{
  "abstract": "Introduction Cardiac function is governed by dynamic interactions between cardiomyocytes (CM) and fibroblasts. Beyond providing structural support, fibroblasts actively regulate CM electrophysiology, calcium handling, and contractile behaviour through direct cell-cell contact, paracrine signalling, and extracellular matrix remodelling. Disruption of this crosstalk contributes to maladaptive structural and electrical remodelling in cardiac disease.In the ventricle, myofibroblasts are well established modulators of CM resting membrane potential, action potential morphology and calcium transients. Contrastingly, the functional role of myofibroblasts in the atria remains poorly understood. Although atrial fibrosis strongly correlates with atrial fibrillation burden and persistence, whether atrial myofibroblasts directly modulate atrial CM calcium handling and electrophysiological behaviour beyond structural remodelling remains unclear.To address this gap, we generated multicellular three-dimensional (3D) spheroids composed of human induced pluripotent stem cell-derived atrial CM (hiPSC-aCM) and primary human atrial fibroblasts or myofibroblasts. This 3D co-culture platform recapitulates key features of atrial cell-cell interactions and provides a human-relevant model to investigate how fibroblast phenotypic state influences atrial CM function.Methods Human induced pluripotent stem cells were differentiated into atrial CM and purified at day 23 of differentiation. Purified atrial CM were assembled into 3D spheroids, using non-adherent microwells, incorporating 15% primary human atrial fibroblasts (control) or 15% atrial myofibroblasts, that had been activated with TGF-β (10ng/ml, 48h) prior to co-culture. Calcium transients were studied using optical mapping, with calcium dye (Fluo-4AM), during field stimulation at 1.5Hz. Transients were analysed using OPTIQ software. Unpaired T-tests were used for statistical analysis of 2 groups.Results Atrial fibroblasts treated with TGF-β acquired a myofibroblast genotype as determined by significant upregulation of fibrotic markers ACTA2 and COL1A1 compared to untreated controls (p=0.0200 and p=0.0204, respectively; n=3). Optical mapping revealed altered calcium handling in spheroids containing atrial myofibroblasts versus spheroids containing control fibroblasts. Calcium transient time to 50% calcium decay and time to 80% calcium decay were both significantly shorter (p=0.008; p=0.0073; n=11), while time to peak was unaffected.Conclusion Our results indicate that primary human atrial fibroblasts can be activated in vitro by exposure to TGF-β. By co-culturing hiPSC-aCM with fibroblasts or myofibroblasts we generated a relevant platform to study the role of fibroblast activation in atrial electrophysiology. The observed changes in calcium transient parameters suggest an active role of myofibroblasts in the electrophysiological remodelling observed in atrial pathologies.",
  "authors": [
    {
      "affiliations": [
        "University of Surrey, Guildford, United Kingdom"
      ],
      "name": "Millie Trowbridge"
    },
    {
      "affiliations": [
        "University of Surrey, Guildford, United Kingdom"
      ],
      "name": "Rahul Sanwlani"
    },
    {
      "affiliations": [
        "University of Surrey, Guildford, United Kingdom"
      ],
      "name": "Rachel Simmonds"
    },
    {
      "affiliations": [
        "University of Surrey, Guildford, United Kingdom"
      ],
      "name": "Patrizia Camelliti"
    }
  ],
  "title": "464 Atrial myofibroblasts alter calcium handling of hiPSC-atrial cardiomyocytes in 3D co-culture spheroids",
  "uid": "0be8c852-e36b-5440-af8f-3caf62208b9f"
}