Search

Background: Electrical remodeling in human persistent atrial fibrillation is believed to result from rapid electrical activation of the atria, but underlying genetic causes may contribute. Indeed, common gene variants in an enhancer region close to PITX2 (paired-like homeodomain transcription factor 2) are strongly associated with atrial fibrillation, but the mechanism behind this association remains unknown. This study evaluated the consequences of PITX2 deletion (PITX2−/−) in human induced pluripotent stem cell–derived atrial cardiomyocytes. Methods: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) was used to delete PITX2 in a healthy human iPSC line that served as isogenic control. Human induced pluripotent stem cell–derived atrial cardiomyocytes were differentiated with unfiltered retinoic acid and cultured in atrial engineered heart tissue. Force and action potential were measured in atrial engineered heart tissues. Single human induced pluripotent stem cell–derived atrial cardiomyocytes were isolated from atrial engineered heart tissue for ion current measurements. Results: PITX2−/− atrial engineered heart tissue beats slightly slower than isogenic control without irregularity. Force was lower in PITX2−/− than in isogenic control (0.053±0.015 versus 0.131±0.017 mN, n=28/3 versus n=28/4, PITX2−/− versus isogenic control; P <0.0001), accompanied by lower expression of CACNA1C and lower L-type Ca2+ current density. Early repolarization was weaker (action potential duration at 20% repolarization; 45.5±13.2 versus 8.6±5.3 ms, n=18/3 versus n=12/4, PITX2−/− versus isogenic control; P <0.0001), and maximum diastolic potential was more negative (−78.3±3.1 versus −69.7±0.6 mV, n=18/3 versus n=12/4, PITX2−/− versus isogenic control; P =0.001), despite normal inward rectifier currents (both IK1 and IK,ACh) and carbachol-induced shortening of action potential duration. Conclusions: Complete PITX2 deficiency in human induced pluripotent stem cell–derived atrial cardiomyocytes recapitulates some findings of electrical remodeling of atrial fibrillation in the absence of fast beating, indicating that these abnormalities could be primary consequences of lower PITX2 levels. [ABSTRACT FROM AUTHOR]

The article deals with the identification of myomasp (Myosin-interacting, M-band-associated stress-responsive protein) as a component of an M-band-associated signaling pathway using a bioinformatic approach. It relates the expression of myomasp in heart and skeletal muscle. It describes the interaction of myomasp with myosin heavy chains. It discusses the role of myomasp in cardiomyocyte function.

The article presents information on a study which described a new miniaturized and automated method based on engineered heart tissue (EHT). The methods employed to produce Sylgard posts and Teflon spacers and to generate fibrin-based mini-EHT are described. It also mentions that video optical analysis was used to monitor the contractile activity. Findings of the study are presented in details. It concludes that the method may be useful for drug screening and drug modeling.

Background: Cardiac repolarization abnormalities in drug-induced and genetic long-QT syndrome may lead to afterdepolarizations and life-threatening ventricular arrhythmias. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) should help to overcome the limitations of animal models based on species differences in repolarization reserve. Here, we compared head-to-head the contribution of IKs (long QT1) and IKr (long QT2) on action potentials (APs) in human left ventricular (LV) tissue and hiPSC-CM-derived engineered heart tissue (EHT).Methods: APs were measured with sharp microelectrodes in EHT from 3 different control hiPSC-CM lines and in tissue preparations from failing LV.Results: EHT from hiPSC-CMs showed spontaneous diastolic depolarization and AP generation that were sensitive to low concentrations of ivabradine. IKr block by E-4031 prolonged AP duration at 90% repolarization with similar half-effective concentration in EHT and LV but larger effect size in EHT (+281 versus +110 ms in LV). Although IKr block alone evoked early afterdepolarizations and triggered activity in 50% of EHTs, slow pacing, reduced extracellular K+, and blocking of IKr, IKs, and IK1 were necessary to induce early afterdepolarizations in LV. In accordance with their clinical safety, moxifloxacin and verapamil did not induce early afterdepolarizations in EHT. In both EHT and LV, IKs block by HMR-1556 prolonged AP duration at 90% repolarization slightly in the combined presence of E-4031 and isoprenaline.Conclusions: EHT from hiPSC-CMs shows a lower repolarization reserve than human LV working myocardium and could thereby serve as a sensitive and specific human-based model for repolarization studies and arrhythmia, similar to Purkinje fibers. In both human LV and EHT, IKs only contributed to repolarization under adrenergic stimulation. [ABSTRACT FROM AUTHOR]

Background: Human engineered heart tissue (EHT) transplantation represents a potential regenerative strategy for patients with heart failure and has been successful in preclinical models. Clinical application requires upscaling, adaptation to good manufacturing practices, and determination of the effective dose., Methods: Cardiomyocytes were differentiated from 3 different human induced pluripotent stem cell lines including one reprogrammed under good manufacturing practice conditions. Protocols for human induced pluripotent stem cell expansion, cardiomyocyte differentiation, and EHT generation were adapted to substances available in good manufacturing practice quality. EHT geometry was modified to generate patches suitable for transplantation in a small-animal model and perspectively humans. Repair efficacy was evaluated at 3 doses in a cryo-injury guinea pig model. Human-scale patches were epicardially transplanted onto healthy hearts in pigs to assess technical feasibility., Results: We created mesh-structured tissue patches for transplantation in guinea pigs (1.5×2.5 cm, 9-15×10 6 cardiomyocytes) and pigs (5×7 cm, 450×10 6 cardiomyocytes). EHT patches coherently beat in culture and developed high force (mean 4.6 mN). Cardiomyocytes matured, aligned along the force lines, and demonstrated advanced sarcomeric structure and action potential characteristics closely resembling human ventricular tissue. EHT patches containing ≈4.5, 8.5, 12×10 6 , or no cells were transplanted 7 days after cryo-injury (n=18-19 per group). EHT transplantation resulted in a dose-dependent remuscularization (graft size: 0%-12% of the scar). Only high-dose patches improved left ventricular function (+8% absolute, +24% relative increase). The grafts showed time-dependent cardiomyocyte proliferation. Although standard EHT patches did not withstand transplantation in pigs, the human-scale patch enabled successful patch transplantation., Conclusions: EHT patch transplantation resulted in a partial remuscularization of the injured heart and improved left ventricular function in a dose-dependent manner in a guinea pig injury model. Human-scale patches were successfully transplanted in pigs in a proof-of-principle study.

Abstract: Atrial tachypacing is an accepted model for atrial fibrillation (AF) in large animals and in cellular models. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CM) provide a novel human source to model cardiovascular diseases. Here, we investigated whether optogenetic tachypacing of atrial-like hiPSC-CMs grown into engineered heart tissue (aEHT) can induce AF-remodeling. After differentiation of atrial-like cardiomyocytes from hiPSCs using retinoic acid, aEHTs were generated from ∼1 million atrial-like hiPSC-CMs per aEHT. AEHTs were transduced with lentivirus expressing channelrhodopsin-2 to enable optogenetic stimulation by blue light pulses. AEHTs underwent optical tachypacing at 5 Hz for 15 seconds twice a minute over 3 weeks and compared with transduced spontaneously beating isogenic aEHTs (1.95 ± 0.07 Hz). Force and action potential duration did not differ between spontaneously beating and tachypaced aEHTs. Action potentials in tachypaced aEHTs showed higher upstroke velocity (138 ± 15 vs. 87 ± 11 V/s, n = 15-13/3; P = 0.018), possibly corresponding to a tendency for more negative diastolic potentials (73.0 ± 1.8 vs. 68.0 ± 1.9 mV; P = 0.07). Tachypaced aEHTs exhibited a more irregular spontaneous beating pattern (beat-to-beat scatter: 0.07 ± 0.01 vs. 0.03 ± 0.004 seconds, n = 15-13/3; P = 0.008). Targeted expression analysis showed higher RNA levels of KCNJ12 [Kir2.2, inward rectifier (IK1); 69 ± 7 vs. 44 ± 4, P = 0.014] and NPPB (NT-proBNP; 39,690 ± 4834 vs. 23,671 ± 3691; P = 0.024). Intermittent tachypacing in aEHTs induces some electrical alterations found in AF and induces an arrhythmic spontaneous beating pattern, but does not affect resting force. Further studies using longer, continuous, or more aggressive stimulation may clarify the contribution of different rate patterns on the changes in aEHT mimicking the remodeling process from paroxysmal to persistent atrial fibrillation., Competing Interests: T. Eschenhagen and A. Hansen are co-founders of EHT Technologies, Hamburg. The other authors report no conflicts of interest., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell-derived cardiomyocytes., Methods: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell-derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward., Results: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding., Conclusion: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.

The engineering of 3-dimensional (3D) heart muscles has undergone exciting progress for the past decade. Profound advances in human stem cell biology and technology, tissue engineering and material sciences, as well as prevascularization and in vitro assay technologies make the first clinical application of engineered cardiac tissues a realistic option and predict that cardiac tissue engineering techniques will find widespread use in the preclinical research and drug development in the near future. Tasks that need to be solved for this purpose include standardization of human myocyte production protocols, establishment of simple methods for the in vitro vascularization of 3D constructs and better maturation of myocytes, and, finally, thorough definition of the predictive value of these methods for preclinical safety pharmacology. The present article gives an overview of the present state of the art, bottlenecks, and perspectives of cardiac tissue engineering for cardiac repair and in vitro testing.