Evaluation of cancer-derived myocardial impairments using a mouse model

Abstract

Myocardial damage in cancer patients is emphasized as a cause of death; however, there are not many murine cachexia models to evaluate cancer-derived heart disorder. Using the mouse cachexia model that we established previously, we investigated myocardial damage in tumor-bearing mice. In cachexic mice, decreased heart weight and myocardial volume, and dilated left ventricular lumen, and atrophied cardiomyocytes were noted. The cardiomyocytes also showed accumulated 8-hydroxydeoxyguanosine, decreased leucine zipper and EF-hand-containing transmembrane protein-1, and increased microtubule-associated protein light chain3-II. Levels of tumor necrosis factor-α and high-mobility group box-1 proteins in the myocardium were increased, and nuclear factor κB, a signaling molecule associated with these proteins, was activated. When rat cardiomyoblasts (H9c2 cells) were treated with mouse cachexia model ascites and subjected to flux analysis, both oxidative phosphorylation and glycolysis were suppressed, and the cells were in a quiescent state. These results are in good agreement with those previously reported on cancerous myocardial damage. The established mouse cachexia model can therefore be considered useful for analyzing cancer-derived myocardial damage.

Keywords: atrophy; cachexia; mitochondria; myocardium; oxidative stress.

Figure 1. Cachexic features of CT26-inoculated BALB/c mice.

(A) Body weight, (B) tumor weight, (C) fat pad weight, and (D) weight of QFM (2 muscles per mouse) in each group at euthanasia. Error bar, standard error of data collected from 3 mice. Significant differences were calculated using Student t-test. Control, no tumor control; CT26, mice inoculated with CT26 cells intraperitoneally; QFM, quadriceps femoris muscle.

Figure 2. Alterations in the myocardium of CT26-inoculated BALB/c mice.

(A) Cardiac weight. (B) Image of the cut surface of the heart; H&E staining. Scale bar, 0.5 mm (C) Myocardial area. (D) Percentage of the luminal area to the myocardial area. (E) Photomicrogram of the left ventricle. H&E staining. Scale bar, 50 μm. (F) Average area of cardiomyocytes. Error bar, standard error of data collected from 3 mice. Significant differences were calculated using Student t-test. Control, no tumor control; CT26, mice inoculated with CT26 cells intraperitoneally; H&E, hematoxylin and eosin.

Figure 3. Oxidative stress, mitochondria, and autophagy in the myocardium of CT26-inoculated BALB/c mice.

(A) Immunostaining of 8-OHdG (oxidative stress). Scale bar, 50 μm. (B) Western blot analysis of LETM1 (mitochondria). (C) Western blot analysis of LC3. LC3-II is a marker for autophagy. (D) Semi-quantification of LETM1 and LC3-II. (E) SDS-soluble myosin light chain-2 (MYL1) measured using ELISA. The signals were standardized with the β-actin signal. Error bar, standard error of data collected from 3 mice. Significant differences were calculated using Student t-test. 8-OHdG, 8-hydroxy-2′-deoxyguanosine; LETM1, leucine zipper and EF-hand-containing transmembrane protein 1; LC3, microtubule-associated protein light chain-3; Control, no tumor control; CT26, mice inoculated with CT26 cells intraperitoneally; ELISA, enzyme-linked immunosorbent assay.

Figure 4. Myocardial cytokines and signals in the CT26-inoculated BALB/c mice.

(A) Serum HMGB1. (B) HMGB1 and TNFα in myocardium. (C) Levels of proteins in the signaling pathway of HMGB1 or TNFα. The signals were standardized with the β-actin signal. (D) H9c2 cells treated with the ascites of CT26-inoculated BALB/c mice (Ascites) with or without a IKKbeta inhibitor IMD0354. Then SDS-soluble myosin light chain-2 (MYL1) measured using ELISA. Error bar, standard error of data collected from 3 mice. Significant differences were calculated using Student t-test. HMGB, high-mobility group box; TNF, tumor necrosis factor; RAGE, receptor for glycation end products; IKK, inhibitor κB kinase; pIKK, phosphorylated IKK; RelA, v-rel avian reticuloendotheliosis viral oncogene homolog A (p65); Control, no tumor control; CT26, mice inoculated with CT26 cells intraperitoneally.

Figure 5. Energy metabolism in the myocardium of CT26-inoculated BALB/c mice.

(A) Protein levels of HMGB1 and TNFα in the ascites of CT26-inoculated BALB/c mice or H9c2 cultured medium (CM). (B) Cell proliferation of H9c2 cells treated with the ascites of CT26-inoculated BALB/c mice (Ascites) or H9c2 cultured medium (Control). (C) Mitochondrial stress test (Seahorse assay) of H9c2 cells. (D) Basal OCR and spare OCR. (E) Proton leak and ATP production. (F) Oxidative stress examined by DHR. (G) Glycolytic stress test of H9c2 cells. (H) Matrix analysis of OCR and ECAR. Error bar, standard error from 3 mice. Significant differences were calculated using Student t-test. HMGB, high-mobility group box; TNF, tumor necrosis factor; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; DHR, dihydrorhodamine 123.