Fig 2: Comparison of the levels of TRPM6 and TRPM7 in IHD vs. non-IHD. (a–d, e–h) Quantification of the intensity of fluorescence in cardiomyocytes obtained from patients with IHD (filled symbols) and without such diagnosis (unfilled symbols) expressed in arbitrary units (a.u.) in presence/absence of divalent cations, 2 h and 12 h, respectively. In all cells used P < 0.001 IHD vs. non-IHD. Other notations are the same as in Fig. 1. IHD increases the fluorescence level of the immunodetected TRPM7 (green) and TRPM6 (red) proteins expression.

Fig 3: Immunofluorescence images depicting co-expression of TRPM6 and TRPM7 proteins in human cardiomyocytes. (a) The immunofluorescence of TRPM7 (green) and TRPM6 (red) in the same LA, RA, LV, and RV cardiomyocyte when using conjugated antibodies (the arrowheads indicate the localization of TRPM6 protein in perinuclear area). (b) Quantification of the staining intensity of the immunodetected conjugated antibodies (spotty) and non-conjugated antibodies (smooth) for both proteins in cardiomyocytes from the four chambers of the heart as indicated. *P < 0.001 2 h vs. 12 h, #P < 0.001 right-sided vs. left-sided heart chambers. Other notations are the same as in Fig. 1.

Fig 4: Immunofluorescence images suggesting the presence of TRPM6 and TRPM7 proteins in pig cardiomyocytes from different cardiac chamber walls. (A–D) Immunofluorescence of TRPM7 (left) and TRPM6 (right) in the left atrium (LA), right atrium (RA), left ventricle (LV), and right ventricle (RV) cardiomyocytes when using Alexa Fluor 488 for the TRPM7 and TRPM6 proteins (stained in green), Alexa Fluor 546 for the F-actin cytoskeleton (stained in red), and Hoechst 33342 for the nuclei (stained in blue). Scale bars indicate 20 µm. (E) Example of a negative control, where the primary antibody for TRPM6 and/or TRPM7 is not added, but the cardiomyocyte was subjected to Hoechst 33342 and Alexa Fluor 546. Under such conditions, only immunofluorescence of the nuclei (stained in blue) and F actin cytoskeleton (stained in red) is detected. Note: same cardiomyocyte in the left and right (merged image) panels (F) Quantification of the staining intensity of the immunodetected fluorescence of TRPM7 and TRPM6 in the four cardiac chamber walls: LA, RA, LV, and RV. The mean data is provided in arbitrary units (a.u.) (see Table 1). A blinded study design (with the origin or treatment of cells unknown to the investigator) was used for the detection of immunofluorescence during the various experimental conditions.

Fig 5: Effect on incubating human cardiomyocytes in acidic solution on the immunofluorescence of TRPM6 and TPRM7. (a, b) Quantification of the intensity of fluorescence at pH = 7.4 (open symbols) and at pH = 5.0 (filled symbols) expressed in arbitrary units (a.u.) with and without divalent cations, respectively. *P < 0.001 pH 5.0 vs. pH 7.4. Other notations are the same as in Fig. 1. Extracellular acidification differently changes the fluorescence level of the immunodetected TRPM7 (filled diamonds) and TRPM6 (filled circles) proteins in human cardiomyocytes depending on the presence of divalent cations in the milieu.