Studying monogenic mitochondrial cardiomyopathies may yield insights into mitochondrial roles in cardiac development and disease. Here, we combine patient-derived and genetically engineered iPSCs with tissue engineering to elucidate the pathophysiology underlying the cardiomyopathy of Barth syndrome (BTHS), a mitochondrial disorder caused by mutation of the gene Tafazzin (TAZ). Using BTHS iPSC-derived cardiomyocytes (iPSC-CMs), we defined metabolic, structural, and functional abnormalities associated with TAZ mutation. BTHS iPSC-CMs assembled sparse and irregular sarcomeres, and engineered BTHS “heart on chip” tissues contracted weakly. Gene replacement and genome editing demonstrated that TAZ mutation is necessary and sufficient for these phenotypes. Sarcomere assembly and myocardial contraction abnormalities occurred in the context of normal whole cell ATP levels. Excess levels of reactive oxygen species mechanistically linked TAZ mutation to impaired cardiomyocyte function. Our study provides new insights into the pathogenesis of Barth syndrome, suggests new treatment strategies, and advances iPSC-based in vitro modeling of cardiomyopathy.

SUMMARY Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research.

Identification of genomic regions that control tissue-specific gene expression is currently problematic. ChIP and high-throughput sequencing (ChIP-seq) of enhancer-associated proteins such as p300 identifies some but not all enhancers active in a tissue. Here we show that co-occupancy of a chromatin region by multiple transcription factors (TFs) identifies a distinct set of enhancers. GATA-binding protein 4 (GATA4), NK2 transcription factor-related, locus 5 (NKX2-5), T-box 5 (TBX5), serum response factor (SRF), and myocyte-enhancer factor 2A (MEF2A), here referred to as “cardiac TFs,” have been hypothesized to collaborate to direct cardiac gene expression. Using a modified ChIP-seq procedure, we defined chromatin occupancy by these TFs and p300 genome wide and provided unbiased support for this hypothesis. We used this principle to show that co-occupancy of a chromatin region by multiple TFs can be used to identify cardiac enhancers. Of 13 such regions tested in transient transgenic embryos, seven (54%) drove cardiac gene expression. Among these regions were three cardiac-specific enhancers of Gata4 , Srf , and swItch/sucrose nonfermentable-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 3 ( Smarcd3 ), an epigenetic regulator of cardiac gene expression. Multiple cardiac TFs and p300-bound regions were associated with cardiac-enriched genes and with functional annotations related to heart development. Importantly, the large majority (1,375/1,715) of loci bound by multiple cardiac TFs did not overlap loci bound by p300. Our data identify thousands of prospective cardiac regulatory sequences and indicate that multiple TF co-occupancy of a genomic region identifies developmentally relevant enhancers that are largely distinct from p300-associated enhancers.