Monoamine oxidase-dependent endoplasmic reticulum-mitochondria dysfunction and mast cell degranulation lead to adverse cardiac remodeling in diabetes

Fig. 1. Effects of MAO inhibition on HG and pro-inflammatory cytokine IL-1β induced ROS formation in NRVMs. ROS formation was measured with genetically encoded H2O2 sensitive probe HyPer targeted either to mitochondria (a, b) or to the cytosol (d, e) upon NRVMs treatment with NG or HG in the absence or presence of IL-1β, and with or without pargyline. Effects of HM, as osmotic control, were assessed on mitochondrial (c) and cytosolic (f) H2O2 formation. MAO-A expression was assessed at different time-points following control or MAO-A siRNA treatment (g, h). ROS formation in control or MAO-A siRNA-treated cells was assessed upon treatment with NG or HG in the absence or presence of IL-1β (i). Approximately 100 cells were analyzed per condition in each experiment and all the experiments were performed at least three times. Data are expressed as mean ± SEM. Two-way ANOVA test followed by post hoc Tukey’s (b and i) or Dunn’s (e) multiple comparison test (*p < 0.05 vs NG vehicle, #p < 0.05 vs HG vehicle and §p < 0.05 vs HG-IL1β)

Fig. 2. Effects of MAO inhibition on ROS formation and ΔΨm in cardiac myocytes exposed to HG and/or IL-1β. ROS formation was assessed with the fluorescent dye MTR in adult cardiomyocytes treated with NG or HG in the absence or presence of IL-1β, and with or without pargyline (a, b). Effects of the osmotic control HM on ROS formation (c, f). ΔΨm was measured in adult cardiomyocytes with the fluorescent dye TMRM in the same conditions as above (d–f). In neonatal cardiomyocytes, ΔΨm was measured following 48 h treatment with NG or HG in the absence or presence of IL-1β (g). Kinetic measurements were performed to follow TMRM fluorescence intensity following additions of oligomycin and FCCP, indicated by arrows (h). Approximately 100 cells were analyzed per condition in each experiment and all the experiments were performed three times. Data are expressed as mean ± SEM. Two-way ANOVA test followed by post hoc Tukey’s (b) or Dunn’s (e) multiple comparison test (*p < 0.05 vs NG vehicle, #p < 0.05 vs HG and §p < 0.05 vs HG + IL-1β vehicle)

Fig. 3. Mitochondrial respiration and PTP opening in cardiomyocytes exposed to HG and/or IL-1β. Oxygen consumption at baseline (S4) and following addition of ADP (S3) was measured in permeabilized neonatal (a, n = 3) or adult cardiomyocytes, using either glutamate/malate (b, n = 4) or succinate in the presence of rotenone (c, n = 3) as substrates. The effect of the PTP inhibitor CsA on ΔΨm was assessed in neonatal cardiomyocytes (d) upon 48 h treatment with NG or HG and IL-1β in presence of oligomycin. Adult cardiomyocytes (e) were treated with NG or HG and IL-1β for 5 h, in the presence and absence of CsA. For TMRM experiments, between 50 and 100 cells were analyzed per condition in each experiment and all the experiments were performed at least three times. Data are expressed as mean ± SEM. Two-way ANOVA test followed by post hoc Dunn’s multiple comparison test (*p < 0.005 vs NG, #p < 0.05 vs NG + IL-1β, §p < 0.005 vs respective group treated with vehicle)

Fig. 4. Effects of MAO inhibition on ER stress in adult cardiomyocytes exposed to HG and/or IL-1β. Representative western blots (a) and quantification of the protein expression of ER stress markers GRP78 (b), GADD34 (c), ATF4 (d), and phosphorylation levels of IRE1α (e) were assessed in adult cardiomyocytes treated with HG and IL-1β with (white) or without (black) pargyline for 48 h. Protein expression was normalized to total protein determined by Red Ponceau staining. Effects of the MAO inhibitor pargyline and the chemical chaperone TUDCA on tunicamycin-induced upregulation of ER stress markers (f, n = 3 per each group). All the experiments were performed at least three times. Data are expressed as mean ± SEM. Two-way ANOVA test followed by post hoc Tukey’s multiple comparison test (*p < 0.05 vs NG vehicle, #p < 0.05 vs HG and §p < 0.05 vs HG + IL-1β vehicle, †p < 0.005 vs respective control, ‡p < 0.005 vs Tunica)

Fig. 5. Characterization of LV function, oxidative and ER stress in vehicle- and pargyline-treated STZ-mice. Representative pressure-volume loops from control (blue, n = 6), STZ (black, n = 8), control + pargyline (green, n = 4) and STZ + pargyline (red, n = 10) treated mice (a). Ejection fraction (b) and diastolic stiffness (c) were determined in vehicle (black bars) or pargyline (white bars) treated control and STZ mice. Levels of 4-HNE (d, e) were assessed in heart tissue lysates from control, STZ and STZ+pargyline treated mice. Protein levels were normalized to total protein content determined by Red Ponceau staining (n = 5 per each group). Representative Western blot for ER stress markers performed in heart tissue lysates from control, STZ and STZ+pargyline treated mice (f). ATF4 (g) and GADD34 (h) band intensities were quantified and normalized to total protein content determined by Red Ponceau staining (n = 5 per each group). PV loop analysis was carried out by an observer blinded to the experimental conditions. Data are expressed as mean ± SEM. Two-way ANOVA test followed by post hoc Tukey’s multiple comparison test (*p < 0.05 vs CT vehicle, #p < 0.05 vs STZ vehicle)

Fig. 6. Effects of MAO inhibition on cardiac fibrosis and mast cell degranulation in STZ-mice. Representative images of Masson’s trichrome staining from control and STZ mice, showing collagen deposition in blue (a). The quantification data are expressed as percentage of fibrotic vs total cardiac tissue (b). Black and white bars represent vehicle and pargyline treated mice, respectively (Control n = 10, control+pargyline n = 5, STZ n = 12, STZ+pargyline n = 15). Representative images of toluidine blue staining of cardiac tissue from indicated experimental groups (c). Red arrows indicate intact mast cells, and black arrows indicate actively degranulating mast cells. Quantification of mast cell density (d) and mast cell degranulation (e). Results are expressed as number of mast cells per field and percentage of degranulating mast cells vs total number of mast cells, respectively. (Control n = 10, control+pargyline n = 5, STZ n = 12, STZ+pargyline n = 15). Analysis was carried out by an observer blinded to the experimental conditions. Data are expressed as mean ± SEM. Two-way ANOVA test followed by post hoc Tukey’s (d and e) or Dunn’s (b) multiple comparison test (*p < 0.05 vs control…

Fig. 7. Schematic representation of the effects elicited by MAO-dependent oxidative stress in diabetic cardiomyopathy. Hyperglycemia and pro-inflammatory stimuli result in enhanced mitochondrial MAO-dependent H2O2 formation that, in turn, targets mitochondria to induce mitochondrial dysfunction. These events occur upstream of the impairment in ER homeostasis and accumulation of unfolded proteins in the ER lumen. The consequent activation of the UPR through three ER stress branches, namely IRE1α, PERK and ATF6, leads to protein translation attenuation, upregulation of chaperones and activation of ERAD machinery. Moreover, MAO-dependent oxidative stress in vivo contributes to mast cell degranulation and cardiac fibrosis, ultimately resulting in diastolic dysfunction in type 1 diabetes. Administration of the MAO inhibitor pargyline prevents exacerbated ROS formation and restores mitochondrial and ER homeostasis. In addition, MAO inhibition abolishes mast cell degranulation and fibrosis, thus improving LV diastolic function. ATF4 activation transcription factor 4, ATF6 activation transcription factor 6, BiP binding immunoglobulin protein, ER endoplasmic reticulum, ERAD ER associated…