RNA was extracted from heart tissue using the RNeasy isolation kit (Qiagen). cDNA was synthesized from total RNA using RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific). For each replicate in each experiment, RNA from tissue samples of different animals was used. Primers were designed corresponding to mouse mRNA sequences using Primer3 (Table S1). Reactions contained PowerUp SYBR green quantitative polymerase chain reaction Master Mix (Applied Biosystems), 500 nmol/L of each primer, and 0.2 µL of template RNA solution. Amplification was carried out using an ABI 7300 real-time polymerase chain reaction system (Applied Biosystems) with uracil-DNA glycosylases activation at 50 °C for 2 minutes, initial denaturation at 95 °C for 2 minutes, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 minute. Relative levels of mRNA expression were calculated using the ∆∆CT method.14 (link) Values were normalized to Gapdh mRNA.