RESUMO
BACKGROUND: The muscular dystrophies exemplify a class of systemic disorders for which widespread protein replacement in situ is essential for treatment of the underlying genetic disorder. Somatic gene therapy will require efficient, scale-independent transport of DNA-containing macromolecular complexes too large to cross the continuous endothelia under physiological conditions. Previous studies in large-animal models have revealed a trade-off between the efficiency of gene transfer and the inherent safety of the required surgical and pharmacological interventions to achieve this. METHODS AND RESULTS: Rats and dogs underwent limb or hemibody isolation via atraumatic tourniquet placement or myocardial isolation via heterotopic transplantation. Recombinant adenovirus (10(13) particles per kilogram) or recombinant adeno-associated virus (10(14) genome copies/kg) encoding the lacZ transgene was delivered through pressurized venous infusion without pharmacological mediators. Muscle exhibited almost 100% myofiber transduction in rats and dogs by X-galactosidase staining and significantly higher beta-galactosidase levels compared with nonpressurized delivery. No significant difference was seen in beta-galactosidase levels between 100- or 400-mm Hg groups. The <50-mm Hg group yielded inhomogeneous and significantly lower transgene expression. CONCLUSIONS: Uniform scale- and vector-independent skeletal and cardiac myofiber transduction is facilitated by pressurized venous infusion in anatomic domains isolated from the central circulation without pharmacological interference with cardiovascular homeostasis. We provide the first demonstration of uniform gene transfer to muscle fibers of an entire extremity in the dog, providing a firm foundation for further translational studies of efficacy in canine models for human diseases.
Assuntos
Dependovirus/genética , Músculo Esquelético/fisiologia , Animais , Cães , Técnicas de Transferência de Genes , Vetores Genéticos , Coração , Transplante de Coração/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , beta-Galactosidase/genéticaRESUMO
BACKGROUND: Several 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. METHODS: We 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. RESULTS: In 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. CONCLUSION: This 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.
Assuntos
Adaptação Fisiológica , Diafragma/fisiopatologia , Enfisema/induzido quimicamente , Enfisema/fisiopatologia , Cadeias Pesadas de Miosina/metabolismo , Elastase Pancreática , Animais , ATPases Transportadoras de Cálcio/metabolismo , Diafragma/metabolismo , Enfisema/metabolismo , Imuno-Histoquímica , Medidas de Volume Pulmonar , Contração Muscular , Cadeias Pesadas de Miosina/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Transportadoras de Cálcio do Retículo SarcoplasmáticoRESUMO
BACKGROUND: The available techniques for intravascular gene delivery to the heart are inefficient and not organ-specific. Yet, effective treatment of heart failure will likely require transgene expression by the majority of cardiac myocytes. To address this problem, we developed a novel cannulation technique that achieves efficient isolation of the heart in situ using separate cardiopulmonary bypass (CPB) circuits for the heart and body in dogs. METHODS: The arterial inflow and venous effluent from the two circuits were physically isolated. The efficiency of separation was 98% to 99% in three preliminary experiments using Evans Blue dye-labeled albumin. In 6 dogs, the cardiac circuit was perfused with oxygenated crystalloid cardioplegia at 37 degrees C containing approximately 4 x 10(11) particles of an adenovirus encoding LacZ (AdCMVLacZ) with a perfusion pressure of 170 to 200 mm Hg for 15 minutes allowing virus to recirculate through the heart approximately 15 times. Cross-clamp time was 26 +/- 2 minutes and CPB time was 90 +/- 3 minutes. RESULTS: Five animals survived and were euthanized at 7 days. Beta-galactosidase activities measured using a chemiluminescent assay were three orders of magnitude higher in all areas of the heart than in the liver. Histological analyses revealed heterogeneous X-Gal staining of myocytes in all areas of the myocardium. CONCLUSIONS: Despite using a constitutive promoter, this technique yields relatively cardiac-specific transgene expression and is potentially translatable to clinical applications. Future studies will allow for further optimization of the conditions necessary for vector-mediated gene delivery to the heart.
Assuntos
Ponte Cardiopulmonar/métodos , Miocárdio , Transgenes , Adenoviridae/genética , Albuminas , Animais , Cães , Técnicas de Transferência de Genes , Coração/diagnóstico por imagem , Óperon Lac/genética , Miocárdio/patologia , CintilografiaRESUMO
BACKGROUND: Some patients develop recrudescence after a malignant hyperthermia (MH) reaction, but it is not clear which patients are at risk. The authors analyzed clinical variables associated with recrudescence after a clinical MH episode. METHODS: Data were obtained from Adverse Metabolic Reaction to Anesthesia reports in the North American Malignant Hyperthermia Registry. Patients who underwent general anesthesia and with an MH clinical grading score of 20 or greater, indicating a likely MH reaction, were included in this analysis. Patient characteristics, anesthetic agents, MH reaction clinical details, and postoperative outcomes were compared in the recrudescence and no recrudescence groups using chi-square tests and Z tests for categorical variables and the Student t test for continuous variables. Associations of clinical variables with recrudescence were assessed with univariate and multivariate logistic regression. RESULTS: Of 308 patients, 63 (20%) had recrudescence. The mean time from initial reaction to recrudescence was 13 h (SD = 13 h). Patients with recrudescence were more likely to have a muscular body type (P < 0.01), malignant hyperthermia score of 35 or greater (P < 0.01), a temperature increase (P < 0.01), and more than 150 min from induction to adverse reaction (P < 0.05). Muscular body type, a temperature increase, and a longer time from induction to initial malignant hyperthermia reaction were associated with recrudescence on multivariate logistic regression analysis. CONCLUSIONS: Recrudescence occurred in 20% of patients. Muscular body types had a higher rate of recrudescence, perhaps associated with increased muscle mass. The risk of recrudescence increased as time from induction to the initial MH reaction increased, perhaps as a result of greater muscle exposure to triggering agents.
Assuntos
Hipertermia Maligna/etiologia , Adolescente , Adulto , Idoso , Temperatura Corporal , Estudos de Casos e Controles , Criança , Pré-Escolar , Dantroleno/uso terapêutico , Feminino , Humanos , Lactente , Recém-Nascido , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Recidiva , Fatores de TempoRESUMO
The mammalian skeletal myosin heavy chain locus is composed of a six-membered family of tandemly linked genes whose complex regulation plays a central role in striated muscle development and diversification. We have used publicly available genomic DNA sequences to provide a theoretical foundation for an experimental analysis of transcriptional regulation among the six promoters at this locus. After reconstruction of annotated drafts of the human and murine loci from fragmented DNA sequences, phylogenetic footprint analysis of each of the six promoters using standard and Bayesian alignment algorithms revealed unexpected patterns of DNA sequence conservation among orthologous and paralogous gene pairs. The conserved domains within 2.0 kilobases of each transcriptional start site are rich in putative muscle-specific transcription factor binding sites. Experiments based on plasmid transfection in vitro and electroporation in vivo validated several predictions of the bioinformatic analysis, yielding a picture of synergistic interaction between proximal and distal promoter elements in controlling developmental stage-specific gene activation. Of particular interest for future studies of heterologous gene expression is a 650-base pair construct containing modules from the proximal and distal human embryonic myosin heavy chain promoter that drives extraordinarily powerful transcription during muscle differentiation in vitro.
Assuntos
Família Multigênica , Músculo Esquelético/fisiologia , Cadeias Pesadas de Miosina/genética , Animais , Sequência de Bases , Sequência Conservada , DNA/química , DNA/genética , Primers do DNA , Eletroporação , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/embriologia , Filogenia , Regiões Promotoras Genéticas , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Ativação TranscricionalRESUMO
Sarcomeric myosin heavy chain (MyHC) is the major contractile protein of striated muscle. Six tandemly linked skeletal MyHC genes on chromosome 17 and two cardiac MyHC genes on chromosome 14 have been previously described in the human genome. We report the identification of three novel human sarcomeric MyHC genes on chromosomes 3, 7, and 20, which are notable for their atypical size and intron-exon structure. Two of the encoded proteins are structurally most like the slow-beta MyHC, whereas the third one is closest to the adult fast IIb isoform. Data from pairwise comparisons of aligned coding sequences imply the existence of ancestral genomes with four sarcomeric genes before the emergence of a dedicated smooth muscle MyHC gene. To further address the evolutionary relationships of the distinct sarcomeric and nonsarcomeric rod sequences, we have identified and further annotated human genomic DNA sequences corresponding to 14 class-II MyHCs. An extensive analysis provides a timeline for intron gain and loss, gene contraction and expansion, and gene conversion among genes encoding class-II myosins. One of the novel human genes is found to have introns at positions shared only with the molluscan catchin/MyHC gene, providing evidence for the structure of a pre-Cambrian ancestral gene.