Development of a Novel Fluorescence Indicator System for the Characterization of Sarcomere Organization an Function in Human Cardiomyocytes
von Til Driehorst
Datum der mündl. Prüfung:2022-03-15
Betreuer:Prof. Dr. Wolfram-Hubertus Zimmermann
Gutachter:Prof. Dr. Stephan E. Lehnart
Gutachter:Prof. Dr. Margarete Schön
EnglischIn this work, we describe a novel fluorescence indicator system for the in-vitro characterization of human stem cell-derived cardiomyocytes (hPSC-CM). We hypothesized that a) Z-lines in hPSC-CM can be reliably labeled via an endogenous expression of an ACTN2-Citrine-fusion protein, b) endogenous labeling of Z-lines allows for longitudinal studies on sarcomere contractility and sarcomere development, c) fluorescently labeled CM can be plated for high-throughput analysis under defined experimental conditions via micropatterning on substrates of defined elasticity, and d) the indicator system can be applied in drug testing. Employing the CRISPR/Cas9 system, we generated two human embryonic stem cell lines with a heterozygous knock-in of Citrine into the sarcomeric actinin gene locus. We were then able to derive an, in principal, unlimited number of functional cardiomyocytes with a stable and bright fluorescent label of Z-lines from both cell lines. We turned to micropatterning and shaped the fluorescing myocytes to defined single-cell geometries on both, very stiff glass substrates, and 15 kPa elastomers. In so-called contractility assays, we then video-recorded the beating micropatterned CM at high spatial and temporal resolution using our high-speed resonant-scanning confocal microscope. Finally, we employed a custom-developed Python®-based video analysis algorithm, which enabled us to derive relevant parameters of sarcomere contractility including inter-sarcomeric coherence of contractions. The combination of the above techniques provided unprecedented insight into sarcomere dynamics at the subcellular level. We observed a severe impairment of contractile function on very stiff glass substrates, which resulted in a 50% decrease in sarcomere shortening amplitude, as compared to 15 kPa. Further we found a strikingly lower level of inter-sarcomeric coherence of contractions on these stiff substrates, which we attributed to the boundary conditions imposed by the microenvironment of the cells. Next, we demonstrated proof of concept that the indicator system can contribute to drug screening assays by evaluating the effects of the cardiac myosin activator omecamtiv mecarbil on CM contractility. Last, we showed proof-of-principle applicability of the indicator system in the studying of myofibrillogenesis.
Keywords: Cardiomyocytes; CRISPR; Stem cell-derived cardiomyocytes; endogenous fluorescence labeling; Confocal microscopy