Cardiac Gene Expression Analysis

Total RNA was extracted from the heart (n=5-9/genotype/timepoint) using TRIzol/chloroform, followed by the RNeasy Fibrous Tissue Mini Kit (Qiagen) as per the manufacturer's instructions. The concentration and quality of RNA samples were determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Real-time reverse transcription PCR was performed as described previously (Murphy et al., 2019 (link); Swiderski et al., 2016 (link)) using the following forward and reverse primer sequences: F4/80, 5′-CATCAGCCATGTGGGTACAG-3′ and 5′-CATCACTGCCTCCACTAGCA-3′; TGFβ, 5′-TGAGTGGCTGTCTTTTGACG-3′ and 5′-TCTCTGTGGAGCTGAAGCAA-3′; Col1a1, 5′-CACCCTCAAGAGCCTGAGTC-3′ and 5′-GTTCGGGCTGATGTACCAGT-3′; Col3a1, 5′-ACCAAAAGGTGATGCTGGAC-3′ and 5′-GACCTCGTGCTCCAGTTAGC-3′; Col6a1, 5′-CCCCATTGGACCTAAAGGAT-3′ and 5′-TCTCCCACTTCACCCTCATC-3′; Runx2, 5′-GCCTTCAAGGTTGTAGCCCT-3′ and 5′-GTTCTCATCATTCCCGGCCA-3′; Ocn, 5′-TTCTGCTCACTCTGCTGACC-3′ and 5′-GGGACTGAGGCTCCAAGGTA-3′; and Alpl, 5′-CAGGCCGCCTTCATAAGCA-3′ and 5′-AATTGACGTTCCGATCCTGC-3′. Gene expression was quantified using a cycle threshold (C_T) method. Relative gene expression was calculated using the expression 2^−ΔCT, normalized to total cDNA content as determined using the Quant-iT OliGreen ssDNA assay kit and Quant-iT OliGreen ssDNA reagent (Thermo Fisher Scientific).