Scott E Gordon

Impaired overload‐induced muscle growth is associated with diminished translational signalling in aged rat fast‐twitch skeletal muscle

Impaired overload‐induced protein synthesis and growth in aged fast‐twitch skeletal muscle may result from diminished responsiveness of signalling intermediates controlling protein translation. Yet, potential age‐related signalling decrements have never been examined in direct parallel with impaired overload‐induced muscle growth in any model. To this end, we used Western blotting to examine the contents and phosphorylation states of mammalian target of rapamycin (mTOR) and its downstream translational signalling intermediates, 70 kDa ribosomal protein S6 kinase (S6k), ribosomal protein S6 (rpS6), eukaryotic elongation factor 2 (eEF2), and eukaryotic initiation factor 4E‐binding protein 1 (4E‐BP1), in conjunction with impaired growth in 1 week overloaded fast‐twitch plantaris muscles (via unilateral gastrocnemius ablation) of old (O; 30 months) versus young adult (YA; 8 months) male Fischer344 × Brown Norway rats. The significantly (P≤ 0.05) diminished growth (assessed by total muscle protein content) in overloaded O muscles (5.6 ± 1.7 versus 19.3 ± 2.9% in YA) was accompanied by significant impairments in the phosphorylation states of mTOR (Ser2448), S6k (impaired at the mTOR‐specific Thr389 residue but not at Thr421/Ser424), rpS6 (Ser235/236) and 4E‐BP1 (gel shift), as well as deficits in total eEF2 accretion. Moreover, in overloaded muscles across both age groups, phospho‐S6k at Thr389 (but not at Thr421/Ser424), 4E‐BP1 phosphorylation status, and total eEF2 accretion were all positively correlated with percentage muscle hypertrophy, and negatively correlated with the phosphorylation (Thr172) of 5′‐AMP‐activated protein kinase (AMPK; which inhibits translational signalling and protein synthesis in young muscle at rest). As previously published by ourselves, AMPK was hyperphosphorylated in O versus YA muscles used in the current investigation. The present results provide solid evidence that impaired overload‐induced growth in aged fast‐twitch muscle may partly result from multiple‐level decrements in signalling pathway(s) controlling protein translation, and also provide an initial indication that AMPK hyperactivation with age may potentially lie upstream of these decrements.

AMP-activated protein kinase response to contractions and treatment with the AMPK activator AICAR in young adult and old skeletal muscle

One characteristic of ageing skeletal muscle is a decline in mitochondrial function. Activation of AMP‐activated protein kinase (AMPK) occurs in response to an increased AMP/ATP ratio, which is one potential result of mitochondrial dysfunction. We have previously observed higher AMPK activity in old (O; 30 months) vs young adult (YA; 8 months) fast‐twitch muscle in response to chronic overload. Here we tested the hypothesis that AMPK would also be hyperactivated in O vs YA fast‐twitch extensor digitorum longus muscles from Fischer344× Brown Norway (FBN) rats (n= 8 per group) in response to high‐frequency electrical stimulation of the sciatic nerve (HFES) or injection of AICAR, an activator of AMPK. Muscles were harvested immediately after HFES (10 sets of six 3‐s contractions, 10 s rest between contractions, 1 min rest between sets) or 1 h after AICAR injection (1 mg (g body weight)−1 subcutaneously). The phosphorylations of AMPKα and acetyl‐CoA carboxylase (ACC2; a downstream AMPK target) were both greatly increased (P≤ 0.05) in response to HFES in O muscles, but were either unresponsive (AMPK α) or much less responsive (ACC) in YA muscles. AMPK α2 activity was also greatly elevated in response to HFES in O muscles (but not YA muscles) despite a lower total AMPK α2 protein content in O vs YA muscles. In contrast, AMPK α2 activity was equally responsive to AICAR treatment in both age groups. Since mitochondrial content and/or efficiency could potentially underlie AMPK hyperactivation, we measured levels of mitochondrial proteins as well as citrate synthase (CS) activity. While CS activity was increased by 25% in O vs YA muscles, uncoupling protein‐3 (UCP‐3) protein level was upregulated with age by 353%. Thus, AMPK hyperactivation in response to contractile activity in aged fast‐twitch muscle may be the result of compromised cellular energetics and not necessarily due to an inherent defect in responsiveness of the AMPK molecule per se.

Effects of 14 days of microgravity on fast hindlimb and diaphragm muscles of the rat

The purpose of the present study was to determine the effects of 14xa0days of microgravity on specific rat fast-twitch muscles, and to compare these data with previous data from rat fast-twitch muscles exposed to microgravity for 10xa0days (Kraemer et al. 2000). Hindlimb muscles containing predominately fast fibers [extensor digitorum longus (EDL), superficial “white” (GSW) and deep “red” (GDR) gastrocnemius] and the diaphragm (DIA) were removed from flight and ground-based control animals and analyzed for: muscle mass, fiber type distribution, cross-sectional area, and myosin heavy chain (MHC) isoform content. Gravitational unloading for 14xa0days caused significant decreases in muscle mass (8–9%) and cross-sectional area of almost all fiber types (10–35%) from both EDL and gastrocnemius muscles. However, microgravity had little effect on fiber type composition in these muscles with significant changes occurring only in the EDL type IID fiber population (9.5% decrease). Similarly, relative MHC isoform content was only slightly altered by exposure to microgravity (increased content of MHCIIa in flight EDL). No changes in area, fiber type percentages, or MHC isoform content were detected in the DIA following the 14-day spaceflight. Similar to data gathered following a 10-day spaceflight (Kraemer et al. 2000), the 14-day flight did not appear to cause significant slow-to-fast (Ixa0→xa0IIA) or fast-to-faster (IIAxa0→xa0IIDxa0→xa0IIB) transformations in hindlimb muscles containing predominantly fast-twitch fibers. However, the longer period of gravitational unloading did result in additional loss in muscle fiber cross-sectional area with involvement of more major fiber types.

Moderate Aging Does Not Modulate Morphological Responsiveness of the Neuromuscular System to Chronic Overload in Fischer 344 Rats

The purpose of this investigation was to determine the effect of aging on neuromuscular adaptations to chronic overload. Eight young adult (8 months old) and eight aged (22 months old) Fischer 344 rats underwent unilateral synergist ablation to overload the plantaris and soleus muscles of that hindlimb and to provide control muscles from the contralateral hindlimb. Cytofluorescent staining and confocal microscopy were used to quantify pre- and post-synaptic features of neuromuscular junctions (NMJs). Histochemical staining and light microscopy were used to assess adaptations of myofibers to chronic overload. Results demonstrate that NMJs of young adult and aged muscles did not undergo morphological remodeling as a result of 4 weeks of chronic overload. In contrast, myofibers of young and aged rats displayed significant (P<0.05), but similar hypertrophy ( approximately 18%) following that 4 week intervention. In both age groups, however, this hypertrophy was detected in the plantaris, but not the soleus. These data indicate that moderate aging (the equivalent of 65 years in human lifetime) does not modify the sensitivity of the neuromuscular system to chronic overload.