Engineered Heart Tissues for Standard 96-Well Tissue Culture Plates.

Engineered heart tissues (EHTs) have been shown to be a valuable platform for disease investigation and therapeutic testing by increasing human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturity and better recreating the native cardiac environment. The protocol detailed in this chapter describes the generation of miniaturized EHTs (mEHTs) incorporating hiPSC-CMs and human stromal cells in a fibrin hydrogel. This platform utilizes an array of silicone posts designed to fit in a standard 96-well tissue culture plate. Stromal cells and hiPSC-CMs are cast in a fibrin matrix suspended between two silicone posts, forming an mEHT that produces synchronous muscle contractions. The platform presented here has the potential to be used for high throughput characterization and screening of disease phenotypes and novel therapeutics through measurements of the myocardial function, including contractile force and calcium handling, and its compatibility with immunostaining.

Correcting dilated cardiomyopathy with fibroblast-targeted p38 deficiency.

Inherited mutations in contractile and structural genes, which decrease cardiomyocyte tension generation, are principal drivers of dilated cardiomyopathy (DCM)- the leading cause of heart failure 1,2 . Progress towards developing precision therapeutics for and defining the underlying determinants of DCM has been cardiomyocyte centric with negligible attention directed towards fibroblasts despite their role in regulating the best predictor of DCM severity, cardiac fibrosis 3,4 . Given that failure to reverse fibrosis is a major limitation of both standard of care and first in class precision therapeutics for DCM, this study examined whether cardiac fibroblast-mediated regulation of the heart's material properties is essential for the DCM phenotype. Here we report in a mouse model of inherited DCM that prior to the onset of fibrosis and dilated myocardial remodeling both the myocardium and extracellular matrix (ECM) stiffen from switches in titin isoform expression, enhanced collagen fiber alignment, and expansion of the cardiac fibroblast population, which we blocked by genetically suppressing p38α in cardiac fibroblasts. This fibroblast-targeted intervention unexpectedly improved the primary cardiomyocyte defect in contractile function and reversed ECM and dilated myocardial remodeling. Together these findings challenge the long-standing paradigm that ECM remodeling is a secondary complication to inherited defects in cardiomyocyte contractile function and instead demonstrate cardiac fibroblasts are essential contributors to the DCM phenotype, thus suggesting DCM-specific therapeutics will require fibroblast-specific strategies.

Engineered Heart Tissues for Contractile, Structural, and Transcriptional Assessment of Human Pluripotent Stem Cell-Derived Cardiomyocytes in a Three-Dimensional, Auxotonic Environment.

Three-dimensional, human engineered heart tissue promotes maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and provides a useful platform for in vitro cardiac development and disease modeling. This protocol describes the generation of fibrin-based engineered heart tissues (EHTs) containing hiPSC-CMs and human stromal cells. The platform makes use of racks of silicone posts that fit a standard 24-well dish. Stromal cells and hiPSC-CMs are cast in a fibrin hydrogel suspended between two silicone posts, forming an engineered tissue that generates synchronous contractions. The platform described herein is amenable to various measures of cardiac function including measurement of contractile force and calcium handling, as well as molecular biology assays and immunostaining.