Shreya Arunkumar, Lynbrook High School, San Jose, California, USA
Abstract
As research progresses on COVID-19, scientists observe that the SARS-CoV-2 virus’ effects have been primarily associated with the lungs, but its effects on the heart remain unknown. Cardiovascular disease, being a global health threat, has shown to be linked to the rise of the SARS-CoV-2 pandemic due to the implication of cardiac fibrosis seen in key signaling pathways. The TGFβ pathway is directly related to cardiac fibrosis, being an influencing factor on myofibroblast differentiation and influences the p38 mitogen activated protein kinase pathway (MAPK) through the renin-angiotensin system. MAPK has demonstrated a connection with COVID-19 because angiotensin receptors have been identified to be upregulated in the pathway with which myocardial injury occurs. Additionally, atherosclerosis — characterized by lipid accumulation in sections of the artery — is an associated factor of the COVID-19 virus. With that said, through the use of bioinformatics resources/tools (DAVID, KEGG, NCBI, Geo2R, etc.), this research study is aimed to identify genetic determinants of heart failure and cardiac fibrosis associated with COVID-19. This study’s results have indeed proven that SARS-COV-2's effects on our hearts are shown through fibrotic pathways, specifically through important genes in the TGFβ and p38 MAPK pathways. Moreover, this study resulted with another theory that COVID-19 causes atherosclerosis via the TGFβ/TLR4 pathway combination, which could impact the heart indirectly by producing cardiac fibrosis. Observing a key gene of interest, TGFBR2 has shown relevance in several pathways involved in COVID-19 and cardiovascular disease. The relevance of this study includes studying these pathways and genes of interest in more detail to reveal prominent connections between our ongoing pandemic and heart disease.
Introduction
On 30 January 2020, the World Health Organization officially declared the COVID-19 pandemic as a public health emergency of international concern. As of September 2021, over 210 million worldwide infections, 17 million active infections, and 4 million worldwide deaths have been the result of this pandemic, thus we can see the severity of this global issue (WHO, 2021). As scientists begin determining the mechanisms of COVID-19 infections in the heart leading to heart failure, myocarditis has been prominently noted as the connection between cardiovascular disease and COVID-19 (Nishiga et al., 2020). Myocarditis is an inflammatory condition of the cardiomyocytes (heart muscle cells), which is typically caused by a virus - in this situation, being COVID-19 (Siripanthong et al., 2020).
SARS-CoV-2 enters human cells by attaching its spike protein to the ACE2 receptor. Because ACE2 receptors are present in cardiomyocytes (Thum, 2020), SARS-CoV-2 can enter the heart. This is greatly observed in cases of heart failure in which cardiomyocytes are upregulated along with ACE2 receptors. However, to bind to ACE2, the spike protein must first enter cardiomyocytes. The enzyme, TMPRSS2, facilitates this entry (Siripanthong et al., 2020).
Figure 1. Shows the presence of ACE2 receptors within the myocardium of the heart and SARS-CoV-2 spike proteins (Siripanthong et al., 2020).
As shown in Figure 1, COVID-19 has effects on the heart due to the host factor, ACE2, making cardiovascular disease inevitable. Cardiac fibrosis, a long-term tissue repair program that results in heart scar tissue, has been linked to nearly every cause of cardiovascular disease, including myocarditis. Cardiac fibrosis occurs when the buildup of the extracellular matrix (ECM) is found in cardiac muscles, leading to blockage and thickening of the heart that eventually results in cardiomyopathy and heart failure (Siripanthong et al., 2020). Cardiac fibroblasts are important cells that reside in between cardiac muscle fibers in the heart responsible for the fibrosis mechanism. Our heart consists of 75% cardiomyocytes by volume, with the rest being primarily fibroblasts cells (Camelliti et al., 2005).
Figure 2. Process of myocardial infarction leading to cardiac fibrosis within myocardium (Talman et al., 2016).
Fibroblasts (shown in Figure 2 above in green) residing within the healthy myocardium maintain the homeostasis of the extracellular matrix (ECM), thus providing structural support to the cardiomyocyte cells shown in red. However, injury such as myocardial infarction or heart attack leads to cardiomyocyte death, which is shown in orange, and the expansion and differentiation of fibroblast cells to myofibroblast (Talman and Ruskoaho, 2016). These transformations are consequential, as fibroblasts secrete and remodel the ECM. Prolonged activation of these fibroblasts leads to excess deposition of ECM and forms scar tissue, eventually leading to cardiac fibrosis (Souders et al., 2009; Zeisberg and Kalluri, 2010; Pinto et al. 2015).
Cardiac fibroblasts are highly adaptable cells that use a complex array of intracellular and extracellular communication pathways. The TGFβ and p38 MAP Kinase (MAPK) have been identified as an essential signaling pathway in cardiac fibrosis (Saadat et al., 2021). The TGFβ pathway is involved in cell differentiation and has been linked to the development of a variety of illnesses, including myocardial infarction (Parichatikanond et.al, 2020). Concerning cardiac fibrosis, the pathway is responsible for fibroblast to myofibroblast conversion and then affects the MAPK pathway (Ma et al., 2018). The MAPK pathway is another pathway involving a family of kinases that regulates multiple aspects of cardiac fibroblast function. It is activated in response to stress, inflammatory stimuli, and injury (Braicu et al., 2019).
Figure 3. Shows the p38 MAPK as a part of the TGFβ pathway and the RAS as a part of the p38 MAPK pathway. Moreover, showing the involvement of both in myocardial damage leading to fibrosis (Braicu et al., 2019; Li et al., 2017).
In cardiac fibrosis and myocardial infarction, the renin-angiotensin system (RAS) plays a key role. The RAS is made up of two counter-regulatory branches called ACE and ACE2, which regulate cardiovascular processes and contain angiotensin receptors. As noted before, ACE2 receptors are important in the spread of the COVID-19 as the virus binds directly to the ACE2 for entry into cardiomyocyte cells and fibroblast cells. Myocardial injury, which activates the MAPK pathway, disrupts the ACE/ACE2 ratio and throws off the entire RAS. Additionally, ANG2, which is downstream of ACE, is a widely pro-fibrotic factor. Since myocardial infarction throws the equilibrium off, this results in cardiac fibrosis (Turner and Blythe, 2019; Li et al., 2017). This shows the interconnection between the TGFβ pathway as it influences MAPK and the MAPK pathway influences COVID-19 through the ACE2 receptors in the RAS (shown in the diagram above).
Hypothesis
Cardiovascular disease is linked to the SARS-CoV-2 pandemic due to the implication of cardiac fibrosis seen in key signaling pathways. The TGFβ pathway is directly related to cardiac fibrosis due to myofibroblast differentiation as well as its influence on the MAPK pathway. MAPK has demonstrated links with COVID-19 as Angiotensin (ACE/ACE2) receptors have been identified to be upregulated in the pathway. Therefore: COVID-19 and cardiac fibrosis may be related through these 2 pathways.
With that said, this study hypothesizes that the effects of SARS-COV-2 on our heart can be seen through fibrotic pathways, particularly the TGFβ and MAPK pathways.
Methods
The study's research started with PubMed literature studies to learn more about the pandemic and cardiovascular illness, especially the pathways involved and cardiac fibrosis. The data was then gathered using the NCBI and Gene Weaver databases. GEO2r from NCBI was used to examine datasets aimed at cardiac fibrosis, myocarditis, and heart failure. Gene Weaver was largely utilized to collect data on genes associated with COVID-19 since the database was younger and had more up-to-date information. Datasets were then merged using Gene Weaver’s “combine genesets” analysis tool. After gathering the datasets and gene lists, everything was entered into Google Sheets and Excel, where advanced tools such as VLOOKUP and filters were utilized to curate and narrow down the results. Finally, DAVID and KEGG were used to do pathway analysis. Steps 3 and 4 were repeated several times to narrow down and focus on key genes implicated in CVD and COVID-19.
Figure 4. Research study simplified process.
Over 15 datasets with 5000+ genes were collected and analyzed to a curated gene list of 156 genes. Additionally, over 36 spreadsheets of data were collected, organized, and analyzed for hits between cardiac fibrosis, COVID-19, and cardiovascular disease.
Results
Once analyzing 156 key genes through the KEGG pathway analysis tool (shown below), 50 genes were found in common pathways.
Figure 5. Post-bioinformatic analysis gene list of 156 genes organized for relevancy in cardiovascular disease and COVID-19.
The gene list was further organized into MAPK and TGFβ pathways along with a general cardiovascular disease and results from an atherosclerosis pathway (shown below). The majority signaling of the curated genes were part of the MAPK pathway proving the importance of the pathway to COVID-19 infections in the heart and the ACE2 receptor identified as a host receptor of COVID-19 was differentially regulated in the MAPK pathway of fibrosis as well. With 24 MAP-Kinase Genes, 6 TGFβ Genes, 11 Cardiovascular disease general genes, 19 Atherosclerosis Genes, and, the presence of cardiac fibrosis could be highly implicated. Additionally, key genes were observed to have overlapped in 3-4 aspects of heart disease including CD40, NFKB1, IL6, and TGFBR2.
Figure 6. Results chart categorizing key genes into pathways of interest including p38 MAPK, TGFβ, CVD, and atherosclerosis. Repeated genes are highlighted and taken as genes of interest.
A very prominent, yet unexpected pathway that kept coming up every time analysis was conducted with different gene lists was lipid and fluid/shear stress atherosclerosis pathways. 19 genes regarding atherosclerosis were observed, showing its potential prevalence in COVID-19 and CVD.
Atherosclerosis is characterized by lipid accumulation in sections of the artery, as well as smooth muscle cell and fibrous matrix growth, which eventually leads to the creation of an atherosclerotic plaque. It is a leading cause of many CVD deaths as it leads to cardiac fibrosis, myocardial infarction, and eventually heart failure (Mayo Clinic, 2021). As shown in the diagram below, at a damaged site, TGFβ signaling leads to myofibroblast differentiation and eventually leading to atherosclerosis or myocardial fibrosis (Orlandi et al., 2004). Additionally, many studies have linked atherosclerosis to cardiac fibrosis through another pathway known as toll-like receptor 4 signaling pathway which has a key gene observed in atherosclerosis and MAPK pathway known as TLR4, suggesting links between cardiac fibrosis and atherosclerosis. Taken together, there is a possibility that COVID-19 leads to atherosclerosis through the TGFβ pathway or the TLR4 pathway and could indirectly affect the heart by causing cardiac fibrosis.
Figure 7. This diagram visualizes a damaged site present in atherosclerosis and the impact of the TGFβ and TLR4 pathways on myofibrosis.(Huang, 2020).
Finally, TGFBR2 was a prominent gene found during the research study part of all 4 gene lists, with links to the TGFβ pathway, the MAPK Pathway, several cardiovascular diseases such as cardiomyopathy, and atherosclerosis. TGFBR2 is a gene that codes for a protein known as transforming growth factor-beta receptor type 2. It is moderately regulated within the heart and highly regulated in the lungs which suggest potential involvement with COVID-19 (NCBI, 2021).
Figure 8. Chromosome location, 3p24.1, of TGFBR2 (NCBI, 2021).
Figure 9. Bar graph illustrating the presence of TGFBR2 in body organs (NCBI, 2021).
TGFBR2 crosses the cell membrane to carry out its signaling activity. The TGFβ molecule binds to the TGFBR2 and activates it, leading to signaling within the cell (NIH, 2020). The combination of proteins activates all receptors part of the TGFβ pathway. As shown in the 3 KEGG pathway maps below, this gene is upstream of all signaling genes in each pathway (highlighted in pink), thus having a major influence.
Figure 10. TGFBR2 in the TGFβ pathway (KEGG, 2021).
Figure 11. TGFBR2 in a diabetic cardiomyopathy pathway (KEGG, 2021).
Figure 12. TGFBR2 in the p38 MAPK pathway. (KEGG, 2021).
Conclusions
COVID-19 infections can affect several genes present in cardiovascular diseases as shown by 156 genes curated list. Moreover, genes involved in cardiovascular disease are impacted in the vital pathways including, 24 MAPK Genes, 6 TGFβ Genes, 11 Cardiovascular disease general genes, 19 Atherosclerosis Genes. Furthermore, key genes involved in COVID-19 and cardiovascular disease were identified and further analyzed (D40, NFKB1, IL6, TLR4, TGFBR2, ACE2). Of this curated list, a particular gene of interest is TGFBR2, present in all pathways and diseases analyzed.
Taken together, the effects of SARS-COV-2 on our heart are seen through fibrotic pathways, particularly through key genes in the TGFβ and MAPK pathways. An alternate hypothesis is that COVID-19 leads to atherosclerosis through the TGFβ/TLR4 pathway and could indirectly affect the heart by causing cardiac fibrosis.
Although minor, I hope my modest contribution can play a role in uncovering the effects of SARS-CoV-2 on the heart. Future directions could include cloning genes including TGFBR2, CD40, NFKB1, IL6, TLR4 that commonly occur within key pathways into plasmids to study their function. Localization studies can be carried out by tagging these genes with green fluorescent proteins (GFPs) and finally, transcriptomic profiling can be conducted by knockout of genes of interest.
Figure 13. TGFBR2 plasmid (Addgene, 2021).
The significance of this study is understanding specific genes implicated in cardiac fibrosis and their relationship to COVID-19 damaged pathways around the cardiovascular system as it can lead to the development of new treatments targeting the development of heart failure. Understanding the mechanisms of COVID-19's impact on our hearts will enable us to better understand and combat the infection. Stay safe and play your part in halting the spread of this virus.
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