*2.2. Experimental Protocol*

The 22-week study phase was preceded by a two-week adaptation period to accustomize the animals to drug-free tablets and to the procedure of BP measurement. The animals were 60.5 ± 0.25 weeks of age (BW 404 ± 31 g) at the beginning and 81.9 ± 0.44 weeks at the end of the 22-week study phase. They were subdivided into three groups (*n* = seven per group). The group size was calculated for a medium effect size of systolic blood pressure (SBP) reduction (with Cohen's f = 0.25). Two groups received antihypertensive treatment with CAP (60 mg kg−<sup>1</sup> d−1, Axxora, Lörrach, Germany) or CAP + NIF (CAP 60 + NIF 10 mg kg−<sup>1</sup> d−1, Sigma-Aldrich Chemie, Steinheim, Germany), while the third group received drug-free tablets and served as untreated control (CTRL). The drugs were added to commercially available rodent sweets (Vitakraft-Werke, Wührmann & Sohn GmbH & Co., KG, Bremen, Germany) and formed to tablets. The tablets were given into the cages for oral uptake along with chow once daily between 9:00 a.m. and 10:00 a.m. After the two-week adaptation period, non-invasive BP measurements were carried out every two to three weeks. At the end of the experimental period, animals were sacrificed, and their hearts were removed for further analyses.

#### *2.3. Non-Invasive Blood Pressure Measurement*

SBP was measured non-invasively using the tail-cuff-method (TSE Blood Pressure Monitor, Series 209002, TSE Systems GmbH, Bad Homburg, Germany). Measurements were performed in awake animals about 1 to 2 h after the administration of the drugs or the drug-free tablets. The animals were placed on a heated plate (36 ◦C) and were allowed to move relatively freely, and were only held by the experimenter's hand on their back or tail. We avoided the use of a conventional restraint box to minimize stress to the animals. After six to eight preliminary tests, the animals were familiar with the environment and the experimenters. The procedure of SBP measurement was well tolerated by the animals. To ensure reproducible results, for each SBP measurement a mean was calculated from two to three tests with each test containing 10 single readings.

#### *2.4. Further Analyses on Heart Tissue*

After extraction, HW was determined as a measure of cardiac hypertrophy. As body weight (BW) developed differently in the SHR groups, the ratio of HW/BW was calculated by relating HW to baseline BW. The apex was separated and fixated in formalin for histological examination. Pieces of the LV were frozen and stored at −80 ◦C for biochemical analyses.

#### *2.5. Ribonuclease Protection Assay*

A ribonuclease protection assay was performed as previously described [14] to determine mRNA expression of atrial natriuretic peptide (ANP), transforming growth factor-β<sup>1</sup> (TGF-β1), TGF-β<sup>2</sup> and TGF-β3, matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinases 2 (TIMP-2) and collagen types I (Coll I) and III (Coll III) in the LV.

In brief, total RNA was isolated according to the method of Chomczynski and Sacchi [18] using TRIZOL (Invitrogen GmbH, Karlsruhe, Germany) as described in the manufacturer's protocol. For investigation of TGF-β, we isolated 2.5 μg of total TGF-β RNA, and for the extracellular matrix (ECM) markers ANP, MMP-2, TIMP-2, Coll I, and Coll III, 5 μg of total RNA was isolated. The isolated RNA was hybridized overnight with the template sets rTGF-β and ECM-3, respectively, and labelled with an RiboQuant® In vitro Transcription Kit (Pharmingen, Hamburg, Germany) and [α-32P]-UTP as described by the manufacturer. After hybridization and digestion of the unprotected probes, the protected radioactive RNA was displayed on a denaturing polyacrylamide gel. For densitometric evaluation we used the Molecular Imager (BioRad, München, Germany). mRNA expression was semi-quantitatively determined in percent of glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) mRNA.
