Engineering Myocardial Anisotropy to Model Electrical Dysfunction and Pharmacological Responsiveness in Arrhythmogenic Cardiomyopathy

The development of engineered cardiac tissues has significantly advanced our understanding of cardiac disease mechanisms and improved therapeutic screening for cardiomyopathies. In arrhythmogenic cardiomyopathy (ACM), an inherited disorder characterized by defective intercellular adhesion and mislocalization of connexin-43 (Cx43) due to mutations in desmosomal proteins like plakophilin-2 (PKP2) and plakoglobin (PKG), the alignment of cardiomyocytes is crucial for influencing electrical propagation and therapeutic outcomes. To explore the effects of myocardial anisotropy on ACM phenotypes, we designed and built cardiac tissue chips using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs). These cells were cultured on micromolded gelatin substrates that mimicked longitudinal, transverse, or isotropic myocardial structures. Tissues with PKP2 or PKG mutations exhibit typical ACM characteristics, such as slowed conduction, disrupted Cx43 localization, and increased arrhythmic risk. Treatment with glycogen synthase kinase-3β inhibitor SB216763 successfully restored Cx43 localization and conduction velocity, particularly in longitudinally aligned tissues, but showed limited effectiveness in transverse and isotropic configurations. These results emphasize the necessity of incorporating physiological myocardial anisotropy into engineered cardiac models to accurately recapitulate ACM pathophysiology and improve the predictive accuracy of therapeutic screenings. This methodology provides a promising foundation for precision medicine, facilitating better predictions of therapeutic efficacy and personalized treatment strategies for patients with ACM.