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. 2011 Aug;301(2):C373-82.
doi: 10.1152/ajpcell.00206.2010. Epub 2011 May 11.

Deletion of Drosophila muscle LIM protein decreases flight muscle stiffness and power generation

Kathleen A Clark  1 , Heather Lesage-Horton, Cuiping Zhao, Mary C Beckerle, Douglas M Swank
Affiliations

Deletion of Drosophila muscle LIM protein decreases flight muscle stiffness and power generation

Kathleen A Clark et al. Am J Physiol Cell Physiol. 2011 Aug.

Abstract

Muscle LIM protein (MLP) can be found at the Z-disk of sarcomeres where it is hypothesized to be involved in sensing muscle stretch. Loss of murine MLP results in dilated cardiomyopathy, and mutations in human MLP lead to cardiac hypertrophy, indicating a critical role for MLP in maintaining normal cardiac function. Loss of MLP in Drosophila (mlp84B) also leads to muscle dysfunction, providing a model system to examine MLP's mechanism of action. Mlp84B-null flies that survive to adulthood are not able to fly or beat their wings. Transgenic expression of the mlp84B gene in the Mlp84B-null background rescues flight ability and restores wing beating ability. Mechanical analysis of skinned flight muscle fibers showed a 30% decrease in oscillatory power production and a slight increase in the frequency at which maximum power is generated for fibers lacking Mlp84B compared with rescued fibers. Mlp84B-null muscle fibers displayed a 25% decrease in passive, active, and rigor stiffness compared with rescued fibers, but no significant decrease in isometric tension generation was observed. Muscle ultrastructure of Mlp84B-null muscle fibers is grossly normal; however, the null fibers have a slight decrease, 11%, in thick filament number per unit cross-sectional area. Our data indicate that MLP contributes to muscle stiffness and is necessary for maximum work and power generation.

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Figures

Fig. 1.
Fig. 1.
Mlp84B expression and distribution in indirect flight muscles (IFMs). A: immunolocalization of muscle LIM protein (MLP) at the Z-disk of IFM sarcomeres. One isolated myofibril from homogenized wild-type IFMs, labeled with antibodies against Mlp84B (top) and α-actinin (middle), is shown. The two proteins display coincident distribution at the Z-disk. B: Western blot analysis of Mlp84B expression. A representative blot is shown with protein lysates from isolated thoracies of indicated genotypes (Df/Df, mlp84BDf/Df; WT, wild type; 2X Rsc, 2-copy rescue). Identities of immunoreactive bands are indicated on the right side of the blot. The graph depicts the relative expression levels of Mlp84B for different genotypes determined from two independent blots. Loading for each sample was first normalized to the tubulin signal, and the average wild-type Mlp84B signal for each blot was set to 100 to normalize for signal intensity between blots. Each value on the graph represents the average Mlp84B band intensity for 3–6 samples from the two blots. Error bars show SEs. Scale bar in A=5 µm.
Fig. 2.
Fig. 2.
Muscle ultrastructure. Cross-sectional views are shown of Mlp84B rescue (A and C) and Mlp84B-null (B and D) IFMs. A and B: cross-sectional view at ×17,592 magnification. C and D: cross-sectional view of a single myofibril, ×70,000 magnification. Note the orderly arrays of myosin and actin filaments present in both the rescued and the mlp84BDf/Df myofibril.
Fig. 3.
Fig. 3.
Muscle stiffness. Mlp84B-null fibers had lower relaxed (A), active (B), and rigor (C) instantaneous stiffness (elastic modulus) values than Mlp84B-rescue fibers. Measurements were made at 15°C. Values are means ± SE. *Span of frequencies over which there is a significant difference (one-way ANOVA, P < 0.05).
Fig. 4.
Fig. 4.
Complex stiffness properties of maximally calcium-activated fibers. A: complex stiffness as a function of frequency for Mlp84B-null and Mlp84B-rescue fibers from 2- to 3-day-old adults. B: phase of vectors from the Nyquist plot origin to each corresponding frequency point shows that there are minor muscle kinetic differences, between 120 and 240 Hz. Values are means ± SE. *Span of frequencies over which there is a significant difference (one-way ANOVA, P < 0.05).
Fig. 5.
Fig. 5.
Work per cycle and power generation of maximally calcium-activated IFM fibers. A: small amplitude sinusoidal analysis of skinned Mlp84B-null fibers showed a decrease in work-producing and work-absorbing (negative work values) properties compared with Mlp84B-rescue fibers. B: Mlp84B-null fibers generated less power, Pmax, and displayed a rightward shift in the frequency at which maximum power is generated (fmax), denoted by the solid vertical line (the position of which was determined from averaged individual fiber values shown in Table 3), compared with MLP rescue fibers, denoted by the dashed vertical line. Measurements were made at 15°C. Values are means ± SE. *Span of frequencies over which there is a significant difference (one-way ANOVA, P < 0.05).
Fig. 6.
Fig. 6.
Stretch activation of IFM fibers at pCa 5.0. A 1% muscle-lengthening step over 0.5 ms was imposed on a Mlp84B-null fiber and a Mlp84B-rescue fiber. Starting tension for each fiber was set to zero by subtracting isometric tension. Inset shows tension from the same two fibers after subtracting its passive tension response at pCa 8.0. FSA, stretch-activated tension.
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