TPM1-p.E181K Mutation Suppresses CaMKII/HDAC4 Signaling Pathway Leading to Pediatric Restrictive Cardiomyopathy

Jia Fu¹Jing Zhang¹Youxian Zhang²Dongming Sun¹Kun Xia¹Ruigeng Wang¹Xiaoyuan Feng³Aiguo Zhai³Yufeng Huang³Xiaobin Li⁴Wenjun Yu^5,6*Yong Zhang^1*

• ¹Department of Cardiology, Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Wuhan, China
• ²Department of Genaral Surgery, Hubei Provincial Hospital of Integrated Chinese&Western Medicine, Wuhan, China
• ³Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
• ⁴College of Food Science and Engineering, Shaanxi University of Science and Technology, Xi'an, China
• ⁵Department of Cardiovascular Surgery, Zhongnan Hospital of Wuhan University, Wuhan, China
• ⁶Wuhan Clinical Research Center for Minimally Invasive Treatment of Structural Heart Disease, Wuhan, China

Background This study aims to elucidate the pathogenicity of the TPM1 mutation (NM_001018005.2:c.541G>A, p.Glu181Lys) in restrictive cardiomyopathy (RCM), establish its ACMG pathogenicity classification, and report for the first time its association with sporadic RCM and underlying molecular mechanisms. The research focuses on delineating how this mutation triggers myocardial pathology via disruption of the CaMKII/HDAC4 signaling pathway. Methods Protein 3D modeling predicted structural alterations induced by the mutation. TPM1-wild-type (WT) and mutant (p.E181K) plasmids were transfected into AC16 cardiomyocyte cell lines. Quantitative PCR (qPCR) and Western blotting (WB) analyzed gene/protein expression levels. Intracellular calcium transients were detected using Rhod-2 AM fluorescent probes. F-actin cytoskeletal reorganization was assessed by Phalloidin-488 staining. Phosphorylation status of key CaMKII/HDAC4 pathway components and troponin (Tn) activity were evaluated to define functional mechanisms. Results Bioinformatic analysis revealed disruption of hydrogen bonding and electrostatic potential at the mutation site. TPM1-p.E181K did not alter overall protein expression or mitochondrial activity but significantly suppressed intracellular Ca²⁺ transients and inhibited CaMKII/HDAC4 phosphorylation. Impaired troponin activity and abnormal cardiomyocyte contractility were observed. Conclusion This study establishes a novel link between TPM1-p.E181K and sporadic RCM. We demonstrate that its pathogenesis is mediated through a cascade of events: calcium dyshomeostasis leads to the suppression of CaMKII/HDAC4 phosphorylation, which subsequently causes sarcomere structural disruption, and ultimately results in myocardial hypercontractility. This identified signaling axis may represent a promising therapeutic target for RCM.