Muscle-specific Drp1 overexpression impairs skeletal muscle growth via translational attenuation

Abstract

Mitochondrial fission and fusion are essential processes in the maintenance of the skeletal muscle function. The contribution of these processes to muscle development has not been properly investigated in vivo because of the early lethality of the models generated so far. To define the role of mitochondrial fission in muscle development and repair, we have generated a transgenic mouse line that overexpresses the fission-inducing protein Drp1 specifically in skeletal muscle. These mice displayed a drastic impairment in postnatal muscle growth, with reorganisation of the mitochondrial network and reduction of mtDNA quantity, without the deficiency of mitochondrial bioenergetics. Importantly we found that Drp1 overexpression activates the stress-induced PKR/eIF2α/Fgf21 pathway thus leading to an attenuated protein synthesis and downregulation of the growth hormone pathway. These results reveal for the first time how mitochondrial network dynamics influence muscle growth and shed light on aspects of muscle physiology relevant in human muscle pathologies.

Figure 1

Mice overexpressing Drp1 (Drp/MC) in muscle have a severe reduction of muscle mass. (a) Transgene construct. (b) Left: Drp1 and Myc proteins were analysed by western blot in EDL and soleus muscles and liver of WT and transgenic animals, right: Drp1 expression in mitochondrial extract was analysed by western blot. Cyclophilin D (CypD) is used as a loading control. (c) Mitofusin 1 (Mfn1), Mitofusin 2 (Mfn2) and Opa1 expression was analysed by western blot. Quantification is given in the right chart (n=4 animals per genotype). (d) Growth curve of WT and Drp/MC animals (n=5 animals per group). (e) Pictures of WT and Drp/MC Tibialis anterior (TA), EDL and soleus muscles. (f) Speed and distance performed by P200 WT and transgenic mice on treadmill tests evaluating power and resistance (n=7 animals per genotype). Error bars represent S.E.M. ***P<0.001 versus wt

Figure 2

Muscle-specific Drp1 overexpression impairs postnatal muscle growth. (a, b, c) Representative images of immunofluorescence on TA sections from P1 (a), P7 (b) and P25 (c) mice by using a laminin-specific antibody. Original magnification x20, scale bar 50um. Frequency histogram showing the distribution of myofibre CSA in WT and Drp/MC TA muscles from P1 (a), P7 (b) and P25 (c) mice. Per condition, at least 500 fibres were counted. Mean myofibre CSA in WT and Drp/MC from P1 (a), P7 (b) and P25 (c) mice (n≥3 animals per genotype). (d) Representative sections of WT and Drp/MC TA muscles from P100 mice stained with H&E. Representative images of immunofluorescence on TA sections by using a laminin-specific antibody. Frequency histogram showing the distribution of myofibre CSA in WT and Drp/MC TA muscles from P100 mice. Mean myofibre CSA in WT and Drp/MC (n=3 animals per genotype). (e) Mean number of myofibres muscle (n=3 animals per genotype). (f) Mean number of myonuclei and Pax7 positive cells in WT and Drp/MC single fibres (n=4 preparations per genotype). Original magnification x20, scale bar: 50 μm. Error bars represent S.E.M. **P<0.01; *P<0.05 versus WT

Figure 3

Muscle-specific Drp1 overexpression induces a drastic remodelling of mitochondrial network. (a) SDH staining of transversal sections from WT and Drp/MC from P1, P7, P25 TA muscles. Original magnification × 20, scale bar: 50 μm. (b) SDH staining of transversal and longitudinal sections from WT and Drp/MC P100 TA muscle. Low magnification, × 4; high magnification, × 40. (c) Representative confocal images of mitochondrial network in PhAM and Drp/MC/PhAM TA myofibres from P100 animals. Transversal and longitudinal optical sections are also shown. Original magnification x40, scale bar: 10 μm. (d) Western blot analysis of Tim23 and Cyclophilin D (CypD) protein expression levels in mitochondrial protein lysates from quadriceps of adult WT and Drp/MC mice. Gapdh was used as internal control. (e) Electron microscopy analysis of TA muscles from P100 Drp/MC and WT mice. Arrows point to swollen mithocondria, with none or fewer than normal cristae. (f) Western blot analysis of Opa1 GTPase activity after pull-down experiments using GTP-conjugated beads

Figure 4

Mitochondrial bioenergetics remained unchanged but mtDNA content is reduced and a mtUPR response is activated in Drp/MC muscle. (a) Fibres were loaded with the mitochondrial potentiometric dye TMRM (25 nM) and the fluorescence was recorded for 40 min. The arrows indicate the time of addition of oligomycin (Oligo; 5 μM) and FCCP (4 μM). (b) Mitochondrial respiration in permeabilised TA myofibres was measured by using high-sensitivity respirometer (n=8 animals per genotype); complex I (CI), complex II (CII) and complex IV (CIV). (c) Isolated mitochondria from TA muscle were incubated with luciferin-luciferase and the ATP generated through oxidative phosphorylation was measured (n=9 animals per genotype). (d) Mitochondrial DNA (mtDNA) content in TA muscle from P7 and P100 animals (n=8 per genotype). (e) RT-qPCR analysis of mtDNA and nuclear DNA encoded respiratory chain subunits in P100 quadriceps muscle. (f) RT-qPCR analysis of Chop mRNA levels in quadriceps from P7, P25 and P100 WT and Drp/MC mice (n=8 per genotype). (g) Western blot analysis of Hsp60 and ClpP protein expression levels in quadriceps from adult WT and Drp/MC mice (n=8 per genotype). Actin was used as control. (h) RT-qPCR analysis of Atpif1 mRNA levels in quadriceps from P100 WT and Drp/MC mice (n=8 per genotype). (i) Western blot analysis of ATPIF1 protein expression levels in quadriceps from adult WT and Drp/MC mice (n=8 per genotype). Tim23 and Complex II (CII) were used as control. Error bars represent S.E.M. *P<0.05, **P<0.01, ***P<0.001 versus WT; ^##P<0.01, ^###P<0.001 versus P7 mice

Figure 5

eIF2α/Atf4 pathway is activated in Drp/MC muscle. (a) Polysome profile in quadriceps from young (P40) WT and Drp/MC mice. Lysates were analysed by centrifugation in a 10–50% sucrose gradient, and the profiles were measured by absorbance at 254 nm. The panel is representative of three independent experiments. (b) Representative western blot analysis of phosphorylated and total eIF2α protein expression levels in quadriceps from P7, P25 and adult (P100) WT and Drp/MC mice. Quantification is given in the right chart (n=5 per genotype). (c) Western blot analysis of phosphorylated PKR protein levels after immunoprecipitation of total PKR protein from gastrocnemius protein lysate of adult (P100) WT and Drp/MC mice (n=3 per genotype). (d) Atf4 mRNA distribution in the fractions collected from the sucrose gradients was determined by RT-qPCR. Percentage of Atf4 transcripts present in polysomal fractions (6–12) (n=3 per genotype). (e) RT-qPCR analysis of representative stress-response genes mRNA levels in quadriceps from adult WT and Drp/MC mice (n=8 per genotype). (f) Plasma levels of Fgf21 in adult WT and Drp/MC mice (n=11 per genotype). (g) RT-qPCR analysis of Cish mRNA levels in quadriceps from adult WT and Drp/MC mice 4 h after GH treatment (1.5 mg/kg i.p., n=7 per group). Error bars represent S.E.M. *P<0.05, **P<0.01, ***P<0.001 versus WT