Pathogenetic relevance of matrix metalloproteinase-2 and its potential as a pharmacological target in a patient-specific Anthracycline-induced cardiotoxicity stem cell model

Background and Aims: Doxorubicin (Dox) is used as an effective chemotherapeutic agent for the treatment of various oncological entities. Both, solid and hematopoietic cancers respond to the drug. However, acute and chronic damage to the heart, known as Anthracycline-induced cardiotoxicity (ACT), can occur as a serious side effect. On the one hand, ACT limits the use of Dox; on the other hand, it means an additional burden of disease for those treated, in the worst case, terminal heart failure with a high mortality risk. The exact pathomechanism of ACT is still unclear, meaning that pharmacological intervention is not yet possible. 

We developed a patient-specific stem cell model of DOX-induced cardiac dysfunction from patients with B-cell lymphoma and showed that DOX-induced stress resulted in arrhythmogenic events associated with contractile dysfunction and, finally, in heart failure. This study was performed to identify the role of matrix metalloproteinase-2 (MMP2) and its isoforms in the development of ACT and prove MMP2 as a suitable therapeutic patient-specific treatment target in vitro. 

 

Methods and Results: We used induced pluripotent stem cell-derived ventricular cardiomyocytes from individuals with CD20+ B-cell lymphoma who had received high doses of DOX and suffered cardiac dysfunction (ACT-iPSC-CMs) and compared these to control-iPSC-CMs from cancer survivors without cardiac symptoms. We were able to show that Dox-treatment caused an increase of MMP2-expression in all patient- and control-iPSC-CMs with significantly higher expression at Dox-concentrations of 0.5µM and 1µM (24 hours) (p < 0.05 at 0.5µM and 1µM Dox vs. 0µM Dox) in patient 2 compared to non-treated cells and to control-iPSC-CMs at the same conditions. Previous data have shown the existence of the N-terminal-76 amino acids truncated MMP2-isoform NTT76-MMP2 in addition to the full-length-MMP2-transcript in cardiomyocytes, responsible for cleaveage of its substrates Titin, TroponinI or SERCA.

We established a qPCR-based method to directly analyze the expression of the NTT76-MMP2 isoform in ACT-iPSC-CMs and detected a potential further human MMP2 isoform, lacking the 39 N-terminal amino acids. A comparison of all isoforms showed with increasing concentrations of DOX a decreasing expression of the full-length protein. However, shorter isoforms are increased expressed with DOX treatment in patient 2. Since it is known that reactive Oxygen Species (ROS) are induced by DOX treatment, we analyzed DOX-dependently H2O2 amounts in ACT-iPSC-CMs and found a significant increase in ROS at 0.5µM Dox. The elevated H2O2 could be reduced by the concurrent treatment with the MMP2-inhibitor Ono-4817 or by the ROS-scavanger N-Acetylcysteine suggesting a MMP2-dependent ROS production. As elevated ROS are known as a stimulus for MMP2-activation and NTT76-MMP2-De-novo-expression, ROS-reduction can protect cellular proteins from strong MMP2-cleavage. Since we have shown in a former study that DOX also influences the mitochondrial network, we tested the protective effect of ONO. We were able to show, that DOX-induced decreased mitochondrial network can be ameliorated by Ono. 

Conclusion: 

In conclusion, we demonstrated the considerable role of MMP2 in the context of patient-specific Dox-induced ACT. Therefore, MMP2 and its newly identified isoforms might be suitable therapeutic patient-specific treatment targets in anthracycline-induced cardiotoxicity.