*2.3. RNA Isolation and Quantitative RT-PCR*

Total RNA isolation and quantitative RT-PCR analysis were performed as described previously [23]. All samples were measured in triplicate and normalized to two housekeeping genes (*Dpm1 and Txnl4a*). The qPCR results were obtained and evaluated with the software qPCRsoft 4.1 (V4.1.3.0, Analytik Jena, Jena, Germany), and then analyzed using the standard delta delta Cq method.

## *2.4. Echocardiography*

Echocardiography was performed one day before the necropsies using a 14MHz linear transducer (Siemens Accuson s2000, Munich, Germany) for evaluation of cardiac function. Mice were under 1% isoflurane anesthesia with a stable body temperature of 37 ◦C. Mmode in short-axis at the level of the papillary muscles was used to evaluate LV thickness, fractional shortening, and internal diameters in systole and diastole. At least four cardiac cycles were analyzed for each parameter.

#### *2.5. Central Arterial and Cardiac Pressure Measurements and Augmentation Index*

Central arterial pressure was measured by inserting a micro-tip catheter (1.4 Millar Instruments, Houston, TX, USA) into the ascending aorta via the right carotid artery under 1.5% isoflurane anesthesia. The catheter was then further advanced into the left ventricle to obtain cardiac pressure parameters. Traces were recorded for at least three minutes and analyzed via LabChart7 software (ADInstruments, Dunedin, New Zealand). The aortic augmentation index was identified from the late systolic portion of the arterial pressure wave as described previously [24]. The augmentation index was defined as the height from the augmentation point to the systolic peak of the pressure wave divided by the pulse pressure, and was expressed as a percentage.
