Natriuretic Peptide Receptor B modulates the proliferation of the cardiac cells expressing the Stem Cell Antigen-1

Figure 1. BNP stimulates Sca-1+ cell proliferation. (A) Non myocyte cells (NMCs) were isolated from neonatal hearts of C57BL/6 mice, cultured 4 and 11 days with or without BNP (untreated cells) and counted. (B) Percentages of c-kit+ and Sca-1+ cells obtained by flow cytometry analysis on BNP treated or untreated NMCs. (C) Number of cells expressing the c-kit or the Sca-1 protein in NMCs treated or not with BNP for 4 and 11 days calculated with the total number of cells and the percentages of the c-kit+ and Sca-1+ cells. (A–C) n = 8 and 16 different experiments after 4 and 11 days of culture, respectively. (D) Representative histogram of NMC sorting for Sca-1 expression. The numbers represent the percentage of the cells compared to the total number of sorted NMCs. n = 18–43 different experiments. (E) Number of sorted Sca-1− and Sca-1high+ cells treated or not with BNP for 11 days. n = 6 and 12 for Sca-1− and Sca-1high+ cells, respectively. (F) Percentages of Sca-1+ cells among sorted Sca-1− cells treated or not with BNP for 9 days. n = 4 different experiments. (A–E) All results expressed as fold-increase above the results obtained in untreated cells. All the results are means ± SEM. *p <…

Figure 2. Molecular characterization of sorted Sca-1−, Sca-1low+ and Sca-1high+ cells directly after cell sorting. Quantitative polymerase chain reactions were performed for genes expressed in differentiated cells (A) or in undifferentiated cells (B). Densities of expression were represented as 2−ΔCT and results are means ± SEM of 6 different cell sortings. Enlargements were represented rights for some genes. *p < 0.05 versus Sca-1− cells and °p < 0.05 versus Sca-1low+.

Figure 3. BNP treatment increases the number of Nkx2.5+ cells among sorted Sca-1high+ cells. (A) Representative immunostainings of sorted Sca-1high+ cells treated or not with BNP for up to 11 days and stained with antibodies against Nkx2.5 (pink), Proliferating Cell Nuclear Antigen (PCNA) (green) and DAPI (blue). White arrows represent proliferating Nkx2.5+ cells. Scale bars represent 100 μm. (B) Quantification of the number of Nkx2.5+ cells among Sca-1high+ cells treated or not with BNP for up to 11 days. The number of Nkx2.5+ cells was related to the total number of nuclei stained with DAPI. n = 5–6 different experiments and in total 3607 cells in untreated and 3867 cells in BNP treated cells were evaluated for the Nkx2.5 expression. (C) Quantitative polymerase chain reaction in untreated (n = 13 different experiments) and BNP treated Sca-1high+ sorted cells (n = 14). Results are expressed as 2−ΔΔCT with untreated cells as reference. (B and C) Results expressed as fold-increase above the results in untreated cells. All the results are means ± SEM. *p < 0.05.

Figure 4. Pluripotency of sorted Sca-1high+ cells. (A) Quantitative relative expression of mRNAs coding for cardiomyocyte specific genes (Nkx2.5, ventricular Myosin Light Chain, Troponin T, beta and alpha Myosin Heavy Chain) in cells cultured 11 or 21 days in two different media. Results expressed as fold-increase above the levels in cells directly after sorting, n = 4–8 different experiments per group. (B) Representative pictures of cells cultured 21 days in medium B and expressing α actinin and Troponin I proteins. (C) Representative immunostainings of cells cultured 7 days in medium A and stained for the expression of CD31, von Willbrand Factor (vWF) and smooth muscle actin. (D) Quantitative relative expression of mRNAs coding for cardiomyocyte specific genes in cells cultured 21 days in differentiating medium C with or without BNP. Results expressed as fold-increase above the levels in untreated cells. n = at least 7 different experiments. (E) Representative immunostainings of cells cultured 21 days in medium C with BNP and stained with Troponin I (green) antibody and DAPI (blue). (F) The number of cells positive for Troponin I were related to the number of nuclei. n = 4–7 different…

Figure 5. BNP binds to NPR-B to stimulate Sca-1high+ cell proliferation. (A) Non-myocyte cells (NMCs) isolated from the hearts of neonatal NPR-A or NPR-B deficient mice were cultured with and without BNP. The total number of cells, the percentages of Sca-1+ cells obtained by flow cytometry analysis as well as the number of Sca-1+ cells were determined 11 days after the onset of BNP treatment. n = 9 different experiments for NPR-A and NPR-B deficient cells. (B and D) Sorted Sca-1high+ cells isolated from wild type hearts were cultured with BNP or CNP in presence or not of NPR-B receptor antagonist (P19, 0.5–1 microM) and of an ANF antagonist (Anantin, 0.2 microM). The number of cells was counted after 9–11 days of culture and the results were related to untreated cells. n = at least 4 different experiments. (C) NMCs isolated from wild type hearts were treated with C-natriuretic peptide (CNP) for up to 11 days. The total number of cells as well as the number of c-kit+ and Sca-1+ cells were determined after counting and flow cytometry analysis. n = 6 different experiments (D). (A–D) Results expressed as fold-increase above the results in the related untreated cells. All the results are means…

Figure 6. BNP treatment increases cGMP level and activates the protein Kinase G and the p38 MAP kinase. (A) cGMP levels were measured in the non myocyte cell supernatants (n = 4–7) and in the EDTA treated plasma of mice (n = 4 mice per group) 1h after BNP or CNP treatment (1 μM). (B) Representative western blots of sorted Sca-1high+ cells isolated from B6+ neonatal hearts and stimulated with or without BNP or CNP (1 μM both). Blots were stained with antibodies against phospho phospholamban (pPLB), phospholamban (PBL), phospho p38 (pp38), p38 and Tubulin (used as loading control). Only the bands at the adequate molecular weight were represented here: tubulin 55 kDa, pp38 and p38 about 43 kDa, pPLB between 21 and 26 kDa and PLB 25 kDa. (C) Quantification of the data from western blot analysis expressed relatively to the average of untreated cells. Sorted Sca-1high+ cells were or not treated 1h with BNP or CNP. n = 4–6 different experiments. All the results are means ± SEM, *p < 0.05 versus untreated cells.

Figure 7. Schematic representation of BNP signaling pathway leading to Sca-1+ cell proliferation. (A) Number of sorted Sca-1high+ cells after 11 days of culture with or without BNP or CNP and PKG or p38 inhibitor. n = at least 6 different experiments per group. The data were related to the average of untreated cells. All the results are means ± SEM, *p < 0.05 versus untreated cells and °p < 0.05 versus BNP or CNP treated cells. (B) Summary of the hypothetical signaling pathway induced by BNP to stimulate Sca-1+ cell proliferation. BNP secreted by cardiac cells binds to NPR-B on Sca-1+ cells which increases the cGMP level and activates the protein kinase G (PKG). Furthermore, BNP phosphorylates the p38 MAP kinase. Addition of the SB203580 increases cell proliferation.