Shane B. Kanatous

Activation of MEF2 by muscle activity is mediated through a calcineurin‐dependent pathway

Gene expression in skeletal muscles of adult vertebrates is altered profoundly by changing patterns of contractile work. Here we observed that the functional activity of MEF2 transcription factors is stimulated by sustained periods of endurance exercise or motor nerve pacing, as assessed by expression in trans genic mice of a MEF2‐dependent reporter gene (desMEF2‐lacZ). This response is accompanied by transformation of specialized myofiber subtypes, and is blocked either by cyclosporin A, a specific chemical inhibitor of calcineurin, or by forced expression of the endogenous calcineurin inhibitory protein, myocyte‐enriched calcineurin interacting protein 1. Calcineurin removes phosphate groups from MEF2, and augments the potency of the transcriptional activation domain of MEF2 fused to a heterologous DNA binding domain. Across a broad range, the enzymatic activity of calcineurin correlates directly with expression of endogenous genes that are transcriptionally activated by muscle contractions. These results delineate a molecular pathway in which calcineurin and MEF2 participate in the adaptive mechanisms by which skeletal myofibers acquire specialized contractile and metabolic properties as a function of changing patterns of muscle contraction.

Hypoxia reprograms calcium signaling and regulates myoglobin expression

Myoglobin is an oxygen storage molecule that is selectively expressed in cardiac and slow-twitch skeletal muscles that have a high oxygen demand. Numerous studies have implicated hypoxia in the regulation of myoglobin expression as an adaptive response to hypoxic stress. However, the details of this relationship remain undefined. In the present study, adult mice exposed to 10% oxygen for periods up to 3 wk exhibited increased myoglobin expression only in the working heart, whereas myoglobin was either diminished or unchanged in skeletal muscle groups. In vitro and in vivo studies revealed that hypoxia in the presence or absence of exercise-induced stimuli reprograms calcium signaling and modulates myoglobin gene expression. Hypoxia alone significantly altered calcium influx in response to cell depolarization or depletion of endoplasmic reticulum calcium stores, which inhibited the expression of myoglobin. In contrast, our whole animal and transcriptional studies indicate that hypoxia in combination with exercise enhanced the release of calcium from the sarcoplasmic reticulum via the ryanodine receptors triggered by caffeine, which increased the translocation of nuclear factor of activated T-cells into the nucleus to transcriptionally activate myoglobin expression. The present study unveils a previously unrecognized mechanism where the hypoxia-mediated regulation of calcium transients from different intracellular pools modulates myoglobin gene expression. In addition, we observed that changes in myoglobin expression, in response to hypoxia, are not dependent on hypoxia-inducible factor-1 or changes in skeletal muscle fiber type. These studies enhance our understanding of hypoxia-mediated gene regulation and will have broad applications for the treatment of myopathic diseases.

The ontogeny of aerobic and diving capacity in the skeletal muscles of Weddell seals.

SUMMARY Our objective was to determine the ontogenetic changes in the skeletal muscles of Weddell seals that transform a non-diving pup into an elite diving adult. Muscle biopsies were collected from pups, juveniles and adults and analyzed for changes in fiber type, mitochondrial density, myoglobin concentrations and aerobic, lipolytic and anaerobic enzyme activities. The fiber type results demonstrated a decrease in slow-twitch oxidative (Type I) fibers and a significant increase in fast-twitch oxidative (Type IIA) fibers as the animals mature. In addition, the volume density of mitochondria and the activity of lipolytic enzymes significantly decreased as the seals matured. To our knowledge, this is the first quantitative account describing a decrease in aerobic fibers shifting towards an increase in fast-twitch oxidative fibers with a significant decrease in mitochondrial density as animals mature. These differences in the muscle physiology of Weddell seals are potentially due to their three very distinct stages of life history: non-diving pup, novice diving juvenile, and elite deep diving adult. During the first few weeks of life, pups are a non-diving terrestrial mammal that must rely on lanugo (natal fur) for thermoregulation in the harsh conditions of Antarctica. The increased aerobic capacity of pups, associated with increased mitochondrial volumes, acts to provide additional thermogenesis. As these future elite divers mature, their skeletal muscles transform to a more sedentary state in order to maintain the low levels of aerobic metabolism associated with long-duration diving.

Emerging roles for myoglobin in the heart

Myoglobin (Mb) is an intensely studied hemoprotein that is restricted mainly to the heart and oxidative myofibers in skeletal muscle. Previous physiologic and pharmacologic studies have supported a role for Mb in facilitated oxygen transport or as an oxygen reservoir in striated muscle. Transgenic and gene disruption technologies have been utilized to produce mice that lack Mb. Studies utilizing these transgenic mouse models support the notion that Mb may have multiple, diverse functions in the heart. Future studies using these emerging technologies will further enhance the understanding of the role of Mb and other hemoproteins in cardiovascular biology.

Regulation of myoglobin expression.

SUMMARY Myoglobin is a well-characterized, cytoplasmic hemoprotein that is expressed primarily in cardiomyocytes and oxidative skeletal muscle fibers. However, recent studies also suggest low-level myoglobin expression in various non-muscle tissues. Prior studies incorporating molecular, pharmacological, physiological and transgenic technologies have demonstrated that myoglobin is an essential oxygen-storage hemoprotein capable of facilitating oxygen transport and modulating nitric oxide homeostasis within cardiac and skeletal myocytes. Concomitant with these studies, scientific investigations into the transcriptional regulation of myoglobin expression have been undertaken. These studies have indicated that activation of key transcription factors (MEF2, NFAT and Sp1) and co-activators (PGC-1α) by locomotor activity, differential intracellular calcium fluxes and low intracellular oxygen tension collectively regulate myoglobin expression. Future studies focused on tissue-specific transcriptional regulatory pathways and post-translational modifications governing myoglobin expression will need to be undertaken. Finally, further studies investigating the modulation of myoglobin expression under various myopathic processes may identify myoglobin as a novel therapeutic target for the treatment of various cardiac and skeletal myopathies.

Cytoglobin Is a Stress-responsive Hemoprotein Expressed in the Developing and Adult Brain

Cytoglobin (Cygb) is a novel tissue hemoprotein relatively similar to myoglobin (Mb). Because Cygb shares several structural features with Mb, we hypothesized that Cygb functions in the modulation of oxygen and nitric oxide metabolism or in scavenging free radicals within a cell. In the present study we examined the spatial and temporal expression pattern of Cygb during murine embryogenesis. Using in situ hybridization, RT-PCR, and Northern blot analyses, limited Cygb expression was observed during embryogenesis compared with Mb expression. Cygb expression was primarily restricted to the central nervous system and neural crest derivatives during the latter stages of development. In the adult mouse, Cygb is expressed in distinct regions of the brain as compared with neuroglobin (Ngb), another globin protein, and these regions are responsive to oxidative stress (i.e., hippo-campus, thalamus, and hypothalamus). In contrast to Ngb, Cygb expression in the brain is induced in response to chronic hypoxia (10% oxygen). These results support the hypothesis that Cygb is an oxygen-responsive tissue hemoglobin expressed in distinct regions of the normoxic and hypoxic brain and may play a key role in the response of the brain to a hypoxic insult.

The effects of water temperature on the energetic costs of juvenile and adult California sea lions (Zalophus californianus): the importance of skeletal muscle thermogenesis for thermal balance.

SUMMARY As highly mobile marine predators, many pinniped species routinely encounter a wide range of water temperatures during foraging and in association with seasonal, geographical and climatic changes. To determine how such variation in environmental temperature may impact energetic costs in otariids, we determined the thermal neutral zone of adult and juvenile California sea lions (Zalophus californianus) by measuring resting metabolic rate using open-flow respirometry. Five adult female (body mass range =82.2-107.2 kg) and four juvenile (body mass=26.2-36.5 kg) sea lions were examined over experimental water temperatures ranging from 0 to 20°C (adults) or 5 to 20°C (juveniles). The metabolic rate of adult sea lions averaged 6.4±0.64 ml O2 kg−1 min−1 when resting within the thermal neutral zone. The lower critical temperature of adults was 6.4±2.2°C, approximately 4°C lower than sea surface temperatures routinely encountered off coastal California. In comparison, juvenile sea lions did not demonstrate thermal neutrality within the range of water temperatures examined. Resting metabolic rate of the younger animals, 6.3±0.53 ml O2 kg−1 min−1, increased as water temperature approached 12°C, and suggested a potential thermal limitation in the wild. To determine whether muscle thermogenesis during activity could mitigate this limitation, we measured the active metabolic rate of juveniles swimming at water temperature (Twater)=5, 12 and 20°C. No significant difference (F=0.377, P=0.583) in swimming metabolic rate was found among water temperatures, suggesting that thermal disadvantages due to small body size in juvenile sea lions may be circumvented by recycling endogenous heat during locomotor activity.

In the face of hypoxia: myoglobin increases in response to hypoxic conditions and lipid supplementation in cultured Weddell seal skeletal muscle cells

SUMMARY A key cellular adaptation to diving in Weddell seals is enhanced myoglobin concentrations in their skeletal muscles, which serve to store oxygen to sustain a lipid-based aerobic metabolism. The aim of this study was to determine whether seal muscle cells are inherently adapted to possess the unique skeletal muscle adaptations to diving seen in the whole animal. We hypothesized that the seal skeletal muscle cells would have enhanced concentrations of myoglobin de novo that would be greater than those from a C2C12 skeletal muscle cell line and reflect the concentrations of myoglobin observed in previous studies. In addition we hypothesized that the seal cells would respond to environmental hypoxia similarly to the C2C12 cells in that citrate synthase activity and myoglobin would remain the same or decrease under hypoxia and lactate dehydrogenase activity would increase under hypoxia as previously reported. We further hypothesized that β-hydroxyacyl CoA dehydrogenase activity would increase in response to the increasing amounts of lipid supplemented to the culture medium. Our results show that myoglobin significantly increases in response to environmental hypoxia and lipids in the Weddell seal cells, while appearing similar metabolically to the C2C12 cells. The results of this study suggest the regulation of myoglobin expression is fundamentally different in Weddell seal skeletal muscle cells when compared with a terrestrial mammalian cell line in that hypoxia and lipids initially prime the skeletal muscles for enhanced myoglobin expression. However, the cells need a secondary stimulus to further increase myoglobin to levels seen in the whole animal.

Age-related differences in skeletal muscle lipid profiles of Weddell seals: clues to developmental changes

SUMMARY Our objective was to elucidate age-related changes in lipids associated with skeletal muscle of Weddell seals and to suggest possible physiological implications. Muscle biopsies were collected from pups, juveniles and adults in McMurdo Sound, Antarctica and analyzed for intramuscular lipid (IML) and triacylglyceride (IMTG) amounts, fatty acid groups, as well as individual fatty acid profiles. The results from this study suggest a switch from primarily saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) in the skeletal muscle of young pups to increases in polyunsaturated fatty acids (PUFAs) as the percentage of blubber increases, resulting in possible thermoregulatory benefits. As Weddell pups continue to develop into juveniles, fatty acids associated with the skeletal muscle changes such that MUFA levels are relatively higher, which may be in response to energy depletion associated with their restricted diving ability and rapid growth. As juveniles transform into adults, a reduction in n-3 PUFA levels in the muscle as the percentage of blubber increases may be indicative of a trigger to prepare for deep diving or could be a mechanism for oxygen conservation during long-duration dives. We speculate that the observed change in lipids associated with the skeletal muscle of Weddell seals is related to ontogenetic differences in thermoregulation and locomotion.

Adaptations to diving hypoxia in the heart, kidneys and splanchnic organs of harbor seals (Phoca vitulina)

SUMMARY Pinnipeds (seals and sea lions) have an elevated mitochondrial volume density [VV(mt)] and elevated citrate synthase (CS) andβ -hydroxyacyl-CoA dehydrogenase (HOAD) activities in their swimming muscles to maintain an aerobic, fat-based metabolism during diving. The goal of this study was to determine whether the heart, kidneys and splanchnic organs have an elevated VV(mt) and CS and HOAD activities as parallel adaptations for sustaining aerobic metabolism and normal function during hypoxia in harbor seals (Phoca vitulina). Samples of heart, liver, kidney, stomach and small intestine were taken from 10 freshly killed harbor seals and fixed in glutaraldehyde for transmission electron microscopy or frozen in liquid nitrogen for enzymatic analysis. Samples from dogs and rats were used for comparison. Within the harbor seal, the liver and stomach had the highest VV(mt). The liver also had the highest CS activity. The kidneys and heart had the highest HOAD activities, and the liver and heart had the highest lactate dehydrogenase (LDH) activities. Mitochondrial volume densities scaled to tissue-specific resting metabolic rate [VV(mt)/RMR] in the heart, liver, kidneys, stomach and small intestine of harbor seals were elevated (range 1.2-6.6×) when compared with those in the dog and/or rat. In addition, HOAD activity scaled to tissue-specific RMR in the heart and liver of harbor seals was elevated compared with that in the dog and rat (3.2× and 6.2× in the heart and 8.5× and 5.5× in the liver, respectively). These data suggest that organs such as the liver, kidneys and stomach possess a heightened ability for aerobic, fat-based metabolism during hypoxia associated with routine diving. However, a heightened LDH activity in the heart and liver indicates an adaptation for the anaerobic production of ATP on dives that exceed the animals aerobic dive limit. Hence, the heart, liver, kidneys and gastrointestinal organs of harbor seals exhibit adaptations that promote an aerobic, fat-based metabolism under hypoxic conditions but can provide ATP anaerobically if required.