5-Hydroxytryptophan acts as a gap junction inhibitor to limit the spread of chemotherapy-induced cardiomyocyte injury and mitochondrial dysfunction

Abstract

Anthracycline chemotherapeutics like doxorubicin (DOX) are widely used against various cancers but are accompanied by severe cardiotoxic effects that can lead to heart failure. Through whole transcriptome sequencing and pathological tissue analysis in a murine model, our study has revealed that DOX impairs collagen expression in the early phase, causing extracellular matrix anomalies that weaken the mechanical integrity of the heart. This results in ventricular wall thinning and dilation, exacerbating cardiac dysfunction. In this work, we have identified 5-hydroxytryptophan (5-HTP) as a potent inhibitor of gap junction communication. This inhibition is key to limiting the spread of DOX-induced cardiotoxicity. Treatment with 5-HTP effectively countered the adverse effects of DOX on the heart, preserving ventricular structure and ejection fraction. Moreover, 5-HTP enhanced mitochondrial respiratory function, as shown by the O2k mitochondrial function assay, by improving mitochondrial complex activity and ATP production. Importantly, the cardioprotective benefits of 5-HTP did not interfere with DOX's ability to combat cancer. These findings shed light on the cardiotoxic mechanisms of DOX and suggest that 5-HTP could be a viable strategy to prevent heart damage during chemotherapy, offering a foundation for future clinical development. This research opens the door for 5-HTP to be considered a dual-purpose agent that can protect the heart without compromising the oncological efficacy of anthracycline chemotherapy.

Keywords: 5-hydroxytryptophan; cardiotoxicity; chemotherapy; collagen; gap junction.

Figure 1

DOX inhibits collagen synthesis in cardiomyocytes and causes progressive thinning of the ventricular wall. (A) heat map of differential expressed collagen genes in the control group versus DOX group; (B) scatter plot of Reactome enrichment analysis of down regulated genes affected by DOX; (C) scan of heart tissue sections after HE staining; (D) statistical illustration of ventricular wall thickness and ventricular size in the control and DOX group. ^*P < 0.05 vs. the Control group, ^#P < 0.05 vs. the DOX group.

Figure 2

5-HTP ameliorates DOX-induced cardiac dysfunction of myocardial tissue in mice. (A) Bodyweight of mice on the last day of the experiment; (B) Morphology of the mice heart; (C) Representative echocardiographic images; (D) Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS); (E) Left ventricular internal diastolic end-systolic (LVIDs) and left ventricular internal diastolic end-diastolic (LVIDd); (F) Heart rate (HR); ^*P < 0.05 vs. the Control group, ^#P < 0.05 vs. the DOX group. (L-5-HTP group: L-5-HTP+DOX; M-5-HTP group: M-5-HTP+DOX; H-5-HTP group: H-5-HTP+DOX).

Figure 3

5-HTP ameliorates DOX-induced morphological damage of myocardial tissue in mice. (A) Scans of heart tissue sections; (B) The results from HE staining, bar = 100 μm; (C, D) The results and extent of fibrosis from MASSON staining, bar = 50 μm; ^*P < 0.05 vs. the Control group, ^#P < 0.05 vs. the DOX group. (L-5-HTP group: L-5-HTP+DOX; M-5-HTP group: M-5-HTP+DOX; H-5-HTP group: H-5-HTP+DOX).

Figure 4

5-HTP ameliorates doxorubicin-induced cardiotoxicity through inhibition of gap junctions. (A) Scatter plot of KEGG enrichment analysis of down-regulated genes by H-5-HTP group; (B) Scatter plot of Reactome enrichment analysis of down-regulated genes by H-5-HTP group; (C) Relative levels of mRNA for gap junction-related genes; (D) IL-1β immunohistochemistry results, bar = 100 μm; (E) Cx43 immunofluorescence results, bar = 100 μm; (F) Fluorescence intensity of Cx43 immunofluorescence; ^*P < 0.05 vs. the Control group, ^#P < 0.05 vs. the DOX group. (L-5-HTP group: L-5-HTP+DOX; M-5-HTP group: M-5-HTP+DOX; H-5-HTP group: H-5-HTP+DOX).

Figure 5

5-HTP significantly improves doxorubicin-induced oxidative damage in H9c2 cells. (A) Fluorescence microscopy image of intracellular calcium ions, bar = 100 μm; (B) DCFH-DA probe detects reactive oxygen species in each group of cells; (C) Intracellular MDA levels in different treatment groups; (D) LDH release in the supernatant is shown. ^*P < 0.05 vs. the Control group, ^#P < 0.05 vs. the DOX group.

Figure 6

5-HTP significantly improves doxorubicin-induced mitochondrial dysfunction in mice hearts. (A) Scatter plot of KEGG enrichment analysis of upregulated genes by H-5-HTP group; (B) Changes in oxygen consumption in mouse heart tissue in the different intervention groups; (C) Respiration rate of mouse heart tissue in state 4, state 3 and after addition of oligomycin in different treatment groups; (D) Changes in control rate of mouse heart respiration. ^*P < 0.05 vs. the Control group, ^#P < 0.05 vs. the DOX group. (H-5-HTP group: H-5-HTP+DOX).

Figure 7

Cell viability of MCF-7 and U2OS cells with different DOX and 5-HTP treatments. (A) DOX-induced antineoplastic activities of 5-HTP in MCF-7 cells. (B) DOX-induced antineoplastic effects of 5-HTP in U2OS cells. ^*P < 0.05 vs. the Control group, ^#P < 0.05 vs. the DOX group.