Atrial Natriuretic Factor

RNA from C57/BL6 mice was isolated from snap-frozen right ventricles using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA from FHL-1^−/− and WT Swiss mice was extracted from formalin-fixed paraffin-embedded heart tissue using the deparaffinization solution (Qiagen) and the RNeasy FFPE Kit (Qiagen). For cDNA synthesis the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Dreieich, Germany) was used. Real-time PCR was performed in a CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). The PCR reactions were set up using iTaq™ Universal SYBR^® Green Supermix (Bio-Rad). The following cycling conditions were chosen: 3 min at 95 °C, [5 s (sec) at 95 °C, 10 s at 59 °C, and 10 s at 72 °C] × 40. As the SYBR^®Green I dye can bind non-selectively to the double-stranded DNA, melting curve analysis and gel electrophoresis were performed to confirm the exclusive amplification of the expected PCR product. The ΔCt values for each target gene were calculated by ΔCt = Ct reference gene—Ct target gene. Primer sequences are given from (5′–3′): mouse B2m (β2-microglobulin; FP: ATG CTA TCC AGA AAA CCC CTC A; RP: GCA GTT CAG TAT GTT CGG CT), mouse Anp (atrial natriuretic peptide; FP: GCT TCC AGG CCA TAT TGG AG; RP: GTC TAG CAG GTT CTT GAA ATC CA), mouse Bnp (brain natriuretic peptide; FP: TAT CTC AAG CTG CTT TGG GC; RP: ACA ACT TCA GTG CGT TAC AGC), mouse Atp2a2 (sarco(endo)plasmic reticulum calcium-ATPase 2; FP: GCC TTT GTA GAG CCG TTT GT; RP: TTT CTT TCC TGC CAC ACA CC), mouse Plb (phospholamban; FP: ATA CAG CTT CAT GCT CTG CAC; RP: TCT TCA CCT GCT TCT GTC TTG), mouse Ryr2 (ryanodine receptor 2; FP: CGA GCG TGT CCT GGG TAT AG; RP: TTG AGG ATG TTC CAC CAG GC), mouse αMhc (α myosin heavy chain; FP: TGT GGT GCC TCG TTC CA; RP: TTT CGG AGG TAC TGG GCT G), mouse βMhc (β myosin heavy chain; FP: GCA TTC TCC TGC TGT TTC CTT; RP: TGG ATT CTC AAA CGT GTC TAG TGA), mouse collagen 1a1 (FP: CTG ACG CAT GGC CAA GAA GA; RP: TAC CTC GGG TTT CCA CGT CT), mouse collagen 1a2 (FP: GCT TGC AGT AAC TTC GTG CCT; RP: CAG TGG GGC CCT TTC GTA CT), mouse collagen 3a1 (FP: AAA ACC CTG CTC GGA ACT G; RP: CTT GCA GCC TTG GTT AGG AT), mouse FHL-2 (FP: GGA AGG GCT CTG ACC TCT AAC A; RP: CAT TGC AGT GGT GGC AGT CAA), and mouse FHL-3 (FP: CCT GGC CCA CAG GTA GGA; RP: CGG TGC CCA GTG AGC C). The primers were intron spanning. B2m served as a reference gene.

The total RNA in heart tissue was extracted by the animal RNA Isolation Kit and then the RNA concentration was determined. The reverse transcription kit was used to reverse transcribe the RNA into cDNA. The cDNA was used as a template and then the target genes were amplified by PCR. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal reference primer for the target genes. The expression of target genes was analyzed by the 2^−ΔΔCT method [23 (link)]. The Primer sequences: GAPDH forward primer (5′-AATGGATTTGGACGATTGGT-3′) and reverse primer (5′-TTTGCACTGGTACGTGTTGAT-3′), brain natriuretic peptide (BNP) forward primer (5′-GAGGTCACTCCTATCCTCTGG-3′) and reverse primer (5′-GCCATTTCCTCCGACTTTTCTC-3′), atrial natriuretic peptide (ANP) forward primer (5′-GCTTCCAGGCCATATTGGAG-3′), and reverse primer (5′-GGGGGCATGACCTCATCTT-3′).

Using the Total RNA Isolation Reagent (BS259A, Biosharp, China), total RNA was extracted from heart tissues or cells. After that, the total RNA underwent reverse transcription and quantification on a Real-Time PCR system (7500, ThermoFisher, USA) using the One-Step SYBR Green qRT-PCR Kit (BL643B, Biosharp, China). Under the adoption of the 2^−ΔΔCT method, B-type natriuretic peptide (BNP), atrial natriuretic peptide (ANP), major histocompatibility complex-B (B-MHC), GSK3A, CD36, PDK4 (Pyruvate dehydrogenase kinase 4), and Cpt1b (carnitine palmitoyltransferase 1B) expressions were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [12 (link)]. Supplementary Table 3 lists the primer sequences.