BACKGROUND:Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia with a strong genetic predisposition. Genome-wide association studies have highlighted CAV1 (caveolin 1), a caveolar protein involved in various signaling pathways, as a candidate for cardiac conduction disorders. METHODS:We explored the role of CAV1 in AF in various models to dissect possible disease mechanisms. First, CAV1 expression was examined together with the AF risk gene SHOX2 in a porcine model of induced AF. Then we screened a cohort of 282 patients with early-onset AF to identify genetic variants within CAV1 and found 1 coding and 5 noncoding variants. The coding variant was functionally investigated in zebrafish, and a comprehensive analysis panel was applied to investigate the noncoding variants. RESULTS:In the porcine AF model, CAV1 and SHOX2 were significantly downregulated in the right atrium and atrioventricular node. Cardiac-specific overexpression of the coding variant in zebrafish increased heart rate and caused fibrillatory waves and loss of the PR interval, supporting a pathogenic effect. Four of the 5 novel identified noncoding variants showed an association with AF and PR interval in published data sets, including 1 with genome-wide significance. The noncoding variants localized to binding sites of transcription factors EOMES, RFX5, TEAD4 and MAX. Luciferase reporter gene assays demonstrated that 3 variants significantly altered the ability of those transcription factors to activate reporter gene expression. CONCLUSIONS:This work underscores CAV1 as an AF susceptibility gene and highlights the critical role of coding and noncoding variants in AF disease mechanisms.

Short stature homeobox (SHOX) and its close paralogue SHOX2 are important transcriptional regulators of developmental processes. To understand how they function both individually and as an ensemble, we examined their mutual interdependence using zebrafish as a model. As both genes maintain important roles in various tissues, we assessed their expression patterns in the whole embryo as well as separately in fins, heart and brain. Fibroblast growth factor receptor 3 (FGFR3) is a direct target of SHOX, and FGFR3 mutations cause achondroplasia, a condition treatable with C-type natriuretic peptide (CNP) analogues. Building on this knowledge, we investigated how altered shox and shox2 expression relates to fgfr3 expression and the natriuretic peptide family (nppa, nppb, nppc) in zebrafish. Here, we demonstrate that shox and shox2 mutually regulate each other’s expression in a tissue-specific manner. In brain and whole embryos their effects are additive and synergistic, while in fins and heart their combined actions become partially antagonistic. Distinct tissue-specific effects of shox and shox2 were also noted on natriuretic peptide gene activity. In the fin, shox knockdown reduces nppc transcript levels, whereas in heart tissue, deficiency of shox and shox2, particularly in the double knockdown, results in an increased expression of nppb, a known marker for cardiac stress. We also show that shox2 deficiency increases fgfr3 levels, which are reduced by CNP treatment. These results in zebrafish may have implications for other vertebrates, including humans with SHOX deficiency, who should be monitored for cardiac disease in later life, and those who have an inadequate response to growth hormone may profit from CNP therapy.

Human induced pluripotent stem cell (hiPSC)-based disease modelling has significantly advanced the field of cardiogenetics, providing a precise, patient-specific platform for studying genetic causes of heart diseases. Coupled with genome editing technologies such as CRISPR/Cas, hiPSC-based models not only allow the creation of isogenic lines to study mutation-specific cardiac phenotypes, but also enable the targeted modulation of gene expression to explore the effects of genetic and epigenetic deficits at the cellular and molecular level. hiPSC-based models of heart disease range from two-dimensional cultures of hiPSC-derived cardiovascular cell types, such as various cardiomyocyte subtypes, endothelial cells, pericytes, vascular smooth muscle cells, cardiac fibroblasts, immune cells, etc., to cardiac tissue cultures including organoids, microtissues, engineered heart tissues, and microphysiological systems. These models are further enhanced by multi-omics approaches, integrating genomic, transcriptomic, epigenomic, proteomic, and metabolomic data to provide a comprehensive view of disease mechanisms. In particular, advances in cardiovascular tissue engineering enable the development of more physiologically relevant systems that recapitulate native heart architecture and function, allowing for more accurate modelling of cardiac disease, drug screening, and toxicity testing, with the overall goal of personalised medical approaches, where therapies can be tailored to individual genetic profiles. Despite significant progress, challenges remain in the maturation of hiPSC-derived cardiomyocytes and the complexity of reproducing adult heart conditions. Here, we provide a concise update on the most advanced methods of hiPSC-based disease modelling in cardiogenetics, with a focus on genome editing and cardiac tissue engineering.

SHOX2 is a homeobox transcription factor associated with atrial fibrillation (AF) and sinus node dysfunction. Here, we generated two homozygous SHOX2 knock-out hiPSC lines from a healthy control line and a corrected AF patient line (disease-specific SHOX2 mutation corrected to WT) using CRISPR/Cas9. These cell lines maintained pluripotency, an ability to differentiate into all three germlayers and a normal karyotype, presenting a valuable tool to investigate the impact of a full SHOX2 knock-out with respect to arrhythmogenic diseases on a cellular level.

Very tall people attract much attention and represent a clinically and genetically heterogenous group of individuals. Identifying the genetic etiology can provide important insights into the molecular mechanisms regulating linear growth. We studied a three-generation pedigree with five isolated (non-syndromic) tall members and one individual with normal stature by whole exome sequencing; the tallest man had a height of 211 cm. Six heterozygous gene variants predicted as damaging were shared among the four genetically related tall individuals and not present in a family member with normal height. To gain insight into the putative role of these candidate genes in bone growth, we assessed the transcriptome of murine growth plate by microarray and RNA Seq. Two (Ift140, Nav2) of the six genes were well-expressed in the growth plate. Nav2 (p-value 1.91E-62) as well as Ift140 (p-value of 2.98E-06) showed significant downregulation of gene expression between the proliferative and hypertrophic zone, suggesting that these genes may be involved in the regulation of chondrocyte proliferation and/or hypertrophic differentiation. IFT140, NAV2 and SCAF11 have also significantly associated with height in GWAS studies. Pathway and network analysis indicated functional connections between IFT140, NAV2 and SCAF11 and previously associated (tall) stature genes. Knockout of the all-trans retinoic acid responsive gene, neuron navigator 2 NAV2, in Xenopus supports its functional role as a growth promotor. Collectively, our data expand the spectrum of genes with a putative role in tall stature phenotypes and, among other genes, highlight NAV2 as an interesting gene to this phenotype.

The elucidation of molecular mechanisms that restrict the potential of pluripotent stem cells and promote cardiac lineage differentiation is of crucial relevance, since embryonic stem cells (ESCs) hold great potential for cell based heart therapies. The homeodomain transcription factor Shox2 is essential for the development and proper function of the native cardiac pacemaker, the sinoatrial node. This prompted us to develop a cardiac differentiation model using ESC lines isolated from blastocysts of Shox2-deficient mice. The established cell model provides a fundamental basis for the investigation of molecular pathways under physiological and pathophysiological conditions for evaluating novel therapeutic approaches.

The homeodomain transcription factor Shox2 controls the development and function of the native cardiac pacemaker, the sinoatrial node (SAN). Moreover, SHOX2 mutations have been associated with cardiac arrhythmias in humans. For detailed examination of Shox2-dependent developmental mechanisms in SAN cells, we established a murine embryonic stem cell (ESC)-based model using Shox2 as a molecular tool. Shox2+/+ and Shox2−/− ESC clones were isolated and differentiated according to five different protocols in order to evaluate the most efficient enrichment of SAN-like cells. Expression analysis of cell subtype-specific marker genes revealed most efficient enrichment after CD166-based cell sorting. Comparative cardiac expression profiles of Shox2+/+ and Shox2−/− ESCs were examined by nCounter technology. Among other genes, we identified Nppb as a novel putative Shox2 target during differentiation in ESCs. Differential expression of Nppb could be confirmed in heart tissue of Shox2−/− embryos. Taken together, we established an ESC-based cardiac differentiation model and successfully purified Shox2+/+ and Shox2−/− SAN-like cells. This now provides an excellent basis for the investigation of molecular mechanisms under physiological and pathophysiological conditions for evaluating novel therapeutic approaches.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia with a strong genetic component. Molecular pathways involving the homeodomain transcription factor Shox2 control the development and function of the cardiac conduction system in mouse and zebrafish. Here we report the analysis of human SHOX2 as a potential susceptibility gene for early-onset AF. To identify causal variants and define the underlying mechanisms, results from 378 patients with early-onset AF before the age of 60 years were analyzed and compared to 1870 controls or reference datasets. We identified two missense mutations (p.G81E, p.H283Q), that were predicted as damaging. Transactivation studies using SHOX2 targets and phenotypic rescue experiments in zebrafish demonstrated that the p.H283Q mutation severely affects SHOX2 pacemaker function. We also demonstrate an association between a 3′UTR variant c.*28T>C of SHOX2 and AF ( p = 0.00515). Patients carrying this variant present significantly longer PR intervals. Mechanistically, this variant creates a functional binding site for hsa - miR - 92b - 5p . Circulating hsa - miR - 92b - 5p plasma levels were significantly altered in AF patients carrying the 3′UTR variant ( p = 0.0095). Finally, we demonstrate significantly reduced SHOX2 expression levels in right atrial appendages of AF patients compared to patients with sinus rhythm. Together, these results suggest a genetic contribution of SHOX2 in early-onset AF.

BACKGROUND:The short stature homeodomain transcription factors SHOX and SHOX2 play key roles in limb formation. To gain more insight into genes regulated by Shox2 during limb development, we analyzed expression profiles of WT and Shox2-/- mouse embryonic limbs and identified the T-Box transcription factor Tbx4 as a potential downstream target. Tbx4 is known to exert essential functions in skeletal and muscular hindlimb development. In humans, haploinsufficiency of TBX4 causes small patella syndrome, a skeletal dysplasia characterized by anomalies of the knee, pelvis, and foot.RESULTS:Here, we demonstrate an inhibitory regulatory effect of Shox2 on Tbx4 specifically in the forelimbs. We also show that Tbx4 activates Shox2 expression in fore- and hindlimbs, suggesting Shox2 as a feedback modulator of Tbx4. Using EMSA studies, we find that Tbx4/TBX4 is able to bind to distinct T-box binding sites within the mouse and human Shox2/SHOX2 promoter.CONCLUSIONS:Our data identifies Tbx4 as a novel transcriptional activator of Shox2 during murine fore- and hindlimb development. Tbx4 is also regulated by Shox2 specifically in the forelimb bud possibly via a feedback mechanism. These data extend our understanding of the role and regulation of Tbx4 and Shox2 in limb development and limb associated diseases.

The heart's rhythm is initiated and regulated by a group of specialized cells in the sinoatrial node (SAN), the primary pacemaker of the heart. Abnormalities in the development of the SAN can result in irregular heart rates (arrhythmias). Although several of the critical genes important for SAN formation have been identified, our understanding of the transcriptional network controlling SAN development remains at a relatively early stage. The homeodomain transcription factor Shox2 is involved in the specification and patterning of the SAN. While the Shox2 knockout in mice results in embryonic lethality due to severe cardiac defects including improper SAN development, Shox2 knockdown in zebrafish causes a reduced heart rate (bradycardia). In order to gain deeper insight into molecular pathways involving Shox2, we compared gene expression levels in right atria of wildtype and Shox2 −/− hearts using microarray experiments and identified the LIM homeodomain transcription factor Islet1 (Isl1) as one of its putative target genes. The downregulation of Isl1 expression in Shox2 −/− hearts was confirmed and the affected region narrowed down to the SAN by whole-mount in situ hybridization. Using luciferase reporter assays and EMSA studies, we identified two specific SHOX2 binding sites within intron 2 of the ISL1 locus. We also provide functional evidence for Isl1 as a transcriptional target of Shox2 by rescuing the Shox2-mediated bradycardia phenotype with Isl1 using zebrafish as a model system. Our findings demonstrate a novel epistatic relationship between Shox2 and Isl1 in the heart with important developmental consequences for SAN formation and heart beat.