Sebastian Gehlert

Ca2+-Dependent Regulations and Signaling in Skeletal Muscle: From Electro-Mechanical Coupling to Adaptation

Calcium (Ca2+) plays a pivotal role in almost all cellular processes and ensures the functionality of an organism. In skeletal muscle fibers, Ca2+ is critically involved in the innervation of skeletal muscle fibers that results in the exertion of an action potential along the muscle fiber membrane, the prerequisite for skeletal muscle contraction. Furthermore and among others, Ca2+ regulates also intracellular processes, such as myosin-actin cross bridging, protein synthesis, protein degradation and fiber type shifting by the control of Ca2+-sensitive proteases and transcription factors, as well as mitochondrial adaptations, plasticity and respiration. These data highlight the overwhelming significance of Ca2+ ions for the integrity of skeletal muscle tissue. In this review, we address the major functions of Ca2+ ions in adult muscle but also highlight recent findings of critical Ca2+-dependent mechanisms essential for skeletal muscle-regulation and maintenance.

Induction and adaptation of chaperone-assisted selective autophagy CASA in response to resistance exercise in human skeletal muscle

Chaperone-assisted selective autophagy (CASA) is a tension-induced degradation pathway essential for muscle maintenance. Impairment of CASA causes childhood muscle dystrophy and cardiomyopathy. However, the importance of CASA for muscle function in healthy individuals has remained elusive so far. Here we describe the impact of strength training on CASA in a group of healthy and moderately trained men. We show that strenuous resistance exercise causes an acute induction of CASA in affected muscles to degrade mechanically damaged cytoskeleton proteins. Moreover, repeated resistance exercise during 4 wk of training led to an increased expression of CASA components. In human skeletal muscle, CASA apparently acts as a central adaptation mechanism that responds to acute physical exercise and to repeated mechanical stimulation.

Skeletal Muscle Function during Exercise—Fine-Tuning of Diverse Subsystems by Nitric Oxide

Skeletal muscle is responsible for altered acute and chronic workload as induced by exercise. Skeletal muscle adaptations range from immediate change of contractility to structural adaptation to adjust the demanded performance capacities. These processes are regulated by mechanically and metabolically induced signaling pathways, which are more or less involved in all of these regulations. Nitric oxide is one of the central signaling molecules involved in functional and structural adaption in different cell types. It is mainly produced by nitric oxide synthases (NOS) and by non-enzymatic pathways also in skeletal muscle. The relevance of a NOS-dependent NO signaling in skeletal muscle is underlined by the differential subcellular expression of NOS1, NOS2, and NOS3, and the alteration of NO production provoked by changes of workload. In skeletal muscle, a variety of highly relevant tasks to maintain skeletal muscle integrity and proper signaling mechanisms during adaptation processes towards mechanical and metabolic stimulations are taken over by NO signaling. The NO signaling can be mediated by cGMP-dependent and -independent signaling, such as S-nitrosylation-dependent modulation of effector molecules involved in contractile and metabolic adaptation to exercise. In this review, we describe the most recent findings of NO signaling in skeletal muscle with a special emphasis on exercise conditions. However, to gain a more detailed understanding of the complex role of NO signaling for functional adaptation of skeletal muscle (during exercise), additional sophisticated studies are needed to provide deeper insights into NO-mediated signaling and the role of non-enzymatic-derived NO in skeletal muscle physiology.

Intense Resistance Exercise Induces Early and Transient Increases in Ryanodine Receptor 1 Phosphorylation in Human Skeletal Muscle

Background While ryanodine receptor 1 (RyR1) critically contributes to skeletal muscle contraction abilities by mediating Ca2+ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK) senses contraction-induced energetic stress by phosphorylation at Thr172. Phosphorylation of RyR1 at serine2843 (pRyR1Ser2843) results in leaky RyR1 channels and impaired Ca2+homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca2+-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise. Purpose Determine the effects and early time-course of resistance exercise on pRyR1Ser2843 and pAMPKThr172 in type I and II myofibers. Methods 7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg) performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser2843 and pAMPKThr172 levels were determined by western blot and semi-quantitative immunohistochemistry techniques. Results While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser2843 increased 15 min and peaked 30 min (p<0.01) post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser2843 levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05). pAMPKThr172 also increased 15 to 30 min post exercise (p<0.01) in type I and II myofibers and in whole skeletal muscle. Conclusion Resistance exercise induces acutely increased pRyR1Ser2843 and concomitantly pAMPKThr172 levels for up to 30 min in resistance exercised myofibers. This provides a time-course by which pRyR1Ser2843 can mechanistically impact Ca2+handling properties and consequently induce reduced myofiber contractility beyond immediate fatiguing mechanisms.

Lactate regulates myogenesis in C2C12 myoblasts in vitro

Satellite cells (SCs) are the resident stem cells of skeletal muscle tissue which play a major role in muscle adaptation, e.g. as a response to physical training. The aim of this study was to examine the effects of an intermittent lactate (La) treatment on the proliferation and differentiation of C2C12 myoblasts, simulating a microcycle of high intensity endurance training. Furthermore, the involvement of reactive oxygen species (ROS) in this context was examined. C2C12 myoblasts were therefore repeatedly incubated for 2 h each day with 10 mM or 20 mM La differentiation medium (DM) and in some cases 20 mM La DM plus different antioxidative substances for up to 5 days. La free (0 mM) DM served as a control. Immunocytochemical staining, Western blot analysis and colorimetric assays were used to assess oxidative stress, proliferation, and differentiation. Results show that La induces oxidative stress, enhances cell-cycle withdrawal, and initiates early differentiation but delays late differentiation in a timely and dose-dependent manner. These effects can be reversed by the addition of antioxidants to the La DM. We therefore conclude that La has a regulatory role in C2C12 myogenesis via a ROS-sensitive mechanism which elicits implications for reassessing some aspects of training and the use of nutritional supplements.

The cochaperone BAG3 coordinates protein synthesis and autophagy under mechanical strain through spatial regulation of mTORC1

The cochaperone BAG3 is a central protein homeostasis factor in mechanically strained mammalian cells. It mediates the degradation of unfolded and damaged forms of the actin-crosslinker filamin through chaperone-assisted selective autophagy (CASA). In addition, BAG3 stimulates filamin transcription in order to compensate autophagic disposal and to maintain the actin cytoskeleton under strain. Here we demonstrate that BAG3 coordinates protein synthesis and autophagy through spatial regulation of the mammalian target of rapamycin complex 1 (mTORC1). The cochaperone utilizes its WW domain to contact a proline-rich motif in the tuberous sclerosis protein TSC1 that functions as an mTORC1 inhibitor in association with TSC2. Interaction with BAG3 results in a recruitment of TSC complexes to actin stress fibers, where the complexes act on a subpopulation of mTOR-positive vesicles associated with the cytoskeleton. Local inhibition of mTORC1 is essential to initiate autophagy at sites of filamin unfolding and damage. At the same time, BAG3-mediated sequestration of TSC1/TSC2 relieves mTORC1 inhibition in the remaining cytoplasm, which stimulates protein translation. In human muscle, an exercise-induced association of TSC1 with the cytoskeleton coincides with mTORC1 activation in the cytoplasm. The spatial regulation of mTORC1 exerted by BAG3 apparently provides the basis for a simultaneous induction of autophagy and protein synthesis to maintain the proteome under mechanical strain.

Exercise-Induced Decline in the Density of LYVE-1-Positive Lymphatic Vessels in Human Skeletal Muscle

BACKGROUND The investigation of lymphatic function and biology and its microvascular influence on tissue integrity, development and failure in various disease conditions constitutes an important field of medical research. To date several investigations were carried out investigating alterations of lymphatic vessel density under medical conditions e.g. in transplanted or failing heart. However, only few studies investigated aspects of exercise induced plasticity of lymphatic vessels. STUDY OBJECTIVE It was investigated, if alterations in lymphatic density can be observed in human skeletal muscle as a response to endurance exercise and if potential changes might be related to the distribution of myofibres. METHODS Muscle biopsies were taken from vastus lateralis muscle of male cyclists under resting conditions. Lymphatic capillary density and myofibre distribution were determined prior, as well as over the time course of a cycling training intervention. Lymphatic capillaries were stained by immunohistochemistry using LYVE-1 and Podoplanin antibodies. Myofibre distribution was classified by myofibrillar ATPase staining. RESULTS The density of LYVE-1/+ capillaries in skeletal muscle was observed to decrease significantly over the time course of the exercise intervention. It was further noticed that in consecutive cross sections a small part of vessels however showed either podoplanin or LYVE-1 staining. We did not recognize correlations of LYVE-1/+ vessel density to the distribution of the myofibre spectrum in trained skeletal muscle. CONCLUSION It was concluded that lymphatic vessels are rather normally distributed in skeletal muscle not dependent on a predominant myofibre type. The partial not observed co staining of LYVE-1 and podoplanin might be influenced by a shift in vessel phenotype. The finding of significantly decreased LYVE-1/+ capillary density over the time course of the applied exercise intervention gives rise to the assumption that exercise induced stimuli were able to induce alterations of lymphangiogenetic responses on a structural level.

Corrigendum: Training-induced alterations of skeletal muscle mitochondrial biogenesis proteins in non-insulin-dependent type 2 diabetic men.

This study investigates whether regular physical activity (moderate endurance or resistance training twice a week for 3 months) influences the key regulatory molecules of mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α), nuclear respiratory factor-1 (NRF1), and mitochondrial transcription factor A (TFAM)) in patients suffering from non-insulin-dependent type 2 diabetes mellitus (T2DM) (n = 16, years = 62 ± 7, body mass index (BMI) = 30 ± 4 kg/m(2)). Seven T2DM men took part in endurance training, and 9 men participated in resistance training. BMI-matched non-diabetic male control subjects (CON) (n = 7, years = 53 ± 6, BMI = 30 ± 4 kg/m(2)) were studied for comparison. The protein contents of PGC1α, NRF1, and TFAM were determined using immunohistochemical staining methods on biopsies taken from the musculus vastus lateralis. At baseline, no differences were observed in NRF1-density between the T2DM men and the CON, while the contents of PGC1α and TFAM were decreased in the T2DM men. PGC1α and TFAM contents were not changed in the T2DM patients after the training period, but NRF1 was decreased. The down-regulation of mitochondrial signaling molecules might explain the patho-physiological reduction in mitochondrial biogenesis found in T2DM. Physical training, as performed in our study, did not reverse the down-regulation of mitochondrial signaling molecules--at least not after 3 months. [corrected].

Microcirculation of skeletal muscle adapts differently to a resistive exercise intervention with and without superimposed whole-body vibrations

Whole‐body vibration (WBV) training is commonly practiced and may enhance peripheral blood flow. Here, we investigated muscle morphology and acute microcirculatory responses before and after a 6‐week resistive exercise training intervention without (RE) or with (RVE) simultaneous whole‐body vibrations (20 Hz, 6 mm peak‐to‐peak amplitude) in 26 healthy men in a randomized, controlled parallel‐design study. Total haemoglobin (tHb) and tissue oxygenation index (TOI) were measured in gastrocnemius muscle (GM) with near‐infrared spectroscopy (NIRS). Whole‐body oxygen consumption (VO2) was measured via spirometry, and skeletal muscle morphology was determined in soleus (SOL) muscle biopsies. Our data reveal that exercise‐induced muscle deoxygenation both before and after 6 weeks training was similar in RE and RVE (P = 0·76), although VO2 was 20% higher in the RVE group (P<0·001). The RVE group showed a 14%‐point increase in reactive hyperaemia (P = 0·007) and a 27% increase in blood volume (P<0·01) in GM after 6 weeks of training. The number of capillaries around fibres was increased by 15% after 6 weeks training in both groups (P<0·001) with no specific effect of superimposed WBV (P = 0·61). Neither of the training regimens induced fibre hypertrophy in SOL. The present findings suggest an increased blood volume and vasodilator response in GM as an adaptation to long‐term RVE, which was not observed after RE alone. We conclude that RVE training enhances vasodilation of small arterioles and possibly capillaries. This effect might be advantageous for muscle thermoregulation and the delivery of oxygen and nutrients to exercising muscle and removal of carbon dioxide and metabolites.

Intense Resistance Exercise Promotes the Acute and Transient Nuclear Translocation of Small Ubiquitin-Related Modifier (SUMO)-1 in Human Myofibres

Protein sumoylation is a posttranslational modification triggered by cellular stress. Because general information concerning the role of small ubiquitin-related modifier (SUMO) proteins in adult skeletal muscle is sparse, we investigated whether SUMO-1 proteins will be subjected to time-dependent changes in their subcellular localization in sarcoplasmic and nuclear compartments of human type I and II skeletal muscle fibers in response to acute stimulation by resistance exercise (RE). Skeletal muscle biopsies were taken at baseline (PRE), 15, 30, 60, 240 min and 24 h post RE from 6 male subjects subjected to a single bout of one-legged knee extensions. SUMO-1 localization was determined via immunohistochemistry and confocal laser microscopy. At baseline SUMO-1 was localized in perinuclear regions of myonuclei. Within 15 and up to 60 min post exercise, nuclear SUMO-1 localization was significantly increased (p < 0.01), declining towards baseline levels within 240 min post exercise. Sarcoplasmic SUMO-1 localization was increased at 15 min post exercise in type I and up to 30 min post RE in type II myofibres. The changing localization of SUMO-1 proteins acutely after intense muscle contractions points to a role for SUMO proteins in the acute regulation of the skeletal muscle proteome after exercise.