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Characterizing the Effect of Myh7b ASO Treatment in Cardiomyocytes Maturation in Cella

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Characterizing the Effect of Myh7b ASO Treatment in Cardiomyocytes Maturation in Cella Characterizing the Effect of Myh7b ASO Treatment in Cardiomyocytes Maturation In Cella Jose Santos Navarro Arriola Molecular, Cellular, and Developmental Biology Departmental Honors Thesis University of Colorado Boulder Defense Date: April 7th, 2021 Thesis Advisor: Dr. Leslie Leinwand (MCDB & BioFrontiers Institute) Honors Council Representative: Dr. Joel KralJ (MCDB) Outside Department Advisor: Member: Dr. Katharine Semsar (Arts & Sciences) Abstract Myh7b is an ancient gene encoding a Myosin Heavy Chain (MyHC) protein that has been repurposed for alternative regulatory roles in cardiomyocytes due to an alternative spliced exon skipping mechanism. To better understand the role of Myh7b gene in cardiomyocytes, this project investigated cardiomyocytes differentiation and maturation. The goal was to expand our knowledge of Myh7b’s roles in cardiomyocytes. A study prior to this project showed that the prominent gene product candidate of the Myh7b gene was the long non-coding RNA (lncMyh7b) produced by the exon-skipping mechanism. This was achieved by eliminating miR-499 and Myh7b short peptide (Myh7b_sp) as candidates. A portion of this project focused on the western blotting for Myh7b_sp, which was found to be undetectable in the human heart. A CRISPR knockout system was developed, but an ASO knockdown system was pursued instead for reducing lncMyh7b as it proved more versatile for experimental design. The lncMyh7b ASO treated hiPSCs showed a heterogenous phenotype, in contrast to the control ASO treated cells which successfully differentiated into cardiomyocytes. Only one well sample of lncMyh7b ASO showed signs of delayed differentiation, whereas all of the control ASO cells followed the established cardiomyocyte differentiation timeline. Along with previous studies. the data of this project points to lncMyh7b ASO treatment disrupting normal cardiomyocyte differentiation and maturation. Specifically, we hypothesiZed that lncMyh7b may affect the Wnt pathway, as temporal control of Wnt is essential for successful differentiation. Keywords: Myh7b, lncMyh7b, Myh7b_sp, Anti Sense Oligos, hiPSC, Wnt Introduction Proper heart development and maturation is dictated by finely-tuned regulations in sarcomeric proteins like those from the myosin family of motor proteins involved in heart contraction. Furthermore, one of the hallmarks of human heart disease are changes to the myosin ratio between the two most common myosin heavy chain (MyHC) proteins: β-MyHC and α- MyHC. In the human heart, β-MyHC comprises 90% of MyHC proteins and α-MyHC makes up the remaining 10% (Nakao et al. 1997). These two MyHC proteins’ differences in speed and energy efficiency contribute to different properties of the sarcomeric unit’s contractility. Therefore, changes to the β-MyHC/α-MyHC ratio has drastic consequences on the heart overall. The third MyHC gene expressed in the heart is called Myh7b, which is predicted to have a slow- twitch property like β-MyHC. However, Myh7b protein is not produced in the heart, due to a post-transcriptional exon-skipping mechanism that prevents translation of the RNA. Despite this, knockdown of Myh7b has been shown to reduce β-MyHC protein expression (Broadwell et al. 2020 Sep 6). Myh7b has a gene product with some regulatory role with the β-MyHC/α-MyHC ratio While previous research with Myh7b has used in vivo mice models, mice have the opposite ratio of β-MyHC/α-MyHC proteins (Krenz and Robbins 2004), and therefore we decided to use human induced pluripotent stem cells (hiPSCs) to examine this regulation. hiPSCs better pertain to human specific regulatory networks. The focus of previous hiPSCs projects looked into the role of acute changes of Myh7b expression in mature cardiomyocytes. No research has explored in the role of Myh7b during cardiomyocyte differentiation and maturation process. This is a perfect opportunity to further shed light on the role of Myh7b in cardiomyocytes. Myh7b is an ancient myosin gene that has been repurposed for novel regulatory roles outside of the typical protein interactions, and currently the gene encodes an RNA mediated regulation in cardiomyocyte. We hypothesiZe that this regulation may play some role in cardiomyocyte differentiation and maturation. My project hopes to explore this possibility. Background Cardiomyocytes Cardiomyocytes (CMs) are the terminally differentiated cells that comprise the part of the heart tissue that provide contractile force. This is because of sarcomeric units pulling in on each other through electrophysiological work by Ca2+ exchange. When millions of sarcomeric units work in unison, the tissue level contractions provide enough force generation to keep the heart beating and blood pumped throughout the organism. The sarcomere is comprised of repeating units of different structural and enzymatic proteins including myosin proteins. Myosin heavy chain (MyHC) proteins are motor proteins that convert chemical energy into kinetic energy. The sarcomeric MyHC proteins involved in contraction are a part of a hexameric complex. Two heavy chains will dimeriZe proteins via their rod domains, which will further self-assemble into the thick filament, leaving the ATPase head domains free to pull on the actin thin filament. Each heavy chain is further decorated by two regulatory myosin light chains. Human cardiomyocytes express two isoforms of MyHC protein, β-MyHC (Myh7 gene) and α-MyHC (Myh6 gene). In humans, 90% of the heart’s myosin protein are β-MyHC and the remaining 10% are α-MyHC (Nakao et al. 1997). This β-MyHC/α-MyHC ratio is maintained in healthy hearts but in human heart failure there is a total shift to 100% β-MyHC. This dysregulation in the ratio exacerbates the toll on the heart (Nakao et al. 1997). Myosin Heavy Chain 7b Myosin Heavy Chain 7b (Myh7b) is another slow twitch and energy efficient MyHC isoform similar to β-MyHC. Full length Myh7b is expressed in human brain, inner ear tissues, and specialiZed muscles but is the primary cardiac myosin in chickens and reptiles (Bell et al. 2010). In human hearts, no full length Myh7b protein can be detected because of an exon-skipping mechanism relegating the transcript to become non-coding for full length protein translation, but Myh7b RNA expression was present and correlated with β-MyHC protein expression (Broadwell et al. 2020 Sep 6). The Myh7b locus has the potential to produce multiple products of interest due to an exon-skipping mechanism. We will focus on the three that can be produced by the exon-skipped transcript, as there is no evidence of full-length transcript in the heart. One of the products is an intronic miRNA called miR-499 cut from Myh7b pre-mRNA transcript intron 19. miRNAs are class of small non-coding RNAs that can have cis and trans regulation of genes. Unlike the corresponding miRNAs from β-MyHC and α-MyHC genes that did have regulatory effects on the β-MyHC/α-MyHC ratio in a mouse model (van RooiJ et al. 2009), miR- 499 does not regulate β-MyHC expression in human cardiomyocytes (Broadwell et al. 2020 Sep 6). The second product is a 27 kDa theoretical alternatively spliced Myh7b short peptide (Myh7b_sp) that can be produced by the shifted open reading frame (ORF) created by the exon skipping mechanism. In contrast to protein prediction, this project showed there were no detectable levels of Myh7b_sp in human hearts and the corresponding RNA transcript is non- coding at least in the human heart. The final product is the long non-coding Myh7b RNA (lncMyh7b) transcript with exon 8 skipped (lncMyh7b) based on current annotation. Previous research has referred the skipping mechanism to exon 7, but the rest of the data remains valid (Bell et al. 2010). lncRNAs can have cis regulatory roles in neighboring coding genes (Batista and Chang 2013; EngreitZ et al. 2016) and trans regulation on more distant genes (Nagano et al. 2008; Batista and Chang 2013). Anti-Sense Oligonucleotides Anti-Sense Oligonucleotides (ASO) are short single stranded RNA that attaches to target mRNAs through Watson-Crick pairing. After attachment, the RNA duplex is marked for degradation by RNase H activity, stall further transcription, or stop translation through steric hindrance to the ribosome (Cavagnari 2011; Kole et al. 2012). ASO treatment is a transient, selective mRNA knockdown that modulates the target gene’s mRNA concentration and, moreover, affects protein expression. The benefits of using ASO is that it is cheap, vector-less, high modularity, and appropriate for studying essential proteins due to transient inhibition. CRISPR KnocKout Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is a form of targeted genome editing that uses a Cas9 endonuclease with a single guided RNA (sgRNA). The sgRNA is composed of a scaffold structure and the 20-mer spacer sequence that will bind to the target sequence adjacent to a PAM sequence (ngg). After finding the target sequence, the CRISPR system makes a double stranded break (DSB). In response to the break, the cell will attempt to repair through non-homologous end joining (NHEJ) or use a template (sister chromosome or template plasmid) for homology directed repair (HDR). Knockouts occurs through frame shifts in the open reading frame caused by indels. Two near breaks in the DNA will “cutout” the region and could completely disrupt the targeted gene. Specific Aims Question: How is Myh7b expression involved in cardiomyocyte maturation? The aim of this project is to further understand the contemporary role of the ancient Myh7b gene in cardiomyocyte differentiation and maturation. The collected data will help to answer the three aims set by the project and provide more knowledge on Myh7b and potential therapeutics to cardiac afflictions caused by dysregulated β-MyHC/α-MyHC ratio. Aim 1: What gene products from Myh7b are involved in mature cardiomyocytes? Aim 2: Is Myh7b expression essential to normal cardiomyocyte maturation? Aim 3: Is Myh7b expression involved in the Wnt pathway crucial for cardiomyocyte maturation? Materials and Methods Whole Cell Lysates & Western Blotting Western blotting was used to investigate the presence of the Myh7b short peptide, one of the three gene products of Myh7b, from human tissue samples.
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