We explored the involvement of Oxytocin receptor (Oxtr)/ Transient-receptor-potential-vanilloid-1 (TRPV1) genes and Oxytocin (Oxt) on the adaptation of skeletal muscle to cold stress challenge in mice. Oxtr expression in hypothalamic paraventricular (PVN), supraoptic nuclei (SON), and hippocampus (HIPP) were evaluated by immunohistochemistry in parallel with the measurement of circulating Oxt. The Oxtr and TRPV1 gene expression in Soleus (SOL) and Tibialis Anterior (TA) muscles were investigated by RT-PCR. Histological studies of the cardiac muscle after cold stress were also performed. Male mice (n=15) were divided into controls maintained at room temperature (RT=24°C), exposed to cold stress (CS) at T=4°C for 6 hours (6h), and 5 days (5d). Immunohistochemical studies showed that Oxtr protein expression increased by 2-fold (p=0.01) in PVN and by 1.5-fold (p=0.0001) in HIPP after 6h and 5d CS, but decreased by 2-fold (p=0.026) in SON at 5d. Both Oxtr and TRPV1 gene expression increased after 6h and 5d CS in SOL and TA muscles. Oxtr vs TRPV1 gene expression in SOL and TA muscles evaluated by regression analysis was linearly correlated following CS at 6h and 5d but not at control temperature of 24+1°C, supporting the hypothesis of coupling between these genes. The circulating levels of Oxt are unaffected after 6h CS but decreased by 0.2-fold (p=0.0141) after 5d CS. This is the first report that Oxtr and TRPV1 expression are upregulated in response to cold acclimation in skeletal muscle. The up-regulation of Oxtr in PVN and HIPP balances the decrease of circulating Oxt.

Cantù syndrome (CS) arises from mutations in ABCC9 and KCNJ8 genes that lead to gain of function (GOF) of ATP-sensitive potassium (KATP) channels containing SUR2A and Kir6.1 subunits, respectively, of KATP channels. Pathological consequences of CS have been reported for cardiac and smooth muscle cells but consequences in skeletal muscle are unknown. Children with CS show muscle hypotonia and adult manifest fatigability. We analyzed muscle properties of Kir6.1[V65M] CS mice, by measurements of forelimb strength and ultrasonography of hind-limb muscles, as well as assessing KATP channel properties in native Flexor digitorum brevis (FDB) and Soleus (SOL) fibers by the patch-clamp technique in parallel with histopathological, immunohistochemical and Polymerase Chain Reaction (PCR) analysis. Forelimb strength was lower in Kir6.1wt/VM mice than in WT mice. Also, a significant enhancement of echodensity was observed in hind-limb muscles of Kir6.1wt/VM mice relative to WT, suggesting the presence of fibrous tissue. There was a higher KATP channel current amplitude in Kir6.1wt/VM FDB fibers relative to WT and a reduced response to glibenclamide. The IC50 of glibenclamide to block KATP channels in FDB fibers was 1.3 ± 0.2 × 10−7 M in WT and 1.2 ± 0.1 × 10−6 M in Kir6.1wt/VM mice, respectively; and it was 1.2 ± 0.4 × 10−7 M in SOL WT fibers but not measurable in Kir6.1wt/VM fibers. The sensitivity of the KATP channel to MgATP was not modified in Kir6.1wt/VM fibers. Histopathological/immunohistochemical analysis of SOL revealed degeneration plus regressive-necrotic lesions with regeneration, and up-regulation of Atrogin-1, MuRF1, and BNIP3 mRNA/proteins in Kir6.1wt/VM mice. Kir6.1wt/VM mutation in skeletal muscle leads to changes of the KATP channel response to glibenclamide in FDB and SOL fibers, and it is associated with histopathological and gene expression changes in slow-twitch muscle, suggesting marked atrophy and autophagy.

Bisphosphonates (BPs) reduce bone pain and fractures by balancing the osteoblast/osteoclast ratio. The behavior of ion channels in the presence of BPs is not known. To investigate this, the effect of zoledronic acid BP (ZOL) (3 × 10−8 to 5 × 10−4 M) treatment, on ion channels, cell proliferation, and mineralization, has been investigated on preosteoclast-like cells, RAW264.7, preosteoblast-like cells MC3T3-E1, and rat/mouse native bone marrow-derived osteoblasts. In whole-cell patch clamp on cell line- and bone marrow-derived osteoblasts, ZOL potentiated outward currents. On RAW264.7, ZOL (10−4 M)-evoked current was reduced by the Kv channel blocker tetraethylammonium hydrochloride (TEA), but not by the selective TRPV1-channel antagonist capsazepine. On MC3T3-E1 cells and bone marrow-derived osteoblasts, ZOL-evoked current (5 × 10−8 to 10−4 M) was reduced by capsazepine, whereas the selective TRPV1-channel agonist capsaicin potentiated the control current. In the cell proliferation assay, 72 h incubation of RAW264.7 and MC3T3-E1 cells with ZOL reduced proliferation, with IC50 values of 2.62 × 10−7 M and 2.02 × 10−5 M, respectively. Mineralization of MC3T3-E1 cells and bone marrow-derived osteoblasts was observed in the presence of capsaicin and ZOL (5 × 10−8–10−7 M); ZOL effects were antagonized by capsazepine. In summary, the ZOL-induced activation of TRPV1 channel mediates the mineralization of osteoblasts and counterbalances the antiproliferative effects, increasing the IC50. This mechanism is not operative in osteoclasts lacking the TRPV1 channel.

Zoledronic acid (ZOL) is used as a bone-specific antiresorptive drug with antimyeloma effects. Adverse drug reactions (A.D.R.) are associated with ZOL-therapy, whose mechanics are unknown. ZOL is a nitrogen-containing molecule whose structure shows similarities with nucleotides, ligands of ATP-sensitive K+ (KATP) channels. We investigated the action of ZOL by performing in vitro patch-clamp experiments on native KATP channels in murine skeletal muscle fibers, bone cells, and recombinant subunits in cell lines, and by in silico docking the nucleotide site on KIR and SUR, as well as the glibenclamide site. ZOL fully inhibited the KATP currents recorded in excised macro-patches from Extensor digitorum longus (EDL) and Soleus (SOL) muscle fibers with an IC50 of 1.2 ± 1.4 × 10−6 and 2.1 ± 3.7 × 10−10 M, respectively, and the KATP currents recorded in cell-attached patches from primary long bone cells with an IC50 of 1.6 ± 2.8 × 10−10 M. ZOL fully inhibited a whole-cell KATP channel current of recombinant KIR6.1-SUR2B and KIR6.2-SUR2A subunits expressed in HEK293 cells with an IC50 of 3.9 ± 2.7 × 10−10 M and 7.1 ± 3.1 × 10−6 M, respectively. The rank order of potency in inhibiting the KATP currents was: KIR6.1-SUR2B/SOL-KATP/osteoblast-KATP > KIR6.2-SUR2A/EDL-KATP >>> KIR6.2-SUR1 and KIR6.1-SUR1. Docking investigation revealed that the drug binds to the ADP/ATP sites on KIR6.1/2 and SUR2A/B and on the sulfonylureas site showing low binding energy <6 Kcal/mol for the KIR6.1/2-SUR2 subunits vs. the <4 Kcal/mol for the KIR6.2-SUR1. The IC50 of ZOL to inhibit the KIR6.1/2-SUR2A/B channels were correlated with its musculoskeletal and cardiovascular risks. We first showed that ZOL blocks at subnanomolar concentration musculoskeletal KATP channels and cardiac and vascular KIR6.2/1-SUR2 channels.

The large-conductance Ca-activated K (BK) channel is broadly expressed in various mammalian cells and tissues such as neurons, skeletal muscles (sarco-BK), and smooth muscles. These channels are activated by changes in membrane electrical potential and by increases in the concentration of intracellular calcium ion (Ca). The BK channel is subjected to many mechanisms that add diversity to the BK channel α-subunit gene. These channels are indeed subject to alternative splicing, auxiliary subunits modulation, posttranslational modifications, and protein-protein interactions. BK channels can be modulated by diverse molecules that may induce either an increase or decrease in channel activity. The linkage of these channels to many intracellular metabolites and pathways, as well as their modulation by extracellular natural agents, have been found to be relevant in many physiological processes. BK channel diversity is obtained by means of alternative splicing and modulatory β- and γ-subunits. The association of the α-subunit with β- or with γ-subunits can change the BK channel phenotype, functional diversity, and pharmacological properties in different tissues. In the case of the skeletal muscle BK channel (sarco-BK channel), we established that the main mechanism regulating BK channel diversity is the alternative splicing of the gene encoding for the α-subunit generating different splicing isoform in the muscle phenotypes. This finding helps to design molecules selectively targeting the skeletal muscle subtypes. The use of drugs selectively targeting the skeletal muscle BK channels is a promising strategy in the treatment of familial disorders affecting muscular skeletal apparatus including hyperkalemia and hypokalemia periodic paralysis.