residue to histidine at position 307 in CASQ2 has been linked to catecholaminergic polymorphic ventricular tachycardia. In this study we made a transgenic mouse model expressing the mutant CASQ2^D307H protein in a CASQ2 null background and investigated if the disease is caused by accelerated degradation of the mutant protein. Our data suggest that the mutant protein can be expressed, is relatively stable, and targets appropriately to the junctional sarcoplasmic reticulum. Moreover, it partially normalizes the ultrastructure of the sarcoplasmic reticulum, which was altered in the CASQ2 null background. In addition, overexpression of the mutant protein does not cause any pathology and/or structural changes in the myocardium. We further demonstrate, using purified protein, that the mutant protein is very stable under chemical and thermal denaturation but shows abnormal Ca²⁺ buffering characteristics at high calcium concentrations. In addition, trypsin digestion studies reveal that the mutant protein is more susceptible to protease activity only in the presence of high Ca²⁺. These studies collectively suggest that the D307H mutation can compromise the dynamic behavior of CASQ2 including supramolecular rearrangement upon Ca²⁺ activation.     

Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection

Laser-capture microdissection was coupled with PCR to define the mitochondrial genotype of aged muscle fibers exhibiting mitochondrial enzymatic abnormalities. These electron transport system (ETS) abnormalities accumulate with age, are localized segmentally along muscle fibers, are associated with fiber atrophy and may contribute to age-related fiber loss. DNA extracted from single, 10 µm thick, ETS abnormal muscle fibers, as well as sections from normal fibers, served as templates for PCR-based deletion analysis. Large mitochondrial (mt) DNA deletion mutations (4.4–9.7 kb) were detected in all 29 ETS abnormal fibers analyzed. Deleted mtDNA genomes were detected only in the regions of the fibers with ETS abnormalities; adjacent phenotypically normal portions of the same fiber contained wild-type mtDNA. In addition, identical mtDNA deletion mutations were found within different sections of the same abnormal region. These findings demonstrate that large deletion mutations are associated with ETS abnormalities in aged rat muscle and that, within a fiber, deletion mutations are clonal. The displacement of wild-type mtDNAs with mutant mtDNAs results in concomitant mitochondrial enzymatic abnormalities, fiber atrophy and fiber breakage.     

Distribution and threshold expression of the tRNA(Lys) mutation in skeletal muscle of patients with myoclonic epilepsy and ragged-red fibers (MERRF).

We investigated the distribution and expression of mutant mtDNAs carrying the A-to-G mutation at position 8344 in the tRNA(Lys) gene in the skeletal muscle of four patients with myoclonus epilepsy and ragged-red fibers (MERRF). The proportion of mutant genomes was greater than 80% of total mtDNAs in muscle samples of all patients and was associated with a decrease in the activity of cytochrome c oxidase (COX). The vast majority of myoblasts, cloned from the satellite-cell population in the same muscles, were homoplasmic for the mutation. The overall proportion of mutant mtDNAs in this population was similar to that in differentiated muscle, suggesting that the ratio of mutant to wild-type mtDNAs in skeletal muscle is determined either in the ovum or during early development and changes little with age. Translation of all mtDNA-encoded genes was severely depressed in homoplasmic mutant myoblast clones but not in heteroplasmic or wild-type clones. The threshold for biochemical expression of the mutation was determined in heteroplasmic myotubes formed by fusion of different proportions of mutant and wild-type myoblasts. The magnitude of the decrease in translation in myotubes containing mutant mtDNAs was protein specific. Complex I and IV subunits were more affected than complex V subunits, and there was a rough correlation with both protein size and number of lysine residues. Approximately 15% wild-type mtDNAs restored translation and COX activity to near normal levels. These results show that the A-to-G substitution in tRNA(Lys) is a functionally recessive mutation that can be rescued by intraorganellar complementation with a small proportion of wild-type mtDNAs and explain the steep threshold for expression of the MERRF clinical phenotype.