Sensing Cardiac Electrical Activity With a Cardiac Myocyte--Targeted Optogenetic Voltage Indicator.

RATIONALE: Monitoring and controlling cardiac myocyte activity with optogenetic tools offer exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level. OBJECTIVE: To test the hypothesis that cardiac myocyte-targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electric activity in isolated cardiac myocytes and the whole heart as well as function and maturity in induced pluripotent stem cell-derived cardiac myocytes. METHODS AND RESULTS: We first generated mice with cardiac myocyte-restricted expression of VSFP2.3 and demonstrated distinct localization of VSFP2.3 at the t-tubulus/junctional sarcoplasmic reticulum microdomain without any signs for associated pathologies (assessed by echocardiography, RNA-sequencing, and patch clamping). Optically recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch clamping. The use of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiac myocytes. Optical cardiograms could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in induced pluripotent stem cell-derived cardiac myocytes. CONCLUSIONS: We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof of concept for its use in optogenetic sensing of physiological and pathological excitation in mature and immature cardiac myocytes in vitro and in vivo.

Preservation of left ventricular function and morphology in volume-loaded versus volume-unloaded heterotopic heart transplants.

Total mechanical unloading of the heart in classical models of heterotopic heart transplantation leads to cardiac atrophy and functional deterioration. In contrast, partial unloading of failing human hearts with left ventricular (LV) assist devices (LVADs) can in some patients ameliorate heart failure symptoms. Here we tested in heterotopic rat heart transplant models whether partial volume-loading (VL; anastomoses: aorta of donor to aorta of recipient, pulmonary artery of donor to left atrium of donor, superior vena cava of donor to inferior vena cava of recipient; n = 27) is superior to the classical model of myocardial unloading (UL; anastomoses: aorta of donor to aorta of recipient, pulmonary artery of donor to inferior vena cava of recipient; n = 14) with respect to preservation of ventricular morphology and function. Echocardiography, magnetic resonance imaging, and LV-pressure-volume catheter revealed attenuated myocardial atrophy with ~30% higher LV weight and better systolic contractile function in VL compared with UL (fractional area shortening, 34% vs. 18%; maximal change in pressure over time, 2,986 ± 252 vs. 2,032 ± 193 mmHg/s). Interestingly, no differences in fibrosis (Picrosirus red staining) or glucose metabolism (2-[18F]-fluoro-2-deoxy-D-glucose-PET) between VL and UL were observed. We conclude that the rat model of partial VL attenuates atrophic remodelling and shows superior morphological as well as functional preservation, and thus should be considered more widely as a research model.

Terminal differentiation, advanced organotypic maturation, and modeling of hypertrophic growth in engineered heart tissue.

RATIONALE: Cardiac tissue engineering should provide "realistic" in vitro heart muscle models and surrogate tissue for myocardial repair. For either application, engineered myocardium should display features of native myocardium, including terminal differentiation, organotypic maturation, and hypertrophic growth. OBJECTIVE: To test the hypothesis that 3D-engineered heart tissue (EHT) culture supports (1) terminal differentiation as well as (2) organotypic assembly and maturation of immature cardiomyocytes, and (3) constitutes a methodological platform to investigate mechanisms underlying hypertrophic growth. METHODS AND RESULTS: We generated EHTs from neonatal rat cardiomyocytes and compared morphological and molecular properties of EHT and native myocardium from fetal, neonatal, and adult rats. We made the following key observations: cardiomyocytes in EHT (1) gained a high level of binucleation in the absence of notable cytokinesis, (2) regained a rod-shape and anisotropic sarcomere organization, (3) demonstrated a fetal-to-adult gene expression pattern, and (4) responded to distinct hypertrophic stimuli with concentric or eccentric hypertrophy and reexpression of fetal genes. The process of terminal differentiation and maturation (culture days 7-12) was preceded by a tissue consolidation phase (culture days 0-7) with substantial cardiomyocyte apoptosis and dynamic extracellular matrix restructuring. CONCLUSIONS: This study documents the propensity of immature cardiomyocytes to terminally differentiate and mature in EHT in a remarkably organotypic manner. It moreover provides the rationale for the utility of the EHT technology as a methodological bridge between 2D cell culture and animal models.

Heart muscle engineering: an update on cardiac muscle replacement therapy.

Cardiac muscle engineering aims at providing functional myocardium to repair diseased hearts and model cardiac development, physiology, and disease in vitro. Several enabling technologies have been established over the past 10 years to create functional myocardium. Although none of the presently employed technologies yields a perfect match of natural heart muscle, it can be anticipated that human heart muscle equivalents will become available after fine tuning of currently established tissue engineering concepts. This review provides an update on the state of cardiac muscle engineering and its utilization in cardiac regeneration. We discuss the application of stem cells including the allocation of autologous cell material, transgenic technologies that may improve tissue structure as well as in vivo engraftment, and vascularization concepts. We also touch on legal and economic aspects that have to be considered before engineered myocardium may eventually be applied in patients and discuss who may be a potential recipient.

Parthenogenetic stem cells for tissue-engineered heart repair.

Uniparental parthenotes are considered an unwanted byproduct of in vitro fertilization. In utero parthenote development is severely compromised by defective organogenesis and in particular by defective cardiogenesis. Although developmentally compromised, apparently pluripotent stem cells can be derived from parthenogenetic blastocysts. Here we hypothesized that nonembryonic parthenogenetic stem cells (PSCs) can be directed toward the cardiac lineage and applied to tissue-engineered heart repair. We first confirmed similar fundamental properties in murine PSCs and embryonic stem cells (ESCs), despite notable differences in genetic (allelic variability) and epigenetic (differential imprinting) characteristics. Haploidentity of major histocompatibility complexes (MHCs) in PSCs is particularly attractive for allogeneic cell-based therapies. Accordingly, we confirmed acceptance of PSCs in MHC-matched allotransplantation. Cardiomyocyte derivation from PSCs and ESCs was equally effective. The use of cardiomyocyte-restricted GFP enabled cell sorting and documentation of advanced structural and functional maturation in vitro and in vivo. This included seamless electrical integration of PSC-derived cardiomyocytes into recipient myocardium. Finally, we enriched cardiomyocytes to facilitate engineering of force-generating myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial infarction. Collectively, our data demonstrate pluripotency, with unrestricted cardiogenicity in PSCs, and introduce this unique cell type as an attractive source for tissue-engineered heart repair.