dna-pkcs br Results br Discussion In this

Results
Discussion In this study we have shown that activating BRAF mutations leading to increased RAS/MAPK pathway signaling induce a hypertrophic phenotype in hiPSC-derived CMs. While BRAF-mutant CMs display intrinsic defects in Ca2+ handling, several aspects of their phenotype require paracrine TGFβ secretion by activated, pro-fibrotic BRAF-mutant FLCs. Inhibition of TGFβ or RAS/MAPK signaling rescues the hypertrophic phenotype. Interestingly, while patient 3 did not show clinical evidence of HCM, we detected underlying defects indistinguishable from those in patients 1 and 2, both diagnosed with HCM. Similar subclinical pathology has been demonstrated in other hiPSC models of HCM (Lan et al., 2013). However, it is also possible that more complex factors active in 3D multi-organ systems such as hemodynamic load, which are inadequately modeled using the 2D hiPSC system, may play a role in disease progression. We recently generated a 3D human engineered cardiac tissue (hECT) model, in which BRAF-mutant hECTs displayed increased twitch force and contraction and relaxation rates, and a lower excitation threshold compared with WT (Cashman et al., 2016). In the future, these hECT models may be helpful for investigating more complex factors such as tissue perfusion, flow dynamics, and mechanical stress, to enable higher-fidelity physiologic measurements of muscle function. Some aspects of the intrinsic CM phenotype we document have been associated with enhanced cellular maturation in culture, including organized sarcomeres and increased cellular area (Yang et al., 2014). However, matured stem cell-derived CMs develop into elongated rods with dna-pkcs arranged parallel to the long axis of the cell, and do not display the irregular, generalized increase in cellular area that we observed. In addition, immature derived CMs possess sophisticated excitation-contraction coupling and do not display increased Ca2+ transient amplitude, irregularity, or increased SR Ca2+ stores upon maturation (Lundy et al., 2013). Although genotype-specific influences on CM maturation may contribute to the CM phenotype, the HCM phenotype we observe in its totality cannot be attributed to them. In addition, the variations in cardiac differentiation efficiencies we document do not segregate WT and mutant populations and are unrelated to CM maturation, as SIRPα expression is detected in stem cell-derived CMs between days 7 and 8 of differentiation (Dubois et al., 2011). Our data also reveal variability in behavior among hiPSC lines (Figures 2, 3, and 4), often attributed to variations that occur during re-programming (Toivonen et al., 2013) and to distinct genetic backgrounds. This variability cannot be attributed to a single cell line and is not replicated across distinct experiments. To strengthen our conclusions, we have utilized six independent patient samples and provided inhibitory and overexpression studies, which support our claims. To date, hiPSC models for cardiac disease have utilized mixed cell populations (Lan et al., 2013; Zanella et al., 2014), obscuring possible contributions of neighboring cells to the disease phenotype. Here, we developed an effective dual-purification method to study cell interactions underpinning human HCM. By combining CM and non-CM markers, we increased hiPSC-derived CM purity from <70% (Dubois et al., 2011) to >95% while simultaneously purifying the non-CM fraction. While CD90 is a well-described marker for human fibroblasts (Kisselbach et al., 2009), it has also been shown to label stem cells, lymphocytes, neurons, and activated endothelial cells (Herrera-Molina et al., 2013). As CM differentiation protocols direct mesodermal lineage formation (Mummery et al., 2012), robust stem cell differentiation into many of these cell types is unlikely. The expression of multiple fibroblast- and fibrosis-associated genes in CD90+ cells supports their likely identity as FLCs.