Mitochondrial adaptation in parvalbumin knockout muscle fibres

Mitochondrial Ca2+ homeostasis plays a fundamental role in the regulation of several biological processes, ranging from the regulation of ATP production to the control of cell death. Recent studies have identified the multimolecular complex responsible for Ca2+ entry into mitochondria: the mitochondrial calcium uniporter (MCU) complex [1]. It is widely accepted that mitochondria actively participate in the regulation of cellular Ca2+ homeostasis by dictating the spatio-temporal properties of [Ca2+]cyt rises [2]. Mitochondrial calcium uptake, in specific cells, contributes to regulate cellular Ca2+ homeostasis acting as high-capacity fixed buffer, sequestering large amounts of Ca2+ from a subcellular domain[2]. Furthermore, one of the most important roles of mitochondrial Ca2+ uptake is the mitochondrial Ca2+-dependent control of the rate of mitochondrial adenosine triphosphate (ATP) production, the main fuel for sustaining cellular functions [3], [4]. This general picture is particularly relevant in skeletal muscle, a tissue where mitochondria produce most of the ATP required to sustain muscle contraction [3], [5]. It is thus not surprising that skeletal muscle mitochondria display the highest mitochondrial Ca2+ transients, as demonstrated by the measurement of the MCU current by patch-clamp from IMM-derived mitoplasts from different tissues [6]. Moreover, pivotal findings have highlighted the role of mitochondria as key players in the dynamic regulation of crucial signalling pathways in skeletal muscle [7], [8], involved not only in muscle contraction but also in skeletal muscle homeostasis [9], [10]. However, whether skeletal muscle mitochondria act also as a possible high capacity Ca2+ buffer remains a fundamental question on muscle physiology and diseases. Our research investigated the regulatory processes that modulate mitochondrial Ca2+ signalling in skeletal muscle. In detail, to understand the impact of changes in cytosolic Ca2+ concentrations ([Ca2+]cyt) on mitochondrial Ca2+ uptake and muscle physiology, we explored a condition where intra-fiber Ca2+ kinetics have been profoundly altered by removing parvalbumin (PV), one of the crucial cytosolic Ca2+ buffers in skeletal muscle, specifically expressed in fast twitch muscle fibers [11], [12]. To this end, as study tool, we used a PV knockout (KO) mouse model obtained from the laboratory of Prof. Beat Schwaller (Dept. of Medicine, University of Fribourg, Switzerland) [13]. PV plays an important role in skeletal muscle, acting as a temporary Ca2+ buffer (e.g. increasing the relaxation rate of fast twitch muscle contraction) [14]. To investigate the physiological role of PV in muscle fibers and in Ca2+ homeostasis, we investigated cytosolic and mitochondrial Ca2+ transients in PV KO mice compared. We observed that basal [Ca2+]cyt was not affected in PV knockout fibers, but kinetics of Ca2+ transients and Ca2+ clearance were altered. In detail, consistently with the role of PV in buffering cytosolic Ca2+, the time-to-peak and the half-relaxation time was increased in PV KO FDB fibers. Unexpectedly, however, under tetanic stimulation, PV KO FDB muscle fibers showed a decrease in [Ca2+]cyt. To explain this result we asked whether the lack of PV could induce rearrangements of one of the two main Ca2+ stores, the sarcoplasmic reticulum (SR) and mitochondria. SR Ca2+ measurements demonstrated that lack of PV increases SR Ca2+ release during stimulation. Therefore, we concluded that SR is not causative of the effect of PV removal on cytosolic Ca2+ transients. Consistently, we found no difference in the mRNA levels of RyR1, the main Ca2+ releasing channel in muscle, and on the expression of two different isoforms of SERCA in PV KO muscles compared to WT. We then focused our attention on mitochondrial Ca2+ homeostasis. The data obtained demonstrated that the lack of PV induces an increase of mitochondrial Ca2+ uptake and this is accompanied by the induction of the expression of MCU complex components, the channel responsible for Ca2+ entry in mitochondria [1], [2], [15], [16]. In addition, electron microscopy analysis demonstrated that the volume of PV KO mitochondria was doubled compared to WT with an increase of mitochondria associated to the Ca2+ release units (CRUs), suggesting a tight connection of PV expression with mitochondrial morphology and function in muscle cells. Furthermore, to further prove that mitochondria are responsible for cytosolic Ca2+ buffering in fibers lacking PV, we silenced MCU on WT and PV KO FDB fibers and we measured [Ca2+]cyt.. In WT animals, [Ca2+]cyt was not affected by the absence of MCU, while MCU silencing in PV KO fibers resulted in a significant higher [Ca2+]cyt, reinforcing the hypothesis that, while in WT animals mitochondria do not significantly buffer [Ca2+]cyt, mitochondria of fibers lacking PV adapt to buffer [Ca2+]cyt increases. Moreover, since PV is one of the most downregulated “atrogenes”, the genes commonly up- and down-regulated during both disuse and systemic types of atrophy [17], [18] and that mitochondrial Ca2+ controls skeletal muscle trophism [10], the role of PV in the regulation of muscle mass was investigated through denervation experiments. In PV KO muscles, loss of muscle mass caused by denervation is reduced compared to WT fibers, demonstrating that the lack of PV can partially protect muscles from denervation-induced atrophy. Since the effect of PV ablation on denervated muscle was modest and the effect on innervated muscles was negligible, we decided to perform PV acute silencing and overexpression in adult WT tibialis anterior (TA) muscles and we monitored fiber size. We demonstrated that the acute modulation of PV protein controls skeletal muscle size. In detail, we observed an increase of fiber size in PV silenced muscles and coherently, PV overexpressing muscles displayed an atrophic phenotype. Since the regulation of muscle size involves a precise transcriptional program [18], [19], we focused our attention on PGC-1α4, a splicing variant of the PGC-1α gene, that plays a key role in triggering muscle hypertrophy as adaptive response to exercise [20]. Intriguingly, we found an up-regulation of PGC-1α4 mRNA in PV KO skeletal muscles, suggesting the activation of this hypertrophic pathway. Of note, our data are in accordance with previous studies showing that mitochondrial Ca2+ positively regulates skeletal muscle mass by impinging also on PGC-1α4 pathway [10]. Our results show that the lack of PV in skeletal muscle leads to morphological and functional adaptations of mitochondria. In particular, mitochondria of fibers lacking PV, either constitutively or transiently, adapt to take up more Ca2+ to control [Ca2+]cyt increases. Furthermore, we demonstrated that the absence of PV partially counteracts denervation atrophy by triggering the expression of PGC-1α4. Our hypothesis is that PV ablation, leading to an increase of mitochondrial Ca2+ uptake, activates mitochondrial Ca2+-dependent pathways to control skeletal muscle trophism.