Growth hormone secretagogues hexarelin and JMV2894 protect skeletal muscle from mitochondrial damages in a rat model of cisplatin-induced cachexia

Abstract

Chemotherapy can cause cachexia, which consists of weight loss associated with muscle atrophy. The exact mechanisms underlying this skeletal muscle toxicity are largely unknown and co-therapies to attenuate chemotherapy-induced side effects are lacking. By using a rat model of cisplatin-induced cachexia, we here characterized the mitochondrial homeostasis in tibialis anterior cachectic muscle and evaluated the potential beneficial effects of the growth hormone secretagogues (GHS) hexarelin and JMV2894 in this setting. We found that cisplatin treatment caused a decrease in mitochondrial biogenesis (PGC-1α, NRF-1, TFAM, mtDNA, ND1), mitochondrial mass (Porin and Citrate synthase activity) and fusion index (MFN2, Drp1), together with changes in the expression of autophagy-related genes (AKT/FoxO pathway, Atg1, Beclin1, LC3AII, p62) and enhanced ROS production (PRX III, MnSOD). Importantly, JMV2894 and hexarelin are capable to antagonize this chemotherapy-induced mitochondrial dysfunction. Thus, our findings reveal a key-role played by mitochondria in the mechanism responsible for GHS beneficial effects in skeletal muscle, strongly indicating that targeting mitochondrial dysfunction might be a promising area of research in developing therapeutic strategies to prevent or limit muscle wasting in cachexia.

Figure 1

Effect of cisplatin and GHS treatments on the level of factors regulating the mitochondrial biogenesis. (A) The histogram shows the relative content of PGC-1α protein normalized with respect to β-actin content. Cisplatin treatment decreases the level of the protein; the administration of hexarelin and, to a lesser extent of JMV, tends to restore the control value. (B) The histogram shows the relative content of NRF-1 protein normalized with respect to β-actin content. Cisplatin treatment decreases the level of the protein; the administration of hexarelin and JMV tends to restore the control value. (C) The histogram shows the relative content of TFAM normalized with respect to β-actin. Cisplatin treatment lowers the level of TFAM; the administration of the two segretagogues brings the protein level near to that of controls. The reported results are the average (±SEM) of experiments performed in quadruplicate. CTR, controls; CIS, rats treated with cisplatin; HEX, rats treated with cisplatin and hexarelin; JMV, rats treated with cisplatin and JMV2894. All data are normalized to control samples set as 1. n, number of animals of each group. *p < 0.05; **p < 0.01. A representative western blot is also shown. White spaces between blots indicate not adjacent lanes deriving from different parts of the same gel. The whole blot is reported in Supplementary Figure S2. The position of molecular weight markers is reported at the right of each blot.

Figure 2

Changes of mitochondrial biogenesis and mitochondrial mass following cisplatin and GHS treatments. (A) MtDNA copy number. The mtDNA copy number decreases in the rats treated with cisplatin, while the administration of hexarelin and JMV brings the level to that of control samples. (B) Content of MHC1 and MHC2A mRNAs. The histograms show the relative content of the two mRNAs normalized with respect to β-actin mRNA. No significant changes following cisplatin and GHS treatments were observed. (C) ND1 content. The histogram shows the relative content of ND1 normalized with respect to β-actin. Cisplatin treatment causes a decrease of ND1 while the administration of hexarelin and at a higher extent of JMV2894 prevents such decrease. (D) Porin content. The histogram shows the relative content of Porin normalized with respect to β-actin. Cisplatin treatment causes a decrease of Porin and that the administration of hexarelin and at a higher extent of JMV2894 prevents such decrease. (E) Citrate Synthase activity. The histogram shows that cisplatin treatment causes a decrease of citrate synthase and that the administration of hexarelin and JMV prevents such decrease. The reported results are the average (±SEM) of experiments performed in quadruplicate. CTR, controls; CIS, rats treated with cisplatin; HEX, rats treated with cisplatin and hexarelin; JMV, rats treated with cisplatin and JMV2894. All data are normalized to control samples. n, number of animals of each group. *p < 0.05; **p < 0.01). For the proteins a representative western blot is also shown. White spaces between blots indicate not adjacent lanes deriving from different parts of the same gel. The whole blot is reported in Supplementary Figure S2. The position of molecular weight markers is reported at the right of each blot.

Figure 3

Mitochondrial oxidative stress in cisplatin-treated rats and after GHS administration. (A) The relative content of overoxidized PRXs (PRX-SO₃) is higher in the cisplatin-treated rats; hexarelin and JMV treatment prevent such increase. (B) The relative content of MnSOD is lower in the cisplatin-treated rats; the two segretagogues prevent the decrease. (C) The relative content of PRX III is lower in the cisplatin-treated rats; hexarelin administration prevents the decrease while JMV does not. The reported results are the average ( ± SEM) of experiments performed in quadruplicate. All data are normalized to control samples. n, number of animals of each group. *p < 0.05; **p < 0.01. CTR, controls; CIS, rats treated with cisplatin; HEX, rats treated with cisplatin and hexarelin; JMV, rats treated with cisplatin and JMV2894. A representative western blot is also shown. White spaces between blots indicate not adjacent lanes deriving from different parts of the same gel. The whole blot is reported in Supplementary Figure S2. The position of molecular weight markers is reported at the right of each blot.

Figure 4

Mitochondrial dynamics in cisplatin-treated rats and after GHS administration. (A) The relative content of MFN2 increases in cisplatin-treated rats; treatment with hexarelin, but not with JMV2894, prevented such increase. (B top part) Drp1 increases in cisplatin-treated rats; the administration of the two GHS tends to lower such increase. (B bottom part) Cisplatin induced a decrease of the phosphorylation of Drp1 at S637 residue, whereas GHS prevent such changes. (C) The ratio between MFN2 and Drp1 (Fusion Index) decreases in cisplatin-treated rats. Hexarelin and more effectively JMV2894 administration tend to bring back the value of the ratio to that of control. Results are the average (mean ± SEM) of experiments performed in quadruplicate. All data have been normalized to control samples. n, number of animals of each group. *p < 0.05; **p < 0.01. A representative western blot is shown (CTR, controls; CIS, rats treated with cisplatin; HEX, rats treated with cisplatin and hexarelin; JMV, rats treated with cisplatin and JMV2894). White spaces between blots indicate not adjacent lanes deriving from different parts of the same gel. The whole blot is reported in Supplementary Figure S2. The position of molecular weight markers is reported at the right of each blot.

Figure 5

Phosphorylation level of AKT and FoxO3a in cisplatin-treated rats and after GHS administration. (A) The AKT phosphorylation level decreases in cisplatin-treated rats and increases after the administration of the two segretagogues. (B) FoxO3a phosphorylation decreases in cisplatin-treated rats and increases after the administration of the two segretagogues. Results are the average (mean ± SEM) of experiments performed in quadruplicate. All data have been normalized to control samples. n, number of animals of each group. *p < 0.05; **p < 0.01. CTR, controls; CIS, rats treated with cisplatin; HEX, rats treated with cisplatin and hexarelin; JMV, rats treated with cisplatin and JMV2894. A representative western blot is also shown. The whole blot is reported in Supplementary Figure S2. The position of molecular weight markers is reported at the right of each blot.

Figure 6

Changes in the level of autophagy marker proteins in cisplatin-trated rats and after GHS administration. (A) Atg1 increases in the cisplatin-treated rats. The administration of hexarelin or JMV2894 prevents such a change. (B) Beclin1 content increases in cisplatin groups; hexarelin and JMV2894 treatments bring back the value to that of control samples. (C) LC3AII slightly increases in the cisplatin-treated rats. JMV2894 treatment prevents such a change. (D) p62 increases in the cisplatin-treated rats. The administration of hexarelin or JMV2894 prevents such a change leading to a value lower than control. Results are the average (mean ± SEM) of experiments performed in quadruplicate. All data have been normalized to control samples. n, number of animals of each group. *p < 0.05; **p < 0.01. CTR, controls; CIS, rats treated with cisplatin; HEX, rats treated with cisplatin and hexarelin; JMV, rats treated with cisplatin and JMV2894. A representative western blot is also shown. White spaces between blots indicate not adjacent lanes deriving from different parts of the same gel. The whole blot is reported in Supplementary Figure S2. The position of molecular weight markers is reported at the right of each blot.