Pyridoxamine Limits Cardiac Dysfunction in a Rat Model of Doxorubicin-Induced Cardiotoxicity

Abstract

The use of doxorubicin (DOX) chemotherapy is restricted due to dose-dependent cardiotoxicity. Pyridoxamine (PM) is a vitamin B6 derivative with favorable effects on diverse cardiovascular diseases, suggesting a cardioprotective effect on DOX-induced cardiotoxicity. The cardioprotective nature of PM was investigated in a rat model of DOX-induced cardiotoxicity. Six-week-old female Sprague Dawley rats were treated intravenously with 2 mg/kg DOX or saline (CTRL) weekly for eight weeks. Two other groups received PM via the drinking water next to DOX (DOX+PM) or saline (CTRL+PM). Echocardiography, strain analysis, and hemodynamic measurements were performed to evaluate cardiac function. Fibrotic remodeling, myocardial inflammation, oxidative stress, apoptosis, and ferroptosis were evaluated by various in vitro techniques. PM significantly attenuated DOX-induced left ventricular (LV) dilated cardiomyopathy and limited TGF-β1-related LV fibrotic remodeling and macrophage-driven myocardial inflammation. PM protected against DOX-induced ferroptosis, as evidenced by restored DOX-induced disturbance of redox balance, improved cytosolic and mitochondrial iron regulation, and reduced mitochondrial damage at the gene level. In conclusion, PM attenuated the development of cardiac damage after DOX treatment by reducing myocardial fibrosis, inflammation, and mitochondrial damage and by restoring redox and iron regulation at the gene level, suggesting that PM may be a novel cardioprotective strategy for DOX-induced cardiomyopathy.

Keywords: anthracyclines; cardioprotection; cardiotoxicity; inflammation; mitochondria; preclinical study; pyridoxamine; redox biology.

Figure 1

PM reduces DOX-induced fibrotic remodeling. (a) Representative images of LV tissue stained with H&E and quantification of cross-sectional cardiomyocyte area (CSA) in CTRL (N = 7), DOX (N = 5), DOX+PM (N = 9), and CTRL+PM (N = 7) animals. The dashed line represents an elliptical representation of one cardiomyocyte. (b) Quantification of the expression of pro-apoptotic genes p53 and BAX in LV tissue from CTRL (N = 3–4), DOX (N = 5), DOX+PM (N = 8), and CTRL+PM (N = 5) animals. (c–e) Representative images of LV tissue stained with Sirius Red/Fast Green (c,d) and quantification of the percentage of interstitial and perivascular collagen deposition (e) in CTRL (N = 7), DOX (N = 5), DOX+PM (N = 9), and CTRL+PM (N = 7) animals. (f) Quantification of the expression of type I collagen, type III collagen, LOX, and TGF-β1 in LV tissue from CTRL (N = 4), DOX (N = 4–5), DOX+PM (N = 8), and CTRL+PM (N = 5) animals. The data are shown as mean ± SEM. DOX, doxorubicin. LV, left ventricular. PM, pyridoxamine.

Figure 2

PM reduces DOX-induced myocardial inflammation. (a,b) Representative images of LV tissue from DOX-treated rats stained with H&E (a) and semi-quantification of leukocytes in LV tissue from CTRL (N = 7), DOX (N = 5), DOX+PM (N = 7), and CTRL+PM (N = 7) animals (b). (c) Representative images and semi-quantification of CD68+ cells in LV tissue sections from CTRL (N = 6), DOX (N = 5), DOX+PM (N = 9), and CTRL+PM (N = 7) animals. (d) Quantification of CD68, IL-6, and IL-1β gene expression in LV tissue from CTRL (N = 4–5), DOX (N = 4–5), DOX+PM (N = 8), and CTRL+PM (N = 5–6) animals. The data are shown as mean ± SEM. DOX, doxorubicin. LV, left ventricular. PM, pyridoxamine.

Figure 3

PM restores DOX-induced disturbances of redox and iron regulation. (a,b) Representative images and quantification of nitrotyrosine staining (a) and 4-hydroxynonenal (4-HNE) staining (b) in LV tissue from CTRL (N = 7), DOX (N = 5), DOX+PM (N = 9), and CTRL+PM (N = 7) animals. (c–e) Quantification of the expression of genes involved in redox regulation (NF-κB, NRF-2, NQO1, and SOD2) (c,d) and iron handling (ZIP14, FTH1, MFRN-1, and FPN) (e) in LV tissue from CTRL (N = 4–5), DOX (N = 4–5), DOX+PM (N = 8), and CTRL+PM (N = 4–6) animals. The data are shown as mean ± SEM. DOX, doxorubicin. LV, left ventricular. PM, pyridoxamine.

Figure 4

PM attenuates DOX-induced mitochondrial damage. (a) Representative images of toluidine blue-stained LV tissue showing clear distortion of cardiomyocyte organization in the DOX group. (b) Quantification of mitochondrial area relative to total cell area in CTRL (ncells = 108), DOX (ncells = 44), DOX+PM (ncells = 77), and CTRL+PM (ncells = 60) cardiomyocytes. (c) Representative transmission electron microscopy (TEM) pictures of LV cardiomyocytes. In the DOX group, cardiomyocytes showed swollen mitochondria, disrupted cristae, autophagic vacuoles containing damaged mitochondria, cytoplasmic vacuoles, and condensed chromatin, which was less pronounced in the DOX+PM group. Cardiomyocytes of CTRL and CTRL+PM groups showed typical intracellular organization of myofilaments (light grey), mitochondria (dark grey), and cytoplasm (white). Magnification: 6000×. Scale bars: 2 μm. (d) Quantification of percentage mitochondrial density, calculated as the mitochondrial number relative to the total cell area, and mitochondrial fraction, expressed as a percentage of total grid points in CTRL (ncells = 32–34), DOX (ncells = 20), DOX+PM (ncells = 44), and CTRL+PM (ncells = 34–35) cardiomyocytes. (e) Quantification of myofilament and cytoplasm fraction, expressed as a percentage of total grid points, in CTRL (ncells = 34), DOX (ncells = 20), DOX+PM (ncells = 43–44), and CTRL+PM (ncells = 33–35) cardiomyocytes. For (b,d,e): NCTRL = 7, NDOX = 4, NDOX+PM = 9, NCTRL+PM = 5–7. The data are shown as mean ± SEM. DOX, doxorubicin. LV, left ventricular. PM, pyridoxamine.

Figure 5

PM partially prevents the DOX-induced shift of genes related to inflammation, apoptosis, fibrosis, and oxidative stress. (a) PCA score plots of the first two principal components (PC) for qPCR gene expression of LV tissue samples from CTRL (N = 4), DOX (N = 4–5), DOX+PM (N = 8), and CTRL+PM (N = 4–5) animals. (b) Heatmap and hierarchical clustering show differentially expressed genes in LV tissue of the four experimental groups. Each column contains the average gene expression in LV tissue samples from CTRL (N = 4), DOX (N = 4–5), DOX+PM (N = 8), and CTRL+PM (N = 4–5) animals. Relative gene expression is indicated by color code: up-regulation (red) and down-regulation (blue). DOX, doxorubicin. LV, left ventricular. PCA, principle components analysis. PM, pyridoxamine.