Time-dependent effects of BRAF-V600E on cell cycling, metabolism, and function in engineered myocardium.

Candidate cardiomyocyte (CM) mitogens such as those affecting the extracellular signal-regulated kinase (ERK) signaling pathway represent potential targets for functional heart regeneration. We explored whether activating ERK via a constitutively active mutant of B-raf proto-oncogene (BRAF), BRAF-V600E (caBRAF), can induce proproliferative effects in neonatal rat engineered cardiac tissues (ECTs). Sustained CM-specific caBRAF expression induced chronic ERK activation, substantial tissue growth, deficit in sarcomeres and contractile function, and tissue stiffening, all of which persisted for at least 4 weeks of culture. caBRAF-expressing CMs in ECTs exhibited broad transcriptomic changes, shift to glycolytic metabolism, loss of connexin-43, and a promigratory phenotype. Transient, doxycycline-controlled caBRAF expression revealed that the induction of CM cycling is rapid and precedes functional decline, and the effects are reversible only with short-lived ERK activation. Together, direct activation of the BRAF kinase is sufficient to modulate CM cycling and functional phenotype, offering mechanistic insights into roles of ERK signaling in the context of cardiac development and regeneration.

Temporal perturbation of histone deacetylase activity reveals a requirement for HDAC1-3 in mesendoderm cell differentiation.

Histone deacetylases (HDACs) are a class of enzymes that control chromatin state and influence cell fate. We evaluated the chromatin accessibility and transcriptome dynamics of zinc-containing HDACs during cell differentiation in vitro coupled with chemical perturbation to identify the role of HDACs in mesendoderm cell fate specification. Single-cell RNA sequencing analyses of HDAC expression during human pluripotent stem cell (hPSC) differentiation in vitro and mouse gastrulation in vivo reveal a unique association of HDAC1 and -3 with mesendoderm gene programs during exit from pluripotency. Functional perturbation with small molecules reveals that inhibition of HDAC1 and -3, but not HDAC2, induces mesoderm while impeding endoderm and early cardiac progenitor specification. These data identify unique biological functions of the structurally homologous enzymes HDAC1-3 in influencing hPSC differentiation from pluripotency toward mesendodermal and cardiac progenitor populations.

Frame-Hydrogel Methodology for Engineering Highly Functional Cardiac Tissue Constructs.

Engineered cardiac tissues hold tremendous promise for in vitro drug discovery, studies of heart development and disease, and therapeutic applications. Here, we describe a versatile "frame-hydrogel" methodology to generate engineered cardiac tissues with highly mature functional properties. This methodology has been successfully utilized with a variety of cell sources (neonatal rat ventricular myocytes, human and mouse pluripotent stem cell-derived cardiomyocytes) to generate tissues with diverse 3D geometries (patch, bundle, network) and levels of structural and functional anisotropy. Maturation of such engineered cardiac tissues is rapidly achieved without the need for exogenous electrical or mechanical stimulation or use of complex bioreactors, with tissues routinely reaching conduction velocities and specific forces of 25 cm/s and 20 mN/mm2, respectively, and forces per input cardiomyocyte of up to 12 nN. This method is reproducible and readily scalable to generate small tissues ideal for in vitro testing as well as tissues with large, clinically relevant dimensions.

Biomaterializing the promise of cardiac tissue engineering.

During an average individual's lifespan, the human heart pumps nearly 200 million liters of blood delivered by approximately 3 billion heartbeats. Therefore, it is not surprising that native myocardium under this incredible demand is extraordinarily complex, both structurally and functionally. As a result, successful engineering of adult-mimetic functional cardiac tissues is likely to require utilization of highly specialized biomaterials representative of the native extracellular microenvironment. There is currently no single biomaterial that fully recapitulates the architecture or the biochemical and biomechanical properties of adult myocardium. However, significant effort has gone toward designing highly functional materials and tissue constructs that may one day provide a ready source of cardiac tissue grafts to address the overwhelming burden of cardiomyopathic disease. In the near term, biomaterial-based scaffolds are helping to generate in vitro systems for querying the mechanisms underlying human heart homeostasis and disease and discovering new, patient-specific therapeutics. When combined with advances in minimally-invasive cardiac delivery, ongoing efforts will likely lead to scalable cell and biomaterial technologies for use in clinical practice. In this review, we describe recent progress in the field of cardiac tissue engineering with particular emphasis on use of biomaterials for therapeutic tissue design and delivery.

Single-Cell Transcriptomic Analysis of Cardiac Differentiation from Human PSCs Reveals HOPX-Dependent Cardiomyocyte Maturation.

Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes.