Raghu Nagalingam ¹ Farah Jayousi ¹ Homa Hamledari ¹ Dina Hosseini ¹ Saif Dababneh ² Chloe Lindsay ³ Ramon I. Klein Geltink ⁴ Philipp F Lange ¹ Ian Michael Dixon ⁵ Robert Alan Rose ⁶ Michael Czubryt ⁵ Glen Findlay Tibbits ^1,2,3*

• ¹ Cellular and Regenerative Medicine Centre, BC Children's Hospital Research Institute, Vancouver, British Columbia, Canada
• ² University of British Columbia, Vancouver, British Columbia, Canada
• ³ Simon Fraser University, Burnaby, British Columbia, Canada
• ⁴ BC Children's Hospital Research Institute, Vancouver, British Columbia, Canada
• ⁵ University of Manitoba, Winnipeg, Manitoba, Canada
• ⁶ University of Calgary, Calgary, Alberta, Canada

Mechanical stress and pathological signaling trigger the activation of fibroblasts to myofibroblasts, which impacts extracellular matrix composition, disrupts normal wound healing, and can generate deleterious fibrosis. Myocardial fibrosis independently promotes cardiac arrhythmias, sudden cardiac arrest, and contributes to the severity of heart failure. Fibrosis can also alter cell-to-cell communication and increase myocardial stiffness which eventually may lead to lusitropic and inotropic cardiac dysfunction. Human induced pluripotent stem cell derived cardiac fibroblasts (hiPSC-CFs) have the potential to enhance clinical relevance in precision disease modeling by facilitating the study of patient-specific phenotypes. However, it is unclear whether hiPSC-CFs can be activated to become myofibroblasts akin to primary cells, and the key signaling mechanisms in this process remain unidentified. We hypothesize that the passaging of hiPSC-CFs, like primary cardiac fibroblasts, induces specific genes required for myofibroblast activation and increased mitochondrial metabolism. Passaging of hiPSC-CFs from passage 0 to 3 (P0 to P3) and treatment of P0 with TGFβ1 was associated with a gradual induction of genes to initiate the activation of these cells to myofibroblasts, including collagen, periostin, fibronectin, and collagen fiber processing enzymes with concomitant downregulation of cellular proliferation markers. Most importantly, canonical TGFβ1 and Hippo signaling component genes including TAZ were influenced by passaging hiPSC-CFs. Seahorse assay revealed that passaging and TGFβ1 treatment increased mitochondrial respiration, consistent with fibroblast activation requiring increased energy production, whereas treatment with the glutaminolysis inhibitor BPTES completely attenuated this process. Based on these data, the hiPSC-CF passaging enhanced fibroblast activation, activated fibrotic signaling pathways, and enhanced mitochondrial metabolism approximating what has been reported in primary cardiac fibroblasts. Thus, hiPSC-CFs may provide an accurate in vitro preclinical model for the cardiac fibrotic condition, which may facilitate the identification of putative anti-fibrotic therapies, including patient-specific approaches.