ONLINESUPPLEMENT-Hypertension.docVIP

Online supplement: Expanded Methods RESULTS: Hemodynamic evaluation: Body temperature was monitored using a rectal probe and was maintained between 37oC and 38oC. Heart rate was monitored by taping paws to electrodes (Indus Instruments, Houston, TX). With appropriate body temperature and anesthetic depth, heart rate was kept above 400 min-1. A 20MHz pulsed Doppler system (model VF-1; Valpey Fisher, Hopkinton, MA) with a hand-held probe (Matec Instrument; Northborough, MA) was used to obtain transcutaneous blood velocity waveforms over three second time intervals from the ascending thoracic aorta and mitral orifice. Ten consecutive waveforms for each animal were saved and later analyzed (Doppler Signal Processing Workstation, Indus Instruments, Houston, TX). Aortic blood velocity and ECG waveforms were analyzed to obtain: 1) heart rate; 2) mean velocity; 3) stroke distance; 4) peak velocity; 5) peak acceleration; 6) ejection time; 7) rise time; and 8) pre-ejection time. Ascending aortic diameter was measured during systole from an image of the long-axis of the left ventricular outflow tract obtained using an Ultrasound Biomicroscope (UBM) (Model VS40; VisualSonics, Toronto, ON) with a 19 MHz transducer (resolution ~100 μm). Cardiac output and stroke volume were calculated as the product of aortic luminal cross-sectional area [( (diameter/2)2] and mean velocity and stroke distance, respectively. In a separate group of 7 isoflurane-anesthetized, non-pregnant adult B6 mice, the coefficient of variation of aortic diameter measurements obtained using this method daily for 4 consecutive days was 2%. Left ventricular geometry: In a separate series of animals, a newer model UBM (Model Vevo660, 30 MHz transducer) was used to measure left ventricular geometry using the parasternal long-axis view. The following 2D M-mode measurements were obtained (Figure 1C): LV end-diastolic (LVED) and end-systolic (LVES) dimensions, anterior (AW) and posterior wall (PW) thick