Robert Wade

Use of thapsigargin to study Ca2+ homeostasis in cardiac cells

Several reports have documented that thapsigargin is a potent inhibitor of the SR Ca2+ ATPase isolated from cardiac or skeletal muscle. We have characterized the specificity of this agent in intact rat cardiac myocytes using cells maintained in the whole cell voltage clamp configuration. We have shown that thapsigargin decreases the magnitude of the Ca2+ transient and the twitch by about 80% while it slows the decay rate for these responses. These changes were not accompanied by any alterations in sarcolemmal currents or in the trigger Ca2+ generated by the inward calcium current. Taken together these results reveal that the action of thapsigargin is restricted to the SR Ca2+ ATPase in intact cardiac myocytes. Furthermore, it is demonstrated unambiguously that SR intracellular Ca2+ stores are an absolute requirement for the development of contractile tension in rat heart myocytes. It is shown that thapsigargin is a valuable probe to examine the importance of SR pools of Ca2+ and the role of the Ca2+ ATPase in intact myocytes as well as in genetically altered heart cells.

Differential patterns of transcript accumulation during human myogenesis

We evaluated the extent to which muscle-specific genes display identical patterns of mRNA accumulation during human myogenesis. Cloned satellite cells isolated from adult human skeletal muscle were expanded in culture, and RNA was isolated from low- and high-confluence cells and from fusing cultures over a 15-day time course. The accumulation of over 20 different transcripts was compared in these samples with that in fetal and adult human skeletal muscle. The expression of carbonic anhydrase 3, myoglobin, HSP83, and mRNAs encoding eight unknown proteins were examined in human myogenic cultures. In general, the expression of most of the mRNAs was induced after fusion to form myotubes. However, several exceptions, including carbonic anhydrase and myoglobin, showed no detectable expression in early myotubes. Comparison of all transcripts demonstrated little, if any, identity of mRNA accumulation patterns. Similar variability was also seen for mRNAs which were also expressed in nonmuscle cells. Accumulation of mRNAs encoding alpha-skeletal, alpha-cardiac, beta- and gamma-actin, total myosin heavy chain, and alpha- and beta-tubulin also displayed discordant regulation, which has important implications for sarcomere assembly. Cardiac actin was the only muscle-specific transcript that was detected in low-confluency cells and was the major alpha-actin mRNA at all times in fusing cultures. Skeletal actin was transiently induced in fusing cultures and then reduced by an order of magnitude. Total myosin heavy-chain mRNA accumulation lagged behind that of alpha-actin. Whereas beta- and gamma-actin displayed a sharp decrease after initiation of fusion and thereafter did not change, alpha- and beta-tubulin were transiently induced to a high level during the time course in culture. We conclude that each gene may have its own unique determinants of transcript accumulation and that the phenotype of a muscle may not be determined so much by which genes are active or silent but rather by the extent to which their transcript levels are modulated. Finally, we observed that patterns of transcript accumulation established within the myotube cultures were consistent with the hypothesis that myoblasts isolated from adult tissue recapitulate a myogenic developmental program. However, we also detected a transient appearance of adult skeletal muscle-specific transcripts in high-confluence myoblast cultures. This indicates that the initial differentiation of these myoblasts may reflect a more complex process than simple recapitulation of development.

Differential control of tropomyosin mRNA levels during myogenesis suggests the existence of an isoform competition-autoregulatory compensation control mechanism☆

We have isolated tropomyosin cDNAs from human skeletal muscle and nonmuscle cDNA libraries and constructed gene-specific DNA probes for each of the four functional tropomyosin genes. These DNA probes were used to define the regulation of the corresponding mRNAs during the process of myogenesis. Tropomyosin regulation was compared with that of beta- and gamma-actin. No two striated muscle-specific tropomyosin mRNAs are coordinately accumulated during myogenesis nor in adult striated muscles. Similarly, no two nonmuscle tropomyosins are coordinately repressed during myogenesis. However, mRNAs encoding the 248 amino acid nonmuscle tropomyosins and beta- and gamma-actin are more persistent in adult skeletal muscle than those encoding the 284 amino acid nonmuscle tropomyosins. In particular, the nonmuscle tropomyosin Tm4 is expressed at similar levels in adult rat nonmuscle and striated muscle tissues. We conclude that each tropomyosin mRNA has its own unique determinants of accumulation and that the 248 amino acid nonmuscle tropomyosins may have a role in the architecture of the adult myofiber. The variable regulation of nonmuscle isoforms during myogenesis suggests that the different isoforms compete for inclusion into cellular structures and that compensating autoregulation of mRNA levels bring gene expression into alignment with the competitiveness of each individual gene product. Such an isoform competition-autoregulatory compensation mechanism would readily explain the unique regulation of each gene.

Differential expression of slow and fast skeletal muscle troponin C: slow skeletal muscle troponin C is expressed in human fibroblasts

We have isolated and sequenced the cDNAs for human slow and fast skeletal muscle troponin C (TnC). Each cDNA is encoded by one of the two TnC genes in the human genome. The fast skeletal muscle TnC gene appears to be expressed exclusively in skeletal muscle. Only the slow TnC gene is expressed in human cardiac ventricle. The slow skeletal TnC gene is also expressed in skeletal muscle and, surprisingly, in several human fibroblast cell lines. Thus, at least one of the three proteins of the troponin complex appears to be expressed in non-muscle cells of higher vertebrates. The relative steady-state amounts of the slow and fast skeletal TnC mRNAs in various adult and embryonic striated muscles are similar to the expected amounts of the corresponding protein, suggesting that the expression of TnC genes is controlled predominantly by the production or accumulation of mRNA rather than by translational or post-translational mechanisms.

Regulation of contractile protein gene family mRNA pool sizes during myogenesis.

During myogenesis, muscle contractile protein gene expression is induced and the products are used to assemble the contractile apparatus characteristic of striated muscle. The different muscle proteins are accumulated in a fixed stoichiometric ratio related to their organization in the contractile apparatus. We have examined the relationship between contractile protein gene expression and the maintenance of stoichiometry at different stages of human myogenesis. Essentially all of the known components of adult human skeletal muscle thick and thin filaments have been cloned in the form of cDNAs and used to generate isoform-specific DNA probes. The expression of fast, slow, and cardiac isoforms was measured in human myogenic primary culture and in fetal and adult human skeletal muscle. We observed that neither fast nor slow nor cardiac isoforms are coordinately regulated at the level of comparative transcript accumulation throughout myogenesis. Thus, the stoichiometry of contractile protein levels cannot be explained by coordination of expression in each of these isoform classes. However, we find that the stoichiometry of mRNA accumulation of each gene family is very similar among three developmental stages: myotubes, fetal skeletal muscle, and adult skeletal muscle. This is consistent with the possibility that the maintenance of stoichiometry between the contractile proteins could be largely regulated by the total accumulation of mRNA from each of these gene families.

Isolation and Characterization of an Avian Slow Myosin Heavy Chain Gene Expressed during Embryonic Skeletal Muscle Fiber Formation

We have isolated and begun characterization of the quail slow myosin heavy chain (MyHC) 3 gene, the first reported avian slow MyHC gene. Expression of slow MyHC 3 in skeletal muscle is restricted to the embryonic period of development, when the fiber pattern of future fast and slow muscle is established. In embryonic hindlimb development, slow MyHC 3 gene expression coincides with slow muscle fiber formation as distinguished by slow MyHC-specific antibody staining. In addition to expression in embryonic appendicular muscle, slow MyHC 3 is expressed continuously in the atria. Transfection of slow MyHC 3 promoter-reporter constructs into embryonic myoblasts that form slow MyHC-expressing fibers identified two regions regulating expression of this gene in skeletal muscle. The proximal promoter, containing potential muscle-specific regulatory motifs, permits expression of a reporter gene in embryonic slow muscle fibers, while a distal element, located greater than 2600 base pairs upstream, further enhances expression 3-fold. The slow muscle fiber-restricted expression of slow MyHC 3 during embryonic development, and expression of slow MyHC 3 promoter-reporter constructs in embryonic muscle fibers in vitro, makes this gene a useful marker to study the mechanism establishing the slow fiber lineage in the embryo.

Sublytic Terminal Complement Attack on Myotubes Decreases the Expression of mRNAs Encoding Muscle‐Specific Proteins

Abstract: Activation of inflammatory and cytotoxic complement effectors that include the C5b‐9 complex plays an important pathogenic role in myasthenia gravis, an inflammatory autoimmune disease of the muscle. Altered muscle‐specific gene expression has been observed in experimental myasthenic rats. In this study, we have examined the effect of sublytic C5b‐9 on myotubes differentiated from C2C12 myoblasts, by generating C5b‐9 with C7‐deficient serum with or without C7. Within 2 h, C7‐deficient serum plus C7, compared with C7‐deficient serum alone, induced markedly decreased levels of mRNAs encoding α‐actin, troponin I slow twitch isoform, acetylcholine receptor α, and muscle aldolase A, whereas the heat shock protein 83 mRNA level remained constant, by northern analysis. Because the half‐life of the acetylcholine receptor α was estimated to be >8 h, the C5b‐9 effect was, in part, due to enhanced mRNA decay. Because C5b‐9 also induced c‐jun mRNA and reduced the myoD mRNA level, a possible inhibition of muscle gene transcription by C5b‐9 was examined in myotubes transfected with troponin promoter‐luciferase gene constructs. Luciferase activity was reduced to 50% in response to C5b‐9 at 2 h. Thus, C5b‐9 appears to inhibit the muscle‐specific gene expression by stimulating mRNA decay and by decreasing the transcription process. The data also indicate a possible pathogenic role of C5b‐9 in immune‐mediated inflammatory muscle disorders in which complement activation has been implicated.

Sequencing of a cDNA encoding the human fast-twitch skeletal muscle isoform of troponin I.

A cDNA encoding the human fast-twitch skeletal muscle isoform of troponin I (TnIfast) has been sequenced. This cDNA is 701 base pairs in length, and encodes a protein of 182 amino acids. TnIfast is the last of the three known human TnI isoforms to be sequenced. Comparison of the deduced human TnIfast protein sequence with a variety of troponin I isoforms from other species has revealed a high degree of cross-species sequence conservation between TnIfast proteins.

Sequence and expression of human myosin alkali light chain isoforms

SummaryIn order to initiate the study of the functional differences between myosin alkali light chain isoforms and to investigate the mechanisms of their differential expression, we have isolated cDNA clones for two human alkali light chain isoforms. Here we report DNA sequence and RNA blotting analyses that demonstrate that these cDNAs represent transcripts encoding human MLC3F and MLClSb. The sequence of the human MLClSb cDNA offers the first fully characterized example of a slow-fiber skeletal muscle alkali light chain isoform from any species. The sequence analysis of these two cDNAs allows an examination of evolutionarily conserved features of mammalian alkali light chain genes. Examination of the genomic organization of the human alkali light chain isoform genes revealed that, in contrast with some strains of mice, both are single copy genes. RNA blot analysis conclusively demonstrates that the human skeletal muscle MLClSb gene is also expressed in the heart ventricle but not the atria. In addition, we examined the expression of alkali light chain isoforms during the in vitro differentiation of a variety of human and rodent myogenic cells and found striking variation in the pattern of alkali light chain isoform gene expression in different myogenic cells.

Ca2+-dependent interaction of the inhibitory region of troponin I with acidic residues in the N-terminal domain of troponin C

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96-116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95-116, 95-119 or 95-124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117-124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.