Fig 2: Expression of isoenzyme mRNAs in skeletal muscle tissue and cells. (A) The expression of isoenzymes was assessed and compared in human muscle (red, n = 2), liver (blue, n = 2), and kidney (yellow, n = 2) biopsies by Q-PCR, underlining the presence of ALDH1A1, 1B1, 1L1, 2, 3A2, 5A1, 6A1, and 9A1. (B) The expression was analysed in the total population of freshly dissociated cells from human biopsies highlighting the presence of isoenzymes involved in the metabolism of retinoic acid (ALDH1A1, 1A2, 1A3, and 1B1) and of ALDH2, 3A2, 6A1, 7A1, and 9A1. (C) The expression was analysed and compared in primary cell cultures in proliferation and in differentiation (n = 4), underlining the presence of ALDH1A1, 1A3, 1B1, 2, 3A2, 7A1, and 9A1. No significant differences were observed between the proliferation and differentiation stages. Data are presented as mean and SD.

Fig 3: Identification of isoenzymes expressed by human freshly dissociated cells. Human cells were fixed using PFA and permeabilized using saponin, then incubated with antibodies directed against the ALDH isoenzymes indicated, then incubated with a secondary antibody labelled with FITC, and analysed by flow cytometry. Some isoenzymes were present in a high proportion of cells (up to 30% regarding ALDH2), while other isoenzymes were detected in smaller percentages of cells, with values around 5% but raising 10% (ALDH1A1 and ALDH9A1). A baseline was set arbitrarily at 5% to illustrate a maximum percentage of human cells detected using the ALDEF assay. This illustrates the potential discrepancy between the identification of the protein content using Ab, and the enzymatic activity detected by a functional assay. Data are presented as mean and SD (n = 2–7).

Fig 4: (A and B) Immunohistological localization of isoenzyme-expressing cells in situ on cryostat sections. Human skeletal muscle sections were fixed with acetone and labelled for expression of several isoenzymes (red) and laminin delineating the basal lamina of muscle fibres (green). Nuclei were stained in blue with DAPI. Arrowheads point nuclear localization of isoenzymes (intranuclear, perinuclear, or both). Arrows point whole cells regardless of their localization (endothelial, interstitial, neural, myogenic, etc.). Asterisks underline global areas of interest (concentration of cells). The most representative isoenzymes are presented from healthy donors (8A), or from DMD donors (8B) when qualitative differences were perceived between the two groups. Isoenzymes were associated with myogenic-like cells (ALDH1A1, 1B1, 1L1, and 3B1), to endothelial or angiogenic structures (ALDH1A1, 1L1, 2, 3A2, 3B2, and 9A1), to interstitial cells (ALDH1A1, 1A2, 1A3, 1L1, 3A1, 3A2, and 9A1), to neural structures (ALDH1L1 and 9A1), or to connective tissue (ALDH1L1, 3A2, and 9A1). Centronucleation is frequently observed in DMD biopsies (B) (ALDH1A1, 1B1, and 3B1). Original magnification is ×100, and it is ×25 when indicated on the scale bar. Scale bars are 40 µm (×100) and 140 µm (×25).