Charles T. Putman

Preferential motor unit loss in the SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis

The present study investigated motor unit (MU) loss in a murine model of familial amyotrophic lateral sclerosis (ALS). The fast‐twitch tibialis anterior (TA) and medial gastrocnemius (MG) muscles of transgenic SOD1G93A and SOD1WT mice were studied during the presymptomatic phase of disease progression at 60 days of age. Whole muscle maximum isometric twitch and tetanic forces were 80% lower (P < 0.01) in the TA muscles of SOD1G93A compared to SOD1WT mice. Enumeration of total MU numbers within TA muscles showed a 60% reduction (P < 0.01) within SOD1G93A mice (38 ± 7) compared with SOD1WT controls (95 ± 12); this was attributed to a lower proportion of the most forceful fast‐fatigable (FF) MU in SOD1G93A mice, as seen by a significant (P < 0.01) leftward shift in the cumulative frequency histogram of single MU forces. Similar patterns of MU loss and corresponding decreases in isometric twitch force were observed in the MG. Immunocytochemical analyses of the entire cross‐sectional area (CSA) of serial sections of TA muscles stained with anti‐neural cell adhesion molecule (NCAM) and various monoclonal antibodies for myosin heavy chain (MHC) isoforms showed respective 65% (P < 0.01) and 28% (P < 0.05) decreases in the number of innervated IIB and IID/X muscle fibres in SOD1G93A, which paralleled the 60% decrease (P < 0.01) in the force generating capacity of individual fibres. The loss of fast MUs was partially compensated by activity‐dependent fast‐to‐slower fibre type transitions, as determined by increases (P < 0.04) in the CSA and proportion of IIA fibres (from 4% to 14%) and IID/X fibres (from 31% to 39%), and decreases (P < 0.001) in the CSA and proportion of type IIB fibres (from 65% to 44%). We conclude that preferential loss of IIB fibres is incomplete at 60 days of age, and is consistent with a selective albeit gradual loss of FF MUs that is not fully compensated by sprouting of the remaining motoneurons that innervate type IIA or IID/X muscle fibres. Our findings indicate that disease progression in fast‐twitch muscles of SOD1G93A mice involves parallel processes: (1) gradual selective motor axon die‐back of the FF motor units that contain large type IIB muscle fibres, and of fatigue‐intermediate motor units that innervate type IID/X muscle fibres, and (2) activity‐dependent conversion of motor units to those innervated by smaller motor axons innervating type IIA fatigue‐resistant muscle fibres.

AMPK activation increases uncoupling protein‐3 expression and mitochondrial enzyme activities in rat muscle without fibre type transitions

The present study examined the effect of chronic activation of 5′‐AMP‐activated protein kinase (AMPK) on the metabolic profile, including uncoupling protein‐3 (UCP‐3) and myosin heavy chain (MHC)‐based fibre phenotype of rodent fast‐twitch tibialis anterior muscle. Sprague‐Dawley rats were given daily injections of 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR), a known activator of AMPK, or vehicle (control) for 28 days. After AICAR treatment, UCP‐3 expression at the mRNA level was elevated 1.6 ± 0.1‐fold (P < 0.006) and corresponded to a 3.3 ± 0.2‐fold increase in UCP‐3 protein content (P < 0.0001). In addition, the activities of the mitochondrial reference enzymes citrate synthase (EC 4.1.3.7) and 3‐hydroxyacyl‐CoA‐dehydrogenase (EC 1.1.1.35), which are known to increase in proportion to mitochondrial volume density, were elevated 1.6‐fold (P < 0.006), while the activity of lactate dehydrogenase (EC 1.1.1.27) was reduced to 80 % of control (P < 0.02). No differences were detected after AICAR treatment in the activities of the glycolytic reference enzymes glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) or phosphofructokinase (EC 2.7.1.11), nor were MHC‐based fibre‐type transitions observed, using immunohistochemical or electrophoretic analytical methods. These changes could not be attributed to variations in inter‐organ signalling by metabolic substrates or insulin. We conclude that an AMPK‐dependent pathway of signal transduction does mimic some of the metabolic changes associated with chronic exercise training, but does not affect expression of the MHC‐based structural phenotype. Thus, the metabolic and MHC‐based fibre types do not appear to be regulated in a co‐ordinated way, but may be independently modified by different signalling pathways.

Intraspinal microstimulation preferentially recruits fatigue‐resistant muscle fibres and generates gradual force in rat

Intraspinal microstimulation (ISMS), a novel rehabilitative therapy consisting of stimulation through fine, hair‐like microwires targeted at the ventral spinal cord, has been proposed for restoring standing and walking following spinal cord injury. This study compared muscle recruitment characteristics of ISMS with those produced by peripheral nerve cuff stimulation (NCS). Thirty‐three minutes of either ISMS or NCS at 1, 20 or 50 s−1 and 1.2 × threshold (T) amplitude depleted glycogen from muscle fibres of vastus lateralis and rectus femoris. ISMS and NCS were also carried out at 20 s−1 and 3.0T. Muscle serial sections were stained for glycogen and for myosin heavy chain (MHC)‐based fibre types using a panel of monoclonal antibodies. The results of this study show that ISMS recruited fatigue‐resistant (FR) fibres at 2.9, 1.9, 1.7 and 2.5 times their relative MHC content at 1, 20 and 50 s−1 1.2T and 20 s−1 3.0T, respectively. In contrast, NCS recruited FR fibres at 1.2, 1.0, 2.1 and 0.0 times their MHC content at 1, 20 and 50 s−1 1.2T and 20 s−1 3.0T, respectively. The proportion of FR fibres recruited by ISMS and NCS was significantly different in the 20 s−1 3.0T condition (P < 0.0001). We also report that force recruitment curves were 4.9‐fold less steep (P < 0.019) for ISMS than NCS. The findings of this study provide evidence for the efficacy of ISMS and further our understanding of muscle recruitment properties of this novel rehabilitative therapy.

Effects of short-term training on plasma acid-base balance during incremental exercise in man

The present study examined the effect of short‐term submaximal training on plasma acid‐base balance during exercise. The influence of water and ion exchange between plasma, active muscles and erythrocytes in the response to training were also studied. The contributions of independent physicochemical variables (i.e. strong ion difference ([SID]), total concentration of weak acids ([Atot]) and PO2) to changes in arterial (a) and femoral venous (v) plasma [H+] were examined in six subjects (age 24 ± 1.5 years; maximum oxygen consumption rate (V̇O2,max), 3.67 ± 0.24 l min−1) during steady‐state cycling for 15 min at each of 30, 65 and 75 % of V̇O2,max before (pre) and after (post) training for 7 days on a cycle ergometer (2 h daily at 60 % V̇O2,max). The rise in [H+]a during exercise was attenuated post‐training by 3 and 5 nequiv l−1 (P < 0.05) at 65 and 75 % V̇O2,max, respectively, due first to less decrease in [SID]a, secondary to lower [Cl−]a and [Lac−]a; and second, to a reduction in [Atot]a, due to greater plasma volume and less plasma water flux (Jv) into leg muscle (P < 0.05). The rise in [H+]v was also less in post‐training by 4.5 and 6 nequiv l−1 (P < 0.05) at 65 and 75 % V̇O2,max, respectively, and attributed solely to lower [Atot]v (P < 0.05). Attenuation of exercise induced decreases in plasma [SID]a and [SID]v from rest to 75 % V̇O2,max was accompanied by reductions in erythrocyte Lac− and Cl− uptake (P < 0.05), and smaller increases in erythrocyte K+ release (P < 0.05). We conclude that the training‐induced attenuation of the rise in plasma [H+]a and [H+]v during incremental exercise resulted from adaptive changes within muscles (less Lac− production and less water uptake) and erythrocytes (less uptake of Lac−, Cl− and K+), leading to greater [SID] and lower [Atot] in both arterial and femoral venous plasma.

Progressive motor unit loss in the G93A mouse model of amyotrophic lateral sclerosis is unaffected by gender

We examined whether there are gender differences in the progressive loss of functional motor units in SOD1G93A transgenic mice. Isometric muscle and motor unit twitch contractions were recorded in fast‐ and slow‐twitch muscles in response to stimulation of the sciatic nerve. Using a modified motor unit number estimation technique (ITS‐MUNE), we found that motor unit numbers declined rapidly from 40 to 90 days of age during the asymptomatic phase of ALS in fast‐ but not slow‐twitch hindlimb muscles of both male and female mice. There was a corresponding decline in twitch and tetanic contractile forces of the fast‐twitch muscles. Gender did not affect the progressive loss of motor units and associated decline in force production. We conclude that gender does not alter progressive, muscle‐specific motor unit loss in ALS, even though gender does influence disease onset. Muscle Nerve 39: 318–327, 2009

Effect of satellite cell ablation on low‐frequency‐stimulated fast‐to‐slow fibre‐type transitions in rat skeletal muscle

The purpose of this study was to determine whether satellite cell ablation within rat fast‐twitch muscles exposed to chronic low‐frequency stimulation (CLFS) would limit fast‐to‐slow fibre‐type transitions. Twenty‐nine male Wistar rats were randomly assigned to one of three groups. Satellite cells of the left tibialis anterior were ablated by weekly exposure to a 25 Gy dose of γ‐irradiation during 21 days of CLFS (IRR‐Stim), whilst a second group received only 21 days of CLFS (Stim). A third group received weekly doses of γ‐irradiation (IRR). Non‐irradiated right legs served as internal controls. Continuous infusion of 5‐bromo‐2′‐deoxyuridine (BrdU) revealed that CLFS induced an 8.0‐fold increase in satellite cell proliferation over control (mean ±s.e.m.: 23.9 ± 1.7 versus 3.0 ± 0.5 mm−2, P < 0.0001) that was abolished by γ‐irradiation. M‐cadherin and myogenin staining were also elevated 7.7‐ and 3.8‐fold (P < 0.0001), respectively, in Stim compared with control, indicating increases in quiescent and terminally differentiating satellite cells; these increases were abolished by γ‐irradiation. Myonuclear content was elevated 3.3‐fold (P < 0.0001) in Stim, but remained unchanged in IRR‐Stim. Immunohistochemical analyses revealed attenuation of fast‐to‐slow fibre‐type transitions in IRR‐Stim compared with Stim. Comparable changes were observed at the protein level by SDS‐PAGE. It is concluded that although considerable adaptive potential exists within myonuclei, satellite cells play a role in facilitating fast‐to‐slow fibre‐type transitions.

Transient expression of myosin heavy chain MHCI alpha in rabbit muscle during fast-to-slow transition.

The expression of an α-cardiac-like myosin heavy chain, MHCIα, was investigated at both the mRNA and protein levels in rabbit tibialis anterior muscle undergoing fast-to-slow transition by continuous chronic low-frequency stimulation (CLFS). According to sequence analyses of the PCR product, the MHCIα isoform was found to be identical to the α-cardiac MHC expressed in rabbit atrium. In muscles at different degrees of transformation, the upregulation of MHCIα mRNA preceded that of the MHCIβ mRNA. At more advanced stages of the transformation, MHCIα mRNA decayed while MHCIβ mRNA persisted at high levels. The expression of MHCIα, therefore, was transitory. Studies at the protein level were based on immunoblotting using a monoclonal antibody (F88 12F8,1), characterized to be specific to MHCIα in rabbit muscle. These studies revealed a similar relationship between initial increase and successive decline of the MHCIα protein as seen at the␣mRNA level. Immunohistochemistry of 30-day stimulated muscle revealed that up to 65% of the fibres expressed the MHCIα isoform in combination with other adult MHC isoforms. The most frequent patterns of coexistence were MHCIIa+MHCIα + MHCIβ (28%), MHCIα+MHCIβ (18%), and MHCIIa + MHCIα (11%). According to these combinations, the upregulation of MHCIα may be assigned as an intermediate step in the transformation of existing fibres during the␣MHCIIa → MHCIβ transition. A small fraction of fibres contained, in addition to the MHCIα + MHCIβ and MHCIIa + MHCIα combinations, developmental myosin, suggesting that MHCIα was also expressed in regenerating fibres originating from satellite cell-derived myotubes.

α-cardiac-like myosin heavy chain MHCIα is not upregulated in transforming rat muscle

The expression of MHCIα, an α-cardiac-like myosin heavy chain isoform, was studied in extensor digitorum longus (EDL) and tibialis anterior (TA) rat muscles undergoing fast-to-slow transition by chronic low-frequency stimulation (CLFS), a condition inducing a transient upregulation of MHCIα in rabbit muscle. In order to enhance the transformation process, CLFS was applied to hypothyroid rats. mRNA analyses were performed by RT-PCR, and studies at the protein level by immunoblotting and immunohistochemistry, using the F88 antibody (F88 12F8,1) demonstrated in the accompanying paper to be specific for MHCIα. In total RNA preparations from slow- and fast-twitch muscles, MHCIα mRNA was present at minute levels, at least three orders of magnitude lower than in cardiac atrium. As verified immunohistochemically, MHCIα is present only in intrafusal fibres of rat muscle. Moreover, MHCIα is not expressed in extrafusal fibres and, contrary to the rabbit, was not upregulated at both the mRNA and protein levels by CLFS. These results support our notion of species-specific responses to CLFS. Another antibody reported to be specific to MHCIα, BA-G5, was also investigated by immunoblot and immunohistochemical analyses. Its specificity could not be validated for skeletal muscles of the rat. BG-A5 was shown to cross-react with MHCIIb and MHCIβ. These results question an upregulation of MHCIα in transforming rat muscles as reported in studies based on the use of this antibody.

Nitric oxide synthase inhibition prevents activity-induced calcineurin–NFATc1 signalling and fast-to-slow skeletal muscle fibre type conversions

•  Exercise is known to trigger skeletal muscle structural and functional adaptations. •  Control of these adaptive alterations is a complex process involving multiple signalling pathways and levels of regulation. •  The well‐characterized calcineurin–nuclear factor of activated T‐cells (NFATc1) signalling pathway is involved in the regulation of activity‐dependent alterations in skeletal muscle myosin heavy chain expression. Myosin heavy chain is a contractile protein that largely dictates a muscles speed of contraction. •  We show that a signalling molecule called nitric oxide may be regulating alterations in myosin heavy chain expression via activity‐modulated calcineurin–NFATc1 signalling. •  These findings increase our understanding of how skeletal muscle adaptive alterations are regulated.

Reliability of isolated isometric function measures in rat muscles composed of different fibre types

The present study investigated the absolute reliability (RAb) of isometric measures of time‐to‐peak tension (TTP), half‐rise time (½RT), half‐fall time (½FT), twitch force (TWf) tetanic force (TETf) and the sag ratio as applied to the slow soleus (SOL) and the fast‐twitch extensor digitorum longus (EDL) and medial gastrocnemius (MG) muscles of the rat hindlimb. In addition, the relationship of each individual isometric measure was examined with regard to the pattern of myosin heavy chain (MHC) isoform expression. Measures of TTP, ½RT, ½FT and sag ratio were negatively correlated with MHCIId(x) and MHCIIb (P < 0.0001), and positively correlated with MHCI (P < 0.0001). TWf and TETf were negatively correlated with MHCI content (P < 0.0001) and positively with MHCIId(x) (P < 0.0001) and MHCIIb (P < 0.001). Comparisons of isometric measures using a paired Students t test revealed that they were not different between the right and left legs; all measures displayed high correlations between the left and right legs (r= 0.71–0.85, P < 0.0001). In contrast to standard tests of statistical significance, these functional measures exhibited a considerable range of RAb when individual muscles were studied in only one hindlimb. When averaged across all muscles, however, the ½FT, ½RT, TWf and TTP measures possessed high overall reliability; measures of TETf and sag ratio were moderately reliable. The results of this study show that the isometric measures studied possess significant predictive value with regard to MHC isoform content; the left and right legs are interchangeable but display a considerable range of reliability when only one hindlimb is studied.