Disconnecting mitochondrial content from respiratory chain capacity in PGC-1-deficient skeletal muscle

Abstract

The transcriptional coactivators PGC-1α and PGC-1β are widely thought to be required for mitochondrial biogenesis and fiber typing in skeletal muscle. Here, we show that mice lacking both PGC-1s in myocytes do indeed have profoundly deficient mitochondrial respiration but, surprisingly, have preserved mitochondrial content, isolated muscle contraction capacity, fiber-type composition, in-cage ambulation, and voluntary running capacity. Most of these findings are recapitulated in cell culture and, thus, are cell autonomous. Functional electron microscopy reveals normal cristae density with decreased cytochrome oxidase activity. These data lead to the following surprising conclusions: (1) PGC-1s are in fact dispensable for baseline muscle function, mitochondrial content, and fiber typing, (2) endurance fatigue at low workloads is not limited by muscle mitochondrial capacity, and (3) mitochondrial content and cristae density can be dissociated from respiratory capacity.

Figure 1. PGC-1α and β are critical for ETC capacity

A–C.) Relative mRNA expression of indicated genes from quadriceps (quad) of Myo-DKO mice (blue bars) and littermate controls (white bars). PGC-1 isoforms (A), nuclear-encoded electron transport chain (ETC) genes (B) and mitochondrial-encoded ETC genes (C). D&E.) Western blot analyses of indicated ETC proteins from quads of Myo-DKO mice F.) Relative activity of the indicated ETC complexes in quad muscles from DKO and control animals. G) Schematic of exercise exhaustion protocol. H–J) Maximal time (H), work (I), and distance (J) achieved on running treadmills prior to exhaustion. Error bars indicate SEM; n > 3 per group in all panels. * - P < 0.05. N.S. – not statistically significant.

Figure 2. PGC-1s are dispensable for baseline activity and muscle function

A.) In-cage locomotor activity over 72hrs of Myo-DKO mice and control littermates. B–C.) Samples of voluntary wheel activity over 11 days. D–F.) Quantification of B–C, represented as percent time spent on wheels (D) total distance run (E) or average revolutions per minute (F). G–I.) Maximal tetanic (G) and isometric twitch forces (H), and contraction-stimulated fatigability (I), of explanted extensor digitorum longus muscle from Myo-DKO and control animals. J–K.) Fiber type composition in plantaris of DKO and control animals. (J) Representative images of immunostaining: MHC Ia (red), MHC IIa (blue) and MHC IIb (green); unstained fibers were counted as MHC IId/x. (K), Quantification of immunostaining. Error bars indicate SEM; n > 6 per group in all panels. N.S. – not statistically significant.

Figure 3. Mitochondrial content and ETC capacity are separable in DKO muscle and cultured myotubes

A–B.) Representative images (A) and morphometric quantification of mitochondrial density (B) of transmission electron micrographs of transverse sections of mid-portion of quadriceps (quad) of Myo-DKO mice and control littermates. Sample mitochondria labeled with a white (*). C.) Mitochondrial-to-nuclear DNA content ratio in quad muscles from Myo-DKO and control animals. D.) Relative expression of indicated genes from the quads of the Myo-DKO and control animals. E–L.) Differentiated myotubes from primary myoblasts isolated from DKO (blue) versus control (white) muscles. (E) Relative mRNA expression of indicated genes. (F) Western blot analysis of indicated ETC proteins. (G) Basal and uncoupled respiration rates of DKO and control differentiated myotubes. (H) State 3 and state 4o respiration rates in permeabilized DKO and control differentiated myotubes. (I) TEMs of DKO and control differentiated myotubes. J–K.) Quantification of mitochondrial content by mitotracker (J) and mitochondrial-to-nuclear DNA content ratio (K) in DKO and control cells. L.) Relative expression of indicated genes in primary DKO and control myotubes. n > 4 fields from 6 animals per group. Error bars indicate SEM; n > 3 per group in all panels. * - P < 0.05 compared to control. # - P < 0.05 compared to basal state N.S. – not statistically significant.

Figure 4. Decreased ultrastructural ETC activity in Myo-PGC-1DKO animals

A.) Schematic of 3,3′-diaminobenzidine (DAB) polymerization. B.) Transmission electron microscopy ultrastructural analysis of mitochondrial cyctochrome c oxidase (COX) enzymatic activity in quadricep muscle of Myo-DKO animals and control littermates. Punctated mitochondrial cristae labeled with white arrowhead.