AbstractHypertrophic cardiomyopathy (HCM), which clinically manifests as an enlarged heart is a highly prevalent cardiac disorder which could result in arrhythmia-induced sudden cardiac death. The mechanism of HCM remains poorly defined, necessitating further understanding of the disease for improved therapeutic strategies. As it is challenging to obtain cardiac biopsies, using induced pluripotent stem cells (iPSCs) technology, we generated cardiomyocytes (CMs) in a dish from HCM patients. These HCM-CMs presented the clinical manifestation in that they were significantly larger in size in comparison to control (healthy)-CMs. Furthermore, gene expression profiling of cardiac ion channels revealed increased transcripts encoding for calcium channels which resulted in calcium-mediated arrhythmias. Our humanized HCM model recapitulated the diseased phenotype and the subsequent findings lay the foundation for understanding the in-depth mechanism of HCM for therapeutic intervention. IntroductionHCM is a genetic, common cardiac disease, estimated to be the most prevalent hereditary heart disease in the world. It is a major cause of disability and sudden cardiac death (SCD) in patients of all ages, particularly youths, and several other clinical complications such as progressive heart failure. In Singapore, it is estimated that every 1 in 500 people are known to suffer from the disease. Some of its symptoms include chest pains, shortness of breath and arrhythmias – abnormal heart rhythms due to the electrical system of the heart being impaired.The disease is linked to mutations in any 1 of 10 genes encoding the cardiac sarcomeric proteins 1, where the heart cells are enlarged and the left ventricular wall of the heart thickens in the presence of a nondilated cavity. However, it has proven complex in both diagnosis and treatment; The pathways by which sarcomeric mutations bring about the associated disease phenotypes, symptoms and abnormalities are not well understood. As for treatment strategies, due to the disease’s heterogeneity in its disease-causing mutations, symptoms and prognosis, it is therefore dependent on several factors as well, which vary from patient to patient. Presently, treatments can be classified under drug and surgical treatments, whereby the standard of care for severe refractory symptoms associated with marked outflow obstruction consists of drug treatment for exertional dyspnea (?-blockers, verapamil, disopyramide) and the septal myotomy-myectomy operation. Alternatively, alcohol septal ablation and pacing have also been sought as treatments in place of surgery for certain patients. High-risk patients may be treated effectively for sudden death prevention with the implantable cardioverter-defibrillator. However, these treatments have proved inefficient due to their respective side effects and the fact that they are largely non-specific. Verapamil, for example, may be deleterious to some patients with severe outflow gradients and heart failure. The inefficiencies of existing treatments point towards the need for more research into newer and better alternative therapeutic strategies. In 2006, Shinya Yamanaka and Kazutoshi Takahashi discovered induced pluripotent stem cells (iPSCs), impacting several fields in science and medicine. Unlike before, pluripotent stem cells could now be obtained not only from the inner cell mass of an embryo in its blastocyst-stage but also from obtaining and reprogramming somatic adult tissue by subjecting it to transcription factors Oct4, Klf4, Sox2, and c-Myc (OKSM). This allowed for the ethical issues arising from the use of discarded human embryos to no longer be applicable. As such, human induced pluripotent stem cells (hiPSCs) have risen over the years to become a highlight and major invention of modern science research, establishing a strong presence in the field of medicine2. Due to the limited regenerative ability of the human heart, cardiomyocytes derived from iPSCs (iPSC-CMs) have the added promising potential of providing cell replacements, and have already proven a novel and valuable tool for drug efficacy and toxicity testing, as well as in vitro disease modelling, in order to achieve deeper understanding of cellular mechanisms of complex cardiovascular diseases 3. In our research project, a humanized model of hypertrophic cardiomyopathy cardiomyocytes using Induced Pluripotent Stem Cells (iPSCs) was created to better study and understand the molecular mechanisms and pathways in HCM. We generated Induced Pluripotent Stem Cells (iPSC) from patients exhibiting HCM, then induced the differentiation of Induced Pluripotent Stem Cell-derived Cardiomyocytes (iPSC-CMs) after embryoid bodies were formed. (Embryoid bodies (EB) are the three-dimensional aggregates formed in suspension by pluripotent stem cells. 4 EB differentiation is a common platform to generate specific cell lineages from iPSCs.) We validated our model through immunostaining, which displayed the increased cell size. Real time PCR was subsequently used to study how HCM gene expression differs from wild type, healthy cells.2. HypothesisDue to SCD, caused by arrhythmia, being a significant consequence of HCM, we hypothesize that ion channel defects are involved in the disease mechanism and hence manifestation, whereby ion irregularities are responsible for the respective disease phenotypes and symptoms.3. Methodology3.1 Embryoid body formation and cardiomyocyte differentiationiPSC colonies were obtained and maintained.5 The iPSC colonies were dispersed into small clumps with dispase (1 mg/ml) and placed in low adhesion culture dishes in Embryoid body(EB) medium 8 days. Subsequently, EBs were plated on 0.1% gelatin coated dishes in EB media. Beating areas were typically observed around day 11–14 from EB formation. These cells were then used for immunostaining and real time PCR. 63.2 Immunostaining Next we conducted immunostaining for alpha (?)-actinin using a monoclonal antibody and the nuclei of the iPSC-CMs with DAPI. For immunostaining, iPSCs (Figure 1.) and iPSC-CMs(Figure 3.) were seeded on matrigel- and gelatin-coated glass slides respectively. All cell types were fixed with 4% PFA(Perfluoroalkoxy alkanes), permeabilized with 0.3% Triton X-100 , blocked with 5% BSA(bovine serum albumin) and stained with mouse monoclonal antibodies from sigma to stain sarcomeric alpha (?)-actinin.7 Cells were washed, probed with respective fluorophore-conjugated secondary antibodies and counterstained with DAPI to stain the nuclei of the cells the following day. The cells which were stained were studied under a confocal microscope6.3.3 Calcium ImagingiPSC-CMs were dissociated from beating EBs formed earlier and seeded in gelatin-coated dishes. 8 Cells were loaded with 5 mM Fluo-4 AM and imaged a confocal microscope. Spontaneous Ca2+transients were recorded at 37°C using standard line-scan methods 9. A total of 1500 line scans were acquired for durations of 1 second 13. These images were then stacked to plot a graph of intensity versus time, which showed the contraction and relaxation patterns of the iPSC-CMs. 14 3.4 Real-time Polymerase Chain ReactionThe real-time polymerase chain reaction (qRT-PCR) analysis first involved isolating RNA and converting to complementary DNA (cDNA) 10 We then added added five nanograms of cDNA template for each gene used ( KCNJ2, KCNE1, KCNQ1, RYR2, ATP2A2, CACNA1D, CACNA1C, TNNT2 and MYL2). Thereafter, SYBR green qRT-PCR were performed as per the manufacturer’s instructions. 10 Samples were cycled using RotorGene Q (Qiagen) in the following order: 95°C for 5 minutes, followed by a total of 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Using an endogenous control gene, GAPDH, relative quantification was calculated according to ??Ct method 6. Finally, Heat-maps for a panel of 9 genes were generated using normalized Ct values using Genesis software 6. 2 wild type controls and 2 iPSC-HCM cell lines were used in the experiment.4. Results 4.1 Control and HCM-iPSCs are pluripotent Oct-4 is a stem cell pluripotency and transcription factor, and PSC-specific protein expressed on the surface of undifferentiated human embryonic stem cells. (ES) We stained both control IPSCs and HCM-IPSCs with Oct-4 and Tra-1-60 antibodies for confirmation of the HCM-IPSCs pluripotency. DAPI staining was also carried out. Figure 1 shows the generated HCM-IPSCs stained positively for both surface antigen,Tra-1-60, and transcription factor, Oct-4, which are pluripotency markers of ES cells.Figure 1. Immunofluorescence staining of IPSCs Left; staining of Control-IPSCs. Right; staining of HCM-IPSCs. Both control and HCM IPSCs were stained with Oct-4 and Tra-1-60, followed by staining with DAPI.4.2 Cell aggregation mediates efficient cardiac differentiation IPSCs were dissociated into individual cells and seeded into aggrewells for the formation of 5000-cell embryoid bodies (EB) and the subsequent differentiation into cardiomyocytes. Day 0 shows individual IPSCs in microwells of a single aggrewell. 6 The dotted yellow circle shows an EB which formed from aggregated IPSCs. From figure 2B, the area and perimeter of the EB increased over time but only statistically significant increase occurred between day 2 and day 4 and between day 8 and day 14.Figure 2. IPSCs differentiation into cardiomyocyte cluster and EB growth Top panel; Pictures shows growth of EB in an aggrewell over 14 days, culminating in a cardiomyocyte cluster. Bottom panel, Left; Area of EBs over 14 days shown on left graph. Right; graph shows perimeter of EBs over 14 days. *p<0.05 for comparison between days4.3 HCM-CMs exhibit a larger phenotype as compared to control-CMs After the formation cardiomyocytes, we wanted to compare the phenotypic differences between control-CMs and HCM-CMs. We found differences in striation patterns, nucleation patterns and cell size. The sarcomere is the basic contractile unit within the cardiomyocyte; this highly organized cytoskeletal structure contains actin filaments anchored to the Z-disc by sarcomeric alpha (?)-actinin.15 We performed immunofluorescence staining using mouse monoclonal antibodies to stain sarcomeric alpha (?)-actinin and DAPI to stain the nuclei. The alpha (?)-actinin stain (green) shows disarrangement of sarcomeric ?-actinin in HCM-CMs. The striation patterns are more disjointed and discontinuous as compared to control-CMs. (Figure 3. left panel, top) The DAPI stain (blue) shows enlarged nucleus in HCM-CMs. (Figure 3. left panel, middle) This indicates that HCM-CMs exhibits the enlarged nucleus phenotype more commonly than control-CMs. Next, we also investigated the difference in cell size of the HCM-CMs as compared to control-CMs, since enlarged cell size is a hallmark of HCM. 11 Control-CMs are on average about 1500 and HCM-CMs are significantly larger at about 2500. (Figure 3. right panel) Figure 3. Phenotypic differences in HCM-CMs as compared to control-CMs Left panel; Images showing immunofluorescence stains of sarcomeric alpha (?)-actinin and nuclei. Top; The antibody for sarcomeric alpha (?)-actinin (green) reveals a distinct cross-striation in control-CMs (left) and in HCM-CMs (right). Middle; Nuclei are stained with DAPI which binds strongly to A-T rich regions in DNA (blue) reveals the nucleation patterns of control-CMs (left) and HCM-CMs (right). Bottom; Merged images of both stains. Right panel; Graph shows the difference in cell size between control-CMs and HCM-CMs (n=100). *p<0.05 in comparison to control-CMs. Scale bar 50 µM4.4 HCM-CMs have severe calcium irregularities Having established an HCM-CM model, we moved on to finding out the difference in gene expression of ion channels and sarcomeric proteins. We found the difference in gene expression of genes encoding for 3 potassium channels(KCNQ1, KCNE1, KCNJ2), 2 calcium channels which resides on the sarcoplasmic reticulum(SR) of the myocytes(RYR2, ATP2A2), 2 L-type voltage dependant calcium channels (CACNA1D, CACNA1C) and 2 sarcomeric proteins(TNNT2, MYL2). The sarcoplasmic reticulum is a specialized type of smooth endoplasmic reticulum that regulates the calcium ion concentration in the cytoplasm of striated muscle cells. Calcium ions regulate the contraction and relaxation of the cardiomyocytes. Figure 4A shows that there is a significant increase in gene expression of genes encoding for calcium channel and sarcomeric proteins in HCM-CMs. It is also apparent that there is a larger increase in gene expression of genes encoding for calcium channels which reside on the SR as compared to those encoding for L-type voltage dependant calcium channels. Due to the significant increase in the gene expression of genes encoding for calcium channels, we investigated the Ca2+ transients to see if there are any irregularities in calcium handling within the HCM-CMs. Figure 4B shows that there are significantly more iPSC-CMs exhibiting irregular Ca2+ transients among the cells affected with HCM as compared to the control cells. Figure 4B(left panel) shows an arrhythmic cytoplasmic Ca2+ transient (CaT) trace of a HCM-CMs, compared to a non-arrhythmic CaT trace of a control-CMs. From Figure 4B(right panel) about 50% of iPSC-CMs affected with HCM exhibit irregular Ca2+ transients while only about 10% of control iPSC-CMs exhibit irregular Ca2+ transients. These results suggests that HCM-CMs have abnormalities in the gene expression of genes encoding for calcium channels and also exhibit abnormal calcium handling properties as compared to control iPSC-CMs. Figure 4. Gene expression and calcium handling properties (A) Graph showing difference in gene expression of 9 various genes in control-CMs and HCM-CMs. Genes coding for calcium channels are annotated in red while genes coding for potassium channels are annotated in black under ion channels. (B) Left Panel; Spontaneous CaT traces in affected and control in iPSC-CMs is shown in the left panel. Note the calcium irregularities (red arrowheads) in HCM-CMs. Right; Graph shows difference in prevalence of irregularities in CaT between affected and control iPSC-CMs. *p<0.05 in comparison to control-CMs5. DiscussionThe aim of the proposed study was to investigate and find out the molecular mechanism in disease manifestation of HCM. We used iPSC-CMs as a disease model to investigate the molecular mechanism of the disease. Using iPSCs as a disease model offers great promise as a viable human model to study HCM. The properties of iPSCs, its pluripotency: being able to proliferate into almost all cell lines and its ability to self-renew, allows a viable human model of almost all cell types that can be studied for long periods of time. In addition, iPSCs are able to recapitulate the disease phenotype accurately and are easily accessible and portable, making it an ideal human model to study diseases In particular, using iPSCs negates most ethical concerns as the cells are generated using reprogramming techniques (viral or RNA) of somatic cells rather than involving the generation of an embryo as in embryonic stem cells. The iPSCs used in our experiment were generated from a patient diagnosed with HCM after reprogramming with viral vectors. In order to generate beating cardiomyocyte clusters, upon confirmation of the iPSCs pluripotency through immunofluorescence staining (from figure 1), the iPSCs were first seeded to form cell aggregates known as embryoid bodies, before undergoing differentiation. From figure 2, the embryoid bodies underwent a statistically significant increase in size between day 2 and day 4, which is when the cells first start committing to the mesodermal lineage according to the developmental ontogeny 12, and the largest increase in size between day 8 and day 14 as the cells initiate differentiation from progenitors to form cardiomyocytes. Beating clusters were observed between day 11 and day 14. Our results confirms that iPSC-CMs with HCM exhibit hypertrophy (increased cell size), a hallmark phenotype of HCM.11 It can be seen through the sarcomeric alpha actinin stain, as well as the graph showing the cell size difference between control-CMs and HCM-CMs. (Figure 3.) This can also be seen from the increased gene expression of genes encoding for sarcomeric proteins which indicates an increase in sarcomeric proteins within the HCM-CMs. This suggests that the affected iPSC-CMs indeed experience hypertrophy. Our results also indicate that HCM-CMs have enhanced gene expression for genes which are responsible for encoding calcium channels, mainly those on the sarcoplasmic reticulum of the myocyte while expression of genes encoding for potassium channels didn't display a significant difference. Then we analysed the cytoplasmic Ca2+ concentrations in HCM-CMs and control-CMs which indicates that HCM-CMs are more likely prone to arrhythmia and irregular Ca2+ transients as compared to control iPSC-CMs (Figures 4A and 4B). 6. ConclusionIn conclusion, our results show that iPSC-CMs are indeed a good human model for HCM, allowing us to observe the disease at a molecular level to understand its mechanisms and manifestations. Our results have proven through examination of the relevant gene expressions that we can indeed confirm our hypothesis; HCM-CMs have been shown to have a significantly enhanced gene expression of genes encoding calcium channels, and abnormal calcium handling properties, leading to significant calcium irregularities.7. Future Work and Limitations The use of iPSCs as disease models have proved effective, novel and significantly insightful to aspects of diseases initially difficult or impossible to research due to several issues ranging from ethical (being unable to study and conduct tests on human models) to practical issues (limited human biopsies). Our research project has concluded on calcium irregularities being a significant factor of HCM, however, there remains much more that can be researched into with the disease models. Using our models, future work can be done to establish what and how treatments can stop or resolve the calcium irregularities, and whether they effectively lead to a decrease in the disease phenotypes. In terms of limitations, both hESC-CMs and iPSC-CMs are developmentally immature and are characterized by gene expression profiles similar to fetal cardiomyocytes, leading to results and conclusions that may not be entirely representative of adult CMs. iPSC-CMs are also not able to model disease phenotypes that present at the tissue level such as interstitial fibrosis, scarring, and myocyte disarray. Additionally, due to the heterogeneity of the disease from its symptoms to its causes, our findings may be not be representative for all manifestations. However, our research has set a foundation and direction for potential treatments and research to be done, pointing towards multiple extensions of our work that will increase our knowledge and efficiency at treating this disease and many others.8. Bibliography1 Tardiff, J. C. (2005, September). Sarcomeric proteins and familial hypertrophic cardiomyopathy: linking mutations in structural proteins to complex cardiovascular phenotypes. 2Takahashi, K., & Yamanaka, S. (2006, August 25). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.3 Mura, M., Mehta, A., Ramachandra, C., Pisano, F., Ciuffreda, M., Crotti, L., . . . Gnecchi, M. (2017). The KCNH2-IVS9-28A/G mutation causes aberrant isoform expression and hERG trafficking defect in cardiomyocytes derived from patients affected by long QT syndrome type 2.4 Rungarunlert, S., Techakumphu, M., Pirity, M. K., & Dinnyes, A. (2009, December 31). 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