A mutation in alpha-tropomyosin(slow) affects muscle strength, maturation and hypertrophy in a mouse model for nemaline myopathy.

Nemaline myopathy is a hereditary disease of skeletal muscle defined by a distinct pathology of electron-dense accumulations within the sarcomeric units called rods, muscle weakness and, in most cases, a slow oxidative (type 1) fiber predominance. We generated a transgenic mouse model to study this disorder by expressing an autosomal dominant mutant of alpha-tropomyosin(slow) previously identified in a human cohort. Rods were found in all muscles, but to varying extents which did not correlate with the amount of mutant protein present. In addition, a pathological feature not commonly associated with this disorder, cytoplasmic bodies, was found in the mouse and subsequently identified in human samples. Muscle weakness is a major feature of this disease and was examined with respect to fiber composition, degree of rod-containing fibers, fiber mechanics and fiber diameter. Hypertrophy of fast, glycolytic (type 2B) fibers was apparent at 2 months of age. Muscle weakness was apparent in mice at 5-6 months of age, mimicking the late onset observed in humans with this mutation. The late onset did not correlate with observed changes in fiber type and rod pathology. Rather, the onset of muscle weakness correlates with an age-related decrease in fiber diameter and suggests that early onset is prevented by hypertrophy of fast, glycolytic fibers. We suggest that the clinical phenotype is precipitated by a failure of the hypertrophy to persist and therefore compensate for muscle weakness.

Identification of a novel slow-muscle-fiber enhancer binding protein, MusTRD1.

The molecular mechanisms which are responsible for restricting skeletal muscle gene expression to specific fiber types, either slow or fast twitch, are unknown. As a first step toward defining the components which direct slow-fiber-specific gene expression, we identified the sequence elements of the human troponin I slow upstream enhancer (USE) that bind muscle nuclear proteins. These include an E-box, a MEF2 element, and two other elements, USE B1 and USE C1. In vivo analysis of a mutation that disrupts USE B1 binding activity suggested that the USE B1 element is essential for high-level expression in slow-twitch muscles. This mutation does not, however, abolish slow-fiber specificity. A similar analysis indicated that the USE C1 element may play only a minor role. We report the cloning of a novel human USE B1 binding protein, MusTRD1 (muscle TFII-I repeat domain-containing protein 1), which is expressed predominantly in skeletal muscle. Significantly, MusTRD1 contains two repeat domains which show remarkable homology to the six repeat domains of the recently cloned transcription factor TFII-I. Furthermore, both TFII-I and MusTRD1 bind to similar but distinct sequences, which happen to conform with the initiator (Inr) consensus sequence. Given the roles of MEF2 and basic helix-loop-helix (bHLH) proteins in muscle gene expression, the similarity of TFII-I and MusTRD1 is intriguing, as TFII-I is believed to coordinate the interaction of MADS-box proteins, bHLH proteins, and the general transcription machinery.

Reappearance of the minor alpha-sarcomeric actins in postnatal muscle.

The postnatal expression profiles of alpha-sarcomeric actin transcripts and protein are quantified in mouse striated muscles from birth to postnatal day 56 by Northern and Western blot analyses. alpha-Cardiac actin (alpha-CA) transcripts transiently increase between 12 and 21 days after birth in the quadriceps muscle, reaching approximately 90% that found in the adult mouse heart. Although alpha-CA is the alpha-sarcomeric actin isoform expressed in the immature fiber, the expression profiles of other contractile protein isoforms indicate that this postnatal period is not reflective of an immature phenotype. alpha-Skeletal actin (alpha-SA) transcripts accumulate to approximately 32% of the total alpha-sarcomeric actin transcripts in the adult heart. Our study shows that 1) there is a simultaneous reappearance of alpha-CA and alpha-SA in postnatal skeletal and heart muscles, respectively, and 2) the contractile protein gene expression profile characteristic of adult skeletal muscle is not achieved until after 42 days postnatal in the mouse. We propose there is a previously uncharacterized period of postnatal striated muscle maturation marked by the reappearance of the minor alpha-sarcomeric actins.

The human troponin I slow promoter directs slow fiber-specific expression in transgenic mice.

Troponin I (TnI) is a muscle-specific protein involved in the calcium-mediated contraction of striated muscle. Three TnI isoforms have been identified, each encoded by a separate gene and expressed in specific striated muscles in the adult. The slow isoform gene (TnIs) is transcriptionally regulated during skeletal muscle development such that its expression in the adult is restricted to muscle fibers innervated by a slow nerve. To delineate regions of this gene that are responsive to information imparted by the slow nerve, we generated transgenic mice carrying -4,200 to +12 bp of the human TnIs gene linked to the bacterial chloramphenicol acetyltransferase (CAT) coding region. By Northern blot analysis, we detected transgene transcripts only in muscles containing slow-twitch fibers. CAT histochemical analysis revealed that expression of the transgene is restricted solely to slow-twitch fibers as characterized by type I myosin heavy-chain (MyHC) expression. Using regeneration as a model for neural influenced expression, we show that this gene construct also contains sequences necessary to respond to cues from the central nervous system.

Delineation of a slow-twitch-myofiber-specific transcriptional element by using in vivo somatic gene transfer.

Contractile proteins are encoded by multigene families, most of whose members are differentially expressed in fast- versus slow-twitch myofibers. This fiber-type-specific gene regulation occurs by unknown mechanisms and does not occur within cultured myocytes. We have developed a transient, whole-animal assay using somatic gene transfer to study this phenomenon and have identified a fiber-type-specific regulatory element within the promoter region of a slow myofiber-specific gene. A plasmid-borne luciferase reporter gene fused to various muscle-specific contractile gene promoters was differentially expressed when injected into slow- versus fast-twitch rat muscle: the luciferase gene was preferentially expressed in slow muscle when fused to a slow troponin I promoter, and conversely, was preferentially expressed in fast muscle when fused to a fast troponin C promoter. In contrast, the luciferase gene was equally well expressed by both muscle types when fused to a nonfiber-type-specific skeletal actin promoter. Deletion analysis of the troponin I promoter region revealed that a 157-bp enhancer conferred slow-muscle-preferential activity upon a minimal thymidine kinase promoter. Transgenic analysis confirmed the role of this enhancer in restricting gene expression to slow-twitch myofibers. Hence, somatic gene transfer may be used to rapidly define elements that direct myofiber-type-specific gene expression prior to the generation of transgenic mice.

Developmental regulation of troponin I isoform genes in striated muscles of transgenic mice.

The differentiation and diversification of striated muscle is a complex process involving numerous temporal and spatial alterations in the pattern of contractile protein isoform gene expression. In order to gain insight into the regulation of contractile protein isoform changes during skeletal and cardiac muscle formation, the expression of a transgene comprising a chloramphenicol acetyltransferase (CAT) reporter gene linked with sequences from -4200 to +12 of the human slow skeletal troponin I (TnIs) gene, and all three endogenous mouse troponin I (TnI) isoform genes, was investigated in embryonic, neonatal, and postnatal mice. The -4200 TnIsCAT transgene was properly activated in the limb and trunk skeletal muscle primordia and the early embryonic atrium and ventricle of the heart. Along with the endogenous mouse TnIs gene, expression of the CAT transgene began to segregate into the presumptive slow-twitch myofibers at late fetal stages and expression declined in the neonatal and postnatal heart except for the conductive tissues, in which expression persisted into adulthood. However, expression of the CAT transgene during development did not completely follow the endogenous mouse TnIs gene. The expression of the CAT transgene was aberrantly low in the embryonic cardiac outflow tract and the ventricles of the fetal heart. In addition to its expression in striated muscles, the transgene was expressed aberrantly in the primordial axial skeleton. We conclude that the upstream sequences from the human TnIs gene contain sufficient regulatory information to confer appropriate transgene expression during the early differentiation of skeletal muscles and during the establishment of fiber type upon the maturation of myofibers. However, additional regulatory elements are likely to be required for correct temporal and spatial regulation in the heart and somitic mesoderm during development. In vitro DNA transfection of cultured skeletal and cardiac muscle cells identified a cell type-specific enhancer element within the first intron of the TnIs gene whose absence in the transgene may account for the aberrant expression observed in vivo. In addition, we provide the first evidence that the fast-twitch skeletal muscle isoform of troponin I, TnIf, is transiently expressed during early cardiac muscle development.

Multiple regions of the human cardiac actin gene are necessary for maturation-based expression in striated muscle.

In order to elucidate mechanisms involved in striated muscle contractile protein isoform expression, we have defined regulatory elements in the cardiac actin gene necessary for postnatal expression at the level of transcript accumulation in the heart and hindlimb muscles of transgenic mice. During this developmental period in the rodent, cardiac actin expression essentially remains constant in the heart, but declines significantly in skeletal muscle. We determined that a 13-kilobase human cardiac actin gene fragment contains sufficient information to direct this maturation-based developmental expression, as well as striated muscle-specific and high level expression. We localized an element responsible for maturation-based down-regulation in the 3' flank of the gene between approximately 950 and 2120 base pairs downstream of the polyadenylation site. Furthermore, we determined that -800 base pairs of 5'-flanking DNA, which contains multiple MyoD1 binding sites, as well as serum response element and AP1 binding sites, can account for striated muscle-specific expression, but not high level expression. Findings indicate that sequence(s) responsible for high level expression of the gene must be located within the body of the gene. We conclude that the human cardiac actin gene contains distinct sequences which confer developmental, tissue-specific, and high level expression.

Production of heat-stable enterotoxin II by chicken clinical isolates of Escherichia coli.

Enterotoxigenic Escherichia coli isolated from diarrhea stools of chickens were examined for production of heat-stable enterotoxin II which is considered to be implicated only in diarrhea of pigs. Seven out of 38 strains examined were found to contain heat-stable enterotoxin II gene, determined by colony hybridization and the polymerase chain reaction. The culture supernatants of these strains caused fluid accumulation in the mouse intestinal loop test. This fluid accumulation activity was not lost by heating at 100 degrees C and was neutralized by anti-heat-stable enterotoxin II antiserum. Purified heat-stable enterotoxin II caused fluid accumulation in the chicken intestinal loop assay. These results indicate that STII-producing E. coli is implicated in chicken diarrhea.

Demonstration of enterotoxigenic Escherichia coli in diarrheic broiler chicks.

An investigation was made to survey the possible presence of enterotoxigenic Escherichia coli (ETEC) in the stools of diarrheal chicks. We analyzed two outbreaks of diarrhea in broiler chicks at two independent farms in the Philippines, from which no pathogens other than Escherichia coli were found. In one outbreak at Farm #1, all 42 isolates produced heat-labile enterotoxin (LT), with 3 of these isolates also producing heat-stable enterotoxin (ST). The O serotypes of 15 strains tested randomly could not be identified as any known serotype (0-antigen; 1-170). In another outbreak at Farm #2, 7 out of 52 isolates produced only LT, their subtypes being identified as O-149 or O-8, common serotypes in pig ETEC. Strains from Farm #1 did not produce any pili usually found in human ETEC. We believe this to be the first isolation of ETEC from diarrheal chicks.

A heat-labile enterotoxin (LT) purified from chicken enterotoxigenic Escherichia coli is identical to porcine LT.

We have previously reported that the heat-labile enterotoxin (LTc) isolated from a chicken enterotoxigenic Escherichia coli (ETEC) was identical to LTh produced by human ETEC (Tsuji et al. (1988) FEMS Microbiol Lett. 52, 79-84). In this study, we purified an LTc-like toxin (LTc') from another strain isolated from a chicken that developed diarrhea at a different place and time to the previously reported chicken. Its molecular weight and antigenicity were compared with those of purified LTs from porcine and human ETEC (LTp and LTh). The A subunit of LTc' was identical to those of the purified LTs in mobility on SDS-polyacrylamide gel electrophoresis. The Ouchterlony test demonstrated that LTc' was antigenically identical to LTp. The isoelectric point and amino acid composition of LTc' were also identical to those of LTp. These data suggest that chicken ETEC can be grouped with both the porcine and human types on the basis of the LTs produced.

Charge heterogeneity of heat-labile enterotoxins from human enterotoxigenic Escherichia coli.

We purified heat-labile enterotoxins (LThs) from YT3, H-10407 and YT240 strains isolated from human diarrheal patients. These LThs were immunologically identical to each other. The molecular weights of their A and B subunits were also the same by means of SDS-polyacrylamide gel electrophoresis. However, the ionic charges of the molecular surfaces of these LThs were different as shown by polyacrylamide gel isoelectric focusing. Though the pI points of B subunits of the LThs were identical to each other, the pI points of A subunits were found to be different. These data suggest that the ionic charge differences among A subunits cause differences in holo LThs in their charge, and that there is heterogeneity among A subunits produced by strains of human enterotoxigenic Escherichia coli.