Essential Role of Obscurin in Cardiac Myofibrillogenesis and Hypertrophic
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Histochem Cell Biol (2006) 125: 227–238 DOI 10.1007/s00418-005-0069-x 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- sarcomeric myosin labeling, and an occasional irregular domain muscle protein (800 kDa) whose structural periodicity of sarcomere spacing were typical of obscu- and regulatory functions remain to be defined. The goal rin siRNA-treated cells. These results suggest that ob- of this study was to examine the effect of obscurin gene scurin is indispensable for spatial positioning of silencing induced by RNA interference on the dynamics contractile proteins and for the structural integration of myofibrillogenesis and hypertrophic response to and stabilization of myofibrils, especially at the stage of phenylephrine in cultured rat cardiomyocytes. We found myosin filament incorporation and A-band assembly. that that the adenoviral transfection of short interfering This demonstrates a vital role for obscurin in myofib- RNA (siRNA) constructs targeting the first coding exon rillogenesis and hypertrophic growth. of obscurin sequence resulted in progressive depletion of cellular obscurin. Confocal microscopy demonstrated Keywords Obscurin Æ Cardiac myocytes Æ that downregulation of obscurin expression led to the Myofibrillogenesis Æ siRNA Æ a-Actinin Æ Myosin Æ impaired assembly of new myofibrillar clusters and Titin Æ Hypertrophy considerable aberrations of the normal structure of the contractile apparatus. While the establishment of the initial periodic pattern of a-actinin localization remained Introduction mainly unaffected in siRNA-transfected cells, obscurin depletion did cause the defective lateral alignment of The molecular mechanisms underlying the proper myofibrillar bundles, leading to their abnormal bifur- assembly and three-dimensional integration of sarco- cation, dispersal and multiple branching. Bending of meric filaments within nascent myofibrils during cardiac immature myofibrils, apparently associated with the loss differentiation and hypertrophy are not completely of their rigidity, a modified titin pattern, the absence of understood. The studies performed over the last decade well-formed A-bands in newly formed contractile clearly demonstrated that loss-of-function mutations in structures as documented by a diffuse localization of 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 A. B. Borisov (&) Æ S. B. Sutter Æ M. W. Russell (&) Thornell 2001; Roberts and Sigwart 2001; Sehnert et al. Division of Pediatric Cardiology Department of Pediatrics and 2002; Mohapatra et al. 2003;Ba¨r et al. 2004; Stefanelli Communicable Diseases, University of Michigan Medical School, et al. 2004; Kasahara 2005; Van Driest et al. 2005). Room 8200, MSRB III, Ann Arbor, MI 48109, USA E-mail: [email protected] Recently, a novel component of the sarcomere, a E-mail: [email protected] large multidomain 800 kDa protein named obscurin, Tel.: +1-734-9368663 was identified in cardiac and skeletal muscle. Obscurin, Fax: +1-734-6151386 localized mainly to M- and Z-lines in mature myofi- A. Kontrogianni-Konstantopoulos Æ R. J. Bloch brils, became the third known member of the family of Department of Physiology, University of Maryland giant muscle proteins that includes titin and nebulin/ School of Medicine, Baltimore, MD 21201, USA nebulette. Sequence analysis of the obscurin gene demonstrated that it encodes about 67 linked immu- M. V. Westfall Department of Molecular and Integrative Physiology and noglobulin domains and two fibronectin-3-like domains Department of Surgery, University of Michigan Medical School, (Bang et al. 2001; Young et al. 2001; Russell et al. Ann Arbor, MI 48109, USA 2002). The structural organization of immunoglobulin 228 domains of obscurin resembles the elastic region of ti- To determine the functional role of obscurin in the tin, and the fibronectin-3 domains share some similarity assembly and maintenance of the contractile apparatus, with the adhesive domains of several other contractile in this study we examined the structure of nascent and proteins (Young et al. 2001). Obscurin also contains mature myofibrils and the progression of myofibrillo- Rho-guanine nucleotide exchange factor (Rho-GEF) genesis induced by phenylephrine in cardiac myocytes and a calmodulin-binding IQ motif, which suggests its following siRNA-mediated obscurin gene silencing in involvement in signal transduction in sarcomeres and cell culture. regulation of the contractile function (Young et al. 2001; Bang et al. 2001; Russell et al. 2002). Earlier we found that the expression of obscurin is upregulated in vivo in hypertrophic murine hearts subjected to Materials and methods ascending aortic banding and that it is rapidly incor- porated in newly forming myofibrils in vitro following Preparation of adenoviral-RNAi constructs stimulation of the hypertrophic response (Borisov et al. 2003). This protein appears to be the vertebrate Short interfering RNA constructs were designed to in- homologue of the unc-89 gene (Sutter et al. 2004) that hibit the expression of the first coding exon of the rat has been demonstrated to be involved in the M-band obscurin gene (nucleotides 1–600 of the coding se- assembly in Caenorhabditis elegans (Benian et al. 1996; quence). Target siRNA sequences were selected based Small et al. 2004). on Tuschl’s principles (Elbashir et al. 2002). BLASTn It has been recently shown that mutations in the sequence homology searches [http://www.ncbi.nlm.nih.- genes encoding other giant muscle proteins, titin and gov/BLAST/databases:non-redundant and EST] were nebulin, have significant functional consequences and used to confirm unique specificity of the RNAi con- are associated with the development of cardiac and structs for the rat obscurin transcript. Potential target skeletal myopathies in humans (Hein and Schaper 2002; sequences that demonstrated greater than 70% sequence Itoh-Satoh et al. 2002; McElhinny et al. 2003; Anderson similarity with a rat cDNA transcript other than ob- et al. 2004; Tskhovrebova and Trinick 2005). This sug- scurin were excluded from further consideration. Three gests that the abnormal functional expression of these different siRNA target sites and one control sequence proteins has direct clinical implications and can explain provided by Ambion, Inc., Austin, TX, USA were the etiology and mechanisms of at least some types of selected: ratObs5¢BH143 (GTGGAGGCAGGAG- muscle pathology. CACGCT), ratObs5¢BH373 (GACGCCACCTTCCGA- The precise functional role of obscurin in muscle TGTC), ratObs5¢BH416 (GCTGTGAGCTGGTCC tissue is still poorly characterized. It remains unclear AAAGA), and NEGCTL (ACTACCGTTGTTATA how important is obscurin for progression of myofib- GGTG). The siRNA target sites were converted into rillogenesis and for the stability of structural organiza- oligonucleotide sequences per instructions supplied with tion and integrity of myofibrils. Cell cultures of cardiac the pSilencerTM kit (Ambion Inc., Austin, TX, USA). myocytes represent an established experimental model For each template, 2 lg of the target and complemen- that has been successfully used in studies of normal, tary oligonucleotides were heated at 95° for 5 min, then induced, and genetically modified patterns of sarcomeric cooled to room temperature in annealing buffer protein expression (e.g., Eppenberger et al. 1994; (100 mM potassium acetate, 30 mM HEPES-potassium Mitcheson et al. 1998; Person et al. 2000; Weikert et al. Hydroxide pH 7.4, 2 mM magnesium acetate). Duplex 2003). The heart cells isolated in vitro actively respond DNA was directionally cloned into the U6 promoter to a number of physiological and pharmacological driven expression vector, pSilencer 2.0-U6 (Ambion stimuli including the hypertrophic ones, and the molec- Inc., Austin, TX, USA). The U6 promoter and RNAi ular mechanisms mediating the hypertrophic response construct were then subcloned into the EcoRI and are very similar in situ and cell culture (for literature see HindIII sites of the adenoviral shuttle vector, pAC- Hefti et al. 1997; Schaub et al. 1997, 1998; Komuro and pL+loxP-SSP. AdBH143, AdBH373, AdBH416 and Yazaki 2002; Opie 2004). AdNEGCTL were generated by Cre-lox recombination During the last several years, short interfering RNAs in vitro according to earlier published procedures (Aoki (siRNAs) became a new tool for the analysis of gene et al. 1999). Recombination occurred in a cell-free sys- functions by selective and specific silencing of targeted tem consisting of only the viral and shuttle plasmids and homologous nucleotide sequences at the transcriptional Cre recombinase. Viral particles were produced in 293 level (reviewed in Chi et al. 2003; Montgomery 2004; cell line by transfection. After harvesting and lysing the Dorsett and Tuschl 2004; Campbell and Choy 2005; infected cells, adenoviral lysates were sterilized by pas- Huppi et al. 2005; Tomari and Zamore 2005). Trans- sage through 45 lm filters and titered using standard fection with replication-defective viral vectors has been methods. The detailed characterization of the