Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, primarily occurring with advanced age, and is associated with an increased risk of stroke and heart failure. Common variation in the non-coding region of PITX2, the left-sided determining transcription factor, is the strongest genetic signature of AF risk. As PITX2 risk alleles further enhance age-associated risk and are associated with decreased expression of PITX2 in the left atrium (LA), we utilized our well-characterized Pitx2 mouse model to study the effect of advanced age at single-cell resolution. We performed single nuclear RNA-sequencing (snRNA-seq) on the LA of 24-month-old heterozygous and littermate controls and compared with our previously reported 6-month-old datasets. We identified a gene signature of decreased Wnt-signaling inhibition from Pitx2-deficient cardiomyocytes and increased Wnt-associated activation of fibroblasts. Transgenic Wnt reporter mice demonstrate that Pitx2 deficiency increases LA Wnt-signaling to right atrial levels. Confirmed using a cre-dependent overexpression allele of Pitx2, PITX2 was able to drive Sfrp1 and Sfrp2, two secreted inhibitors of Wnt signaling, in right atrial cardiomyocytes. Histological analysis of aged Pitx2-deficient mice had increased LA fibroblast proliferation and interstitial fibrosis not previously seen in young animals. Furthermore, aged Pitx2-deficient mice were susceptible to atrial arrhythmogenesis when challenged by transesophageal pacing. Lastly, we demonstrate that pharmacological Wnt inhibition is sufficient to reduce fibroblast activation and proliferation in the Pitx2-deficient LA. Collectively, these data indicate that reduced LA Pitx2 leads to increased left-sided Wnt signaling, resulting in LA remodeling and increased arrhythmia susceptibility with age. Furthermore, in functionally validating the Pitx2-Wnt-fibrosis signaling axis in the aging LA, our data provides new insights for both AF pathogenesis and therapeutic strategies.

Treatments for ventricular tachycardia (VT), a life-threatening arrhythmia, have limited efficacy and significant morbidity. Stereotactic arrhythmia radiotherapy (STAR) has emerged as a highly efficacious novel treatment. Clinical data and murine models from our group have contradicted the originally purported mechanism behind the antiarrhythmic effects of STAR—scar homogenization to ablate re-entry circuits—and instead have suggested that STAR favorably reprograms the cardiac electrical substrate by increasing conduction velocity (CV) via durable increases in sodium channel (Nav1.5) and gap junction (Cx43) protein levels. The persistence of these protein and functional changes, as well as the durability of the clinical effect, led us to hypothesize that a single dose of radiation may lead to epigenetically mediated cardiomyocyte reprogramming. Using combined ATAC-seq and RNA-seq approaches, we observed dynamic changes in chromatin accessibility and gene expression in the murine heart after irradiation (IR). These accessibility changes included peaks responsive at both early (2 day) and late (6 weeks) time points. Interestingly, we also identified acutely emerging peaks that were inaccessible in sham mice but became accessible after IR, with a subset of these emergent peaks remaining persistently accessible 6 weeks after IR. These findings demonstrate that after DNA damage, rather than returning to its original state, there are some changes in the chromatin landscape which persist as an ‘epigenetic scar.’ Similar reprogramming phenomena were observed across multiple human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) lines after IR. Using in vitro models including 2D multielectrode array cell culture systems and optical mapping of engineered heart tissues, we demonstrated increased CV after IR, suggesting this physiologic effect is cell autonomous to cardiomyocytes. Although additional studies are needed to better understand the mechanisms and clinical implications of these findings, this study implicates radiation-induced chromatin dynamics in the response to STAR for VT and may also suggest IR as a novel strategy for in vitro hiPSC-CM maturation in tissue engineering models.

Located at the sinoatrial region, the primary cardiac pacemaker rhythmically depolarizes the surrounding working myocardium to initiate the heartbeat. Pacemaker malfunction results in cardiac defects including the development of arrhythmias. In this study, we aim to understand how different celltypes influence pacemaker formation and function in the zebrafish. Using 3D imaging, we investigated epicardial derived cells (EPDCs) and neurons of the autonomous nervous system, both sympathetic and parasympathetic, at the pacemaker cardiomyocytes (PCs). As early as 48 hours post fertilization (hpf), we identified a cluster of EPDCs that colocalizes and migrates with the PCs over time, while parasympathetic axons extend along and intertwine with the EPDC cluster around 80 hpf. Sympathetic innervation followed later in development. In addition, functional studies identified changes in EPDC coverage in the pacemaker-associated isl1a mutant, as well as defective innervation after epicardial ablation. Using scRNAseq data of several developmental time points, we identified distinct cell populations involved in pacemaker regulation. Further investigations will look into potential intercellular interactions influencing cardiac contraction. Altogether, we show celltype specific pacemaker regulation, thereby offering a way to help understand the role of each celltype on impulse generation.

Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy. Thus far, more than 50 genes have been associated with DCM, of which LMNA (lamin A/C) is ranked as the most highly associated, representing 5-10% of the cases. Currently, in many cases of LMNA-related DCM (LMNA-DCM) the direct link between the LMNA variants and DCM is not fully understood. Furthermore, there are no pharmaceutical therapeutics designed specifically for LMNA-DCM patients. We have generated an in vitro model of LMNA-DCM using patient-specific human induced pluripotent stem cells (hiPSCs). Nine laminopathy patients, eight harboring five different pathogenic LMNA variants, and one with an EMD (emerin) variant were selected. Five healthy individuals were recruited as controls. hiPSCs from all the individuals were differentiated into cardiomyocytes, purified, and assessed based on their gene expression and function. Laminopathy hiPSC-derived cardiomyocytes (hiPSC-CMs) showed a significantly higher level of nuclear deformation compared to controls which was exacerbated after mechanical stress. RNA-sequencing analysis revealed more than 300 genes differentially expressed between controls and patients-derived hiPSC-CMs which were potentially associated with the DCM phenotype. Pathway enrichment analysis also confirmed prior findings that mTOR pathway genes are involved. Analysis of intracellular calcium dynamics revealed prolongation of calcium transient durations and arrhythmia in laminopathy hiPSC-CMs. Contractility analysis also revealed significantly lower beat rate and higher pulse width in hiPSC-CMs from LMNA variant carriers compared to controls. mTOR inhibition by sirolimus led to the correction of calcium transient abnormalities. These results suggested that high-throughput assays can be used to find drugs which can alleviate the abnormal cellular phenotype in LMNA hiPSC-CMs. Based on that, in an unbiased high-throughput functional drug screen analysis performed with hiPSC-CMs we tested 1280 FDA-approved drugs and identified one drug as the only compound to successfully correct the defective function of all LMNA hiPSC-CMs. In conclusion, our findings show significant electrophysiological and transcriptome differences between LMNA and control hiPSC-CMs, which can be attenuated by drug treatment. The in vitro functional trial performed in this study can provide human cell-based data for further development of effective treatments for LMNA-DCM.