Powerlab hardware

Heart rate tachograms were obtained from data collected at the Charles Sturt Diabetes Complications Screening Clinic (DiScRi), Australia [52 (link)] and were approved by the Charles Sturt University Human Ethics Committee. Written informed consent was obtained from all participants. A 20-min lead II ECG recording was taken from participants attending the clinic, using Powerlab hardware with Chart 7 software (ADInstruments, Sydney) during the morning in an ambient temperature room and after the participants were relaxed. Participants were comparable for age, gender, and heart rate, and after initial screening, those with heart disease, presence of a pacemaker, kidney disease or polypharmacy (including multiple anti-arrhythmic medications) were excluded from the study. The status of CAN was defined using the Cardiac Autonomic Reflex Test battery criteria [53 (link)]. Each participant was assigned as either without CAN (71 participants), early CAN (67 participants) or definite CAN (NN participants) [54 (link),55 (link)].

Rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), a polyethylene catheter connected with a pressure transducer was inserted into the left ventricular cavity via the right carotid artery [25 (link)]. Left ventricular systolic pressure (LVSP) and left ventricular diastolic pressure (LVDP) were digitally processed via hemodynamic analyzing system (Powerlab Hardware, AD Instruments) [29 ]. Maximal positive and negative values of the instantaneous first derivative of LVP (+dP/dt_max⁡ and −dP/dt_max⁡) were derived by computer algorithms.

Urothelium-intact DSM strips (~4 mm long and ~2 mm wide) were connected to isometric force transducers and to a micrometer screw in a temperature-controlled (37°C) chamber (DMT-820MS, Aarhus, Denmark) containing 5 ml PSS aerated with 95% O₂ and 5% CO₂ to reach a pH = 7. The signal was recorded using data acquisition PowerLab hardware and LabChart v5.3 software (ADInstruments, Heidelberg, Germany). Strips were subjected to a tension of 1.2 g and allowed to equilibrate during 60 min. Samples were then incubated for 30 min with propranolol (10 μM) and N^G-nitro-L-arginine (L-NOARG, 100 μM), to block, respectively, beta-adrenoceptor- and NO-mediated DSM relaxation. EFS was performed on strip basal tension by generating rectangular pulses (1 ms duration and 20 s trains) with 75 mA constant current output (Cibertec CS20 stimulator, Barcelona, Spain). For each strip, the first control curve was obtained by increasing EFS frequencies from 0.5 to 8 Hz at 4 min intervals. Then, the bath PSS was replaced every 15 min in a total period of 90 min and incubated with ASP 7663 [20 (link)], TRPA1 agonist, for 10 min, and the second curve was constructed. Strips were then washed out again and treated with HC 030031 [21 (link)], TRPA1 antagonist plus ASP 7663, for 30 min, and the third curve was performed.