Mitochondria are crucial for cardiomyocytes, providing the energy required for their contractile activity and playing pivotal roles in cellular homeostasis. The constitution of mitochondrial networks in cardiomyocytes is highly dynamic through continuous fusion and fission. Moreover, the clearance of damages/oxidized mitochondria via mitophagy is important for maintaining cellular function. Disturbances in these processes eventually lead to elevated production of reactive oxygen species (ROS) and subsequent cell damage.
The AAA-domain-containing ATPase 3A (ATAD3A) is a key regulator of mitochondrial dynamics, including fusion, fission, mitophagy, and cristae morphology. It also contributes to the maintenance of mitochondrial function by modulating the respiratory chain and mitochondrial DNA (mtDNA) dynamics. Structurally, ATAD3A comprises two monomeric subunits that span both mitochondrial membranes, allowing it to interact with various regulators of mitochondrial homeostasis. Mutations in the ATAD3A gene have been associated with neurological disorders and cardiomyopathies.
In this project, we aim to investigate the impact of the newly identified point mutation ATAD3A(R109P), located in the structurally crucial coiled-coil domain 1 (CC1), on mitochondrial structure and function in cardiomyocytes.
Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) served as a model system. Patient-specific iPSCs were reprogrammed from skin fibroblasts and differentiated into cardiomyocytes using an established protocol. These were compared to iPSC-CMs derived from healthy donors (WT). Flow cytometry analysis confirmed a high purity of 98.2% cTnT-positive cells in the patient-derived iPSC-CMs. Afterwards, iPSC-CMs were matured in fatty acid-supplemented maturation medium for 4 weeks. Video-based contraction analyses showed significant alterations in patient-derived iPSC-CMs, including increased beating rate, shortened relaxation time, and reduced maximum contraction velocity compared to WT iPSC-CMs. In addition, patient-derived iPSC-CMs showed a higher expression of dynamin-related protein 1 (DRP1) and PTEN-induced kinase 1 (PINK1), important regulators of mitochondrial dynamics. Live-cell imaging demonstrated increased mitochondrial-lysosomal colocalization in the patient-derived iPSC-CMs compared to WT iPSC-CMs, although lysosomal density and mitochondrial structures appeared comparable between WT- and patient-derived iPSC-CMs. Given the central role of mitophagy in removing dysfunctional mitochondria, we assessed ROS levels and mitochondrial function. Live-cell imaging with H2DCFDA revealed elevated ROS production in patient-derived iPSC-CMs compared to WT iPSC-CMs. Analysis of mitochondrial function in Seahorse MitoStress Test assays revealed an elevated proton leak in patient-derived iPSC-CMs, while ATP-production, as well as basal and maximal respiration remained comparable to WT cells.
Taken together, these results indicate that the ATAD3A(R109P) mutation induces mitochondrial dysfunction, characterized by elevated ROS production and enhanced mitophagy in iPSC-CMs. As mitophagy represents a key for mitochondrial quality control, especially in energy-demanding tissues such as the heart, these molecular defects may contribute to the development of patient cardiomyopathy. Ongoing studies aim to correct the ATAD3A mutation in the patient cells to examine the causal relationship between mutation and mitochondrial dysfunction.
