Animals

Neonatal C57BL/6 mice from specific families (P1, P4, and P7) were euthanized with pentobarbital and their hearts were excised for protein extraction.

Protein extraction, digestion, and liquid chromatography with tandem mass spectrometry (LC–MS/MS)

Ventricular tissue samples from the P1 (n = 4), P4 (n = 3), and P7 (n = 3) groups were homogenized in RIPA lysis buffer containing a protease inhibitor cocktail (Roche). The homogenates were centrifuged, and the Bradford assay was used to measure the protein concentration in the supernatant. To minimize biological variations, we used a pooled approach to extract protein. Equal weight ventricular tissues from the same day (day 1 n = 4, day 4 n = 3, day 7 n = 3) were pooled together as described in previous studies [19, 20], and then homogenized in RIPA lysis buffer containing a protease inhibitor cocktail.

From each group, 500 µg of the protein from the solution digest was used according to the method described previously [21]. The protein in the supernatant was precipitated and centrifuged. The pellets were resuspended and reduced with DTT and then alkylated using iodoacetamide. Lastly, trypsin (Promega) was added to the protein solution in a 1:50 ratio (trypsin/protein, w/w) and incubated at 37°C for 12 h.

Stable isotope dimethyl labeling, a reliable, cost-effective, and undemanding procedure method was used to relative quantification. Dimethyl labeling was performed as previously described with minor modifications [22]. Briefly, P1 samples were labeled with 4% CH2O and NaBH3CN (light labeling), P4 samples were labeled with 4% CD2O and NaBH3CN (middle labeling), and P7 samples were labeled with 4% 13CD2O and NaBD3CN (heavy labeling). Equal amounts of peptides from the light-, middle-, and heavy-labeled samples were mixed and desalted using C18 columns (Waters). Strong cation–exchange chromatography was used to separate the mixed samples. Eight fractions were collected and desalted using gradient elution before MS analysis. MS/MS analysis was performed using a QE-HF mass spectrometer (Thermo Fisher Scientific) coupled with a nano-HPLC system (SCIEX). The suspended peptides were first loaded onto a C18 trap column (0.5 mm × 2 mm, MICHROM Bioresources, Inc.) and eluted from the trap column to a C18 analytical column (100 μm × 150 mm, 100 Å pore size, 3 μm particle size, MICHROM Bioresources, Inc.) at a flow rate of 300 nL/min. Liquid chromatography was performed using a 100-min gradient (mobile phase A: 3% DMSO, 97% H2O, 0.1% formic acid; mobile phase B: 3% DMSO, 97% ACN, 0.1% formic acid; gradient: 0 min in 5% B, 65 min of 5–23% B, 20 min of 23–52% B, 1 min of 52–80% B, 80% B for 4 min, 0.1 min of 80–5% B, 5% B for 9.9 min). Proteome Discoverer Software Version 2.1 (PD2.1, Thermo Scientific) was used for the relative quantification of proteins. The UniProt database (Mus musculus.20160318.fasta, Proteome ID: UP000000589, Download date: 2016 Mar 18) was used. Parameters were set as follows: parent ion tolerance 10 ppm; fragment ion mass tolerance 0.02 Da; carbamidomethyl cysteine as a fixed modification; methionine oxidation and dimethylation 3plex (C2H4, C2D4, 13C2D4) were variable modifications. The peptide charge was set to 2 + or 3 + , and up to two missed cleavages were allowed. The proteins and peptides denoted with high FDR confidence (FDR < 1%).

Bioinformatics analysis

The threshold of “significantly changed” ratios was set by analyzing the Gaussian distribution of the dimethyl-labeling ratio (logarithmized to base 2). To place a threshold at the 95% confidence limit (p ≤ 0.05), we set mean ± 1.96 standard deviations as the “significantly changed” threshold [23]. Protein ratios were uploaded to the PANTHER website (http://www.pantherdb.org/) for statistical enrichment, which can identify specific protein classes that are significantly changed during the development of the heart. Intracellular pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database. Significantly changed KEGG pathways were identified based on the hypergeometric distribution of genes with p < 0.05.

Cell culture

Briefly, one-day-old C57BL/6 mice were anesthetized with pentobarbital and were subjected to thoracotomy to expose the heart. Hearts were quickly isolated and cardiomyocyte isolation and cell culture were performed following a previously reported protocol with slight modifications [24]. Cardiomyocytes were cultured for future experiments.

5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay

EdU assays were used to determine cardiomyocyte replication. Neonatal mouse cardiomyocytes were treated with the vehicle and the PKA agonist bucladesine (MedChemExpress, HY-15979) or the PKA inhibitor H-89 (MedChemExpress, HY-15979). Six hours later, EdU (50 μM) was added to cardiomyocytes and cultured for 24 h. EdU staining was conducted using the Cell-Light EdU Apollo567 in vitro kit according to the manufacturer’s instructions (RIBOBIO, C10310-1). Cardiomyocytes were stained with an anti-cTnT antibody (Abcam catalog no. ab8295) combined with Alexa Fluor® 488 conjugate second antibody (CST catalog no. 4480). Next, 4′,6-diamidino-2-phenylindole (DAPI) counterstaining (Vector lab, H1200) was performed. Stained sections were photographed using a Zeiss microscope and EdU-positive cardiomyocytes (red, green, and blue merge) were quantified using ImageJ software (3 random fields for each well, 3 replicate wells for each experiment). Three independent experiments were used for data analysis.

Ki67 detection

Neonatal mouse cardiomyocytes were treated with the drugs as described above. Ki67 was stained using the anti-Ki67 antibody (Abcam catalog no. ab15580) combined with Alexa Fluor® 594 Conjugate (CST catalog no. 8889). Cardiomyocytes and nuclei were stained with cTnT and DAPI as described above. Ki67-positive cardiomyocytes (red, green, and blue merge) were quantified using ImageJ software (3 random fields for each well, 3 replicate wells for each experiment). Three independent experiments were used for data analysis.

RT-PCR assays

Ventricular tissue samples from neonatal mice were homogenized in TRIzol reagent (Invitrogen) and the total RNA was extracted according to the manufacturer’s protocols. One μg of total RNA (1 mg) was used for reverse transcription (TaKaRa). qRT-PCR was carried out in a 10-μL reaction volume containing SYBR Green Master Mix (TaKaRa) and primers. The sequences of PCR primer are shown as follows: Ctbp2 5′- AGTCTCCACACACCTCATTCA-3′, and 5′-GGGGCGGATACTTGGTTC-3′; Grb10 5′– CACCTGCCAAGCATTTCCCT-3′, and 5′-GTGTCGGTTGGACACTGGTT-3′; Erbin 5′- GCACATTTTTCGACATCCGCA-3′, and 5′-CAGGCTGTCTGGAAGACCTC-3′; Pkn2 5′- CACCCGTTTTTCCGGCTAAC-3′, and 5′-TGGTTCTCGAGGTGGAGTCA-3′; Tsfm 5′- GGCCCTAGCGATTGGTAAACT-3′, and 5′-GCCGACATAGAACCCAGAGG-3′; Stat3 5′- GCCCCGTACCTGAAGACCAA-3′, and 5′-ACGTGAGCGACTCAAACTGC-3′; Acox1 5′- CGTCGAGAAATCGAGAACTTG-3′, and 5′-GGTTCCACAAAATTGACCATATGTA-3′; Lipe 5′- CCAGGGAGGGCCTCAGC-3′, and 5′-TGTCTTCTGCGAGTGTCACC-3′; Adcy5 5′- TGGTGGACCGTGTTCTTCATC-3′, and 5′-ATGAGGACATTGGAGACAAGCTGTT-3′; Acox3 5′- GACAAAGCAGGTCGGTGACA-3′, and 5′- CAGCTCCCCAGAGTTGAAGG-3’, GAPDH 5′- AATGGGCAGCCGTTAGGAAA-3′, and 5′- GCCCAATACGACCAAATCAGAG-3′.

Preparation of whole-cell extracts and western blotting

Whole-cell extracts were prepared, and western blotting was conducted as described previously [25]. Briefly, ventricular tissue samples from neonatal mice or the neonatal cardiomyocytes were homogenized in RIPA lysis buffer (Thermo Scientific™) containing a protease inhibitor cocktail and phosphatase inhibitor PhosStop (Roche). The homogenates were centrifuged at 12 000 xg for 15 min and protein concentrations were determined using a BCA protein assay kit (Pierce). Proteins were normalized to the same amount and treated with SDS-PAGE loading buffer. Then the proteins were separated using SDS-PAGE and electrotransferred onto a nitrocellulose membrane (Bio-Rad). Antibodies against PKA α/β/γ pT197 (Abcam catalog no. ab75991), PKA α/β/γ (Santa Cruz catalog no. sc-365615), CDK4 (ABclonal catalog no. A11136), cyclin D1 (ABclonal catalog no. A19038), cyclin E1 (ABclonal catalog no. A12000), Rb (bimake.cn, catalog no. A5073), phosph-Rb(s807) antibody (bimake.cn, catalog no. A5735), Ctbp2 (Proteintech Cat No. 10346–1-AP), Grb10 (Proteintech Cat No. 23591–1-AP), Stat3 (Proteintech Cat No. 10253–2-AP) and GAPDH (ABclonal catalog no. A19056) were used as primary antibodies. Protein signals were determined using an electrochemiluminescence western blotting substrate (Pierce) and analyzed using Image Lab software (version 5.2.1, Bio-Rad).

Statistical enrichment test

The statistical enrichment test is a statistical analysis tool offered by the PANTHER website (http://www.pantherdb.org/) [26]. The submitted proteins were grouped according to the GO or PANTHER classification, and the expression ratio of a particular group of proteins was compared with that of all proteins in the list to assess the likelihood of a significant change in a particular group of proteins.

Pathway analysis

The KEGG pathway database was used for pathway research (http://www.kegg.jp/). This analysis divides proteins in the KEGG pathway and sets all qualitative proteins as the background. The KEGG pathway analysis is based on supergeometric testing and reveals protein enrichment levels of each pathway to determine significant impacts of metabolism and signal transduction pathways.

Statistical analysis

All statistical analyses were performed using SPSS software (version 20.0, IBM Corp., USA). Values are presented as mean ± standard error of the mean (SEM [n ≥ 3]). P < 0.05 was considered significant. The significance of differences was determined using independent samples t-tests or one-way analysis of variance followed by Student-Newmann-Keuls multiple comparison tests.