Induced pluripotent stem cell-derived cardiomyocytes as models for genetic cardiovascular disorders

Purpose of review: The development of induced pluripotent stem cell (iPSC) technology has led to many advances in the areas of directed cell differentiation and characterization. New methods for generating iPSC-derived cardiomyocytes provide an invaluable resource for the study of certain cardiovascular disorders. This review highlights the current technology in this field, its application thus far to the study of genetic disorders of the RAS/MAPK pathway and long-QT syndrome (LQTS), and future directions for the field. Recent findings: Enhanced methods increase the efficiency of generating and stringently purifying iPSC-derived cardiomyocytes. The use of cardiomyocytes derived from patients with LEOPARD syndrome and LQTS has shed light on the molecular mechanisms of disease and validated their use as reliable human disease models. Summary: The use of iPSC-derived cardiomyocytes to study genetic cardiovascular disorders will enable a deeper and more applicable understanding of the molecular mechanisms of human disease, as well as improving our ability to achieve successful cell-based therapies. Methods to efficiently generate these cells are improving and provide promise for future applications of this technology. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Human Engineered Cardiac Tissues Created Using Induced Pluripotent Stem Cells Reveal Functional Characteristics of BRAF-Mediated Hypertrophic Cardiomyopathy

<div><p>Hypertrophic cardiomyopathy (HCM) is a leading cause of sudden cardiac death that often goes undetected in the general population. HCM is also prevalent in patients with cardio-facio-cutaneous syndrome (CFCS), which is a genetic disorder characterized by aberrant signaling in the RAS/MAPK signaling cascade. Understanding the mechanisms of HCM development in such RASopathies may lead to novel therapeutic strategies, but relevant experimental models of the human condition are lacking. Therefore, the objective of this study was to develop the first 3D human engineered cardiac tissue (hECT) model of HCM. The hECTs were created using human cardiomyocytes obtained by directed differentiation of induced pluripotent stem cells derived from a patient with CFCS due to an activating BRAF mutation. The mutant myocytes were directly conjugated at a 3:1 ratio with a stromal cell population to create a tissue of defined composition. Compared to healthy patient control hECTs, BRAF-hECTs displayed a hypertrophic phenotype by culture day 6, with significantly increased tissue size, twitch force, and atrial natriuretic peptide (ANP) gene expression. Twitch characteristics reflected increased contraction and relaxation rates and shorter twitch duration in BRAF-hECTs, which also had a significantly higher maximum capture rate and lower excitation threshold during electrical pacing, consistent with a more arrhythmogenic substrate. By culture day 11, twitch force was no longer different between BRAF and wild-type hECTs, revealing a temporal aspect of disease modeling with tissue engineering. Principal component analysis identified diastolic force as a key factor that changed from day 6 to day 11, supported by a higher passive stiffness in day 11 BRAF-hECTs. In summary, human engineered cardiac tissues created from BRAF mutant cells recapitulated, for the first time, key aspects of the HCM phenotype, offering a new <i>in vitro</i> model for studying intrinsic mechanisms and screening new therapeutic approaches for this lethal form of heart disease.</p></div

Twitch force characteristics of wild-type and BRAF-mutant hECTs.

<p>(<b>A</b>) Cartoon of iPSC-derived hECT force data illustrating twitch characteristic parameters. (<b>B-G</b>) Mean (±SD) twitch parameters for wild-type (open bars, n = 7) and BRAF mutant (solid bars, n = 5) iPSC-hECTs tested on culture days 6 and 11, including developed force (<b>B</b>), 50% twitch duration (<b>C</b>), time to 50% contraction (<b>D</b>), time to 50% relaxation (<b>E</b>) and the maximum rates of contraction (<b>F</b>) and relaxation (<b>G</b>). * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Investigating longitudinal changes in hECT functional phenotype.

<p>(<b>A</b>) Principal component analysis and k-means clustering of tissues paced at 2 Hz on day 6 (left) and day 11 (right). (<b>B</b>) Developed force of the wild-type tissues increased significantly over day 6 and day 11, while mutant tissues did not. The lack of a significant change from day 6 to day 11 appeared to be due to a greater increase in diastolic force of mutants from day 6 to 11 than wild-type tissues, while systolic force increased approximately by the same amount for both tissue types. (<b>C</b>) Passive Young’s modulus determined by uniaxial stretch measurements on days 12–15 (n = 4 per tissue type). * p < 0.05 between tissues types, † p < 0.05 between day 6 and day 11 for wild-type tissues.</p

Electrical properties of of wild-type and BRAF-mutant hECTs.

<p>(<b>A</b>) Developed force versus frequency relationship of iPSC-derived hECTs at culture day 6 (left) and day 11 (right); (<b>B</b>) maximum pacing frequency of wild-type and mutant tissues; (<b>C</b>) spontaneous beating frequency; (<b>D</b>) The minimum voltage necessary for pacing for both tissues; (<b>E</b>) Relative chronotropic response to isoproterenol (normalized to baseline beating frequency) with fitted nonlinear regression model. (<b>F</b>) Twitch rate variability plots of each tissue type with a paced control for comparison. Each color represents a different tissue. (<b>G</b>) Quantification of twitch rate variability by the mean distance of each point on the twitch rate variability plots from each cluster centroid. Error bars represent standard error (<b>A</b>) or standard deviation (<b>B-G</b>) Open symbols and bars are wild-type hECTS (n = 7), filled symbols and bars are BRAF-mutant hECTs (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Characterization of wild-type and BRAF iPSC-hECTs.

<p>(<b>A</b>) Sorting paradigm for construction of defined iPSC-hECTs; (<b>B</b>) Photograph of wild-type (left) and mutant (right) tissues after 6 days in culture; (<b>C</b>) longitudinal section of wild-type (left) and mutant (right) tissues stained with hematoxylin and eosin after 12 days in culture; (<b>D</b>) Post deflections of both tissue types on day 6 at 2 Hz pacing frequency; (<b>E</b>) Cross-sectional area (mean±SD) of wild-type (open bars, n = 7) and mutant (filled bars, n = 4) tissues; (<b>F</b>) Molecular analysis of mutant and wild-type tissues performed on day 12 (n = 3 for each tissue type).</p

Abstract 19583: Autonomous and Non-Autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes

Hypertrophic cardiomyopathy (HCM) is a pathological disorder predominantly due to mutations in sarcomeric components. Germline mutations in BRAF cause a developmental syndrome called cardio-facio-cutaneous syndrome (CFCS), in which 40% of patients also develop HCM. Since the role of the RAS/MAPK pathway in HCM is still unclear, we generated a human induced pluripotent stem cell model for CFCS from three patients with activating BRAF T599R or Q257R mutations. In order to examine hiPSC-derived cell-type specific phenotypes and cellular interactions underpinning HCM, we generated a method to purify cardiomyocytes and non-cardiomyocytes simultaneously by cell sorting based on SIRPα and CD90 expression. Purified BRAF-mutant SIRPα+/CD90- cells were &gt;95% cardiomyocytes, displayed cellular hypertrophy with pro-hypertrophic gene expression, and dysregulation of the RAS/MAPK pathway. BRAF-mutant cardiomyocytes also displayed intrinsic calcium handling defects, including increased calcium transient irregularity and increased stored calcium within the sarcoplasmic reticulum. In addition, purified BRAF-mutant SIRPα-/CD90+ cells, which were fibroblast-like, displayed activation of the RAS/MAPK pathway and exhibited a pro-fibrotic phenotype. Cross-culture studies revealed that BRAF-mutant fibroblast-like cells critically modulate cardiomyocyte hypertrophy through TGFβ paracrine signaling, as TGFβ inhibition prevented cardiomyocyte hypertrophy induced by BRAF-mutant fibroblast-like cells. Additionally, inhibition of RAS/MAPK signaling was capable of rescuing BRAF-mutant cardiomyocyte hypertrophy and cardiomyocyte-intrinsic calcium handling abnormalities. Thus, we show for the first time that cell autonomous and non-autonomous defects underlie RASopathy-associated HCM. As fibroblast activation has been documented previously in sarcomeric HCM, our findings suggest that cardiac fibroblasts may contribute to pathologic hypertrophy in addition to causing fibrosis in primary forms of HCM. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies

Autonomous and Non-autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes

Germline mutations in BRAF cause cardio-facio-cutaneous syndrome (CFCS), whereby 40% of patients develop hypertrophic cardiomyopathy (HCM). As the role of the RAS/MAPK pathway in HCM pathogenesis is unclear, we generated a human induced pluripotent stem cell (hiPSC) model for CFCS from three patients with activating BRAF mutations. By cell sorting for SIRPα and CD90, we generated a method to examine hiPSC-derived cell type-specific phenotypes and cellular interactions underpinning HCM. BRAF-mutant SIRPα+/CD90− cardiomyocytes displayed cellular hypertrophy, pro-hypertrophic gene expression, and intrinsic calcium-handling defects. BRAF-mutant SIRPα−/CD90+ cells, which were fibroblast-like, exhibited a pro-fibrotic phenotype and partially modulated cardiomyocyte hypertrophy through transforming growth factor β (TGFβ) paracrine signaling. Inhibition of TGFβ or RAS/MAPK signaling rescued the hypertrophic phenotype. Thus, cell autonomous and non-autonomous defects underlie HCM due to BRAF mutations. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies