Heart failure is still a major cause of death and one of the most common reasons for hospital admission in the western world. It is known that calcium hemostasis and regular L-type Ca²⁺channel (LTCC) function plays a key role in patients with dilated cardiomyopathy (DCM) and heart failure.

In previous experiments we have shown that Myoscape - a member of the mammalian Striatin-interacting phosphatase and kinase (STRIPAK) complex – directly interacts with the LTCC to stabilize LTCC surface expression. Loss of Myoscape leads to endocytotic internalization of LTCC and subsequent heart failure in vivo. Interestingly, the QT-time is also shortened in Myoscape knockout mice. Thus, we searched for Myoscape gene sequence variations found in both patients with DCM and patients with Short-QT syndrome (SQTS).

One promising mutation that was found in both patient cohorts was a stop mutation (R217ter), which is found in only one of about 84,000 healthy individuals in the gnomAD database and leads to expression of a truncated Myoscape protein.

In preliminary experiments, this stop mutation, which was generated by site strand mutagenesis and preceded with a heart-specific promoter, was injected into zebrafish embryos together with the enzyme transposase and thereby successfully integrated into the genome. These transgenic fish were compared with fish injected with the wildtype (WT) Myoscape and with non-injected control fish.

48 hours after fertilization, heart rate was significantly decreased in Myoscape R217ter fish and Myoscape wildtype fish compared with non-injected fish (149/min vs 171/min, two independent injections with n=16 (not-injected) and n=20 (Myoscape R217ter), p=0.015). Most strikingly, even though heart rate was reduced, cardiac output was significantly increased in fish overexpressing wildtype Myoscape (n=21) compared with non-injected fish (66.2 nL/min vs. 53.5 nL/min, p=0.035) whereas cardiac output in Myoscape R217ter fish (49.9 nL/min) showed no alteration when compared to control fish (49.9 nL/min vs. 53.5nL/min, p=0.771), indicating that could be a “loss of function” mutation.

72 hours after fertilization, the hearts of the fish were isolated and the cell borders of the cardiomyocytes were labeled with a β-catenin antibody in order to measure cell sizes in the ventricle. The cell sizes did not vary significantly in the three groups (not injected = 96.83 µm² (n=152 cells) vs. Myoscape WT = 107.5 µm² (n=183 cells) vs. Myoscape R217ter = 109.6µm² (n=148 cells)).

Beyond that, in in vitro experiments with neonatal rat cardiomyocytes (NRVCMs) we could show that after PE stress (100µM for 48h) only after adenoviral overexpression of the wildtype Myoscape, the mRNA expression of the hypertrophic gene Nppa is significantly reduced in comparison with LacZ (4.98-fold vs. 10.28-fold, n=2, p=0.028). However, Myoscape R217ter overexpression was not able to reduce Nppa mRNA Expression significantly after PE stress (7-fold vs. 10.28-fold, n=2, p=0.254).

Along with these results, we could show that wildtype Myoscape overexpression significantly reduced NFAT Luciferase activity in NRVCM after PE treatment, whereas Myoscape R217ter could not show this effect.

Further understanding of the function of wildtype and mutated Myoscape could contribute to a better understanding of the pathophysiology of DCM and cardiac hypertrophy, possibly leading to new therapeutic targets in the future.