Langendorff Perfusion.

Mice were injected with heparin (1,000 U/kg, i.p.) and anesthetized with ketamine (80 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). Depth of anesthesia was monitored by toe pinch reflex, and mice were killed by cervical dislocation. Hearts were rapidly excised and arrested in ice-cold Krebs–Henseleit buffer. After cannulation of the aorta with a 20-gauge needle, retrograde perfusion was established at a constant flow of 20 mL/min/g wet weight with a modified Krebs–Henseleit bicarbonate buffer (in mM: 118 NaCl, 25 NaHCO₃, 1.2 KH₂PO₄, 4.8 KCl, 1.2 MgSO₄, 1.25 CaCl₂, 11 glucose, pH 7.4) using the ISO-HEART perfusion system (Hugo Sachs Elektronik) as described previously with slight modifications (37). The perfusate was filtered through a 5-µm filter before reaching the heart and continuously gassed with carbogen (95% O₂, 5% CO₂). Heart temperature was maintained at 37 °C throughout the experiments, monitored with a physitemp probe (Physitemp Instruments). After removal of the left auricle, a tiny fluid-filled balloon made of a small square of polyethylene film was inserted into the left ventricle and connected to a pressure transducer via a 4F biluminal monitoring catheter (Vygon). An epicardial ECG was recorded throughout the experimental period with the use of two stainless steel electrodes attached directly to the base of the right atrium and left ventricle, respectively. Hearts were perfused for 30 min to establish stable baseline conditions, followed by 15-min perfusion with 0.1 µM CCh.

RR intervals (1,000 baseline and 1,000 after 10 min of CCh perfusion) were extracted using HRV module of LabChart 7 Pro software (ADInstruments). Each RR interval was plotted as a function of time (tachogram) or as a function of the preceding RR (Poincaré plot) to select the normal RR intervals excluding artifact and ectopic beats, as reported previously (38). Heart rate and time-domain statistical measures of HRV (SDNN, RMSSD, and pNN6) were calculated from RR interval using MATLAB™. SDNN, the SD of all normal RR intervals (in ms), estimates the total HRV arising from autonomic nervous system. RMSSD denotes the square root of the mean of the sum of the squares of differences between adjacent normal RR intervals (in ms), quantifying short-term variation between two successive beats as a measure of parasympathetic regulation. pNN6 is the percentage of consecutive normal RR intervals differing more than 6 ms, and reflects the relatively abrupt and fast changes in HR, attributed to cardiac parasympathetic activity.