Molecular plasticity and functional enhancements of leg muscles in response to hypergravity in the fruit fly Drosophila melanogaster

Abstract

Studies of organismal and tissue biomechanics have clearly demonstrated that musculoskeletal design is strongly dependent on experienced loads, which can vary in the short term, as a result of growth during life history and during the evolution of animal body size. However, how animals actually perceive and make adjustments to their load-bearing musculoskeletal elements that accommodate variation in their body weight is poorly understood. We developed an experimental model system that can be used to start addressing these open questions, and uses hypergravity centrifugation to experimentally manipulate the loads experienced by Drosophila melanogaster We examined effects of this manipulation on leg muscle alternative splicing of the sarcomere gene troponin T (Dmel\up; Fbgn0004169, herein referred to by its synonym TnT), a process that was previously demonstrated to precisely correlate with quantitative variation in body weight in Lepidoptera and rat. In a similar fashion, hypergravity centrifugation caused fast (i.e. within 24 h) changes to fly leg muscle TnT alternative splicing that correlated with body weight variation across eight D. melanogaster lines. Hypergravity treatment also appeared to enhance leg muscle function, as centrifuged flies showed an increased negative geotaxis response and jump ability. Although the identity and location of the sensors and effectors involved remains unknown, our results provide further support for the existence of an evolutionarily conserved mechanism that translates signals that encode body weight into appropriate skeletal muscle molecular and functional responses.

Keywords: Alternative splicing; Body mass; Body weight; Centrifugation; Muscle performance; Musculoskeletal; Troponin T; Weight sensing.

Fig. 1.

Troponin T splice variants and experimental setup. (A) Sample trace from DNA fragment analyses of D. melanogaster 5′ troponin T gene (TnT) splice variants. (B) Right, hypergravity centrifuge illustration; left, generally observed fly positions post-24 h, 12 g treatment, i.e. flies aligned themselves along a relatively thin strip of the outer vial wall.

Fig. 2.

Hypergravity treatment causes significant changes to D. melanogaster leg muscle TnT alternative splicing. (A) Least squares means estimated by linear modeling (2-way ANOVA, total N=32, N=16 per treatment) of arcsine-transformed TnT_A relative abundance, illustrating the main effect of 12 g treatment (after adjusting for effects of fly line). Asterisk indicates a significant difference. (B) Actual mean (non-transformed) relative abundance of TnT_A for each fly line and treatment. Error bars represent s.e.m. (N=2 per treatment group).

Fig. 3.

TnT­_A reaction norm and body mass. (A) Illustration of the method (see Materials and methods) used to calculate the TnT_A reaction norm for three imaginary fly lines exposed to control and 12 g treatments. (B) Mean TnT_A reaction norm for eight fly lines plotted against their mean body mass. Linear fitting (see Results for R²) was performed on the eight mean values. Body mass means were determined from all individuals (i.e. control and 12 g-treated groups combined) of a given fly line. Line designations are indicated for clarity. Error bars represent s.e.m. (N=4 groups of 20 per fly line for body mass means, and N=2 groups of 20 per fly line for TnT_A reaction norm means). See also Table 3.

Fig. 4.

Hypergravity treatment significantly increases overall mean negative geotaxis responses across the eight D. melanogaster fly lines used. (A) Least squares means estimated by linear modeling (2-way ANOVA, total N=42 vials measured, N=3 vial means per treatment group; see Results for details) of negative geotaxis data (i.e. percentage of flies residing in the top quadrant of the vial), illustrating the main effect of 12 g treatment (after adjusting for effects of fly line). Asterisk indicates a significant difference. (B) Actual mean (non-transformed) percentages for each fly line and treatment. Error bars represent s.e.m. (C) Mean negative geotaxis reaction norm plotted against mean fly line body mass, determined for all individuals (i.e. control and 12 g-treated groups combined) of a given fly line. Fly line identifications are indicated for clarity; note that data for fly line 25199 are missing (see Materials and methods). Error bars represent s.e.m. (N=6 vial means per line for body mass means, N=3 vial means per line for negative geotaxis reaction norm means).

Fig. 5.

Jump distance is significantly increased in response to hypergravity treatment across the eight D. melanogaster fly lines used. (A) Least squares means estimated by linear modeling (two-way ANOVA, N=78; see Results for details) of jump distance, illustrating the main effect of 12 g treatment (after adjusting for effects of fly line). Asterisk indicates a significant difference. (B) Actual mean jump distance for each line and treatment used in the linear model. Error bars represent s.e.m. (N=5 males per treatment group, except for 12 g-treated 25190, and control 25199 groups, where N=4 males).

Fig. 6.

Mass-specific resting metabolic rate (V̇_CO2) is unaffected by hypergravity treatment. (A) Sample V̇_CO2 traces obtained for control (blue trace) and experimental (red trace) groups of five males from seven D. melanogaster lines. The square-wave appearance of the trace is due to the animal chambers and reference chamber being measured in sequence. Note that these traces have not yet been normalized to mean fly body mass in each of the respirometer chambers. Thus, the y-axis represents V̇_CO2 in ml h⁻¹, whereas the x-axis represents time in s. (B) Least squares means estimated by linear modeling (2-way ANOVA, total N=28; see Results for details) of mass-specific resting metabolic rate (after adjusting for effects of fly line). (C) Actual mass-specific resting metabolic rate for each line and treatment used in the linear model; note that data for line 25199 are missing (see Materials and methods). Error bars represent s.e.m. (N=4 per line, N=2 per treatment).

Fig. 7.

Drosophila body weight sensor location. (A) Hypergravity treatment (24 h, 12 g) did not appear to affect leg muscle TnT alterative splicing in male flies whose legs were not supporting body weight (‘unloaded’, see B). Flies were attached dorsally (see Discussion) so that their legs were suspended. Moist cotton was provided to prevent desiccation. (B) TnT_A relative abundance in legs from control and unloaded flies. This experiment was performed once using two of the original eight DGRP lines, hence the absence of a standard error bar (i.e. N=1/treatment). (C) Hypergravity treatment (24 h, 12 g) did not appear to affect whole-thorax muscle TnT alternative splicing in fly line 25175 males (N=2/treatment). Because of low sample sizes, no formal statistical analyses were performed on these data.