Calcium homeostasis workgroup

Group leader: Prof. Dr. László Csernoch

Post docs: Beatrix Dienes, Nóra Dobrosi, János Fodor, Mónika Gönczi, Péter Szentesi, Mónika Sztretye, Andrea Telek

Ph.D. students: Ágnes Angyal, Bernadett Bákány, Norbert Balogh, Zsolt Ráduly, Zoltán Singlár, László Szabó

Technicians: Tamara Lővei, Róza Őri, Anita Szabóné Jeney

Selected ongoing projects

Selenium supplementation improves skeletal muscle performance

As an essential trace element selenium plays an important role in many physiological functions of the organs. It is found within muscles as selenocystein in selenoprotein N, which is involved in redox-modulated calcium homeostasis and in protection against oxidative stress. We tested the effects of two different selenium compounds (selenate and NanoSe) on muscle properties of mice. Selenium diets significantly increased the speed of voluntary running and the daily distance covered. Both forms of selenium increased significantly the amplitude of single twitches in EDL and SOL muscle. The amplitude of the calcium transients evoked by KCl depolarization increased significantly in the presence of selenate in FDB fibers. In parallel, the rate of calcium release during short depolarizations increased significantly in the presence of NanoSe and selenate. Both selenium compounds increased significantly the selenoprotein N expression in EDL muscle (Figure 1). Thus we concluded, that selenium supplementation augments calcium release from the sarcoplasmic reticulum and improves skeletal muscle performance. The increased selenoprotein N expression in the muscles could result in increased oxidative stress tolerance in case of long lasting contraction.

Figure 1. Representative Western blot image showing the expression of selenoprotein N (SelN) in EDL and SOL muscles from a control mouse and a mouse fed with 0.5 ppm NanoSe or selenate for two weeks

Hypermuscular mice display impared EC coupling

Myostatin, a member of the transforming growth factor β superfamily has emerged as a potent negative regulator of skeletal muscle growth. It is strongly expressed in skeletal muscle and Cmpt mice have a great increase in muscle mass (Figure 2) demonstrating that myostatin is a muscle-specific negative regulator of skeletal muscle size and it also regulates muscle mass in adult mice.

Figure 2. A control C57/BL6 (left) and a Cmpt (right) mouse.

Cmpt mice display excessive muscle mass and this is associated with a profound loss of oxidative metabolic properties. In grip tests the myostatin-deficient mice showed higher average absolute force than the control mice. In voluntary wheel running the control mice performed better showing higher average and maximal speed, and total running distance. The amplitude of single twitches in EDL and SOL is higher in the control strain. The Ca²⁺-sensitivity of force production was not significantly difference between the two mouse strains. While resting intracellular Ca2+ concentration measured on single intact flexor digitorum brevis (FDB) muscle fibres was identical to control, the amplitude of depolarization-evoked calcium transients was smaller in the mutant strain (Figure 3). SR calcium release flux, calculated from these transients showed a reduced peak and steady level with no change in the peak-to-steady ratio. The amplitude and spatial spread of spontaneous elementary calcium release events detected on permeabilized FDB fibres were also significantly smaller in mutant mice. These results indicate that alterations in calcium signaling may underlie the reduced muscle performance in Cmpt animals.

Figure 3. Calcium transients were elicited by a supra-threshold pulse in intact FDB fibres from a control (A) and Cmpt (B) mouse.

SOCE is important during normal EC coupling

We are interested in the characterization of store operated calcium entry (SOCE) in the Cmpt mouse model. Our aim is to understand the role of SOCE in refilling SR Ca²⁺ stores in skeletal muscle which is currently fairly controversial. We recently demonstrated that the naturally occurring Cmpt mutation leads to reduced Ca²⁺ store content (Figure 4) that could be responsible for the reduced specific force. The most likely explanation for how the mutation leads to this reduction is the reduced expression of the SOCE partner proteins and the concomitant lower SOCE activity. We propose that SOCE has a role in maintaining and refilling SR Ca²⁺ stores not only in repetitive tetanic stimulation, as it was previously reported, but on an immediate basis in agreement with latest observations.

Figure 4. Representative xy images showing the response to various solution exchanges and the consequent SOCE activation in enzymatically isolated FDB fibers of C57/BL6 wild type (A) and Cmpt (C) mouse. The averaged profile of fluo-8 fluorescence over the fibers was used to assess the cytoplasmic [Ca²⁺]. Intracellular Ca²⁺ stores were emptied using a depleting cocktail in a Ca²⁺-free medium resulting in a massive Ca²⁺ -release from the SR via RyRs. (B, D) During manual solution exchanges the fluorescence profile vs. elapsed time was plotted. Each confocal image from panel A and C is depicted by the cyan squares. When returning to the 1.8 mM Ca²⁺ in the external solution, a secondary increase in fluorescence was detected, indicating perhaps SOCE activation and Ca²⁺-influx via activated Orai1 channels in the sarcolemma. (E) The ratio distribution between the peak of the slow Ca²⁺ transient highlighting the activation of SOCE (P2) and the depleting cocktail induced Ca²⁺ transient´s peak (P1). 1 µM nifedipine (L-type Ca²⁺ channel blocker) the Ca²⁺ influx was abolished in the mutant suggesting some contribution via the DHPR, a process also known as excitation-coupled calcium entry (ECCE). The number of cells examined is given in parenthesis. For statistical analysis, the paired T-test was used, and p ≤0.05 was considered significant. (F) Representative Western blot illustrating the relative expression levels of STIM1 and Orai1, identified as the key proteins involved in SOCE. Note: the presence of two distinct STIM1 proteins: the well-known 90 kDa STIM1S (S denoting small) and the newly identified, widely expressed 115 kDa STIM1L isoform (L for long). 20 µg of whole FDB muscle homogenate was loaded into each lane and immunoreactivity with actin was used as an internal control. (G) In 4 independent experiments the STIM1 and Orai1 endogenous proteins level distribution was assessed as a percentage of control. On average, both STIM1 and Orai1 were decreased by 40 % and 27% respectively, in the Cmpt muscles.

Selected references:

Bodnár D., Geyer N., Ruzsnavszky O., Oláh T., Hegyi B., Sztretye M., Fodor J., Dienes B., Balogh Á., Papp Z., Szabó L., Müller G., Csernoch L., Szentesi P. (2014) Hypermuscular mice with mutation in the myostatin gene display altered calcium signaling. Journal of Physiology, 592: 1353-1365. doi: 10.1113/jphysiol.2013.261958.

Bodnár D., Ruzsnavszky O., Oláh T., Dienes B., Balatoni I., Ungvári É., Benkő I.., Babka B., Prokisch J., Csernoch L., Szentesi P. (2016) Dietary selenium augments sarcoplasmic calcium release and mechanical performance in mice. Nutrition and Metabolism, 13:76. doi: 10.1186/s12986-016-0134-6

Sultana N., Dienes B., Benedetti A., Tuluc P., Szentesi P., Sztretye M., Rainer J., Hess M.W., Schwarzer C., Obermair G.J., Csernoch L., Flucher B.E. (2016) Restricting calcium currents is required for correct fiber type specification in skeletal muscle. Development, 143: 1547-1559. doi: 10.1242/dev.129676.

Sztretye M., Geyer N., Vincze J., Al-Gaadi D., Oláh T., Szentesi P., Kis G., Balatoni I., Csernoch L., Dienes B. (2017) Store-operated calcium entry is important for maintaining sarcoplasmic calcium content and release in mammalian skeletal muscle fibers. Biophysical Journal, 113:2496-2507. doi: 10.1016/j.bpj.2017.09.023.

Pierantozzi E., Szentesi P., Al-Gaadi D., Oláh T., Dienes B., Sztretye M., Rossi D., Sorrentino V., Csernoch L. (2019) Calcium homeostasis is modified in skeletal muscle fibers of small Ankyrin1 knockout mice. International Journal of Molecular Sciences, 20(13):pii: E3361. doi: 10.3390/ijms20133361.

Fodor J., Al-Gaadi D., Czirják T., Oláh T., Dienes B., Csernoch L., Szentesi P. (2020) Improved calcium homeostasis and force by selenium treatment and training in aged mouse skeletal muscle. Scientific Reports, 10(1):1707. doi: 10.1038/s41598-020-58500-x.

Collaborations:

Claude Collet, National Institute for Agricultural Research, Avignon, France

Laszlo Dux, University of Szeged, Hungary

Bernhard Flucher, University of Innsbruck, Austria

Vincent Jacquemond, University of Lyon, France

Vincenzo Sorrentino, University of Siena, Italy

Available techniques:

Intracellular ion concentration measurement with fluorescence dyes on confocal microscope

Electrophysiological measurement on isolated single skeletal muscle fibers

In vivo muscle grip force, voluntary and forced treadmill running on mice

In vitro muscle force measurement on isolated skeletal muscle

Immunohistochemistry measurement on tissues and cells

Molecular biology techniques