Abstract
The inability of the adult heart to repair or regenerate is manifested in prevalent morbidity and mortality related to myocardial infarction and heart failure. However, the primer cue to the reactivation of cardiomyocyte proliferation in the adult remains largely unknown. In the present study, three independent datasets were explored using bioinformatics analysis methods to address the problem. Our results revealed that atrium genes were upregulated in response to the injury, which indicates the possible cell type withdraw and re-initiation of proliferation capability. Our findings might provide an alternative viewpoint on the cardiomyocyte regeneration or myocardial infarction.
Introduction
Cardiovascular diseases have long been the leading cause of death in both industrialized and developing countries. Underlying basis of the diseases is cardiomyocyte loss. The notion that it might be possible to rebuild the cardiac muscle paves the way of new concept, heart regeneration, which is one of the most active and controversial areas of biomedical research. Clinical practice at the bench-side proposes three strategies, including cell-based therapy1, bioengineering methods2,3 and synthetic organogenesis4–6.
However, a major barrier against myocardial regeneration appears to be cell-cycle arrest of adult mammalian cardiomyocytes. One theory is that mitosis of incessantly contractile cells is not feasible7. Another theory is that neonatal heart regeneration in mammals is simply a remnant of developmental programs that are lost after birth8,9. Researchers around the world now trace the answer from a wide variety of animal models10.
In zebrafish model, differentiated atrial cardiomyocytes can transdifferentiate into ventricular cardiomyocytes to contribute to cardiac ventricular regeneration11. A subpopulation of cardiomyocytes that transiently express atrial myosin heavy chain (amhc) contributes to specific regions of the ventricle12. The finding suggests an unappreciated level of plasticity during chamber formation. These observations prompt us to hypothesize there might be a kind of heart atrium progenitor cell exists in the mammalian system, which will play a role in heart regeneration in response to injury stimuli. We designed the following study using public data to explore the possibility of the above hypothesis.
Methods
Data sources
All three datasets used in the present study were downloaded from NIH GEO center. The dataset GSE1479 is a benchmark set for early cardiac development. At stage 10.5, the rostral and caudal parts of the mouse embryo were removed and the middle part, which includes the heart, was subjected to expression analysis. From embryonic day 11.5 on, embryonic hearts were isolated and the ventricular from the atrial chambers were separated. The GSE775 dataset is a time series experiment design intended to compare normal functioning left ventricles(lv) with infarcted (ilv) and non-infarcted left ventricles(nilv). Ilv samples are taken from the region between the LAD artery and the apex on a mouse with myocardial infarction. Nilv samples are taken from the region above the infarction and the left ventricle(lv) samples mimic that region in a sham mouse. Both GSE1479 and GSE775 were released by the CardioGenomics project13. The GSE64403 was published by Boyer’s lab on the subject of transcriptional reversion of cardiac myocyte fate during mammalian cardiac regeneration14. Primary myocardial tissues sampled from neonatal mice and murine hearts undergoing post-injury regeneration were harvested according to the original description.
Collation of atrium specific genes
Atrium (specific) genes are highly expressed in heart atrium compared with these in the ventricle. Therefore, atrium genes play import roles in chamber physiological function or morphogenesis during heart development. The GSE1479 is a benchmark set for early cardiac development. From embryonic day 11.5 on, embryonic hearts were isolated and separated to the ventricular from the atrial chambers. In the bioinformatic curation procedure, the microarray data from respective chambers were pooled together to perform gene differential expression analysis using Limma package15. The expression value contrast above 1 (logFC > 1) and p-value above 0.001 were chosen as the complete chamber specific genes. After filtering, the hierarchical clustering was used to separate the atrium and ventricle sets. Manual curation of chamber specific genes was undertaken to construct an independent evaluation set.
Source code management
The source code of the present project was deposited in the GitLab (atriumization_source_code.R, 45888b43aaa717aea3c40c70ed289fa455719872). The development process followed the etiquette of the scientific reproducible research. Every step of revision or update was recorded and uploaded to the remote repository at GitLab.
Data analysis tools
Data was analysis using the R/Bioconductor packages. Picture and graph layout were generated by ggplot2 and cowplot packages. Differential gene expression analysis was conducted using limma if the data type is Affymetrix microarray, or DESeq2 if the data is high-throughput sequencing. Gene Set Enrichment Analysis (GSEA) is a computational method that determines whether an a priori defined set of genes shows statistically significant, concordant differences between two biological states. This approach was conducted using ClusterProfiler package. Heatmap analysis was generated using the pheatmap package. Principle component analysis results were generated using the factoextra and FactoMineR packages. Most packages were from Bioconductor or CRAN16.
Results
Atrium specific genes curated from the public dataset
Principle component analysis (PCA) was performed to check the data quality (GSE1479). The original experiment claimed to monitor changes in gene expression related to heart development and maturation. From embryonic day 11.5 on, the expression profiling by array also reported the gene expression respectively from ventricular and the atrial chambers. In the PCA results, components PC1 and PC2 can perfectly separate these two events, cardiac maturation and chamber differentiation although the maturation as an independent variable was collineared partially with chamber differentiation (Figure 1A). To minimize the co-founder of the maturation event, the chamber specific samples at different developmental stages were pooled together and gene differential expression analysis was performed to curate the chamber specific genes and then hierarchical clustering was conducted to obtain these two groups. After above filtering, the atrium and ventricle specific gene sets were generated accordingly. The chamber specific set was then used in the next round of PCA analysis. This resulted in the increase percentage of PC1 component and about 70 % variability can be explained by the new PC1. In the atrium genes, sarcolipin (Sln, gene ID: 66402) is included, which is a hallmark of heart atrium17,18. To further confirm these findings, panorama view by the David functional analysis was performed (Figure 1C & Figure 1D). The result revealed that these genes are involved in heart morphogenesis and secretion as the key term was enriched in protein function in consistent with the previous observation that atrium is a secretory organ19.
Expression of atrium gene set was rekindled in myocardial infarction
Data set GSE775 were re-analyzed using above defined set for the atrium genes. This set included spatiotemporal result by the microarray design. The original study included the time-serials samples and the experiment also included three myocardial regions13. Reactivation of natriuretic peptide A (Nppa) and smooth muscle actin alpha 2 (Acta2) is the golden standard to assess the quality of myocardial infarction model. The increased expression of both indicated that the mouse model is qualified to downstream analysis (Figure 2A & Figure 2B). Expression of the atrium hallmark gene Sln was not statistically significant, however. The manual curated atrium genes plus Nppa were plotted using the heatmap. Only heart stress indicator, Nppa, was fluctuated with time change. The GSEA analysis using the defined set was then performed to assess the phenotype change. The result showed that the increased expression of atrium genes (Figure 2D) with statistical significance.
Expression of atrium gene set was enlightened in heart regeneration
Data set GSE66403 was used as the biological and technique replicate to infer the phenotype transformation during the heart regeneration. However, the new data set is much more sensitive as the RNA-seq have more accurate measurement. The chamber specific gene set, which was manually curated from the published literature, was used as an independent set to deduce the phenotype change. In the myocardial infarction dataset, the expression of atrium and ventricle genes was not altered. However, the GSEA analysis indicated that the expression of atrium genes are increased, showing that the cell physiological character tend to be atrium cell instead of ventricle cell. While as an independent replicate, the isolated ventricle cells in the heart regeneration dataset showed that the expression of this atrium gene marker was increased and statistically significant. Particularly, expression of the Sln was significantly increased (P < 0.05, Figure 3D).
RA signaling upstream of patterning was activated
To tackle the upstream event, retinoid acid pathway was explored in both datasets. Retinoic acid (RA) is a vitamin A metabolite that acts as a morphogen during body development. The retinoid pathway was monitored in the present analysis since it plays a role in cardiac chamber patterning. Aldh1a2 (also named Radlh2), component of the RA signaling was activated in the epicardium during zebrafish heart regeneration20. Aldh1a2 was increased in these two datasets. The expression of Aldh1a2 in the infarction site compared with ventricle sham site or non-injury site was increased significantly (Figure 4A). However, in the regeneration model, the expression of Aldh1a2 was only significantly increased in the surgery heart compared with the normal heart (Figure 4B).
Discussions
We rigorously defined the atrium gene sets, including ventricle gene set using a benchmark dataset GSE1479. The myocardial infarction (GSE775) and heart regeneration (GSE64403) datasets were explored using data-mining methods. The results showed that the expression of atrium genes was increased in the harvested heart ventricle tissue. This observation can be explained by three interpretations. One possible mechanism is ventricular-to-atrial trans-differentiation, leading to the local increase of atrium cells. The second is atrium-like progenitor cell proliferation. The third may be the atrium cell migration. We cannot exclude the other possibility. The discovery of this phenomena is solely dependent on the re-analysis of these public data.
Verification of chamber specific cell requires three pieces of evidence in wet procedure. They include gene expression pattern, electrophysiological properties and calcium sparks21,22. In the present study, a predefined atrium set was used in GSEA analysis and in facto atrium cell marker, Sln, was used as a second evidence. Adolfo J. de Bold proposed that, based upon the association of Nppa with the atrial-specific granules, the concept of the heart as an endocrine organ23. We also found in the present unbiased bioinformatics approach that atrial cardiomyocytes displayed secretory phenotype. The heart is regarded as a endocrine organ thirty years ago. Expression of Nppa was an early and specific marker for the differentiating working myocardium of the atria and ventricles of the developing heart24. However, our statistical analysis did not support Nppa as the hallmark of atrium phenotype. Instead, Nppa is a true marker in clinical investigation of cardiovascular diseases. Two separated enhancers which govern independent response during development and disease24. Instead, recent study revealed that Sln is the true lineage marker of atrium cells and have a conserved expression pattern. Early work showed that Sln was up-regulated during development and down-regulated by cardiac hypertrophy17. Thus, expression of Sln is a perfect inner control in our study. We found that the expression of Sln mRNA was detected selectively in the atria and not in myocardial infarction.
We hypothesized that the expression of atrium genes would be increased in response to the cardiac damage and myocardial infarction (MI) is permanent damage to the heart muscle. The microarray data, GSE775, was recruited to support our assumption. This mouse dataset is a time series intended to compare normal functioning left ventricles with infarcted (ilv) and non-infarcted left ventricles (nilv). Ilv samples are taken from the region between the LAD artery and the apex on a mouse with myocardial infarction. Nilv samples are taken from the region above the infarction and the left ventricle (lv) samples mimic that region in a sham mouse. To double check the quality of the dataset and analysis pipeline, the Nppa expression was assayed as it is the golden standard of cardiac diseases. Reactivation of ventricular Nppa expression is part of a conserved adaptive change in molecular phenotype in response to heart failure that serves both diagnostic and potential therapeutic options25,26. The expression levels of Nppa was regressed across the whole time points and upregulated in the ilv or nilv samples while that in lv sample (sham counterpart) was kept same.
To further confirm the finding that the myocardial infarction model was successful, the expression of Acta2 was checked as it is an alternative marker which was used to corroborate the finding27. The upregulation of both Nppa and Acta2 at early stages indicates that the mouse model truly exhibits the disease progression. To test expression level of the atrium genes, Nppa and other manually curated atrium genes17,28,29 were plotted in the same heatmap. The wave-like expression pattern of Nppa (Figure 2C) is in consistent with the previous analysis (Figure 2A). However, the atrium genes were kept no change. The Sln expression is consistent with the previous finding that the expression in the heart ventricle is not upregulated during cardiac hypertrophic remodeling. GSEA analysis was then undertaken to increase the power the detect the expression direction. The result showed that the expression levels of the atrium dataset were increased significantly.
To further confirm our finding that expression of atrium gene set was upregulated, atrium gene expression pattern was verified in an independent dataset GSE64403, which utilized deep-sequencing method and adopted to heart regeneration model. In the heart ventricle tissues, the atrium genes were not scientifically increased and the same were ventricle genes (Figure 3A). GSEA analysis was conducted and showed that the increased expression pattern of atrium genes is above statistical significance. However, the harvested ventricle tissues included other cell lineages besides cardiac ventricle cells. To exclude this influence and identify the true source, the same group of chamber genes was analyzed in the isolated heart ventricle cells. The increased expression pattern can be identified in the authentic atrium markers, Sln, Myl7 and Myl4 (Figure 3C). The golden marker of atrium gene, Sln was analysis and compared between surgical and sham group at day 1. The upregulated Sln expression was statistically significant (P < 0.05). In the present study, we examined the effects of development and cardiac hypertrophy on the expression of Sln mRNA in heart. We found that the expression of Sln mRNA was detected selectively in the atria. Furthermore, Sln was up-regulated during development and down-regulated by cardiac hypertrophy17.
To ask what the upstream event of atrium gene reactivation, multiple comparisons of Aldh1a2 expression between different groups in data set GSE64403 were performed. Early study in zebrafish revealed that the injuried heart at the epicardium exhibited increased expression of Radhl2 using mRNA probe hybridization. The P values were adjusted using the nbinomTest method in DESeq2 package. vAd was the mouse sample from the region of adult ventricular myocardium. vD7S was the mouse sample from the region of 7 days post-sham surgery ventricular myocardium. vD7R was the sample from the region of 7 days post-sham surgery ventricular myocardium. Expression of Aldh1a2 from vD7R or vD7S was increased compared with that from vAd (Padj < 0.001). Hox genes and retinoid receptors were also checked and they were at different stages upregulated. These results indicate that at earliest stage of myocardial infarction or heart regeneration, immune response elicited the patterning process to be reactivated through retinoid acid signaling pathway, which mirrors the developmental progress in cardiac development.
Numerous studies propose that the heart regeneration involved in re-activation of the development pathways. But this conclusion is not based upon the quantitative data. We here used the bioinformatic analysis to infer that the ventricle-atrium trans-differentiation might occur during the heart regeneration or myocardial infarction in mouse model.
Conclusions
The present approach using the public datasets and data-mining methods indicated that the increased expression of atrium specific genes is the hallmark of local response to the heart injures. The atriumization then revealed possible phenotype transformation during heart regeneration. This kind of gene expression upregulation suggested that the heart ventricle cells preferred to withdrawing their default physiological character and make an attempt by the outside morphogen signal to turn into atrium-like cells. The cell-level reprogramming might be the fundamental mechanism of priming heart regeneration. Impediment of the process probably resulted in cardiomyocyte loss, leading to myocardial infarction or regeneration failure. Our work is only based upon the public data and needs to be addressed by wet verification. The preliminary results will shed light on the future investigation on regeneration medicine.
Sources of Funding
This work was supported by the National Natural Science Foundation of China (Grant number 31000644 to Y.Z.) and China Medical Board Award (to Y.Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Disclosures
The author declares that he has no competing interests.
Acknowledgments
The author is grateful to Prof. Shou, Weinian (Indiana University School of Medicine) and Prof. Xiong, Jing-wei (Peking University) for their expertise in mouse development and zebrafish development respectively.