Journal: Scientific reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart.

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Figure 1. Immunofluorescence of TRPM7 and TRPM6 proteins in all cells used. Image acquisition performed using confocal laser scanning microscope (a, atria; b, ventricle). Immunofluorescence of confocal z-stack of cardiomyocytes with immunodetected TRPM7 and TRPM6 proteins, respectively. Alexa Fluor 488 and Alexa Fluor 546 for the TRPM7 and TRPM6 protein appear in green and red, respectively. Alexa Fluor 405 for F-actin cytoskeleton appears in surrogate grey. Hoechst 33342 for nuclei appears in blue (the arrowheads indicate the localization of TRPM6 protein in the perinuclear area). (c, d) Quantification of immunofluorescence levels of the TRPM7 (green) and TRPM6 (red) proteins in cardiomyocytes from four chambers of the heart (left atrium, LA; right atrium, RA; left ventricle, LV; and right ventricle, RV), under experimental conditions with (c) and without (d) divalent cations in the extracellular milieu, respectively. Cardiomyocytes were fixed following 2 h (filled columns) or 12 h (unfilled columns) after cell isolation. Mean data provided in arbitrary units (a.u.) (Supplementary Table 1 online). A blinded study-design (with the investigator reading the fluorescence not knowing the cell incubation conditions) was used for the detection of protein concentration during various experimental conditions. *P < 0.05 2 h vs. 12 h and #P < 0.05 right-sided vs. left-sided heart chambers. Scale bars indicate 20 µm.

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Immunofluorescence, Laser-Scanning Microscopy, Cell Isolation, Fluorescence, Incubation, Protein Concentration

Journal: Scientific reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart.

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Figure 2. 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.

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Immunofluorescence, Expressing, Staining

Journal: Scientific reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart.

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Figure 3. Effect of 2-APB and CAR on the immunofluorescence of TRPM6 and TPRM7 in human cardiomyocytes. (a–f): Cardiomyocyte staining with anti-TRPM7 (a, c, e) or anti-TRPM6 (b, d, f) in the absence (a, b) of drugs and in the presence of either 2-APB (c, d) or CAR (e, f). (g–j): Quantification of the intensity of fluorescence without drugs (open) and with the drugs (filled) expressed in arbitrary units (a.u.). Note lack of influence of the solvent, DMSO, at 500 μmol/L (triangles) but opposite change with 2-APB and CAR on TRPM7 vs. TRPM6, i.e. decrease of the immunofluorescence level of TRPM7 but increase of the TRPM6 fluorescence level. *P < 0.001 drug vs. no drug. Other notations are the same as in Fig. 1.

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Immunofluorescence, Staining, Fluorescence, Solvent

Journal: Scientific reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart.

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Figure 5. 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.

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Comparison, Fluorescence, Biomarker Discovery, Expressing

Journal: Scientific reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart.

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Figure 4. 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.

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Immunofluorescence, Fluorescence

Journal: Scientific reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart.

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Figure 6. TRPM7 and TRPM6 protein levels and RT-qPCR mRNA relative expression levels in human heart tissue homogenates. (a, b) TRPM7 and TRPM6 proteins are increased in the walls of all heart chambers with IHD (filled columns) vs. non-IHD (unfilled columns). A blinded study-design (with the diagnosis unknown to the investigator) was used for the detection of protein concentration in the various samples. Values (mean ± SEM) are in pg/mL and from 3–33 heart tissue homogenates. *P < 0.05 IHD vs. non-IHD, #P < 0.05 right-sided vs. left-sided using ANOVA with LSD test (Least Significant Difference). (c) mRNA relative expression levels of human Trpm6 and Trpm7 genes detected in the four cardiac chambers from explanted human hearts (n = 3, for each). *P < 0.05 Trpm7 vs. Trpm6, #P < 0.05 right-sided vs. left-sided.

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Quantitative RT-PCR, Expressing, Biomarker Discovery, Protein Concentration

Journal: Scientific reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart.

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Figure 8. Representative histotopograms of TRPM6 images in the four chamber walls of the human heart. (a) Control subject after traffic accident. (b) IHD patient. The intensity of the brownish pigments shows areas with expression of TRPM6 in the heart (× 40 magnification). (c) Negative control (left) in the LV, and positive control (right) of TRPM6 in tumour of the colon (× 40 magnification). Scale bars indicate 100 µm. Other notations are the same as in Fig. 7.

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Control, Expressing, Negative Control, Positive Control