Characterizing Smyd5's functions in the heart

Heart disease has long been an issue in medicine of importance and is a leading cause of death worldwide. Understanding how heart disease manifests itself is crucial to the development of novel treatment. One particular characteristic of all forms of heart disease the cardiac hypertrophy, a phenomenon where individual cardiomyocytes grow in size to meet the increased demand for cardiac output. While beneficial to a certain extent, uncontrolled hypertrophy of the heart can lead to things such as ventricular remodeling causing problems with cardiac output and leading to heart failure. In this investigation Smyd5, a member of the histone methyltransferase Smyd family, was studied in the context of the heart using histological techniques. The results have revealed a general increase in cell area in mouse hearts 5 weeks after Smyd5 activity was abolished. These observations suggest Smyd5 plays a role in cardiac hypertrophy and the progression towards heart disease, warranting further study.

Characterizing Smyd5’s functions in the heart by Nick Santa Ana A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Biology Approved: ___ Sarah Franklin, Ph.D. Thesis Faculty Supervisor Denise Dearing, Ph.D. Chair, Department of Biology ________ Michael Bastiani, Ph.D. Honors Faculty Advisor _____________________________ Sylvia D. Torti, PhD Dean, Honors College April 2020 Copyright © 2020 All Rights Reserved ABSTRACT Heart disease has long been an issue in medicine of importance and is a leading cause of death worldwide. Understanding how heart disease manifests itself is crucial to the development of novel treatment. One particular characteristic of all forms of heart disease the cardiac hypertrophy, a phenomenon where individual cardiomyocytes grow in size to meet the increased demand for cardiac output. While beneficial to a certain extent, uncontrolled hypertrophy of the heart can lead to things such as ventricular remodeling causing problems with cardiac output and leading to heart failure. In this investigation Smyd5, a member of the histone methyltransferase Smyd family, was studied in the context of the heart using histological techniques. The results have revealed a general increase in cell area in mouse hearts 5 weeks after Smyd5 activity was abolished. These observations suggest Smyd5 plays a role in cardiac hypertrophy and the progression towards heart disease, warranting further study. ii TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 METHODS 9 RESULTS 11 DISCUSSION 17 REFERENCES 21 iii INTRODUCTION Heart disease remains one of the leading causes of death world-wide. In 2016, of the approximately 2.7 million deaths registered in the United States that year, the leading cause was heart disease, followed by cancer and unintentional injuries.1 Based on 20132016 data from the National Health and Nutrition Examination Survey (NHANES), an estimated 1 in 3 adults in the United States suffered from at least one form of cardiovascular disease. Cardiovascular disease has been and currently remains a crucial issue in the world of medicine. One of the distinct characteristics shared by all forms of heart disease is cardiac hypertrophy. Hypertrophy is usually beneficial (referred to as adaptive or eccentric) but when uncontrolled can become detrimental (referred to as maladaptive or concentric). The heart is able to respond to an increased requirement of blood flow by allowing its cells (cardiomyocytes) to grow in size. This improved performance, however, comes at a cost. Hypertrophy can cause the ventricular walls to become too thick, causing the heart to have trouble pumping blood through the ventricle leading to a dysfunctional system. For these reasons, cardiac hypertrophy has become an area of interest to researchers. One method of achieving this state is through the use of epigenetic mechanisms. Over the past century, epigenetics has risen as a new field in biology and explains ways for the alteration of phenotype long after organismal development. Understanding heart disease in the context of epigenetics is crucial to developing novel methods of treatment. Smyd1, a protein of the Smyd family of methyltransferases, was identified by the Franklin Lab as a regulator of mammalian heart growth and thus a potential regulatory in the pathway towards heart disease.2 This study tested the effects of Smyd1 knockout in transgenic mice and demonstrated that mice with cardiac-specific Smyd1 knockout exhibit a heart disease phenotype with symptoms of cardiac hypertrophy on a macroscopic and cellular level. In this thesis, similar experiments will be conducted with Smyd5, another member of the Smyd family. Preliminary data from the Franklin lab has indicated that by week 5 after inducing cardiac failure, mice hearts have already exhibited changes in gross morphology and increased fibrosis indicating hypertrophic growth. Moreover, trichrome staining has revealed increased cell infiltration of macrophages and fibroblasts potentially implicating Smyd5 is related to immune function. Heart samples will be analyzed using wheat germ agglutinin (WGA) staining to detect hypertrophic growth at the cellular level. For future experiments, the TUNEL assay will be used to identify any cells undergoing apoptosis. Finally, a selection of markers for macrophages and fibroblasts (cd45, IL6, CXCL10, vimentin and CCL4) will be stained in order to gauge immune response upon inducing heart failure via Smyd5 KO. Gaining an improved understanding of the Smyd family and their role in heart development can pave the way for viable treatment options of heart disease. Heart Disease Initially, it was thought that heart disease was a more modern condition due to rapid changes in lifestyle over the past century. However, conditions such as coronary heart disease and atherosclerosis date as far back to ancient Egypt as mummies studied with computerized tomography have shown signs of atherosclerosis.3 While Homer acknowledged the medicine of the Egyptians in the Odyssey, there was no evidence of any attempt to correlate these symptoms with pathology.3It was not until Leonardo da Vinci when atherosclerosis was first characterized. da Vinci described the condition by 2 stating that “vessels in the elderly restrict the transit of blood through thickening of the tunics.”3 By 1768, William Heberden had begun to describe angina pectoris as a pain in the breast area.3 He originally coined the term angina pectoris meaning “strangling chest” in Latin. In next few centuries, many more advancements in the characterization of heart disease were made. Anatomist Giovanni Morgagni first mentioned the hardening of arteries and physician Edward Jenner correlated angina pectoris to this hardening.3 Rudolf Virchow, considered the “father of pathology”, developed the concept of thrombosis, which is still clinically relevant in modern medicine.3 Heart disease encompasses a variety of conditions and is generally characterized by problems with the structure of the heart and blood vessels. These problems can complicate and eventually lead to heart failure in which the heart is no longer able to maintain sufficient cardiac output (CO) that is able to meet metabolic requirements.4 Typical heart diseases include stroke, congenital cardiovascular defects, arrhythmia, atherosclerosis, coronary heart disease, and cardiomyopathy.1 Conditions such as heart attacks and strokes result from blockage in the vessels that obstructs blood supply to places such as the heart or brain.5 When a blockage is met, the heart can bypass it by pumping harder which while mildly beneficial can serve as a cause for more serious heart problems. This leads to cardiac hypertrophy, a common characteristic of all forms of heart disease. Common causes of heart disease are largely lifestyle related and include use of tobacco, physical inactivity, poor nutrition which can lead to diabetes mellitus, hypertension, and obesity.1 Initial signs and risk factors for cardiovascular disease 3 include increased blood pressure, increased blood glucose, increase blood lipids, and being overweight or obese.5 Measures have been taken in response to these general causes such as a 1 liter per excise tax on sugar-sweetened drinks in Mexico and policy level interventions such as the Tobacco 21 law but these have had marginal effects.1 Globally, there were around 17.6 million deaths attributed to cardiovascular disease in 2016 marking an increase of around 14.5% since 2006.1 This number represented 31% of all global deaths that year.5 In addition, 81% of the people who died from a cardiovascular disease died from a heart attack or stroke.5 Over the past century, current lifestyles have changed drastically and this is reflected in the increasing prevalence of cardiovascular disease globally. Despite advances in medicine, cardiovascular disease remains an important issue in modern medicine. A variety of methods exist today to combat cardiovascular disease. The form of treatment can vary depending on the severity of the disease with the patient and among the different types of cardiovascular disease can be somewhat similar. When diagnosing a patient, a physician often time will consider a 10-year risk calculation as well as things such as family history and other risk factors.6 One of the most common recommendations is to make healthy lifestyle changes. These include things such as losing weight, eating healthy, maintaining physical activity, managing stress, and quitting smoking.6 These changes combat heart disease by also alleviating risk factors such as high blood cholesterol, hypertension, and being overweight or obese.6 Unhealthy diets and smoking can contribute to plaque buildup in circulatory vessels while being active can promote beneficial heart growth 4 If the condition is more serious, certain medicines may be administered. ACE (angiotensin converting enzyme) inhibitors and beta blockers help by decreasing blood pressure.6 ACE is an important component of the renin-angiontensin-aldosterone system which is used primarily to regulate blood pressure and volume. Similarly, calcium channel blockers can be utilized to relax blood vessels.6 Other medicines include metaformin to aid with plaque buildup, nitrates and ranolazine to aid with pain from angina, and statin therapy.6 Lastly, there exist medical procedures in order to treat advanced forms of heart disease. Procedures used to treat coronary heart disease include percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG), and transmyocardial laser revascularization.6 These methods involve surgical intervention to restore flow to the coronary arteries of the heart. Generally, it is better to utilize medicine and lifestyle changes before resorting to using these techniques. Cardiac Hypertrophy Cardiac hypertrophy is a characteristic that manifests itself in all forms of heart disease and is a common link between them. In order to compensate for added stress on the heart, the heart undergoes hypertrophy, that is the largening of cardiomyocytes. Cardiac hypertrophy is generally considered an adaptive response to hemodynamic stress and needed to enhance cardiac performance.7 Without this mechanism, heart failure may occur in which the heart cannot adequately sustain the supply of oxygenated blood to the body.8 The progression of cardiac hypertrophy can be divided into 3 phases: development of hypertrophy, the compensatory phase, and heart failure.9 When cardiac load exceeds cardiac output, the heart enacts hypertrophy to reconcile this difference. The equalization 5 of cardiac workload and resting cardiac output is the compensatory phase. However, if the hypertrophy becomes maladaptive or continues to occur beyond its necessary amount, then heart failure may occur as a result of ventricular dilation effectively reducing cardiac output. Thus, there is a distinction between hypertrophy that is beneficial to the heart and hypertrophy that may develop into more serious conditions. Hypertrophy can be split into two categories: eccentric and concentric.9 Eccentric hypertrophy, sometimes referred to as adaptive or physiological hypertrophy, occurs in athletes or pregnant women. This type of hypertrophy is characterized by longitudinal cell growth and additional sarcomeres as a result of volume overload.9 Eccentric hypertrophy is natural and beneficial in that it allows for increased cardiac output for a mother who needs to provide for her growing child or the athlete who wishes to improve performance. Concentric hypertrophy, also known as maladaptive or pathological hypertrophy, generally results from stressors that cause pressure overload and unlike eccentric hypertrophy, sarcomeres are added in parallel, facilitating lateral cell growth.9 As a result, the ventricular remodeling occurs. When the ventricular walls of the heart grow to big, the heart has trouble successfully ejecting blood from its chambers. Increased ventricular wall size can decrease chamber volume and decrease cardiac output. This is detrimental as any plaque in the blood vessels generally requires the heart to pump more blood in order to bypass the blockage and continue to provide areas such as the brain and other tissue the necessary amount of blood. Knowing the distinction between both forms of hypertrophy is critical as hypertrophy of the heart can be normal and expected in some individuals. 6 Most conditions associated with heart failure require increased cardiac output to meet the needs of the body. Some of the compensatory mechanisms the heart can use to respond to these conditions include the Frank-Starling mechanism and neurohormonal activation.4 By controlling the degree of muscle contractility, the heart can regulate stroke volume in in response to changes in venous return. As left ventricular end diastolic volume (LVEDV, also referred to as preload) increases, the LV end diastolic pressure increases leading to a stretch in the myocardium allowing for increased cardiac output.4 The Frank-Starling mechanism can only serve as a compensatory mechanism until a certain point where a patient begins to exhibit systolic dysfunction.4 As this type of dysfunction worsens, the stroke volume only increases slightly in response to increased contractility suggesting decreased effectiveness of the Frank-Starling mechanism as a compensatory mechanism.4 Here, adaptive hypertrophy may shift to maladaptive. The other mechanism that can be used to counteract a drop in CO as a result of heart failure is neurohormonal activation. Neurohormonal systems such as the renin-angiotensinaldosterone system can also increase mean arterial pressure (MAP) during HF but ultimately leads to ventricular remodeling. 4 Ventricular remodeling is the most detrimental to the heart and results from increased CO.4 Chronic stress on the heart leads to changes in the structure and ventricular function.4 The gross geometry of the ventricle becomes more spherical as myocardial wall thickness and overall ventricular mass increase allowing for improved contractility.4 To enlarge the ventricle, the myocardium undergoes cellular hypertrophy. Once ventricular walls grow too large, increased wall tension and fibrosis hinder contractility leading to myocardial apoptosis.4 The change in chamber volume also 7 prevents the heart from efficiently ejecting all the blood from the ventricle. Generally, hypertrophy of the left ventricle, which supplies the entire body with oxygenated blood, is more common than hypertrophy of the right ventricle (which sends blood through the pulmonary circuit). It is when this phenomenon occurs that heart failure becomes a potential problem as the heart begins to have issues supplying the rest of the body with oxygenated blood. Epigenetics While conditions such as heart disease can be passed down genetically, they can also arise as responses to external stimuli such as poor nutrition, smoking, and other factors. These changes can be explained by epigenetics, which is defined as the study of heritable changes in gene function that do not involve changes in DNA sequence.10 The term “epigenetics” was first coined by geneticist Conrad Hal Waddington in 1941 as a portmanteau of the words “genetics” and “epigenesis”.11 Originally used as a term to describe variations in the phenotype of organisms during their developmental stage, epigenetics has become better understood as a mechanism to control gene expression in order to adapt to the environment. Known epigenetic mechanisms today include DNA methylation, acetylation, and forms of chromatin remodeling. In 1975, researchers proposed the idea that DNA methylation could serve as a marker for the inactivation of genes.12 In 1996, it was discovered that a histone acetyltransferase in Tetrahymena was homologous to a transcriptional regulator found in yeast, supporting the idea that histone acetylation was linked to gene expression.12 Mechanisms like these were shown to regulate what parts of 8 the genome were expressed. The initial idea of epigenetics serving as a developmental mechanism later shifted to the idea of epigenetics as more of an adaptational system. In the context of heart disease, changes in the pattern of DNA methylation were observed with a number of conditions, including cardiovascular disease.13 Epigenetic modifications were also found to have affected the development of heart failure.13 Overexpression of the transcriptional co-activator CREB binding protein (CBP), which is activated by histone methyltransferase activity, resulted in hypertrophy but overexpression of a mutant CBP without histone methyltransferase activity did not exhibit this phenotype.13 Similarly, in another mouse study, hydroxylmethylation of the epidermal growth factor receptor gene led to ventricular hypertrophy.13 These are just two examples of common epigenetic mechanisms and how they affect heart physiology. In order for the heart to maintain sufficient cardiac output in response to lifestyle changes, it can enact these epigenetic mechanisms to change its ventricular structure. 9 Smyd5 and the Smyd Family The Smyd family of proteins is a group containing five lysine methyltransferases.14 The name “Smyd” comes from the the Su(var)3-9, Enhancer-ofzeste and Trithorax (SET) domain, which is split by a Myeloid-Nervy-DEAF1 (MYND) domain. Of the five proteins, Smyd1 is known to be the most expressed in the fetal heart out of all the members.14 Smyd1 was identified through proteomic studies as a muscle-specific regulator of chromatin.2 In a mature heart, Smyd1 decreases expression in the heart potentially indicating Smyd1’s potential role in heart development. Smyd1 has been associated with heart development and cardiomyogenesis and has been shown to have an effect on cardiomyocyte maturation when deleted in mice.15 Upon heart failure, Smyd1 appeared to have been upregulated.2 This event, accompanied by ventricular dilation (suggesting cardiac hypertrophy), suggested Smyd1 may be acting as an inhibitor to certain genes that prevent the adult heart from reverting to a transcriptome associated with a developing heart. In general, Smyd1 has been shown to inhibit mammalian heart growth by binding to chromatin and thus preventing uncontrollable hypertrophy.2 Smyd1 also has its role in the adult heart as well. It has been shown to regulate cardiac energetics in the adult hearts of mice.16 Smyd1 KO in these adult mice not only leads to down regulation of mitochondrial energetics but also down regulation in gene expression of certain regulators of cardiac energetics (PGC-1α, PPARα, and RXRα).16 Smyd5 remains one of least characterized members of the Smyd family. One quality that separates Smyd5 from the other members of the Smyd family is its lack of a C-terminal domain.14 It is also the most expressed member in adult human tissue.14 10 Smyd5 has also been shown to play a role in the differentiation of embryonic stem cells by targeting lysine 4 of histone 20 (H4K20) through trimethylation of heterochromatin regions.17,18 Through trimethylation of H4K20, Smyd5 has also been shown to be involved in hematopoiesis and inflammatory response.19,20 Figure 1: Expression of Smyd proteins across various human tissue and cells. Measurements of expression were taken by Kim et al. using mass spectrometry.14 All the listed proteins play some role in the fetal stage and all but Smyd4 are expressed in the adult heart. 11 Experimental Approach In order to begin characterizing Smyd5’s function in the context of the heart, histological studies will be performed to acquire visual and tangible data. Previous histology images with Smyd1 KO hearts have shown evidence of cardiac hypertrophy, fibrosis, and ventricular modeling. The primary technique that will be used in this project is wheat germ agglutinin staining (WGA). WGA staining works by binding to the glycoproteins of the cell membrane and can be used to stain cardiac sarcolemma in order to determine cross sectional area.21 By staining the cell membranes of cardiomyocytes, local changes in cell size can be quantified and visualized. Having visual evidence is an easy method of confirming whether or not cardiac hypertrophy and ventricular remodeling is occurring before beginning studies on a molecular level. It is expected that Smyd5 KO samples will experience changes in gross morphology, fibrosis, and local changes in cell area size just as Smyd1 has shown in previous literature. 12 Methods Transgenic Mice All protocols involving animals conform to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Utah. Cardiac specific inducible Smyd5 KO mice will made by crossing floxxed Smyd5 with α-MHC-MerCreMer mice. To induce recombination, mice will be fed a diet containing Tamoxifen (Tmx), 0.4 mg per gram of chow diet. Cardiac tissue was harvested after 5 weeks after treating the mice with Tamoxifen. Figure 2: Diagram showing loss of functional smyd5 allele in inducible cardiac-specific Smyd5 knockout mice. Exon 2 is flanked by two loxP sites. Upon expression of Cre recombinase via the inducible cardiac-specific MerCreMer allele, exon 2 is lost resulting in the loss of functional smyd5. 13 Histological analysis Tissue samples from mice were collected from control and transgenic mice to examine gross morphology and cell characteristics. The hearts have been fixed in 4% paraformaldehyde and submitted to the Histology core at the University of Utah for paraffin embedding and sectioning (4μm). The samples were then returned to the Franklin lab and were examined Texas red-X-conjugated wheat germ agglutinin (WGA) to quantify local changes in cell size. Tissue sections were deparaffinized with xylenes and rehydrated with various ethanol concentrations. WGA stain was applied to each section after rehydration. Slides were imaged using an Olympus BX51WI microscope with the 20x objective lens and the Olympus CellSens software to individually measure each cardiomyocyte in terms of area (μm). Prior to image capture, images will be adjusted using ISO, exposure, and black balance to emphasize the WGA stain. Four images were taken of each heart and 50 cells were measure per imaged. After taking each measurement, cell area data was aggregated and analyzed based on sample (WT vs. KO). A T-test was performed to determine if any statistically significant change occurred between WT and KO area measurements. 14 Results Wheat Germ Agglutinin Staining To test for Smyd5 involvement in cardiac hypertrophy, a cardiac specific inducible mouse model was used. If Smyd5 was removed by feeding cre expressing mice tamoxifen, then it would be expected that these mice would undergo cardiac hypertrophy and thus heart failure. Mice were fed tamoxifen chow and after 5 weeks, heart tissue was harvested for experiments. Wheat germ agglutinin (WGA) staining was performed on heart samples from week 5 cardiac specific inducible Smyd5 KO mice. Slides were deparaffinized using a series of ethanol washes (ranging from 100% to 50%) along with xylene, 10X PBS, and citric acid. After being treated with WGA mixed with 2% BSA in PBS, slides were imaged 24-48 hours post staining. Quantification of local cardiomyocytes revealed larger cell sizes in mice positive for cre expression (indicating Smyd5 KO). Figure 2 depicts the average cell areas for WT and KO mice which were 243.45μm and 359.04 μm respectively, marking a statistically significant increase (p = 3.053e-121). This change in average was generally apparent among the 16 other heart sections imaged. These results are similar to WGA experiments in previous work done by Franklin et. al with Smyd1 where KO of Smyd1 also resulted in increased local cell size.2 15 Figure 3a: WGA image of a WT mouse heart section (SD5F 05-03-4). Imaged using 40x objective. Cells appear to be uniform in size in their respective regions. 16 Fig 3b: Quantification of WT mouse section (SD5F 05-03-4). 50 cells were selected and measured using the close polygon tool of the Olympus CellSens softwares. Average cardiomyocyte area measured in this section is 248.86μm. 17 Figure 4a: WGA image of a KO mouse heart section ((SD5F 05-02-3)). Imaged using 40x objective. Abnormally large cells begin to appear throughout the section with no clear pattern. 18 Figure 4b: Quantification of KO mouse section (SD5F 05-02-3). 50 cells were selected and measured using the close polygon tool of the Olympus CellSens softwares. Average cardiomyocyte area measured in this section is 312.30 μm, marking an increase from the WT average. 19 Discussion Heart disease remains a critical issue in medicine, especially as it is the number one cause of death in America. Changes in lifestyle over the past century have greatly increased the prevalence of the disease around the world. Understanding how cardiovascular disease manifests itself is critical to developing better treatment and methods of prevention. One key characteristic of heart disease is maladaptive cardiac hypertrophy, where cardiomyocytes enlarge causing ventricular remodeling, which can inhibit the heart from successfully emptying its chambers. The purpose of this investigation was to better understand Smyd5 and its role in the context of the heart. Previous data has shown that Smyd1, another member of the Smyd family, has played a role in cardiac hypertrophy.2 An inducible Smyd5 knockout mouse model was used in this study to test the effects of Smyd5 KO against WT mice. By feeding the mice tamoxifen, Smyd5 KO was expressed. After five weeks, hearts from KO and WT mice were harvested and fixed with formalin and placed on slides embedded with paraffin. The results indicate a general local cell area increase in KO mice slides compared to WT, suggesting Smyd5’s involvement in cardiac hypertrophy. Imaging heart sections by use of wheat germ agglutinin staining proved useful as it was easy to detect and visualize changes in local cell size when looking for instances of cardiac hypertrophy. The cardiomyocytes of WT mice generally appeared to be normally distributed and of normal size (see Fig. 3a) across each of the WT slides. Individual and surrounding cells had similar cell area measurements and any larger cells only had a small increase in area size. These larger cells are most likely the result of adaptive hypertrophy and are to be expected. By contrast, a majority of the KO slides imaged had 21 regions where cells were obviously larger than surrounding cells (Fig 4a.). Quantification of KO and WT slides revealed a significant change in cell area size (Fig 5.) The results suggest that Smyd5 is directly involved in this pathway towards hypertrophy. Because loss of Smyd5 leads to cardiac hypertrophy, it may be reasonable to assume that Smyd5 is preventing hypertrophy from occurring, much like Smyd1 restricts growth of the heart.2 If the mechanism surrounding Smyd5’s ability to potentially restrict growth in a similar manner to Smyd1 was elucidated, then perhaps treatment could developed to specifically target Smyd5 and slow the effects of heart disease. The findings of the experiment implicate the need to further study Smyd5. While it is one thing to observe changes in heart morphology on a gross level, it is also important to understand how Smyd5 works on a molecular basis. Mice that are subject to Smyd5 KO are already undergoing heart failure by week 5. Another method of studying Smyd5 in the context of heart disease would be to measure the apoptotic activity it causes using the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. One key feature of cell death is double stranded DNA breaks. In future experiments, heart sections would be fixed on slides the same way they were for WGA staining. Instead, after deparaffinization, these sections would be treated with the TUNEL reagents to stain for regions of apoptosis. By performing a counterstain with DAPI, regions of apoptosis due to hypertrophy can be visualized underneath the microscope. To ensure that the TUNEL solution is working as intended, two slides from each experimental group will be designated as either the positive or negative control. The positive control will be treated with a DNA recombinant that will for sure induce double stranded breaks and always show up during imaging. The negative control will only be treated with the labeling 22 solution and will come up as blank upon imaging. Acquiring this kind of data will further support the claim of Smyd5’s involvement with heart disease. Preliminary data from the Franklin Lab has also revealed that loss of Smyd5 in the heart has led to increased cell infiltration. Other literature has also shown that Smyd5 has been involved with inflammatory response.20 If Smyd5 is involved with immune response, then it’s certainly possible that modulation of Smyd5 expression could be used to induce or reduce immune response. One way to measure immune response would be to perform qPCR to detect expression for certain markers. Some markers of interest include cd45 (related to hematopoetic cell infiltration and general immune response), cxcl10 (related to inflammatory response via chemokine activity), Il6 (related to chemokine activity), ccl4 (related to chemotaxis and inflammation), tnf (related to inflammatory response), and vimentin (related to fibroblasts). If Smyd5 KO is achieved and these markers are related to Smyd5 in some way, it is likely that these markers would have their expression affected. WT and KO tissue would be tested for expression of these markers. Heart tissue would be homogenized and RNA would be isolated for qPCR. Expression of Smyd5 would also be tested for. The qPCR experiments would produce an expression curve for each primer used (one for each protein of interest). KO tissue would show a plot where the associated Ct values of each primer would be smaller, indicating a loss of expression. Similarly, each of the aforementioned markers could be stained using immunohistochemistry. Tissue sections would be deparaffinized and boiled in a citrate buffer in order to aid with antigen retrieval, as fixing tissue in formalin can form protein cross-links that mask antigenic sites. By incubating the slides in primary antibodies 23 overnight then again with the secondary antibodies, images can be produced that show the prevalence of the markers of interest. This method would similar to the proposed qPCR experiments but would allow for tangible data that may directly show and increase in immune response in the heart upon heart failure. Further study of Smyd5 can pave the path for new treatments of heart disease. A screen could be developed to determine the expression of members of the Smyd family in a patient to assess whether or not a patient may be more prone to heart disease. The proposed experiments regarding immune function would be of utmost importance in the study of Smyd5 as Smyd5 could potentially be used to modulate immune response as well. Elucidating Smyd5’s role in immune response may allow it to become a target of novel drugs that may be developed to aid in treating heart disease. 24 References 1. Benjamin EJ, Muntner P, Alonso A, et al. Heart Disease and Stroke Statistics2019 Update: A Report From the American Heart Association. Vol 139.; 2019. doi:10.1161/CIR.0000000000000659 2. Franklin S, Kimball T, Rasmussen TL, et al. The chromatin-binding protein Smyd1 restricts adult mammalian heart growth. Am J Physiol - Hear Circ Physiol. 2016;311(5):H1234-H1247. doi:10.1152/ajpheart.00235.2016 3. Hajar R. Coronary Heart Disease: From Mummies to 21st Century. Hear Views. 2017;18(2):68-74. doi:Hajar R. Coronary Heart Disease: From Mummies to 21st Century. Heart Views. 2017;18(2):68–74. doi:10.4103/HEARTVIEWS.HEARTVIEWS_57_17 4. Kemp CD, Conte J V. The pathophysiology of heart failure. Cardiovasc Pathol. 2012;21(5):365-371. doi:10.1016/j.carpath.2011.11.007 5. Cardiovascular diseases (CVDs). https://www.youtube.com/watch?v=KIQUo69YWc. 6. Coronary Heart Disease. https://www.nhlbi.nih.gov/health-topics/coronary-heartdisease. 7. Samak M, Fatullayev J, Sabashnikov A, et al. Cardiac Hypertrophy: An Introduction to Molecular and Cellular Basis. Med Sci Monit Basic Res. 2016;22:75-79. doi:10.12659/MSMBR.900437 8. Tham YK, Bernardo BC, Ooi JYY, Weeks KL, McMullen JR. Pathophysiology of cardiac hypertrophy and heart failure: signaling pathways and novel therapeutic targets. Arch Toxicol. 2015;89(9):1401-1438. doi:10.1007/s00204-015-1477-x 25 9. Frey N, Katus HA, Olson EN, Hill JA. Hypertrophy of the Heart: A New Therapeutic Target? Circulation. 2004;109(13):1580-1589. doi:10.1161/01.CIR.0000120390.68287.BB 10. Merriam Webster. https://www.merriam-webster.com/dictionary/epigenetics. 11. Choudhuri S. From Waddington’s epigenetic landscape to small noncoding RNA: Some important milestones in the history of epigenetics research. Toxicol Mech Methods. 2011;21(4):252-274. doi:10.3109/15376516.2011.559695 12. Felsenfeld G. A Brief History of Epigenetics. Cold Spring Harb Lab Press. 2004;6:12-14. doi:10.1101/cshperspect.a018200 13. Abi Khalil C. The emerging role of epigenetics in cardiovascular disease. Ther Adv Chronic Dis. 2014;5(4):178-187. doi:10.1177/2040622314529325 14. Tracy CM, Warren JS, Szulik M, et al. The Smyd family of methyltransferases: role in cardiac and skeletal muscle physiology and pathology. Curr Opin Physiol. 2018;1:140-152. doi:10.1016/j.cophys.2017.10.001 15. Du SJ, Tan X, Zhang J. SMYD proteins: Key regulators in skeletal and cardiac muscle development and function. Anat Rec. 2014;297(9):1650-1662. doi:10.1002/ar.22972 16. Warren JS, Tracy CM, Miller MR, et al. Histone methyltransferase Smyd1 regulates mitochondrial energetics in the heart. Proc Natl Acad Sci U S A. 2018;115(33):E7871-E7880. doi:10.1073/pnas.1800680115 17. Kidder BL, Hu G, Cui K, Zhao K. SMYD5 regulates H4K20me3-marked heterochromatin to safeguard ES cell self-renewal and prevent spurious differentiation. Epigenetics and Chromatin. 2017;10(1):1-20. doi:10.1186/s1307226 017-0115-7 18. Kidder BL, He R, Wangsa D, et al. SMYD5 controls heterochromatin and chromosome integrity during embryonic stem cell differentiation. Cancer Res. 2017;77(23):6729-6745. doi:10.1158/0008-5472.CAN-17-0828 19. Fujii T, Tsunesumi SI, Sagara H, et al. Smyd5 plays pivotal roles in both primitive and definitive hematopoiesis during zebrafish embryogenesis. Sci Rep. 2016;6(April 2015):1-11. doi:10.1038/srep29157 20. Stender JD, Pascual G, Liu W, et al. Control of Proinflammatory Gene Programs by Regulated Trimethylation and Demethylation of Histone H4K20. Mol Cell. 2012;48(1):28-38. doi:10.1016/j.molcel.2012.07.020 21. Emde B, Heinen A, Gödecke A, Bottermann K. Wheat germ agglutinin staining as a suitable method for detection and quantification of fibrosis in cardiac tissue after myocardial infarction. Eur J Histochem. 2014;58(4):315-319. doi:10.4081/ejh.2014.2448 27