GATA6 is a regulator of sinus node development and heart rhythm Lara Gharibeha, Abir Yamaka,b,1, Jamieson Whitcomba,1, Aizhu Luc, Mathieu Joyala, Hiba Komatia, Wenbin Liangc, Céline Fisetd,e, and Mona Nemera,2 aMolecular Genetics and Cardiac Regeneration Laboratory, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON, Canada K1H 8M5; bCardiovascular Research Center, Harvard Medical School, Boston, MA 02115; cCardiovascular Electrophysiology Laboratory, University of Ottawa Heart Institute, University of Ottawa, Ottawa, ON, Canada K1Y 4W7; dResearch Center, Montreal Heart Institute, Montreal, QC, Canada H1T 1C8; and eFaculty of Pharmacy, Université de Montréal, Montréal, QC, Canada H3T 1J4 Edited by Christine E. Seidman, Howard Hughes Medical Institute, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, and approved October 19, 2020 (received for review April 16, 2020) The sinus node (SAN) is the primary pacemaker of the human heart, reported in several cases of familial AF (8, 9). The mutations and abnormalities in its structure or function cause sick sinus frequently lead to loss of function, but gain of function mutations syndrome, the most common reason for electronic pacemaker have also been found. Analysis of mice lacking a Tbx5 or an implantation. Here we report that transcription factor GATA6, whose Nkx2.5 allele confirmed that decreased levels of these TFs is mutations in humans are linked to arrhythmia, is highly expressed in sufficient to produce conduction abnormalities, including AV the SAN and its haploinsufficiency in mice results in hypoplastic SANs block (10). Decreased levels of another T-box family member, and rhythm abnormalities. Cell-specific deletion reveals a require- TBX3 in different regions of the developing heart, also leads to ment for GATA6 in various SAN lineages. Mechanistically, GATA6 lethal arrythmias (11). TBX5 and NKX2.5 cooperatively activate directly activates key regulators of the SAN genetic program in con- transcription of several genes important for cardiac conduction, duction and nonconduction cells, such as TBX3 and EDN1, respec- including connexin 40 (Cx40) and the DNA-binding protein in- tively. The data identify GATA6 as an important regulator of the hibitor Id2 (10, 12). In cardiomyocytes, TBX5 and NKX2.5 also SAN and provide a molecular basis for understanding the conduction interact with GATA4 (13, 14); mutations in GATA4 are found in abnormalities associated with GATA6 mutations in humans. They association with human AF, and GATA4 was shown to regulate also suggest that GATA6 may be a potential modifier of the conduction genes such as Cx40 and Cx30.2 (15). DEVELOPMENTAL BIOLOGY cardiac pacemaker. A role for GATA6 in CCS regulation was first suggested by the finding that an enhancer region upstream of the Gata6 pro- GATA proteins | sinus node | cardiac conduction system | TBX3 | moter is specifically active in the AV conduction system (16). The heart development presence of GATA6 in the ventricular CCS was subsequently confirmed, and its contribution to AV development and function he cardiac conduction system (CCS) consists of muscle cells was inferred from analysis of mice with myocardial-specific dele- Tand conducting fibers that ensure the initiation of the im- tion of its carboxyl zinc finger domain using Mlc2v-cre (17). In this pulse and its propagation throughout the heart. The CCS is com- model, a truncated GATA6 protein containing the N-terminal posed of the sinus node (SAN), known as the pacemaker of the activation domain and the first zinc finger can still be produced heart since it generates the electrical impulse, and the atrioven- and retains the ability to bind GATA sites and interact with known tricular node (AVN), the His or atrioventricular bundle (AV GATA cofactors. Of note, the mutant protein was restricted to bundle), and the Purkinje fibers, which propagate the signal to the MLC2V-expressing myocytes, while intact GATA6 is expressed in ventricular myocytes (1). Despite remarkable progress in the past atrial cells and in other cell types that may contribute to the decade, the cellular origins of the CCS components remain in- proximal CCS. completely elucidated. The SAN is thought to be composed mainly of secondary heart field cells derived from ISL1- and TBX18- Significance expressing cells (2), while the AVN and His bundle are thought to segregate from precursors of the working myocardium and are – GATA6 is a key regulator of sinus node (SAN) development. positive for transcription factors TBX2 and NKX2.5 (3 5). Loss of one Gata6 allele disrupts patterning and size of the Abnormalities affecting the generation or propagation of the SAN. GATA6 plays cell autonomous as well as non-cell auton- electrical impulses lead to conduction defects ranging from be- omous functions in several cell types required for proper SAN nign to fatal arrhythmias. Impaired impulse generation in the development and function. Haploinsufficiency of Gata6 in mice SAN causes sick sinus syndrome (SSS), manifesting as sinus leads to electrophysiological alterations and increases suscep- pause/arrest or bradycardia. Blockage of the impulse conduction tibility to develop arrhythmias. GATA6 is critical for pacemaker to the AVN or Purkinje fibers results in atrioventricular block. In cell differentiation and cardiac conduction. humans, atrial fibrillation (AF), a condition involving a rapid and irregular cardiac rhythm, is the most common type of arrhyth- Author contributions: L.G. and M.N. designed research; L.G., A.Y., J.W., A.L., and M.J. mia, affecting 2.7 million individuals in the United States alone performed research; W.L. and C.F. contributed new reagents/analytic tools; L.G., A.Y., (6). Its frequency increases with aging, and it occurs in isolation J.W., A.L., M.J., H.K., W.L., C.F., and M.N. analyzed data and interpreted results; L.G. or in association with other complications such as dementia, and M.N. wrote the paper. stroke, and heart failure (7). Atrial fibrillation can be triggered The authors declare no competing interest. by alterations in atrial electrophysiology or calcium handling, This article is a PNAS Direct Submission. genetic factors, and contractile and structural remodeling in the This open access article is distributed under Creative Commons Attribution-NonCommercial- heart that increase with aging. Human genetic studies as well as NoDerivatives License 4.0 (CC BY-NC-ND). analysis of genetically modified mouse models have identified 1A.Y. and J.W. contributed equally to this work. several genes encoding critical cardiac transcription factors (TFs) 2To whom correspondence may be addressed. Email: [email protected]. that contribute to rhythm regulation disturbances. For example, This article contains supporting information online at https://www.pnas.org/lookup/suppl/ mutations in the GATA family of TFs (GATA4/5/6), the T-box doi:10.1073/pnas.2007322118/-/DCSupplemental. factor TBX5, and the homeodomain protein NKX2.5 have been Published December 28, 2020. PNAS 2021 Vol. 118 No. 1 e2007322118 https://doi.org/10.1073/pnas.2007322118 | 1of11 Downloaded by guest on October 2, 2021 To determine the contribution of GATA6 to the CCS, we such as atrial dilation and impaired ventricular function are analyzed CCS structure and function in mice haploinsufficient common in patients with chronic AF (19). We previously reported for Gata6. We found that loss of one Gata6 allele leads to that adult mice with 50% reduction in GATA6 have reduced left electrophysiological alterations and increased susceptibility to ventricular function as assessed by echocardiography, and several arrhythmias. Furthermore, hypocellularity and reduction in display a dilated right atrium (RA) and right ventricle (20). As +/− HCN4+/TBX3+ conduction cells marked the SAN of Gata6 shown in Fig. 2 A and B, these structural defects are evident at mice. Cell-specific deletion of Gata6 revealed its requirement birth. Further examination of Masson trichrome–stained sections +/− in different cell types that contribute to SAN development and revealed a hypoplastic SAN with fewer myocytes in all Gata6 CCS function, namely, conduction, second heart field (SHF), neonates compared to control littermates (Fig. 2C). To determine and endothelial cells. The results support a key function for whether this phenotype is associated with differentiation defects, GATA6 in pacemaker cell differentiation and in cardiac con- expression of key atrial and CCS markers and regulators, including duction. They also suggest that GATA6 may be an arrhythmia- NKX2.5, TBX5, ANP, and HCN4, was examined. Maintenance of causing or -modifying gene. the SAN and atrial identities involves transcriptional repression of atrial and SAN genes, respectively. For example, TBX3 represses Results the ANP gene in the SAN, while in the adjacent atrial myocar- Loss of One Allele of Gata6 in Mice Results in Electrocardiogram dium, NKX2.5 activates ANP and represses HCN4 and TBX3, as Alterations and Hypoplastic SAN. A surface electrocardiogram evidenced by ectopic expression of TBX3 and HCN4 in the em- +/− +/− (ECG) was performed on Gata6 and control littermates to bryonic myocardium of Nkx2.5 null mice (1). In Gata6 mice, assess whether these mice display any electrophysiological ab- NKX2.5 and TBX5 were up-regulated in the SAN relative to normalities. The ECG profiling showed significant increases in control littermates (Fig. 2 D, Top and Bottom). Interestingly, ANP +/− both PR segment and QT