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Sarcolemmal membrane excitability is often evaluated by considering the peak-to-peak amplitude of the compound muscle action potential (M wave). However, the first and second M-wave phases represent distinct properties of the muscle action potential, which are differentially affected by sarcolemma properties and other factors such as muscle architecture. Contrasting with previous studies in which voluntary contractions have been used to induce muscle fatigue, we used repeated electrically induced tetanic contractions of the adductor pollicis muscle and assessed the kinetics of M-wave properties during the course of the contractions. Eighteen participants (24 ± 6 yr; means ± SD) underwent 30 electrically evoked tetanic contractions delivered at 30 Hz, each lasting 3 s with 1 s intervals. We recorded the amplitudes of the first and second M-wave phases for each stimulation. During the initial stimulation train, the first and second M-wave phases exhibited distinct kinetics. The first phase amplitude showed a rapid decrease to reach ∼59% of its initial value (P < 0.001), whereas the second phase amplitude displayed an initial transient increase of ∼19% (P = 0.007). Within subsequent trains, both the first and second phase amplitudes consistently decreased as fatigue developed with a reduction during the last train reaching ∼47% of its initial value (P < 0.001). Analyzing the first M wave of each stimulation train unveiled different kinetics for the first and second phases during the initial trains, but these distinctions disappeared as fatigue progressed. These findings underscore the interplay of factors affecting the M wave and emphasize the significance of separately scrutinizing its first and second phases when assessing membrane excitability adjustments during muscle contractions. NEW & NOTEWORTHY: Our understanding of how the first and second phases of the compound muscle action potential (M wave) behave during fatigue remains incomplete. Using electrically evoked repeated tetanic contractions of the adductor pollicis, we showed that the first and second phases of the M wave followed distinct kinetics only during the early stages of fatigue development. This suggests that the factors affecting the M-wave first and second phases may change as fatigue develops. [ABSTRACT FROM AUTHOR]

Understanding the gross organization of skeletal muscle is critical to understanding the mechanism of action of muscle physiology. Due to coronavirus disease-19 (COVID-19), many colleges have had to discontinue or curtail teaching and laboratory activities. Whether students are in the classroom or learning online, it is important for them to understand the basics of skeletal muscle organization that allows for movement. Manipulatives have been shown to enhance student learning and understanding in many fields, including physiology. This gives instructors an easy-to-follow tool for making a manipulative that allows students to see the organization of the skeletal muscle. Students can make this manipulative themselves from supplies commonly found in the home or office. [ABSTRACT FROM AUTHOR]

Angiotensin-converting enzyme inhibitors (ACEi) and mineralocorticoid receptor (MR) antagonists are FDA-approved drugs that inhibit the renin-angiotensin-aldosterone system (RAAS) and are used to treat heart failure. Combined treatment with the ACEi lisinopril and the nonspecific MR antagonist spironolactone surprisingly improves skeletal muscle, in addition to heart function and pathology in a Duchenne muscular dystrophy (DMD) mouse model. We recently demonstrated that MR is present in all limb and respiratory muscles and functions as a steroid hormone receptor in differentiated normal human skeletal muscle fibers. The goals of the current study were to begin to define cellular and molecular mechanisms mediating the skeletal muscle efficacy of RAAS inhibitor treatment. We also compared molecular changes resulting from RAAS inhibition with those resulting from the current DMD standard-of-care glucocorticoid treatment. Direct assessment of muscle membrane integrity demonstrated improvement in dystrophic mice treated with lisinopril and spironolactone compared with untreated mice. Short-term treatments of dystrophic mice with specific and nonspecific MR antagonists combined with lisinopril led to overlapping gene-expression profiles with beneficial regulation of metabolic processes and decreased inflammatory gene expression. Glucocorticoids increased apoptotic, proteolytic, and chemokine gene expression that was not changed by RAAS inhibitors in dystrophic mice. Microarray data identified potential genes that may underlie RAAS inhibitor treatment efficacy and the side effects of glucocorticoids. Direct effects of RAAS inhibitors on membrane integrity also contribute to improved pathology of dystrophic muscles. Together, these data will inform clinical development of MR antagonists for treating skeletal muscles in DMD. [ABSTRACT FROM AUTHOR]

Unique to striated muscle cells, transverse tubules (t-tubules) are membrane organelles that consist of sarcolemma penetrating into the myocyte interior, forming a highly branched and interconnected network. Mature t-tubule networks are found in mammalian ventricular cardiomyocytes, with the transverse components of t-tubules occurring near sarcomeric z-discs. Cardiac t-tubules contain membrane microdomains enriched with ion channels and signaling molecules. The microdomains serve as key signaling hubs in regulation of cardiomyocyte function. Dyad microdomains formed at the junctional contact between t-tubule membrane and neighboring sarcoplasmic reticulum are critical in calcium signaling and excitation-contraction coupling necessary for beat-to-beat heart contraction. In this review, we provide an overview of the current knowledge in gross morphology and structure, membrane and protein composition, and function of the cardiac t-tubule network. We also review in detail current knowledge on the formation of functional membrane subdomains within t-tubules, with a particular focus on the cardiac dyad microdomain. Lastly, we discuss the dynamic nature of t-tubules including membrane turnover, trafficking of transmembrane proteins, and the life cycles of membrane subdomains such as the cardiac BIN1-microdomain, as well as t-tubule remodeling and alteration in diseased hearts. Understanding cardiac t-tubule biology in normal and failing hearts is providing novel diagnostic and therapeutic opportunities to better treat patients with failing hearts. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine whether plasma lactate and skeletal muscle glucose regulatory pathways, specifically PDH dephosphorylation, are impaired during hyperinsulinemic conditions in middle- to older-aged individuals and determine whether exercise training could improve key variables responsible for skeletal muscle PDH regulation. Eighteen young (19-29 yr; n = 9 males and 9 females) and 20 middle- to older-aged (57-82 yr; n = 10 males and 10 females) individuals underwent a 2-h euglycemic hyperinsulinemic clamp. Plasma samples were obtained at baseline and at 30, 50, 90, and 120 min for analysis of lactate, and skeletal muscle biopsies were performed at 60 min for analysis of protein associated with glucose metabolism. In response to insulin, plasma lactate was elevated in aged individuals when normalized to insulin action. Insulin-stimulated phosphorylation of skeletal muscle PDH on serine sites 232, 293, and 300 decreased in young individuals only. Changes in insulin-stimulated PDH phosphorylation were positively related to changes in plasma lactate. No age-related differences were observed in skeletal muscle phosphorylation of LDH, GSK-3α, or GSK-3β in response to insulin or PDP1, PDP2, PDK2, PDK4, or MPC1 total protein. Twelve weeks of endurance- or strength-oriented exercise training improved insulin-stimulated PDH dephosphorylation, which was related to a reduced lactate response. These findings suggest that impairments in insulin-induced PDH regulation in a sedentary aging population contribute to impaired glucose metabolism and that exercise training is an effective intervention for treating metabolic inflexibility. [ABSTRACT FROM AUTHOR]

Diseases of striated muscle linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus and its links to costameres, which connect the sarcomeres to the cell membrane. Here we study the role in skeletal muscle of synemin, a type IV IF protein, by examining mice null for synemin (synm-null). Synm-null mice have a mild skeletal muscle phenotype. Tibialis anterior (TA) muscles show a significant decrease in mean fiber diameter, a decrease in twitch and tetanic force, and an increase in susceptibility to injury caused by lengthening contractions. Organization of proteins associated with the contractile apparatus and costameres is not significantly altered in the synm-null. Elastimetry of the sarcolemma and associated contractile apparatus in extensor digitorum longus myofibers reveals a reduction in tension consistent with an increase in sarcolemmal deformability. Although fatigue after repeated isometric contractions is more marked in TA muscles of synm-null mice, the ability of the mice to run uphill on a treadmill is similar to controls. Our results suggest that synemin contributes to linkage between costameres and the contractile apparatus and that the absence of synemin results in decreased fiber size and increased sarcolemmal deformability and susceptibility to injury. Thus synemin plays a moderate but distinct role in fast twitch skeletal muscle. [ABSTRACT FROM AUTHOR]

Cholesterol- rich caveolar microdomains and associated caveolins influence sarcolemmal ion channel and receptor function and protective stress signaling. However, the importance of membrane cholesterol content to cardiovascular function and myocardial responses to ischemiareperfusion (I/R) and cardioprotective stimuli are unclear. We assessed the effects of graded cholesterol depletion with methyl-β- cyclodextrin (MβCD) and lifelong knockout (KO) or overexpression (OE) of caveolin-3 (Cav-3) on cardiac function, I/R tolerance, and opioid receptor (OR)-mediated protection. Langendorff-perfused hearts from young male C57Bl/6 mice were untreated or treated with 0.02-1.0 mM MβCD for 25 min to deplete membrane cholesterol and disrupt caveolae. Hearts were subjected to 25-min ischemia/45-min reperfusion, and the cardioprotective effects of morphine applied either acutely or chronically [sustained ligand-activated preconditioning (SLP)] were assessed. MβCD concentration dependently reduced normoxic contractile function and postischemic outcomes in association with graded (10-30%) reductions in sarcolemmal cholesterol. Cardioprotection with acute morphine was abolished with ≥20 μM MβCD, whereas SLP was more robust and only inhibited with ≥200 μM MβCD. Deletion of Cav-3 also reduced, whereas Cav-3 OE improved, myocardial I/R tolerance. Protection via SLP remained equally effective in Cav-3 KO mice and was additive with innate protection arising with Cav-3 OE. These data reveal the membrane cholesterol dependence of normoxic myocardial and coronary function, I/R tolerance, and OR-mediated cardioprotection in murine hearts (all declining with cholesterol depletion). In contrast, baseline function appears insensitive to Cav-3, whereas cardiac I/R tolerance parallels Cav-3 expression. Novel SLP appears unique, being less sensitive to cholesterol depletion than acute OR protection and arising independently of Cav-3 expression. [ABSTRACT FROM AUTHOR]

Activation of AMP-activated protein kinase (AMPK) in cardiomyocytes induces translocation of glucose transporter GLUT4 and long-chain fatty acid (LCFA) transporter CD36 from endosomal stores to the sarcolemma to enhance glucose and LCFA uptake, respectively. Ca2+/calmodulinactivated kinase kinase-β (CaMKKβ) has been positioned directly upstream of AMPK. However, it is unknown whether acute increases in [Ca2+]i stimulate translocation of GLUT4 and CD36 and uptake of glucose and LCFA or whether Ca2+ signaling converges with AMPK signaling to exert these actions. Therefore, we studied the interplay between Ca2+ and AMPK signaling in regulation of cardiomyocyte substrate uptake. Exposure of primary cardiomyocytes to inhibitors or activators of Ca2+ signaling affected neither AMPK-Thr172 phosphorylation nor basal and AMPK-mediated glucose and LCFA uptake. Despite their lack of an effect on substrate uptake, Ca2+ signaling activators induced GLUT4 and CD36 translocation. In contrast, AMPK activators stimulated GLUT4/CD36 translocation as well as glucose/LCFA uptake. When cardiomyocytes were cotreated with Ca2+ signaling and AMPK activators, Ca2+ signaling activators further enhanced AMPK-induced glucose/LCFA uptake. In conclusion, Ca2+ signaling shows no involvement in AMPK-induced GLUT4/CD36 translocation and substrate uptake but elicits transporter translocation via a separate pathway requiring CaMKKβ/ CaMKs. Ca2+-induced transporter translocation by itself appears to be ineffective to increase substrate uptake but requires additional AMPK activation to effectuate transporter translocation into increased substrate uptake. Ca2+-induced transporter translocation might be crucial under excessive cardiac stress conditions that require supraphysiological energy demands. Alternatively, Ca2+ signaling might prepare the heart for substrate uptake during physiological contraction by inducing transporter translocation. [ABSTRACT FROM AUTHOR]

The mitochondrion has been implicated in the development of diabetic cardiomyopathy. Examination of cardiac mitochondria is complicated by the existence of spatially distinct subpopulations including subsarcolemmal (SSM) and interfibrillar (IFM). Dysfunction to cardiac SSM has been reported in murine models of type 2 diabetes mellitus; however, subpopulationbased mitochondrial analyses have not been explored in type 2 diabetic human heart. The goal of this study was to determine the impact of type 2 diabetes mellitus on cardiac mitochondrial function in the human patient. Mitochondrial subpopulations from atrial appendages of patients with and without type 2 diabetes were examined. Complex I- and fatty acid-mediated mitochondrial respiration rates were decreased in diabetic SSM compared with nondiabetic (P ≤ 0.05 for both), with no change in IFM. Electron transport chain (ETC) complexes I and IV activities were decreased in diabetic SSM compared with nondiabetic (P ≤ 0.05 for both), with a concomitant decline in their levels (P ≤ 0.05 for both). Regression analyses comparing comorbidities determined that diabetes mellitus was the primary factor accounting for mitochondrial dysfunction. Linear spline models examining correlative risk for mitochondrial dysfunction indicated that patients with diabetes display the same degree of state 3 and electron transport chain complex I dysfunction in SSM regardless of the extent of glycated hemoglobin (HbA1c) and hyperglycemia. Overall, the results suggest that independent of other pathologies, mitochondrial dysfunction is present in cardiac SSM of patients with type 2 diabetes and the degree of dysfunction is consistent regardless of the extent of elevated HbA1c or blood glucose levels. [ABSTRACT FROM AUTHOR]

Mena, a member of the Ena/VASP family of actin regulatory proteins, modulates microfilaments and interacts with cytoskeletal proteins associated with heart failure. Mena is localized at the intercalated disc (ICD) of adult cardiac myocytes, colocalizing with numerous cytoskeletal proteins. Mena's role in the maintainence of mechanical myocardial stability at the cardiomyocyte ICD remains unknown. We hypothesized that Mena may modulate signals from the sarcolemma to the actin cytoskeleton at the ICD to regulate the expression and localization of connexin 43 (Cx43). The small GTPase Rac1 plays a pivotal role in the regulation of actin cytoskeletal reorganization and mediating morphological and transcriptional changes in cardiomyocytes. We found that Mena is associated with active Rac1 in cardiomyocytes and that RNAi knockdown of Mena increased Rac1 activity significantly. Furthermore, Mena knockdown increased Cx43 expression and altered Cx43 localization and trafficking at the ICD, concomitant with faster intercellular communication, as assessed by dye transfer between cardiomyocyte pairs. In mice overexpressing constitutively active Rac1, left ventricular Mena expression was increased significantly, concomitant with lateral redistribution of Cx43. These results suggest that Mena is a critical regulator of the ICD and is required for normal localization of Cx43 in part via regulation of Rac1. [ABSTRACT FROM AUTHOR]

The Type 2 diabetic db/db mouse experiences vascular dysfunction typified by changes in the contraction and relaxation profiles of small mesenteric arteries (SMAs). Contractions of SMAs from the db/db mouse to the [[alpha].sub.1]-adrenoceptor agonist phenylephrine (PE) were significantly enhanced, and acetylcholine (ACh)-induced relaxations were significantly depressed. Drug treatment of db/db mice with a nonthiazolidinedione peroxisome prolifetor-activated receptor-[gamma] agonist and insulin sensitizing agent 2-[2-(4-phenoxy-2-propylphenoxy)ethyl]indole-5-acetic acid (COOH) completely prevented the changes in endothelium-dependent relaxation, but, with the discontinuation of therapy, endothelial dysfunction returned. Dysfunctional SMAs were found to specifically upregulate the expression of a 35-kDa isoform of sarcolemmal membrane-associated protein (SLMAP), which is a component of the excitation-contraction coupling apparatus and implicated in the regulation of membrane function in muscle cells. Real-time PCR revealed high SLMAP mRNA levels in the db/db microvasculature, which were markedly downregulated during COOH treatment hut elevated again when drug therapy was discontinued. These data reveal that the microvasculature in db/db mice undergoes significant changes in vascular function with the endothelial component of vascular dysfunction specifically correlating with the over-expression of SLMAP. Thus changes in SLMAP expression may be an important indicator for microvascular disease associated with Type 2 diabetes. mice; microvascular function; gene expression; sarcolemmal membrane-associated protein

Costameres are mechanosensory sites of focal adhesion in the sarcolemma that reinforce the muscle-fiber composite and provide an anchor for myofibrillogenesis. We hypothesized that elevated content of the integrinassociated regulator of costamere turnover in culture, focal adhesion kinase (FAK), drives changes in costamere component content in antigravity muscle in a load-dependent way in correspondence with altered muscle weight. The content of FAK in soleus muscle being phosphorylated at autoregulatory tyrosine 397 (FAK-pY397) was increased after 20 s of stretch. FAK-pY397 content remained elevated after 24 h of stretch-overload due to upregulated FAK content. Overexpression of FAK in soleus muscle fibers by means of gene electrotransfer increased the β1-integrin (+56%) and meta-vinculin (+88%) content. α7-Integrin (P = 0.46) and γ-vinculin (P = 0.18) content was not altered after FAK overexpression. Co-overexpression of the FAK inhibitor FAK-related nonkinase (FRNK) reduced FAKpY397 content by 33% and increased the percentage of fast-type fibers that arose in connection with hybrid fibers with gene transfer. Transplantation experiments confirmed the association of FRNK expression with slow-to-fast fiber transformation. Seven days of unloading blunted the elevation of FAK-pY397, β1-integrin, and metavinculin content with FAK overexpression, and this was reversed by 1 day of reloading. The results highlight that the expression of components for costameric attachment sites of myofibrils is under load- and fiber type-related control via FAK and its inhibitor FRNK. [ABSTRACT FROM AUTHOR]

Peripheral arterial disease (PAD), which affects 10 million Americans, is characterized by atherosclerosis of the noncoronary arteries. PAD produces a progressive accumulation of ischemic injury to the legs, manifested as a gradual degradation of gastrocnemius histology. In this study, we evaluated the hypothesis that quantitative morphological parameters of gastrocnemius myofibers change in a consistent manner during the progression of PAD, provide an objective grading of muscle degeneration in the ischemic limb, and correlate to a clinical stage of PAD. Biopsies were collected with a Bergström needle from PAD patients with claudication (n = 18) and critical limb ischemia (CLI; n = 19) and control patients (n = 19). Myofiber sarcolemmas and myosin heavy chains were labeled for fluorescence detection and quantitative analysis of morphometric variables, including area, roundness, perimeter, equivalent diameter, major and minor axes, solidity, and fiber density. The muscle specimens were separated into training and validation data sets for development of a discriminant model for categorizing muscle samples on the basis of disease severity. The parameters for this model included standard deviation of roundness, standard deviation of solidity of myofibers, and fiber density. For the validation data set, the discriminant model accurately identified control (80.0% accuracy), claudicating (77.7% accuracy), and CLI (88.8% accuracy) patients, with an overall classification accuracy of 82.1%. Myofiber morphometry provided a discriminant model that establishes a correlation between PAD progression and advancing muscle degeneration. This model effectively separated PAD and control patients and provided a grading of muscle degeneration within clinical stages of PAD. [ABSTRACT FROM AUTHOR]

Lactate is an important intermediate metabolite in human bioenergetics and is oxidized in many different tissues including the heart, brain, kidney, adipose tissue, liver, and skeletal muscle. The mechanism(s) explaining the metabolism of lactate in these tissues, however, remains unclear. Here, we analyze the ability of skeletal muscle to respire lactate by using an in situ mitochondrial preparation that leaves the native tubular reticulum and subcellular interactions of the organelle unaltered. Skeletal muscle biopsies were obtained from vastus lateralis muscle in 16 human subjects. Samples were chemically permeabilized with saponin, which selectively perforates the sarcolemma and facilitates the loss of cytosolic content without altering mitochondrial membranes, structure, and subcellular interactions. High-resolution respirometry was performed on permeabilized muscle biopsy preparations. By use of four separate and specific substrate titration protocols, the respirometric analysis revealed that mitochondria were capable of oxidizing lactate in the absence of exogenous LDH. The titration of lactate and NAD+ into the respiration medium stimulated respiration (P ≤ 0.003). The addition of exogenous LDH failed to increase lactate-stimulated respiration (P = 1.0). The results further demonstrate that human skeletal muscle mitochondria cannot directly oxidize lactate within the mitochondrial matrix. Alternately, these data support previous claims that lactate is converted to pyruvate within the mitochondrial intermembrane space with the pyruvate subsequently taken into the mitochondrial matrix where it enters the TCA cycle and is ultimately oxidized. [ABSTRACT FROM AUTHOR]

Glucose uptake across the sarcolemma is regulated by a family of membrane proteins called glucose transporters (GLUTs), which includes GLUT4 (the major cardiac isoform) and GLUT12 (a novel, second insulin-sensitive isoform). Potential regional patterns in glucose transport across the cardiac chambers have not been examined; thus, we hypothesized that insulin-responsive GLUT4 and -12 protein and gene expression would be chamber specific in healthy subjects and during chronic heart failure (HF). Using a canine model of tachypacing-induced, progressive, chronic HF, total GLUT protein and messenger RNA in both ventricles and atria (free wall and appendage) were investigated by immunoblotting and real-time PCR. In controls, GLUT4, but not GLUT12, protein content was significantly higher in the atria compared with the ventricles, with the highest content in the right atrium (RA; P < 0.001). GLUT4 and GLUT12 mRNA levels were similar across the cardiac chambers. During chronic HF, GLUT4 and GLUT12 protein content was highest in the left ventricle (LV; by 2.5- and 4.2-fold, respectively, P < 0.01), with a concomitant increase in GLUT4 and GLUT12 mRNA (P < 0.001). GLUT4, but not GLUT12, protein content was decreased in RA during chronic HF (P = 0.001). In conclusion, GLUT4 protein was differentially expressed across the chambers in the healthy heart, and this regional pattern was reversed during HF. Our data suggest that LV was the primary site dependent on both GLUT4 and GLUT12 during chronic HF. In addition, the paradoxical decrease in GLUT4 content in RA may induce perturbations in atrial energy production during chronic HF. [ABSTRACT FROM AUTHOR]

Cardioprotection by ischemic preconditioning (IP) was abolished in connexin 43 (Cx43)-deficient mice due to loss of Cx43 located in mitochondria rather than at the sarcolemma. IP is lost in hyperlipidemic rat hearts as well. Since changes in mitochondrial Cx43 in hyperlipidemia have not yet been analyzed, we determined total and mitochondrial Cx43 levels in male Wistar rats fed a laboratory chow enriched with 2% cholesterol or normal chow for 12 wk. Hearts were isolated and perfused according to Langendorff. After a 10-min perfusion, myocardial tissue cholesterol, superoxide, and nitrotyrosine contents were measured and Cx43 content in whole heart homogenate and a mitochondrial fraction determined. In the cholesterol-fed group, tissue cholesterol and superoxide formation was increased (P < 0.05), while total Cx43 content remained unchanged. Mitochondrial total and dephosphorylated Cx43 content decreased. Hearts were subjected to an IP protocol (3 × 5 min ischemia-reperfusion) or time-matched aerobic perfusion followed by 30-min global ischemia and 5-min reperfusion. IP reduced infarct size in normal but not in cholesterol-fed rats. At 5-min reperfusion following 30-min global ischemia, the total and dephosphorylated mitochondrial Cx43 content was increased, which was abolished by IP in both normal and high-cholesterol diet. In conclusion, loss of cardioprotection by IP in hyperlipidemia is associated with a redistribution of both sarcolemmal and mitochondrial Cx43. [ABSTRACT FROM AUTHOR]

Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. Our previous results show that the tibialis anterior (TA) muscles of mice lacking keratin 19 (K19) lose costameres, accumulate mitochondria under the sarcolemma, and generate lower specific tension than controls. Here we compare the physiology and morphology of TA muscles of mice lacking K19 with muscles lacking desmin or both proteins [double knockout (DKO)]. K19-/- mice and DKO mice showed a threefold increase in the levels of creatine kinase (CK) in the serum. The absence of desmin caused a larger change in specific tension (-40%) than the absence of K19 (-19%) and played the predominant role in contractile function (-40%) and decreased tolerance to exercise in the DKO muscle. By contrast, the absence of both proteins was required to obtain a significantly greater loss of contractile torque after injury (-48%) compared with wild type (-39%), as well as near-complete disruption of costameres. The DKO muscle also showed a significantly greater misalignment of myofibrils than either mutant alone. In contrast, large subsarcolemmal gaps and extensive accumulation of mitochondria were only seen in K19-null TA muscles, and the absence of both K19 and desmin yielded milder phenotypes. Our results suggest that keratin filaments containing K19- and desmin-based intermediate filaments can play independent, complementary, or antagonistic roles in the physiology and morphology of fast-twitch skeletal muscle. [ABSTRACT FROM AUTHOR]

The glycosylation of dystroglycan is required for its function as a high-affinity laminin receptor, and loss of dystroglycan glycosylation results in congenital muscular dystrophy. The purpose of this study was to investigate the functional defects in slow- and fast-twitch muscles of glycosylation-deficient Largemyd mice. While a partial alteration in glycosylation of dystrogly- can in heterozygous Largemyd/+ mice was not sufficient to alter muscle function, homozygous Largemyd/myd mice demonstrated a marked reduction in specific force in both soleus and extensor digitorum longus (EDL) muscles. Although EDL muscles from Largemyd/myd mice were highly susceptible to lengthening contraction-induced injury, Large myd/myd soleus muscles surprisingly showed no greater force deficit compared with wild-type soleus muscles even after five lengthening contractions. Despite no increased suscepti- bility to injury, Largemyd/myd soleus muscles showed loss of dystroglycan glycosylation and laminin binding activity and dys- trophic pathology. Interestingly, we show that soleus muscles have a markedly higher sarcolemma expression of β1-containing inte- grins compared with EDL and gastrocnemius muscles. Therefore, we conclude that β1-containing integrins play an important role as matrix receptors in protecting muscles containing slow-twitch fibers from contraction-induced injury in the absence of dystrogly- can function, and that contraction-induced injury appears to be a separable phenotype from the dystrophic pathology of muscular dystrophy. [ABSTRACT FROM AUTHOR]

The aim of this research was the analysis of structural changes in various parts of the sarcolemma and contractile apparatus of muscle fibers by measuring their transversal stiffness by atomic force microscopy under gravitational unloading. Soleus, medial gastrocnemius, and tibialis anterior muscles of Wistar rats were the objects of the study. Gravitational unloading was carded out by antiorthostatic suspension of hindlimbs for 1, 3, 7, and 12 days. It was shown that the transversal stiffness of different parts of the contractile apparatus of soleus muscle fibers decreases during gravitational unloading in the relaxed, calcium-activated, and rigor states, the fibers of the medial gastrocnemius show no changes, whereas the transversal stiffness of tibialis anterior muscle increases. Thus the transversal stiffness of the sarcolemma in the relaxed state is reduced in all muscles, which may be due to the direct action of gravity as an external mechanical factor that can influence the tension on a membrane. The change of sarcolemma stiffness in activated fibers, which is due probably to the transfer of tension from the contractile apparatus, correlates with the dynamics of changes in the content of desmin. [ABSTRACT FROM AUTHOR]

Fatty acid oxidation is highly regulated in skeletal muscle and involves several sites of regulation, including the transport of fatty acids across both the plasma and mitochondrial membranes. Transport across these membranes is recognized to be primarily protein mediated, limited by the abundance of fatty acid transport proteins on the respective membranes. In recent years, evidence has shown that fatty acid transport proteins move in response to acute and chronic perturbations; however, in human skeletal muscle the localization of fatty acid transport proteins in response to training has not been examined. Therefore, we determined whether high-intensity interval training (HIIT) increased total skeletal muscle, sarcolemmal, and mitochondrial membrane fatty acid transport protein contents. Ten untrained females (22 ± 1 yr, 65 ± 2 kg; V̇o2peak: 2.8 ± 0.1 l/min) completed 6·wk of HIIT, and biopsies from the vastus lateralis muscle were taken before training, and following 2 and 6 wk of HIIT. Training significantly increased maximal oxygen uptake at 2 and 6 wk (3.1 ± 0.1, 3.3 ± 0.1 l/min). Training for 6 wk increased FAT/CD36 at the whole muscle (10%) and mitochondrial levels (51%) without alterations in sarcolemmal content. Whole muscle plasma membrane fatty acid binding protein (FABPpm) also increased (48%) after 6 wk of training, but in contrast to FAT/CD36, sarcolemmal FABPpm increased (23%). whereas mitochondrial FABPpm was unaltered. The changes on sarcolemmal and mitochondrial membranes occurred rapidly, since differences (≤2 wk) were not observed between 2 and 6 wk. This is the first study to demonstrate that exercise training increases fatty acid transport protein content in whole muscle (FAT/CD36 and FABPpm) and sarcolemmal (FABPpm) and mitochondrial (FAT/CD36) membranes in human skeletal muscle of females. These results suggest that increases in skeletal muscle fatty acid oxidation following training are related in part to changes in fatty acid transport protein content and localization. [ABSTRACT FROM AUTHOR]

The mechanisms responsible for anti-arrhythmic protection during ischemia-reperfusion (IR) in exercised hearts are not fully understood. The purpose of this investigation was to examine whether the ATP-sensitive potassium channels in the mitochondria (mito KATP) and sarcolemma (sarc KATP) provide anti-arrhythmic protection in exercised hearts during IR. Male Sprague-Dawley rats were randomly assigned to cardioprotective treadmill exercise or sedentary conditions before IR (I = 20 min, R = 30 min) in vivo. Subsets of exercised animals received pharmacological inhibitors for mito KATP (5-hydroxydecanoate) or sarc KATP (HMR1098) before IR. Blinded analysis of digital ECG tracings revealed that mito KATP inhibition blunted the anti-arrhythmic effects of exercise, while sarc KATP inhibition did not. Endogenous antioxidant enzyme activities for total, CuZn, and Mn superoxide dismutase, catalase, and glutathione peroxidase from ischemic and perfused ventricular tissue were not mitigated by IR, although oxidative stress was elevated in sedentary and mito KATP-inhibited hearts from exercised animals. These findings suggest that the mito KATP channel provides anti-arrhythmic protection as part of exercise-mediated cardioprotection against IR. Furthermore, these data suggest that the observed anti-arrhythmic protection may be associated with preservation of redox balance in exercised hearts. [ABSTRACT FROM AUTHOR]

To sustain cardiac muscle contractility relatively independent of temperature, some fish species are capable of temporarily altering excitation-contraction coupling processes to meet the demands of their environment. The Pacific bluefin tuna, Thunnus orientalis, is a partially endothermic fish that inhabits a wide range of thermal niches. The present study examined the effects of temperature and thermal acclimation on sarcolemmal K+ currents and their role in action potential (AP) generation in bluefin tuna cardiomyocytes. Atrial and ventricular myocytes were enzymatically isolated from cold (14°C)- and warm (24°C)-acclimated bluefin tuna. APs and current-voltage relations of K+ channels were measured using the whole cell current and voltage clamp techniques, respectively. Data were collected either at the cardiomyocytes' respective acclimation temperature of 14 or 24°C or at a common test temperature of 19°C (to reveal the effects of acclimation). AP duration (APD) was prolonged in cold-acclimated (CA) cardiomyocytes tested at 14°C compared with 19°C and in warm-acclimated (WA) cardiomyocytes tested at 19°C compared with 24°C. This effect was mirrored by a decrease in the density of the delayed-rectifier current (IKr), whereas the density of the background inward-rectifier current (lK1) was unchanged. When CA and WA cardiomyocytes were tested at a common temperature of 19°C, no significant effects of temperature acclimation on AP shape or duration were observed, whereas IKr density was markedly increased in CA cardiomyocytes. IK1 density was unaffected in CA ventricular myocytes but was significantly reduced in CA atrial myocytes, resulting in a depolarization of atrial resting membrane potential. Our results indicate the bluefin AP is relatively short compared with other teleosts, which may allow the bluefin heart to function at cold temperatures without the necessity for thermal compensation of APD. [ABSTRACT FROM AUTHOR]

We investigated whether impaired sarcolemmal excitability causes severe fatigue during repeated tetani in isolated mouse skeletal muscle. Slow-twitch soleus or fast-twitch extensor digitorum longus (EDL) muscles underwent intensive stimulation (standard protocol: 125 Hz for 500 ms, every second, parallel plate electrodes, 20 V, 0.1-ms pulses). Interventions with altered stimulation characteristics were tested either on the entire fatigue profile or after 90- to 100-s stimulation. D-tubocurarine did not alter the fatigue profile in soleus thereby eliminating impaired neuromuscular transmission. Lower stimulation frequencies partially restored peak force, especially in soleus. The twitch force-stimulation strength relationship shifted towards higher voltages in both muscle types, with a much larger shift in EDL. Augmenting pulse strength restored tetanic force from 29% (4.4 V) to 79% (20 V), or slowed fatigue in soleus. Increasing pulse duration (0.1 to 1.0 ms) restored tetanic force from 8 to 46% in EDL and from 41 to 90% in soleus; 0.25-ms pulses restored tetanic force to 83% in soleus. Switching from transverse wire to parallel plate stimulation increased tetanic force from 34 to 63%, and fatigue was exacerbated with wires compared with plates in soleus. The combined data suggest that impaired excitability (disrupted action potential generation) within trains is the main contributor (∼50% initial force) to severe fatigue in both muscle types, the surface rather than t-tubular membrane is the main site of impairment during wire stimulation, and extreme fatigue in EDL includes an increased action potential threshold leading to inexcitable fibers. Moreover, mathematical modeling discounts anoxia as the major contributor to fatigue during our stimulation regime in isolated muscles. [ABSTRACT FROM AUTHOR]

The α7β1 integrin is increased in skeletal muscle in response to injury-producing exercise, and transgenic overexpression of this integrin in mice protects against exercise- induced muscle damage. The present study investigates whether the increase in the α7β1 integrin observed in wild-type mice in response to exercise is due to transcriptional regulation and examines whether mobilization of the integrin at the myotendinous junction (MTJ) is a key determinant in its protection against damage. A single bout of downhill running exercise selectively increased transcription of the α7 integrin gene in 5-wk-old wild-type mice 3 h postexercise, and an increased α7 chain was detected in muscle sarcolemma adjacent to tendinous tissue immediately following exercise. The α7B, but not α7A isoform, was found concentrated and colocalized with tenascin-C in muscle fibers lining the MTJ. To further validate the importance of the integrin in the protection against muscle damage following exercise, muscle injury was quantified in α7-/- mice. Muscle damage was extensive in α7-/- mice in response to both a single and repeated bouts of exercise and was largely restricted to areas of high MTJ concentration and high mechanical force near the Achilles tendon. These results suggest that exercise-induced muscle injury selectively increases transcription of the α7 integrin gene and promotes a rapid change in the α7β integrin at the MTJ. These combined molecular and cellular alterations are likely responsible for integrin-mediated attenuation of exercise-induced muscle damage. [ABSTRACT FROM AUTHOR]

Totzeck A, Boengler K, van de Sand A, Konietzka I, Gres P, Garcia-Dorado D, Heusch G, Schulz R. No impact of protein phosphatases on connexin 43 phosphorylation in ischemic preconditioning. Am J Physiol Heart Circ Physiol 295: H2106-H21 12, 2008. First published October 3, 2008; doi:10.1152/ajpheart.00456.2008. Cardiac connexin 43 (Cx43) is involved in infarct propagation, and the uncoupling of Cx43-formed channels reduces infarct size. Cx43formed channels open upon Cx43 dephosphorylation, and ischemic preconditioning (IP) prevents the ischemia-induced Cx43 dephosphorylation. In addition to the sarcolemma, Cx43 is also present in the cardiomyocyte mitochondria. We now examined the interaction of Cx43 with protein phosphatases PPIα, PP2Aα, and PP2Bα and the role of such interaction for Cx43 phosphorylation in preconditioned myocardium. Infarct size (in %area at risk) in left ventricular anterior myocardium of Gottinger minipigs subjected to 90 mm of low-flow ischemia and 120 mm of reperfusion was 23.1 ± 2.7 [n = 7, nonpreconditioned (NIP) group] and was reduced by IP to 10.0 ± 3.2 (n = 6, P < 0.05). Mitochondrial and gap junctional Cx43 dephosphorylation increased after 85 mm of ischemia in NIP myocardium, whereas Cx43 phosphorylation was preserved with IP. PP2As and PP1α, but not PP2Bα, were detected by Western blot analysis in the left ventricular myocardium. Cx43 coprecipitated with PP2Aα but not with PPlα. Although the total PP2Aα immunoreactivity (confocal laser scan) was increased to 154 ± 24% and 194 ± 13% of baseline (P < 0.05) after 85 mm of ischemia in NIP and IP myocardium, respectively, the PP2A activities were similar between the groups. The amount of PP2Aα coimmunoprecipitated with Cx43 remained unchanged. Only PP2Aα coprecipitates with Cx43 in pig myocardium. This interaction is not affected by IP, suggesting that PP2Aα is not involved in the prevention of the ischemia-induced Cx43 dephosphorylation by IP. [ABSTRACT FROM AUTHOR]

We examined male and female glucose transporter isoform-3 (GLUT3; placenta)-null heterozygous+/- mutation-carrying mice and compared them with age- and sex-matched wild-type4+/- littermates. No difference in postnatal (1-2 days, 6-7 days, 12-13 days, 20-21 days), postsuckling (1-2 mo), and adult (3-6 mo) growth pattern was seen except for an increase in body weight of 9- to 11-mo-old male but not female GLUT3+/- mice. This change in male mutant mice was associated with increased total body fat mass, perirenal and epididymal white adipose tissue weight, and hepatic lipid infiltration. These minimally glucose-intolerant male mutant mice demonstrated no change in caloric intake but a decline in basal metabolic rate and insulin resistance. No perturbation in basal circulating glucose concentrations but an increase in insulin concentrations, triglycerides, and total cholesterol was observed in GLUT3+/- male mice. Tissue analysis in males and females demonstrated diminished GLUT3 protein in GLUT3+/- brain and skeletal muscle with no change in brain and adipose tissue GLUT I protein concentrations. Furthermore, the male GLUT3+/- mice expressed decreased insulin-responsive GLUT4 in white adipose tissue and skeletal muscle sarcolemma. We conclude that the GLUT3+/- male mice develop adult-onset adiposity with insulin resistance. [ABSTRACT FROM AUTHOR]

Previous studies in our laboratory suggest that an acute inhibition of glycogen synthase kinase 3 (GSK3) by SB-216763 (SB21) is cardioprotective when administered just before reperfusion. However, it is unknown whether the OSK inhibitor SB21 administered 24 h before ischemia is cardio-protective and whether the mechanism involves ATP-sensitive potassium (KATP) channels and the mitochondrial permeability transition pore (MPTP). Male Sprague-Dawley rats were administered the GSK inhibitor SB21 (0.6 mg/kg) or vehicle 24 h before ischemia. Subsequently, the rats were acutely anesthetized with mactin and underwent 30 mm of ischemia and 2 h of reperfusion followed by infarct size determination. Subsets of rats received either the sarcolemmal KATP channel blocker HMR-1098 (6 mg/kg), the mitochondrial KATP channel blocker 5-hydroxydecanoic acid (5-HD; 10 mg/kg), or the MPTP opener atractyloside (5 mg/kg) either 5 mm before SB21 administration or 5 mm before reperfusion 24 h later. The infarct size was reduced in SB21 compared with vehicle (44 ± 2% vs. 61 ± 2%, respectively; P < 0.01). 5-HD administered either before SB21 treatment or 5 mm before reperfusion the following day abrogated SB21-induced protection (54 ± 4% and 61 ± 2%, respectively). HMR-1098 did not affect the SB21-induced infarct size reduction when administered before the SB21 treatment (43 ± 1%); however, HMR-1098 partially abrogated the SB21-induced infarct size reduction when administered just before reperfusion 24 h later (52 ± 1%). The MPTP opening either before SB21 administration or 5 min before reperfusion abrogated the infarct size reduction produced by SB21 (61 ± 2% and 62 ± 2%, respectively). Hence, GSK inhibition reduces infarct size when given 24 h before the administration via the opening KATP channels and MPTP closure. [ABSTRACT FROM AUTHOR]

From time of their discovery, sarcolemmal ATP-sensitive K+ (sarcKATP) channels were thought to have an important protective role in the heart during stress whereby channel opening protects the heart from stress-induced Ca2+ overload and resulting damage. In contrast, some recent studies indicate that sarcKATP channel closing can lead to cardiac protection. Also, the role of the sarcKATP channel in apoptotic cell death is unclear. In the present study, the effects of channel inhibition on apoptosis and the specific interaction between the sarcKATP channel and mitochondria were investigated. Apoptotic cell death of cultured HL-1 and neonatal cardiomyocytes following exposure to oxidative stress was significantly increased in the presence of sarcKATP channel inhibitor HMR-1098 as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and caspase-3,7 assays. This was paralleled by an increased release of cytochrome c from mitochondria to cytosol, suggesting activation of the mitochondrial death pathway. SarcKATP channel inhibition during stress had no effect on Bcl-2, Bad, and phospho-Bad, indicating that the increase in apoptosis cannot be attributed to these modulators of the apoptotic pathway. However, monitoring of mitochondrial Ca2+ with rhod-2 fluorescent indicator revealed that mitochondrial Ca2+ accumulation during stress is potentiated in the presence of HMR-1098. In conclusion, this study provides novel evidence that opening of sarcKATP channels, through a specific Ca2+-related interaction with mitochondria, plays an important role in preventing cardiomyocyte apoptosis and mitochondrial damage during stress. [ABSTRACT FROM AUTHOR]

Airway smooth muscle (ASM) cells may contribute to asthma pathogenesis through their capacity to switch between a synthetic/proliferative and a contractile phenotype. The multimeric dystrophin-glycoprotein complex (DGC) spans the sarcolemma, linking the actin cytoskeleton and extracellular matrix. The DGC is expressed in smooth muscle tissue, but its functional role is not fully established. We tested whether contractile phenotype maturation of human ASM is associated with accumulation of DGC proteins. We compared subconfluent, serum-fed cultures and confluent cultures subjected to serum deprivation, which express a contractile phenotype. Western blotting confirmed that β-dystroglycan, β-δ-, and ϵ-sarcoglycan, and dystrophin abundance increased six- to eightfold in association with smooth muscle myosin heavy chain (5mMHC) and calponin accumulation during 4-day serum deprivation. Immunocytochemistry showed that the accumulation of DGC subunits was specifically localized to a subset of cells that exhibit robust staining for smMHC. Laminin competing peptide (YIGSR, 1 μM) and phosphatidylinositol 3-kinase (P13K) inhibitors (20 μM LY-294002 or 100 nM wortmannin) abrogated the accumulation of smMHC, calponin, and DGC proteins. These studies demonstrate that the accumulation of DCC is an integral feature for phenotype maturation of human ASM cells. This provides a strong rationale for future studies investigating the role of the DCC in ASM smooth muscle physiology in health and disease. [ABSTRACT FROM AUTHOR]

To determine whether sarcolemmal and/or mitochondrial ATP-sensitive potassium (KATP) channels (sarcKATP, mitoKATP) are involved in stretch-induced protection, isolated isovolumic rat hearts were assigned to the following protocols: nonstretched hearts were subjected to 20 min of global ischemia (Is) and 30 min of reperfusion, and before Is stretched hearts received 5 min of stretch + 10 min of no intervention. Stretch was induced by a transient increase in left ventricular end-diastolic pressure (LVEDP) from 10 to 40 mmHg. Other hearts received 5-hydroxydecanoate (5-HD; 100 µM), a selective inhibitor of mitoKATP, or HMR-1098 (20 µM), a selective inhibitor of sarcKATP, before the stretch protocol. Systolic function was assessed through left ventricular developed pressure (LVDP) and maximal rise in velocity of left ventricular pressure (+dP/dtmax) and diastolic function through maximal decrease in velocity of left ventricular pressure (-dP/dtmax) and LVEDP. Lactate dehydrogenase (LDH) release and ATP content were also measured. Stretch resulted in a significant increase of postischemic recovery and attenuation of diastolic stiffness. At 30 min of reperfusion LVDP and +dP/dtmax were 87 ± 4% and 92 ± 6% and -dP/dtmax and LVEDP were 95 ± 9% and 10 ± 4 mmHg vs. 57 ± 6%, 53 ± 6%, 57 ± 10%, and 28 ± 5 mmHg, respectively, in nonstretched hearts. Stretch increased ATP content and did not produce LDH release. 5-HD did not modify and HMR-1098 prevented the protection achieved by stretch. Our results show that the beneficial effects of stretch on postischemic myocardial dysfunction, cellular damage, and energetic state involve the participation of sarcKATP but not mitoKATP. [ABSTRACT FROM AUTHOR]

The expression of carbonic anhydrase (CA) XIV was investigated in mouse skeletal muscles. Sarcoplasmic reticulum (SR) and sarcolemmal (SL) membrane fractions were isolated from wild-type (WT) and CA XIV knockout (KO) mice. The CA XIV protein of 54 kDa was present in SR and SL membrane fractions as shown by Western blot analysis. CA activity measurements of WT and KO membrane fractions showed that CA XIV accounts for ~50% and 66% of the total CA activities determined in the SR and SL fractions, respectively. This indicates the presence of at least one other membrane-associated CA isoform in these membranes, e.g., CA IV, CA IX, or CA XII. Muscle fibers of the extensor digitorum longus (EDL) muscle were immunostained with anti-CA XIV/FITC and anti-sarco(endo)plasmic reticulum Ca2+-ATPase 1/TRITC, with anti-CA XIV/FITC and anti-ryanodine receptor/TRITC, or with anti-CA XIV/FITC and anti-monocarboxylate transporter-4/TRITC. CA XIV was expressed in the plasma membrane and in the longitudinal SR but not in the terminal SR. Isometric contraction measurements of single twitches and tetani and a fatigue protocol applied to fiber bundles of the fast-twitch EDL and of the slow-twitch soleus muscle from WT and KO mice showed that the lack of SR membrane-associated CA XIV did not affect maximum force, rise and relaxation times, and fatigue behavior. Thus, it is concluded that a reduction of the total SR CA activity by ~50% in CA XIV KO mice does not lead to an impairment of SR function. [ABSTRACT FROM AUTHOR]

The Na+/K+-ATPase (NKA) is the main route for Na+ extrusion from cardiac myocytes. Different NKA α-subunit isoforms are present in the heart. NKA-α1 is predominant, although there is a variable amount of NKA-α2 in adult ventricular myocytes of most species. It has been proposed that NKA-α2 is localized mainly in T-tubules (TF), where it could regulate local Na+/Ca2+ exchange and thus cardiac myocyte Ca2+. However, there is controversy as to where NKA-α1 vs. NKA-α2 are localized in ventricular myocytes. Here, we assess the TT vs. external sarcolemma (ESL) distribution functionally using formamide-induced detubulation of rat ventricular myocytes, NKA current (IPump) measurements and the different ouabain sensitivity of NKA-α1 (low) and NKA-α2 (high) in rat heart. Ouabain-dependent IPump inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-α2, K1/2 = 0.38 ± 0.16 µM) that accounts for 29.5 ± 1.3% of IPump and a low-affinity isoform (NKA-α1, K1/2 = 141 ± 17 µM) that accounts for 70.5% of IPump. Detubulation decreased cell capacitance from 164 ± 6 to 120 ± 8 pF and reduced IPump density from 1.24 ± 0.05 to 1.02 ± 0.05 pA/pF, indicating that the functional density of NKA is significantly higher in TT vs. ESL. In detubulated myocytes, NKA-α2 accounted for only 18.2 ± 1.1% of IPump. Thus, ~63% of IPump generated by NKA-α2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-α2/NKA-α1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-α2 is ~4.5 times higher in the T-tubules vs. ESL, whereas NKA-α1 is almost uniformly distributed between the TT and ESL. [ABSTRACT FROM AUTHOR]

Background: intracellular Na+ accumulation during ischemia and reperfusion leads to cytosolic Ca2+ overload through reverse-mode operation of the sarcolemmal Na+- Ca2+ exchanger. Cytosolic Ca2+ accumulation promotes mitochondrial Ca2+ (Ca2+m) overload, leading to mitochondrial injury. We investigated whether limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation (VF) attenuates Ca2+m overload and lessens myocardial dysfunction in a rat model of VF and closed-chest resuscitation. Methods: hearts were harvested from 10 groups of 6 rats each representing baseline, 15 mm of untreated VF, 15 mm of VF with chest compression given for the last 5 mm (VF/CC), and 60 mm postresuscitation (PR). VF/CC and PR included four groups each randomized to receive before starting chest compression the new NHE-1 inhibitor AVE4454B (1.0 mg/kg), the Na+ channel blocker lidocaine (5.0 mg/kg), their ëombination, or vehicle control. The left ventricle was processed for intracellular Na+ and Ca2+m measurements. Results: limiting sarcolemmal Na+ entry attenuated cytosolic Na+ increase during VF/CC and the PR phase and prevented Ca2+, overload yielding levels that corresponded to 77% and 71% of control hearts at VF/CC and PR, without differences among specific Na+- limiting interventions. Limiting sarcolemmal Na+ entry attenuated reductions in left ventricular compliance during VF and prompted higher mean aortic pressure (110 ± 7 vs. 95 ± 11 mmHg, P < 0.001) and higher cardiac work index (159 ± 34 vs. 126 ± 29 g·m·min-1kg-1, P < 0.05) with lesser increases in circulating cardiac troponin 1 at 60 min PR. Conclusions: Na+-limiting interventions prevented excess Ca2+m accumulation induced by ischemia and reperfusion and ameliorated myocardial injury and dysfunction. [ABSTRACT FROM AUTHOR]

When muscle fibers are repeatedly stimulated, they may become depolarized and force output decline. Excitation of the transverse tubular system (T-system) is critical for activation, but its role in muscle fatigue is poorly understood. Here, mechanically skinned fibers from rat fast-twitch muscle were used, because the sarcolemma is absent but the T-system retains normal excitability and its properties can be studied in isolation. The T-system membrane was fully polarized by bathing the skinned fiber in an internal solution with 126 mM K+ (control solution) or set at partially depolarized levels (approximately -63 and -58 mV) in solutions with 66 or 55 mM K+, respectively, and action potentials (APs) were triggered in the sealed T-system by field stimulation. Prolonged depolarization of the T-system reduced tetanic force proportionately more than twitch force, with greater effect at higher stimulation frequency (responses at 20 and 100 Hz reduced to 71 and 62% in 66 mM K+ and to 54 and 35% in 55 mM K+, respectively). Double-pulse stimulation showed that depolarization increased the repriming period (estimated minimum time before a second AP can be produced) from ~4 ms to ~7.5 and 15 ms in the 66 and 55 mM K+ solutions, respectively. These results demonstrate that T-system depolarization reduces tetanic force by impairing AP repriming, rather than by preventing AP generation per se or by inactivating the T-system voltage sensors. The findings also explain why it is advantageous to reduce the rate of motoneuron stimulation to muscles during repeated or prolonged periods of activity. [ABSTRACT FROM AUTHOR]

The present study was conducted to determine whether the infarct sparing effect of short- term exercise is dependent on the operation of the myocardial sarcolemmal ATP-sensitive K+ (KATP) channel. Adult male and female Sprague-Dawley rats were exercised on a motorized treadmill for 5 days. Twenty-four hours following the training or sedentary period, hearts were isolated and exposed to 1 h of regional ischemia followed by 2 h of reperfusion on a modified Langendorf apparatus in the presence or absence of the sarcolemmal KATP channel antagonist HMR-1098 (30 µM). Following the ischemia-reperfusion protocol, infarct size was determined as a percentage of the total ischemic zone at risk (ZAR). Short-term exercise reduced infarct size by 24% in males (32 ± 2% of ZAR; P < 0.01) and by 18% in females (26 ± 2% of ZAR; P < 0.05). Sarcolemmal KATP channel blockade abolished the training-induced cardioprotection in both males and females, increasing infarct size to 43 ± 3% and 52 ± 4% of ZAR, respectively. In the absence of HMR-1098, infarct size was significantly lower in sedentary females than in males (33 ± 4% vs. 42 ± 2% of ZAR, respectively; P < 0.01). However, the presence of HMR-1098 abolished this sex difference, increasing infarct size by 58% in the sedentary females (P < 0.01) but having no effect on infarct size in sedentary males. This study demonstrates that the sex-specific and exercise-acquired resistance to myocardial ischemia-reperfusion injury is dependent on sarcolemmal KATP activity during ischemia. [ABSTRACT FROM AUTHOR]

Mammalian skeletal muscles generate marked amounts of superoxide (O2·-) at 37°C, but it is not well understood which is the main source of O2·- production in the muscle fibers and how this interferes with muscle function. To answer these questions, O2·- production and twitch force responses were measured at 37°C in mechanically skinned muscle fibers of rat extensor digitorum longus (EDL) muscle. In mechanically skinned fibers, the sarcolemma is removed avoiding potential sources of O2·- production that are not intrinsically part of the muscle fibers, such as nerve terminals, blood cells, capillaries and other blood vessels in the whole muscle. O2·- production was also measured in split single EDL muscle fibers, where part of the sarcolemma remained attached, and small bundles of intact isolated EDL muscle fibers at rest, in the presence and absence of modifiers of mitochondrial function. The results lead to the conclusion that mitochondrial production of O2·- accounts for most of the O2·- measured intracellularly or extracellularly in skeletal muscle fibers at rest and at 37°C. Muscle fiber excitability at 37°C was greatly improved in the presence of a membrane permeant O2·- dismutase mimetic (Tempol), demonstrating a direct link between O2·- production in the mitochondria and muscle fiber performance. This implicates mitochondrial O2·- production in the down-regulation of skeletal muscle function, thus providing a feedback pathway for communication between mitochondria and plasma membranes that is not directly related to the main function of mitochondria as the power plant of the mammalian muscle cell. [ABSTRACT FROM AUTHOR]

The effect of diabetes on sarcolemmal Na+-K+ pump function is important for our understanding of heart disease associated with diabetes and design of its treatment. We induced diabetes characterized by hyperglycemia but no other major metabolic disturbances in rabbits. Ventricular myocytes isolated from diabetic rabbits and controls were voltage clamped and internally perfused with the whole cell patch-clamp technique. Electrogenic Na+-K+ pump current (Ip, arising from the 3:2 Na+-to-K+ exchange ratio) was identified as the shift in holding current induced by Na+-K+ pump blockade with 100 µmol/l ouabain in most experiments. There was no effect of diabetes on Ip recorded when myocytes were perfused with pipette solutions containing 80 mmol/l Na+ to nearly saturate intracellular Na+-K+ pump sites. However, diabetes was associated with a significant decrease in I+, measured when pipette solutions contained 10 mmol/l Na+. The decrease was independent of membrane voltage but dependent on the intracellular concentration of K+. There was no effect of diabetes on the sensitivity of lp, to extracellular K+. Pump inhibition was abolished by restoration of euglycemia or by in vivo angiotensin II receptor blockade with losartan. We conclude that diabetes induces sarcolemmal Na+-K+ pump inhibition that can be reversed with pharmacological intervention. [ABSTRACT FROM AUTHOR]

Chronic contractile activity of skeletal muscle induces an increase in mitochondria located in proximity to the sarcolemma [subsarcolemmal (SS)] and in mitochondria interspersed between the myofibrils [intermyofibrillar (IMF)]. These are energetically favorable metabolic adaptations, but because mitochondria are also involved in apoptosis, we investigated the effect of chronic contractile activity on mitochondrially mediated apoptotic signaling in muscle. We hypothesized that chronic contractile activity would provide protection against mitochondrially mediated apoptosis despite an elevation in the expression of proapoptotic proteins. To induce mitochondrial biogenesis, we chronically stimulated (10 Hz; 3 h/day) rat muscle for 7 days. Chronic contractile activity did not alter the Bax/Bcl-2 ratio, an index of apoptotic susceptibility, and did not affect manganese superoxide dismutase levels. However, contractile activity increased antiapoptotic 70-kDa heat shock protein and apoptosis repressor with a caspase recruitment domain by 1.3- and 1.4-fold (P < 0.05), respectively. Contractile activity elevated SS mitochondrial reactive oxygen species (ROS) production 1.4- and 1.9-fold (P < 0.05) during states IV and III respiration, respectively, whereas IMF mitochondrial state IV ROS production was suppressed by 28% (P < 0.05) and was unaffected during state III respiration. Following stimulation, exogenous ROS treatment produced less cytochrome c release (25-40%) from SS and IMF mitochondria, and also reduced apoptosis-inducing factor release (≈30%) from IMF mitochondria, despite higher inherent cytochrome c and apoptosis-inducing factor expression. Chronic contractile activity did not alter mitochondrial permeability transition pore (mtPTP) components in either subfraction. However, SS mitochondria exhibited a significant increase in the time to Vmax of mtPTP opening. Thus, chronic contractile activity induces predominantly antiapoptotic adaptations in both mitochondrial subfractions. Our data suggest the possibility that chronic contractile activity can exert a protective effect on mitochondrially mediated apoptosis in muscle. [ABSTRACT FROM AUTHOR]

Although the Na+/H+ exchanger (NHE) is considered to be involved in regulation of intracellular Ca2+ concentration ([Ca2+J+) through the Na+ICa2+ exchanger, the exact mechanisms of its participation in Ca2+ handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca2+ movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N- isobutyl)amiloride (MIA). [Ca2+]i in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca2+]+ and augmented the KCl-induced increase in [Ca2+]+ in a concentration-dependent manner. The MIA-induced increase in basal [Ca2+]i was unaffected by extracellular Ca2+, antagonists of the sarcolemmal (SL) L-type Ca2+ channel, and inhibitors of the SL Na+/Ca2+ exchanger, SL Ca2+ pump ATPase and mitochondrial Ca2+ uptake. However, the MIA-induced increase in basal [Ca2+]i was attenuated by inhibitors of SL Na+-K+-ATPase and sarcoplasmic reticulum (SR) Ca2+ transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca2+ concentration and attenuated by agents that inhibit SL L-type Ca2+ channels, the SL Na+ICa2+ exchanger, SL Na+-K+-ATPase, and SR Ca2+ release channels and the SR Ca2+ pump. However, the effect of MIA on the KCl-induced increase in [Ca2+]i remained unaffected by treatment with inhibitors of SL Ca2+ pump ATPase and mitochondrial Ca2+ uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal +Ca2+]i, whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCI-induced increase in [Ca2+J1 in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca2+I1 and that MIA-induced increases in basal [Ca2+]+, as well as augmentation of the KCI-induced increase in [Ca2+I+, in cardiomyocytes are regulated differentially. [ABSTRACT FROM AUTHOR]

Annexin A5 is a Ca2+-dependent phospholipid binding protein well known for its high phosphatidylserine affinity. In vitro, translocation to sarcolemma and externalization of endogenous annexin A5 in the cardiomyocyte has recently been demonstrated to exert a proapoptotic effect. To determine whether these in vitro findings occurred in vivo, we performed myocardial infarction (MI) and studied the time course of apoptosis and annexin A5 localization (0.5 to 8 h) in the border zone around the infarcted area. This zone that was defined as Evans blue unstained and triphenyltetrazolium chloride (TFC) stained, represented 42.3 ± 5.5% of the area at risk and showed apoptotic characteristics (significant increases in caspase 3 activity 2.3-fold at 0.5 h; P < 0.05), transferase-mediated dUTP nick-end labeling-positive cardiomyocytes (15.8 ± 0.8% at 8 h), and DNA ladder. When compared with sham-operated rats, we found that in this area, annexin A5 was translocated to the sarcolemma as early as 0.5 h after MI and that translocation increased with time. Moreover, the amount of annexin A5 was unchanged in the border zone and decreased in the infarcted area after 1 h (77.1 ± 4.8%; P < 0.01 vs. perfused area), suggesting a release in the latter but not in the former. In conclusion, we demonstrated that annexin A5 translocation is an early and rapid event of the whole border zone, likely due to Ca2+ increase. Part of this translocation occurred in areas where apoptosis was later detected and suggests that in vivo as in vitro annexin A5 might be involved in the regulation of early apoptotic events during cardiac pathological situations. [ABSTRACT FROM AUTHOR]

Strength loss following disuse may result from alterations in muscle and/or neurological properties. In this paper, we report our findings on human plantar flexor muscle properties following 4 wk of limb suspension (unilateral lower limb suspension), along with the effect of applied ischemia (Isc) on these properties. In the companion paper (Part II), we report our findings on the changes in neurological properties. Measurements of voluntary and evoked forces, the compound muscle fiber action potential (CMAP), and muscle cross-sectional area (CSA) were collected before and after 4 wk of unilateral lower limb suspension in adults (n = 18; 19–28 yr). A subset of subjects (n = 6) received applications of Isc 3 days/wk (3 sets; 5-min duration). In the subjects not receiving Isc, the loss in CSA and strength was as expected (∼9 and 14%). We observed a 30% slowing in the duration of the CMAP, a 10% decrease in evoked doublet force, a 12% increase in the twitch-to-doublet force ratio, and an altered postactivation potentiation response (11% increase in the postactivation potentiation-to-doublet ratio). We also detected a 10% slowing in the ability of the plantar flexor to develop force during the initial phase of an evoked contraction, along with a 6% reduction in in vivo specific doublet force. In the Isc subjects, no preservation was observed in strength or the evoked muscle properties. However, the Isc group did maintain CSA of the lateral gastrocnemius, as the control subjects' lateral gastrocnemius atrophied 10.2%, whereas the subjects receiving Isc atrophied 4.7%. Additionally, Isc abolished the unweighting-induced slowing in the CMAP. These findings suggest that unweighting alters the contractile properties involved in the excitation-contraction coupling processes and that Isc impacts the sarcolemma. [ABSTRACT FROM AUTHOR]

The FXYD protein family consists of several small, single-span membrane proteins that exhibit a high degree of homology. The best-known members of the family include the γ-subunit of the Na+ -K+ -ATPase and phospholemman (PLM), a phosphoprotein of cardiac sarcolemma. Other members of the family include corticosteroid hormone-induced factor (CHIF), mammary tumor protein of 8 kDa (Mat-8), and related to ion channels (RIC). The exact physiological roles of the FXYD proteins remain unknown. To better characterize the function of the members of the FXYD protein family, we expressed several members of the family in Madin-Darby canine kidney (MDCK) cells. All of the FXYD proteins, with the exception of PLM, were primarily found in the basolateral plasma membrane. Surprisingly, PLM, a previously characterized plasma membrane protein, was found to colocalize with the endoplasmic reticulum marker protein disulfide isomerase. Treatment of MDCK cells expressing PLM with an agonist of PKC caused some of the PLM to be redistributed to the plasma membrane. Site-directed mutagenesis of residues within the cytoplasmic domain of PLM indicated that a negative charge at Ser69 is necessary to shift the localization of PLM to the plasma membrane. In addition, other regions of PLM necessary for either its endoplasmic reticulum or plasma membrane localization have been elucidated. In contrast to PLM, the plasma membrane localization of CHIF and RIC was not altered by mutation of potential cytoplasmic phosphorylation sites. Overall, these results suggest that phosphorylation of specific residues of PLM may direct PLM from an intracellular compartment to the plasma membrane. [ABSTRACT FROM AUTHOR]

Acute activation of the serine-threonine kinase Akt is cardioprotective and increases glucose uptake, at least in part, through enhanced expression of GLUT4 on the sarcolemma. The effects of chronic Akt activation on glucose uptake in the heart remain unclear. To address this issue, we examined the effects of chronic Akt activation on glucose uptake, glycogen storage, and relevant glucose transporters in the hearts of transgenic mice. We found that chronic cardiac activation of Akt led to a substantial increase in the rate of basal glucose uptake (P < 0.05) but blunted the response to insulin (1.9 vs. 18.1-fold increase compared with baseline) using NMR in ex vivo perfused heart. Basal glucose uptake was also increased in Akt transgenic mice in vivo (P < 0.005). These changes were associated with an increase on glycogen deposition, examined with histochemical staining, biochemical (>6-fold, P < 0.001) and in vivo radioactive (5-fold, P < 0.01) assays. Studies in chimeric hearts of female X-linked transgenic Akt mice suggested that increased glycogen deposition occurred as a cell autonomous effect of transgene expression. Interestingly, although sarcolemmal GLUT1 was not significantly altered, chronic Akt activation actually decreased plasma membrane GLUT4. Moreover, intracellular pools of GLUT1 were modestly reduced, whereas intracellular GLUT4 was substantially reduced. It seems likely that neither GLUT1 nor GLUT4 explains the increase in basal glucose uptake but that these reductions contribute to the loss of insulin responsiveness that we observed. These data demonstrate that chronic Akt activation increases basal glucose uptake and glycogen deposition while inhibiting the response to insulin. [ABSTRACT FROM AUTHOR]