A Catecholaldehyde Metabolite of Norepinephrine Induces Myofibroblast Activation and Toxicity via the Receptor for Advanced Glycation Endproducts: Mitigating Role of l-Carnosine

Abstract

Monoamine oxidase (MAO) is rapidly gaining appreciation for its pathophysiologic role in cardiac injury and failure. Oxidative deamination of norepinephrine by MAO generates H₂O₂ and the catecholaldehyde 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), the latter of which is a highly potent and reactive electrophile that has been linked to cardiotoxicity. However, many questions remain as to whether catecholaldehydes regulate basic physiological processes in the myocardium and the pathways involved. Here, we examined the role of MAO-derived oxidative metabolites in mediating the activation of cardiac fibroblasts in response to norepinephrine. In neonatal murine cardiac fibroblasts, norepinephrine increased reactive oxygen species (ROS), accumulation of catechol-modified protein adducts, expression and secretion of collagens I/III, and other markers of profibrotic activation including STAT3 phosphorylation. These effects were attenuated with MAO inhibitors, the aldehyde-scavenging dipeptide l-carnosine, and FPS-ZM1, an antagonist for the receptor for advanced glycation endproducts (RAGE). Interestingly, treatment of cardiac fibroblasts with a low dose (1 μM) of DOPEGAL-modified albumin phenocopied many of the effects of norepinephrine and also induced an increase in RAGE expression. Higher doses (>10 μM) of DOPEGAL-modified albumin were determined to be toxic to cardiac fibroblasts in a RAGE-dependent manner, which was mitigated by l-carnosine. Collectively, these findings suggest that norepinephrine may influence extracellular matrix remodeling via an adrenergic-independent redox pathway in cardiac fibroblasts involving the MAO-mediated generation of ROS, catecholaldehydes, and RAGE. Furthermore, since elevations in the catecholaminergic tone and oxidative stress in heart disease are linked with cardiac fibrosis, this study illustrates novel drug targets that could potentially mitigate this serious disorder.

Figure 1

Catechol-modified adducts and oxidative stress in CFs following NE treatment. Reaction schematic showing norepinephrine (NE) is metabolized by MAO-A to produce DOPEGAL and H₂O₂, and then the catechol-modified protein adducts in cell lysates are captured using aminophenylboronic acid (APBA)-coated resin and quantified by a BCA assay (A). Cardiac fibroblasts (CFs) were treated for 96 h with NE (20 μM) alone or concurrently with MAOI (clorgiline + selegiline, 1 μM) or l-carnosine (10 μM), and the catechol-modified proteins from the lysate were isolated with APBA resin and quantified by a BCA assay (n = 3) (B). CFs were treated with NE (5 μM) for 48 h alone or concurrently with RAGE-antagonist FPS-ZM1 (223 nM), MAOIs, or l-carnosine as above, and the cytosolic ROS was visualized with a CellROX Deep Red reagent and then quantified using ImageJ and normalized to the nuclear Hoechst stain (n = 3) (C). *P < 0.05 versus vehicle control and †P < 0.05 versus the NE-treated group.

Figure 2

Redox-dependent effects of NE on CF activation and profibrotic phenotype. Representative immunoblot of STAT3 phosphorylation in the cell lysate from CFs treated for 6 h with NE (1 μM) alone or concurrently with MAOIs (clorgiline + selegiline, 1 μM), FPS-ZM1 (223 nM), or l-carnosine (10 μM) (A). CF proliferation after NE treatment for 48 h alone or concurrently with MAOIs or l-carnosine (B). Expression of Col1a1 (C), Cola1a3 (D), and α smooth muscle actin (E) in CFs treated for 6 h with NE alone or concurrently with MAOIs, FPS-ZM1, or l-carnosine. Collagen I (F) and III (G) secretion by CFs following treatment with NE alone for 96 h or concurrently with MAOIs, FPS-ZM1, or l-carnosine. *P < 0.05 versus vehicle control and ^†P < 0.05 versus the NE alone group. Data are representative of N = 3–6 with a minimum of 2 replicates for each experiment.

Figure 3

Proinflammatory, profibrotic effect of exogenous DOPEGAL–BSA adducts in CFs. The proinflammatory effect after treatment of CFs with DOPEGAL–BSA adducts (1 μM) alone or concurrently with FPS-ZM1 (223 nM) or l-carnosine (10 μM) on RAGE protein after 72 h (A) and mRNA (B) and TNFα expression after 48 h (C) are shown. Expression of Col1a1 (D), Cola1a3 (E), and αSMA (F) in CFs treated for 3 h with DOPEGAL–BSA alone or concurrently with FPS-ZM1 or l-carnosine. Collagen I (G) and III (H) secretion by CFs following treatment with DOPEGAL–BSA alone for 96 h or concurrently with FPS-ZM1, NOX inhibitor apocynin (2 μM), or l-carnosine. *P < 0.05 versus vehicle control and †P < 0.05 versus the DOPEGAL–BSA-treated group. Data are representative of N = 3–4 with a minimum of 2 replicates for each experiment.

Figure 4

Toxicity of exogenous DOPEGAL–BSA adducts in CFs. Representative images (A) showing the dose-dependent toxicity of DOPEGAL–BSA in CFs using calcein AM (green) to stain living cells and ethidium homodimer-1 (red) to stain dead cells, and the overall ratio of live/dead cells was quantified at each DOPEGAL–BSA concentration (B). CFs were treated with a high dose of DOPEGAL–BSA adduct (200 μM) alone or concurrently with FPS-ZM1 (223 nM), apocynin (2 μM), or l-carnosine (100 μM), and the cells were imaged (C) and quantified (D) using the same technique. *P < 0.001 versus vehicle control, **P < 0.0001 versus vehicle control, and †P < 0.05 versus the DOPEGAL–BSA-treated group. Data are representative of N = 3–4 with a minimum of 2 replicates for each experiment.