Human Cachexia Induces Changes in Mitochondria, Autophagy and Apoptosis in the Skeletal Muscle

Abstract

Cachexia is a wasting syndrome characterized by the continuous loss of skeletal muscle mass due to imbalance between protein synthesis and degradation, which is related with poor prognosis and compromised quality of life. Dysfunctional mitochondria are associated with lower muscle strength and muscle atrophy in cancer patients, yet poorly described in human cachexia. We herein investigated mitochondrial morphology, autophagy and apoptosis in the skeletal muscle of patients with gastrointestinal cancer-associated cachexia (CC), as compared with a weight-stable cancer group (WSC). CC showed prominent weight loss and increased circulating levels of serum C-reactive protein, lower body mass index and decreased circulating hemoglobin, when compared to WSC. Electron microscopy analysis revealed an increase in intermyofibrillar mitochondrial area in CC, as compared to WSC. Relative gene expression of Fission 1, a protein related to mitochondrial fission, was increased in CC, as compared to WSC. LC3 II, autophagy-related (ATG) 5 and 7 essential proteins for autophagosome formation, presented higher content in the cachectic group. Protein levels of phosphorylated p53 (Ser46), activated caspase 8 (Asp384) and 9 (Asp315) were also increased in the skeletal muscle of CC. Overall, our results demonstrate that human cancer-associated cachexia leads to exacerbated muscle-stress response that may culminate in muscle loss, which is in part due to disruption of mitochondrial morphology, dysfunctional autophagy and increased apoptosis. To the best of our knowledge, this is the first report showing quantitative morphological alterations in skeletal muscle mitochondria in cachectic patients.

Keywords: apoptosis; autophagy; cancer cachexia; mitochondria; skeletal muscle.

Figure 1

Skeletal muscle photomicrograph and intermyofibrillar mitochondrial area of WSC and CC. Image J software was used to assess mitochondrial area (WSC, n = 3; CC, n = 4). Mitochondrial area was compared using Student’s t test and data are expressed as mean and standard error. WSC—weight-stable cancer patients; CC—cachectic cancer patients.

Figure 2

Mitochondrial DNA copy number of WSC and CC subjects. Total DNA was amplified using RT-PCR to assess nuclear DNA and mtDNA copy number (WSC, n = 12; CC, n = 13). Data are expressed as mean and standard error and Student’s t test was used to compare WSC and CC groups. WSC—weight-stable cancer patients; CC—cachectic cancer patients.

Figure 3

Autophagy-related proteins in skeletal muscle of WSC and CC patients. Western blot analysis of (a) LCB3 I (WSC, n = 8; CC, n = 10); (b) LC3B II (WSC, n = 8; CC, n = 10); (c) p62 (WSC, n = 8; CC, n = 10); (d) ATG5 (WSC, n = 6; CC, n = 8) and (e) ATG7 (WSC, n = 6; CC, n = 8). Data are expressed as mean and standard error and were compared using Student’s t test or were represented in box plots and compared using Mann–Whitney test. WSC—weight-stable cancer patients; CC—cachectic cancer patients.

Figure 4

Apoptosis-related protein in skeletal muscle of WSC and CC patients. Multiplex-based analysis of phosphorylated (a) JNK (WSC, n = 9; CC, n = 12); (b) phosphorylated Akt (WSC, n = 8; CC, n = 12); (c) phosphorylated Bcl-2 (WSC, n = 7; CC, n = 11); (d) activated caspase 8 (WSC, n = 5; CC, n = 11); (e) activated caspase 9 (WSC, n = 7; CC, n = 11) and (f) phosphorylated p53 (WSC, n = 7; CC, n = 11). Data are expressed as mean and standard error and were compared using Student’s t test or were represented in box plots and compared using Mann–Whitney test. WSC—weight-stable cancer patients; CC—cachectic cancer patients.