Identification Of Cardiomyocytes Research Articles

Identification of Cardiomyocytes' Inner Workings Responsible for Dynamical Changes in Calcium Profile in Response to Mechanical Load

Background: We embedded cardiomyocytes in a cross-linked hydrogel to study the effect of increased afterload on in the heart. We observed that mechanically-loaded cardiomyocytes undergo changes in their Ca2+ dynamics that can compensate for the increase in load. We have shown that these changes are mediated by the upregulation of nitric oxide (NO) signalling, which in turns affects Ca2+ handling. Because many different Ca2+ pathways could be affected we used an agnostic approach based on mathematical modelling to tease out changes in cell characteristics responsible for the differences in calcium and contraction profiles between load-free and after-load environment. Mathematical Method: We coupled the Shannon-Bers ventricular action potential model to a viscoelastic model to simulate the myocyte contracting in either a load-free condition (Tyrode solution) or under load (in the gel matrix). The mathematical model establishes a closed feedback loop between the calcium system and the extracellular environment that gives rise to the self-regulation we observed. We ran extensive simulations where parameters associated with the influx and efflux of Ca2+ were modulated such that they are either up-regulated or down-regulated by NO. In silico results are filtered out to qualitatively match cell-in-gel in vitro results. The filtering process is based on measures that capture multiple properties of calcium profiles. Conclusion: Our approach of identification hints that the upregulation of NO has the effect of simultaneously modulating multiple parameters of the Ca2+ handling pathway. Of the modulated parameters, the L-type current amplitude has to be consistently increased. Coupled with the increase in L-type current, parameters associated with release and uptake of calcium by the SR have to be modulated in opposite directions.

Identification of cardiomyocytes' characteristics responsible for dynamical changes in calcium profile in response to mechano-chemo transduction

Background: We embedded cardiomyocytes in a cross-linked hydrogel to study the effect of increased afterload on in the heart. We observed that mechanically-loaded cardiomyocytes undergo noticeable changes in their Ca2+ dynamics that can compensate for the increase in load. We have shown that these changes are mediated by the upregulation of nitric oxide (NO) signaling, which in turns affects Ca2+ handling. Because many different Ca2+ pathways could be affected we used an agnostic approach based on mathematical modeling to tease out changes in cell characteristics responsible for the difference in calcium and contraction profiles between load-free and after-load environments. Mathematical Method: We use a mathematical model to simulate the extracellular environment, and use the simulation results coupled with the experimental observations to identify parameters that change between load-free and after-load conditions. The mathematical model details a closed feedback loop between the calcium system and the extracellular environment to give rise to the self-regulation we observed. We ran extensive simulations where parameters associated with the influx and efflux of calcium are modulated such that they are either up-regulated or down-regulated by NO. In silico results are filtered out to qualitatively match cell-in-gel in vivo results. The filtering process is based on measures that capture multiple properties of calcium profiles. Conclusion: Our agnostic approach of identification hints that upregulation of NO modulates multiple parameters of the Ca2+ handling pathways simultaneously. Of the modulated parameters, the L-type current amplitude has to be consistently increased. Coupled with the increase in L-type current, cell’s parameters associated with release of calcium from the SR and uptakes of calcium by the SR have to be modulated in opposite directions.

Abstract 6: Novel Tools for Generation, Purification, and Analysis of Pluripotent Stem Cell Derived Cardiomyocytes

Pure and well-characterized cardiac cells derived from human pluripotent stem cells (hPSCs) are of high interest for cardiovascular disease modeling, drug safety studies and development of cell replacement strategies. Although several protocols for cardiac differentiation of hPSCs have been developed, major limitations are clone-to-clone variations in differentiation efficacy as well as heterogeneity of generated cardiomyocyte populations. Therefore, we have developed novel tools for cardiomyocyte differentiation, magnetic cell sorting-based purification and cell analysis using new antibody-conjugates enabling for flow cytometry- and immunofluorescence-based identification of cardiomyocytes and subtypes. hPSCs were maintained under xeno-free conditions in our recently developed StemMACS iPS-Brew XF medium to keep pluripotency for more than >20 passages and enable for efficient cardiac differentiation. We chose a monolayer differentiation protocol based on the timely regulated activation and inhibition of Wnt signaling by small molecules. In order to identify antibodies suitable for cardiomyocyte enrichment or depletion of non-myocytes, we performed a surface marker screen with more than 400 antibodies between days 10-20 of differentiation. Besides identification of new surface markers, our screen confirmed expression of recently published markers like CD172a and CD106. In order to evaluate kinetics of their expression and correlation with intracellular markers of cardiomyocytes and subtypes, we performed a flow cytometry-based analysis of marker expression. Our data indicate a dynamic expression pattern for both CD172a and CD106, either completely or partially overlapping with intracellular cardiomyocyte marker expression. Based on these data we developed magnetic cell separation procedures for the isolation of cardiomyocytes. Magnetically enriched cardiomyocytes initiated contractions after replating and could be stably maintained in culture. Taken together, we have developed novel tools supporting the workflow for efficient generation of PSC-derived cardiomyocytes, magnetic purification and flow cytometry or immunofluorecence-based characterization of cardiomyocytes.

Abstract P036: Regional Characterization of Myocardial Renewal in Humans

We and other groups have reported cardiomyocyte renewal in adult human hearts. However, there is no consensus about magnitude and regional heterogeneity in myocardial turnover. Important requirements to quantify cellular turnover are the accurate identification of cardiomyocytes and ploidy level. We present a regional analysis of turnover in the adult myocardium, using the integration of atmospheric radiocarbon (14C) into genomic myocardial DNA. Cardiomyocyte nuclei were labeled with pericentriolar protein 1 (PCM-1) and isolated by flow cytometry. In order to improve estimates on cardiomyocyte turnover, nuclei were isolated according to their DNA content. A substantial fraction of cardiomyocyte nuclei in the right and left ventricle were polyploid: 28.9%±10.6% diploid, 58.9%±8.5% tetraploid and 12.1%±7.4% octaploid in the left ventricle and 47.9%±10.6% diploid, 46.6%±8.3% tetraploid and 5.6%±3.2% octaploid in the right ventricle. Cardiomyocyte turnover was analyzed according to their ploidy distribution in the left and right ventricle. Data from this ongoing study indicates a limited regenerative potential in both ventricles. A comprehensive mathematical analysis of regional turnover distribution will be presented. Non-cardiomyocytes were in average 18.0±2.3 years younger than the respective subjects, indicating a substantial turnover in the whole myocardium. The human heart has the capability to regenerate cardiomyocytes. However, the magnitude of this process does not allow for the renewal of the whole myocardium under homeostatic condition in a lifetime. Understanding the underlying mechanisms of cardiac renewal will open up new avenues to treat cardiac injuries.