Intact Muscle Fibers Research Articles

Localized nuclear and perinuclear Ca(2+) signals in intact mouse skeletal muscle fibers.

Nuclear Ca2+ is important for the regulation of several nuclear processes such as gene expression. Localized Ca2+ signals (LCSs) in skeletal muscle fibers of mice have been mainly studied as Ca2+ release events from the sarcoplasmic reticulum. Their location with regard to cell nuclei has not been investigated. Our study is based on the hypothesis that LCSs associated with nuclei are present in skeletal muscle fibers of adult mice. Therefore, we carried out experiments addressing this question and we found novel Ca2+ signals associated with nuclei of skeletal muscle fibers (with possibly attached satellite cells). We measured localized nuclear and perinuclear Ca2+ signals (NLCSs and PLCSs) alongside cytosolic localized Ca2+ signals (CLCSs) during a hypertonic treatment. We also observed NLCSs under isotonic conditions. The NLCSs and PLCSs are Ca2+ signals in the range of micrometer [FWHM (full width at half maximum): 2.75 ± 0.27 μm (NLCSs) and 2.55 ± 0.17 μm (PLCSs), S.E.M.]. Additionally, global nuclear Ca2+ signals (NGCSs) were observed. To investigate which type of Ca2+ channels contribute to the Ca2+ signals associated with nuclei in skeletal muscle fibers, we performed measurements with the RyR blocker dantrolene, the DHPR blocker nifedipine or the IP3R blocker Xestospongin C. We observed Ca2+ signals associated with nuclei in the presence of each blocker. Nifedipine and dantrolene had an inhibitory effect on the fraction of fibers with PLCSs. The situation for the fraction of fibers with NLCSs is more complex indicating that RyR is less important for the generation of NLCSs compared to the generation of PLCSs. The fraction of fibers with NLCSs and PLCSs is not reduced in the presence of Xestospongin C. The localized perinuclear and intranuclear Ca2+ signals may be a powerful tool for the cell to regulate adaptive processes as gene expression. The intranuclear Ca2+ signals may be particularly interesting in this respect.

Intracellular Ca(2+)-handling differs markedly between intact human muscle fibers and myotubes.

BackgroundIn skeletal muscle, intracellular Ca2+ is an important regulator of contraction as well as gene expression and metabolic processes. Because of the difficulties to obtain intact human muscle fibers, human myotubes have been extensively employed for studies of Ca2+-dependent processes in human adult muscle. Despite this, it is unknown whether the Ca2+-handling properties of myotubes adequately represent those of adult muscle fibers.MethodsTo enable a comparison of the Ca2+-handling properties of human muscle fibers and myotubes, we developed a model of dissected intact single muscle fibers obtained from human intercostal muscle biopsies. The intracellular Ca2+-handling of human muscle fibers was compared with that of myotubes generated by the differentiation of primary human myoblasts obtained from vastus lateralis muscle biopsies.ResultsThe intact single muscle fibers all demonstrated strictly regulated cytosolic free [Ca2+] ([Ca2+]i) transients and force production upon electrical stimulation. In contrast, despite a more mature Ca2+-handling in myotubes than in myoblasts, myotubes lacked fundamental aspects of adult Ca2+-handling and did not contract. These functional differences were explained by discrepancies in the quantity and localization of Ca2+-handling proteins, as well as ultrastructural differences between muscle fibers and myotubes.ConclusionsIntact single muscle fibers that display strictly regulated [Ca2+]i transients and force production upon electrical stimulation can be obtained from human intercostal muscle biopsies. In contrast, human myotubes lack important aspects of adult Ca2+-handling and are thus an inappropriate model for human adult muscle when studying Ca2+-dependent processes, such as gene expression and metabolic processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0050-x) contains supplementary material, which is available to authorized users.

Acute Hypoxia Differentially Modifies Ca 2+ Waves in Pulmonary Arterial Smooth Muscle Cells of Intact Arteries from Fetal and Adult Sheep

Ca2+ signaling is important to pulmonary arterial (PA) contraction during acute hypoxia. To better understand Ca2+ signals under acute hypoxia in fetal and adult sheep, we performed confocal imaging of Fluo-4 loaded intact ovine PA Smooth Muscle Cells (PASMCs). Recordings were made in a controlled atmospheric chamber at ~37°C under normoxic conditions at ~300 m or during acute hypoxia, induced by exposing arteries to ~4% O2 for 30 min. Automated analysis procedures were used to examine Ca2+ waves to determine the magnitude, duration, and the number of regions of interest that had events at similar times (“friends”). We tested the hypothesis that acute hypoxia differentially modifies Ca2+ signaling in PASMCs of fetuses versus adults. Adult normoxic PASMC Ca2+ waves had lower AUC and shorter duration compared to fetuses, comparable amplitudes, but greater number of friends. In fetal PASMCs, acute hypoxia decreased Ca2+ wave duration, amplitude, and the area under the curve (AUC) but did not alter the number of friends. In adult sheep, acute hypoxia reduced Ca2+ wave amplitude and the number of friends, but did not impact wave duration or AUC. These results indicate that acute hypoxia modifies fetal and adult PASMC Ca2+ waves in unique ways. These differences in the Ca2+ responses may help define the resultant vasoreactivity in response to acute hypoxia in fetal and adult PASMCs. (Support from NIH and NSF)

Statin Induced Myopathy: A Role for Mitochondrial Ca2+ and No in Enhanced Sarcoplasmic Reticulum Ca2+ Leak

The need to understand the mechanism of statin-induced myopathy is increasing as the cardiovascular risk threshold for statin prescription is reduced across the globe. Here we determine for the first time the effect of chronic statin treatment in vivo (simvastatin 40 mg/kg/day by oral gavage over a 4 week period) on Ca2+ homeostasis in intact (non-permeabilised) type II rat skeletal muscle fibres, and investigate a role for mitochondria and NO. In fluo-4 loaded FDB fibres, sarcoplasmic reticulum (SR) Ca2+ leak (indexed in the presence of the SERCA inhibitor cyclopiazonic acid) was significantly higher (P<0.05) following statin treatment. This could be explained by the observed increase (P<0.001) in the frequency and duration of Ca2+ sparks/embers in statin fibres. Interestingly, inhibition of the mitochondrial Ca2+ uniporter by Ru360 in statin fibres normalised spark frequency and duration to control levels (P<0.05), suggesting that mitochondrial Ca2+ uptake contributes to statin effects on SR Ca2+ release. NOS inhibition with L-NAME had a greater impact (P<0.05) on NO (indexed with DAF-2) in statin fibres compared with controls, suggesting increased NOS activity with statin treatment. This is consistent with reduced expression (P<0.05) of GAPDH-normalised caveolin 1 and 3 in gastrocnemius muscle from statin treated animals; caveolins are the main constitutive inhibitors of nNOS and eNOS. Together these data show for the first time that statin treatment enhances SR Ca2+ leak in intact muscle fibres. We propose that Ca2+-dependent mitochondrial ROS production and NO modify RyR to effect this leak. Defining the cellular processes that underlie statin induced myopathy is the first step in the development of co-therapies to improve statin compliance. Sponsored by the British Heart Foundation.

Calcium Sparklets in Intact Mammalian Skeletal Muscle Fibers Expressing the Embryonic CaV1.1 Splice Variant

The embryonic splice variant of the voltage-gated L-type calcium channel (CaV1.1e) displays an altered voltage-dependence and gating kinetics as compared to that expressed in adult skeletal muscle. Because the adult CaV1.1a only opens slowly at strong depolarizations, its contribution as a source of calcium influx during action potentials is negligible. In contrast, calcium influx through the embryonic CaV1.1e substantially contributes to depolarization-induced calcium transients in fetal muscles and in cultured myotubes. In a genetically modified mouse (CaV1.1αE29), which exclusively expresses the embryonic CaV1.1e variant also in adult muscle the calcium influx component is maintained throughout life. Utilizing this mouse model, calcium release events - calcium sparklets - were recorded in enzymatically isolated, intact adult skeletal muscle fibers from the m. flexor digitorum longus using the fluorescent calcium probe fluo-8 and the fast confocal scanner (ZeissLive) in the x-y mode. While control animals did not display such events, CaV1.1αE29 mice spontaneously generated sparklets with a frequency of 9.2∗10-4±6∗10-4 Hz/μm2 (19 fibers; mean±SEM). The role of external calcium as the trigger was tested by either removing calcium from the external solution or by the application of 5μM nizoldipine to block the calcium current through CaV1.1e. Both interventions resulted in a complete loss of the events. Identified sparklets (n=311) were characterized by an average amplitude (αF/F0) of 0.287±0.005, a full-width at half-maximum of 3.05±0.05 µm, and duration of 235±4 ms, clearly different from the properties of calcium sparks on saponin-permeabilized adult mammalian skeletal muscle fibers. These findings indicate that the sustained expression of the CaV1.1e splice variant gives rise to spontaneous calcium entry events (sparklets) in adult muscle fibers and that their properties are distinct from calcium sparks arising from ryanodine receptors. Support: OTKA-NN107765, FWF P23479, W1101, TAMOP-4.2.4.A./2-11-1-2012-0001.

Subcellular distribution of glycogen and decreased tetanic calcium in fatigued single intact mouse muscle fibres.

Subcellular distribution of glycogen and decreased tetanic calcium in fatigued single intact mouse muscle fibres.

Comparison of force and shortening velocity of fast and slow rabbit muscle fibers at different temperatures

The temperature dependence of force, maximal shortening velocity and power of maximally activated single permeabilized fibers from fast and slow muscles of the rabbit were recorded in a temperature range from 10 to 35 degrees C with 5 degrees C step. It was found that temperature dependence of force of both types of fibers is identical. Averaged maximal shortening velocity in the slow fibers, unlike the fast fibers, had no statistically significant temperature dependence that is not in agreement with the data obtained on intact rat muscle fibers and in an in vitro motility assay. However maximal shortening velocity in each individual slow fiber did depend on temperature. The temperature dependence of power of the slow fibers was lower than that of the fast ones. Because of large data scattering the average temperature dependence of power of the slow fibers was significantly lower than that in individual slow fibers.

The force-velocity relationship at negative loads (assisted shortening) studied in isolated, intact muscle fibres of the frog.

The study was undertaken to explore the force-velocity relationship under conditions where the myofilament system is subjected to an external force that serves as a negative load and assists the shortening movement. The experiments were carried out on single muscle fibres isolated from the anterior tibialis muscle of Rana temporaria. The fibres, being operated under load-clamp control, were released to shorten during tetanic stimulation at sarcomere lengths where the fibres carried different degrees of passive tension. The shortening thus occurred while the sarcomeres were subjected to a force that may be characterized as a 'negative load', that is, a force assisting the shortening movement. The force-velocity relationship below zero load was found to be a smooth continuation of the force-velocity curve recorded at positive loads with the shortening velocity increasing steeply at loads below zero. A negative load amounting to merely 10% of the isometric force, thus raised the shortening velocity to a level two to three times higher than V0 , the velocity recorded at zero load. The results provide evidence that, even in the presence of a longitudinal compressive force, the speed of shortening of the muscle fibre is determined by the cycling rate of the interacting cross-bridges. The force-velocity relationship at negative loads may play a relevant part during fast movements of striated muscle as pointed out in the discussion.

In vivo effects of propyl gallate, a novel Ca2 + sensitizer, in a mouse model of dilated cardiomyopathy caused by cardiac troponin T mutation

AimsWe have previously demonstrated that propyl gallate has a Ca2+ sensitizing effect on the force generation in membrane-permeabilized (skinned) cardiac muscle fibers. However, in vivo beneficial effects of propyl gallate as a novel Ca2+ sensitizer remain uncertain. In the present study, we aim to explore in vivo effects of propyl gallate. Main methodsWe compared effects of propyl gallate on ex vivo intact cardiac muscle fibers and in vivo hearts in healthy mice with those of pimobendan, a clinically used Ca2+ sensitizer. The therapeutic effect of propyl gallate was investigated using a mouse model of dilated cardiomyopathy (DCM) with reduced myofilament Ca2+ sensitivity due to a deletion mutation ΔK210 in cardiac troponin T. Key findingsPropyl gallate, as well as pimobendan, showed a positive inotropic effect. Propyl gallate slightly increased the blood pressure without changing the heart rate in healthy mice, whereas pimobendan decreased the blood pressure probably through vasodilation via inhibition of phosphodiesterase and increased the heart rate. Propyl gallate prevented cardiac remodeling and systolic dysfunction and significantly improved the life-expectancy of knock-in mouse model of DCM with reduced myofilament Ca2+ sensitivity due to a mutation in cardiac troponin T. On the other hand, gallate, a similarly strong antioxidant polyphenol lacking Ca2+ sensitizing action, had no beneficial effects on the DCM mice. SignificanceThese results suggest that propyl gallate might be useful for the treatment of inherited DCM caused by a reduction in the myofilament Ca2+ sensitivity.

IL-1α Reversibly Inhibits Skeletal Muscle Ryanodine Receptor. A Novel Mechanism for Critical Illness Myopathy?

Critical illness myopathies in patients with sepsis or sustained mechanical ventilation prolong intensive care treatment and threaten both patients and health budgets; no specific therapy is available. Underlying pathophysiological mechanisms are still patchy. We characterized IL-1α action on muscle performance in "skinned" muscle fibers using force transducers and confocal Ca(2+) fluorescence microscopy for force/Ca(2+) transients and Ca(2+) sparks. Association of IL-1α with sarcoplasmic reticulum (SR) release channel, ryanodine receptor (RyR) 1, was investigated with coimmunoprecipitation and confocal immunofluorescence colocalization. Membrane integrity was studied in single, intact fibers challenged with IL-1α. IL-1α reversibly stabilized Mg(2+) inhibition of Ca(2+) release. Low Mg(2+)-induced force and Ca(2+) transients were reversibly abolished by IL-1α. At normal Mg(2+), IL-1α reversibly increased caffeine-induced force and Ca(2+) transients. IL-1α reduced SR Ca(2+) leak via RyR1, as judged by (1) increased SR Ca(2+) retention, (2) increased IL-1α force transients being reproduced by 25 μM tetracaine, and (3) reduced Ca(2+) spark frequencies by IL-1α or tetracaine. Coimmunoprecipitation confirmed RyR1/IL-1 association. RyR1/IL-1 immunofluorescence patterns perfectly colocalized. Long-term, 8-hour IL-1α challenge of intact muscle fibers compromised membrane integrity in approximately 50% of fibers, and confirmed intracellular IL-1α deposition. IL-1α exerts a novel, specific, and reversible interaction mechanism with the skeletal muscle RyR1 macromolecular release complex without the need to act via its membrane IL-1 receptor, as IL-1R membrane expression levels were not detectable in Western blots or immunostaining of single fibers. We present a potential explanation of how the inflammatory mediator, IL-1α, may contribute to muscle weakness in critical illness.

A novel quantitative morphometry approach to assess regeneration in dystrophic skeletal muscle

Duchenne muscular dystrophy is an inherited degenerative muscle disease with progressive weakness of skeletal and cardiac muscle. Disturbed calcium homeostasis and signalling pathways result in degeneration/regeneration cycles with fibrotic remodelling of muscle tissue, sustained by chronic inflammation. In addition to altered microarchitecture, regeneration in dystrophic muscle fibres is often only classified by centrally located nuclei but correlation of the regeneration process to nuclear volumes, myosin amounts, architecture and functional quality are missing, in particular in old muscles where the regenerative capacity is exhausted. Such information could yield novel regeneration-to-function biomarkers. Here we used second harmonic generation and multi photon fluorescence microscopy in intact single muscle fibres from wild-type, dystrophic mdx and transgenic mdx mice expressing an Δex 17–48 mini-dystrophin to determine the percentage of centronucleated fibres and nucleus-to-myosin volume ratio as a function of age. Based on this ratio we define a ‘biomotoric efficiency’ as an optical measure for fibre maturation, which is close to unity in adult wild-type and mini-dystrophin fibres, but smaller in very young and old mdx mice as a result of ongoing cell maturation (young) and regeneration (aged). With these parameters it is possible to provide a quantitative measure about muscle fibre regeneration.

Subcellular distribution of glycogen and decreased tetanic Ca2+ in fatigued single intact mouse muscle fibres

In skeletal muscle fibres, glycogen has been shown to be stored at different subcellular locations: (i) between the myofibrils (intermyofibrillar); (ii) within the myofibrils (intramyofibrillar); and (iii) subsarcolemmal. Of these, intramyofibrillar glycogen has been implied as a critical regulator of sarcoplasmic reticulum Ca(2+) release. The aim of the present study was to test directly how the decrease in cytoplasmic free Ca(2+) ([Ca(2+)]i) during repeated tetanic contractions relates to the subcellular glycogen distribution. Single fibres of mouse flexor digitorum brevis muscles were fatigued with 70Hz, 350ms tetani given at 2s (high-intensity fatigue, HIF) or 10s (low-intensity fatigue, LIF) intervals, while force and [Ca(2+)]i were measured. Stimulation continued until force decreased to 30% of its initial value. Fibres were then prepared for analyses of subcellular glycogen distribution by transmission electron microscopy. At fatigue, tetanic [Ca(2+)]i was reduced to 70±4% and 54±4% of the initial in HIF (P<0.01, n=9) and LIF (P<0.01, n=5) fibres, respectively. At fatigue, the mean inter- and intramyofibrillar glycogen content was 60-75% lower than in rested control fibres (P<0.05), whereas subsarcolemmal glycogen was similar to control. Individual fibres showed a good correlation between the fatigue-induced decrease in tetanic [Ca(2+)]i and the reduction in intermyofibrillar (P=0.051) and intramyofibrillar (P=0.0008) glycogen. In conclusion, the fatigue-induced decrease in tetanic [Ca(2+)]i, and hence force, is accompanied by major reductions in inter- and intramyofibrillar glycogen. The stronger correlation between decreased tetanic [Ca(2+)]i and reduced intramyofibrillar glycogen implies that sarcoplasmic reticulum Ca(2+) release critically depends on energy supply from the intramyofibrillar glycogen pool.

To understand muscle you must take it apart

Striated muscle is an elegant system for study at many levels. Much has been learned about the mechanism of contraction from studying the mechanical properties of intact and permeabilized (or skinned) muscle fibers. Structural studies using electron microscopy, X-ray diffraction or spectroscopic probes attached to various contractile proteins were possible because of the highly ordered sarcomeric arrangement of actin and myosin. However, to understand the mechanism of force generation at a molecular level, it is necessary to take the system apart and study the interaction of myosin with actin using in vitro assays. This reductionist approach has lead to many fundamental insights into how myosin powers muscle contraction. In addition, nature has provided scientists with an array of muscles with different mechanical properties and with a superfamily of myosin molecules. Taking advantage of this diversity in myosin structure and function has lead to additional insights into common properties of force generation. This review will highlight the development of the major assays and methods that have allowed this combined reductionist and comparative approach to be so fruitful. This review highlights the history of biochemical and biophysical studies of myosin and demonstrates how a broad comparative approach combined with reductionist studies have led to a detailed understanding of how myosin interacts with actin and uses chemical energy to generate force and movement in muscle contraction and motility in general.

Chemical Uncoupling the DHPR-RyR1 Complex by Substituted Halogenated Biphenyls and Diphenylethers

Ca2+ release units (CRU) of skeletal muscle have two components necessary for excitation-contraction coupling (ECC); the DHPR and RyR1 that physically interact to engage conformational coupling that involves both orthograde and retrograde regulation of Ca2+ dynamics during ECC and SR Ca2+ leak when the muscle is at rest. We previously identified that 2,2',3,5',6-pentachlorobiphenyl (PCB95) promotes long-lived stability of the full open state of RyR1 channels (Samso et al 2009). Further investigations PCB95 and its 4- and 5-hydroxy (-OH) derivatives toward rabbit skeletal muscle ryanodine receptor (RyR1) were performed using [3H]ryanodine binding and SR Ca2+ flux analyses. 5-OH metabolites have comparable activity to their respective parent in both assays; however, 4-OH derivatives are unable to trigger Ca2+ release from SR microsomes in the presence of Ca2+-ATPase activity. PCB95 and derivatives are investigated using single channel voltage-clamp and primary murine embryonic muscle cells. Like PCB95, 5-OH-PCB95 quickly and persistently increases channel open probability (Po>0.9) by stabilizing the full-open channel state, whereas 4-OH-PCB95 transiently enhances Po. Ca2+ imaging of myotubes show acute exposure to PCB95 (5μM) potentiates ECC and caffeine responses and partially depletes SR Ca2+ stores. Exposure to 5-OH-PCB95 (5μM) increases cytoplasmic Ca2+, leading to ECC failure in 50% of myotubes with the remainder retaining negligible responses. 4-OH-PCB95 neither increases baseline Ca2+ nor causes ECC failure but depresses ECC and caffeine responses by 50%. With longer (3 h) exposure to 300nM PCB95, 5-OH-PCB95, or 4-OH-PCB95 decreases the number of ECC responsive myotubes by 22%, 81%, and 51% compared with control by depleting SR Ca2+ and/or uncoupling ECC. The position of hydroxylation of PCB95 and chemically related diphenylethers differentially influence RyR1 channel gating kinetics, SR Ca2+ leak, and the pattern of ECC impairment, including ECC uncoupling in intact muscle cells. P01 AR52354.

743: Pharmacological blockade of anoctamin-1 on human pregnant uterine smooth muscle attenuates oxytocin induced muscle force and pacing frequency

Anoctamins are a group of calcium-activated chloride channels that play a role in the pacing of intestinal smooth muscle, and which can be identified in myometrial cells. The goal was to assess the ability of tannic acid, an anoctamin-1 antagonist, to suppress intact human uterine smooth muscle cell contractility

Effect of temperature on crossbridge force changes during fatigue and recovery in intact mouse muscle fibers.

Repetitive or prolonged muscle contractions induce muscular fatigue, defined as the inability of the muscle to maintain the initial tension or power output. In the present experiments, made on intact fiber bundles from FDB mouse, fatigue and recovery from fatigue were investigated at 24°C and 35°C. Force and stiffness were measured during tetani elicited every 90 s during the pre-fatigue control phase and recovery and every 1.5 s during the fatiguing phase made of 105 consecutive tetani. The results showed that force decline could be split in an initial phase followed by a later one. Loss of force during the first phase was smaller and slower at 35°C than at 24°C, whereas force decline during the later phase was greater at 35°C so that total force depression at the end of fatigue was the same at both temperatures. The initial force decline occurred without great reduction of fiber stiffness and was attributed to a decrease of the average force per attached crossbridge. Force decline during the later phase was accompanied by a proportional stiffness decrease and was attributed to a decrease of the number of attached crossbridge. Similarly to fatigue, at both 24 and 35°C, force recovery occurred in two phases: the first associated with the recovery of the average force per attached crossbridge and the second due to the recovery of the pre-fatigue attached crossbridge number. These changes, symmetrical to those occurring during fatigue, are consistent with the idea that, i) initial phase is due to the direct fast inhibitory effect of [Pi]i increase during fatigue on crossbridge force; ii) the second phase is due to the delayed reduction of Ca2+ release and /or reduction of the Ca2+ sensitivity of the myofibrils due to high [Pi]i.

AICAR inhibits oxygen consumption by intact skeletal muscle cells in culture

Activation of 5' adenosine monophosphate-activated protein kinase (AMPK) with aminoimidazole carboxamide ribonucleotide (AICAR) increases skeletal muscle glucose uptake and fatty acid oxidation. The purpose of these experiments was to utilize AICAR to enhance palmitate consumption by mitochondria in cultured skeletal muscle cells. In these experiments, we treated C2C12 myotubes or adult single skeletal muscle fibers with varying concentrations of AICAR for different lengths of time. Surprisingly, acute AICAR exposure at most concentrations (0.25-1.5 mM), but not all (0.1 mM), modestly inhibited oxygen consumption even though AICAR increased AMPK phosphorylation. The data suggest that AICAR inhibited oxygen consumption by the cultured muscle in a non-specific manner. The results of these experiments are expected to provide valuable information to investigators interested in using AICAR in cell culture studies.

Aging increases the oxidation of dichlorohydrofluorescein in single isolated skeletal muscle fibers at rest, but not during contractions

An increase in the activity of reactive oxygen species (ROS) has been implicated in the mechanisms of loss of skeletal muscle that occurs during aging, but few studies have attempted to directly assess activities in intact muscle fibers. The current project used the nonspecific fluorescent probe for ROS and reactive nitrogen species, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (CM-DCFH), in single, isolated, mature skeletal muscle fibers from adult and old mice in addition to biochemical measurements of key regulatory proteins for ROS in muscles of these animals. Data confirmed the changes in key regulatory processes for ROS (increased glutathione peroxidase 1 and catalase activities and reduced total glutathione content) previously reported in muscle from old mice and showed increased CM-DCFH oxidation in muscle fibers from old mice at rest and indicate that these changes are likely due to an increase in generation of oxidants rather than a lack of scavenging capacity. The increased CM-DCFH oxidation persisted even when cellular defenses against oxidants were increased by loading fibers from young and old mice with glutathione. During contractile activity, and in contrast to the increase observed in fibers from young mice, there was no further increase in CM-DCFH oxidation in muscle fibers from old mice. These data also suggest that the defect in short-term adaptations to contractions that occurs in old mice may be related to a diminished, or absent, increase in the muscle generation of ROS and/or reactive nitrogen species that normally accompanies contractile activity in young mice.

Physical and functional association of lactate dehydrogenase with mitochondria in intact and permeabilized skeletal muscle fibers

To clarify whether mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO2) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat red (RG) and white (WG) gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO2 in either RG or WG. However, subsequent addition of NAD+ significantly increased JO2, and was abolished by the inhibitor of mitochondrial pyruvate transport, α‐hydroxycinnamate. In parallel experiments with subsarcolemmal (SS) and intramyofibrillar (IMF) mitochondria isolated from RG, only SS exhibited significant NAD+ ‐ dependent lactate oxidation. To determine whether lactate dehydrogenase (LDH) is physically associated with mitochondria in RG, confocal microscopy was used to detect immunohistochemically stained LDH and mitochondrial proteins. LDH clearly colocalized with mitochondria in intact fibers. In permeabilized fibers, most of the LDH signal was lost in IMF but not SS regions. The LDH signal was absent in SS regions following trypsin treatment; however, colocalization was augmented in both SS and IMF regions with exogenous LDH + washing, suggesting LDH preferentially associates with muscle mitochondria. Collectively, these results suggest that LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.

Developmental changes in diaphragm muscle function in the preterm and postnatal lamb

The preterm diaphragm is structurally and functionally immature, potentially contributing to an increased risk of respiratory distress and failure. We investigated developmental changes in contractile function and susceptibility to fatigue of the costal diaphragm in the fetal lamb to understand factors contributing to the risk of developing diaphragm dysfunction and respiratory disorders. We hypothesized that the functional capacity of the diaphragm will vary with maturational stage as will its susceptibility to fatigue. Lambs were studied at 75, 100, 125, 145, 154, 168, and 200 days postconceptional age (term = 147 days). Lambs were euthanized (sodium pentobarbitone, 100 mg/kg) either at delivery or immediately prior to post-mortem for postnatal lambs. Contractile function was assessed on longitudinal strips of intact muscle fibers and the remaining tissue frozen in liquid nitrogen for analysis of myosin heavy chain (MHC) mRNA expression and protein content. Fetal development of diaphragm function was characterized by a significant increase in maximum specific force, increased susceptibility to fatigue, reduced twitch contraction times, and a progressive increase in MHCI and MHCII protein content. Postnatally, there was a progressive decrease in the susceptibility to fatigue that coincided with an increase in the MHC I:II protein ratio. These data indicate that the functional capacity of the diaphragm varies with maturational age and may be an important determinant of the susceptibility to preterm respiratory failure.