Striatin-gene loss of function induces functional alterations in a haploid stem cell mouse model for cardiomyogenic differentiation

Background Striatin (Strn) is a ubiquitous Ca2+-dependent scaffolding protein that in cardiomyocytes (CMs) co-localizes at the intercalated disc region with desmosomal proteins. Recent studies demonstrated that a deletion in the 3’ untranslated region of the STRN gene was associated with arrhythmogenic right ventricular cardiomyopathy and dilated cardiomyopathy in dogs. Moreover, the Striatin gene was also recently associated with cardiac abnormalities in Drosophila melanogaster. Mouse embryonic stem cells (mESCs) with a haploid chromosomal set have recently been suggested as a good tool for genetic screens. They retain the capacity to differentiate into the three germ layers in vitro and, possessing just one copy of each gene, they facilitate the generation of loss-of-function mutants that represent a powerful tool to investigate the role of new target genes. Preliminary evidence obtained in our laboratory indicates that Strn knock out could impact the electrophysiological properties of haploid mESC-derived cardiomyocytes (CMs). Objectives This thesis tries to elucidate how the Strn cellular depletion affects the functional characteristics of haploid mESC-derived CMs. To this aim, a Strn-mutant line of haploid mESC line (namely 377C05) generated by gene trapping by J. Penninger and co-workers was used. This mutant and the corresponding wild-type (WT) line were used as cell models for this project. Methods The differentiation protocol based on the hanging drop technique was modified to optimize the appearance of beating embryoid bodies (EBs) from haploid mESCs. The cardiomyogenic differentiation process was evaluated at the functional, morphological and molecular level in the Strn-mutants and compared to WTs. Specifically, the contractility properties of the beating areas were assessed by video tracking analysis, spontaneous action potentials (APs) were recorded by current clamp, and intracellular calcium concentration was simultaneously monitored in Fluo-4 loaded EBs. Structural changes of haploid mESC-derived CMs immunostained for Troponin-T were evaluated by confocal microscope, while changes in the expression of pluripotency genes, cardiac differentiation markers, Strn interacting proteins and ion channels were estimated by quantitative Real time-PCR. All the data were analyzed using non-parametric statistical tests. Results At day 12 of differentiation, we found a significant decrease in the number and the dimension of beating areas in Strn-mutant as compared with WT, together with a significant lower frequency of contraction and a dysregulated (arrhythmic) contraction behavior. However, when the frequency of APs was evaluated, it appeared higher in Strn-mutant. Furthermore, the AP duration was decreased, as well as the duration of the diastolic depolarization phase, possibly suggesting an alteration in the EC-coupling process. Thus, the calcium-induced calcium release process was investigated by simultaneous recordings of spontaneous APs and their induced calcium transients. Intriguingly, no differences were observed: in fact, every AP was aligned with a calcium spike. However, when we evaluated the delay between each AP peak and its correspondent calcium peak, we observed a trend of increased delay in the mutants versus WTs. In addition, the amount of Ca2+ released after spontaneous APs in the mutant was about twice lower than in the WTs. Further, the sarcomere structure analysis showed that 49% of CMs, obtained from Strn-mutant, presented sarcomeres with an absence of the striated configuration for Troponin-T. When gene expression was investigated, we found a significant upregulation of β-catenin and HCN4 expression in Strn-mutants. Conclusions Here we provide evidence that Strn-mutants are characterized by: (i) alteration in APs frequency and action potential duration, (ii) reduced contraction frequency and beating area dimension, (iii) delayed Ca2+ release (iv) reduced amplitude of Ca2+ transients and (v) structural alteration of the sarcomere. Overall, these results indicate that Strn has a strong impact on CMs function, possibly representing a new molecular target for the identification of new causes and therapies of cardiovascular disease.