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Biomedical subjects

Francisco H Andrade

Publications and source records attributed to Francisco H Andrade.

15 recordsLinked to original sources
PubMed2006

Nebulin isoforms of extraocular muscle.

The extraocular muscles (EOMs), which are responsible for reflexive and voluntary eye movements, have many unique biochemical, physiological, and ultrastructural features that set them apart from other skeletal muscles. For example, rodent EOMs lack M-lines and express EOM-specific myosin heavy chain (MYH13) and alpha-cardiac myosin heavy chain. Recent gene-expression profiling studies indicate the presence of other cardiac-specific proteins in adult EOMs. This interesting mixture of myofibrillar and cytoskeletal proteins poses the questions as to whether nebulette, as opposed to nebulin, might be expressed in EOM, and what isoforms of titin are expressed in the EOM. We have performed gel electrophoresis and immunological analyses to determine the titin and nebulin isoforms expressed in the EOM. We have found that the mass of the titin isoforms expressed in the EOM most closely resemble those found in the skeletal muscles tested, viz., the soleus and extensor digitorum longus (EDL). We also demonstrate that, although the EOM expresses cardiac isoforms of myosin, it does not express nebulette and contains a nebulin isoform with a mass consistent with that found in the prototypical fast hindlimb muscle EDL.

Animals↗
PubMed2005

Lactate is a metabolic substrate that sustains extraocular muscle function.

Lactic acid is considered the end product of glycolysis and is a major cause of muscle fatigue. However, the lactate dehydrogenase (LDH) reaction is bidirectional: Lactate can be oxidized to pyruvate and used as a substrate for the Krebs cycle. Therefore, our hypothesis was that lactate sustains the contractile function of rat extraocular muscles during periods of increased activity. The study used extraocular and extensor digitorum longus (EDL) muscles from adult Sprague-Dawley rats to determine LDH isoform expression, total LDH activity, and contractile function in vitro. To evaluate the role of lactate on fatigue, we tested the effect of cinnamate, a blocker of lactate transport, and exogenous lactate on fatigue resistance. Cinnamate accelerated fatigue in the extraocular muscles: Endurance and residual force decreased significantly. Conversely, cinnamate did not affect the endurance or residual force of EDL muscles. Replacing glucose with exogenous lactate increased EDL fatigability but had no effect on the extraocular muscles. However, the extraocular muscles fatigued faster when exposed to exogenous lactate combined with cinnamate. The LDH-A and LDH-C isoforms were expressed at lower levels in extraocular muscle; LDH-B was equally abundant in the EDL and extraocular muscles. Total LDH activity in the extraocular muscles was only approximately 32% of the level in EDL. These results support the hypothesis that lactate sustains the contractile performance of the extraocular muscles.

Animals↗
PubMed2005

Contractile dysfunction and altered metabolic profile of the aging rat thyroarytenoid muscle.

The larynx and its muscles are important for ventilation, coughing, sneezing, swallowing, Valsalva's maneuver, and phonation. Because of their functional demands, the intrinsic laryngeal muscles have a unique phenotype: very small and fast fibers with high mitochondrial content. How aging affects their function is largely unknown. In this study, we tested the hypothesis that an intrinsic laryngeal muscle (thyroarytenoid muscle, a vocal fold adductor) would become weaker, slower, and fatigable with age. Muscles from Fischer 344 x Brown Norway F1 hybrid rats (6, 18, and 30 mo of age) were used for in vitro contractile function and histology. Thyroarytenoid muscles generated significantly lower twitch and tetanic forces at 30 mo vs. 6 and 18 mo. Maximal shortening velocity decreased by 20% at 30 mo (vs. 6 mo), and velocity of unloaded shortening was slower at 18 and 30 mo by 19 and 27% vs. 6 mo. There was no histochemical evidence of altered myosin ATPase activity at 18 or 30 mo of age. Fatigue resistance was significantly decreased at 18 and 30 mo. We also found abundant mitochondrial clusters and ragged red fibers in the muscles of 30-mo-old rats, and there was an age-related increase in glycogen-positive fibers. We conclude that rat thyroarytenoid muscles become weaker, slower, and more fatigable with age. These functional changes are not due to alterations in myosin ATPase activity, but a switch in the expression of myosin isoforms remains a possibility. Finally, the alterations in mitochondrial and glycogen content indicate a shift in the metabolic characteristics of these muscles with age.

Aging↗
PubMed2005

Fatigue resistance of rat extraocular muscles does not depend on creatine kinase activity.

BACKGROUND: Creatine kinase (CK) links phosphocreatine, an energy storage system, to cellular ATPases. CK activity serves as a temporal and spatial buffer for ATP content, particularly in fast-twitch skeletal muscles. The extraocular muscles are notoriously fast and active, suggesting the need for efficient ATP buffering. This study tested the hypotheses that (1) CK isoform expression and activity in rat extraocular muscles would be higher, and (2) the resistance of these muscles to fatigue would depend on CK activity. RESULTS: We found that mRNA and protein levels for cytosolic and mitochondrial CK isoforms were lower in the extraocular muscles than in extensor digitorum longus (EDL). Total CK activity was correspondingly decreased in the extraocular muscles. Moreover, cytoskeletal components of the sarcomeric M line, where a fraction of CK activity is found, were downregulated in the extraocular muscles as was shown by immunocytochemistry and western blotting. CK inhibition significantly accelerated the development of fatigue in EDL muscle bundles, but had no major effect on the extraocular muscles. Searching for alternative ATP buffers that could compensate for the relative lack of CK in extraocular muscles, we determined that mRNAs for two adenylate kinase (AK) isoforms were expressed at higher levels in these muscles. Total AK activity was similar in EDL and extraocular muscles. CONCLUSION: These data indicate that the characteristic fatigue resistance of the extraocular muscles does not depend on CK activity.

Adenylate Kinase↗
PubMed2005

Eliminating the Ant1 isoform produces a mouse with CPEO pathology but normal ocular motility.

PURPOSE: The adenine nucleotide transporter 1 gene (ANT1) encodes an inner mitochondrial membrane protein that transports ATP into the cell. Mutations within ANT1 produce a syndrome of chronic progressive external ophthalmoplegia (CPEO) in humans. Ant1 knockout (Ant1-/-) mice develop cardiomyopathy and mitochondrial myopathy of limb muscles. Because the extraocular muscles (EOM) are preferentially affected in human CPEO, the objective of this study was to determine whether Ant1-/- mice also exhibit an EOM mitochondrial myopathy. METHODS: ANT isoform expression of isolated EOMs, EOM morphology and mitochondrial content, mitochondrial structure and function, ocular motility in intact mice, and contractile performance in isolated muscle preparations were examined. RESULTS: Ant1-/- EOMs had the typical appearance of mitochondrial myopathy, including increase in mitochondrial size, number, and oxidative phosphorylation (OXPHOS) staining. However, there were no measurable ocular motor abnormalities in intact Ant1-/- mice, and their isolated EOMs did not show evidence of increased fatigability. EOMs of wild-type mice exhibited higher levels of Ant2 mRNA compared with hindlimb muscle, which may compensate for the Ant1 loss in mutant mouse EOMs and account for the normal EOM function. CONCLUSIONS: The Ant1-/- mice provide a model in which to study CPEO pathology and compensatory mechanisms.

Adenine Nucleotide Translocator 1↗
PubMed2005

Mitochondria are fast Ca2+ sinks in rat extraocular muscles: a novel regulatory influence on contractile function and metabolism.

PURPOSE: The ultrafast extraocular muscles necessitate tight regulation of free cytosolic Ca2+ concentration ([Ca2+]i). Mitochondrial Ca2+ influx may be fast enough for this role. In the present study, three hypotheses were tested: (1) Mitochondrial Ca2+ uptake regulates [Ca2+]i and production of force in extraocular muscle; (2) mitochondrial content correlates with their use as Ca2+ sinks; and (3) mitochondrial content in extraocular muscle is determined by the transcription factors and coactivators that initiate muscle adaptation to aerobic exercise. METHODS: Extraocular and extensor digitorum longus (EDL) muscles from adult Sprague-Dawley rats were used to examine how the Ca2+ release agonists caffeine and 4-chloro-3-ethylphenol (CEP), calcimycin (a Ca2+ ionophore) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP; a mitochondrial uncoupler) alter [Ca2+]i and force transients. Mitochondrial volume density and capillary density were analyzed by stereology and citrate synthase and cytochrome c oxidase by biochemical assays. Real-time PCR measured mRNAs of genes involved in mitochondrial biogenesis. RESULTS: Caffeine, CEP, and calcimycin increased resting [Ca2+]i to a greater extent in EDL. Peak tetanic [Ca2+]i increased in extraocular muscle with caffeine and CEP. CCCP augmented peak tetanic and submaximum [Ca2+]i and force significantly more in extraocular muscles. Mitochondrial volume density and capillary density were three times greater, and citrate synthase and cytochrome c oxidase were only approximately 2-fold higher in extraocular muscle. Calcineurin Aalpha, calcineurin B, and peroxisome proliferator activated receptor (PPAR)gamma were more abundant in extraocular muscle. CONCLUSIONS: These data support the hypothesis that mitochondria serve as Ca2+ sinks in extraocular muscles. The high mitochondrial content of these muscles may partly reflect this additional function. It is likely that mitochondrial Ca2+ influx increases the dynamic response range of the extraocular muscles and matches metabolic demand to supply.

Animals↗
PubMed2004

Carbonic anhydrase isoform expression and functional role in rodent extraocular muscle.

Carbonic anhydrase (CA) accelerates contractile function, particularly in fast-twitch skeletal muscles. Since the extraocular muscles are considered to be amongst the fastest skeletal muscles in mammals, this study tested two hypotheses: (1) CA is expressed at higher levels in rat extraocular muscles than in extensor digitorum longus (EDL, a fast limb muscle), and (2) inhibition of CA activity increases twitch duration and force in the extraocular muscles to a greater extent than in EDL. By real-time quantitative PCR we determined that the expression of CA3 isoform, typically high in skeletal muscles, is significantly depressed in extraocular muscles. Message levels for the CA2 and CA4 isoforms were higher in the extraocular muscles, while CA5 expression was equivalent in both muscles. Strong CA activity was demonstrated by histochemistry in frozen EDL muscle sections, in particular along the sarcolemma and in capillaries. By contrast, extraocular muscle had very low sarcolemmal or cytosolic CA activity. CA inhibition with 6-ethoxyzolamide (ETZ) reversibly increased twitch duration and force in EDL muscle bundles. In the extraocular muscles, ETZ did not alter twitch kinetics. Based on these results, we reject our initial hypotheses and conclude that CA does not influence the fast contractile kinetics characteristic of the extraocular muscles.

Animals↗
PubMed2004

Functional and genomic changes in the mouse ocular motor system in response to light deprivation from birth.

Previous studies have suggested that abnormal visual experience early in life induces ocular motor abnormalities. The purpose of this study was to determine how visual deprivation alters the function and gene expression profile of the ocular motor system in mice. We measured the effect of dark rearing on eye movements, gene expression in the oculomotor nucleus, and contractility of isolated extraocular muscles. In vivo eye movement recordings showed decreased gains for optokinetic and vestibulo-ocular reflexes, confirming an effect of dark rearing on overall ocular motor function. Saccade peak velocities were preserved, however, arguing that the quantitative changes in these reflexes were not secondary to limitations in force generation. Using microarrays and quantitative PCR, we found that dark rearing shifted the oculomotor nucleus transcriptome to a state of delayed/arrested development. The expression of 132 genes was altered by dark rearing; these genes fit in various functional categories (signal transduction, transcription/translation control, metabolism, synaptic function, cytoskeleton), and some were known to be associated with neuronal development and plasticity. Extraocular muscle contractility was impaired by dark rearing to a greater extent than expected from the in vivo ocular motility studies: changes included decreased force and shortening speed and evidence of abnormal excitability. The results indicate that normal development of the mouse ocular motor system and its muscles requires visual experience. The transcriptional pattern of arrested development may indicate that vision is required to establish the adult pattern, but it also may represent the plastic response of oculomotor nuclei to abnormal extraocular muscles.

Animals↗
PubMed2003

Paradoxical absence of M lines and downregulation of creatine kinase in mouse extraocular muscle.

The M lines are structural landmarks in striated muscles, necessary for sarcomeric stability and as anchoring sites for the M isoform of creatine kinase (CK-M). These structures, especially prominent in fast skeletal muscles, are missing in rodent extraocular muscle, a particularly fast and active muscle group. In this study, we tested the hypotheses that 1). myomesin and M protein (cytoskeletal components of the M lines) and CK-M are downregulated in mouse extraocular muscle compared with the leg muscles, gastrocnemius and soleus; and 2). the expression of other cytosolic and mitochondrial CK isoforms is correspondingly increased. As expected, mouse extraocular muscles expressed lower levels of myomesin, M protein, and CK-M mRNA than the leg muscles. Immunocytochemically, myomesin and M protein were not detected in the banding pattern typically seen in other skeletal muscles. Surprisingly, message abundance for the other known CK isoforms was also lower in the extraocular muscles. Moreover, total CK activity was significantly decreased compared with that in the leg muscles. Based on these data, we reject our second hypothesis and propose that other energy-buffering systems may be more important in the extraocular muscles. The downregulation of major structural and metabolic elements and relative overexpression of two adenylate kinase isoforms suggest that the extraocular muscle group copes with its functional requirements by using strategies not seen in typical skeletal muscles.

Adenylate Kinase↗
PubMed2003

Constitutive properties, not molecular adaptations, mediate extraocular muscle sparing in dystrophic mdx mice.

Extraocular muscle (EOM) is spared in Duchenne muscular dystrophy. Here, we tested putative EOM sparing mechanisms predicted from existing dystrophinopathy models. Data show that mdx mouse EOM contains dystrophin-glycoprotein complex (DGC)-competent and DGC-deficient myofibers distributed in a fiber type-specific pattern. Up-regulation of a dystrophin homologue, utrophin, mediates selective DGC retention. Counter to the DGC mechanical hypothesis, an intact DGC is not a precondition for EOM sarcolemmal integrity, and active adaptation at the level of calcium homeostasis is not mechanistic in protection. A partial, fiber type-specific retention of antiischemic nitric oxide to vascular smooth muscle signaling is not a factor in EOM sparing, because mice deficient in dystrophin and alpha-syntrophin, which localizes neuronal nitric oxide synthase to the sarcolemma, have normal EOMs. Moreover, an alternative transmembrane protein, alpha7beta1 integrin, does not appear to substitute for the DGC in EOM. Finally, genomewide expression profiling showed that EOM does not actively adapt to dystrophinopathy but identified candidate genes for the constitutive protection of mdx EOM. Taken together, data emphasize the conditional nature of dystrophinopathy and the potential importance of nonmechanical DGC roles and support the hypothesis that broad, constitutive structural cell signaling, and/or biochemical differences between EOM and other skeletal muscles are determinants of differential disease responsiveness.

Adaptation, Physiological↗
PubMed2003

Persistent over-expression of specific CC class chemokines correlates with macrophage and T-cell recruitment in mdx skeletal muscle.

Prior studies and the efficacy of immunotherapies provide evidence that inflammation is mechanistic in pathogenesis of Duchenne muscular dystrophy. To identify putative pro-inflammatory mechanisms, we evaluated chemokine gene/protein expression patterns in skeletal muscle of mdx mice. By DNA microarray, reverse transcription-polymerase chain reaction, quantitative polymerase chain reaction, and immunoblotting, convergent evidence established the induction of six distinct CC class chemokine ligands in adult MDX: CCL2/MCP-1, CCL5/RANTES, CCL6/mu C10, CCL7/MCP-3, CCL8/MCP-2, and CCL9/MIP-1gamma. CCL receptors, CCR2, CCR1, and CCR5, also showed increased expression in mdx muscle. CCL2 and CCL6 were localized to both monocular cells and muscle fibers, suggesting that dystrophic muscle may contribute toward chemotaxis. Temporal patterns of CCL2 and CCL6 showed early induction and maintained expression in mdx limb muscle. These data raise the possibility that chemokine signaling pathways coordinate a spatially and temporally discrete immune response that may contribute toward muscular dystrophy. The chemokine pro-inflammatory pathways described here in mdx may represent new targets for treatment of Duchenne muscular dystrophy.

Animals↗
PubMed2003

Postnatal suppression of myomesin, muscle creatine kinase and the M-line in rat extraocular muscle.

The M-line and its associated creatine kinase (CK) M-isoform (CK-M) are ubiquitous features of skeletal and cardiac muscle. The M-line maintains myosin myofilaments in register, links the contractile apparatus to the cytoskeleton for external force transfer and localizes CK-based energy storage and transfer to the site of highest ATP demand. We establish here that the muscle group responsible for movements of the eye, extraocular muscle (EOM), is divergent from other striated muscles in lacking both an M-line and its associated CK-M. Although an M-line forms during myogenesis, both in vivo and in vitro, it is actively repressed after birth. Transcripts of the major M-line structural proteins, myomesin 1 and myomesin 2, follow the same pattern of postnatal downregulation, while the embryonic heart-specific EH-myomesin 1 transcript is expressed early and retained in adult eye muscle. By immunocytochemistry, myomesin protein is absent from adult EOM sarcomeres. M-line suppression does not occur in organotypic co-culture with oculomotor motoneurons, suggesting that the mechanism for suppression may lie in muscle group-specific activation or workload patterns experienced only in vivo. The M-line is, however, still lost in dark-reared rats, despite the developmental delay this paradigm produces in the visuomotor system and EOMs. EOM was low in all CK isoform transcripts except for the sarcomeric mitochondrial (Ckmt2) isoform. Total CK enzyme activity of EOM was one-third that of hindlimb muscle. These findings are singularly unique among fast-twitch skeletal muscles. Since EOM exhibits isoform diversity for other sarcomeric proteins, the M-line/CK-M divergence probably represents a key physiological adaptation for the unique energetics and functional demands placed on this muscle group in voluntary and reflexive eye movements.

Adaptation, Physiological↗
PubMed2002

A chronic inflammatory response dominates the skeletal muscle molecular signature in dystrophin-deficient mdx mice.

Mutations in dystrophin cause Duchenne muscular dystrophy (DMD), but absent dystrophin does not invariably cause necrosis in all muscles, life stages and species. Using DNA microarray, we established a molecular signature of dystrophinopathy in the mdx mouse, with evidence that secondary mechanisms are key contributors to pathogenesis. We used variability controls, adequate replicates and stringent analytic tools, including significance analysis of microarrays to estimate and manage false positive rates. In leg muscle, we identified 242 differentially expressed genes, >75% of which have not been previously reported as altered in human or animal dystrophies. Data provide evidence for coordinated activity of numerous components of a chronic inflammatory response, including cytokine and chemokine signaling, leukocyte adhesion and diapedesis, invasive cell type-specific markers, and complement system activation. Selective chemokine upregulation was confirmed by RT-PCR and immunoblot, and may be a key determinant of the nature of the inflammatory response in dystrophic muscle. Up-regulation of secreted phosphoprotein 1 (minopontin, osteopontin) mRNA and protein in dystrophic muscle identified a novel linkage between inflammatory cells and repair processes. Extracellular matrix genes were up-regulated in mdx to levels similar to those in DMD. Since, unlike DMD, mdx exhibits little fibrosis, data suggest that collagen regulation at post-transcriptional stages mediates extensive fibrosis in DMD. Taken together, these data identify a relatively neglected aspect of DMD, suggest new treatment avenues, and highlight the value of genome-wide profiling in study of complex disease processes.

Animals↗