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Essential role of obscurin in cardiac myofibrillogenesis and hypertrophic response: evidence from small interfering RNA-mediated gene silencing

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Abstract

Obscurin is a recently identified giant multidomain muscle protein (∼800 kDa) whose structural and regulatory functions remain to be defined. The goal of this study was to examine the effect of obscurin gene silencing induced by RNA interference on the dynamics of myofibrillogenesis and hypertrophic response to phenylephrine in cultured rat cardiomyocytes. We found that that the adenoviral transfection of short interfering RNA (siRNA) constructs targeting the first coding exon of obscurin sequence resulted in progressive depletion of cellular obscurin. Confocal microscopy demonstrated that downregulation of obscurin expression led to the impaired assembly of new myofibrillar clusters and considerable aberrations of the normal structure of the contractile apparatus. While the establishment of the initial periodic pattern of α-actinin localization remained mainly unaffected in siRNA-transfected cells, obscurin depletion did cause the defective lateral alignment of myofibrillar bundles, leading to their abnormal bifurcation, dispersal and multiple branching. Bending of immature myofibrils, apparently associated with the loss of their rigidity, a modified titin pattern, the absence of well-formed A-bands in newly formed contractile structures as documented by a diffuse localization of sarcomeric myosin labeling, and an occasional irregular periodicity of sarcomere spacing were typical of obscurin siRNA-treated cells. These results suggest that obscurin is indispensable for spatial positioning of contractile proteins and for the structural integration and stabilization of myofibrils, especially at the stage of myosin filament incorporation and A-band assembly. This demonstrates a vital role for obscurin in myofibrillogenesis and hypertrophic growth.

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Acknowledgements

The authors gratefully acknowledge support from grants through the Muscle Dystrophy Association (MDA3803) to M.W.R. and A.B.B., the NIH (R01 HL 075093-01) to M.W.R. and A.B.B., and R.J.B. (R01 HL 64304). We are grateful to Chris Edwards and Bruce Donohoe, the members of the Microscopy and Image Analysis Laboratory, for support of this work. We also thank Pavel Borisov for assistance in preparation of the manuscript.

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  1. Division of Pediatric Cardiology Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Room 8200, MSRB III, Ann Arbor, MI, 48109, USA

    Andrei B. Borisov, Sarah B. Sutter & Mark W. Russell

  2. Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Aikaterini Kontrogianni-Konstantopoulos & Robert J. Bloch

  3. Department of Molecular and Integrative Physiology and Department of Surgery, University of Michigan Medical School, Ann Arbor, MI, 48109, USA

    Margaret V. Westfall

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  1. Andrei B. Borisov
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Borisov, A.B., Sutter, S.B., Kontrogianni-Konstantopoulos, A. et al. Essential role of obscurin in cardiac myofibrillogenesis and hypertrophic response: evidence from small interfering RNA-mediated gene silencing. Histochem Cell Biol 125, 227–238 (2006). https://doi.org/10.1007/s00418-005-0069-x

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