Derangements of amino acids in cachectic skeletal muscle are caused by mitochondrial dysfunction

Abstract

Background: Cachexia is the direct cause of at least 20% of cancer-associated deaths. Muscle wasting in skeletal muscle results in weakness, immobility, and death secondary to impaired respiratory muscle function. Muscle proteins are massively degraded in cachexia; nevertheless, the molecular mechanisms related to this process are poorly understood. Previous studies have reported conflicting results regarding the amino acid abundances in cachectic skeletal muscle tissues. There is a clear need to identify the molecular processes of muscle metabolism in the context of cachexia, especially how different types of molecules are involved in the muscle wasting process.

Methods: New in situ -omics techniques were used to produce a more comprehensive picture of amino acid metabolism in cachectic muscles by determining the quantities of amino acids, proteins, and cellular metabolites. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging, we determined the in situ concentrations of amino acids and proteins, as well as energy and other cellular metabolites, in skeletal muscle tissues from genetic mouse cancer models (n = 21) and from patients with cancer (n = 6). Combined results from three individual MALDI mass spectrometry imaging methods were obtained and interpreted. Immunohistochemistry staining for mitochondrial proteins and myosin heavy chain expression, digital image analysis, and transmission electron microscopy complemented the MALDI mass spectrometry imaging results.

Results: Metabolic derangements in cachectic mouse muscle tissues were detected, with significantly increased quantities of lysine, arginine, proline, and tyrosine (P = 0.0037, P = 0.0048, P = 0.0430, and P = 0.0357, respectively) and significantly reduced quantities of glutamate and aspartate (P = 0.0008 and P = 0.0124). Human skeletal muscle tissues revealed similar tendencies. A majority of altered amino acids were released by the breakdown of proteins involved in oxidative phosphorylation. Decreased energy charge was observed in cachectic muscle tissues (P = 0.0101), which was related to the breakdown of specific proteins. Additionally, expression of the cationic amino acid transporter CAT1 was significantly decreased in the mitochondria of cachectic mouse muscles (P = 0.0133); this decrease may play an important role in the alterations of cationic amino acid metabolism and decreased quantity of glutamate observed in cachexia.

Conclusions: Our results suggest that mitochondrial dysfunction has a substantial influence on amino acid metabolism in cachectic skeletal muscles, which appears to be triggered by diminished CAT1 expression, as well as the degradation of mitochondrial proteins. These findings provide new insights into the pathobiochemistry of muscle wasting.

Keywords: Amino acids; Cancer cachexia; MALDI; Mass spectrometry imaging; Mitochondrial dysfunctions.

Figure 1

In situ analysis of amino acids in cachectic mouse skeletal muscle tissues. (A) Quantities of amino acids in skeletal muscles from cachectic and non‐cachectic mice. All intensity values were determined by mass spectrometry imaging. Quantities of lysine, arginine, proline, and tyrosine were significantly higher in cachectic mice than in non‐cachectic ones. The intensities for alanine, asparagine, glutamine, leucine/isoleucine, methionine, phenylalanine, threonine, tryptophan, and valine also revealed a higher relative mean intensity in cachectic mice than in non‐cachectic ones. Glutamate and aspartate intensities were significantly decreased in cachectic mouse muscles. Whiskers of the boxplots illustrate the minimal and maximum intensity values. (B) False colour visualization of amino acids in mouse skeletal muscle tissues. Lysine, arginine, and proline were increased, and glutamate and aspartate were decreased in cachectic mouse muscles, compared with non‐cachectic ones. ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.005. H&E, haematoxylin and eosin.

Figure 2

Evaluation of potential protein breakdown targets. (A) Visualization of Spearman's rank correlation analysis results examining the relationships between amino acids and proteins. Square sizes represent the magnitude of the Spearman's rank correlation coefficient. Blue squares indicate positive correlations, and red squares indicate negative correlations. Non‐significant correlations (P > 0.05) are indicated by empty squares. Many OXPHOS proteins seemed to be degraded. (B) False colour visualization of COX6B1. A reduced quantity of COX6B1 was observed in cachectic mouse tissues, compared with non‐cachectic ones. (C) Statistical analysis of the false colour visualization results depicted in (B). COX6B1 intensities were significantly lower in cachectic mouse muscle tissues than in non‐cachectic ones (P = 0.0008). Boxplot whiskers represent the minimum and maximum intensities. ^*** P < 0.005.

Figure 3

Energy changes in cancer cachexia. (A) Heatmap visualization and statistical analysis of the calculated energy charge. Calculation of the energy charge revealed a significantly lower charge in cachectic mice (P = 0.0101). (B) AMP, ADP, and ATP distribution in cachectic and non‐cachectic mouse skeletal muscle tissues. No significant differences were detected between cachectic and non‐cachectic mouse tissues. (C) Heatmap visualization and statistical analysis of changes in molecules of the tricarboxylic acid cycle. Cachectic mice exhibited significantly higher quantities of malate (P = 0.0295) and lower quantities of oxaloacetate (P = 0.0448) than non‐cachectic mice. Whiskers of the boxplots represent the lowest and highest peak intensities in each group. ^* P < 0.05.

Figure 4

Impact of proteins and amino acids on the energy charge. Correlation plot depicting the results of Spearman's rank analysis examining the associations between amino acid and protein expression and the calculated energy charge. Square size represents the magnitude of the Spearman's rank correlation coefficient. Blue squares indicate positive correlations, and red squares indicate negative correlations. Non‐significant correlations (P > 0.05) are symbolized by empty squares. Energy charge was significantly correlated with 58 proteins and five amino acids

Figure 5

Changes in CAT1 expression in cachexia. (A) Digital image analysis of the CAT1 immunohistochemistry (IHC) results. Definiens Software Developer XD2 was used to detect the CAT1 expression in all tissue sections. (B) The detected CAT1 abundance was significantly lower in skeletal muscles of cachectic mice than in non‐cachectic ones (P = 0.0133). Whiskers of the boxplots represent the lowest and highest CAT1 expression in each group. (C) CAT1 expression in skeletal muscle tissues from humans with cancer for all and only female patients. CAT1 was slightly lower in patients with cachexia, compared with non‐cachectic patients, but the difference did not reach statistical significance. Horizontal lines represent the mean intensity of each group. (D) Number of mitochondria in mouse skeletal muscle tissues determined by transmission electron microscopy (TEM) at 1600× magnification. The red colour represents the mitochondria. Intact mitochondria were observed in tissues from both cachectic and non‐cachectic mice. (E) Boxplots illustrating the number of mitochondria in mouse skeletal muscle tissues. No significant difference was detected between cachectic and non‐cachectic mice. (F) Correlation analysis between the number of mitochondria determined by electron microscopy and the intensity of voltage‐dependent anion channel (VDAC) staining detected by IHC. The number of mitochondria was significantly correlated with VDAC expression (P = 0.0033). (G) Statistical analysis of VDAC staining in muscle tissues from cachectic and non‐cachectic mice. No significant difference was detected. ^* P < 0.05.

Figure 6

Hypothesis regarding the molecular changes in skeletal muscles during cachexia, focusing on mitochondrial dysfunction. The small red and green arrows indicate molecules for which significant changes were detected in the current study. (A) Proteins in muscle tissues of cachectic mice are degraded and subsequently processed to amino acids. Individual amino acids are then transported into the mitochondria for further metabolism. (B) (1) Lysine (Lys), arginine (Arg), and ornithine (data not shown) are transported via CAT1 into the mitochondria. (2) Specific transaminase proteins metabolize Lys, Arg, ornithine, and other amino acids and produce glutamate. Glutamate is decreased in cachexia because of reduced CAT1 expression. (3) Cytosolic NADH reduces oxaloacetate to malate; it is hypothesized that an increased quantity of cytosolic NADH in cachexia increases the quantity of malate. (4) Malate is exchanged with α‐ketoglutarate in the mitochondrial matrix by the malate‐α‐ketoglutarate transporter.