15 INTRODUCTION Hypertrophic and dilated cardiomyopathies are major causes of human heart failure and mortality. Familial forms of these pathologies have led to investigation of the genetic causes, and there is a growing body of evidence that suggests a role for mutations in cardiac cytoskeletal genes (Chien, 2000; Seidman and Seidman, 2001). Cytoskeletal components located in the Z- disc have previously been recognized as critical for normal cardiac function. The Z-discs define the lateral boundaries of sarcomeres and constitute anchoring sites for actin and titin filaments, crosslinked by -actinin. It is now well established that Z-disc components are essential for mechanical stretch sensing and sarcomere integrity (Clark et al., 2002). Loss or mutation of Z-disc proteins such as -actinin-associated LIM protein (Pashmforoush et al., 2001), Cypher/ZASP (Vatta et al., 2003; Zhou et al., 2001), muscle LIM protein/CRP3 (Arber et al., 1997; Geier et al., 2003; Knöll et al., 2002; Mohapatra et al., 2003), titin (Gerull et al., 2002) and Tcap, also known as telethonin (Hayashi et al., 2004; Moreira et al., 2000), all lead to hypertrophic or dilated cardiomyopathy in mouse models as well as in human patients. Loss of Drosophila MLP (Mlp84B) leads to developmental arrest at the pupal stage. Specifically, the muscle-dependent morphogenetic movements necessary for pupation are severely compromised in Mlp84B mutants (Clark et al., 2007). These defects may be explained by muscle weakness, and the observation that the few mlp84B –/– adult escapers fail in a flight test is consistent with this hypothesis (Clark et al., 2007). However, the cause of lethality remains unclear. Mlp84B shares many features with vertebrate MLPs including muscle-specific expression and localization at the Z-disc I-band interface (Stronach et al., 1999). Since vertebrate studies have demonstrated a critical requirement for MLP in cardiac function (Arber et al., 1997) and the MLP/CRP3 residues mutated in human patients are conserved in Drosophila Mlp60A and Mlp84B (Fig.·1A), we hypothesized that Mlp84B is essential for normal cardiac function in Drosophila. Here we show that Mlp84B is expressed in the Drosophila heart from late embryonic stages to adulthood and that mlp84B-deficient Drosophila display bradycardia and heart rhythm abnormalities. Thus, Drosophila provides a new model system in which genetic and physiological tools can be applied in combination to investigate the cardiac stretch-sensing response in vivo. MATERIALS AND METHODS Fly strains w 1118 flies (Drosophila melanogaster) were used as the wild-type control. Crosses were done at 25°C unless otherwise specified. Expression of mlp84B RNA interference (RNAi) was achieved using the standard Gal4/UAS system (Brand and Perrimon, 1993). The drivers used were Daughterless (Da)-Gal4 and tinC4-Gal4 (Lo and Frasch, 2001). UAS-mlp84B-RNAi flies were obtained from a genome-wide RNAi collection (B. Dickson, Institute of Molecular Pathology, Vienna, Austria). The sequence of the RNAi construct was as follows: ACCGATGGCTTGACCGAGGATCA- GATCAGCGCCAACAGGCCCTTCTATAACCCGGACACCA- CGTCAATTAAGGCCCGTGACGGCGAAGGCTGTCCCCGG- TGCGGAGGAGCCGTATTCGCCGCCGAGCAACAGCTGTC- CAAGGGCAAGGTGTGGCACAAGAAGTGCTACAACTGC- SUMMARY Muscle LIM protein (MLP) is a cytoskeletal protein located at the Z-disc of sarcomeres. Mutations in the human MLP gene are associated with hypertrophic and dilated cardiomyopathy. MLP has been proposed to be a key player in the stretch-sensing response, but the molecular mechanisms underlying its function in normal and diseased cardiac muscle have not been established. A Drosophila homolog, Mlp84B, displays a similar subcellular localization at the Z-disc of sarcomeres throughout development and in the adult, suggesting Drosophila as a model to study MLP function. Here we employed genetic ablation and cardiac-specific RNA interference (RNAi) knockdown of mlp84B to investigate its role in heart function. We found that Mlp84B- deficient or heart-specific RNAi knockdown flies exhibit diastolic interval prolongation, heart rhythm abnormalities and a reduced lifespan, while showing no obvious structural phenotype. Our data demonstrate that Mlp84B is essential for normal cardiac function and establish the Drosophila model for the investigation of the mechanisms connecting defective cardiac Z-disc components to the development of cardiomyopathy. Supplementary material available online at http://jeb.biologists.org/cgi/content/full/211/1/15/DC1 Key words: Drosophila, muscle LIM protein, cardiomyopathy, cardiac function, arrhythmia, cytoskeleton, sarcomere. The Journal of Experimental Biology 211, 15-23 Published by The Company of Biologists 2008 doi:10.1242/jeb.012435 The Drosophila muscle LIM protein, Mlp84B, is essential for cardiac function Annabelle Mery 1 , Ouarda Taghli-Lamallem 1 , Kathleen A. Clark 2 , Mary C. Beckerle 2 , Xiushan Wu 3 , Karen Ocorr 1 and Rolf Bodmer 1, * 1 Development and Aging Program, Neuroscience, Aging and Stem Cell Research Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA, 2 Hunstman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA and 3 Center for Heart Development, College of Life Science, Hunan Normal University, Changsha 410081, Hunan Province, Peopleʼs Republic of China *Author for correspondence (e-mail: rolf@burnham.org) Accepted 16 October 2007