The influence of skeletal muscle cell volume on the regulation of carbohydrate uptake and muscle metabolism

This study investigated the regulation of carbohydrate metabolism and glucose uptake through changes in skeletal muscle cell volume. Using an established invitro isolated whole muscle model, soleus (SOL) and extensor digitorum longus (EDL) muscles were dissected from male rats and incubated in an organ bath containing Sigma medium-199 with 8 mM D-glucose altered to target osmolality (hypo-osmotic: HYPO, iso-osmotic: ISO, hyper-osmotic: HYPER; 190, 290, 400 mmol/kg). Muscles were divided into two groups; metabolite (MM) and uptake (MU). MM (N=48) were incubated for 60 minutes and were then immediately flash frozen. MU (N=24) were incubated for 30 minutes and then the extracellular fluid was exchanged for media containing ^H-glucose and ^'*C-mannitol and incubated for another 30 minutes. After the incubation, the muscles were freeze clamped. Results demonstrated a relative water decrease and increase in HYPER and HYPO, respectively. EDL and SOL glucose uptakes were found to be significantly greater in HYPER conditions. The HYPER condition resulted in significant alterations in muscle metabolite concentrations (lower glycogen, elevated lactate, and G-6-P) suggesting a catabolic cell state, and an increase in glycogen synthase transformation when compared to the HYPO group. In conclusion, skeletal muscle cell volume alters rates of glucose uptake with further alterations in muscle metabolites and glycogen synthase transformation.

Adaptations of skeletal muscle pyruvate dehydrogenase kinase in response to food-restriction in mitochondrial subpopulations

University, 2006 Dr. Sandra J. Peters Pyruvate dehydrogenase (PDH) catalyses the decarboxylation of pyruvate, to form acetyl-CoA. PDH activity is down-regulated by intrinsic PDH kinases (predominantly PDK2 and PDK4 isoforms), but the understanding of the PDK isoform distribution and adaptation to nutritional stresses has been restricted to mixed mitochondrial populations, and not delineated between subsarcolemmal (SS) and intermyofibrillar (IMF) subpopulations. SS and IMF mitochondria exhibit distinct morphological and biochemical properties; however the functional differences are not well understood. This study investigated the effect of fed (FED) versus 48 h total foodrestriction (FR) on rat red gastrocnemius muscle PDK2 and 4 isoform content in SS and IMF mitochondria. PDK4 content was ~3-5 fold higher in SS mitochondria compared to IMF (p=0.001), and increased with FR -3-4- fold in both subpopulations (p<0.001). PDK2 was -2.5-4 fold higher in SS mitochondria compared to IMF (p=0.001), but PDK2 was unaltered with FR. Citrate synthase activity (|imol/min/mg mitochondrial protein) was not different between either subpopulation. As well there were no significant differences between mitochondrial subpopulations in PDH complex components in both fed and FR states. These results demonstrate that there is a markedly higher content of both PDK isofonns in SS compared to IMF mitochondria. Although PDK2 does not increase in either subpopulation in response to FR, PDK4 increases to a similar extent in both SS and IMF after 48 h food-restriction.

The effect of elevated muscle fluid volume on indices of muscle damage following an acute bout of eccentric exercise

The primary purpose of the current investigation was to develop an elevated muscle fluid level using a human in-vivo model. The secondary purpose was to determine if an increased muscle fluid content could alter the acute muscle damage response following a bout of eccentric exercise. Eight healthy, recreationally active males participated in a cross-over design involving two randomly assigned trials. A hydration trial (HYD) consisting of a two hour infusion of a hypotonic (0.45%) saline at a rate of 20mL/minVl .73m"^ and a control trial (CON), separated by four weeks. Following the infusion (HYD) or rest period (CON), participants completed a single leg isokinetic eccentric exercise protocol of the quadriceps, consisting of 10 sets of 10 repetitions with a one minute rest between each set. Muscle biopsies were collected prior to the exercise, immediately following and at three hours post exercise. Muscle analysis included determination of wet-dry ratios and quantification of muscle damage using toluidine blue staining and light microscopy. Blood samples were collected prior to, immediately post, three and 24 hours post exercise to determine changes in creatine kinase (CK), lactate dehydrogenase (LD), interleukin-6 (IL-6) and Creactive protein (CRP) levels. Results demonstrated an increased muscle fluid volume in the HYD condition following the infusion when compared to the CON condition. Isometric peak torque was significantly reduced following the exercise in both the HYD and CON conditions. There were no significant differences in the number of areas of muscle damage at any of the time points in either condition, with no differences between conditions. CK levels were significantly greater 24hour post exercise compared to pre, immediately and three hours post similarly in both conditions. LD in the HYD condition followed a similar trend as CK with 24 hour levels higher than pre, immediately post and three hours post and LD levels were significantly greater 24 hours post compared to pre levels in the CON condition, with no differences between conditions. A significant main effect for time was observed for CRP (p<0.05) for time, such that CRP levels increased consistently at each subsequent time point. However, CRP and IL-6 levels were not different at any of the measured time points when comparing the two conditions. Although the current investigation was able to successfully increase muscle fluid volume and an increased CK, LD and CRP were observed, no muscle damage was observed following the eccentric exercise protocol in the CON or HYD conditions. Therefore, the hypotonic infusion used in the HYD condition proved to be a viable method to acutely increase muscle fluid content in in-vivo human skeletal muscle.

The influence of skeletal muscle cell volume on carbohydrate metabolism in contracting skeletal muscle

This study investigated the regulation of carbohydrate metabolism through changes in skeletal muscle cell volume immediately post contraction and during recovery. Using an established in vitro isolated muscle strip model, soleus (SOL) and extensor digitorum longus (EDL) were dissected from male rats and incubated in an organ bath (perfused with 95% O2; 5% CO2, pH 7.4, temperature 25°C) containing medium- 199 altered to a target osmotic condition (iso-, hypo- or hyper-osmotic; 290, 1 80, 400 mmol/kg). Muscles were stimulated for 10 minutes (40 Hz SOL; 30 Hz EDL) and then either immediately flash frozen or allowed to recover for 20 minutes before subsequent metabolite and enzyme analysis. Results demonstrated a relative water decrease in HYPER vs. HYPOosmotic condition (n=8/group; p<0.05) regardless of muscle type. Specifically, the SOL HYPER condition had elevated metabolite concentrations after 10 minutes of stimulation in comparison to both HYPO and ISO (p<0.05), while EDL muscle did not show any significant difTerences between the HYPER or HYPO conditions. After 20 minutes of recovery, metabolic changes occurred in both SOL and EDL with the SOL HYPER condition showing greater relative changes in metabolite concentrations versus HYPO. The results of the current study have demonstrated that osmotic imbalance induces metabolic change within the skeletal muscle cell and muscle type may influence the mechanisms utilized for cell volume regulation.

Attenuation of skeletal muscle FFA-induced insulin resistance by the polyphenol resveratrol. Elucidation of the mechanisms involved

Excess plasma free fatty acids (FFA) are correlated with insulin resistance and are a risk factor for the development of type 2 diabetes. In this study we examined the effect of the polyphenol resveratrol on FF A-induced insulin resistance in skeletal muscle cells and the mechanisms involved. Incubation of L6 myotubes with the FF A palmitate significantly decreased the insulin-stimulated glucqse uptake. Importantly, the effect of palmitate was ameliorated by resveratrol. Palmitate significantly increased serine phosphorylation of IRS..; 1 and reduced insulin-stimulated Akt phosphorylation, an effect that was abolished by resveratrol. We then investigated the effect of palmitate and resveratrol on the expression and phosphorylation of JNK, mTOR, p70-S6K, and AMPK kinases. The results demonstrated that our treatments had no effect on the expression of these proteins. However, palmitate increased the phosphorylation of mTOR and p70- S6K, whereas resveratrol abolished this effect and increased the phosphorylation of AMPK. Furthermore, all effects of resveratrol were abolished with sirtuin inhibitors, sirtinol and nicotinamide. These results indicate that resveratrol ameliorated FF A-induced insulin resistance by regulating mTOR and p70-S6K phosphorylation in skeletal muscle cells, through a mechanism involving sirtuins.

Phosphorylation of Skeletal Muscle Pyruvate Dehydrogenase Phosphatase in Response to Insulin Stimulation

Pyruvate dehydrogenase phosphatase (PDP) regulates carbohydrate oxidation through the pyruvate dehydrogenase (PDH) complex. PDP activates PDH, enabling increased carbohydrate flux towards oxidative energy production. In culture myoblasts, both PDP1 and PDP2 undergo covalent activation in response to insulin–stimulation by protein kinase C delta (PKCδ). Our objective was to examine the effect of insulin on PDP phosphorylation and PDH activation in skeletal muscle. Intact rat extensor digitorum longus muscles were incubated (oxygenated at 25°C, 1g of tension) for 30min in basal or insulin–stimulated (10 mU/mL) media. PDH activity increased 58% following stimulation, (p=0.057, n=11). Serine phosphorylation of PDP1 (p=0.047) and PDP2 (p=0.006) increased by 29% and 48%, respectively (n=8), and mitochondrial PKCδ protein content was enriched by 45% in response to stimulation (p=0.0009, n=8). These data suggest that the insulin–stimulated increase in PDH activity in whole tissue is mediated through mitochondrial migration of PKCδ and subsequent PDP phosphorylation.

The role of skeletal muscle PLIN proteins at rest and following lipolytic stimulation

This thesis investigated the subcellular location of skeletal muscle PLIN proteins (PLIN2, PLIN3, and PLIN5) as well as protein interactions with ATGL and HSL at rest and following lipolytic stimulation. In addition, the serine phosphorylation state of PLIN2, PLIN3, and PLIN5 was determined at rest and following lipolytic stimulation. An isolated whole muscle technique was used to study the effects of contraction and epinephrine-induced lipolysis. This method allowed for the examination of the effects of contraction and epinephrine alone and in combination. Further, the soleus was chosen for investigating the role of PLIN proteins in skeletal muscle lipolysis due to its suitability for isolated incubation, and the fact that it is primarily oxidative in nature (~80% type I fibres). It has also been previously shown to have the greatest reliance on lipid metabolism and for this reason is ideal for investigating the role of PLIN proteins in lipolysis. Immunofluorescence microscopy revealed that skeletal muscle lipid droplets are partially co-localized to both PLIN2 and PLIN5 and that contraction does not affect the amount of colocalization, indicating that PLIN5 is not recruited to lipid droplets with contraction (PLIN2 ~65%; PLIN5 ~56%). Results from the immunoprecipitation studies revealed that with lipolysis in skeletal muscle the interaction between ATGL and CGI-58 is increased (study 2: 128% with contraction, p<0.05; study 3: 50% with contraction, 25% epinephrine, 80% contraction + epinephrine, p>0.05). Further PLIN2, PLIN3, and PLIN5 all interact with ATGL and HSL, while only PLIN3 and PLIN5 interact with CGI-58. Among these interactions, the association between PLIN2 and ATGL decreases with lipolytic stimulation (study 2: 21% with contraction, p<0.05). Finally our results demonstrate that PLIN3 and PLIN5 are serine phosphorylated at rest and that the level of phosphorylation remains unchanged in the face of either contractile or adrenergic stimulation. In summary, the regulation of skeletal muscle lipolysis is a complex process involving multiple proteins and enzymes. The skeletal muscle PLIN proteins likely play a role in skeletal muscle lipid droplet dynamics, and the data from this thesis indicate that these proteins may work together in regulating lipolysis by interaction with both ATGL and HSL.

Force potentiation as a modulator of contractile performance: Implications for control of skeletal muscle force and energetics of work

This thesis investigated the modulation of dynamic contractile function and energetics of work by posttetanic potentiation (PTP). Mechanical experiments were conducted in vitro using software-controlled protocols to stimulate/determine contractile function during ramp shortening, and muscles were frozen during parallel incubations for biochemical analysis. The central feature of this research was the comparison of fast hindlimb muscles from wildtype and skeletal myosin light chain kinase knockout (skMLCK-/-) mice that does not express the primary mechanism for PTP: myosin regulatory light chain (RLC) phosphorylation. In contrast to smooth/cardiac muscles where RLC phosphorylation is indispensable, its precise physiological role in skeletal muscle is unclear. It was initially determined that tetanic potentiation was shortening speed dependent, and this sensitivity of the PTP mechanism to muscle shortening extended the stimulation frequency domain over which PTP was manifest. Thus, the physiological utility of RLC phosphorylation to augment contractile function in vivo may be more extensive than previously considered. Subsequent experiments studied the contraction-type dependence for PTP and demonstrated that the enhancement of contractile function was dependent on force level. Surprisingly, in the absence of RLC phosphorylation, skMLCK-/- muscles exhibited significant concentric PTP; consequently, up to ~50% of the dynamic PTP response in wildtype muscle may be attributed to an alternate mechanism. When the interaction of PTP and the catchlike property (CLP) was examined, we determined that unlike the acute augmentation of peak force by the CLP, RLC phosphorylation produced a longer-lasting enhancement of force and work in the potentiated state. Nevertheless, despite the apparent interference between these mechanisms, both offer physiological utility and may be complementary in achieving optimal contractile function in vivo. Finally, when the energetic implications of PTP were explored, we determined that during a brief period of repetitive concentric activation, total work performed was ~60% greater in wildtype vs. skMLCK-/- muscles but there was no genotype difference in High-Energy Phosphate Consumption or Economy (i.e. HEPC: work). In summary, this thesis provides novel insight into the modulatory effects of PTP and RLC phosphorylation, and through the observation of alternative mechanisms for PTP we further develop our understanding of the history-dependence of fast skeletal muscle function.

The capillary supply of human skeletal muscle in health and disease

BACKGROUND: Capillaries function to provide a surface area for nutrient and waste exchange with cells. The capillary supply of skeletal muscle is highly organized, and therefore, represents an excellent choice to study factors regulating diffusion. Muscle is comprised of three specific fibre types, each with specific contractile and metabolic characteristics, which influence the capillary supply of a given muscle; in addition, both environmental and genetic factors influence the capillary supply, including aging, physical training, and various disease processes. OBJECTIVE: The present study was undertaken to develop and assess the functionality of a data base, from which virtual experiments can be conducted on the capillary supply of human muscle, and the adaptations of the capillary bed in muscle to various perturbations. METHODS: To create the database, an extensive search of the literature was conducted using various search engines, and the three key words - "capillary, muscle, and human". This search yielded 169 papers from which the data for the 46 variables on the capillary supply and fibre characteristics of muscle were extracted for inclusion in the database. A series of statistical analyses (ANOVA) were done on the capillary database to examine differences in skeletal muscle capillarization and fibre characteristics between young and old individuals, between healthy and diseased individuals, and between untrained, endurance trained, endurance welltrained, and resistance trained individuals, using SAS. RESULTS: There was a significantly higher capillarization in the young compared to the old individuals, in the healthy compared to the diseased individuals, and in the endurance-trained and endurance well-trained compared to the untrained individuals. CONCLUSIONS: The results of this study support the conclusion that the capillary supply of skeletal muscle is closely regulated by factors aimed at optimizing oxygen and nutrient supply and/or waste removal in response to changes in muscle mass and/or metabolic activity.

X-ray diffraction studies of muscle : observations on the anterior byssus retractor muscle of Mytilus edulis and the insect flight muscle from Sarcophaga bullata

The interfilament spacing of the anterior byssus retractor muscle from Mytilus edulis was studied as the muscle was extended. It was found that variations in this spacing were very small and consistent with the hypothesis that the interfilament spacing was independent of the extension of the muscle. It was observed that the interfilament spacing was dependent on the osmolarity of the bathing medium. In concentrated solutions of the artificial seawater, the interfilament spacing decreased; while in dilute solutions of artificial seawater, it was observed that the interfilament spacing was increasing. X-ray diffraction patterns were obtained from fresh, and glutaraldehyde fixed, specimens of insect flight muscle from Sarcophaga bullata. There patterns were in general agreement with previous X-ray diffraction studies of insect flight muscle. A reflexion G at 93A was observed and interpreted as arising from diffraction in the mitochondria. Specimens of dried insect flight muscle produced a diffraction pattern consisting of arc and ring reflexions. This was interpreted as suggesting an ordered arrangement of cristae, in the mitochondria from these muscles.

Myosin Regulatory Light Chain Phosphorylation and Its Effect on the Contractile Economy of Mouse Fast Muscle

Activated by elevations in myoplasmic calcium concentration, myosin light chain kinase (skMLCK) phosphorylates the regulatory light chains (RLCs) of fast muscle myosin. This covalent modification potentiates force production, but requires an investment of ATP. Our objective was to investigate the effect of RLC phosphorylation on the contractile economy (mechanical output:metabolic input) of fast twitch skeletal muscle. Extensor digitorum longus muscles isolated from Wildtype and skMLCK-/- mice mounted in vitro (25°C) were subjected to repetitive low-frequency stimulation (10Hz,15s) known to cause activation of skMLCK, and staircase potentiation of force. With a 3-fold increase in RLC phosphate content, Wildtype generated 44% more force than skMLCK-/- muscles over the stimulation period (P = .002), without an accompanied increase in energy cost (P = .449). Overall, the contractile economy of Wildtype muscles, with an intact RLC phosphorylation mechanism, was 73% greater than skMLCK /- muscles (P = .043), demonstrating an important physiological function of skMLCK during repetitive contractile activity.

The roles of ERK1/2 and PI3K in abnormal vascular functions in angiotensin II-infused hypertensive rats

Background: Ang II plays a major role in cardiovascular regulation. Recently, it has become apparent that vascular superoxide anion may play an important role in hypertension development. Treatment with antisense NAD(P)H oxidase or SOD decreased BP in Ang II-infused rats. Wang et al recently reported mice which lack one of the subunits of NAD(P)H oxidase developed hypertension at a much lower extent when compared to the wild type animals infused with Ang II, indicating that superoxide anion contributes to elevation in BP in the Ang II-infused hypertensive model. In the Ang II-infused hypertensive model, altered reactivity of blood vessels is often associated with the elevation of systolic blood pressure. We have observed abnormal tension development and impaired endothelium-dependent relaxation in the isolated aorta of Ang II-infused and DOCA-salt hypertensive rats. Recently, several other cellular signal molecules, including ERK1I2 and PI3K, have been determined to play important roles in the regulation of smooth muscle contraction and relaxation. ERKl/2 and PI3K pathways are also reported to contribute to Ang II induced cell growth, hypertrophy, remodeling and contraction. Moreover, these signaling pathways have shown ROS-sensitive properties. Therefore, the aim of the present study is to investigate the roles of ERKl12 and PI3K in vascular oxidative stress, spontaneous tone and impaired endothelium relaxation in Ang II-infused hypertensive model. Hypothesis: We hypothesize that the activation of ERKl12 and PI3K are elevated in response to an Ang II infusion for 6 days. The elevated activation of phospho-ERKl/2 and PI3K mediated the increased level of vascular superoxide anion, the abnormal vascular contraction and impaired endothelium-dependent vascular relaxation in Ang II-infused hypertensive rats. Methods: Vascular superoxide anion level is measured by lucigenin chemiluminescence. Spontaneous tone and ACh-induced endothelium-dependent relaxation was measured by isometric tension recording in organ chamber. The activity of ERK pathway will be measured by its Western blot of phosphorylation of ERK. PI3K activity was evaluated indirectly by Western blot of the phosphorylation of PDKl, a downstream protein of PI3K signaling pathway. The role of each pathway was also addressed via comparing the responses to the specific inhibitors. Results: Superoxide anion was markedly increased in the isolated thoracic aorta from Ang II-infused rats. There was spontaneous tone developed in rings from Ang II-induced hypertensive but not sham-operated normotensive rats. ACh-induced endothelium-dependent relaxation function is impaired in Ang II-infused hypertensive rats. Superoxide dismutase and NAD(P)H oxidase inhibitor, apocynin, inhibited the abnormal spontaneous tone and ameliorated impaired endothelium-dependent relaxation. The expression of phopho-ERKII2 was enhanced in Ang II-infused rats, indicating the activity of ERK1I2 could be increased. MEK1I2 inhibitors, PD98059 and U126, but not their inactive analogues, SB203580 and U124, significantly reduced the vascular superoxide anion in aortas from Ang II-infused rats. The MEK1I2 inhibitors reduced the spontaneous tone and improved the impaired endothelium-dependent relaxation in aorta of hypertension. These findings supported the role of ERKII2 signaling pathway in vascular oxidative stress, spontaneous tone and impaired endothelium-dependent relaxation in Ang II-infused hypertensive rats. The amount of phospho-PDK, a downstream protein of PI3K was increased in Ang II rats indicating the activity of PI3K activity was elevated. Strikingly, PI3K significantly inhibited the increase of superoxide anion level, abnormal spontaneous tone and restored endothelium-dependent relaxation in Ang II-infused hypertensive rats. These findings indicated the important role of PI3K in Ang II-infused hypertensive rats. Conclusion: ERKII2 and PI3K signaling pathways are sustained activated in Ang II-infused hypertensive rats. The activated ERKII2 and PI3K mediate the increase of vascular superoxide anion level, vascular abnormal spontaneous tone and impaired endothelium-dependent relaxation.

Active isolated stretching : an investigation of the mechanical mechanisms

The Active Isolated Stretching (AIS) technique proposes that by contracting a muscle (agonist) the opposite muscle (antagonist) will relax through reciprocal inhibition and lengthen without increasing muscle tension (Mattes, 2000). The clinical effectiveness of AIS has been reported but its mechanism of action has not been investigated at the tissue level. Proposed mechanisms for increased range of motion (ROM) include mechanical or neural changes, or an increased stretch tolerance. The purpose of the study was to investigate changes in mechanical properties, i.e. stiffness, of skeletal muscle in response to acute and long-term AIS stretching for the hamstring muscle group. Recreationally active university-aged students (female n=8, male n=2) classified as having tight hamstrings, by a knee extension test, volunteered for the study. All stretch procedures were performed on the right leg, with the left leg serving as a control. Each subject was assessed twice: at an initial session and after completing a 6-week AIS hamstring stretch training program. For both test sessions active knee extension (ROM) to a position of "light irritation", passive resisted torque and stiffness were determined before and after completion of the AIS technique (2x10 reps). Data were collected using a Biodex System 3 Pro (Biodex Medical Systems, NY, USA) isokinetic dynamometer. Surface electromyography (EMG) was used to monitor vastus lateralis (VL) and hamstring muscle activity during the stretching movements. Between test sessions, 2x10 reps of the AIS bent knee hamstring stretch were performed daily for 6-weeks.

The influence of cognitive resources on compensatory arm responses

The purpose of this study was to determine the influence of an ongoing cognitive task on an individual’s ability to generate a compensatory arm response. Twenty young and 16 older adults recovered their balance from a support surface translation while completing a cognitive (counting) task of varying difficulty. Surface electromyographic (EMG) recordings from the shoulders and kinematics of the right arm were collected to quantify the compensatory arm response. Results indicated that the counting task, regardless of its difficulty as well as the age of the individual, had minimal influence on the onset or magnitude of arm muscle activity that occurred following a loss of balance. In contrast to previous research, this study’s findings suggest that the cortical or cognitive resources utilized by the cognitive task are not relied upon for the generation of compensatory arm responses and that older adults are not disproportionately affected by dual-tasking than young adults.

Action of octopamine and tyramine on muscles of Drosophila melanogaster larvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10−5 and 10−4 M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10−5 M OA increased synaptically driven contractions by ∼1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.