ECG Recordings in Conscious Mice and Heart Rate Variability Analysis

Mice undergoing telemetric ECG recordings were anesthetized with 2% isoflurane (Forene^®, Abbott, United Kingdom). A midline incision was made on the back along the spine to insert a telemetric transmitter (ETA-F10, Data Sciences International) into a subcutaneous pocket. Paired wire electrodes were placed over the thorax (chest bipolar ECG lead) in DII derivation against the heart axis. Mice were left to recover for 14 days before ECG recordings. ECG signals were recorded using a telemetry receiver and an analog-to-digital conversion data acquisition system for display and analysis by Dataquest^TM A.R.T.^TM software (Data Sciences International). We recorded ECG for 12 h, before the ramp-up period (basal conditions) and daily (from 20:00 to 08:00 dark period) after ramp-up period until the 28th day of training. Heart rates (HR) were measured from ventricular RR intervals. ECG parameters were measured with ECG Auto 1.5.7 software (EMKA Technologies). HRV analysis was performed on telemetric ECGs by sampling four different 5-min periods of stable ECG segments (first 5-min period 22:55–23:00; second 5-min period 01:55–02:00; third 5-min period 04:55–05:00, and fourth 5-min period 07:55–08:00) at day 0 and at day 28 in WT and Girk4^–/– sedentary and trained animals. The standard deviation of intervals between two consecutive heart beats (SDNN), power spectral density (PSD) of HRV determined by Fast Fourier Transformation analysis (Welch Periodogram method), spectral frequency bands (low frequency spectra 0.15–1.5 Hz, high-frequency spectra 1.5–5 Hz and ratio between LF and HF values), percentage of successive intervals that differ by more than 6 ms (pNN6), standard deviation of instantaneous beat-to-beat interval variability (SD1) and continuous long-term R-R interval variability (SD2) provided by ellipse-fitting technique of the Poincaré scatter-gram obtained in each of the four 5 min period were averaged.

ECGs were also recorded from conscious restrained mice using the non-invasive ecgTUNNEL^® device (Emka Technologies). ECG signals were continuously recorded for 15 min using iOX Software v2.10.5.14 (Emka Technologies) and the heart rate was analyzed with ecgAUTO v3.3.5.12 (Emka Technologies). Each mouse underwent habituation to the setup for 10 min before data collection. ECG measurements started 40 min after intraperitoneal injection of saline or atropine (0.5 mg/kg, Aguettant) and propranolol (5 mg/kg, Sigma Aldrich) solution. This delay was considered as a good compromise between the absence of the artifact due to the stress of the injection and the measurement of the amplitude of the drug effect.