Fiber Type Identification of Human Skeletal Muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

La Trobe University

Through its upregulation and/or translocation, heat shock protein 72 (HSP72) is involved in protection and repair of key proteins after physiological stress. In human skeletal muscle we investigated HSP72 protein after eccentric (ECC1) and concentric (CONC) exercise and repeated eccentric exercise (ECC2; 8 wk later) and whether it translocated from its normal cytosolic location to membranes/myofibrils. HSP72 protein increased ~2-fold 24 h after ECC1, with no apparent change after CONC or ECC2. In resting (nonstressed) human skeletal muscle the total pool of HSP72 protein was present almost exclusively in the cytosolic fraction, and after each exercise protocol the distribution of HSP72 protein remained unaltered. Overall, the amount of HSP72 protein in the cytosol increased 24 h after ECC1, matching the fold increase that was measured in total HSP72 protein. To better ascertain the capabilities and limitations of HSP72, using quantitative Western blotting we determined the HSP72 protein content to be 11.4 μmol/kg wet weight in resting human vastus lateralis muscle, which is comprised of Type I (slow-twitch) and Type II (fast-twitch) fibers. HSP72 protein content was similar in individual Type I or II fiber segments. After physiological stress, HSP72 content can increase and, although the functional consequences of increased amounts of HSP72 protein are poorly understood, it has been shown to bind to and protect protein pumps like SERCA and Na(+)-K(+)-ATPase. Given no translocation of cytosolic HSP72, these findings suggest eccentric contractions, unlike other forms of stress such as heat, do not trigger tight binding of HSP72 to its primary membrane-bound target proteins, in particular SERCA.

Subcellular fractionation reveals HSP72 does not associate with SERCA in human skeletal muscle following damaging eccentric and concentric exercise.

Small heat shock proteins (sHSPs) are a subgroup of the highly conserved family of HSPs that are stress inducible and confer resistance to cellular stress and injury. This study aimed to quantitatively examine whether type of contraction (concentric or eccentric) affects sHSPs, HSP27 and αB-crystallin, localization, and phosphorylation in human muscle. Vastus lateralis muscle biopsies from 11 healthy male volunteers were obtained pre- and 3 h, 24 h, and 7 days following concentric (CONC), eccentric (ECC1), and repeated bout eccentric (ECC2) exercise. No changes were apparent in a control group (n = 5) who performed no exercise. Eccentric exercise induced muscle damage, as evidenced by increased muscle force loss, perceived muscle soreness, and elevated plasma creatine kinase and myoglobin levels. Total HSP27 and αB-crystallin amounts did not change following any type of exercise. Following eccentric exercise (ECC1 and ECC2) phosphorylation of HSP27 at serine 15 (pHSP27-Ser15) was increased approximately 3- to 6-fold at 3 h, and pαB-crystallin-Ser59 increased ~10-fold at 3 h. Prior to exercise most of the sHSP and psHSP pools were present in the cytosolic compartment. Eccentric exercise resulted in partial redistribution of HSP27 (~23%) from the cytosol to the cytoskeletal fraction (~28% for pHSP27-Ser15 and ~7% for pHSP27-Ser82), with subsequent full reversal within 24 h. αB-crystallin also showed partial redistribution from the cytosolic to cytoskeletal fraction (~18% of total) 3 h post-ECC1, but not after ECC2. There was no redistribution or phosphorylation of sHSPs with CONC. Eccentric exercise results in increased sHSP phosphorylation and translocation to the cytoskeletal fraction, but the sHSP translocation is not dependent on their phosphorylation.

Small heat shock proteins translocate to the cytoskeleton in human skeletal muscle following eccentric exercise independently of phosphorylation.

The 5'-AMP-activated protein kinase (AMPK), a heterotrimeric complex that functions as an intracellular fuel sensor that affects metabolism, is activated in skeletal muscle in response to exercise and utilization of stored energy. The diffusibility properties of α- and β-AMPK were examined in isolated skeletal muscle fiber segments dissected from rat fast-twitch extensor digitorum longus and oxidative soleus muscles from which the surface membranes were removed by mechanical dissection. After the muscle segments were washed for 1 and 10 min, ∼60% and 75%, respectively, of the total AMPK pools were found in the diffusible fraction. After in vitro stimulation of the muscle, which resulted in an ∼80% decline in maximal force, 20% of the diffusible pool became bound in the fiber. This bound pool was not associated with glycogen, as determined by addition of a wash step containing amylase. Stimulation of extensor digitorum longus muscles resulted in 28% glycogen utilization and a 40% increase in phosphorylation of the downstream AMPK target acetyl carboxylase-CoA. This, however, had no effect on the proportion of total β2-AMPK that was phosphorylated in whole muscle homogenates measured by immunoprecipitation. These findings suggest that, in rat skeletal muscle, β2-AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate β2-AMPK. These findings question the physiological relevance of the carbohydrate-binding function of β2-AMPK in skeletal muscle.

When phosphorylated at Thr148, the β2-subunit of AMP-activated kinase does not associate with glycogen in skeletal muscle.

Major stores of glucose are found as glycogen in skeletal muscle and liver. Skeletal muscle is a heterogenous tissue, with cellular metabolic and contractile distinctions dependent on whether the cell (fibre) is slow-twitch (Type I) or fast-twitch (Type II). We hypothesised that proteins important for glycogen metabolism would be differentially abundant between these diverse fibres. We further hypothesised that the cellular location of these proteins would be different in muscle samples between control (CON) and individuals with type 2 diabetes (T2D). We dissected individual muscle fibre segments from vastus lateralis skeletal muscle biopsy samples from CON and T2D and used cell-type-specific approaches to address muscle heterogeneity. We measured glycogen and glycogen-related proteins by immunoblotting techniques. A lower proportion of Type I fibres was found in muscle in T2D compared with CON. AMPK-β2, glycogen branching enzyme (GBE), glycogen debranching enzyme (GDE), and glycogen phosphorylase (GP) were differentially localized between fibre types and in fibres from CON and T2D individuals. A key novel finding was that the majority of glycogen is loosely bound or cytosolic in location in human skeletal muscle. The proportion of this diffusible pool of glycogen was significantly lower in Type I fibres in T2D compared to CON. A hyperinsulinaemic, euglycaemic clamp in people with type 2 diabetes had no effect on the proportion of diffusible glycogen. We identify cell-type as an important consideration when assessing glycogen metabolism in muscle. Our findings demonstrate varying glucose handling abilities in specific muscle fibre types in type 2 diabetes. A model is presented to provide an overview of the cell-specific differences in glycogen metabolism in type 2 diabetes.

Noni T. Frankenberg

Department of Biochemistry and Chemistry,
La Trobe Institute for Molecular Science,
School of Agriculture,
Biomedicine and Environment,
Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Science, School of Agriculture, Biomedicine and Environment

La Trobe University

Fiber Type Identification of Human Skeletal Muscle