Evidence for mitochondrial respiratory deficiency in rat rhabdomyosarcoma cells

Abstract

Background: Mitochondria can sense signals linked to variations in energy demand to regulate nuclear gene expression. This retrograde signaling pathway is presumed to be involved in the regulation of myoblast proliferation and differentiation. Rhabdomyosarcoma cells are characterized by their failure to both irreversibly exit the cell cycle and complete myogenic differentiation. However, it is currently unknown whether mitochondria are involved in the failure of rhabdomyosarcoma cells to differentiate.

Methodology/principal findings: Mitochondrial biogenesis and metabolism were studied in rat L6E9 myoblasts and R1H rhabdomyosacoma cells during the cell cycle and after 36 hours of differentiation. Using a combination of flow cytometry, polarographic and molecular analyses, we evidenced a marked decrease in the cardiolipin content of R1H cells cultured in growth and differentiation media, together with a significant increase in the content of mitochondrial biogenesis factors and mitochondrial respiratory chain proteins. Altogether, these data indicate that the mitochondrial inner membrane composition and the overall process of mitochondrial biogenesis are markedly altered in R1H cells. Importantly, the dysregulation of protein-to-cardiolipin ratio was associated with major deficiencies in both basal and maximal mitochondrial respiration rates. This deficiency in mitochondrial respiration probably contributes to the inability of R1H cells to decrease mitochondrial H2O2 level at the onset of differentiation.

Conclusion/significance: A defect in the regulation of mitochondrial biogenesis and mitochondrial metabolism may thus be an epigenetic mechanism that may contribute to the tumoral behavior of R1H cells. Our data underline the importance of mitochondria in the regulation of myogenic differentiation.

Figure 1. Cell cycle analysis and proliferating cell nuclear antigen (PCNA) protein content in L6E9 and R1H cells.

Cell cycle analysis of L6E9 myoblasts and R1H cells cultured in growth medium (A) and differentiation medium for 36 hours (B). Cell cycle analysis was performed after doublet exclusion on morphologically normal living cells. (C) PCNA protein level in L6E9 myoblasts and R1H cells cultured in growth medium (GM) and differentiation medium (DM). PCNA immunolabeling was performed after doublet exclusion on morphologically normal fixed cells. Data are means ± SE from 6 culture dishes. * P<0.05: significantly different from corresponding cells in GM; † P<0.05 and †† P<0.01: significantly different from L6E9 myoblasts.

Figure 2. Cardiolipin content, mitochondrial content and mtDNA content.

(A) Representative image of mitochondrial staining by nonyl acridine orange (NAO) in L6E9 myoblasts and R1H cells cultured in growth medium (GM) and in differentiation medium (DM) for 36 hours. Nuclei were stained with Hoechst 33342. Cells were visualized by fluorescence microscopy (Olympus inverse microscope IX81 system). (B) Cardiolipin content was determined from the geometric means of height NAO fluorescence signal. (C) Mitochondrial content was assessed from the geometric means of Mitotracker deep red fluorescence signal. Flow cytometry analysis was performed on living cells after doublet exclusion as a function of L6E9 and R1H cells position in the cell cycle (G1, S and G2M). Cells were cultured in GM and DM. (D) mtDNA quantity calculated as the ratio of mitochondrial subunit II of cytochrome oxidase to peptidylprolyl isomerase A DNA levels determined by real-time PCR in L6E9 and R1H cultured in GM and DM. Data are means ± SE from 6 culture dishes. *** P<0.001: significantly different from corresponding cells in G1 phase; ††† P<0.001: significantly different from L6E9 myoblasts.

Figure 3. Bioenergetic status of L6E9 and R1H cells.

(A) Mitochondrial proton leak (oligomycin insensitive respiration), mitochondrial ATP synthesis (basal respiration minus oligomycin-insensitive respiration) and mitochondrial respiratory reserve (FCCP-stimulated respiration minus basal respiration) are expressed as a percent of corresponding basal mitochondrial respiration reported in Table 2. Data are means ± SE from 12 independent culture dishes cultured in growth medium (GM) and differentiation medium (DM). (B) Lactate production of L6E9 and R1H cells cultured in GM and DM. Data are means ± SE from 6 independent culture dishes. *P<0.05: significantly different from corresponding cells in GM; †† P<0.01: significantly different from L6E9 myoblasts.

Figure 4. Mitochondrial import of Core 2 subunit of complex III, ATP synthase α (20D6) and citrate synthase.

Lactate dehydrogenase (LDH) activity (A), Core 2 subunit of complex III and 20D6 protein content (B) and citrate synthase (CS) activity (C) were determined in mitochondrial and cytosolic fractions of L6E9 and R1H cells cultured in growth medium (GM) and differentiation medium (DM) for 36 hours. Data are means ± SE from 3 culture flasks.

Figure 5. Mitochondrial H₂O₂ level.

(A) Representative frequency histogram of 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) fluorescence (FL1-H) obtained in L6E9 myoblasts cultured in growth medium. Positive and negative controls were obtained by incubating cells with 100 µM H₂O₂ (right displacement) and 1 mM CCCP (left displacement), respectively. (B) H₂DCFDA fluorescence was determined from the geometric means of fluorescence intensity peaks. Analyses were performed on morphologically normal living cells cultured in growth medium (G1, S and G2M phases) and differentiation medium (DM) for 36 hours. Data are means ± SE from 6 culture dishes. * P<0.05 and *** P<0.001: significantly different from corresponding cells in G1 phase; † P<0.05: significantly different from L6E9 myoblasts.