Model Of Human Cardiomyopathy Research Articles

Abstract 381: Ex vivo Heart Function Analysis in Adult Zebrafish

Zebrafish ( Danio rerio ) is an efficient vertebrate model of human cardiomyopathy which is amenable to the medium throughput screening approaches opening opportunities to search new genetic modifiers via mutagenesis screening and assessing compound-based therapies at larger scale. The advent of genome editing technology enables the generation of a panel of genetic models of cardiomyopathy with mutations in leading cardiomyopathy genes. However, one of the major bottlenecks for adult zebrafish as a cardiomyopathy model is the lack of appropriate cardiac functional assays. Due to small heart size, in vivo methods such as those based on echocardiography, are limited by their insufficient resolution. Here, we report the development of an ex vivo approach aimed to facilitate phenotyping in adult zebrafish. We show that our method is able to quantify parameters of pump function of the heart, including end-diastolic/systolic length/volumes, ejection and shortening fractions, and velocities of contraction/relaxation. We defined the basic parameters of these indices using different wild-type strains, age, and sex, and then demonstrated that our method can be useful in definition of progression of pathogenesis of both acquired (doxorubicin-injected) and inherited cardiomyopathy models. We conclude that our novel approach shall facilitate cardiac phenotyping in adult zebrafish models of heart diseases.

In vivo gene supplementation for the therapy of cardiomyopathy

We have identified the gene defect and breakpoint of δ-sarcoglycan (SG) in cardiomyopathic (CM) BIO 14.6 hamsters, which are a representative model of human cardiomyopathy. The aim of the present study is to explore a gene therapy using gene transfection to in vivo myocardial cells that we have previously established. Liposomes containing a reporter gene (β-galactosidase, β-Gal) with or without the δ-SG gene were coated with UV-inactivated hemagglutinating virus of Japan and injected into the left ventricular free wall in anesthetized open-chest CM hamsters. We prepared site-directed antibody to the extracellular domain of the δ-SG protein, the amino acid sequence of which was deduced from the cloned cDNA. Affinity-purified antibody demonstrated a single band after Western blotting in the normal Syrian hamster heart but no band in the CM heart. On day 7 after the transfection, hearts were removed and fixed. The adjacent cryosections were immunostained with antibody to β-Gal or δ-SG using the ABC procedure. The δ-SG gene product was clearly expressed in both sarcolemma and cytoplasm of cardiomyocytes of CM hamsters, whereas the β-Gal protein was exclusively coexpressed in the cytosol, showing the efficient cotransfection of both β-Gal and δ-SG genes and the slow protein traffic to the sarcolemma after biosynthesis of δ-SG in cytoplasm. We have succeeded in an efficient expression of the δ-SG gene in living CM hamster hearts with deleted δ-SG gene, suggesting that the newly developed in vivo gene transfection might be promising for the rescue of targeted CM cells.

Morphological investigations of the myocardium ofcardiomyopathic hamsters during the postnatal development and experimental hypoxia. A quantitative ultrastructural study

The hereditary cardiomyopathy of the Syrian hamster (strain BIO 8262) as a model of human cardiomyopathy was investigated during postnatal development using ultrastructural morphometric analysis. The aim of the study was to establish basic ultrastructural morphometric data of cardiomyocytes of this model during all stages of the disease in comparison to corresponding stages of normal development, and to compare the ultrastructural resistance of normal and myopathic myocardium against acute hypoxic stress. The postnatal development of this cardiomyopathy is characterized by focal myolytic lesions already in the first postnatal days, extended myofibrillic and mitochondrial damage developing after the 1st month, and features of hypertrophy, disarray of myofibrils, thickening, and clumping of z-lines after the 4th month. Morphometric parameters of myopathic cardiomyocytes differing from those of the control are diminished volume density of myofibrils by about 10% of the control values in middle age, slightly increased volume density of sarcoplasmic reticulum and t-tubules in the 1st months, significant increase of them after the 3rd month, and significant alterations of many mitochondrial parameters indicating degenerations (numeric density, volume density, average volume, volume density of cristae, and of destroyed areas, and ratio of surface to volume density of mitochondrial cristae). Acute isobaric hypoxia was applied to myopathic and healthy hamsters of corresponding age (at day 100). Evaluation of ultrastructural morphometric parameters of cardiomyocytes revealed that especially mitochondria were more affected by hypoxia in the myopathic animals. The average mitochondrial volume was significantly more increased in the myopathic condition, the ratio of surface to volume density of cristae significantly diminished. Our ultrastructural - morphometric study has shown, that morphometric parameters in general can be assigned to the stages of cardiomyopathy, and that the ultrastructural differences are best expressed about the 4th month.