Total RNA was isolated from NRCMs and fresh heart tissues using TRIzol reagent (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer’s instructions. The first-strand cDNA was synthesized from 1 to 2 μg of total RNA by oligo(dT)-primed RT (iScript cDNA synthesis kit; Bio-Rad Laboratories, Hercules, CA, USA). The mRNA levels of atrial natriuretic peptide (ANP)/ANF, BNP, β-MHC, collagen I, collagen III, and EGFR were determined by real-time qPCR analysis on an Applied Biosystems 7500 Real-Time PCR System using SYBR Green (Applied Biosystems/Thermo Fisher Scientific, Foster City, CA, USA) as described previously (43). FAM channel intensity was normalized to ROX intensity, and Ct values were calculated using automatically determined threshold values with SDS software (Applied Biosystems). The levels of detected mRNA were normalized to the amount of endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the qPCR primers are shown in table S2.

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