{
  "abstract": "Introduction Type 2 Diabetes Mellitus (T2DM) affects over 3.7 million individuals in the UK alone with cardiovascular disease (CVD) a major co-morbidity, all resulting from complex cellular dysfunction occurring involving endothelial cell loss, cardiac myocyte damage & subsequent fibrosis resulting in poor clinical outcomes.Studies using 2D cardiac cell cultures fall short in mimicking the structural and functional intricacies of diabetic heart tissue, especially endothelial-cardiac interactions and vascular integration. T2DM animal studies fail to capture similar results when transitioned to clinical studies. We developed a novel 3D vascularised cardiac organoid (VCO) platform derived from diabetic (DB) and non-diabetic (ND) donor-specific induced pluripotent stem cells (iPSCs), enabling the integration of blood vessels during cardiac lineage commitment, resulting in a multicellular 3D structure that more closely emulates the native human myocardium.Cardiac cell mitochondria are susceptible to oxidative stress, superoxide and ROS-mediated damage, during T2DM-assicated CVD leading to severely impaired function and impaired mitophagy. The Forkhead transcription factor-O1 (FOXO1) was shown to negatively modulate mitochondrial function during the presence of high glucose. Whilst overexpression of the TERF1-interactive nuclear factor 2 (TINF2) is associated with mitochondrial dysfunction, leading to the indirect inhibition of antioxidant signaling via mTOR signaling and directly limiting PINK1/PARKIN signaling for mitophagy induction. However, data is lacking regarding the role of TINF2-FOXO1 signaling in cardiac or vascular cell signaling, cardiac toxicity and T2DM. Here we demonstrated the effects of TINF2-FOXO1 using the novel 3D VCO and its novel effects on cardiac and endothelial cell interaction in T2DM.Methods and Results iPSCs from DB and ND donors were differentiated into 3D-VCOs over a 20-day protocol, a novel approach combining mesodermal bodies programmed for either vascular or cardiac specific lineage commitment, allowing for the spontaneous formation of vascular-like networks directly within the cardiac tissue.DB and ND-derived iPSCs successfully differentiated into structurally mature VCOs, showing robust and consistent expression of key cardiac cellular markers (TNNT2, MYLC7, vimentin) and endothelial cellular markers for adhesion junctions and components (CD31, CD144, VWF), confirmed with RT-PCR and western blot analysis. ICC confocal imaging confirmed the presence of vascular-like networks within the 3D organoids, a blood vessel-like structures closely integrated with surrounding cardiac cells. No significant differences were observed in structural or characterization gene or protein expression between DB and ND groups (n3, p>0.05).VCOs were successfully characterised for cellular markers using RT-PCR, western analysis & combined RNA sequencing to demonstrate the T2DM profile, as close to a patient profile as possible, revealing significant changes in molecular signaling of AMPK, mTOR, PTEN, ANP & BNP. (n3, p<0.05-0.0001).Mitochondrial-targeted functional assays demonstrated T2DM-induced mitochondrial-associated depolarization, ROS increase and calcium-influx within the cardiac-endothelial cell population in ND and DB VCO, as well as impaired mitophagy and reduced lysosome clearance (n3, p<0.05-0.0001).TINF2-FOXO1 expression was found to be significantly increased in DB VCOs compared to ND VCOs using RT-PCR, western blot analysis and ICC imaging (n3, p<0.05-0.001). shRNA transgenic knocked-down (KD) of the TINF2 gene within DB VCOs significantly reversing DB-associated effects, cardiac toxicity and mitochondrial loss of function. Crucially, mitophagy signaling was restored, whilst further reversing DB-associated loss of mitophagy key signaling pathways (n3, p<0.05-0.001).Conclusion The VCO demonstrated key functional loss of mitochondria and impairment of mitophagy within cardiac-specific tissue, in a glucose-independent state of T2DM and demonstrated the upregulation of TINF2-FOXO1 within cardiac-specific tissue for the first time. TINF2 was identified as a key gene responsible for the impairment of mitophagy and mitochondria. Here we set the foundation for a novel therapeutic target for T2DM. Data generated will be incorporated into a multi-omics approach and digital twins’ model to vastly understand key epigenetic regulatory genes and improving the understanding of the crucial role of cardiac and vascular-specific cell-cell interaction during T2DM.These functional alterations mirror key features of diabetes-associated cardiac pathology, our platform effectively recapitulates disease phenotypes in vitro. As such, our newly developed, first-of-its-kind, patient-derived 3D VCO model can serve as highly valuable, physiologically relevant models for advancing mechanistic studies and the development of targeted therapeutic strategies for diabetes-related cardiovascular disease.",
  "authors": [
    {
      "affiliations": [
        "The Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, Belfast, United Kingdom"
      ],
      "name": "Refik Kuburas"
    },
    {
      "affiliations": [
        "The Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, Belfast, United Kingdom",
        "Department of Anatomy, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia"
      ],
      "name": "Wiwit Ananda Wahyu Setyaningsih"
    },
    {
      "affiliations": [
        "The Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, Belfast, United Kingdom",
        "Department of Clinical Anatomy, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia"
      ],
      "name": "Asim J Tashkandi"
    },
    {
      "affiliations": [
        "The Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, Belfast, United Kingdom"
      ],
      "name": "Abigail Gorman"
    },
    {
      "affiliations": [
        "The Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, Belfast, United Kingdom"
      ],
      "name": "Andriana Margariti"
    }
  ],
  "title": "201 TINF2 impairs mitophagy in diabetic cardiac cells using the newly developed vascularised-cardiac organoid from T2DM donor iPSCs",
  "uid": "b92a482f-43a3-5d74-aace-7fa79b3509a1"
}