iPSC-Derived Engineered Cardiac Spheroids Reveal the Fibrotic Role of Testosterone in Arrhythmogenic Right Ventricular Cardiomyopathy

Academic Background

Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is an inherited cardiomyopathy characterized by the replacement of myocardial tissue with adipose and fibrous tissue, leading to arrhythmias, ventricular fibrillation, and even sudden cardiac death. The prevalence of ARVC ranges from 1:2000 to 1:5000, with male patients being more susceptible and experiencing more severe symptoms than females. Studies suggest that testosterone may play a significant role in the pathological process of ARVC, but its specific mechanisms remain unclear. In particular, whether testosterone exacerbates ARVC progression by promoting myocardial fibrosis lacks direct evidence.

To address this issue, researchers utilized patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes and cardiac fibroblasts to construct a three-dimensional (3D) engineered cardiac spheroid model, aiming to simulate the pathological process of ARVC and explore the role of testosterone. This study not only provides new insights into the pathological mechanisms of ARVC but also offers a potential experimental platform for drug screening and personalized treatment.

Source of the Study

This research was conducted by a collaborative team from the First Affiliated Hospital of Nanjing Medical University, Southeast University, Fujian Medical University, and other institutions. The primary authors include Hongyi Cheng, Xinrui Wang, Sichong Qian, and others. The paper was published online on January 7, 2025, in the journal Bio-design and Manufacturing, titled Deep phenotyping of testosterone-prompted fibrosis in arrhythmogenic right ventricular cardiomyopathy using iPSC-derived engineered cardiac spheroids.

Research Process and Results

1. Clinical Data Analysis and Feature Selection

The study first retrospectively analyzed clinical data from 60 ARVC patients, 19 of whom underwent 3D electrophysiological mapping. Using the Maximal Information Coefficient (MIC) algorithm for feature selection, the researchers found that gender differences were a significant factor influencing the distribution of low-voltage areas (LVAs). Male patients exhibited larger LVA areas, suggesting a potential link between testosterone and myocardial fibrosis.

2. iPSC Differentiation and Construction of Cardiac Spheroid Models

The research team extracted iPSCs from two ARVC patients with different genetic mutations (PKP2 c.336+1G>A and DSG2 1592T>G) and differentiated them into ventricular cardiomyocytes (VCMs) and iPSC-derived cardiac fibroblasts (iCFBs). By combining collagen type I and Geltrex matrix, the researchers constructed a 3D cardiac spheroid model. This model successfully replicated the pathological features of ARVC, including cardiomyocyte apoptosis, abnormal adipogenesis, and calcium handling dysfunction.

3. Functional Analysis of Cardiac Spheroids

The researchers conducted detailed analyses of the spheroids’ contractile function, calcium transients, and fibrosis levels. The results showed that spheroids carrying PKP2 and DSG2 mutations exhibited higher spontaneous contraction frequencies and irregular contraction patterns. Additionally, after treatment with dihydrotestosterone (DHT), the expression of the fibrosis marker α-smooth muscle actin (α-SMA) significantly increased in the mutant spheroids, indicating that DHT exacerbated the fibrotic process.

4. Gene Mutations and DNA Damage

Through Sanger sequencing and immunofluorescence staining, the researchers discovered that an intronic mutation in the PKP2 gene led to abnormal splicing of exon 2, thereby affecting the expression of intercellular junction proteins. Furthermore, DHT treatment exacerbated DNA damage and reactive oxygen species (ROS) levels in mutant cardiomyocytes, suggesting that testosterone promotes fibrosis through the ROS pathway.

5. Coculture Experiments and Mechanism Exploration

In coculture experiments, the researchers found that ROS released by mutant cardiomyocytes activated cocultured fibroblasts, transforming them into myofibroblasts and thereby exacerbating fibrosis. DHT treatment further enhanced this effect, indicating that testosterone acts as a “trigger” in the fibrotic process of ARVC.

Conclusions and Significance

This study provides the first direct in vitro evidence of the fibrogenic effect of testosterone in ARVC and reveals its molecular mechanism of exacerbating DNA damage and fibrosis through the ROS pathway. Additionally, the 3D cardiac spheroid model constructed in this study offers a powerful tool for pathological research and drug screening in ARVC. This achievement not only deepens the understanding of ARVC’s pathological mechanisms but also provides potential targets for future personalized treatments.

Research Highlights

1. Innovative Model: Utilized iPSC-derived cardiomyocytes and fibroblasts to construct a 3D cardiac spheroid model, successfully replicating the pathological features of ARVC.
2. Mechanistic Insights: First to reveal the molecular mechanism by which testosterone exacerbates ARVC fibrosis through the ROS pathway.
3. Clinical Significance: Provides a new experimental platform for personalized treatment and drug screening in ARVC.

Additional Valuable Information

The study also found that ROS released by mutant cardiomyocytes can activate fibroblasts, suggesting that intercellular communication plays a crucial role in the fibrotic process of ARVC. This discovery offers new directions for future therapeutic strategies, such as inhibiting ROS generation to slow fibrosis progression.

This research not only provides new insights into the pathological mechanisms of ARVC but also lays an important foundation for future clinical treatments and drug development.