|dc.description.abstracteng||Heart failure is one of the most common cause of death in Germany. The current treatment options are known to decelerate disease progression, but it cannot be halted or reversed. To date, the underlying molecular mechanisms of cardiac remodeling are not completely understood but interesting candidate genes have been identified in a preceding study. The mRNA sequencing screen of aortic stenosis patients with preserved and reduced ejection fraction were compared to a corresponding mouse model of transverse aortic constriction identifying the differentially regulated candidate, secreted-frizzled-related protein 5 (SFRP5), which is almost not expressed in compensated hypertrophy and is re-expressed in patients with heart failure. Until today, little is known about the function of SFRP5 in human cardiomyocytes.
To examine the role of SFRP5 in human cardiomyocytes, an AAV-mediated SFRP5 overexpression model and a CRISPR/Cas9-mediated gene knock out was established in two month old hiPSC derived cardiomyocytes (hiPSC-CMs). To gain a first understanding of SFRP5’s role in cardiomyocytes, a mRNA sequencing screen was performed identifying the differentially regulated apoptosis and calcium handling pathways. The expression of pro- and anti-apoptotic genes was regulated in SFRP5 overexpressing hiPSC-CMs, especially intrinsic and extrinsic apoptosis induction as well as increased expression of anti-apoptotic proteins. Furthermore, reduced calcium channel and sarcomere protein expression together with differential microdomain sodium and calcium homeostasis regulation indicates reduced calcium circulation due to increased calcium sequestration. These results were further validated in a functional analysis of SFRP5 overexpressing hiPSC-CMs using confocal calcium imaging. Calcium transients were significantly altered due to SFRP5 overexpression indicating reduced excitation-contraction coupling and smaller sarcoplasmic calcium stores, thereby confirming transcriptional alterations of calcium handling proteins. The calcium handling regulation by SFRP5 overexpression may result in protecting the cell against calcium overload and/or reduce the contractile force. The functional outcome of SFRP5s anti-hypertrophic effects in calcium handling and WNT pathway regulation can only be hypothesized and has to be examined in further experiments.
The CRISPR/Cas9-mediated SFRP5 knock out hiPSC line was efficiently differentiated into SFRP5 knock out hiPSC-CMs. Under basal conditions the gene knock out did not reveal alterations in sarcomere structure, contractility, or expression changes of predicted SFRP5 target genes identified in the mRNA sequencing study, probably due to only moderate expression levels of SFRP5 in hiPSC-CMs.
In the mRNA sequencing screen and following confocal calcium imaging SFRP5 was identified as novel calcium handling modulator in hiPSC-CM, decreasing the expression of calcium channels and myofilaments, as well as decreasing calcium transient amplitudes during overexpression. Under basal conditions a gene knock out of SFRP5 in cardiomyocytes does not display transcriptional calcium handling changes.||de