FHOD3 deficiency disrupts sarcomere organization and activates CaMKII signaling in human stem cell-derived cardiomyocytes

Abstract

Background: Inherited cardiomyopathy (ICM) is a genetic disorder characterized by abnormal myocardial structure and function, often progressing to heart failure. FHOD3, a member of the Formin gene family, plays a crucial role in cardiomyocyte cytoskeletal organization. Mutations in FHOD3 have been associated with various cardiomyopathies, including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and left ventricular noncompaction (LVNC). However, the molecular mechanisms underlying FHOD3 deficiency-induced cardiomyopathy remain elusive. Methods: A FHOD3 knockout (FHOD3-/-) human embryonic stem cell (hESC) line was generated using the CRISPR/Cas9 system and subsequently differentiated into cardiomyocytes (hESC-CMs). Sarcomere structure, calcium handling, mitochondrial function, and contractility were evaluated via immunofluorescence, electron microscopy, Seahorse metabolic analysis, and high-definition video analysis, respectively. Transcriptomic sequencing was performed to identify differentially expressed genes and enriched pathways. Results: FHOD3-deficient hESC-CMs exhibited marked sarcomere disorganization and degradation, impaired calcium handling and compromised mitochondrial function, ultimately leading to reduced contractility. Transcriptomic analysis revealed significant downregulation of sarcomere-related genes and calcium-handling genes, with enrichment in pathways associated with cardiomyopathy and calcium signaling. Furthermore, FHOD3 deficiency triggered the phosphorylation of CaMKII (Thr286), a key regulator of cardiac hypertrophy and remodeling, contributing to the progression of heart failure. Treatment with the myosin activator Omecamtiv mecarbil (OM) partially restored contractility without affecting calcium handling, highlighting its potential as a therapeutic strategy. Conclusions: Our study establishes a valuable human-derived model for investigating the molecular mechanisms of FHOD3 deficiency-induced cardiomyopathy. This model allows for extensive investigation into the phenotypes caused by FHOD3 deficiency and identifies CaMKII activation as a crucial factor contributing to the HF phenotype. Additionally, this model serves as an important tool for discovering novel therapeutic agents, and we demonstrate that OM can partially improve myocardial function in FHOD3 KO hESC-CMs.

1. Introduction

Inherited cardiomyopathy (ICM) is a group of diseases caused by genetic mutations, characterized by abnormal myocardial structure and function, and often leads to heart failure as a severe complication. The incidence of inherited cardiomyopathy in the general population is approximately 0.2% -0.5%, posing a serious threat to human life [1, 2]. Therefore, investigating the function and pathogenic mechanisms of relevant mutated genes may facilitate the provision of personalized treatment, including drug and gene therapy, for patients with inherited cardiomyopathy [3, 4].

FHOD3 protein (Formin Homology 2 Domain Containing 3) is a member of the Formin gene family, which is characterized by its Formin homology domains (FH1 and FH2). It plays a major role in cell cytoskeletal organization, particularly in actin filament assembly and microtubule stabilization. FHOD3 is highly expressed in cardiomyocytes and mediates the nucleation and polymerization of actin filaments through the FH2 domain, thereby regulating actin assembly and sarcomere organization in cardiomyocytes, playing a key role in myofibrillogenesis [5, 6]. Previous studies have shown that mutations in FHOD3 are associated with hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and left ventricular noncompaction (LVNC) in humans [7–10]. FHOD3 mutations are estimated to account for 1–2% of genetically explained hypertrophic cardiomyopathy (HCM) in unselected cohorts and up to 4% in familial cases [7, 8, 11, 12]. Clinically, most of FHOD3-related HCM presents in adulthood with asymmetric septal hypertrophy and carries a markedly elevated arrhythmic burden. Consequently, affected individuals face a significantly higher risk of sudden cardiac death [7, 11]. Notably, homozygosity for the p.Arg637Gln variant observed in several families is associated with a much earlier and more aggressive phenotype, with several patients developing severe disease and multiple complications between the ages of 2 and 4 years [13]. In contrast, FHOD3-related DCM is still sparsely reported and lacks a direct prevalence rate in the general population. The only fully characterized case is a family in Japan with the p.Tyr1249Asn variant, showing slow-progressing adult DCM without arrhythmia [10, 14]. In addition, only one FHOD3-LVNC pedigree has been reported: an aggressive phenotype of massive trabeculation, rapid EF fall and lethal ventricular arrhythmias causing death at 25 and 49 years. The patients produces severe trabeculation, diffuse fibrosis, rapid EF decline, recurrent LV thrombi and lethal arrhythmias, with sudden death at 25 and 49 years, indicating an aggressive phenotype [9].