TY - JOUR
T1 - Hippo-Yap Signaling Maintains Sinoatrial Node Homeostasis
AU - Zheng, Mingjie
AU - Li, Rich G.
AU - Song, Jia
AU - Zhao, Xiaolei
AU - Tang, Li
AU - Erhardt, Shannon
AU - Chen, Wen
AU - Nguyen, Bao H.
AU - Li, Xiao
AU - Li, Min
AU - Wang, Jianxin
AU - Evans, Sylvia M.
AU - Christoffels, Vincent M.
AU - Li, Na
AU - Wang, Jun
N1 - Funding Information: The authors thank the sequencing support from Cancer Genomics Center supported by CPRIT RP180734. The authors also thank Drs Eric N. Olson, James F. Martin, and Randy L. Johnson for mouse lines; Dr Idaliz M. Martínez Traverso for technical support of RNAscope; Drs Stephen N. Palmer and Nicole Stancel of Scientific Publications at the Texas Heart Institute for editorial assistance, and Kaleigh L. Riggs for statistical analysis assistance. Funding Information: This study was supported by the funding from the National Institutes of Health (R56HL142704 and R01HL142704 to Dr Jun Wang; T32HL07676 to Dr R.G. Li; R01HL163277, R01HL136389, and R01HL147108 to Dr N. Li) and American Heart Association (SDG19840000 and TPA970606 to Dr Jun Wang; 902940 to Dr Zheng; 903251 to Dr R.G. Li; EIA936111 to Dr N. Li). Publisher Copyright: © 2022 Lippincott Williams and Wilkins. All rights reserved.
PY - 2022/11/29
Y1 - 2022/11/29
N2 - Background: The sinoatrial node (SAN) functions as the pacemaker of the heart, initiating rhythmic heartbeats. Despite its importance, the SAN is one of the most poorly understood cardiac entities because of its small size and complex composition and function. The Hippo signaling pathway is a molecular signaling pathway fundamental to heart development and regeneration. Although abnormalities of the Hippo pathway are associated with cardiac arrhythmias in human patients, the role of this pathway in the SAN is unknown. Methods: We investigated key regulators of the Hippo pathway in SAN pacemaker cells by conditionally inactivating the Hippo signaling kinases Lats1 and Lats2 using the tamoxifen-inducible, cardiac conduction system-specific Cre driver Hcn4CreERT2with Lats1 and Lats2 conditional knockout alleles. In addition, the Hippo-signaling effectors Yap and Taz were conditionally inactivated in the SAN. To determine the function of Hippo signaling in the SAN and other cardiac conduction system components, we conducted a series of physiological and molecular experiments, including telemetry ECG recording, echocardiography, Masson Trichrome staining, calcium imaging, immunostaining, RNAscope, cleavage under targets and tagmentation sequencing using antibodies against Yap1 or H3K4me3, quantitative real-time polymerase chain reaction, and Western blotting. We also performed comprehensive bioinformatics analyses of various datasets. Results: We found that Lats1/2 inactivation caused severe sinus node dysfunction. Compared with the controls, Lats1/2 conditional knockout mutants exhibited dysregulated calcium handling and increased fibrosis in the SAN, indicating that Lats1/2 function through both cell-autonomous and non-cell-autonomous mechanisms. It is notable that the Lats1/2 conditional knockout phenotype was rescued by genetic deletion of Yap and Taz in the cardiac conduction system. These rescued mice had normal sinus rhythm and reduced fibrosis of the SAN, indicating that Lats1/2 function through Yap and Taz. Cleavage Under Targets and Tagmentation sequencing data showed that Yap potentially regulates genes critical for calcium homeostasis such as Ryr2 and genes encoding paracrine factors important in intercellular communication and fibrosis induction such as Tgfb1 and Tgfb3. Consistent with this, Lats1/2 conditional knockout mutants had decreased Ryr2 expression and increased Tgfb1 and Tgfb3 expression compared with control mice. Conclusions: We reveal, for the first time to our knowledge, that the canonical Hippo-Yap pathway plays a pivotal role in maintaining SAN homeostasis.
AB - Background: The sinoatrial node (SAN) functions as the pacemaker of the heart, initiating rhythmic heartbeats. Despite its importance, the SAN is one of the most poorly understood cardiac entities because of its small size and complex composition and function. The Hippo signaling pathway is a molecular signaling pathway fundamental to heart development and regeneration. Although abnormalities of the Hippo pathway are associated with cardiac arrhythmias in human patients, the role of this pathway in the SAN is unknown. Methods: We investigated key regulators of the Hippo pathway in SAN pacemaker cells by conditionally inactivating the Hippo signaling kinases Lats1 and Lats2 using the tamoxifen-inducible, cardiac conduction system-specific Cre driver Hcn4CreERT2with Lats1 and Lats2 conditional knockout alleles. In addition, the Hippo-signaling effectors Yap and Taz were conditionally inactivated in the SAN. To determine the function of Hippo signaling in the SAN and other cardiac conduction system components, we conducted a series of physiological and molecular experiments, including telemetry ECG recording, echocardiography, Masson Trichrome staining, calcium imaging, immunostaining, RNAscope, cleavage under targets and tagmentation sequencing using antibodies against Yap1 or H3K4me3, quantitative real-time polymerase chain reaction, and Western blotting. We also performed comprehensive bioinformatics analyses of various datasets. Results: We found that Lats1/2 inactivation caused severe sinus node dysfunction. Compared with the controls, Lats1/2 conditional knockout mutants exhibited dysregulated calcium handling and increased fibrosis in the SAN, indicating that Lats1/2 function through both cell-autonomous and non-cell-autonomous mechanisms. It is notable that the Lats1/2 conditional knockout phenotype was rescued by genetic deletion of Yap and Taz in the cardiac conduction system. These rescued mice had normal sinus rhythm and reduced fibrosis of the SAN, indicating that Lats1/2 function through Yap and Taz. Cleavage Under Targets and Tagmentation sequencing data showed that Yap potentially regulates genes critical for calcium homeostasis such as Ryr2 and genes encoding paracrine factors important in intercellular communication and fibrosis induction such as Tgfb1 and Tgfb3. Consistent with this, Lats1/2 conditional knockout mutants had decreased Ryr2 expression and increased Tgfb1 and Tgfb3 expression compared with control mice. Conclusions: We reveal, for the first time to our knowledge, that the canonical Hippo-Yap pathway plays a pivotal role in maintaining SAN homeostasis.
KW - Hippo signaling pathway
KW - calcium homeostasis
KW - fibrosis
KW - sinus node dysfunction
KW - transforming growth factor-β
UR - http://www.scopus.com/inward/record.url?scp=85142940978&partnerID=8YFLogxK
U2 - https://doi.org/10.1161/CIRCULATIONAHA.121.058777
DO - https://doi.org/10.1161/CIRCULATIONAHA.121.058777
M3 - Article
C2 - 36317529
SN - 0009-7322
VL - 146
SP - 1694
EP - 1711
JO - Circulation
JF - Circulation
IS - 22
ER -