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First published online December 14, 2007
Journal of Experimental Biology 211, 15-23 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012435
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The Drosophila muscle LIM protein, Mlp84B, is essential for cardiac function

Annabelle Mery1, Ouarda Taghli-Lamallem1, Kathleen A. Clark2, Mary C. Beckerle2, Xiushan Wu3, Karen Ocorr1 and Rolf Bodmer1,*

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
3 Center for Heart Development, College of Life Science, Hunan Normal University, Changsha 410081, Hunan Province, People's Republic of China


Figure 1
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  		Fig. 1. Developmental cardiac expression and localization of Mlp84B. (A) Alignment of the first LIM domain glycine-rich region from human CRP1, 2 and 3 (CRP3=MLP) and Drosophila Mlp84B and Mlp60A. The first residue highlighted is a mutation associated with human dilated cardiomyopathy (see Knöll et al., 2003), while the other three highlighted residues are associated with cardiac hypertrophy (see Geier et al., 2003Go). (B) Stage 17 embryo stained with Mlp84B antiserum. The heart tube is delineated by arrows. (C) Image montage of a heart tube dissected from a mlp84B::GFP 3rd instar larva. Expression can be seen in sarcomeres as well as in cell nuclei. (D) Posterior part of a wild-type 3rd instar larva heart (segment A6) stained with Mlp84B antiserum. (E–G) Detail of the cardiac myofibrils in the larval heart showing co-localization of {alpha}-actinin and Mlp84B. All scale bars are 50 µm.

 

Figure 2
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  		Fig. 2. Mlp84B co-localizes with {alpha}-actinin at the Z-disc of sarcomeres in the adult heart. (A) Low-magnification image of a cypher-GFP adult Drosophila heart, encompassing two abdominal segments (A3–A4; note the body wall muscles segmentally arranged on either side of the heart tube). (B) Detail of one segment of a cypher-GFP adult heart. Both the inner spiral and outer longitudinal myofibers are clearly visualized with the Cypher-GFP signal. The scale bar is 40 µm. (C–E) High magnification images of the longitudinal myofibrils stained with anti-{alpha}-actinin (C) and anti-Mlp84B (D) antibodies. Mlp84B forms a doublet on either side of the {alpha}-actinin band (E). The scale bar is 20 µm.

 

Figure 3
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  		Fig. 3. Cardiac-specific mlp84B knockdown causes early mortality. (A,B) mlp84B expression level is significantly reduced in mlp84B RNAi-expressing Drosophila. A strong diminution in mlp84B expression level was observed using either a ubiquitous (Daughterless-Gal4, A) or a heart-specific (tinC{Delta}4-Gal4, B) driver. mlp84B expression levels were determined by performing real-time quantitative PCR on reverse-transcribed mRNA from whole fly (A) or heart only (B) and normalized to actin79B expression. (C) Male flies expressing mlp84B RNAi in the heart have a dramatically shortened lifespan compared with controls (UAS-mlp84B-RNAi outcrossed to wild-type). No lifespan reduction was observed in females.

 

Figure 4
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  		Fig. 4. Cardiac myofibril cytoarchitecture in mlp84B-deficient flies. mpl84B–/– (A,B) and mlp84B–/– rescue (C,D) adult Drosophila hearts were co-stained with anti-{alpha}-actinin and anti-Mlp84B antibodies. mlp84B–/– hearts show sarcomeric disorganization of the longitudinal fibers as evidenced by misalignments and gaps in {alpha}-actinin-positive bands (A). Sarcomeric structure is restored in the mlp84B–/– rescue line (C). Cardiac spiral myofibers were visualized in cypher-GFP;mlp84B–/– (E,F) and cypher-GFP;mlp84B–/– rescue (G,H) hearts. The A3 segment is shown, including the ostia in the center. No obvious structural defect was detected (E). All scale bars are 20 µm.

 

Figure 5
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  		Fig. 5. Reduced levels of Mlp84B result in a slower heart beat due to prolongation of the diastolic interval. (A) Heart period is significantly longer in 1 week old mlp84B–/– flies compared with age-matched wild-type (wt) controls. Reintroduction of a mlp84B transgene in the mlp84B–/– background results in partial rescue. (B) Heart-specific mlp84B knockdown prolongs the heart period in 5 week old flies compared with age-matched wild-type or UAS-mlp84B-RNAi outcrossed to wild-type flies. No significant increase in heart period was observed in 1 week old mlp84B RNAi-expressing flies. (C,D) Diastolic interval is significantly longer in 1 week old mlp84B–/– and 5 week old mlp84B RNAi-expressing flies compared with corresponding controls (as in A and B). mlp84B–/– rescue flies show intermediate diastolic interval duration (C). No significant prolongation of the diastolic interval was detected in mlp84B RNAi-expressing flies at 1 week of age (not shown). See supplementary material Fig. S1 for increases in standard deviations of the heart period and diastolic interval of 1 week old mlp84B–/– flies and 5 week old mlp84B RNAi-expressing flies.

 

Figure 6
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  		Fig. 6. Distribution of heart period and intervals is altered when mlp84B is absent or down-regulated in the heart. The frequency of heart period (HP), diastolic interval (DI) and systolic interval (SI) durations is represented for all heartbeats from all analyzed recordings. (A) One week old mlp84B–/– flies display globally longer and more irregular heart period and diastolic interval than wild-type or mlp84B–/– rescue flies. The dashed vertical lines indicate the most frequent duration for each interval in wild-type and mlp84B–/– rescue flies. Systolic interval appears to be unaffected. (B) Cardiac mlp84B mRNA knockdown results in overall longer and highly irregular heart period and diastolic interval in 5 week old flies. Systolic interval is moderately prolonged relative to 1 week old wild-type in A. The heart period and diastolic/systolic interval distributions were not obviously affected in 1 week old mlp84B RNAi-expressing hearts compared with corresponding controls (not shown).

 

Figure 7
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  		Fig. 7. Loss or cardiac knockdown of mlp84B results in heart rhythm abnormalities. (A) M-mode representations of heart contractions recorded in wild-type, mlp84B–/–, UAS-mlp84B-RNAi/+ and tinC{Delta}4>mlp84B RNAi flies. Asystole and tachyarrhythmia are shown by arrowheads and arrows, respectively. (B) The frequency of fibrillation is higher in 5 week old mlp84B RNAi-expressing flies than in age-matched UAS-mlp84B-RNAi/+ or wild-type control flies. (C) Asystole is observed in 1 week old mlp84B–/– flies but not wild-type flies. The occurrence of asystole is reduced when the mlp84B transgene is reintroduced in the mlp84B–/– background. (D) Five week old Drosophila expressing mlp84B RNAi in the heart show a high rate of asystole compared with wild-type or UAS-mlp84B-RNAi/+ flies.

 

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© The Company of Biologists Ltd 2008