Animal experiments and pressure overload-induced cardiac hypertrophy

All animal experiments were carried out after obtaining approval from the Institutional Animal Ethics Committee (IAEC) of Rajiv Gandhi Centre for Biotechnology, Trivandrum (Protocol No. IAEC/529/TRSK/2016). We strictly followed the rules and regulations of the Committee for Control for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

Three months old male Wistar rats were used for our experiments. Animals were maintained on a 12 hours day/light cycle in a temperature and humidity controlled room. The body weights of rats were recorded on a weekly basis. All animals were provided food and water ad libitum and observed for any untoward symptoms.

After an initial assessment of basal echocardiography parameters, 50 male Wistar rats (3 months old) were divided into three groups. Twenty rats (at the age of 3 months) underwent constriction of ascending aorta using titanium clip (aortic constricted approximately 60% of original diameter) for induction of pressure overload-induced left ventricular cardiac hypertrophy. For ensuring successful constriction of the aorta (AC), we recorded pressure gradient across aortic constriction using two-dimensional color Doppler analysis. Twenty rats had a sham operation (SO) as described previously^56,57. Another 10 rats were considered to control young animals. Echocardiography was performed periodically to assess left ventricular hypertrophy.

Cardiac structure and function in rats were assessed by echocardiography. Systolic intra-ventricular septal thickness (IVSs), diastolic intraventricular septal thickness (IVSd), systolic left ventricular internal dimension (LVIDs), diastolic left ventricular internal dimension (LVIDd), systolic left ventricular posterior wall thickness (LVPWs), diastolic left ventricular posterior wall thickness (LVPWd), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) at the beginning of the experiment, 6 months after aortic constriction or sham operation and 15 months after aortic constriction or sham operation.