Neal A. Rubinstein

Rapid Disuse Atrophy of Diaphragm Fibers in Mechanically Ventilated Humans

BACKGROUND The combination of complete diaphragm inactivity and mechanical ventilation (for more than 18 hours) elicits disuse atrophy of myofibers in animals. We hypothesized that the same may also occur in the human diaphragm. METHODS We obtained biopsy specimens from the costal diaphragms of 14 brain-dead organ donors before organ harvest (case subjects) and compared them with intraoperative biopsy specimens from the diaphragms of 8 patients who were undergoing surgery for either benign lesions or localized lung cancer (control subjects). Case subjects had diaphragmatic inactivity and underwent mechanical ventilation for 18 to 69 hours; among control subjects diaphragmatic inactivity and mechanical ventilation were limited to 2 to 3 hours. We carried out histologic, biochemical, and gene-expression studies on these specimens. RESULTS As compared with diaphragm-biopsy specimens from controls, specimens from case subjects showed decreased cross-sectional areas of slow-twitch and fast-twitch fibers of 57% (P=0.001) and 53% (P=0.01), respectively, decreased glutathione concentration of 23% (P=0.01), increased active caspase-3 expression of 100% (P=0.05), a 200% higher ratio of atrogin-1 messenger RNA (mRNA) transcripts to MBD4 (a housekeeping gene) (P=0.002), and a 590% higher ratio of MuRF-1 mRNA transcripts to MBD4 (P=0.001). CONCLUSIONS The combination of 18 to 69 hours of complete diaphragmatic inactivity and mechanical ventilation results in marked atrophy of human diaphragm myofibers. These findings are consistent with increased diaphragmatic proteolysis during inactivity.

Myogenic and neurogenic contributions to the development of fast and slow twitch muscles in rat

Abstract The histochemical ATPase activity and the myosin light chains of a rat fast muscle (extensor digitorum longus, EDL) and a rat slow muscle (soleus) during development have been investigated. Both muscles initially synthesize fast myosin light chains and show the intense histochemical ATPase activity characteristic of adult fast muscle fibers. After birth, the soleus begins to accumulate slow fibers with their characteristic low histochemical ATPase activity, and slow myosin light chains begin to appear. Sciatic neurectomy prevents the development of slow fibers and the synthesis of slow myosin light chains in the soleus, while the EDL is unaffected. Similarly, cordotomy of an adult rat results, in the soleus, in the appearance of fibers with more intense staining for ATPase and an increase in fast myosin light chains. The EDL is unchanged by cordotomy. As a result, we suggest that slow muscle development, but not fast muscle development, is dependent upon the functional activity of the nervous system.

THYROIDAL AND NEURAL CONTROL OF MYOSIN TRANSITIONS DURING DEVELOPMENT OF RAT FAST AND SLOW MUSCLES

Experiments with developing euthyroid, hypothyroid and hyperthyroid rats show that the transition from neonatal to adult fast myosin is orchestrated by thyroid hormones acting directly upon fast muscle cells. Denervation studies reveal the switch from neonatal to adult fast myosin synthesis is independent of the motoneuron. However the synthesis of slow myosin during development is critically dependent on innervation.

Lineages, quantal cell cycles, and the generation of cell diversity

Most theories of determination or differentiation assume that embryonic cells differ from mature cells. Embryonic cells are thought to have metastable control mechanisms. These labile controls are believed to become progressively more stabilized as the cells differentiate. Zygote, blastula, neural plate, limb bud, somite, or ‘stem’ cells are conceived of as undifferentiated, totipotent, or multipotential cells. As such, these cells supposedly have available for activation a larger repertoire of phenotypic programmes than their progeny. A necessary corollary to this view is that the activation of one particular phenotypic programme out of the many available is a function of instructive exogenous inducing molecules.

Human skeletal myosin heavy chain genes are tightly linked in the order embryonic-IIa-IId/x-IIb-perinatal-extraocular

Myosin heavy chain (MyHC) is the major contractile protein of muscle. We report the first complete cosmid cloning and definitive physical map of the tandemly linked human skeletal MyHC genes at 17p13.1. The map provides new information on the order, size, and relative spacing of the genes, and it resolves uncertainties about the two fastest twitch isoforms. The physical order of the genes is demonstrated to contrast with the temporal order of their developmental expression. Furthermore, nucleotide sequence comparisons allow an approximation of the relative timing of five ancestral duplications that created distinct genes for the six isoforms. A firm foundation is provided for molecular analysis in patients with suspected primary skeletal myosinopathies and for detailed modelling of the hypervariable surface loops which dictate myosins kinetic properties.

Myosin heavy chain and physiological adaptation of the rat diaphragm in elastase-induced emphysema

BackgroundSeveral physiological adaptations occur in the respiratory muscles in rodent models of elastase-induced emphysema. Although the contractile properties of the diaphragm are altered in a way that suggests expression of slower isoforms of myosin heavy chain (MHC), it has been difficult to demonstrate a shift in MHCs in an animal model that corresponds to the shift toward slower MHCs seen in human emphysema.MethodsWe sought to identify MHC and corresponding physiological changes in the diaphragms of rats with elastase-induced emphysema. Nine rats with emphysema and 11 control rats were studied 10 months after instillation with elastase. MHC isoform composition was determined by both reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry by using specific probes able to identify all known adult isoforms. Physiological adaptation was studied on diaphragm strips stimulated in vitro.ResultsIn addition to confirming that emphysematous diaphragm has a decreased fatigability, we identified a significantly longer time-to-peak-tension (63.9 ± 2.7 ms versus 53.9 ± 2.4 ms). At both the RNA (RT-PCR) and protein (immunocytochemistry) levels, we found a significant decrease in the fastest, MHC isoform (IIb) in emphysema.ConclusionThis is the first demonstration of MHC shifts and corresponding physiological changes in the diaphragm in an animal model of emphysema. It is established that rodent emphysema, like human emphysema, does result in a physiologically significant shift toward slower diaphragmatic MHC isoforms. In the rat, this occurs at the faster end of the MHC spectrum than in humans.

Quantitative Proteomics Profiling of Sarcomere Associated Proteins in Limb and Extraocular Muscle Allotypes

The sarcomere is the major structural and functional unit of striated muscle. Approximately 65 different proteins have been associated with the sarcomere, and their exact composition defines the speed, endurance, and biology of each individual muscle. Past analyses relied heavily on electrophoretic and immunohistochemical techniques, which only allow the analysis of a small fraction of proteins at a time. Here we introduce a quantitative label-free, shotgun proteomics approach to differentially quantitate sarcomeric proteins from microgram quantities of muscle tissue in a fast and reliable manner by liquid chromatography and mass spectrometry. The high sequence similarity of some sarcomeric proteins poses a problem for shotgun proteomics because of limitations in subsequent database search algorithms in the exclusive assignment of peptides to specific isoforms. Therefore multiple sequence alignments were generated to improve the identification of isoform specific peptides. This methodology was used to compare the sarcomeric proteome of the extraocular muscle allotype to limb muscle. Extraocular muscles are a unique group of highly specialized muscles with distinct biochemical, physiological, and pathological properties. We were able to quantitate 40 sarcomeric proteins; although the basic sarcomeric proteins in extraocular muscle are similar to those in limb muscle, key proteins stabilizing the connection of the Z-bands to thin filaments and the costamere are augmented in extraocular muscle and may represent an adaptation to the eccentric contractions known to normally occur during eye movements. Furthermore, a number of changes are seen that closely relate to the unique nature of extraocular muscle.

Coordinated synthesis and degradation of actin and myosin in a variety of myogenic and non-myogenic cells

Abstract The turnover of myosin and actin in both muscle and non-muscle cells in culture was investigated. By the double-label criterion, myosin and actin were coordinately synthesized and degraded in replicating, mononucleated fibroblasts, chondrocytes, BUdR-suppressed myogenic cells, and in post-mitotic, multinucleated myotubes. Myosin and actin were among the most stable proteins in each cell type. In single label ‘pulse-chase’ experiments, the half-lives of myosin and actin in all replicating, mononucleated cells were 2.5–3 days; in myotubes, however, they were approx. 6 days. Myosin and actin labelled in replicating presumptive myoblasts and chased until the cells ceased replicating and fused into multinucleated myotubes retained the degradation rate of 3 days; this differed from Jhe rate of 6 days shown for myosin and actin newly-synthesized in post-mitotic myotubes. The type of myosin synthesized in the mother presumptive myoblast, then, is transmitted to the postmitotic daughters. This myosin, however, is more rapidly degraded than the definitive myosin that is synthesized in the myotube.

Actin and myosin in a variety of myogenic and non-myogenic cells

Abstract Replicating presumptive myoblasts and bromodeoxyuridine-suppressed myogenic cells, as well as embryonic fibroblasts and chondroblasts, synthesize actin and myosin. The ratio of myosin to actin and the number of myosin light chains synthesized in these cells differ from those in multinucleated myotubes. These findings are discussed in relationship to myogenesis.

Sexual dimorphism in the fibers of a “clasp” muscle of Xenopus laevis

When antibodies specific to fast-twitch, slow-twitch, and slow-tonic myosins were used to stain the clasp muscle, m. sternoradialis, of Xenopus laevis, three predominant fiber types were identified in both males and females. Three fiber types can also be distinguished by the diameters and conduction velocities of their motor nerves. Significant differences in the numbers of slow-tonic fibers were identified between the genders and between control and castrated male animals. This finding suggests that these slow-tonic fibers, which probably dominate the clasp reflex during mating, may be under hormonal control.