BackgroundCachexia is a wasting condition associated with cancer types and, at the same time, is a serious and dose‐limiting side effect of cancer chemotherapy. Skeletal muscle loss is one of the main characteristics of cachexia that significantly contributes to the functional muscle impairment. Calcium‐dependent signaling pathways are believed to play an important role in skeletal muscle decline observed in cachexia, but whether intracellular calcium homeostasis is affected in this situation remains uncertain. Growth hormone secretagogues (GHS), a family of synthetic agonists of ghrelin receptor (GHS‐R1a), are being developed as a therapeutic option for cancer cachexia syndrome; however, the exact mechanism by which GHS interfere with skeletal muscle is not fully understood.MethodsBy a multidisciplinary approach ranging from cytofluorometry and electrophysiology to gene expression and histology, we characterized the calcium homeostasis in fast‐twitch extensor digitorum longus (EDL) muscle of adult rats with cisplatin‐induced cachexia and established the potential beneficial effects of two GHS (hexarelin and JMV2894) at this level. Additionally, in vivo measures of grip strength and of ultrasonography recordings allowed us to evaluate the functional impact of GHS therapeutic intervention.ResultsCisplatin‐treated EDL muscle fibres were characterized by a ~18% significant reduction of the muscle weight and fibre diameter together with an up‐regulation of atrogin1/Murf‐1 genes and a down‐regulation of Pgc1‐a gene, all indexes of muscle atrophy, and by a two‐fold increase in resting intracellular calcium, [Ca2+]i, compared with control rats. Moreover, the amplitude of the calcium transient induced by caffeine or depolarizing high potassium solution as well as the store‐operated calcium entry were ~50% significantly reduced in cisplatin‐treated rats. Calcium homeostasis dysregulation parallels with changes of functional ex vivo (excitability and resting macroscopic conductance) and in vivo (forelimb force and muscle volume) outcomes in cachectic animals. Administration of hexarelin or JMV2894 markedly reduced the cisplatin‐induced alteration of calcium homeostasis by both common as well as drug‐specific mechanisms of action. This effect correlated with muscle function preservation as well as amelioration of various atrophic indexes, thus supporting the functional impact of GHS activity on calcium homeostasis.ConclusionsOur findings provide a direct evidence that a dysregulation of calcium homeostasis plays a key role in cisplatin‐induced model of cachexia gaining insight into the etiopathogenesis of this form of muscle wasting. Furthermore, our demonstration that GHS administration efficaciously prevents cisplatin‐induced calcium homeostasis alteration contributes to elucidate the mechanism of action through which GHS could potentially ameliorate chemotherapy‐associated cachexia.

1 Glaucoma pathophysiology appears to involve vascular deficits, which may contribute to initiation and progression of the disease. 2 Anandamide, the endogenous cannabinoid ligand, and WIN55212-2, a synthetic cannabinoid agonist, are able to evoke concentration-dependent relaxations in bovine ophthalmic artery rings, precontracted with 5-hydroxytryptamine (5-HT) (1 mM). Endothelium removal reduces cannabinoid agonist potency and efficacy. 3 The selective cannabinoid 1 (CB 1 ) receptor antagonists SR141716A (100 nM) and AM251 (100 nM) cause a shift to the right in the concentration-response curves to anandamide and WIN55212-2 in arterial rings both in the presence and in the absence of endothelium. 4 In endothelium-intact arteries, the nitric oxide synthase inhibitor, N G -monomethyl-L-arginine (L-NMMA, 300 mM), completely blocked the anandamide-and WIN55212-2-relaxant responses; by contrast, the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP, 100 mM) induced an increase in vasorelaxant responses to cannabinoid agonists. 5 Relaxations to anandamide and WIN55212-2 were inhibited by iberiotoxin (IbTX, 200 nM), a blocker of large conductance, Ca 2 þ -activated K þ channel (BK Ca ), and by 4-aminopyridine (4-AP; 1 mM), a blocker of delayed rectifier K þ channel, whereas the blockade of K ATP channels by glibenclamide (5 mM) and of small conductance Ca 2 þ -activated K þ channels (SK Ca ) by apamin (100 nM) did not produce any effects. 6 These data suggest that anandamide and WIN55212-2 relax the bovine ophthalmic artery by involving CB 1 the cannabinoid receptor-sensitive pathway. In endothelium-intact arteries, relaxation occurs through activation of nitric oxide synthase cyclic GMP and Ca 2 þ -activated K þ channels. They also cause endothelium-independent relaxation by involving potassium channel opening.

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The correlation between the Ngf/p75ntr-Ntrk1 and Bdnf, Osteocalcin-Ost/Gprc6a and Oxytocin-Oxt/Oxtr genes, was challenged investigating their mRNA levels in 3 months-old mice after cold-stress (CS). Uncoupling protein-1 (Ucp-1) was used as positive control. Control mice were maintained at room temperature T = 25 • C, CS mice were maintained at T = 4 • C for 6 h and 5-days (N = 15 mice). RT-PCR experiments showed that Ucp-1 and Ngf genes were up-regulated after 6 h CS in brown adipose tissues (BAT), respectively, by 2 and 1.5-folds; Ucp-1 was upregulated also after 5-days, while Ngfr (p75ntr) and Ntrk1 genes were downregulated after 6 h and 5-days CS in BAT. NGF and P75NTR were upregulated in bone and testis following 5-days, and P75NTR in testis after 6 h CS. Bdnf was instead up-regulated in bone following 5-days CS and down-regulated in testis. OST was upregulated by 16 and 3-fold in bone and BAT, respectively, following 5-days CS. Gprc6a was upregulated after 6 h in brain, while Bglap (Ost) gene was downregulated. Oxt gene was upregulated by 5-fold following 5-days CS in bone. Oxtr was upregulated by 0.5 and 0.3-fold, respectively, following 6 h and 5-days CS in brain. Oxtr and Oxt were downregulated in testis and in BAT. The changes in the expression levels of control genes vs. genes following 6 h and 5-days CS were correlated in all tissues, but not in BAT. Correlation in BAT was improved eliminating Ngfr (p75ntr) data. The correlation in brain was lost eliminating Oxtr data. In sum, Ucp-1 potentiation in BAT after cold stress is associated with early Ngf-response in the same tissue and trophic action in bone and testis. In contrast, BDNF exerts bone and neuroprotective effects. Similarly to Ucp-1, Bglap (Ost) signaling is enhanced in bone and BAT while it Camerino et al.Neurotrophins and Cold Stress may exert local neuroprotective effects thought its receptor. Ngfr (p75ntr) regulates the adaptation to CS through a feed-back loop in BAT. Oxtr regulates the gene-response to CS through a feed-forward loop in brain. Overall these results expand the understanding of the physiology of these molecules under metabolic thermogenesis.

Oxytocin (Oxt), osteocalcin (Ost), and NGF/BDNF have a role in bone homeostasis, reproduction, and cognition. Oxt/Ost is required for muscle repair. We investigated gene response of muscle and the inter-organ communication following cold stress (CS). The mRNA quantity of Ngf, Ost, Oxt, Bdnf, p75ntr, Ntrk1, Gprc6a, Oxtr, Ntrk2, UCP1, and Il-6 genes in bone, brain, soleus (SOL), and tibialis anterior (TA) muscles from adult mice following CS were investigated. The myosin heavy-chain Mhc2b, Mhc1, Mhc2x, and Mhc2a gene expression were investigated. Mice were maintained at T = 23°C or 4°C for 6 h and 5-days (5d). CS mice did not show signs of muscle degeneration. An upregulation of Ucp1 and Ngf genes by 2 and 1.5 folds, respectively, in TA after 6 h CS and Ntrk1 by 4 and 22 folds in SOL muscle after 6 h and 5d CS, respectively, was observed; while after 6 h CS p75Ntr was downregulated in either muscle. Bdnf was unaffected, while after 5d CS Ntrk2 was upregulated in TA. Ost was downregulated in SOL by 0.9-folds at 5d. Following 5d CS, Oxtr and Il-6 genes were upregulated, respectively, by 1 and 1.5 folds in SOL. A downregulation of Mhc2b, respectively, by 0.96 and 0.88-folds after 6 h and 5d CS in SOL and Mhc2a was also downregulated by 0.88-fold after 5d CS in TA. Mhc1 and Mhc2x were not affected. Changes in the expression levels of genes in TA and SOL muscles, bone, and brain following CS were regulated by IL6 and Oxt. CS potentiates the slow-twitch phenotype of SOL which is in line with the metabolic need of this muscle, and the potentiation of the slow-twitch phenotype in TA. Oxt and IL6 coordinate a phenotype-dependent tonic effect of slow-twitch muscle and Oxt regulates the inter-organ interaction between brain and SOL muscle. Muscle tropism is maintained by NGF signaling following CS.