Thursday, April 18, 2024
4/18/2024
PROJECT SUMMARY Cardiovascular disease accounted for 1 in 3 deaths in the USA in 2016 and was the leading cause of death worldwide (AHA, 2019). Treatment is limited to symptomatic interventions through small molecule drugs or surgery, but these do not address the underlying molecular causes of heart failure. One such mechanism is the dysregulation of myosin heavy chain isoform expression. Myosin heavy chains (MyHC) are the motor proteins that convert chemical energy into kinetic energy to produce the force necessary for muscle contraction. In the heart, three MyHC isoforms are expressed: a-MyHC, b-MyHC, and Myosin Heavy Chain 7b (MYH7b). However, MYH7b is not translated to protein due to a post-transcriptional exon-skipping mechanism that produces a premature stop codon. At the protein level, a-MyHC and b-MyHC exist in a carefully controlled ratio of 10% a-MyHC to 90% b-MyHC. In late-stage heart diseases, the expression of a-MyHC and b-MyHC is dysregulated; the proportion of a-MyHC is reduced to undetectable levels, while b-MyHC expression increases to essentially 100%. This is thought to be a compensatory mechanism to conserve energy, as b-MyHC has a slower ATP- turnover rate; however, it compromises the contractile function of the heart and can thus lead to cardiac death. The mechanism behind this transcriptional shift is not understood. We have recently discovered that expression levels of MYH7b RNA and b-MyHC (both RNA and protein) positively correlate, and that changes in MYH7b expression always precede those of b-MyHC. Furthermore, knockdown of MYH7b by anti-sense oligonucleotides causes a decrease in b-MyHC expression. We have performed RNA-sequencing analysis in cardiomyocytes differentiated from human-derived induced pluripotent stem cells with reduced levels of MYH7b RNA. This lead to a discovery of a proposed pathway where MYH7b controls the expression of focal adhesion kinase, leading to a correlating change in the transcription of TEAD3, a transcription enhancer factor that enhances the expression of b-MyHC. We will validate this pathway through a series of rescue experiments. Then we will determine the epistasis of the pathway using genetic manipulations to determine where each gene resides in the pathway. Finally, we want to fully define this pathway responsible for controlling the transcription of b-MyHC by identifying the molecular partners of MYH7b, using RNA-based affinity purification. We will use a comprehensive approach to identify both protein and nucleic-acid interactions, thus fully defining the interactome of MYH7b. This research will have a large impact on the field of cardiac biology, as it will help solve the age-old puzzle of MyHC transcriptional control in the heart. I hypothesize that MYH7b is acting as a long non-coding RNA (lncMYH7b) in the heart to regulate the transcription of b-MyHC through controlling levels of focal adhesion kinase and TEAD3. In Aim 1 I will validate and define this pathway. In Aim 2, I will identify the molecular partners of lncMYH7b.
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