RESUMO
BACKGROUND: The anthracycline doxorubicin (DOX) is a highly effective anticancer agent, especially in breast cancer and lymphoma. However, DOX can cause cancer therapy-related cardiovascular toxicity (CTR-CVT) in patients during treatment and in survivors. Current diagnostic criteria for CTR-CVT focus mainly on left ventricular systolic dysfunction, but a certain level of damage is required before it can be detected. As diastolic dysfunction often precedes systolic dysfunction, the current study aimed to identify functional and molecular markers of DOX-induced CTR-CVT with a focus on diastolic dysfunction. METHODS: Male C57BL/6J mice were treated with saline or DOX (4 mg/kg, weekly i.p. injection) for 2 and 6 weeks (respectively cumulative dose of 8 and 24 mg/kg) (n = 8 per group at each time point). Cardiovascular function was longitudinally investigated using echocardiography and invasive left ventricular pressure measurements. Subsequently, at both timepoints, myocardial tissue was obtained for proteomics (liquid-chromatography with mass-spectrometry). A cohort of patients with CTR-CVT was used to complement the pre-clinical findings. RESULTS: DOX-induced a reduction in left ventricular ejection fraction from 72 ± 2% to 55 ± 1% after 2 weeks (cumulative 8 mg/kg DOX). Diastolic dysfunction was demonstrated as prolonged relaxation (increased tau) and heart failure was evident from pulmonary edema after 6 weeks (cumulative 24 mg/kg DOX). Myocardial proteomic analysis revealed an increased expression of 12 proteins at week 6, with notable upregulation of SERPINA3N in the DOX-treated animals. The human ortholog SERPINA3 has previously been suggested as a marker in CTR-CVT. Upregulation of SERPINA3N was confirmed by western blot, immunohistochemistry, and qPCR in murine hearts. Thereby, SERPINA3N was most abundant in the endothelial cells. In patients, circulating SERPINA3 was increased in plasma of CTR-CVT patients but not in cardiac biopsies. CONCLUSION: We showed that mice develop heart failure with impaired systolic and diastolic function as result of DOX treatment. Additionally, we could identify increased SERPINA3 levels in the mice as well as patients with DOX-induced CVT and demonstrated expression of SERPINA3 in the heart itself, suggesting that SERPINA3 could serve as a novel biomarker.
RESUMO
Doxorubicin (dox) is an affordable, and highly effective chemotherapeutic agent used in cancer treatment, yet its application is known to cause cumulative cardiac and renal toxicity. In this study, we employed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to evaluate the distribution of dox in mouse heart and kidney after in vivo treatment. To this end, we performed absolute quantification using an isotopically labeled form (13C d3-dox) as an internal standard. Unfortunately, ion suppression often leads to loss of sensitivity in compound detection and can result in hampered drug quantification. To overcome this issue, we developed an on-tissue chemical derivatization (OTCD) method using Girard's reagent T (GirT). With the developed method, dox signal was increased by two orders of magnitude. This optimized sample preparation enabled a sensible gain in dox detection, making it possible to study its distribution and abundance (up to 0.11 pmol/mm2 in the heart and 0.33 pmol/mm2 in the kidney medulla). The optimized approach for on-tissue derivatization and subsequent quantification creates a powerful tool to better understand the relationship between dox exposure (at clinically relevant concentrations) and its biological detrimental effects in various tissues. Overall, this work is a showcase of the added value of MALDI-MSI for pharmaceutical studies to better understand heterogeneity in drug exposure between and within organs.
