Introduction

Type 2 Diabetes Mellitus (T2DM) is known to affect over 5.6 million individuals in the UK alone (1) with cardiovascular disease (CVD) a common comorbidity. During T2DM-CVD, complex cellular dysfunction occurs involving endothelial cell loss, cardiac myocyte damage & subsequent fibrosis resulting in poor clinical outcomes (2, 3). Here we demonstrated for the first time the novel iPSC-derived vascularised cardiac organoid (VCO), derived from non-diabetic (ND) and diabetic (DB)-donors, demonstrating both cardiac and endothelial cells in a 3D structure, more closely emulating the human myocardium. Cardiac-endothelial cell mitochondria are susceptible to damage, leading to impaired function. Damaged mitochondria would be targeted for mitophagy, triggering the renewal of mitochondrial biogenesis, here we investigated the impairment of mitophagy within cells during T2DM. Telomere shortening and dysfunction is established in T2DM, with dysfunction of the telomeric repeat binding factor-interactive nuclear factor (TINF)-2 protein associated with mitochondrial loss in T2DM (4,5).

Aims and objectives

Data is currently lacking regarding the role of TINF2 signaling in cardiac or vascular cell signaling, cardiac toxicity and T2DM. Here we demonstrated the effects of TINF2 using the novel 3D VCO and its novel effects on cardiac and endothelial cell interaction in T2DM, and the re-establishment of mitophagy.

Methods & Results

iPSCs from DB and ND donors (3 individual donors, n3) 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.

Both DB- and ND-derived iPSCs successfully differentiated into structurally mature VCOs, showing robust and consistent expression of key cardiac cellular markers and endothelial cellular markers confirmed with RT-PCR and western blot analysis (n3, p<0.05-0.0001).

Confocal imaging confirmed the presence of vascular-like networks within the VCOs, blood vessel-like structures closely integrated with surrounding cardiac cells. VCOs were successfully characterised for cellular markers using RT-PCR, western analysis & combined large scale RNA sequencing to demonstrate the T2DM profile, revealing significant changes in molecular signaling of AMPK, mTOR, PTEN, ANP & BNP (n3, p<0.05-0.0001).

Moreover, 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). Correspondingly TINF2 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). Using shRNA, we transgenically knocked down (KD) TINF2 within DB VCOs, significantly reversing the previously demonstrated effects of DB, cardiac toxicity and mitochondrial loss of function. Mitophagy signaling was restored, whilst further reversing mitophagy and DB-associated loss of key signaling pathways (n3, p<0.05-0.001).

Conclusion

Using the VCO we demonstrate key functional loss of mitochondria and impairment of mitophagy within cardiac-specific tissue, in a glucose-independent state. We demonstrated the upregulation of TINF2 within cardiac-specific tissue for the first time, and as a key gene responsible for the impairment of mitophagy, setting the foundation for a novel therapeutic target for T2DM.

Ethics statement

All procedures were carried out in accordance with UK legislation.