טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLewis Yair
SubjectMolecular Mechanisms of Cardiac Sarcomere Maintenance
DepartmentDepartment of Medicine
Supervisor Professor Izhak Kehat
Full Thesis textFull thesis text - English Version


Abstract

The cardiomyocyte is a highly-specialized cell that is dedicated to operating and maintaining a macromolecular machine, called the sarcomere - the basic contractile unit of the cardiomyocyte. Changes at the sarcomere level are invariably involved in the pathophysiology of heart failure, yet the basic mechanisms of sarcomere maintenance have yet to be fully delineated, limiting our understanding of heart failure. While cardiomyocytes are long lived cells that generally do not divide, sarcomeric proteins degrade and therefore need to be constantly replaced in order to maintain contractile function, both in quiescence and in hypertrophy. Our study set out to shed light on these mechanisms.

We studied cardiomyocytes using imaging of mRNA and protein synthesis, and gene expression via RT-qPCR. These demonstrated sarcomeric mRNA localization, which is followed by localized protein synthesis at the sarcomere. We also showed that sarcomeric gene transcription and protein synthesis rates vary widely between cells, yet protein content is relatively constant. Unincorporated sarcomeric proteins are rapidly degraded by the localized proteasome system, offsetting the transcription and translation variability. Based on these findings, we propose a model of sarcomere maintenance in cardiomyocytes which involves localization of sarcomeric mRNA transcripts, on-site protein synthesis, and proteasome mediated degradation of unincorporated proteins. These work in unison to buffer the variability in transcription and translation and maintain sarcomeric homeostasis while allowing the cell a high resolution of spatial and temporal control.

A second aspect of this project was further investigation of the transcriptional variability we observed by single-cell RNA-seq of cardiomyocytes. We hypothesized that this variability may stem not only from bursty promotor kinetics, but also due the existence of cardiomyocyte subpopulations. Previous studies have highlighted a number of genes whose expression level is elevated or reduced in cardiac hypertrophy, yet these genomic studies were based on bulk analysis of heart tissue or cardiomyocytes, which assumes a homogenous population. Consequently, cellular subpopulations and variability in gene expression in hypertrophy are not amenable to research with these techniques. Major advances in RNA extraction and sequencing techniques over the past few years have allowed for single-cell analysis of gene expression, but cell isolation techniques are not easily implemented in mature cardiomyocytes. We therefore developed a streamlined experimental method for single-cell RNA-seq of mature cardiomyocytes, and used single-cell transcriptional profiling of cardiomyocytes in hypertrophic vs. control hearts to investigate the cardiac transcriptome in hypertrophy.

Analysis of single-cell RNA-seq of hypertrophic vs. control hearts identified potential cardiomyocyte subpopulations, highlighted genes that were previously not known to play a part in cardiac hypertrophy, and presented novel findings with regard to expression of known markers of hypertrophy. These findings expand on the current understanding of the molecular mechanisms of cardiac hypertrophy and heart failure, and present a large dataset which can be used for further analysis. The results underscore the importance of single-cell studies of cardiomyocytes in healthy and diseased hearts, and their potential to uncover new pathways which may in the future allow for new therapeutic approaches.