ORIGINAL PAPER Andrei B. Borisov Æ Sarah B. Sutter Aikaterini Kontrogianni-Konstantopoulos Robert J. Bloch Æ Margaret V. Westfall Mark W. Russell Essential role of obscurin in cardiac myoﬁbrillogenesis and hypertrophic response: evidence from small interfering RNA-mediated gene silencing Accepted: 2 August 2005 / Published online: 5 October 2005 Ó Springer-Verlag 2005 Abstract Obscurin is a recently identiﬁed giant multi- domain 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 a-actinin localization remained mainly unaﬀected in siRNA-transfected cells, obscurin depletion did cause the defective lateral alignment of myoﬁbrillar bundles, leading to their abnormal bifur- cation, 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 obscu- rin siRNA-treated cells. These results suggest that ob- scurin 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 myoﬁb- rillogenesis and hypertrophic growth. Keywords Obscurin Æ Cardiac myocytes Æ Myoﬁbrillogenesis Æ siRNA Æ a-Actinin Æ Myosin Æ Titin Æ Hypertrophy Introduction The molecular mechanisms underlying the proper assembly and three-dimensional integration of sarco- meric ﬁlaments within nascent myoﬁbrils during cardiac diﬀerentiation and hypertrophy are not completely understood. The studies performed over the last decade clearly demonstrated that loss-of-function mutations in structural muscle proteins result in the impairment of the contractile function and can lead to severe myocar- dial pathology including dilated and hypetrophic car- diomyopathy and congestive heart failure (Carlsson and Thornell 2001; Roberts and Sigwart 2001; Sehnert et al. 2002; Mohapatra et al. 2003; Ba¨r et al. 2004; Stefanelli et al. 2004; Kasahara 2005; Van Driest et al. 2005). Recently, a novel component of the sarcomere, a large multidomain 800 kDa protein named obscurin, was identiﬁed in cardiac and skeletal muscle. Obscurin, localized mainly to M- and Z-lines in mature myoﬁ- brils, became the third known member of the family of giant muscle proteins that includes titin and nebulin/ nebulette. Sequence analysis of the obscurin gene demonstrated that it encodes about 67 linked immu- noglobulin domains and two ﬁbronectin-3-like domains (Bang et al. 2001; Young et al. 2001; Russell et al. 2002). The structural organization of immunoglobulin A. B. Borisov (&) Æ S. B. Sutter Æ M. W. Russell (&) Division of Pediatric Cardiology Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Room 8200, MSRB III, Ann Arbor, MI 48109, USA E-mail: aborisov@umich.edu E-mail: mruss@umich.edu Tel.: +1-734-9368663 Fax: +1-734-6151386 A. Kontrogianni-Konstantopoulos Æ R. J. Bloch Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA M. V. Westfall Department of Molecular and Integrative Physiology and Department of Surgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA Histochem Cell Biol (2006) 125: 227–238 DOI 10.1007/s00418-005-0069-x