Title: Myosin 7b is a regulatory long noncoding RNA (lncMYH7b) in the human heart
Abstract: Myosin heavy chain 7b (MYH7b) is an ancient member of the myosin heavy chain motor protein family that is expressed in striated muscles. In mammalian cardiac muscle, MYH7b RNA is expressed along with two other myosin heavy chains, β-myosin heavy chain (β-MyHC) and α-myosin heavy chain (α-MyHC). However, unlike β-MyHC and α-MyHC, which are maintained in a careful balance at the protein level, the MYH7b locus does not produce a full-length protein in the heart due to a posttranscriptional exon-skipping mechanism that occurs in a tissue-specific manner. Whether this locus has a role in the heart beyond producing its intronic microRNA, miR-499, was unclear. Using cardiomyocytes derived from human induced pluripotent stem cells as a model system, we found that the noncoding exon-skipped RNA (lncMYH7b) affects the transcriptional landscape of human cardiomyocytes, independent of miR-499. Specifically, lncMYH7b regulates the ratio of β-MyHC to α-MyHC, which is crucial for cardiac contractility. We also found that lncMYH7b regulates beat rate and sarcomere formation in cardiomyocytes. This regulation is likely achieved through control of a member of the TEA domain transcription factor family (TEAD3, which is known to regulate β-MyHC). Therefore, we conclude that this ancient gene has been repurposed by alternative splicing to produce a regulatory long-noncoding RNA in the human heart that affects cardiac myosin composition. Myosin heavy chain 7b (MYH7b) is an ancient member of the myosin heavy chain motor protein family that is expressed in striated muscles. In mammalian cardiac muscle, MYH7b RNA is expressed along with two other myosin heavy chains, β-myosin heavy chain (β-MyHC) and α-myosin heavy chain (α-MyHC). However, unlike β-MyHC and α-MyHC, which are maintained in a careful balance at the protein level, the MYH7b locus does not produce a full-length protein in the heart due to a posttranscriptional exon-skipping mechanism that occurs in a tissue-specific manner. Whether this locus has a role in the heart beyond producing its intronic microRNA, miR-499, was unclear. Using cardiomyocytes derived from human induced pluripotent stem cells as a model system, we found that the noncoding exon-skipped RNA (lncMYH7b) affects the transcriptional landscape of human cardiomyocytes, independent of miR-499. Specifically, lncMYH7b regulates the ratio of β-MyHC to α-MyHC, which is crucial for cardiac contractility. We also found that lncMYH7b regulates beat rate and sarcomere formation in cardiomyocytes. This regulation is likely achieved through control of a member of the TEA domain transcription factor family (TEAD3, which is known to regulate β-MyHC). Therefore, we conclude that this ancient gene has been repurposed by alternative splicing to produce a regulatory long-noncoding RNA in the human heart that affects cardiac myosin composition. The myosin family of motor proteins that drives striated muscle contraction consists of ten genes with distinct functions (1Weiss A. Leinwand L. The mammalian myosin heavy chain gene family.Annu. Rev. Cell Dev. Biol. 1996; 12: 417-439Crossref PubMed Scopus (254) Google Scholar). Three of these genes are expressed in mammalian hearts (MYH6, MYH7, and MYH7b). MYH6 (α-MyHC) and MYH7 (β-MyHC) are the major sarcomeric myosin proteins expressed in mammalian hearts. In humans, >90% of the heart’s myosin protein composition is comprised of β-MyHC with the remaining <10% being α-MyHC and the two are antithetically regulated (1Weiss A. Leinwand L. The mammalian myosin heavy chain gene family.Annu. Rev. Cell Dev. Biol. 1996; 12: 417-439Crossref PubMed Scopus (254) Google Scholar, 2Nakao K. Minobe W. Roden R. Bristow M.R. Leinwand L.A. Myosin heavy chain gene expression in human heart failure.J. Clin. Invest. 1997; 100: 2362-2370Crossref PubMed Scopus (315) Google Scholar, 3Miyata S. Minobe W. Bristow M.R. Leinwand L.A. Myosin heavy chain isoform expression in the failing and nonfailing human heart.Circ. Res. 2000; 86: 386-390Crossref PubMed Scopus (364) Google Scholar, 4Lowes B.D. Gilbert E.M. Abraham W.T. Minobe W.A. Larrabee P. Ferguson D. Wolfel E.E. Lindenfeld J.A. Tsvetkova T. Robertson A.D. Quaife R.A. Bristow M.R. Myocardial gene expression in dilated cardiomyopathy treated with beta-blocking agents.N. Engl. J. Med. 2002; 346: 1357-1365Crossref PubMed Scopus (413) Google Scholar, 5Hasegawa K. Lee S.J. Jobe S.M. Markham B.E. Kitsis R.N. cis-Acting sequences that mediate induction of beta-myosin heavy chain gene expression during left ventricular hypertrophy due to aortic constriction.Circulation. 1997; 96: 3943-3953Crossref PubMed Scopus (149) Google Scholar). However, various conditions can shift their relative proportions. The finely tuned balance of these two motors is critical for proper cardiac function since they have very different enzymatic properties, which determine the contractile velocity of the muscle (6Krenz M. Robbins J. Impact of beta-myosin heavy chain expression on cardiac function during stress.J. Am. Coll. Cardiol. 2004; 44: 2390-2397Crossref PubMed Scopus (171) Google Scholar). It has been well established that disturbing the β-MyHC/α-MyHC ratio leads to compromised contractility in cardiomyocytes; in human heart failure, there is a shift to ∼100% β-MyHC and α-MyHC becomes undetectable (2Nakao K. Minobe W. Roden R. Bristow M.R. Leinwand L.A. Myosin heavy chain gene expression in human heart failure.J. Clin. Invest. 1997; 100: 2362-2370Crossref PubMed Scopus (315) Google Scholar, 3Miyata S. Minobe W. Bristow M.R. Leinwand L.A. Myosin heavy chain isoform expression in the failing and nonfailing human heart.Circ. Res. 2000; 86: 386-390Crossref PubMed Scopus (364) Google Scholar, 4Lowes B.D. Gilbert E.M. Abraham W.T. Minobe W.A. Larrabee P. Ferguson D. Wolfel E.E. Lindenfeld J.A. Tsvetkova T. Robertson A.D. Quaife R.A. Bristow M.R. Myocardial gene expression in dilated cardiomyopathy treated with beta-blocking agents.N. Engl. J. Med. 2002; 346: 1357-1365Crossref PubMed Scopus (413) Google Scholar, 7Rundell V.L.M. Manaves V. Martin A.F. de Tombe P.P. Impact of beta-myosin heavy chain isoform expression on cross-bridge cycling kinetics.Am. J. Physiol. Heart Circ. Physiol. 2005; 288: H896-H903Crossref PubMed Scopus (95) Google Scholar). As β-MyHC is the slower, more efficient motor, this shift in expression is thought to be an initial compensatory mechanism to preserve energy in the failing heart. Heart failure patients that show functional improvement upon treatment with β-adrenergic receptor blockers have an increase in α-MyHC expression (4Lowes B.D. Gilbert E.M. Abraham W.T. Minobe W.A. Larrabee P. Ferguson D. Wolfel E.E. Lindenfeld J.A. Tsvetkova T. Robertson A.D. Quaife R.A. Bristow M.R. Myocardial gene expression in dilated cardiomyopathy treated with beta-blocking agents.N. Engl. J. Med. 2002; 346: 1357-1365Crossref PubMed Scopus (413) Google Scholar). This suggests that maintenance of the β-MyHC/α-MyHC ratio is fundamental to proper cardiac function. While MYH6 and MYH7 have been studied for decades, MYH7b was not identified until the sequencing of the human genome and was initially annotated as a sarcomeric myosin based on sequence alignments with other known family members (8Desjardins P.R. Burkman J.M. Shrager J.B. Allmond L.A. Stedman H.H. Evolutionary implications of three novel members of the human sarcomeric myosin heavy chain gene family.Mol. Biol. Evol. 2002; 19: 375-393Crossref PubMed Scopus (60) Google Scholar). Based on phylogenetic analysis, MYH7b was identified as an ancient myosin, indicating that MYH7b was present before the gene duplication events that led to the other sarcomeric myosins (8Desjardins P.R. Burkman J.M. Shrager J.B. Allmond L.A. Stedman H.H. Evolutionary implications of three novel members of the human sarcomeric myosin heavy chain gene family.Mol. Biol. Evol. 2002; 19: 375-393Crossref PubMed Scopus (60) Google Scholar). The human MYH7b gene is located on chromosome 20, separate from the two canonical sarcomeric myosin clusters on chromosomes 14 (cardiac myosins) and 17 (skeletal muscle myosins), supporting the idea that MYH7b may have a specialized role in muscle biology (1Weiss A. Leinwand L. The mammalian myosin heavy chain gene family.Annu. Rev. Cell Dev. Biol. 1996; 12: 417-439Crossref PubMed Scopus (254) Google Scholar, 8Desjardins P.R. Burkman J.M. Shrager J.B. Allmond L.A. Stedman H.H. Evolutionary implications of three novel members of the human sarcomeric myosin heavy chain gene family.Mol. Biol. Evol. 2002; 19: 375-393Crossref PubMed Scopus (60) Google Scholar). In certain species, including snakes and birds, MYH7b is expressed as a typical sarcomeric myosin protein in the heart and skeletal muscle (9Lee L.A. Karabina A. Broadwell L.J. Leinwand L.A. The ancient sarcomeric myosins found in specialized muscles.Skelet. Muscle. 2019; 9: 1-15Crossref PubMed Scopus (8) Google Scholar). However, MYH7b has a unique expression pattern in mammals: the encoded protein is expressed in specialized muscles and nonmuscle tissues (10Bell M.L. Buvoli M. Leinwand L.A. Uncoupling of expression of an intronic microRNA and its myosin host gene by exon skipping.Mol. Cell. Biol. 2010; 30: 1937-1945Crossref PubMed Scopus (102) Google Scholar, 11Rossi A.C. Mammucari C. Argentini C. Reggiani C. Schiaffino S. Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles.J. Physiol. 2010; 588: 353-364Crossref PubMed Scopus (90) Google Scholar, 12Rubio M.D. Johnson R. Miller C.A. Huganir R.L. Regulation of synapse structure and function by distinct myosin II motors.J. Neurosci. 2011; 31: 1448-1460Crossref PubMed Scopus (39) Google Scholar). Furthermore, while MYH7b RNA is expressed in mammalian cardiac and skeletal muscle, an alternative splicing event skips an exon, introducing a premature termination codon (PTC) that prevents full-length protein expression (Fig. 1A, Fig. S1) (10Bell M.L. Buvoli M. Leinwand L.A. Uncoupling of expression of an intronic microRNA and its myosin host gene by exon skipping.Mol. Cell. Biol. 2010; 30: 1937-1945Crossref PubMed Scopus (102) Google Scholar). This raises the question of why MYH7b RNA expression has been conserved in the mammalian heart. One obvious hypothesis is that MYH7b transcription is preserved in mammalian heart and skeletal muscle in order to maintain the expression of an intronic MYH7b microRNA, miR-499, in those tissues (10Bell M.L. Buvoli M. Leinwand L.A. Uncoupling of expression of an intronic microRNA and its myosin host gene by exon skipping.Mol. Cell. Biol. 2010; 30: 1937-1945Crossref PubMed Scopus (102) Google Scholar). However, miR-499 knockout mice have no discernible cardiac or skeletal muscle phenotype (13van Rooij E. Quiat D. Johnson B.A. Sutherland L.B. Qi X. Richardson J.A. Kelm R.J. Olson E.N. A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance.Dev. Cell. 2009; 17: 662-673Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). It is possible that the species-specific regulation of myosin isoforms complicates the interpretation of this mouse model and that miR-499 could play an important role in human muscle (6Krenz M. Robbins J. Impact of beta-myosin heavy chain expression on cardiac function during stress.J. Am. Coll. Cardiol. 2004; 44: 2390-2397Crossref PubMed Scopus (171) Google Scholar, 14Sadayappan S. Gulick J. Klevitsky R. Lorenz J.N. Cardiac myosin binding protein-C phosphorylation in a “humanized” mouse heart.Physiology. 2010; 119: 1253-1262Google Scholar). Another hypothesis is that a heretofore unknown functional molecule is expressed from the MYH7b gene, resulting in its conserved expression in the sarcomeric muscle tissue. In this report, we performed a thorough dissection of this locus in order to discern the role of MYH7b gene expression in human cardiomyocytes and found a novel long noncoding RNA (lncMYH7b) with roles in cardiac gene expression, leading to changes in cardiomyocyte beat rate and sarcomeric organization. The human MYH7b locus is complex. In certain tissues, it encodes a typical striated muscle myosin mRNA and full-length protein. In tissues where exon 7 is skipped, the locus could theoretically produce both a peptide originating from the short open reading frame preceding the PTC in exon 9 (MYH7b_sp) and the exon 7-skipped long-noncoding RNA itself (lncMYH7b, Fig. 1A). In both contexts, the microRNA miR-499 is encoded in intron 19. We analyzed RNA-sequencing (RNA-seq) data from 64 healthy and diseased human heart samples and found that exon 7 (the skipped exon) of the MYH7b RNA is essentially undetectable regardless of disease state, which is consistent with our previous observation that MYH7b has a tissue-specific alternative splicing pattern (Fig. 1, B and C) (10Bell M.L. Buvoli M. Leinwand L.A. Uncoupling of expression of an intronic microRNA and its myosin host gene by exon skipping.Mol. Cell. Biol. 2010; 30: 1937-1945Crossref PubMed Scopus (102) Google Scholar). This transcriptomic analysis suggests that the full-length MYH7b protein is not present in the human heart. This observation is also consistent with our previous results showing that forced expression of full-length MYH7b protein in the mouse heart results in cardiac dilation and dysfunction, despite roles the full-length protein is known to have in other tissues (12Rubio M.D. Johnson R. Miller C.A. Huganir R.L. Regulation of synapse structure and function by distinct myosin II motors.J. Neurosci. 2011; 31: 1448-1460Crossref PubMed Scopus (39) Google Scholar, 15Peter A.K. Rossi A.C. Buvoli M. Ozeroff C.D. Crocini C. Perry A.R. Buvoli A.E. Lee L.A. Leinwand L.A. Expression of normally repressed myosin heavy chain 7b in the mammalian heart induces dilated cardiomyopathy.J. Am. Heart Assoc. 2019; 8e013318Crossref PubMed Scopus (5) Google Scholar, 16Haraksingh R.R. Jahanbani F. Rodriguez-Paris J. Gelernter J. Nadeau K.C. Oghalai J.S. Schrijver I. Snyder M.P. Exome sequencing and genome-wide copy number variant mapping reveal novel associations with sensorineural hereditary hearing loss.BMC Genomics. 2014; 15: 1155Crossref PubMed Scopus (17) Google Scholar). β-MyHC induction, which shifts the β-MyHC/α-MyHC ratio, is a hallmark of chronic heart disease (2Nakao K. Minobe W. Roden R. Bristow M.R. Leinwand L.A. Myosin heavy chain gene expression in human heart failure.J. Clin. Invest. 1997; 100: 2362-2370Crossref PubMed Scopus (315) Google Scholar, 3Miyata S. Minobe W. Bristow M.R. Leinwand L.A. Myosin heavy chain isoform expression in the failing and nonfailing human heart.Circ. Res. 2000; 86: 386-390Crossref PubMed Scopus (364) Google Scholar, 4Lowes B.D. Gilbert E.M. Abraham W.T. Minobe W.A. Larrabee P. Ferguson D. Wolfel E.E. Lindenfeld J.A. Tsvetkova T. Robertson A.D. Quaife R.A. Bristow M.R. Myocardial gene expression in dilated cardiomyopathy treated with beta-blocking agents.N. Engl. J. Med. 2002; 346: 1357-1365Crossref PubMed Scopus (413) Google Scholar). Intriguingly, we observed that MYH7b RNA levels correlate with β-MyHC expression, including the known increase in β-MyHC expression in diseased human hearts (Fig. 1D and Fig. S2). This raised the possibility that there is a regulatory relationship between the MYH7b locus and β-MyHC expression. Although full-length MYH7b protein and mRNA are essentially absent in the human heart, there are several other molecules originating from the MYH7b gene locus, as mentioned above. The presence of these different molecules prompted further investigation into the locus as a whole. One hypothesis is that the MYH7b locus regulates β-MyHC through the miR-499, which is a miRNA encoded in intron 19 of the MYH7b pre-mRNA. There is no evidence to suggest that miR-499 regulates the expression of either α- or β-MyHC, and the miR-499 knockout mouse still induces β-MyHC in the heart under conditions of hypothyroidism (10Bell M.L. Buvoli M. Leinwand L.A. Uncoupling of expression of an intronic microRNA and its myosin host gene by exon skipping.Mol. Cell. Biol. 2010; 30: 1937-1945Crossref PubMed Scopus (102) Google Scholar, 13van Rooij E. Quiat D. Johnson B.A. Sutherland L.B. Qi X. Richardson J.A. Kelm R.J. Olson E.N. A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance.Dev. Cell. 2009; 17: 662-673Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, 17Chistiakov D.A. Orekhov A.N. Bobryshev Y.V. Cardiac-specific miRNA in cardiogenesis, heart function, and cardiac pathology (with focus on myocardial infarction).J. Mol. Cell. Cardiol. 2016; 94: 107-121Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Other microRNAs, such as miR-208a and miR-208b encoded by the α-MyHC and β-MyHC genes, respectively, have been shown to have effects on the β-MyHC/α-MyHC ratio in mouse models (13van Rooij E. Quiat D. Johnson B.A. Sutherland L.B. Qi X. Richardson J.A. Kelm R.J. Olson E.N. A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance.Dev. Cell. 2009; 17: 662-673Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). However, the adult mouse cardiac β-MyHC/α-MyHC ratio is opposite to the human one (5%:95% compared with 90%:10%), demonstrating species-specific regulation (6Krenz M. Robbins J. Impact of beta-myosin heavy chain expression on cardiac function during stress.J. Am. Coll. Cardiol. 2004; 44: 2390-2397Crossref PubMed Scopus (171) Google Scholar, 14Sadayappan S. Gulick J. Klevitsky R. Lorenz J.N. Cardiac myosin binding protein-C phosphorylation in a “humanized” mouse heart.Physiology. 2010; 119: 1253-1262Google Scholar). Therefore, it was important to investigate the β-MyHC response to changes in miR-499 activity in a human context. We employed a cellular model of human induced pluripotent stem cells differentiated to cardiomyocytes (hiPS-CMs), which we treated with an anti-miR that targets miR-499 specifically. We observed no changes in MYH7b or β-MyHC levels (Fig. 2). Therefore, we conclude that miR-499 does not regulate β-MyHC expression in human cardiomyocytes. The elimination of miR-499 as a regulator of the β-MyHC/α-MyHC ratio left both a putative short peptide and the lncMYH7b itself to investigate (Fig. 3A). The MYH7b locus produces full-length protein in the specialized muscles, brain, and inner ear tissues and therefore encodes all of the essential signals for translation (9Lee L.A. Karabina A. Broadwell L.J. Leinwand L.A. The ancient sarcomeric myosins found in specialized muscles.Skelet. Muscle. 2019; 9: 1-15Crossref PubMed Scopus (8) Google Scholar). We previously reported increased exon 7-skipped MYH7b RNA levels in C2C12 mouse myotubes after blocking translation with cycloheximide, indicating that the exon-skipped RNA is either translated or shunted to the nonsense-mediated decay (NMD) pathway (10Bell M.L. Buvoli M. Leinwand L.A. Uncoupling of expression of an intronic microRNA and its myosin host gene by exon skipping.Mol. Cell. Biol. 2010; 30: 1937-1945Crossref PubMed Scopus (102) Google Scholar). Using FISH, we determined that MYH7b RNA is localized to the cytoplasm in hiPS-CMs, consistent with translation (Fig. 3, B and C). We confirmed these results by checking for signal upon knockdown and with biochemical fractionation (Fig. S3, B and C). Furthermore, we saw very few transcripts along the nuclear envelope, which is where mRNAs undergoing NMD typically localize (Fig. 3B) (18Trcek T. Sato H. Singer R.H. Maquat L.E. Temporal and spatial characterization of nonsense-mediated mRNA decay.Genes Dev. 2013; 27: 541-551Crossref PubMed Scopus (79) Google Scholar). This suggests that not all exon 7-skipped MYH7b RNA is degraded by NMD. There is also precedent for small peptides being produced from “non-coding” RNAs, which affect muscle contraction (19Nelson B.R. Makarewich C.A. Anderson D.M. Winders B.R. Troupes C.D. Wu F. Reese A.L. McAnally J.R. Chen X. Kavalali E.T. Cannon S.C. Houser S.R. Bassel-duby R. Olson E.N. A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle.Science. 2016; 351: 271-275Crossref PubMed Scopus (372) Google Scholar, 20Anderson D.M. Anderson K.M. Chang C.L. Makarewich C.A. Nelson B.R. McAnally J.R. Kasaragod P. Shelton J.M. Liou J. Bassel-Duby R. Olson E.N. A micropeptide encoded by a putative long noncoding RNA regulates muscle performance.Cell. 2015; 160: 595-606Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). Based on this, we next investigated whether the short open reading frame prior to the PTC in exon 9 is translated and, if so, whether the short peptide (MYH7b_sp) could regulate the β-MyHC/α-MyHC ratio in human cardiomyocytes. The putative MYH7b_sp is predicted to be 206 amino acids in length and approximately 27 kDa. It is possible that MYH7b_sp has escaped detection in previous studies simply because full-length MyHCs are approximately 250 kDa, and a 27 kDa MyHC has not been described. Therefore, we used our anti-MYH7b antibody, which is directed against an epitope that should present in both the full-length MYH7b (as shown in Fig. 3D, mouse cerebellum) and the putative MYH7b_sp to probe for MYH7b_sp expression (11Rossi A.C. Mammucari C. Argentini C. Reggiani C. Schiaffino S. Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles.J. Physiol. 2010; 588: 353-364Crossref PubMed Scopus (90) Google Scholar, 15Peter A.K. Rossi A.C. Buvoli M. Ozeroff C.D. Crocini C. Perry A.R. Buvoli A.E. Lee L.A. Leinwand L.A. Expression of normally repressed myosin heavy chain 7b in the mammalian heart induces dilated cardiomyopathy.J. Am. Heart Assoc. 2019; 8e013318Crossref PubMed Scopus (5) Google Scholar). As a positive control, we infected C2C12 cells with an adenovirus containing only the MYH7b_sp open reading frame and detected a protein of the right size and immunoreactivity (Fig. 3D). We probed human heart tissue from dilated cardiomyopathy patients, where MYH7b transcripts are easily detectable (Fig. 1B), but did not detect it via western blotting (Fig. 3D). In case MYH7b_sp exists at levels below the detection limit of a western blot but does have a regulatory role, we forced expression of the MYH7b_sp using adenovirus infection of hiPS-CMs and performed RNA-seq (Fig. S4). We found no difference in the β-MyHC/α-MyHC ratio at the RNA level, and many differentially expressed genes were chaperones, which is both consistent with an adenoviral infection and production of a foreign peptide (Fig. S4) (21Zhao H. Dahlö M. Isaksson A. Syvänen A.C. Pettersson U. The transcriptome of the adenovirus infected cell.Virology. 2012; 424: 115-128Crossref PubMed Scopus (40) Google Scholar). Thus, we conclude that MYH7b_sp is not expressed at detectable levels in human cardiomyocytes and has no effect on the expression of α- or β-MyHC, even after forced expression. Having ruled out the possible regulatory roles of miR-499 and MYH7b_sp in β-MyHC expression, we hypothesized that the lncMYH7b transcript itself regulates β-MyHC. We attempted to overexpress the exon-skipped cDNA (MYH7bΔ7 cDNA) via adenoviral infection as we did with MYH7b_sp. Unfortunately, we discovered that this cDNA expresses large quantities of MYH7b_sp, which we have that shown is not produced from endogenous lncMYH7b (Fig. S5). We hypothesize that this is due to the lack of exon junction complexes that would normally trigger NMD and other RNA quality-control pathways. This led us to postulate that having a spliced transcript is essential to lncMYH7b’s function. Therefore, we decided to move forward with a loss-of-function approach. To achieve this, we knocked down MYH7b RNA with locked nucleic acid antisense oligonucleotides (ASOs) and measured the kinetics of lncMYH7b and β-MyHC expression in hiPS-CMs. Upon treatment with 10 μM ASO, MYH7b RNA was knocked down ∼12-fold by 48 h, which was maintained through 7 days (Fig. 4A). Importantly, we observed decreased β-MyHC expression only after loss of MYH7b RNA (Fig. 4A), supporting an upstream role for the exon-skipped MYH7b RNA in regulating β-MyHC expression. To further support the idea that lncMYH7b is regulating β-MyHC/α-MyHC ratios independently of miR-499, we measured the change in myomiR levels after lncMYH7b knockdown (KD). We observed no change in miR-499 levels, nor in the other two myomiRs encoded by α-MyHC and β-MyHC (miR-208a and miR-208b, respectively) upon treatment with the MYH7b ASO (Fig. 4B). Next, we determined that the change observed in β-MyHC RNA upon MYH7b KD was recapitulated at the protein level. As independent validation, we differentiated hiPS-CMs from a different iPSC line and used a different ASO sequence. A myosin separating gel showed a significant decrease of the β-MyHC/α-MyHC ratio upon KD of MYH7b RNA (Fig. 4C). Together, these data demonstrate a role for lncMYH7b in regulating the β-MyHC/α-MyHC ratio in human cardiomyocytes. In order to elucidate the potential regulatory impacts of lncMYH7b in a more global and unbiased manner, we performed RNA-seq on MYH7b ASO-treated hiPS-CMs. We used the publicly available NF-core/RNAseq pipeline to analyze differential gene expression (22Ewels P.A. Peltzer A. Fillinger S. Alneberg J. Patel H. Wilm A. Garcia M.U. Tommaso P. Di Nahnsen S. nf-core: Community curated bioinformatics pipelines.bioRxiv. 2019; https://doi.org/10.1101/610741Crossref Scopus (0) Google Scholar). Differential expression analysis revealed a large bias toward downregulation (535 downregulated versus 224 upregulated genes) with very little overlap with our MYH7b_sp dataset (Fig. 5B). As knockdown of the RNA would also prevent protein expression, this further supports the lack of MYH7b_sp translated from the endogenous locus. We found that the change in the β-MyHC/α-MyHC ratio was also evident at the RNA level, suggesting that lncMYH7b is affecting β-MyHC pretranslationally (Fig. 5A). To gain functional insight into the role of lncMYH7b in regulating cardiac gene expression, we performed a KEGG pathway analysis of our RNA-seq dataset and found that pathways associated with cardiomyopathies were regulated by the reduction in lncMYH7b expression (Fig. 5D). This is consistent with the increases in lncMYH7b levels observed in the tissue from patients with chronic heart disease (Fig. S2). Of interest, expression of two members of the TEA domain (TEAD) transcription factor family, TEAD1 and TEAD3 (log2(fold change) = −0.67 and −1.68, respectively), was decreased in our lncMYH7b KD dataset. As TEAD3 was the most downregulated, and has been shown to directly regulate β-MyHC transcription, we decided to focus on that transcription factor (23Iwaki H. Sasaki S. Matsushita A. Ohba K. Matsunaga H. Essential role of TEA domain transcription factors in the negative regulation of the MYH 7 gene by thyroid hormone and its receptors.PLoS One. 2014; 9e88610Crossref PubMed Scopus (17) Google Scholar). We independently confirmed a decrease in TEAD3 RNA levels using qPCR, which agrees with TEAD3 being the most downregulated transcription factor in our data (Fig. S6). TEAD family members are known to bind to and enhance transcription at the β-MyHC promoter and are upregulated in hypertrophic cardiomyopathy patient hearts (23Iwaki H. Sasaki S. Matsushita A. Ohba K. Matsunaga H. Essential role of TEA domain transcription factors in the negative regulation of the MYH 7 gene by thyroid hormone and its receptors.PLoS One. 2014; 9e88610Crossref PubMed Scopus (17) Google Scholar). TEAD3 specifically has not been well studied, but has a cardiac-enriched expression pattern (24Jin Y. Messmer-Blust A.F. Li J. The role of transcription enhancer factors in cardiovascular biology.Trends Cardiovasc. 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