Elucidating the role of SHOX2 in atrial fibrillation using a patient-derived human induced pluripotent stem cell model

Abstract

The sinoatrial node (SAN) is the primary pacemaker of the heart, where the pulse necessary for cardiac contraction arises to be propagated to the cardiac chambers via the cardiac conduction system. The transcription factor short stature homeobox 2 ( SHOX2 ) is essential for the development and function of the SAN, activating a transcriptional program for pacemaker development while suppressing differentiation towards working myocardium during cardiogenesis. Variants in the SHOX2 gene have been associated with atrial fibrillation (AF), the most common cardiac arrhythmia. However, the pathomechanisms behind SHOX2 dependent AF and the SHOX2 regulatory genetic network have not been fully understood. Until now, the impact of Shox2 deficiency has only been investigated in mouse or zebrafish animal models. Therefore, the aim of this thesis was to gain new insights from a human background and to investigate whether data obtained via animal models can be transferred to a human model system. At the beginning of this work, two induced pluripotent stem cell (iPSC) lines from two different patients with early onset AF were available, carrying a heterozygous single nucleotide variant either in the 3’UTR or in the coding region of SHOX2. A targeted correction of these variants had already been performed to generate isogenic control lines. First, I generated two homozygous SHOX2 KO iPSC lines from the available cell lines and validated them using several methods. Furthermore, I established and adapted previously published differentiation protocols to generate pacemaker and atrial cardiomyocytes (CMs) from iPSCs. To uncover variant specific changes in the two patient lines that contribute to the disease phenotype, I performed comparative profiling with their isogenic controls using gene expression analysis, single cell RNA sequencing and electrical phenotyping. Gene expression profiling of iPSC derived CMs confirmed several deregulated gene expression patterns known from animal models and uncovered additional dysregulation. Data from Single cell RNA sequencing revealed a possibly impaired differentiation capability and disturbed mitochondrial function in the patient cells. In CMs of the 3’UTR variant patient line, changed action potential characteristics were discovered, which notably could be attributed to differential ion channel expression. M ost strikingly, upregulation of the Na+ channel SCN5A led to an increased action potential upstroke velocity and amplitude, and upregulation of several K+ channels caused a shortened repolarization time observed in the patient line. Thus, novel disease mechanisms causing SHOX2 dependent AF as well as underlying molecular mechanisms and potential targets for adapted treatment strategies could be uncovered. Overall, the established human iPSC model and the differentiated cardiac cell types provide a valuable tool to further dissect the detailed molecular mechanisms of SHOX2 dependent AF in the future