Background: Doxorubicin (Dox) is an anti-cancer drug with severe cardiac side effects. Accompanying cardiotoxicity is cardiac fibrosis as a result of the treatment. Of note, Dox treatment increases cardiac long chain ceramide levels with proinflammatory effects complemented with cell death and fibrosis.

Aim: The purpose of our study was to identify ceramide signaling pathways and characterize downstream effects on cell survival and metabolism and the progression of fibrosis.

Methods: Human fibroblasts were treated with 0.7 µM Dox for 24h. In parallel cells were preincubated with MitoTempo (20 µM, 2h, MT) or Fumonisin B (100 µM, 4h, FuB) to mitigate Dox effects. Expression of genes and proteins were measured with immunofluorescence (IF), Western Blot (WB) or qPCR. Ceramide levels were determined with mass spectrometry. Illustration was done with either confocal laser scanning microscopy or electron microscopy. Mitochondrial viability and activity were measured with seahorse analysis.

Results: Protein expression measurement revealed significantly increased level of CerS2 (1.5 ± 0.19, p<0.001 for IF, 2.0 ± 0.2, p=0.002 for WB). Coherently, long chain and very long chain ceramides (C14- C24:1) were increased in Dox treated cells (1.39 ± 0.01, p<0.001) with the highest peak for C24:0 (1.64 ± 0.02, p=0.02). FuB pretreatment reduced most long chain and very long chain ceramides to non- significant level. Dox increased mRNA levels of IL-6 (2.95 ± 0.26, p<0.001) and IL-1β (12.12 ± 0.10, p<0.001), as well as mitochondrial ROS production (3.41 ± 0.10, p<0.001). Cytokine elevation was reduced with FuB (1.66 ± 0.29, p= 0.08 for IL-6, 7.06 ± 0.67, p=0.002 for IL-1β), while mitochondrial ROS level could be decreased using either MT (1.75 ± 0.20, p<0.001) or FuB (1.61 ± 0.29, p<0.001). Mitochondrial impairment was assessed with MitoTracker staining, electron microscopy, qPCR and seahorse analysis. qPCR revealed reduction of the mitochondria fusion genes MFN1 (0.80 ± 0.14, p=0.002) and OPA1 (0.66 ± 0.18, p=0.002), while seahorse analysis verified mitochondrial damage through reduced ATP production (0.31 ± 0.44, p<0.001). MT and FuB pretreatment reduced Dox effects but were not strong enough to completely reverse the observed effects. Fibrosis- associated genes MMP8 (3.30 fold ± 0.2 p=0.0002), MMP9 (2.89 fold ± 0.5 p=0.01) and MMP14 (1.53 fold ± 0.24 p=0.02) showed elevated mRNA expression levels in Dox treated cells.

Conclusion: Our data suggest that doxorubicin impairs mitochondrial function by an increase in proinflammtory long chain ceramides followed by increased mitochondrial ROS production. Further, it promotes fibrosis and fibroblast activation. The regulation of ceramide levels and side effects caused by doxorubicin might control long term effects in patients undergoing anti-cancer treatment and prevent heart failure progression.