subtype-specific-optical-action-potential-recordings-human-induced

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a ...

Regenerative therapies for heart diseases require the understanding of the molecular mechanisms that govern the fates and differentiation of the diverse muscle and nonmuscle cell lineages that form during heart development. During mouse cardiogenesis, the major lineages of the mature heart, cardiomyocytes, smooth muscle, endothelial cells, and cardiac mesenchyme, arise from multipotent cardiovascular progenitors expressing the transcription factors Mesp1, Isl1, Nkx2-5, and Tbx18. Recent identification of stem/progenitor cells of embryonic origin with intrinsic competence to differentiate into multiple lineages of the heart offers exciting new possibilities for cardiac regeneration. When combined with new advances in nuclear reprogramming, the prospect of achieving autologous, cardiomyogenic, stem-cell-based therapy might be within reach.

Multipotent progenitor cells in regenerative cardiovascular medicine.

Ectopic expression of defined sets of genetic factors can reprogram somatic cells to create induced pluripotent stem (iPS) cells. The capacity to direct human iPS cells to specific differentiated lineages and to their progenitor populations can be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. During mouse cardiogenesis, the major lineages of the mature heart, cardiomyocytes, smooth muscle cells, and endothelial cells arise from a common, multipotent cardiovascular progenitor expressing the transcription factors Isl1 and Nkx2.5. Here we show, using genetic fate-mapping, that Isl1(+) multipotent cardiovascular progenitors can be generated from mouse iPS cells and spontaneously differentiate in all 3 cardiovascular lineages in vivo without teratoma. Moreover, we report the identification of human iPS-derived ISL1(+) progenitors with similar developmental potential. These results support the possibility to use patient-specific iPS-generated cardiovascular progenitors as a model to elucidate the pathogenesis of congenital and acquired forms of heart diseases.-Moretti, A., Bellin, M., Jung, C. B., Thies, T.-M., Takashima, Y., Bernshausen, A., Schiemann, M., Fischer, S., Moosmang, S., Smith, A. G., Lam, J. T., Laugwitz, K.-L. Mouse and human induced pluripotent stem cells as a source for multipotent Isl1(+) cardiovascular progenitors.

Mouse and human induced pluripotent stem cells as a source for multipotent Isl1+ cardiovascular progenitors.

Long-QT syndromes are heritable diseases associated with prolongation of the QT interval on an electrocardiogram and a high risk of sudden cardiac death due to ventricular tachyarrhythmia. In long-QT syndrome type 1, mutations occur in the KCNQ1 gene, which encodes the repolarizing potassium channel mediating the delayed rectifier I(Ks) current.

Patient-specific induced pluripotent stem-cell models for long-QT syndrome.

Long QT syndrome (LQTS) is a genetic disease characterized by a prolonged QT interval in an electrocardiogram (ECG), leading to higher risk of sudden cardiac death. Among the 12 identified genes causal to heritable LQTS, ∼90% of affected individuals harbor mutations in either KCNQ1 or human ether-a-go-go related genes (hERG), which encode two repolarizing potassium currents known as I(Ks) and I(Kr). The ability to quantitatively assess contributions of different current components is therefore important for investigating disease phenotypes and testing effectiveness of pharmacological modulation. Here we report a quantitative analysis by simulating cardiac action potentials of cultured human cardiomyocytes to match the experimental waveforms of both healthy control and LQT syndrome type 1 (LQT1) action potentials. The quantitative evaluation suggests that elevation of I(Kr) by reducing voltage sensitivity of inactivation, not via slowing of deactivation, could more effectively restore normal QT duration if I(Ks) is reduced. Using a unique specific chemical activator for I(Kr) that has a primary effect of causing a right shift of V(1/2) for inactivation, we then examined the duration changes of autonomous action potentials from differentiated human cardiomyocytes. Indeed, this activator causes dose-dependent shortening of the action potential durations and is able to normalize action potentials of cells of patients with LQT1. In contrast, an I(Kr) chemical activator of primary effects in slowing channel deactivation was not effective in modulating action potential durations. Our studies provide both the theoretical basis and experimental support for compensatory normalization of action potential duration by a pharmacological agent.

Modulation of hERG potassium channel gating normalizes action potential duration prolonged by dysfunctional KCNQ1 potassium channel.

Induced pluripotent stem cell (iPSC) technology is creating exciting new opportunities for cardiovascular research by providing platforms to study the mechanisms of disease pathogenesis that could lead to new therapies or reveal drug sensitivities. In this review, the potential usefulness of iPSC-derived cardiomyocytes in drug development as well as in drug toxicity testing is discussed, with a focus on the achievements that have been already made in this regard. Moreover, the crucial steps that have to be taken before this technology can be broadly used in drug discovery and toxicology assessments are highlighted. 

Induced pluripotent stem cell-derived cardiomyocytes for drug development and toxicity testing.

Extending human induced pluripotent stem cell technology to infectious diseases: new model for viral myocarditis.

Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided. 

Genetically Encoded Voltage Indicators in Circulation Research.

Long-QT syndrome (LQTS) is an arrhythmogenic disorder characterised by prolongation of the QT interval in the electrocardiogram, which can lead to sudden cardiac death. Pharmacological treatments are far from optimal for congenital forms of LQTS, while the acquired form, often triggered by drugs that (sometimes inadvertently) target the cardiac hERG channel, is still a challenge in drug development because of cardiotoxicity. Current experimental models in vitro fall short in predicting proarrhythmic properties of new drugs in humans. Here, we leveraged a series of isogenically matched, diseased and genetically engineered, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients to test a novel hERG allosteric modulator for treating congenital LQTS, drug-induced LQTS or a combination of the two. By slowing IK r deactivation and positively shifting IK r inactivation, the small molecule LUF7346 effectively rescued all of these conditions, demonstrating in a human system that allosteric modulation of hERG may be useful as an approach to treat inherited and drug-induced LQTS Furthermore, our study provides experimental support of the value of isogenic pairs of patient hiPSC-CMs as platforms for testing drug sensitivities and performing safety pharmacology.

A new hERG allosteric modulator rescues genetic and drug-induced long-QT syndrome phenotypes in cardiomyocytes from isogenic pairs of patient induced pluripotent stem cells.

Diabetes mellitus causes microcirculatory rarefaction and may impair the responsiveness of ischemic myocardium to proangiogenic factors.

Diabetes Mellitus-Induced Microvascular Destabilization in the Myocardium.

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by  fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages.

Article	Year
Multipotent progenitor cells in regenerative cardiovascular medicine. Pediatric cardiology\| PubMed ID: 19415155	2009
Mouse and human induced pluripotent stem cells as a source for multipotent Isl1+ cardiovascular progenitors. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 19850773	2010
Patient-specific induced pluripotent stem-cell models for long-QT syndrome. The New England journal of medicine\| PubMed ID: 20660394	2010
Modulation of hERG potassium channel gating normalizes action potential duration prolonged by dysfunctional KCNQ1 potassium channel. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22745159	2012
Induced pluripotent stem cell-derived cardiomyocytes for drug development and toxicity testing. Pharmacology & therapeutics\| PubMed ID: 24657289	2014
Extending human induced pluripotent stem cell technology to infectious diseases: new model for viral myocarditis. Circulation research\| PubMed ID: 25170088	2014
Genetically Encoded Voltage Indicators in Circulation Research. International journal of molecular sciences\| PubMed ID: 26370981	2015
A new hERG allosteric modulator rescues genetic and drug-induced long-QT syndrome phenotypes in cardiomyocytes from isogenic pairs of patient induced pluripotent stem cells. EMBO molecular medicine\| PubMed ID: 27470144	2016
Diabetes Mellitus-Induced Microvascular Destabilization in the Myocardium. Journal of the American College of Cardiology\| PubMed ID: 28081822	2017
Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity. The EMBO journal\| PubMed ID: 29764980	2018

Alessandra Moretti

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