A Closed Circulation Langendorff Heart Perfusion Method for Cardiac Drug Screening

Abstract

Cardiovascular diseases represent an economic burden for health systems accounting for substantial morbidity and mortality worldwide. Despite timely and costly efforts in drug development, the cardiovascular safety and efficacy of the drugs are not always fully achieved. These lead to the drugs' withdrawal with adverse cardiac effects from the market or in the late stages of drug development. There is a growing need for a cost-effective drug screening assay to rapidly detect potential acute drug cardiotoxicity. The Langendorff isolated heart perfusion technique, which provides cardiac hemodynamic parameters (e.g., contractile function and heart rate), has become a powerful approach in the early drug discovery phase to overcome drawbacks in the drug candidate's identification. However, traditional ex vivo retrograde heart perfusion methods consume a large volume of perfusate, which increases the cost and limits compound screening. An elegant and cost-effective alternative mode for ex vivo retrograde heart perfusion is the constant-flow with a recirculating circuit (CFCC), which allows assessment of cardiac function using a reduced perfusion volume while limiting adverse effects on the heart. Here, we provide evidence for cardiac parameters stability over time in this mode. Next, we demonstrate that our recycled ex vivo perfusion system and the traditional open one yield similar outputs on cardiac function under basal conditions and upon ?-adrenergic stimulation with isoproterenol. Subsequently, we validate the proof of concept of therapeutic agent screening using this efficient method. ?-blocker (i.e., propranolol) infusion in closed circulation countered the positive effects induced by isoproterenol stimulation on cardiac function. Keywords: Drug development, Drug screening, Cardiovascular safety, Langendorff method, Closed circulation.

Fig. 1

Ex vivo retrograde-perfused heart setup in constant-flow and closed circulation (CFCC). (A) Scheme of the retrograde heart perfusion system in CFCC; (B) Left ventricular pressure trace showing the left ventricular end diastole pressure (LVEDP; purple circle), the developed left ventricular pressure (dLVP; dashed red line) and the maximum systolic pressure (SP_max; red circle); (C) Representative traces of left ventricular pressure were recorded post-cardiac stabilization (early phase; light grey) and 1h later (late phase; light blue).

Fig. 2

Perfused rat heart in constant-flow and closed circulation (CFCC) mode shows similar cardiac functional parameters compared to constant-flow and open circulation (CFOC) with or without β-adrenergic stimulation. (A) Time course traces with zoomed sections of left ventricular pressure from perfused rat hearts in closed (dark blue) or open (grey) circulation with or without isoproterenol stimulation (10 nM). (B–F) Amalgamated data extracted from traces showing the heart rate (B), the developed left ventricular pressure (dLVP) (C), the maximal contraction velocity as dP/dt_max (D), the maximal relaxation velocity as dP/dt_min (E), and the rate pressure product (RPP) (F). Data show the mean ± SEM (n=5 independent experiments; ns for non-significant, ** p<0.01).

Fig. 3

Recirculated propranolol reduces cardiac functional parameters of perfused rat hearts in closed circulation under β-adrenergic stimulation. (A) Time course traces with zoomed sections of left ventricular pressure from perfused rat hearts in closed circulation in the presence (green) or absence (dark blue) of propranolol (1 μM) with or without isoproterenol stimulation (10 nM). (B–F) Amalgamated data extracted from traces showing heart rate (B), developed left ventricular pressure (dLVP) (C), maximal velocity of contraction (dP/dt_max) (D), and relaxation (-dP/dt_min) (E), and rate pressure product (RPP) (F). Data show the mean ± SEM (n=5 independent experiments; ns for non-significant, * p<0.1, ** p<0.01 and *** p<0.001).