Postnatal Right Ventricular Developmental Track Changed by Volume Overload

Animal Experiments

C57/BL6 mice were purchased from Xipu'er‐bikai Experimental Animal Co., Ltd (Shanghai, China). At postnatal day 7 (P7), the neonates males were randomized into 2 groups—an experimental group (VO group) and a control group (sham group)—who underwent the same procedure except for the puncture step. The fistula surgery protocol was modified according to previous publication.²⁸ Briefly, the neonatal mice were anesthetized using 4% isoflurane. Under general anesthesia, a midline laparotomy was made, and the abdominal aorta (AA) and inferior vena cava (IVC) were exposed. A 0.07‐mm diameter needle was used to puncture fistula from AA into the IVC. The surrounding connective tissue was used for hemostatic compression for 2 minutes. We closed the abdominal wall and the pain was relieved with local lidocaine treatment, and the pups were returned to their mother. We have provided a surgical video of RV VO model in the supplemental materials to enable others to learn the technique. At the 3 time points: P14, P21, and 12 months, 6 mice from each group were euthanized, the mice were anesthetized with 1.5% isoflurane and then euthanized to obtain the hearts, which were washed with cold PBS and stored at −80°C for further analysis. All of the procedures conformed to the principles outlined in the Declaration of Helsinki and were approved by the Animal Welfare and Human Studies Committee at Shanghai Children's Medical Center (Institutional Review Board number:SCMCIRB‐Y2020094).

Abdominal Ultrasound and Echocardiography

At P14, mice were anesthetized with isoflurane and allowed to breathe spontaneously through a nasal cone (isoflurane/oxygen: 1.5%–2.0% maintenance). The fistula between AA and IVC (AVF) and pulmonary artery (PA) flow were analyzed with a Vevo 2100 imaging system (Visual Sonics, Toronto, Ontario, Canada). For confirmation of an AVF, the waveform in the IVC was recorded using pulse‐wave mode. To confirm the VO, the velocity time integral (VTI) of the PA (PA‐VTI) blood flow and the PA‐velocity were calculated from the mean of 3 consecutive measurements using 2‐dimensional and pulse doppler echocardiography.

Histology

Mouse hearts were fixed in 10% paraformaldehyde overnight at room temperature, then dehydrated in an ethanol series, embedded in paraffin, and sectioned into 6‐µm slices. Hematoxylin and eosin staining were performed according to the manufacturer's instructions.

Total RNA Preparation and Real‐Time Quantitative Polymerase Chain Reaction Analysis

For mRNA quantification, mRNA was extracted from the RV free wall and purified using a PureLink RNA Micro Scale Kit (Catalog No. 12183016; Life Technologies, Carlsbad, CA). Reverse transcriptase polymerase chain reaction (PCR) was performed using the PrimeScript reagent kit (Takara Bio, Kusatsu, Japan). Quantitative real‐time PCR was performed using SYBR Green Power Premix Kits (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Quantitative real‐time PCR was performed with a 7900 Fast Real‐Time PCR System (Applied Biosystems), and the following conditions were used: 1 cycle at 95°C for 10 seconds, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The primers were obtained from Generay Biotech Co., Ltd (Shanghai, China). The relative fold change was then calculated using the ΔΔCT method.

Library Preparation for Transcriptome Sequencing

A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using the NEBNext^® Ultra^™ RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer's recommendations. Index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly‐T oligo‐attached magnetic beads. Fragmentation was performed using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5X). First‐strand cDNA was synthesized using random hexamer primers and M‐MuLV Reverse Transcriptase (RNase H –). Second‐strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3' ends of the DNA fragments, NEBNext Adaptors with hairpin loop structures were ligated to prepare for hybridization. To select cDNA fragments of preferentially 250–300 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, MA). Then 3 μL USER Enzyme (NEB, USA) was used with size‐selected, adaptor‐ligated cDNA at 37°C for 15 minutes followed by 5 minutes at 95°C. Then, PCR was performed with Phusion High‐Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, PCR products were purified (AMPure XP system), and library quality was assessed on an Agilent Bioanalyzer 2100 system.

Clustering and Sequencing

The clustering of the index‐coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3‐cBot‐HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform, and 150 bp paired‐end reads were generated.

Quality Control, Read Mapping, and Quantification of Gene Expression Levels

Raw data (raw reads) in fastq format were first processed through in‐house Perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapters, reads containing poly‐N, and low quality reads from raw data. At the same time, Qphred ≥ 20, Qphred ≥ 30, and Guanine Cytosine content of the clean data were calculated. All of the downstream analyses were based on clean, high‐quality data.

Reference genome and gene model annotation files were downloaded from the genome website directly. The index of the reference genome was built using Hisat2 v2.0.5, and paired‐end clean reads were also aligned to the reference genome using Hisat2 v2.0.5.

The expected number of Fragments Per Kilobase of transcript sequence per Million base pairs sequenced considers the effect of sequencing depth and gene length for the reads counted at the same time. FeatureCounts v1.5.0‐p3 was used to count the number of reads mapped to each gene. Then, Fragments Per Kilobase of transcript sequence per Million of each gene was calculated based on the length of the gene and read counts mapped to each gene.

Differential Expression Analysis and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Enrichment Analysis of Differentially Expressed Genes

Differential expression analysis was performed using the DESeq2 R package (1.16.1). The resulting P values were adjusted using Benjamini and Hochberg approach for controlling the false discovery rate. Genes with an adjusted P value of <0.05 found by DESeq2 were considered differentially expressed.

Gene ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, in which gene length bias was corrected. GO terms with corrected P values under 0.05 were considered to be significantly enriched by differentially expressed genes.

The clusterProfiler R package was used to test the statistical enrichment of differentially expressed genes in Kyoto Encyclopedia of Genes and Genomes pathways.

Immunofluorescence

Slides were washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 minutes, permeated with 0.5% Triton X‐100 for 15 minutes, blocked with 10% donkey serum for 30 minutes, and stained with primary antibodies overnight at 4°C. After washing the slides 3 more times, we incubated the sections or cells with secondary antibodies and 4',6‐diamidino‐2‐phenylindole for 30 minutes. Three researchers who were masked to sample identity quantified cellular Ki67, pHH3 (phospho‐histone H3), and aurora B via either manual counting or digital thresholding. This included image segmentation and creation of a binary image from a grayscale. We analyzed the converted binary images using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA; Laboratory for Optical and Computational Instrumentation, University of Wisconsin, Madison, Wisconsin, USA).

Cardiomyocyte Isolation From RV Free Wall and Calcium Imaging

Cardiomyocytes were isolated with a Langendorff perfusion system as described previously.²⁹ After perfusion, only the RV free wall was removed and cardiomyocytes from the RV were used for calcium image and immunostaining. Calcium imaging was performed according to a previous publication.²⁹ Briefly, before contractility and calcium analyses, calcium was re‐introduced into isolated cardiomyocytes by treating cells with a series of 10‐mL 2,3‐Butanedione monoxime‐free perfusion buffers containing 100 nmol/L, 400 nmol/L, 900 nmol/L, and 1.2 µmol/L CaCl₂. At each step, cardiomyocytes were settled down by gravity for 10 minutes at room temperature before cells were transferred to the next buffer with a higher calcium concentration. Cardiomyocytes were loaded with 5‐µmol/L Rhod‐4 for 20 minutes. The cells were next washed with normal Tyrode solution (NaCl, 140 mmol/L; KCl, 4 mmol/L; MgCl₂, 1 mmol/L; CaCl₂, 1.8 mmol/L; glucose, 10 mmol/L; and HEPES, 5 mmol/L, pH=7.4, adjusted with NaOH) for 20 minutes. The cells were next settled in a laminin‐coated glass‐bottom flow chamber at 30°C for 10 minutes and electrically stimulated at 1 Hz to produce steady‐state conditions. Calcium signals were next acquired through confocal line scanning using a ×60 objective. Line scan was positioned along the long axis of the cell in the cytosol, avoiding the nuclear area. Calcium signal was quantified manually using ImageJ.³⁰

VO Changes the Transcriptome of Postnatal RV Development

To investigate how VO changes gene expression in postnatal RV development from P7 to P21, we selected the RVs of the hearts from AVF and sham‐operated mice at P14 and P21 and performed RNA sequencing. Our results showed that during normal postnatal RV development, there were 3012 differentially expressed genes between from P21 to P14, among which 1405 were downregulated and 1607 were upregulated (Figure 3A). The VO increased this number to 3470, among which 1732 were downregulated and 1738 were upregulated (Figure 3A). The heatmap results showed that the individual mice in the same group were similar to each other but differed noticeably from the mice in the other group (Figure 3B). The Venn diagram shows that the P21_sham group shared 11 459 genes with the P14_sham group, 451 genes were only expressed in the P14_sham group, and 304 genes were only expressed in the P21_sham group. The P21_VO group shared 11 523 genes with P14_VO group, 403 genes were only expressed in the P14_VO group, and 451 genes were only expressed in the P21_VO group (Figure 3C). These results indicate that VO changes the transcriptome of postnatal RV development.

Open in a separate window

Figure 3

The transcriptomic changes of postnatal right ventricular development.

A, Volcano map of differentially expressed genes of postnatal right ventricular development in the normal condition (postnatal day 21 sham vs postnatal day 14 sham) and under the influence of volume overload (postnatal day 21 volume overload and postnatal day 14 volume overload). B, Cluster analysis of differentially expressed genes. Every group had 3 mice. The redder the color, the higher the expression level; the bluer, the lower. The clusters of genes in each group are quite different from each other but similar in the same group. C, Venn diagram of differentially expressed genes. P14 indicates postnatal day 14; and P21, postnatal day 21.

Processes of Normal Postnatal RV Development Were Primarily Associated With Metabolic and Cardiac Muscle Maturation, as Indicated by the GO Analysis

To understand normal postnatal RV development from P14 to P21, the differentially expressed genes in the sham groups were analyzed using GO analysis. The results showed that the top 10 most enriched GO terms of biological process were energy derivation by oxidation of organic compounds, generation of precursor metabolites and energy, cellular respiration, striated muscle tissue development, muscle organ development, muscle tissue development, sterol metabolic process, cholesterol metabolic process, cofactor metabolic process, and lipid homeostasis (Figure 4A and 4B). The top 10 most enriched GO terms of the cellular component were the mitochondrial inner membrane, organelle inner membrane, mitochondrial matrix, mitochondrial protein complex, myelin sheath, myofibrils, extracellular matrix, contractile fiber components, contractile fibers, and sarcomeres (Figure 4A and 4B). The top 10 most enriched GO terms of molecular function were cofactor binding, extracellular matrix structural constituent, coenzyme binding, structural molecule activity, O‐acyltransferase activity, alpha‐actinin binding, oxidoreductase activity, acting on the aldehyde or oxo group of donors, anion transmembrane transporter activity, unfolded protein binding, and active transmembrane transporter activity (Figure 4A and 4B). These results suggested that the processes of normal postnatal RV development were primarily associated with metabolic and cardiac muscle maturation, similar to that of the LV.

Open in a separate window

Figure 4

The processes of normal postnatal right ventricular development were primarily associated with metabolic and cardiac muscle maturation, as indicated by the gene ontology (GO) analysis.

A, From the results of the GO enrichment analysis, the most significant 10 terms are displayed. The abscissa is the GO term, and the ordinate is the significance level of GO term enrichment. The higher the value, the more significant the result, and the different colors represent 3 different GO subclasses: biological process, cellular component, and molecular function. B, From the results of the GO enrichment analysis, we selected the most significant 30 terms to draw scatter plots for display. The abscissa is the ratio of the number of differentially expressed genes on the GO term to the total number of differentially expressed genes, the ordinate is GO term, the size of the dots represents the number of genes annotated to the GO term, and the color from red to purple represents the significance level of GO term enrichment. BP indicates biological process; CC, cellular component; and MF, molecular function; P14, postnatal day 14; P21, postnatal day 2; and VO, volume overload.

Process of Postnatal RV Development Under the Influence of VO Were Partially Changed to Angiogenesis and Cell Cycle Regulation, as Indicated by the GO Analysis

To understand how the VO changed the postnatal RV development from P14 to P21, the differentially expressed genes in the VO groups were analyzed using a GO analysis. The results showed that the top 10 most enriched GO terms of the biological process were angiogenesis, regulation of cytoskeleton organization, regulation of vasculature development, regulation of mitotic cell cycle, regulation of actin filament‐based process, regulation of angiogenesis, regulation of cell morphogenesis, regulation of cell morphogenesis involved in differentiation, monocarboxylic acid metabolic process, and regulation of cell‐substrate adhesion (Figure 5A and 5B). The 10 most enriched GO terms of the cellular component were sarcolemma, extracellular matrix, actin cytoskeleton, extracellular matrix component, proteinaceous extracellular matrix, cell leading edge, cell cortex, contractile fiber part, contractile fiber, and myofibril (Figure 5A and 5B). The top 10 most enriched GO terms of the molecular function were cell adhesion molecule binding, actin binding, cadherin binding, actin filament binding, Rho GTPase binding, integrin binding, ubiquitin protein ligase binding, ubiquitin‐like protein ligase binding, tubulin binding, and protein heterodimerization activity (Figure 5A and 5B). These results suggested that the process of postnatal RV development was partially changed by the VO. During the change process, angiogenesis and cell cycle regulation played a role.

Open in a separate window

Figure 5

The processes of postnatal right ventricular development partially switched to angiogenesis and cell cycle regulation, as indicated by gene ontology (GO) analysis.

A, From the results of the GO enrichment analysis, the most significant 10 terms are displayed. The abscissa is the GO term, and the ordinate is the significance level of the GO term enrichment. The higher the value, the more significant the result, and the different colors represent 3 different GO subclasses: biological process, cellular component, and molecular function. B, From the results of the GO enrichment analysis, the most significant 30 terms were selected to draw scatter plots for display. The abscissa is the ratio of the number of differentially expressed genes on the GO term to the total number of differentially expressed genes, the ordinate is GO term, the size of the dots represent the number of genes annotated to the GO term, and the colors from red to purple represent the significance level of the GO term enrichment. P14 indicates postnatal day 14; BP, biological process; CC, cellular component; MF, molecular function; and P21, postnatal day 2.

Pathways of the Postnatal RV Developmental Trajectory Changed by VO

Kyoto Encyclopedia of Genes and Genomes pathway analysis was applied to find out what types of pathways regulating normal RV development were changed by VO. The results indicated that the top 20 enriched pathways in the normal RV development were the PPAR signaling pathway, citrate cycle (Tricarboxylic acid cycle), fatty acid degradation, valine, leucine and isoleucine degradation, propanoate metabolism, steroid biosynthesis, carbon metabolism, cardiac muscle contraction, fatty acid metabolism, cholesterol metabolism, thermogenesis, fatty acid elongation, aminoacyl‐tRNA biosynthesis, pyruvate metabolism, protein digestion and absorption, AMPK signaling pathway, Alzheimer disease, butanoate metabolism, oxidative phosphorylation, and alanine, aspartate, and glutamate metabolism (Figure 6A and 6C). These results further confirmed that metabolic and cardiac muscle maturation were the primary processes of postnatal RV development, similar to that of the LV.^{23, 24, 25}

Open in a separate window

Figure 6

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes of normal postnatal right ventricular (RV) development and the postnatal RV development under the influence of volume overload.

A, The KEGG pathways of normal RV development. The most significant 20 KEGG pathways from the KEGG enrichment results are displayed. The abscissa is the KEGG pathway, and the ordinate is the significance level of pathway enrichment. The higher the value, the greater the significance. B, The KEGG pathway of postnatal RV development under the influence of volume overload. The most significant 20 KEGG pathways from the KEGG enrichment results are displayed. The abscissa is the KEGG pathway, and the ordinate is the significance level of pathway enrichment. The higher the value, the greater the significance. C, The KEGG pathways of normal RV development. From the KEGG enrichment results, the most significant 20 KEGG pathways were selected for the scatter plots. The abscissa is the ratio of the number of differentially expressed genes on the KEGG pathway to the total number of differentially expressed genes, the ordinate is the KEGG pathway, the size of the dots represents the number of genes annotated to the KEGG pathway, and the color from red to purple the represents significance level of KEGG pathway enrichment. D, The KEGG pathways of postnatal RV development under the influence of volume overload. From the KEGG enrichment results, the most significant 20 KEGG pathways were selected for the scatter plots. The abscissa is the ratio of the number of differentially expressed genes on the KEGG pathway to the total number of differentially expressed genes, the ordinate is the KEGG pathway, the size of the dots represents the number of genes annotated to the KEGG pathway, and the color from red to purple the represents significance level of KEGG pathway enrichment. P14 indicates postnatal day 14; P21, postnatal day 2; and VO, volume overload.

Under the influence of VO, the top 20 enriched pathways were focal adhesion, PI3K‐Akt signaling pathway, pathways in cancer, microRNAs in cancer, extracellular matrix‐receptor interaction, cell cycle, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, valine, leucine and isoleucine degradation, human papillomavirus infection, dilated cardiomyopathy, PPAR signaling pathway, fatty acid degradation, proteoglycans in cancer, fluid shear stress and atherosclerosis, advanced glycation end products and their receptors signaling pathway in diabetic complications, propanoate metabolism, axon guidance, small cell lung cancer, and protein digestion and absorption (Figure 6B and 6D). These results further confirmed that the primary process of postnatal RV development was changed by the VO, in which angiogenesis and cell cycle regulation played a role.