*2.1. Animals and Experimental Design*

The experiments were performed on 9-month-old male and female hairless coisogenic spontaneously hypertensive rats (SHR) harboring the mutant desmoglein 4 (Dsg4) gene (SHRM), as well as age and sex-matched wild type SHR. Besides, male and female Wistar Kyoto rats (WKY) were used as a reference normotensive strain. Animals were obtained from the Institute of Physiology, v.v.i., Academy of Sciences of the Czech Republic, Prague. The Dsg4 mutation was originally found in the SHR.BN-chr.1 congenic strain and was transferred on the SHR genetic background by backcross breeding [16]. The animals were housed at 22 ◦C with 12-h light/dark cycles and ad libitum access to tap water and standard laboratory chow. Experiments were performed in agreement with the Animal Protection Laws of the Czech Republic. The maintenance and handling of the animals were performed in accordance with the "Guide for the Care and Use of Laboratory Animals" published by the U.S. National Institutes of Health (NIH Publication, 8th ed., revised 2011) and approved by the Ethics Committee of the Institute of Physiology v.v.i, Academy of Sciences of the Czech Republic, Prague.

Male and female SHR were randomly divided into 6 experimental groups: male SHR (n = 6), female SHR (n = 6), male hyperthyroid SHR TH (n = 5), female hyperthyroid SHR TH (n = 5), male hypothyroid SHR HY (n = 5) and female hypothyroid SHR HY (n = 5). Male and female SHR<sup>M</sup> were randomly divided into 6 experimental groups: male SHRM (n = 6), female SHR<sup>M</sup> (n = 6), male hyperthyroid SHRM TH (n = 5), female hyperthyroid SHRM TH (n = 5), male hypothyroid SHR<sup>M</sup> HY (n = 5) and female hypothyroid SHRM HY (n = 5). The hyperthyroid status was established by the intraperitoneal injection of 3,3 ,5-triiodo-L-thyronine (T3; Sigma Aldrich, St. Louis, MO, USA) at 0.15 mg/kg b.w. three times weekly, and the hypothyroid status was induced by a 0.05% solution of methimazole (Sigma Aldrich, St. Louis, MO, USA) in the drinking water. Wistar Kyoto (male WKY, n = 5; female WKY, n = 5) nontreated normotensive rats were used as the control reference strain. At the end of the experiment, the animals were euthanized with 100 mg/kg b.w. of ketamine (Narketan; Vetoquinol UK Ltd., Towcester, UK), followed by 10 mg/kg b.w. of myorelaxant xylazine (Xylapan; Vetoquinol UK Ltd., Towcester, UK), and the body weight was registered. Then, the chest was opened, and the heart was quickly excised into ice-cold saline, followed by weight registration and heart tissue sampling. Blood samples were taken from the thoracic aorta. All samples were stored in a freezer at −80 ◦C.
