Teaching old drugs new tricks: high-throughput drug screening identifies a novel, therapeutic combination for the potential treatment of atrial fibrillation-associated cardiac fibrosis
A M Johnston, C H K Yiu, L M Moreira, C Ma, V Millar, P Robinson, R Sayeed, G Krasopoulos, P Sastry, A Kourliouros, N R H Walcot, D Ebner, N Smart, S ReillyAbstract
Introduction
Atrial fibrillation (AF), the most common cardiac arrhythmia, is sustained by structural atrial remodelling, hallmarked by cardiac fibrosis. Current therapies (antiarrhythmics, catheter ablation, rate control strategies) primarily target electrical changes and have little effect on the fibrotic substrate. Therefore, there is an urgent need for disease-modifying treatments that can halt or reverse cardiac fibrosis. We uncovered that calcitonin (CT), produced by atrial cardiomyocytes, signals via the calcitonin receptor (CALCR) on adjacent atrial cardiac fibroblasts (ACFs) to suppress their pro-fibrotic activity.1 However, in AF, myocardial CT production is reduced and ACFs are refractory to CT due to excessive CALCR internalisation relative to sinus rhythm controls (A). We therefore screened for repurposable drugs that restore CALCR localisation, resensitise AF-derived ACFs to CT, and thereby suppress cardiac fibrosis.
Methods
Primary ACFs isolated from 3 persistent-AF donors (AF-ACFs) were screened in 384-well format (1250 cells/well) using an FDA-approved & Pharmacopeial Library (3019 compounds, 10 µM, 24h, in technical replicates). Cells were stained for CALCR, filamin-A, collagen-1, and nuclei. High-content imaging and an automated image analysis pipeline quantified a perinuclear-to-outer-cytoplasmic CALCR intensity ratio (P/C) (B). Compounds reducing cell count by ~50% (≥2 SD) were retested at 5, 1 & 0.25 µM. Hits were defined as compounds that reduced the P/C ratio by 2 SD. Top hits were validated in independent AF-ACFs: 24h drug pre-treatment, 48h co-treatment with CT ± sCT8-32 after which collagen 1 (Western blot) and total secreted collagen (Sirius Red assay) were measured. Data were analysed using the Friedman test.
Results
Compared to sinus rhythm controls, CALCR P/C ratios were significantly higher in the AF donor AF-ACFs with an average Z’-score for each plate of 0.31, indicating assay robustness. Of the 3000+ compounds screened, levamlodipine besylate (the stereoselective preparation of the antihypertensive amlodipine) emerged as a concordant hit in all 3 donors (C). Nine further compounds, including racemic amlodipine, were hits in 2/3 donors. In validation studies, levamlodipine and CT dual treatment for 48h reduced collagen 1 in AF-ACFs (D) and showed a trend for reduced total secreted collagen (E). Levamlodipine alone had no effect, and sCT8-32 (a CALCR antagonist) abrogated the response, confirming CALCR dependence.
Conclusions
This study identifies a clinically repurposable combination – levamlodipine and calcitonin – that restores CALCR signalling to suppress profibrotic activity in human AF-derived ACFs. Given (lev)amlodipine’s extensive clinical use in cardiovascular disease (hypertension, angina pectoris) and favourable safety profile, this strategy has promising translational potential. Ongoing rodent studies aim to further validate this combination therapy for cardiac fibrosis.Figure