Monoamine Oxidase A Contributes to Serotonin—But Not Norepinephrine-Dependent Damage of Rat Ventricular Myocytes

Figures

• 

  Figure 1. Expression of MAO isoforms in rat hearts exposed to pressure overload by aortic banding (AOB) and pulmonary artery banding (PAB). (A) Expression of MAOA in the left ventricle in hearts from AOB and PAB rats with still normal cardiac function (compensated state). (B) Expression of MAOA in the left ventricle in hearts from AOB and PAB rats with reduced cardiac function (decompensated state). (C) Expression of MAOA in the right ventricle in hearts from AOB and PAB rats with still normal cardiac function (compensated state). (D) Expression of MAOA in the right ventricle in hearts from AOB and PAB rats with reduced cardiac function (decompensated state). (E) Expression of MAOB in the left ventricle in hearts from AOB and PAB rats with still normal cardiac function (compensated state). (F) Expression of MAOB in the left ventricle in hearts from AOB and PAB rats with reduced cardiac function (decompensated state). (G) Expression of MAOB in the right ventricle in hearts from AOB and PAB rats with still normal cardiac function (compensated state). (H) Expression of MAOB in the right ventricle in hearts from AOB and PAB rats with reduced cardiac function (decompensated state). Data are…

• 

  Figure 2. Expression of MAO isoforms in rat heart and myocytes and effect of MAO-B activation on mouse and rat myocytes. (A) Western blot indicating the expression of MAO-A and MAO-B in the liver (L), left ventricle (LV), and cardiomyocytes (CM) from rat hearts. GAPDH was used as loading control. (B) mRNA expression in rat hearts of the genes (MAOA; MAOB) encoding for MAO-A and MAO-B. The data are normalized to beta-2-microglobulin (b2m) as a housekeeping gene. The mean of MAOA expression is set as 1. (C) Load-free cell shortening is expressed as % shortening amplitude (dL) normalized to the diastolic cell length (L). Data are full ranges (whiskers) with median and 25 and 75% quartiles (boxes). Cells (isolated ventricular myocytes from mice) were incubated with selegiline (Sel; 1 µM; n = 26), β-phenylethylamine (PEA, 250 µM; n = 18), or combinations thereof (n = 27) for 24 h. Untreated controls (n = 18) were used to control the quality of the preparation. Cells were stimulated at 2 Hz and cell shortening was monitored by a line camera. (D) Similar experiment to C, but with rat myocytes (ARVM; n = 36 each).

• 

  Figure 3. Effect of 5-HT on load-free cell shortening. (A–C) Concentration–response curves for myocytes from MAO-B expressing mice myocytes (MAO-B+/+, n = 54–72), MAO-B knockout myocytes (MAO-B−/−, n = 65–72), and ARVM (n = 28–45). Cells were exposed to 5-HT for 24 h and cell shortening was subsequently analyzed as in Figure 1. (D) Effect of clorgyline (Clorg, 1 µM), 5-HT (100 µM), and combinations thereof (n = 45–108). Data are full ranges (whiskers) with median and 25 and 75% quartiles (boxes). *; p < 0.05 vs. control; Kruskal–Wallis test with Bonferroni (correction for multiple testing) post hoc analysis.

• 

  Figure 4. Effect of 5-HT on mitochondrial ROS production and ROS on cell shortening. (A) Rat mitochondrial ROS production. Mitochondria from rat hearts were isolated and ROS production was measured in the presence of complex I substrate, with 5-HT (concentration 1–100 µM) and/or clorgyline (1 µM) and 5-HT. Data are full ranges (whiskers) with median and 25 and 75% quartiles (boxes). *; p < 0.05 vs. control; Kruskal–Wallis test with Bonferroni (correction for multiple testing) post hoc analysis; n = 4–8. (B) Effect of Tempol (1 mmol/L) on cell shortening. Data are full ranges (whiskers) with median and 25 and 75% quartiles (boxes). *; p = 0.000192 vs. control; Student’s t-test; n = 54.

• 

  Figure 5. Effect of 5-HT on cell structure. (Top) Original photographs obtained from cultures exposed to clorgyline (1 µM), 5-HT (100 µM), or combinations thereof for 24 h (n = 5 each). For greater visibility of rod-shaped and round cells, see Supplementary Information. Data are expressed as the number of rod-shaped cells per mm2. Data are full ranges (whiskers) with median and 25 and 75% quartiles (boxes). *; p < 0.05 vs. control; Kruskal–Wallis test with Bonferroni (correction for multiple testing) post hoc analysis.

• 

  Figure 6. Effect of 5-HT on cell shortening of cardiomyocytes isolated from right (RV) or left (LV) ventricle. Cells (n = 27 (RV or 54 (LV) were exposed to 5-HT (100 µM) for 24 h and cell shortening was subsequently analyzed as in Figure 1. Data are full ranges (whiskers) with median and 25 and 75% quartiles (boxes). One-way ANOVA with Student–Newman–Keuls post hoc analysis. Different letters indicate group differences with p < 0.05.

• 

  Figure 7. Age-dependent regulation of left ventricular mRNA expression of genes involved in 5-HT metabolism or signaling ((A): MAOA, (B): MAOB; (C): SLC6A4; (D): HTR2A; (E): HTR2B; (F): CAT; (G): SSAO; (H): ALDH2). Left ventricular tissue was dissected from rats at the age of 1.5, 7.5, or 11.5 months (n = 6 each). Data are full ranges (whiskers) with median and 25 and 75% quartiles (boxes). One-way ANOVA with Student–Newman–Keuls post hoc analysis or Kruskal–Wallis test with Bonferroni (correction for multiple testing) post hoc analysis where appropriate. * p < 0.05 vs. 1.5 months old.

• 

  Figure 8. Hypertension-dependent regulation of left ventricular mRNA expression of genes involved in 5-HT metabolism or signaling ((A): MAOA, (B): MAOB; (C): SLC6A4; (D): HTR2A; (E): HTR2B; (F): CAT; (G): SSAO; (H): ALDH2). Left ventricular tissue was dissected from normotensive rats (WIS) or spontaneously hypertensive rats (SHR) at the age of 7.5 months (n = 6 each). T-tests or Mann–Whitney U-tests where appropriate. * p < 0.05 vs. WIS.

• 

  Figure 9. Effect of norepinephrine on cell structure and load-free cell shortening. (A) Original photographs obtained from cultures exposed to clorgyline (1 µM), norepinephrine (NOR, 10 µM), atwenolol (10 µM), or combinations thereof for 24 h (n = 4 each). For better visibility of rod-shaped and round cells see Supplementary Information. Data are expressed as the number of rod-shaped cells per mm2. Data are full ranges (whiskers) with median and 25 and 75% quartiles (boxes). One-way ANOVA with Student–Newman–Keuls post hoc analysis. Equal letters indicate no group differences. (B) Load-free cell shortening (see Figure 1 for details) for cells exposed to clorgyline (1 µM), norepinephrine (NOR, 10 µM), or combinations thereof for 24 h (n = 119–123). One-way ANOVA with Student–Newman–Keuls post hoc analysis. Equal letters indicate no group differences. (C) Load-free cell shortening (see Figure 1 for details) for cells exposed to atenolol (Ate, 10 µM), norepinephrine (NOR, 10 µM), or combinations thereof for 24 h (n = 97–121). One-way ANOVA with Student–Newman–Keuls post hoc analysis. Equal letters indicate no group differences.