Cardiac pressure overload exposes hidden anthracycline-induced cardiotoxicity: results from a preclinical large-animal trial
Authors: Galan C , Bautista B , Higuero-Verdejo M I , Cadiz L , Lopez-Martin G , Ayaon A , De Molina Iracheta A , Perez C , Villena R , Oliver E , Sanchez-Gonzalez J , Fuster V , Ibanez B 
Published: October 2022

Abstract

Aim
The purpose of this work is to elucidate if low cumulative doxorubicin doses has a differential cardiac impact in pigs with or without pre-existing LV pressure overload. Impact of interventions on LV anatomy, function and tissue composition was evaluated by serial cardiac magnetic resonance (CMR). At the end of the in vivo protocol, hearts were processed to evaluate differential molecular pathways in both groups.

Methods
A total of 95 pigs were included. 52 animals underwent supravalvular aortic banding to generate LV hypertrophy, while another did not. 4 months after banding (a time where LV hypertrophy is prominent), animals in both groups were randomized to receive intravenous doxorubicin (1 mg/kg once per week, for 5 weeks) or saline. Thus, 4 study groups (N=15–22 per group) comprised the study population (banding yes/no, doxorubicin yes/no). All individuals underwent to serial Cardiac Magnetic Resonance exams every month and were sacrificed fo ex vivo analysisat 4 months after doxorubicin injections. To discriminate between doxorubicin effect in banding/no banding groups but also the effect of banding in doxorubicin protocols, t-test was used for two-group comparisons after two-way ANOVA test. Differences were considered statistically significant at p&lt;0.05. Logrank test was performed to compare survival curves.

Results
Pigs receiving doxorubicin after cardiac pressure overload showed an increased mortality compared with the other groups (p=0.006). Only this group developed a progressive cardiotoxicity phenotype, characterized as a significant decline in cardiac function compared with control operated animals (49.7% ± 8.1 vs 79.6% ± 6.7, p&lt;0.001), but also with non-operated pigs receiving the same anthracycline regime (vs 63.9% ± 4, p&lt;0.001). Cardiac deterioration was based mainly on systolic dysfunction compared with control operated animals (38.2 ml indexed ± 3.5 vs 13.6±6.6, p=0.01) and non-operated pigs treated with doxorubicin (vs 26.6 ml indexed ± 6.6, p&lt;0.001). In-vivo CMR tissue characterization revealed elevation on T2 relaxation times compared with Control animals (46.2 ms ± 3.5 vs 41.8±2.7, p=0.04) with no Extracellular Volume expansion. Ex-vivo histological analyses showed presence of intra-cardiomyocyte vacuolization in the Banding + DOXO group (p=0.01). Regarding mitochondrial phenotype, animals with Banding + DOXO exhibited a fragmented phenotype with extremely damaged mitochondria. Animals with only aortic banding exhibit increasing cardiac hyperkinesia and cardiac hypertrophy at the end of the study.

Conclusion(s)
We demonstrated that a very low dosage of doxorubicin is enough to exert cardiotoxicity in a pressure overload scenario. Characterized as a deterioration in cardiac function, myocardial edema, classical histological lesions and mitochondrial damage. Providing preclinical evidence that modest anthracycline protocols may be harmful for patients with cardiac comorbidities.

Funding Acknowledgement
Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ERC MATRIX