Langendorff Mouse Model

To characterize whether IPA exhibits direct effects on cardiac function, the ex vivo Langendorff mouse model was used (28–30). Animal experiments were approved by the government of North Rhine-Westphalia, Germany. C57BL/6J male mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France). Mice were housed in filtertop cages in a controlled environment (12 h daylight cycle) at the animal facility of the RWTH Aachen University. Mice had free access to water and food. At age of 9–12 weeks (average weight of 22 g) mice were anesthetized with ketamine hydrochloride and xylazine hydrochloride and anticoagulated with 1,000 IE heparin.

The heart was quickly excised and was prepared in ice-cold Krebs–Henseleit buffer. The aorta was cannulated for retrograde perfusion at constant pressure of 90 mmHg using a Langendorff perfusion apparatus (Harvard Apparatus, Massachusetts, USA). The perfusion solution was heated to 37°C and oxygenated with 100% oxygen. A latex balloon filled with distillated water connected to a pressure transducer was inserted into the left ventricle (LV). LV diastolic pressure was set to 12 mmHg. The heart was stimulated resulting in a heart rate of 600 bpm. LV systolic (LVPsys) and LV developed pressure (LVPdp), as well as maximum rate of increase (dLVPmax) and maximum rate of decrease in left ventricular pressure during isovolumic contraction (dLVPmin) were measured continuously (all 2 s) with ISOHEART Isolated Heart Data Acquisition Software (Harvard Apparatus, Massachusetts, USA). Hearts were perfused for a 15 min equilibration period. Following, 200 μL Krebs-Henseleit buffer was administered through the aortic cannula into the heart. After 10 min, dobutamine was administered (1 mg/ml, diluted 1:256). In cycles of 10 min, IPA in different concentrations (1–100 μM) was administered following dobutamine as positive control to ensure proper heart function. Therefore, IPA was dissolved in Krebs-Henseleit buffer and pH-adjusted to 7.4 afterwards. pH-adjusted buffer was used as control to exclude side-effects dependent on buffer administration. In between, washing steps with buffer were performed.