Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2015)

GATA4 is a central transcriptional regulator during cardiac development and for postnatal function. Familial mutations in GATA4 lead to autosomal dominant congenital heart disease and cardiomyopathy. We previously reported a GATA4 G296S mutation that resides near the second zinc finger of GATA4, involved in DNA-binding and protein-protein interactions thought to be dominant due to haploinsufficiency. However, the mechanisms by which this mutation affects the transcriptional and epigenetic landscape as well as the cellular physiology is unknown. Here, we have used genomic approaches as well as cell-based assays to delineate the consequences of the GATA4 G296S mutation in cardiomyocytes.

We generated iPS cells from human patients with or without the GATA4 G296S mutation and differentiated them to a purified population of cardiomyocytes. Using these cells, we conducted investigations into the role of a putative modifier gene identified by bioinformatics analyses for its potential involvement in the development of the GATA4 cardiomyopathy. However we were not able to uncover a disease causing relationship independent of GATA4.

We performed chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq) on endogenous GATA4 protein and revealed hundreds of previously unidentified GATA4-bound loci while confirming known GATA4 targets. We also performed ChIP-Seq on TBX5, a transcription factor associated with congenital heart disease that physically interacts with GATA4 and whose interaction is specifically disrupted by the G296S mutation. Genome-wide we identified thousands of loci where GATA4 and TBX5 co-localized in wild-type cells, but surprisingly this co-occupancy was lost in cardiomyocytes heterozygous for the GATA4 G296S mutation, despite the presence of the wildtype allele. RNA-Seq revealed dysregulation of transcription at many of these loci, along with expected epigenetic changes, with clustered dysregulation of genes involved in cellular respiration, inflammation, and muscle contraction. To investigate the effect of these genes, we undertook a number of cell-based assays. In collaboration with Beth Pruitt’s lab at Stanford, we developed a platform for performing individual cell-based force generation and contractility studies. These contractility and other cell-based studies confirmed defects in contractility and calcium handling in cardiomyocytes heterozygous for the GATA4 G296S mutation. We also verified that the heterozygous Gata4 G295S mouse has an increased susceptibility to pressure-overload induced hypertrophy and demonstrated its limited ability to validate targets dysregulated by RNA-seq.

The deep “-omic” interrogation of iPS-derived cardiomyocytes effectively revealed the transcriptional and cellular consequences of an important human mutation and may explain many of the disease features observed in individuals with GATA4 mutations. Further study is still required to delineate the full consequences of this dysregulated transcriptome, but these observations help to confirm the role of GATA4 in the heart and set a strong foundation for future investigations.

    Cover page: Modeling congenital heart disease with human iPS cell-derived cardiomyocytes in vitro and in vivo using a genomics approach
    • Article
    • Peer Reviewed
    UC Santa Barbara Previously Published Works (2014)
    Introduction: Heart function relies on the contractility of its muscle cells (cardiomyocytes). Human pluripotent stem cells (hPSC) can be differentiated into cardiomyocytes (hPSC-CMs). However, while their myogenic maturity increases with time, they do not resemble adult cardiomyocytes in their morphology, structural organization, mechanical output and electrophysiology. The low maturity of hPSC-CMs limits their potential to model, study and treat heart diseases. Hypothesis: Since the organization of sarcomeres regulates the mechanical output of cardiomyocytes, we hypothesized that enhanced sarcomere maturity correlates with the mechanical output of these cells and the physiology of matured cardiomyocytes. Methods: We cultured single hPSC-CMs on 2000 μm2 rectangular protein patterns on polyacrylamide substrates with a modulus of 10 kPa to resemble the morphology of ventricular cardiomyocytes and match healthy adult ventricular stiffness. We infected seeded cells with rAV CAG-LifeAct-Tag RFP to label actin and image sarcomeres in contracting live cells. We estimated forces generated during single-cell contractile events with traction force microscopy and calculated power (force x cell shortening velocity). We varied cell elongation by changing the cell aspect ratio (1:1 to 7:1) and maintaining constant area. We used substrates having stiffness of 6 kPa (embryonic myocardium), 10 kPa (adult) and 35 kPa (infarcted myocardium). Results: Optimized power output was observed in cells with a 7:1 aspect ratio. The size, organization and dynamics of sarcomeres in these elongated hPSC-CMs are more mature. Optimized power output was observed in cells with a 7:1 aspect ratio. The power generated by hPSC-CMs on 35 kPa substrates was significantly reduced, while reduced variations in the power output across different aspect ratios was observed in 6 kPa. Forces generated by patterned hPSC-CMs were related to the organization, dynamics and size of sarcomeres. Electrophysiological parameters closer to that of mature cardiomyocytes were also observed with patch-clamp measurements in these hPSC-CMs with mature sarcomere organization. Conclusions: Stiffness and shape contribute to the structural and electrophysiological maturation of hPSC-CMs.
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2015)
      Single cardiomyocytes contain myofibrils that harbor the sarcomere-based contractile machinery of the myocardium. Cardiomyocytes differentiated from human pluripotent stem cells (hPSC-CMs) have potential as an in vitro model of heart activity. However, their fetal-like misalignment of myofibrils limits their usefulness for modeling contractile activity. We analyzed the effects of cell shape and substrate stiffness on the shortening and movement of labeled sarcomeres and the translation of sarcomere activity to mechanical output (contractility) in live engineered hPSC-CMs. Single hPSC-CMs were cultured on polyacrylamide substrates of physiological stiffness (10 kPa), and Matrigel micropatterns were used to generate physiological shapes (2,000-µm(2) rectangles with length:width aspect ratios of 5:1-7:1) and a mature alignment of myofibrils. Translation of sarcomere shortening to mechanical output was highest in 7:1 hPSC-CMs. Increased substrate stiffness and applied overstretch induced myofibril defects in 7:1 hPSC-CMs and decreased mechanical output. Inhibitors of nonmuscle myosin activity repressed the assembly of myofibrils, showing that subcellular tension drives the improved contractile activity in these engineered hPSC-CMs. Other factors associated with improved contractility were axially directed calcium flow, systematic mitochondrial distribution, more mature electrophysiology, and evidence of transverse-tubule formation. These findings support the potential of these engineered hPSC-CMs as powerful models for studying myocardial contractility at the cellular level.
        Cover page: Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness.
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2015)
        Single cardiomyocytes contain myofibrils that harbor the sarcomere-based contractile machinery of the myocardium. Cardiomyocytes differentiated from human pluripotent stem cells (hPSC-CMs) have potential as an in vitro model of heart activity. However, their fetal-like misalignment of myofibrils limits their usefulness for modeling contractile activity. We analyzed the effects of cell shape and substrate stiffness on the shortening and movement of labeled sarcomeres and the translation of sarcomere activity to mechanical output (contractility) in live engineered hPSC-CMs. Single hPSC-CMs were cultured on polyacrylamide substrates of physiological stiffness (10 kPa), and Matrigel micropatterns were used to generate physiological shapes (2,000-µm(2) rectangles with length:width aspect ratios of 5:1-7:1) and a mature alignment of myofibrils. Translation of sarcomere shortening to mechanical output was highest in 7:1 hPSC-CMs. Increased substrate stiffness and applied overstretch induced myofibril defects in 7:1 hPSC-CMs and decreased mechanical output. Inhibitors of nonmuscle myosin activity repressed the assembly of myofibrils, showing that subcellular tension drives the improved contractile activity in these engineered hPSC-CMs. Other factors associated with improved contractility were axially directed calcium flow, systematic mitochondrial distribution, more mature electrophysiology, and evidence of transverse-tubule formation. These findings support the potential of these engineered hPSC-CMs as powerful models for studying myocardial contractility at the cellular level.
          • Article
          • Peer Reviewed
          UC Santa Barbara Previously Published Works (2016)
          Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects.
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2016)
            Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects.
              Cover page: Disease Model of GATA4 Mutation Reveals Transcription Factor Cooperativity in Human Cardiogenesis.
              Top