Molecular basis for an attenuated mitochondrial adaptive plasticity in aged skeletal muscle

Abstract

Our intent was to investigate the mechanisms driving the adaptive potential of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in young (6 mo) and senescent (36 mo) animals in response to a potent stimulus for organelle biogenesis. We employed chronic electrical stimulation (10 Hz, 3 h/day, 7 days) to induce contractile activity of skeletal muscle in 6 and 36 mo F344XBN rats. Subsequent to chronic activity, acute stimulation (1 Hz, 5 min) in situ revealed greater fatigue resistance in both age groups. However, the improvement in endurance was significantly greater in the young, compared to the old animals. Chronic muscle use also augmented SS and IMF mitochondrial volume to a greater extent in young muscle. The molecular basis for the diminished organelle expansion in aged muscle was due, in part, to the collective attenuation of the chronic stimulation-evoked increase in regulatory proteins involved in mediating mitochondrial protein import and biogenesis. Furthermore, adaptations in mitochondrial function were also blunted in old animals. However, chronic contractile activity evoked greater reductions in mitochondrially-mediated proapoptotic signaling in aged muscle. Thus, mitochondrial plasticity is retained in aged animals, however the magnitude of the changes are less compared to young animals due to attenuated molecular processes regulating organelle biogenesis.

Keywords: apoptosis; chronic contractile activity; intermyofibrillar mitochondria; performance; protein import; subsarcolemmal mitochondria.

Figure 1.. Chronic contractile activity-evoked increases in skeletal muscle endurance performance and mitochondrial content are reduced in old, compared to young animals. (A).

Fatigue resistance during 5 min of 1 Hz in situ stimulation of the control (CON, open squares) and chronically stimulated (STIM, closed circles) tibialis anterior muscles from young (solid lines) and old (dashed lines) animals (n = 7-8). (B) Electron micrographs depicting skeletal muscle morphology and SS and IMF mitochondrial volumes in young and old, control (CON, open bars) and chronically stimulated (STIM, closed bars) extensor digitorum longus (EDL) muscle sections. All images were taken at the same magnification. Scale bar located at the lower right of each picture represents 1 μm. (C) COX enzyme activity in EDL muscle homogenates (n = 9-13). Data represent the mean ± SEM. * P < 0.05 vs. Young; ¶ P < 0.05 vs. CON.

Figure 2.. Chronic muscle use increases the expression of muscle and mitochondrial regulatory proteins in young and old animals. (A).

Representative Western blots and graphical summary (B) of the effects of chronic contractile activity on the expression levels of proteins important for mitochondrial biogenesis (PGC-1α, Tfam), apoptotic signaling (AIF, HSP70), and aging (SIRT1), in muscles from young (closed bars) and old (open bars) animals expressed as the fold increase in chronically stimulated over control muscles (n = 6-11). (C) HSP70 protein content is shown separately due to the difference in scale, compared to the data in B. Data represent the mean ± SEM. * P < 0.05, stimulated vs. control.

Figure 3.. Subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial protein import machinery components are increased in young, but not old animals in response to chronic contractile activity. (A).

Representative Western blots of mtHSP70, Tim17, and Tim23 proteins in SS and IMF (B) mitochondrial subfractions isolated from the control (C) and chronically stimulated (S) limbs of young and old animals. (C) Graphical summary of the data in panels A and B expressed as the fold difference of the stimulated, over the control legs (n = 7-9). (D) Pooled results of the protein expression data in young, compared to old animals shown above in panel C, and panel B of Figure 3 (n = 74-86). Data represent the mean ± SEM. * P < 0.05, stimulated vs. control, † P < 0.05 vs. Young.

Figure 4.. Mitochondrial import of the matrix protein ornithine carbamoyltransferase (OCT) is induced to a greater extent after chronic muscle use in young animals.

(A) Representative autoradiograms of precursor (pOCT) and mature (mOCT) OCT after 5 and 20 min of the import reaction timecourse in isolated SS (top) and IMF (bottom) mitochondrial subfractions harvested from the control (CON, open bars) and chronically stimulated (STIM, closed bars) limbs of young and old animals (TL, translation lane without mitochondria). (B) and (C) Graphical summaries of the 20 min import data from repeated experiments shown in panel A (n = 9-12). (D) Western blots of MSF-L and HSP90 in isolated cytosolic fractions obtained from the control (C) and chronically stimulated (S) legs of young and old animals. GAPDH was used to confirm equal loading of protein. (E) Summary of repeated experiments shown in panel D (n = 5-7). Data represent the mean ± SEM. * P < 0.05 vs. Young; ¶ P < 0.05 vs. CON.

Figure 5.. Chronic stimulation-induced adaptations in mitochondrial function and anti-apoptotic cell death signaling in young and old animals. (A).

and (B) State 4 (2 μM rotenone and 10 mM succinate as substrates) and state 3 (rotenone and succinate plus 0.44 mM ADP) rates of oxygen consumption (VO₂) in isolated subsarcolemmal (SS; A) and intermyofibrillar (IMF; B) mitochondria from the control (CON, open bars) and chronically stimulated (STIM, closed bars) limbs of young and old animals (n = 6-10). (C) and (D) State 4 and state 3 rates of reactive oxygen species (ROS) production per natom oxygen consumed in SS (C) and IMF (D) mitochondria from the CON and STIM limbs of young and old animals (n = 7-10). (E) Level of fragmented DNA, in the form of mono- and oligonucleosomes, in myonuclei-containing cytosolic extracts isolated from young and old animals (n: STIM = 4, CON = 21). Data represent the mean ± SEM. * P < 0.05 vs. Young; ¶ P < 0.05 vs. CON).