The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that main... more The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that maintains a large Ca(2+) gradient and exchanges Ca(2+) between the extracellular and intracellular environments. Little is known of the Ca(2+) -handling properties of the t-system as the small Ca(2+) fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t-system trapped rhod-5N inside skinned fibres from rat and [Ca(2+) ]t-sys , allowing confocal measurements of Ca(2+) -dependent changes in rhod-5N fluorescence during rapid changes in the intracellular ionic environment to be converted to [Ca(2+) ]t-sys (t). Furthermore, t-system Ca(2+) -buffering power was determined so that t-system Ca(2+) fluxes could be derived from [Ca(2+) ]t-sys (t). With this new approach, we show that rapid depletion of sarcoplasmic reticulum (SR) Ca(2+) induced a robust store-operated Ca(2+) entry (SOCE) in fast- and slow-twitch fibres, reducing [Ca(2+) ]t-sys to <0.1 mm. The rapid activation of SOCE upon Ca(2+) release was consistent with the presence of STIM1L in both fibre types. Abruptly introducing internal solutions with 1 mm Mg(2+) and [Ca(2+) ]cyto (28 nm-1.3 μm) to Ca(2+) -depleted fibres generated t-system Ca(2+) uptake rates dependent on [Ca(2+) ]cyto with [Ca(2+) ]t-sys reaching final plateaus in the mm range. For the same [Ca(2+) ]cyto , t-system Ca(2+) fluxes of fast-twitch fibres were greater than that in slow-twitch fibres. In addition, simultaneous imaging of t-system and SR Ca(2+) -signals indicated that both membrane compartments accumulated Ca(2+) at similar rates and that SOCE was activated early during SR Ca(2+) depletion. This article is protected by copyright. All rights reserved.
Skeletal muscle fibre specialization is a critical step towards the optimization of the contracti... more Skeletal muscle fibre specialization is a critical step towards the optimization of the contractile performance. Molecular structure and functional parameters differ among muscle fibres to obtain the best response to specific functional demands. This versatile diversity has been developed during phylogenesis and evolution, but further refinement still occurs during ontogenesis and even in adult life as muscle fibres are endowed with a powerful plasticity. The diversity among muscle fibres, however, has clear borders defined by the consistency between functional parameters and structural features (Schiaffino & Reggiani, 2011). For example, force generation ability must be matched by the strength of the cytoskeleton, the myo-tendinous connections and the trans-versal interfibre connections to withstand mechanical stress without damage. Another example is the necessary match between the kinetic properties of calcium-mediated activation, myofibrillar contractile response and metabolic s...
Store-operated Ca(2+) entry (SOCE) is a robust mechanism in skeletal muscle, supported by abundan... more Store-operated Ca(2+) entry (SOCE) is a robust mechanism in skeletal muscle, supported by abundant STIM1 and Orai1 in the junctional membranes. The precise role of SOCE in skeletal muscle Ca(2+) homeostasis and excitation-contraction coupling remains to be defined. Regardless, it remains important to determine whether the function and capacity of SOCE changes in aged skeletal muscle. We identified an approximate 40% decline in the expression of the integral SOCE protein, stromal interacting molecule 1 (STIM1), but no such decline in its coupling partner, Orai1, in muscle fibers from aged mice. To determine whether this changed aspects of SOCE functionality in skeletal muscle in aged mice, Ca(2+) in the cytoplasm and t-system were continuously and simultaneously imaged on a confocal microscope during sarcoplasmic reticulum Ca(2+) release and compared to experiments under identical conditions using muscle fibers from young mice. Normal activation, deactivation, Ca(2+) influx, and spat...
American journal of physiology. Cell physiology, 2001
The present study examined the gene expression and cellular localization of the creatine transpor... more The present study examined the gene expression and cellular localization of the creatine transporter (CreaT) protein in rat skeletal muscle. Soleus (SOL) and red (RG) and white gastrocnemius (WG) muscles were analyzed for CreaT mRNA, CreaT protein, and total creatine (TCr) content. Cellular location of the CreaT protein was visualized with immunohistochemical analysis of muscle cross sections. TCr was higher (P < or = 0.05) in WG than in both RG and SOL, and was higher in RG than in SOL. Total CreaT protein content was greater (P < or = 0.05) in SOL and RG than in WG. Two bands (55 and 70 kDa) of the CreaT protein were found in all muscle types. Both the 55-kDa (CreaT-55) and the 70-kDa (CreaT-70) bands were present in greater (P < or = 0.05) amounts in SOL and RG than in WG. SOL and RG had a greater amount (P < or = 0.05) of CreaT-55 than CreaT-70. Immunohistochemical analysis revealed that the CreaT was mainly associated with the sarcolemmal membrane in all muscle type...
Journal of physiology and biochemistry, Jan 15, 2015
Glycogenin, glycogen-debranching enzyme (GDE) and glycogen phosphorylase (GP) are important enzym... more Glycogenin, glycogen-debranching enzyme (GDE) and glycogen phosphorylase (GP) are important enzymes that contribute to glycogen particle metabolism. In Long-Evans Hooded rat whole muscle homogenates prepared from extensor digitorum longus (EDL, fast-twitch) and soleus (SOL, oxidative, predominantly slow twitch), it was necessary to include α-amylase, which releases glucosyl units from glycogen, to detect glycogenin but not GDE or GP. Up to ∼12 % of intramuscular glycogen pool was broken down using either in vitro electrical stimulation or leaving muscle at room temperature >3 h (delayed, post-mortem). Electrical stimulation did not reveal glycogenin unless α-amylase was added, although in post-mortem muscle ∼50 and ∼30 % of glycogenin in EDL and SOL muscles, respectively, was detected compared to the amount detected with α-amylase treatment. Single muscle fibres were dissected from fresh or post-mortem EDL muscles, mechanically skinned to remove surface membrane and the presence ...
Journal of applied physiology (Bethesda, Md. : 1985), 2014
Taurine occurs in high concentrations in muscle and is implicated in numerous physiological proce... more Taurine occurs in high concentrations in muscle and is implicated in numerous physiological processes, yet its effects on many aspects of contractility remain unclear. Using mechanically skinned segments of human vastus lateralis muscle fibers, we characterized the effects of taurine on sarcoplasmic reticulum (SR) Ca2+ accumulation and contractile apparatus properties in type I and type II fibers. Prolonged myoplasmic exposure (>10 min) to taurine substantially increased the rate of accumulation of Ca2+ by the SR in both fiber types, with no change in the maximum amount accumulated; no such effect was found with carnosine. SR Ca2+ accumulation was similar with 10 or 20 mM taurine, but was significantly slower at 5 mM taurine. Cytoplasmic taurine (20 mM) had no detectable effects on the responsiveness of the Ca2+ release channels in either fiber type. Taurine caused a small increase in Ca2+ sensitivity of the contractile apparatus in type I fibers, but type II fibers were unaffect...
Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expressi... more Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70-80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either i...
Inhibition of nitric oxide synthase (NOS) significantly attenuates the increase in skeletal muscl... more Inhibition of nitric oxide synthase (NOS) significantly attenuates the increase in skeletal muscle glucose uptake during contraction/exercise, and a greater attenuation is observed in individuals with type 2 diabetes compared with healthy individuals. Therefore, nitric oxide appears to play an important role in mediating muscle glucose uptake during contraction. In this study, we investigated the involvement of neuronal NOSμ (nNOSμ), the main NOS isoform activated during contraction, on skeletal muscle glucose uptake during ex vivo contraction. Extensor digitorum longus muscles were isolated from nNOSμ(-/-) and nNOSμ(+/+) mice. Muscles were contracted ex vivo in a temperature-controlled (30oC) organ bath with or without the presence of the NOS inhibitor, N-G-Monomethyl-L-arginine (L-NMMA), and the NOS substrate, L-arginine. Glucose uptake was determined using radioactive tracers. Skeletal muscle glucose uptake increased approximately four-fold during contraction in muscles from both nNOSμ(-/-) and nNOSμ(+/+) mice. L-NMMA significantly attenuated the increase in muscle glucose uptake during contraction in both genotypes. This attenuation was reversed by L-arginine suggesting that L-NMMA attenuated the increase in muscle glucose uptake during contraction by inhibiting NOS and not via a non-specific effect of the inhibitor. Low levels of NOS activity (approximately 4%) were detected in muscles from nNOSμ(-/-) mice and there was no evidence of compensation from other NOS isoform or AMPK which is also involved in mediating muscle glucose uptake during contraction. These results indicate that NO regulates skeletal muscle glucose uptake during ex vivo contraction independently of nNOSμ.
The present study examined the validity and reliability of measuring the expression of various ge... more The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (C(T)) values were established for beta-actin, beta2-microglobulin (beta2M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between C(T) values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw C(T) values and the linear value of 2(-C(T)), respectively. Inter...
The cellular role of creatine (Cr) and Cr phosphate (CrP) has been studied extensively in neural,... more The cellular role of creatine (Cr) and Cr phosphate (CrP) has been studied extensively in neural, cardiac and skeletal muscle. Several studies have demonstrated that alterations in the cellular total Cr (Cr + CrP) concentration in these tissues can produce marked functional and/or structural change. The primary aim of this review was to critically evaluate the literature that has examined the regulation of cellular total Cr content. In particular, the review focuses on the regulation of the activity and gene expression of the Cr transporter (CreaT), which is primarily responsible for cellular Cr uptake. Two CreaT genes (CreaT1 and CreaT2) have been identified and their chromosomal location and DNA sequencing have been completed. From these data, putative structures of the CreaT proteins have been formulated. Transcription products of the CreaT2 gene are expressed exclusively in the testes, whereas CreaT1 transcripts are found in a variety of tissues. Recent research has measured the...
Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations ... more Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na+/Cl−-dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to
Journal of negative results in biomedicine, Jan 30, 2014
BackgroundSkeletal muscle undergoes significant atrophy in Type 2 diabetic patients and animal mo... more BackgroundSkeletal muscle undergoes significant atrophy in Type 2 diabetic patients and animal models. We aimed to determine if atrophy of Zucker rat skeletal muscle was due to the activation of intracellular damage pathways induced by excess reactive oxygen species production (specifically those associated with the peroxidation of lipid membranes) and calpain activity. 14 week old obese Zucker rats and littermate lean controls were injected with 1% Evan¿s Blue Dye. Animals were anaesthetised and extensor digitorum longus and soleus muscles were dissected, snap frozen and analysed for ROS-mediated F2-isoprostane production and calpain activation/autolysis. Contralateral muscles were histologically analysed for markers of muscle membrane permeability and atrophy.ResultsMuscle mass was lower in extensor digitorum longus and soleus of obese compared with lean animals, concomitant with reduced fibre area. Muscles from obese rats had a higher proportional area of Evan¿s Blue Dye fluoresc...
The present study investigated whether there were any differences between males and females in re... more The present study investigated whether there were any differences between males and females in respect to creatine transporter (CreaT) gene expression and/or total creatine (TCr) content in human vastus lateralis muscle. Skeletal muscle obtained from young healthy male (n = 13, age: 23.2 +/- 5.0 years) and female subjects (n = 12, age: 21.7 +/- 4.3 years) was analyzed for CreaT mRNA, CreaT protein and TCr content. Total CreaT protein content in the muscle was similar (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05) between the sexes. Two bands (approximately 55 and 73 kDa) of the CreaT protein were detected in all muscle samples. Both the 55 and the 73 kDa bands were present in similar (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05) amounts in males compared with females. The 73 kDa band was in greater abundance (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) than the 55 kDa band, irrespective of gender. In addition, CreaT mRNA expression relative to beta-actin mRNA and the TCr content (males: 117.8 +/- 2.2, females: 125.3 +/- 4.3 mmol.kg(-1) dry mass) were also unaffected (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05) by gender. These data demonstrate that gender does not influence skeletal muscle TCr content and CreaT gene expression in young human subjects.
The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that main... more The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that maintains a large Ca(2+) gradient and exchanges Ca(2+) between the extracellular and intracellular environments. Little is known of the Ca(2+) -handling properties of the t-system as the small Ca(2+) fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t-system trapped rhod-5N inside skinned fibres from rat and [Ca(2+) ]t-sys , allowing confocal measurements of Ca(2+) -dependent changes in rhod-5N fluorescence during rapid changes in the intracellular ionic environment to be converted to [Ca(2+) ]t-sys (t). Furthermore, t-system Ca(2+) -buffering power was determined so that t-system Ca(2+) fluxes could be derived from [Ca(2+) ]t-sys (t). With this new approach, we show that rapid depletion of sarcoplasmic reticulum (SR) Ca(2+) induced a robust store-operated Ca(2+) entry (SOCE) in fast- and slow-twitch fibres, reducing [Ca(2+) ]t-sys to &lt;0.1 mm. The rapid activation of SOCE upon Ca(2+) release was consistent with the presence of STIM1L in both fibre types. Abruptly introducing internal solutions with 1 mm Mg(2+) and [Ca(2+) ]cyto (28 nm-1.3 μm) to Ca(2+) -depleted fibres generated t-system Ca(2+) uptake rates dependent on [Ca(2+) ]cyto with [Ca(2+) ]t-sys reaching final plateaus in the mm range. For the same [Ca(2+) ]cyto , t-system Ca(2+) fluxes of fast-twitch fibres were greater than that in slow-twitch fibres. In addition, simultaneous imaging of t-system and SR Ca(2+) -signals indicated that both membrane compartments accumulated Ca(2+) at similar rates and that SOCE was activated early during SR Ca(2+) depletion. This article is protected by copyright. All rights reserved.
Skeletal muscle fibre specialization is a critical step towards the optimization of the contracti... more Skeletal muscle fibre specialization is a critical step towards the optimization of the contractile performance. Molecular structure and functional parameters differ among muscle fibres to obtain the best response to specific functional demands. This versatile diversity has been developed during phylogenesis and evolution, but further refinement still occurs during ontogenesis and even in adult life as muscle fibres are endowed with a powerful plasticity. The diversity among muscle fibres, however, has clear borders defined by the consistency between functional parameters and structural features (Schiaffino & Reggiani, 2011). For example, force generation ability must be matched by the strength of the cytoskeleton, the myo-tendinous connections and the trans-versal interfibre connections to withstand mechanical stress without damage. Another example is the necessary match between the kinetic properties of calcium-mediated activation, myofibrillar contractile response and metabolic s...
Store-operated Ca(2+) entry (SOCE) is a robust mechanism in skeletal muscle, supported by abundan... more Store-operated Ca(2+) entry (SOCE) is a robust mechanism in skeletal muscle, supported by abundant STIM1 and Orai1 in the junctional membranes. The precise role of SOCE in skeletal muscle Ca(2+) homeostasis and excitation-contraction coupling remains to be defined. Regardless, it remains important to determine whether the function and capacity of SOCE changes in aged skeletal muscle. We identified an approximate 40% decline in the expression of the integral SOCE protein, stromal interacting molecule 1 (STIM1), but no such decline in its coupling partner, Orai1, in muscle fibers from aged mice. To determine whether this changed aspects of SOCE functionality in skeletal muscle in aged mice, Ca(2+) in the cytoplasm and t-system were continuously and simultaneously imaged on a confocal microscope during sarcoplasmic reticulum Ca(2+) release and compared to experiments under identical conditions using muscle fibers from young mice. Normal activation, deactivation, Ca(2+) influx, and spat...
American journal of physiology. Cell physiology, 2001
The present study examined the gene expression and cellular localization of the creatine transpor... more The present study examined the gene expression and cellular localization of the creatine transporter (CreaT) protein in rat skeletal muscle. Soleus (SOL) and red (RG) and white gastrocnemius (WG) muscles were analyzed for CreaT mRNA, CreaT protein, and total creatine (TCr) content. Cellular location of the CreaT protein was visualized with immunohistochemical analysis of muscle cross sections. TCr was higher (P < or = 0.05) in WG than in both RG and SOL, and was higher in RG than in SOL. Total CreaT protein content was greater (P < or = 0.05) in SOL and RG than in WG. Two bands (55 and 70 kDa) of the CreaT protein were found in all muscle types. Both the 55-kDa (CreaT-55) and the 70-kDa (CreaT-70) bands were present in greater (P < or = 0.05) amounts in SOL and RG than in WG. SOL and RG had a greater amount (P < or = 0.05) of CreaT-55 than CreaT-70. Immunohistochemical analysis revealed that the CreaT was mainly associated with the sarcolemmal membrane in all muscle type...
Journal of physiology and biochemistry, Jan 15, 2015
Glycogenin, glycogen-debranching enzyme (GDE) and glycogen phosphorylase (GP) are important enzym... more Glycogenin, glycogen-debranching enzyme (GDE) and glycogen phosphorylase (GP) are important enzymes that contribute to glycogen particle metabolism. In Long-Evans Hooded rat whole muscle homogenates prepared from extensor digitorum longus (EDL, fast-twitch) and soleus (SOL, oxidative, predominantly slow twitch), it was necessary to include α-amylase, which releases glucosyl units from glycogen, to detect glycogenin but not GDE or GP. Up to ∼12 % of intramuscular glycogen pool was broken down using either in vitro electrical stimulation or leaving muscle at room temperature >3 h (delayed, post-mortem). Electrical stimulation did not reveal glycogenin unless α-amylase was added, although in post-mortem muscle ∼50 and ∼30 % of glycogenin in EDL and SOL muscles, respectively, was detected compared to the amount detected with α-amylase treatment. Single muscle fibres were dissected from fresh or post-mortem EDL muscles, mechanically skinned to remove surface membrane and the presence ...
Journal of applied physiology (Bethesda, Md. : 1985), 2014
Taurine occurs in high concentrations in muscle and is implicated in numerous physiological proce... more Taurine occurs in high concentrations in muscle and is implicated in numerous physiological processes, yet its effects on many aspects of contractility remain unclear. Using mechanically skinned segments of human vastus lateralis muscle fibers, we characterized the effects of taurine on sarcoplasmic reticulum (SR) Ca2+ accumulation and contractile apparatus properties in type I and type II fibers. Prolonged myoplasmic exposure (>10 min) to taurine substantially increased the rate of accumulation of Ca2+ by the SR in both fiber types, with no change in the maximum amount accumulated; no such effect was found with carnosine. SR Ca2+ accumulation was similar with 10 or 20 mM taurine, but was significantly slower at 5 mM taurine. Cytoplasmic taurine (20 mM) had no detectable effects on the responsiveness of the Ca2+ release channels in either fiber type. Taurine caused a small increase in Ca2+ sensitivity of the contractile apparatus in type I fibers, but type II fibers were unaffect...
Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expressi... more Recent studies using the mouse showed an inverse correlation between the Caveolin 1 gene expression and lactation, and this was regulated by prolactin. However, current study using mammary explants from pregnant mice showed that while insulin (I), cortisol (F) and prolactin (P) resulted in maximum induction of the β-casein gene, FP and IFP resulted in the downregulation of Caveolin 1. Additionally, IF, FP and IFP resulted in the downregulation of Caveolin 2. Immunohistochemistry confirmed localisation of Caveolin 1 specific to myoepithelial cells and adipocytes. Comparative studies with the tammar wallaby showed Caveolin 1 and 2 had 70-80% homology with the mouse proteins. However, in contrast to the mouse, Caveolin 1 and 2 genes showed a significantly increased level of expression in the mammary gland during lactation. The regulation of tammar Caveolin 1 and 2 gene expression was examined in mammary explants from pregnant tammars, and no significant difference was observed either i...
Inhibition of nitric oxide synthase (NOS) significantly attenuates the increase in skeletal muscl... more Inhibition of nitric oxide synthase (NOS) significantly attenuates the increase in skeletal muscle glucose uptake during contraction/exercise, and a greater attenuation is observed in individuals with type 2 diabetes compared with healthy individuals. Therefore, nitric oxide appears to play an important role in mediating muscle glucose uptake during contraction. In this study, we investigated the involvement of neuronal NOSμ (nNOSμ), the main NOS isoform activated during contraction, on skeletal muscle glucose uptake during ex vivo contraction. Extensor digitorum longus muscles were isolated from nNOSμ(-/-) and nNOSμ(+/+) mice. Muscles were contracted ex vivo in a temperature-controlled (30oC) organ bath with or without the presence of the NOS inhibitor, N-G-Monomethyl-L-arginine (L-NMMA), and the NOS substrate, L-arginine. Glucose uptake was determined using radioactive tracers. Skeletal muscle glucose uptake increased approximately four-fold during contraction in muscles from both nNOSμ(-/-) and nNOSμ(+/+) mice. L-NMMA significantly attenuated the increase in muscle glucose uptake during contraction in both genotypes. This attenuation was reversed by L-arginine suggesting that L-NMMA attenuated the increase in muscle glucose uptake during contraction by inhibiting NOS and not via a non-specific effect of the inhibitor. Low levels of NOS activity (approximately 4%) were detected in muscles from nNOSμ(-/-) mice and there was no evidence of compensation from other NOS isoform or AMPK which is also involved in mediating muscle glucose uptake during contraction. These results indicate that NO regulates skeletal muscle glucose uptake during ex vivo contraction independently of nNOSμ.
The present study examined the validity and reliability of measuring the expression of various ge... more The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (C(T)) values were established for beta-actin, beta2-microglobulin (beta2M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between C(T) values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw C(T) values and the linear value of 2(-C(T)), respectively. Inter...
The cellular role of creatine (Cr) and Cr phosphate (CrP) has been studied extensively in neural,... more The cellular role of creatine (Cr) and Cr phosphate (CrP) has been studied extensively in neural, cardiac and skeletal muscle. Several studies have demonstrated that alterations in the cellular total Cr (Cr + CrP) concentration in these tissues can produce marked functional and/or structural change. The primary aim of this review was to critically evaluate the literature that has examined the regulation of cellular total Cr content. In particular, the review focuses on the regulation of the activity and gene expression of the Cr transporter (CreaT), which is primarily responsible for cellular Cr uptake. Two CreaT genes (CreaT1 and CreaT2) have been identified and their chromosomal location and DNA sequencing have been completed. From these data, putative structures of the CreaT proteins have been formulated. Transcription products of the CreaT2 gene are expressed exclusively in the testes, whereas CreaT1 transcripts are found in a variety of tissues. Recent research has measured the...
Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations ... more Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na+/Cl−-dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to
Journal of negative results in biomedicine, Jan 30, 2014
BackgroundSkeletal muscle undergoes significant atrophy in Type 2 diabetic patients and animal mo... more BackgroundSkeletal muscle undergoes significant atrophy in Type 2 diabetic patients and animal models. We aimed to determine if atrophy of Zucker rat skeletal muscle was due to the activation of intracellular damage pathways induced by excess reactive oxygen species production (specifically those associated with the peroxidation of lipid membranes) and calpain activity. 14 week old obese Zucker rats and littermate lean controls were injected with 1% Evan¿s Blue Dye. Animals were anaesthetised and extensor digitorum longus and soleus muscles were dissected, snap frozen and analysed for ROS-mediated F2-isoprostane production and calpain activation/autolysis. Contralateral muscles were histologically analysed for markers of muscle membrane permeability and atrophy.ResultsMuscle mass was lower in extensor digitorum longus and soleus of obese compared with lean animals, concomitant with reduced fibre area. Muscles from obese rats had a higher proportional area of Evan¿s Blue Dye fluoresc...
The present study investigated whether there were any differences between males and females in re... more The present study investigated whether there were any differences between males and females in respect to creatine transporter (CreaT) gene expression and/or total creatine (TCr) content in human vastus lateralis muscle. Skeletal muscle obtained from young healthy male (n = 13, age: 23.2 +/- 5.0 years) and female subjects (n = 12, age: 21.7 +/- 4.3 years) was analyzed for CreaT mRNA, CreaT protein and TCr content. Total CreaT protein content in the muscle was similar (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05) between the sexes. Two bands (approximately 55 and 73 kDa) of the CreaT protein were detected in all muscle samples. Both the 55 and the 73 kDa bands were present in similar (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05) amounts in males compared with females. The 73 kDa band was in greater abundance (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) than the 55 kDa band, irrespective of gender. In addition, CreaT mRNA expression relative to beta-actin mRNA and the TCr content (males: 117.8 +/- 2.2, females: 125.3 +/- 4.3 mmol.kg(-1) dry mass) were also unaffected (p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05) by gender. These data demonstrate that gender does not influence skeletal muscle TCr content and CreaT gene expression in young human subjects.
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