Mind the gap (junction): cGMP induced by nitric oxide in cardiac myocytes originates from cardiac fibroblasts

Figures

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  Figure 1. cGMP signals of adult primary cardiac myocytes. (a) In cardiac myocytes, ANP did not increase cGMP in the absence or presence of IBMX; CNP was applied as positive control. Values are means ± SEM of myocytes from N = 5 mice. (b) CNP concentration–response curves were recorded with and without IBMX. Values are means ± SEM of cardiac myocytes from N = 5 (10, 1,000 nM) or 6 (0.1, 1, 30–300 nM) mice. (c) Effect of PDE inhibitors added to CNP stimulation (300 nM). Shown are individual values (for each inhibitor, one pair corresponds to measurements from one mouse) before and after application of PDE inhibitors. * P < .05, significant effect of PDE inhibitor. (d) Stimulation of cardiac myocytes by the indicated NO–GC‐activating compounds does not increase cGMP; CNP was applied as positive control; all in the presence of IBMX. Data shown in the time courses as means ± SEM of myocytes from N = 5 or N = 6 (BAY41‐2272) mice. Cilo, cilostamide; Sil, sildenafil.

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  Figure 2. NO‐induced cGMP of cardiac fibroblasts. (a) Primary cardiac fibroblasts were stimulated for 5 min with the indicated GSNO concentrations. Values are means ± SEM of fibroblasts from N = 5 mice. (b–d) Cardiac (b) and dermal (c) fibroblasts and aortic smooth muscle cells (d) were stimulated with GSNO and IBMX as indicated and formed cGMP was determined by RIA. Data shown are individual values obtained from cells of one mouse each, together with means. * P < .05, significantly different as indicated.

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  Figure 3. NO–GC‐induced cGMP signals in cardiac fibroblasts. (a) Stimulation of cardiac fibroblasts by the indicated NO–GC‐activating compounds increased cGMP. CNP was applied as positive control; all in the presence of IBMX. Data shown are the time courses as means ± SEM of fibroblasts from N = 5 mice. (b) Effect of PDE inhibitors added to GSNO stimulation (1 nM). Data shown are individual values (for each inhibitor, one pair corresponds to measurements with fibroblasts from one mouse) before and after application of PDE inhibitors. * P < .05, significant effect of PDE inhibitor. Cilo, cilostamide; Sil, sildenafil. (c) Angiotensin II decreased GSNO‐induced cGMP and the PDE1 inhibitor 8MMX abolished this effect Data shown are means ± SEM of fibroblasts from N = 5 (ctrl, 8MMX) or 6 (Ang II, Ang II + 8MMX) mice; * P < .05, significantly different from ctrl.

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  Figure 4. CNP‐induced cGMP in cardiac fibroblasts. (a) ANP induced a small but significant increase of cGMP in cardiac fibroblasts; CNP was applied as positive control. Values are means ± SEM of N = 5 mice. Data shown are the time courses as means ± SEM of fibroblasts from N = 7 mice. (b) CNP concentration–response curves were recorded with and without IBMX. Values are means ± SEM of fibroblasts from N = 5 mice. (c) Effect of PDE inhibitors added to CNP stimulation (3 nM). Data shown are individual values (for each inhibitor, one pair corresponds to measurements with fibroblasts from one mouse) before and after application of PDE inhibitors. * P < .05, significant effect of PDE inhibitor. Cilo, cilostamide; Sil, sildenafil.

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  Figure 5. NO‐induced cGMP signals in cardiac myocytes co‐cultured with cardiac fibroblasts. (a) To detect cGMP specifically in cardiac myocytes, myocytes expressing the cGMP indicator cGi500 (indicated by green colour) were cultured on cardiac fibroblasts not expressing the indicator. (b) In the co‐cultures, preincubation with the gap junction inhibitor carbenoxolone (CBX) or culturing myocytes and fibroblasts in a non‐touching manner decreased the fraction of GSNO‐responsive cardiac myocytes. Total number of viable myocytes was identified by subsequent CNP stimulation during the same recording. Shown are individual values together with means ± SEM of co‐cultures with myocytes of N = 5 mice; * P < .05, significantly different as indicated. (c) In the co‐cultures, the indicated gap junction inhibitors decreased GSNO‐induced cGMP. Values are normalized to the signal reached after 5 min preincubation with GSNO. Data shown are means ± SEM of co‐cultures with myocytes of N = 7 (ctrl), 6 (GAP26), or 5 (CBX, GAP27, and α/βGlycA) mice. * P < .05, significantly different from control. (d) In the co‐cultures, GAP26 and GAP27 reversed NO‐induced cGMP signals whereas the scrambled versions of the…