Connecting Sarcomere Protein Mutations to Pathogenesis in Cardiomyopathies: The Development of “Disease in a Dish” Models

Recent technological and protocol developments have greatly increased the ability to utilize stem cells transformed into cardiomyocytes as models to study human heart muscle development and how this is affected by disease associated mutations in a variety of sarcomere proteins. In this perspective we provide an overview of these emerging technologies and how they are being used to create better models of ‘disease in a dish’ for both research and screening assays. We also consider the value of these assays as models to explore the seminal processes in initiation of the disease development and the possibility of early interventions

Genetically Engineered Human Stem Cell-Derived Cardiomyocytes to Study the Novel Titin Isoform Cronos in Development and Disease

Thesis (Ph.D.)--University of Washington, 2018The giant sarcomere protein titin plays a number of important roles in the cardiomyocyte, and truncating mutations to the gene that encodes this protein (TTN) are the leading known cause of dilated cardiomyopathy. Despite the frequency with which these mutations occur, significant questions remain about how they act to cause disease. Of particular interest is the clustering of truncating mutations to the A-band of TTN in DCM cohorts. To investigate this phenomenon, we genetically engineered human induced pluripotent stem cell (hiPSC) lines carrying homozygous mutations in the Z-disk (TTN-Z-/-) and A-band (TTN-A-/-) region of the gene. Surprisingly, TTN-Z-/- cardiomyocytes (CMs) visibly contracted and were found to express a C-terminal portion of titin, which we determined to be the isoform Cronos titin. TTN-A-/- CMs produce truncation products of both full-length and Cronos titin and are not able to produce sarcomeres. Using a custom antibody, we demonstrate that TTN-Z-/- CMs only express Cronos titin and are able to form sarcomeres with this isoform in the absence of full-length titin. However, these cells produce drastically reduced contractile force as both single cells and in engineered heart tissues (EHTs). Using live cell imaging, we determine this is due to an inability of TTN-Z-/- CMs to properly bundle myofibrils, resulting in sarcomeric instability. We further investigate the biological relevance of Cronos titin by investigating its expression in human cardiac tissue. Genomic methylation, transcript levels, and immunostaining indicate that Cronos titin is most highly expressed in fetal cardiac tissue and is present at lower but detectable levels in adult left ventricular tissue. This indicates that Cronos titin is predominantly a developmental isoform. Finally, we investigate the role of Cronos titin by generating two Cronos knock-out (KO) hiPSC lines. When differentiated into cardiomyocytes, these cells do not express Cronos but do express full-length titin at comparable levels to wildtype controls. EHTs generated with Cronos KO CMs produce drastically lower force than control cells and exhibit significant myofibrillar disarray, indicating that Cronos titin is necessary for proper cardiomyocyte function. As a whole, this work demonstrates for the first time that Cronos titin is expressed in human cardiomyocytes, is necessary for proper sarcomere function, and is an important isoform of titin that may play a role in dilated cardiomyopathy

Chromatin and Transcriptional Analysis of Mesoderm Progenitor Cells Identifies HOPX as a Regulator of Primitive Hematopoiesis

We analyzed chromatin dynamics and transcriptional activity of human embryonic stem cell (hESC)-derived cardiac progenitor cells (CPCs) and KDR+/CD34+ endothelial cells generated from different mesodermal origins. Using an unbiased algorithm to hierarchically rank genes modulated at the level of chromatin and transcription, we identified candidate regulators of mesodermal lineage determination. HOPX, a non-DNA-binding homeodomain protein, was identified as a candidate regulator of blood-forming endothelial cells. Using HOPX reporter and knockout hESCs, we show that HOPX regulates blood formation. Loss of HOPX does not impact endothelial fate specification but markedly reduces primitive hematopoiesis, acting at least in part through failure to suppress Wnt/β-catenin signaling. Thus, chromatin state analysis permits identification of regulators of mesodermal specification, including a conserved role for HOPX in governing primitive hematopoiesis