Glycolytic Muscle Fibers Research Articles

Meta-analysis of the relationships between beef tenderness and muscle characteristics

Beef tenderness is characterised by a high and uncontrolled variability which depends, at least in part, on differences in muscle characteristics. The aim of this work was to identify general relationships between beef tenderness and muscle characteristics across experiments, using a large set of data available in the BIF-Beef (Integrated and Functional Biology of Beef) database. Tenderness was evaluated by sensory methods with trained panellists and by shear force measurements. Total and insoluble collagen contents, intramuscular fat content (IMF), mean cross sectional fibre area, isocitrate dehydrogenase (ICDH) and lactate dehydrogenase (LDH) activities and the proportion of slow oxidative (SO), fast oxido-glycolytic (FOG) and fast glycolytic (FG) muscle fibres were measured in both Longissimus thoracis (LT) and Semitendinosus (ST) muscles. Total collagen content, IMF content, mean muscle fibre area, LDH and ICDH activities explained respectively, 2%, 0.3%, 1.8%, 1.6% and 1.7% maximum of the variability (r²) in the sensory tenderness score. The total and insoluble collagen contents, the LDH activity and the FG proportion explained, respectively, 6%, 6%, 4% and 5% of the variability in the shear force, essentially in the ST muscle but not in LT muscle. The relationships between different muscle characteristics were confirmed. It was demonstrated that the determinism of tenderness was complex and mainly muscle dependant. The large data set used allowed the statement of general laws and contributed to explain the divergent results in the literature from smaller sets of data originating from specific experiments.

Regulation of Respiration in Permeabilized Muscle Cells: Apparent KM for ADP Shows the Mitochondrial Outer Membrane Permeability

The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers. Despite certain similarities of mitochondrial organization into Intracellular Energetic Units (ICEUs), different muscle types have distinct metabolic pattern with specific regulatory mechanisms of mitochondrial respiration. In this work we measured ADP fluxes through mitochondrial outer membrane (MOM) in permeabilized fibers oxygraphically using pyruvate kinase - phosphoenolpyruvate system for trapping ADP produced in mitochondrial creatine kinase localized behind MOM. These fluxes were high in permeabilized fibers from glycolytic muscles and very low or absent in soleus muscle fibers and cardiac myocytes. These results indicate that the permeability of voltage-dependent anion channel (VDAC) in MOM correlates with the apparent Km for ADP in regulation of respiration. In cardiac cells, the MOM permeability seems to be regulated by the interaction of VDAC with cytoskeletal protein βII tubulin. To ascertain the role of this protein in skeletal muscles we visualized the localization pattern of βII tubulin together with mitochondrial protein VDAC immunocytochemically. We used the Metabolic Control Analysis to evaluate the Flux Control Coefficients of the respiratory chain complexes (I,III, IV), ANT, ATP synthase, and MtCK in permeabilized soleus muscle fibers under conditions of respiration stimulated by exogenous ADP and by endogenous ADP produced in activated MtCK reaction.

Glucocorticoid-induced myopathy: Pathophysiology, diagnosis, and treatment

Glucocorticoid-induced myopathy is the most common type of drug-induced myopathy. Nearly 60% of patients with Cushing's syndrome have muscle weakness. Glucocorticoid-induced muscle atrophy affects mainly fast-twitch glycolytic muscle fibers (type IIb fibers). This brief review will discuss the pathophysiology behind glucocorticoid-induced myopathy, along with diagnostic features and treatment.

Metavinculin: New insights into functional properties of a muscle adhesion protein

Metavinculin is a muscle-specific splice variant of the ubiquitously expressed cytoskeletal adaptor protein vinculin. Both proteins are thought to be co-expressed in all muscle types where they co-localize to microfilament-associated adhesion sites. It has been shown that a metavinculin-specific insertion of 68 amino acids alters the biochemical properties of the five-helix bundle in the tail domain. Here, we demonstrate that the metavinculin-specific helix H1' plays an important role for protein stability of the tail domain, since a point mutation in this helix, R975W, which is associated with the occurrence of dilated cardiomyopathy in man, further decreases thermal stability of the metavinculin tail domain. In striated muscle progenitor cells (myoblasts), both, metavinculin and the R975W mutant show significantly reduced, albeit distinctive residency and exchange rates in adhesion sites as compared to vinculin. In contrast to previous studies, we show that metavinculin is localized in a muscle fiber type-dependent fashion to the costameres of striated muscle, reflecting the individual metabolic and physiological status of a given muscle fiber. Metavinculin expression is highest in fast, glycolytic muscle fibers and virtually absent in M. diaphragmaticus, a skeletal muscle entirely lacking fast, glycolytic fibers. In summary, our data suggest that metavinculin enrichment in attachment sites of muscle cells leads to higher mechanical stability of adhesion complexes allowing for greater shear force resistance.

Tissue-Specific Strategies of the Very-Long Chain Acyl-CoA Dehydrogenase-Deficient (VLCAD−/−) Mouse to Compensate a Defective Fatty Acid β-Oxidation

Very long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency is the most common long-chain fatty acid oxidation disorder presenting with heterogeneous phenotypes. Similar to many patients with VLCADD, VLCAD-deficient mice (VLCAD−/−) remain asymptomatic over a long period of time. In order to identify the involved compensatory mechanisms, wild-type and VLCAD−/− mice were fed one year either with a normal diet or with a diet in which medium-chain triglycerides (MCT) replaced long-chain triglycerides, as approved intervention in VLCADD. The expression of the mitochondrial long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) was quantified at mRNA and protein level in heart, liver and skeletal muscle. The oxidation capacity of the different tissues was measured by LC-MS/MS using acyl-CoA substrates with a chain length of 8 to 20 carbons. Moreover, in white skeletal muscle the role of glycolysis and concomitant muscle fibre adaptation was investigated. In one year old VLCAD−/− mice MCAD and LCAD play an important role in order to compensate deficiency of VLCAD especially in the heart and in the liver. However, the white gastrocnemius muscle develops alternative compensatory mechanism based on a different substrate selection and increased glucose oxidation. Finally, the application of an MCT diet over one year has no effects on LCAD or MCAD expression. MCT results in the VLCAD−/− mice only in a very modest improvement of medium-chain acyl-CoA oxidation capacity restricted to cardiac tissue. In conclusion, VLCAD−/− mice develop tissue-specific strategies to compensate deficiency of VLCAD either by induction of other mitochondrial acyl-CoA dehydrogenases or by enhancement of glucose oxidation. In the muscle, there is evidence of a muscle fibre type adaptation with a predominance of glycolytic muscle fibres. Dietary modification as represented by an MCT-diet does not improve these strategies long-term.

Preservation of metabolic flexibility in skeletal muscle by a combined use of n-3 PUFA and rosiglitazone in dietary obese mice.

Insulin resistance, the key defect in type 2 diabetes (T2D), is associated with a low capacity to adapt fuel oxidation to fuel availability, i.e., metabolic inflexibility. This, in turn, contributes to a further damage of insulin signaling. Effectiveness of T2D treatment depends in large part on the improvement of insulin sensitivity and metabolic adaptability of the muscle, the main site of whole-body glucose utilization. We have shown previously in mice fed an obesogenic high-fat diet that a combined use of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and thiazolidinediones (TZDs), anti-diabetic drugs, preserved metabolic health and synergistically improved muscle insulin sensitivity. We investigated here whether n-3 LC-PUFA could elicit additive beneficial effects on metabolic flexibility when combined with a TZD drug rosiglitazone. Adult male C57BL/6N mice were fed an obesogenic corn oil–based high-fat diet (cHF) for 8 weeks, or randomly assigned to various interventions: cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids (cHF+F), cHF with 10 mg rosiglitazone/kg diet (cHF+ROSI), cHF+F+ROSI, or chow-fed. Indirect calorimetry demonstrated superior preservation of metabolic flexibility to carbohydrates in response to the combined intervention. Metabolomic and gene expression analyses in the muscle suggested distinct and complementary effects of the interventions, with n-3 LC-PUFA supporting complete oxidation of fatty acids in mitochondria and the combination with n-3 LC-PUFA and rosiglitazone augmenting insulin sensitivity by the modulation of branched-chain amino acid metabolism. These beneficial metabolic effects were associated with the activation of the switch between glycolytic and oxidative muscle fibers, especially in the cHF+F+ROSI mice. Our results further support the idea that the combined use of n-3 LC-PUFA and TZDs could improve the efficacy of the therapy of obese and diabetic patients.

Aberrant iron homeostasis, oxidative fiber enrichment, and activation of ketogenesis in muscle tissue of ISCU Myopathy patients

ISCU Myopathy, a disease characterized by life-long exercise intolerance and impaired mitochondrial oxidative metabolism, is caused by deficiency of the Fe-S cluster scaffold protein ISCU. We performed gene expression analysis on muscle biopsies from ISCU Myopathy patients to elucidate which molecular processes were transcriptionally remodeled in response to impaired Fe-S cluster assembly. We found that ISCU depletion led to increased expression of the mitochondrial iron importer MFRN2 and the rate-limiting heme biosynthetic enzyme ALAS1. Gene expression and histologic studies demonstrated that patient muscle composition was shifted towards fewer glycolytic muscle fibers, more oxidative fibers, and increased capillary abundance. Paradoxically, mitochondrial fatty acid uptake and oxidation genes were coordinately up-regulated in patient muscles despite dramatic impairments of aconitase and succinate dehydrogenase activities. The ketogenic enzymes HMGCS2 and BDH1 were also significantly up-regulated, as was the secreted starvation response hormone, FGF-21. We propose that ketogenesis may be initiated to restore free coenzyme A levels and shunt fatty acid oxidation products to distal respiration-competent tissues when TCA cycle and/or respiratory chain function is sufficiently impaired in affected patient muscle fibers. Moreover, our work shows that plasma FGF21 is a sensitive non-invasive biomarker of ISCU Myopathy, in addition to other mitochondrial myopathies.

Mouse Skeletal Muscle Fiber-Type-Specific Macroautophagy and Muscle Wasting Are Regulated by a Fyn/STAT3/Vps34 Signaling Pathway

Skeletal muscle atrophy induced by aging (sarcopenia), inactivity, and prolonged fasting states (starvation) is predominantly restricted to glycolytic type II muscle fibers and typical spares oxidative type I fibers. However, the mechanisms accounting for muscle fiber-type specificity of atrophy have remained enigmatic. In the current study, although the Fyn tyrosine kinase activated the mTORC1 signaling complex, it also induced marked atrophy of glycolytic fibers with relatively less effect on oxidative muscle fibers. This was due to inhibition of macroautophagy via an mTORC1-independent but STAT3-dependent reduction in Vps34 protein levels and decreased Vps34/p150/Beclin1/Atg14 complex 1. Physiologically, in the fed state endogenous Fyn kinase activity was increased in glycolytic but not oxidative skeletal muscle. In parallel, Y705-STAT3 phosphorylation increased with decreased Vps34 protein levels. Moreover, fed/starved regulation of Y705-STAT3 phosphorylation and Vps34 protein levels was prevented in skeletal muscle of Fyn null mice. These data demonstrate a Fyn/STAT3/Vps34 pathway that is responsible for fiber-type-specific regulation of macroautophagy and skeletal muscle atrophy.

Mitochondrial Functional Specialization in Glycolytic and Oxidative Muscle Fibers: Tailoring the Organelle for Optimal Function

A unique evolutionary feature of skeletal muscle is the co‐existence within the same organs of fast (glycolytic, Type II) and slow (oxidative, Type I) muscle fibers. These fiber‐types have specific contractile properties that generate unique intracellular conditions, to which bioenergetics and mitochondrial function must be tailored. However, the extent to which mitochondrial function differs between fast and slow muscle fibers has not clearly been established. Here, we present extensive data supporting the existence of distinct functional phenotypes in mitochondria from slow and fast fibers, including differences in energy substrate preferences, regulation of oxidative phosphorylation, dynamics of reactive oxygen species release, handling of Ca2+ and regulation of cell death via the mitochondrial permeability transition pore (mPTP). The potential physiological implications on muscle function, and the putative mechanisms responsible for establishing and maintaining distinct mitochondrial phenotype across fiber types are also discussed.

Differential adaptation of glycolytic and oxidative muscles to hypoxia

BackgroundCOPD‐associated skeletal muscle atrophy is found in glycolytic rather than oxidative muscle fibers. We hypothesize that hypoxia is a trigger for this fiber type‐specific atrophy.MethodsMice were kept under normoxia or hypoxia. Oxygen levels were reduced stepwise to 8% during 48h, and maintained for 21 days. Food intake was monitored and mice were sacrificed at days 4 and 21.ResultsReduction of oxidative (Soleus) muscle weight was due to hypoxia‐induced hypophagia. Glycolytic (EDL) muscle weight reduction involved additional hypophagia‐independent effects. Cross sectional area (CSA) of individual fibers was reduced in glycolytic but not oxidative muscle. Expression of genes involved in proteasomal and lysosomal protein degradation, ER stress and glucocorticoid receptor (GR) signaling were increased in both muscles. In glycolytic muscle, upregulation could not be explained by hypophagia alone. Capillary contacts per fiber CSA increased in both muscles. In oxidative muscle, this was due to a reduction of the interstitial space, whereas in glycolytic muscle fiber CSA decreased.ConclusionsMuscle mass, muscle fiber CSA, proteolysis, ER‐stress and GR signaling are more affected by hypoxia in glycolytic than oxidative muscle. In oxidative, but not glycolytic muscle, these alterations are accounted for by hypophagia.Supported by the Dutch Top Institute Pharma (project # T1‐201).

Fine Architectonics and Protein Turnover Rate in Myofibrils of Glucocorticoid-Caused Myopathic Rats

Objective: The purpose of this study was to assess the relationship between the synthesis and degradation rate of thick and thin myofilaments, and changes in fine architectonics of myofibrils of glycolytic muscle fibers of glucocorticoid caused myopathic rats. Methods: Male Wistar rats receive dexamethasone to induce myopathy. Protein synthesis rate degradation and structure of myofibrils was measured. Results: Myofibrils from glycolytic muscle fibers of 16-week-old male rats of the Wistar Strain were used for analysis. Myofibrils of glucocorticoid caused myopathic rats were thinner than in the control group (1.10 ± 0.05 m2 and 2.56 ± 0.08 m2, respectively) and thick and thin myofilaments in myopathic muscle disappeared from 22.0 ± 2.7% of the myofibrils cross-section area. In the control group, myofilaments were absent from 1.8 ± 0.5%. In the myopathic group, a positive correlation was found between the area of myofibrils where myofilaments disappeared and the myofibrillar protein degradation rate (r=0.849; p

Abstract 9679: Fast/Glycolytic Skeletal Muscle Growth Attenuates Cardiac Dysfunction and Remodeling After Experimental Myocardial Infarction

Introduction: It is appreciated that aerobic endurance exercise can attenuate unfavorable myocardial remodeling post myocardial infarction (MI). In contrast, little is known about the effect of resistance exercise on these processes, partly because animal models that can assess the relationship between glycolytic muscle fiber growth and cardiac remodeling have been lacking. Hypothesis: Fast/glycolytic skeletal muscle growth attenuates cardiac dysfunction and remodeling after MI. Methods: We utilized skeletal muscle-specific, conditional Akt1 transgenic (TG) mice, that can induce the growth of functional fast/glycolytic skeletal muscle by switching Akt1 signaling on in muscle fibers. Male non-induced TG mice and their non-transgenic littermates (control) were subjected to left anterior descending coronary artery ligation. Two days after surgery, mice were provided doxycycline in their drinking water to activate Akt1 transgene expression in a skeletal muscle-specific manner. Cardiac function and remodeling were assessed by micromanometer catheter, echocardiography, histology and gene expression analyses at 2 and 4 weeks after surgery. Results: Activation of Akt1 signaling in myofibers led to a modest increase in skeletal muscle mass. Skeletal muscle-specific activation of Akt1 led to diminished left ventricular dilation and reduced contractile dysfunction compared with control mice at 4 weeks after MI. The increase in heart weight/BW ratio and lung wet weight/BW ratio was significantly diminished in Akt1 TG mice compared to control mice, consistent with an improvement in heart function. Improved cardiac function in Akt1 TG mice was coupled to diminished myocyte hypertrophy, decreased interstitial fibrosis in the infarct border zone, and increased capillary density in the remote zone. Comprehensive protein array analysis showed that panels of angiogenic factors were increased in skeletal muscle in TG mice. An ELISA analysis demonstrated that serum VEGF levels were upregulated in Akt1 TG mice compared with control mice. Conclusions: Akt1-mediated skeletal muscle growth attenuates cardiac remodeling after MI, and this is associated with increased capillary density in the heart and a reduction in cardiac fibrosis.

Pharmacologic activation of mitochondrial biogenesis exerts widespread beneficial effects in a transgenic mouse model of Huntington's disease

There is substantial evidence that impairment of peroxisome proliferator-activated receptor (PPAR)-γ-coactivator 1α (PGC-1α) levels and activity play an important role in Huntington's disease (HD) pathogenesis. We tested whether pharmacologic treatment with the pan-PPAR agonist bezafibrate would correct a deficiency of PGC-1α and exert beneficial effects in a transgenic mouse model of HD. We found that administration of bezafibrate in the diet restored levels of PGC-1α, PPARs and downstream genes to levels which occur in wild-type mice. There were significant improvements in phenotype and survival. In the striatum, astrogliosis and neuronal atrophy were attenuated and numbers of mitochondria were increased. Bezafibrate treatment prevented conversion of type I oxidative to type II glycolytic muscle fibers and increased the numbers of muscle mitochondria. Finally, bezafibrate rescued lipid accumulation and apparent vacuolization of brown adipose tissue in the HD mice. These findings provide strong evidence that treatment with bezafibrate exerts neuroprotective effects which may be beneficial in the treatment of HD.

Purinergic activation of rat skeletal muscle membranes increases V max and Na+ affinity of the Na,K-ATPase and phosphorylates phospholemman and α1 subunits

Muscle activity is associated with an increase in extracellular purines (ATP, ADP), which are involved in signalling mechanisms. The present study investigates the effect of purines on the function of Na,K-ATPase (Na,K-pump) in rat skeletal muscle. Na,K-ATPase activity was quantified by measuring the release of inorganic phosphate in the presence of ATP and variable Na(+) concentrations. In membranes purified from glycolytic muscle fibres, purinergic stimulation increases V (max) and decreases the K (m) (higher Na(+) affinity) of the Na,K-ATPase. Stimulatory effects were obtained using ATP, ADP, 2-methylthio-ADP and UPT, but not UDP and adenosine. The effect of ADP on V (max) can be inhibited by the non-specific P2Y receptor antagonists, suramin and PPADS. Moreover, the P2Y(13) receptor antagonist MRS 2211 strongly inhibited the response to ADP, whereas the specific P2Y(1) receptor antagonist MRS 2500 had less effect. Based on results from these agonists and antagonists, we conclude that P2Y(13) receptors mediate the main effects observed, that P2Y1 receptors are also involved and that some P2Y(2)/P2Y(4) receptors also appear to be involved. Receptor antagonists had no effect on ADP-induced subunit (phospholemman and α1) phosphorylation and changes in K (m) (Na(+) affinity). Thus, the stimulatory effects of purines are mediated by two independent mechanisms: P2Y receptor-mediated increase in Na,K-ATPase capacity (increased V (max)) and P2Y receptor-independent phosphorylation of Na,K-ATPase phospholemman and α1 subunits, which induce changes in ion affinity. These mechanisms may contribute to up-regulation of Na,K-ATPase during muscle activity.

Mitochondrial functional specialization in glycolytic and oxidative muscle fibers: tailoring the organelle for optimal function

In skeletal muscle, two major types of muscle fibers exist: slow-twitch oxidative (type I) fibers designed for low-intensity long-lasting contractions, and fast-twitch glycolytic (type II) fibers designed for high-intensity short-duration contractions. Such a wide range of capabilities has emerged through the selection across fiber types of a narrow set of molecular characteristics suitable to achieve a specific contractile phenotype. In this article we review evidence supporting the existence of distinct functional phenotypes in mitochondria from slow and fast fibers that may be required to ensure optimal muscle function. This includes differences with respect to energy substrate preferences, regulation of oxidative phosphorylation, dynamics of reactive oxygen species, handling of Ca2+, and regulation of cell death. The potential physiological implications on muscle function and the putative mechanisms responsible for establishing and maintaining distinct mitochondrial phenotype across fiber types are also discussed.

Development of the aerobic dive limit and muscular efficiency in northern fur seals (Callorhinus ursinus)

Northern fur seal (Callorhinus ursinus; NFS) populations have been declining, perhaps due to limited foraging ability of pups. Because a marine mammal's proficiency at exploiting underwater prey resources is based on the ability to store large amounts of oxygen (O(2)) and to utilize these reserves efficiently, this study was designed to determine if NFS pups had lower blood, muscle, and total body O(2) stores than adults. Pups (<1-month old) had a calculated aerobic dive limit only ~40% of adult females due to lower blood and, to a much greater extent, muscle O(2) stores. Development of the Pectoralis (Pec) and Longissimus dorsi (LD) skeletal muscles was further examined by determining their myosin heavy chain (MHC) composition and enzyme activities. In all animals, the slow MHC I and fast-twitch IIA proteins typical of oxidative fiber types were dominant, but adult muscles contained more (Pec ~50%; LD ~250% higher) fast-twitch MHC IID/X protein characteristic of glycolytic muscle fibers, than pup muscles. This suggests that adults have greater ability to generate muscle power rapidly and/or under anaerobic conditions. Pup muscles also had lower aerobic and anaerobic ATP production potential, as indicated by lower metabolically scaled citrate synthase, β-hydroxyacyl CoA dehydrogenase, and lactate dehydrogenase activities (all P values ≤0.001). In combination, these findings indicate that pups are biochemically and physiologically limited in their diving capabilities relative to adults. This may contribute to lower NFS first year survival.

Intraspecific variation in the thermal plasticity of mitochondria in killifish

Populations of the Atlantic killifish (Fundulus heteroclitus) inhabit salt marshes and estuaries along the eastern coast of North America from Newfoundland to northern Florida, and are thus exposed to a large range of temperatures. Previous studies have shown higher whole-organism metabolic rates in the northern subspecies (F. h. macrolepidotus) compared with the southern subspecies (F. h. heteroclitus) of these fish. Here, we examine phenotypic plasticity in the response to cold temperatures between the two subspecies by acclimating fish to 5, 15 and 25°C and comparing several mitochondrial and muscle properties. The relative area of oxidative muscle versus glycolytic muscle fibers was greater in the northern subspecies at the 5 and 15°C acclimation temperatures. However, there were no differences in capillary density between the two subspecies or at different temperatures. Mitochondrial volume and surface densities increased in response to cold temperature acclimation in red and white muscle, but only in the northern killifish. Citrate synthase activities also increased in the northern killifish at 5 and 15°C. The ratio of calculated [free ADP] to [ATP] increased in the 5°C acclimated southern killifish but not in the northern killifish at 5°C when compared with the 15°C acclimation group, suggesting that there are differences in adenylate signaling for mitochondrial respiration between subspecies at low temperature. Taken together, our data indicate that the northern subspecies have a greater ability to increase mitochondrial capacity at colder temperatures compared with the southern subspecies, providing one of the few examples of intraspecific variation in phenotypic plasticity in mitochondrial amount in response to cold temperatures.

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: Manipulating meat tenderness by increasing the turnover of intramuscular connective tissue1,2

Controlled reduction of the connective tissue contribution to cooked meat toughness is an objective that would have considerable financial impact in terms of added product value. The amount of intramuscular connective tissue in a muscle appears connected to its in vivo function, so reduction of the overall connective tissue content is not thought to be a viable target. However, manipulation of the state of maturity of the collagenous component is a biologically viable target; by increasing connective tissue turnover, less mature structures can be produced that are functional in vivo but more easily broken down on cooking at temperatures above 60°C, thus improving cooked meat tenderness. Recent work using cell culture models of fibroblasts derived from muscle and myoblasts has identified a range of factors that alter the activity of the principal enzymes responsible for connective tissue turnover, the matrix metalloproteinases (MMP). Fibroblasts cultured from 3 different skeletal muscles from the same animal show different cell proliferation and MMP activity, which may relate to the different connective tissue content and architecture in functionally different muscles. Expression of MMP by fibroblasts is increased by vitamins that can counter the negative effects of oxidative stress on new collagen synthesis. Preliminary work using in situ zymography of myotubes in culture also indicates increased MMP activity in the presence of epinephrine and reactive oxidative species. Comparison of the relative changes in MMP expression from muscle cells vs. fibroblasts shows that myoblasts are more responsive to a range of stimuli. Muscle cells are likely to produce more of the total MMP in muscle tissue as a whole, and the expression of latent forms of the enzymes (i.e., pro-MMP) may vary between oxidative and glycolytic muscle fibers within the same muscle. The implication is that the different muscle fiber composition of different muscles eaten as meat may influence the potential for manipulation of their connective tissue turnover.

The KATP channel Kir6.2 subunit content is higher in glycolytic than oxidative skeletal muscle fibers

It has long been suggested that in skeletal muscle, the ATP-sensitive K(+) channel (K(ATP)) channel is important in protecting energy levels and that abolishing its activity causes fiber damage and severely impairs function. The responses to a lack of K(ATP) channel activity vary between muscles and fibers, with the severity of the impairment being the highest in the most glycolytic muscle fibers. Furthermore, glycolytic muscle fibers are also expected to face metabolic stress more often than oxidative ones. The objective of this study was to determine whether the t-tubular K(ATP) channel content differs between muscles and fiber types. K(ATP) channel content was estimated using a semiquantitative immunofluorescence approach by staining cross sections from soleus, extensor digitorum longus (EDL), and flexor digitorum brevis (FDB) muscles with anti-Kir6.2 antibody. Fiber types were determined using serial cross sections stained with specific antimyosin I, IIA, IIB, and IIX antibodies. Changes in Kir6.2 content were compared with changes in CaV1.1 content, as this Ca(2+) channel is responsible for triggering Ca(2+) release from sarcoplasmic reticulum. The Kir6.2 content was the lowest in the oxidative soleus and the highest in the glycolytic EDL and FDB. At the individual fiber level, the Kir6.2 content within a muscle was in the order of type IIB > IIX > IIA ≥ I. Interestingly, the Kir6.2 content for a given fiber type was significantly different between soleus, EDL, and FDB, and highest in FDB. Correlations of relative fluorescence intensities from the Kir6.2 and CaV1.1 antibodies were significant for all three muscles. However, the variability in content between the three muscles or individual fibers was much greater for Kir6.2 than for CaV1.1. It is suggested that the t-tubular K(ATP) channel content increases as the glycolytic capacity increases and as the oxidative capacity decreases and that the expression of K(ATP) channels may be linked to how often muscles/fibers face metabolic stress.

Evidence for a stromal-epithelial “lactate shuttle” in human tumors

Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to “feed” adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal physiological function of muscle tissue and brain. These data are consistent with the “reverse Warburg effect,” which states that cancer-associated fibroblasts undergo aerobic glycolysis, thereby producing lactate, which is utilized as a metabolic substrate by adjacent cancer cells. In this model, “energy transfer” or “metabolic-coupling” between the tumor stroma and epithelial cancer cells “fuels” tumor growth and metastasis, via oxidative mitochondrial metabolism in anabolic cancer cells. Most importantly, our current findings provide a new rationale and novel strategy for anti-cancer therapies, by employing MCT inhibitors.