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. 2020 Nov 27;9:e61050. doi: 10.7554/eLife.61050

Figure 1. Intraperitoneal BNP injection acts on cardiac non-myocyte cells (NMCs).

(A) Experimental protocol as described in details in Material and methods section. (B) cGMP level measurement in cardiac tissue of unmanipulated mice injected or not with BNP for 30 min and 1–2 hr. n = at least six mice. (C) cGMP plasma level measurement in unmanipulated or infarcted mice injected or not with BNP. n = 6–7 mice for unmanipulated hearts, n = 6 infarcted mice 24 hr after injection. (D) Representative western blots of total proteins isolated hearts of saline or BNP-injected mice, 3 and 10 days after surgery. Blots were stained with antibodies against phospho phospholamban (pPLB), phospholamban (PBL) and Tubulin (used as loading control). Only the bands at the adequate molecular weight were represented here: Tubulin 55 kDa, pPLB between 17 and 26 kDa and PLB 25 kDa. Quantification of the pPLB/PLB ratio. Data obtained from western blot analysis on unmanipulated (n = 7–11 mice per group) and infarcted hearts of mice treated or not with BNP. Results of BNP-treated hearts expressed relatively to the average of saline-treated hearts. 3 days after MI: n = 5 mice for the ZI+BZ and 7–8 mice for the RZ 10 days after MI: n = 10–11 mice for the ZI+BZ and n = 9–10 mice for the RZ. (E) NMCs were isolated from both areas of infarcted hearts treated or not with BNP 10 days after surgery. Proteins were extracted from these cells (n = 5 independent isolation per group for the ZI+BZ and n = 6–7 for the RZ) and pPLB/PLB ratio was evaluated. Only the western blots obtained for NMCs isolated from the ZI+BZ were represented. For B, C, D and E: Individual values are represented and the means ± SEM are represented in red. Statistical analysis was performed only for groups with n ≥ 6. # p<0.05 for different variance between groups, *p<0.05 using unpaired T tests with or without Welch’s corrections.

Figure 1.

Figure 1—figure supplement 1. Adult cardiac endothelial cells express BNP receptors in both infarcted and border (ZI+BZ) and remote (RZ) zones.

Figure 1—figure supplement 1.

The presence of NPR-A (A) or NPR-B (B) was assessed by immunostainings on adult hearts 24 hr after MI or by flow cytometry analysis (C) on adult non-myocytes cells isolated from hearts 4 days after MI. Antibody against CD31 was used in combination with anti-NPR-A or NPR-B antibodies. The percentages of NPR-A or NPR-B expressing cells were evaluated on CD31+ cells.