Although the importance of mitochondria in patho-physiology has become increasingly evident, it remains unclear whether these organelles play a role in Ca²⁺ handling by skeletal muscle. This undefined situation is mainly due to technical limitations in measuring Ca²⁺ transients reliably during the contraction–relaxation cycle. Using two-photon microscopy and genetically expressed “cameleon” Ca²⁺ sensors, we developed a robust system that enables the measurement of both cytoplasmic and mitochondrial Ca²⁺ transients in vivo. We show here for the first time that, in vivo and under highly physiological conditions, mitochondria in mammalian skeletal muscle take up Ca²⁺ during contraction induced by motor nerve stimulation and rapidly release it during relaxation. The mitochondrial Ca²⁺ increase is delayed by a few milliseconds compared with the cytosolic Ca²⁺ rise and occurs both during a single twitch and upon tetanic contraction.

Significance: The sympathetic nervous system regulates basic body functions such as heartbeat, blood pressure, and gland activities. Whereas hormone secretion from the adrenal medulla modulates these processes systemically, local and fast responses can be mediated by direct sympathetic innervation. Although many effects of the sympathetic system on skeletal muscle physiology and disease are known, direct sympathetic innervation targets in skeletal muscle have been scarcely studied. We investigated this aspect and found that neuromuscular junctions, the contact points between motor neurons and muscle fibers, are innervated by sympathetic neurons, which is of crucial importance for the integrity and function of nerve–muscle contact. Our findings help to understand and refine treatment of neuromuscular diseases, including myasthenic syndromes.

Neuroendocrine secretory granules, the storage organelles for neuropeptides and hormones, are formed at the trans-Golgi network, stored inside the cell and exocytosed upon stimulation. Previously, we have reported that newly formed secretory granules of PC12 cells are transported in a microtubule-dependent manner from the trans-Golgi network to the F-actin-rich cell cortex, where they undergo short directed movements and exhibit a homogeneous distribution. Here we provide morphological and biochemical evidence that myosin Va is associated with secretory granules. Expression of a dominant-negative tail domain of myosin Va in PC12 cells led to an extensive clustering of secretory granules close to the cell periphery, a loss of their cortical restriction and a strong reduction in their motility in the actin cortex. Based on this data we propose a model that implies a dual transport system for secretory granules: after microtubule-dependent delivery to the cell periphery, secretory granules exhibit a myosin Va-dependent transport leading to their restriction and even dispersal in the F-actin-rich cortex of PC12 cells.

Skeletal muscle contraction depends on the release of Ca2+ from the sarcoplasmic reticulum (SR), but the dynamics of the SR free Ca2+ concentration ([Ca2+]SR), its modulation by physiological stimuli such as catecholamines, and the concomitant changes in cAMP handling have never been directly determined. We used two-photon microscopy imaging of GFP-based probes expressed in mouse skeletal muscles to monitor, for the first time in a live animal, the dynamics of [Ca2+]SR and cAMP. Our data, which were obtained in highly physiological conditions, suggest that free [Ca2+]SR decreases by ∼50 μM during single twitches elicited through nerve stimulation. We also demonstrate that cAMP levels rise upon β-adrenergic stimulation, leading to an increased efficacy of the Ca2+ release/reuptake cycle during motor nerve stimulation.

Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles cont...