Hippo-Yap Signaling Maintains Sinoatrial Node Homeostasis.

Abstract

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. We investigated key regulators of the Hippo pathway in SAN pacemaker cells by conditionally inactivating the Hippo signaling kinases <i>Lats1</i> and <i>Lats2</i> using the tamoxifen-inducible, cardiac conduction system-specific Cre driver <i>Hcn4^CreERT2</i> with <i>Lats1</i> and <i>Lats2</i> conditional knockout alleles. In addition, the Hippo-signaling effectors <i>Yap</i> and <i>Taz</i> 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. We found that <i>Lats1/2</i> inactivation caused severe sinus node dysfunction. Compared with the controls, <i>Lats1/2</i> conditional knockout mutants exhibited dysregulated calcium handling and increased fibrosis in the SAN, indicating that <i>Lats1/2</i> function through both cell-autonomous and non-cell-autonomous mechanisms. It is notable that the <i>Lats1/2</i> conditional knockout phenotype was rescued by genetic deletion of <i>Yap</i> and <i>Taz</i> in the cardiac conduction system. These rescued mice had normal sinus rhythm and reduced fibrosis of the SAN, indicating that <i>Lats1/2</i> function through <i>Yap</i> and <i>Taz.</i> Cleavage Under Targets and Tagmentation sequencing data showed that Yap potentially regulates genes critical for calcium homeostasis such as <i>Ryr2</i> and genes encoding paracrine factors important in intercellular communication and fibrosis induction such as <i>Tgfb1</i> and <i>Tgfb3</i>. Consistent with this, <i>Lats1/2</i> conditional knockout mutants had decreased <i>Ryr2</i> expression and increased <i>Tgfb1</i> and <i>Tgfb3</i> expression compared with control mice. We reveal, for the first time to our knowledge, that the canonical Hippo-Yap pathway plays a pivotal role in maintaining SAN homeostasis.