Catherine M. Pollina

Cell and fiber type distribution of dystrophin

Duchenne muscular dystrophy is the result of dystrophin deficiency. We have determined the cell types likely to express the pathogenic effects of this neuromuscular disease by determining the pattern of dystrophin expression in normal cells. We find that all physiological types of muscle cells express dystrophin at similar levels, and that the dystrophin content of various tissues correlates with the myogenic cell population of each tissue. The dystrophin content of brain and spinal cord, however, is found not to correlate with any type of muscle cell, and it is suggested that neurons express dystrophin. The potential involvement of striated muscle fibers, the vasculature, and the nervous system in the etiology of Duchenne muscular dystrophy makes it likely that the disease is a complex disorder of combined pathogenesis. We also find that the dystrophic chicken does not represent an animal model for dystrophin deficiency.

Pre-clinical screening of drugs using the mdx mouse

The genetically dystrophin-deficient mdx mouse, with its characteristic and regular exercise-induced loss of strength, is a useful experimental platform on which to screen potential drug therapies in the treatment of some dystrophic diseases. Pharmacological agents of several chemical and functional classes were examined in their ability to reduce the loss of muscular strength in young exercised mdx mice. Therapeutic intervention over the period 4-10 weeks of age was evaluated in weekly tests of whole-body strength. This age period represents the most severe manifestation of disease in these animals. Significant improvements in whole-body strength were brought about by treatment with the immunosuppressive and anti-inflammatory drugs prednisone (at low dose only, 1 mg/kg body weight), pentoxifylline (100 mg/kg) and tinset (100 mg/kg). The anabolic hormone insulin-like growth factor-1 (5 mg/kg), as well as the amino acids/metabolites glutamine (10 mg/kg), glutamine plus alanine (each 10 mg/kg), and creatinine (10 mg/kg) all improved strength test performance. The mdx mouse is a responsive system for the screening of potential therapeutic treatments for the muscular dystrophies.

Natriuretic peptides in fish physiology.

Natriuretic peptides exist in the fishes as a family of structurally-related isohormones including atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and ventricular natriuretic peptide (VNP); to date, brain natriuretic peptide (or B-type natriuretic peptide, BNP) has not been definitively identified in the fishes. Based on nucleotide and amino acid sequence similarity, the natriuretic peptide family of isohormones may have evolved from a neuromodulatory, CNP-like brain peptide. The primary sites of synthesis for the circulating hormones are the heart and brain; additional extracardiac and extracranial sites, including the intestine, synthesize and release natriuretic peptides locally for paracrine regulation of various physiological functions. Membrane-bound, guanylyl cyclase-coupled natriuretic peptide receptors (A- and B-types) are generally implicated in mediating natriuretic peptide effects via the production of cyclic GMP as the intracellular messenger. C- and D-type natriuretic peptide receptors lacking the guanylyl cyclase domain may influence target cell function through G(i) protein-coupled inhibition of membrane adenylyl cyclase activity, and they likely also act as clearance receptors for circulating hormone. In the few systems examined using homologous or piscine reagents, differential receptor binding and tissue responsiveness to specific natriuretic peptide isohormones is demonstrated. Similar to their acute physiological effects in mammals, natriuretic peptides are vasorelaxant in all fishes examined. In contrast to mammals, where natriuretic peptides act through natriuresis and diuresis to bring about long-term reductions in blood volume and blood pressure, in fishes the primary action appears to be the extrusion of excess salt at the gills and rectal gland, and the limiting of drinking-coupled salt uptake by the alimentary system. In teleosts, both hypernatremia and hypervolemia are effective stimuli for cardiac secretion of natriuretic peptides; in the elasmobranchs, hypervolemia is the predominant physiological stimulus for secretion. Natriuretic peptides may be seawater-adapting hormones with appropriate target organs including the gills, rectal gland, kidney, and intestine, with each regulated via, predominantly, either A- or B-type (or C- or D-type?) natriuretic peptide receptors. Natriuretic peptides act both directly on ion-transporting cells of osmoregulatory tissues, and indirectly through increased vascular flow to osmoregulatory tissues, through inhibition of drinking, and through effects on other endocrine systems.

In Vivo Effects of Protease Inhibitors on Chickens with Hereditary Muscular Dystrophy

Beginning on day 4 ex ovo, and every 3 d thereafter, genetically dystrophic Line 413 chickens were given intraperitoneal injections (4 mg/kg body wt) of a protease inhibitor, leupeptin, pepstatin, or antipain. Experimental chickens received protease inhibitors dissolved in a water:ethanol:dimethyl sulfoxide solution (50:40:10, vol:vol:vol). Control untreated animals received diluent injections. Untreated dystrophic chickens typically reach around day 30 ex ovo a maximum ability to right from the supine position in a standardized functional test for muscle weakness. After day 30 ex ovo, the dystrophic chickens are found to decline progressively in their ability to right, compared with normal, nondystrophic controls, which have an unimpaired ability to right. Concomitantly, dystrophic chickens exhibit characteristically high levels of plasma creatine phosphokinase enzyme activity. In addition, an increased frequency of degenerating, regenerating, and vacuolated myofibers, and inflammatory cells appear in the affected pectoralis major muscles from the dystrophic chicken. Throughout the duration of the trial, there was no major enhancement in the functional righting ability of dystrophic chickens receiving any one of the protease inhibitors tested. However, there was a significant reduction in the abnormally high levels of plasma creatine phosphokinase in the treated chickens. Also, there was an apparent reduction in the mean number of vacuolated fibers in the pectoralis muscle from the protease inhibitor-treated birds. No significant reductions were observed in the relative frequency of degenerating and regenerating myofibers or inflammatory cells. In addition to the plasma creatine phosphokinase decrease, however, therapeutic benefit was seen in 31.0, 30.5, and 14.8% increases in the wet weight (and total noncollagen protein) of pectoralis muscle from dystrophic chickens receiving leupeptin, pepstatin or antipain, respectively.

Extracellular calcium-sensing receptor distribution in osmoregulatory and endocrine tissues of the tilapia.

The extracellular calcium-sensing receptor (CaSR) serves an important detector function in vertebrate Ca(2+) homeostasis. In this study, we surveyed using immunohistochemistry the tissue and cellular distribution of the CaSR protein in the Mozambique tilapia (Oreochromis mossambicus) and the Japanese eel (Anguilla japonica). Specifically, we examined receptor expression in ion-transporting barrier tissues that may be directly responsive to extracellular Ca(2+) levels, and in tissues that are implicated in endocrine signaling to homeostatic effectors such as Ca(2+)-transporting epithelia. In tilapia osmoregulatory tissues, CaSR protein is strongly expressed in proximal segments of renal tubule, but not in distal segments (where Na(+),K(+)-ATPase is prominently expressed) or in glomeruli. The receptor was also localized in the ion-transporting mitochondria-rich cells of gill and in ion- and nutrient-transporting epithelia of middle and posterior intestine. Consistent with our earlier RT-PCR assessment of mRNA expression in tilapia, CaSR protein expression was salinity dependent in some osmoregulatory tissues. In tilapia pituitary gland, CaSR expression was observed in the rostral pars distalis (containing prolactin-secreting cells, and in the pars intermedia (containing somatolactin-secreting and melanocyte-stimulating hormone-secreting cells), with notably greater expression in the latter. In the eel, weak immunostaining was seen in the stanniocalcin-secreting cells of the corpuscles of Stannius. Olfactory lobe CaSR expression suggests an environment-sensing role for the receptor. Altogether, these findings support the involvement of CaSR in piscine Ca(2+) homeostasis at the levels of environmental sensing, of integrative endocrine signaling through both hypercalcemic (prolactin, and perhaps somatolactin) and hypocalcemic (stanniocalcin) hormones, and of direct local regulation of Ca(2+)-transporting tissues.

Duchenne-like myopathy in double-mutant mdx mice expressing exaggerated mast cell activity

Dystrophin-deficient female mdx mice were bred with male Tsk+/+ pa mice to examine the role played by mast cells in the pathophysiology of dystrophin deficiency. Resultant mdx/Tsk double-mutant mice were then examined functionally, biochemically, and histologically. While mdx mice remained as strong as their normal counterparts, mdx/Tsk double-mutant mice became progressively weak with age. Serum creatine kinase activity was significantly elevated in both mdx and mdx/Tsk double-mutant mice over normal controls. However, mast cell-derived plasma tryptase activity was consistently higher in the double-mutant than in mdx mice. In addition, histological examination of gastrocnemius muscle revealed that while necrosis was persistent in both strains of mdx mice from 2 to 8 weeks of age, regeneration was significantly reduced in the double-mutant mice. Of particular interest was the fact that necrosis in the mdx/Tsk double mutant exceeded mdx values at 8 weeks of age, corresponding approximately with a second peak in tryptase activity. Therefore, heightened mast cell activity appears to elicit in the dystrophin-deficient mdx mouse a myopathy not unlike the human Duchenne disease.

MDX Mouse as Therapeutic Model System: Development and Implementation of Phenotypic Monitoring

As Michael Brooke so aptly stated at the beginning of this session on phenotypic monitoring, it is imperative to have a quantifiable test system in place prior to assessing treatment entities for any of the muscular dystrophies (Brooke et al., 1981). In this manner, the various medical, experimental, and ethical considerations have been resolved in advance providing an unequivocal foundation for determining efficacy (regardless of the particular therapeutic approach under consideration). Similarly, in the preclinical study of myopathic animal models, it is equally pertinent to establish reliable phenotypic endpoints before the implementation of a therapeutic study. Hence, efficacy or a lack thereof can be objectively and rationally determined against a backdrop of standardized markers of the disease.

Limited benefit to genetically dystrophic chickens from a synthetic proteinase inhibitor: Ep475

Chickens with inherited muscular dystrophy (Line 413) were treated in two separate trials with daily intraperitoneal injections of 10% DMSO-water solutions containing the proteinase inhibitors, Ep475 and E64. Drug therapy in each case significantly prolonged the functional ability of the treated chickens. Diluent control chickens around day 35 ex ovo characteristically reached a maximum ability to right from the supine position in a standardized functional test for muscle weakness. Subsequently, the control chickens were found to decline progressively in their ability to right. Treatment with the proteinase inhibitors had no effect on the typically elevated levels of plasma creatine kinase activity. In a histological analysis of the affected pectoralis major muscle, drug treatment had no effect on the relative distribution of degenerating, and vacuolated fibers, inflammatory cells, and abnormal fiber diameters. An exception was seen in decreased necrotic fibers of chickens treated with high doses of Ep475. Moreover, both inhibitors had positive effects on two biochemical abnormalities common to the dystrophic pectoralis muscle: increase in noncollagen protein, and reduction in total calcium.

Abnormal expression of the calmodulin gene in muscle from the dystrophic chicken

Compared to that of genetically-related normal chickens, pectoralis muscle from the dystrophic chicken contained increased calmodulin measured by radioimmunoassay. Determined by the dot blot procedure, expression of the calmodulin gene was enhanced in muscle from affected animals. The bioactivity of the gene product was normal. Together with previous studies reporting increased cell Ca2+ content in dystrophic muscle, the current findings of increased sarcoplasmic calmodulin suggest the latter is a cellular response to defective Ca2+ transport at the level of cell efflux or intracellular organelle (sarcoplasmic reticulum) uptake.

Skeletal tissues in Mozambique tilapia (Oreochromis mossambicus) express the extracellular calcium-sensing receptor

Molecular phylogenetic analysis suggests that the extracellular calcium-sensing receptor (CaSR) emerged evolutionarily in association with the chordate-vertebrate lineage. Our studies overall explore the evolution of CaSRs, and the possible historical linkage of CaSRs to vertebrate skeleton as functional components of calcium homeostasis through regulated storage and/or release. We applied both reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) to evaluate Casr gene and CaSR protein expression, respectively, in skeletal tissues of a cichlid teleost, the Mozambique tilapia (Oreochromis mossambicus). By RT-PCR, CaSR gene (Casr) expression was observed in skull and vertebral column (including notochordal tissues). Relative to skeleton, IHC revealed CaSR protein expression in notochordal sheath cells within the vertebral canal, in scleroblasts associated with body surface scales, and in chondrocytes within hyaline cartilage. Although closely apposed cells border the acellular bone in tilapia, these cells were only weakly immunostained. We conclude, therefore, that CaSR is expressed in skeletal tissues of tilapia, an advanced teleost fish, and that Casr may be part of a genetic network associated with vertebrate skeletal system. Our immunohistochemical examination also newly revealed CaSR protein expression in epidermis and red muscle of fishes.