Conditions mimicking obesity result in mitochondrial dysfunction in three-dimensional in vitro cardiovascular spheroid models

The U.S. Food and Drug Administration (FDA) recently announced a significant policy shift aimed at reducing animal testing by using alternative methodologies, including in silico models, spheroids, and organoids for drug development and screening. However, affordable, easy implementable cardiovascular spheroid and organoid models are still largely lacking.

The aims of this study was to 1) characterize a rat and human cardiovascular spheroid model and 2) investigate mitochondrial response to hyper-glycemic, lipidemic and insulinemic conditions.

Rat cardiomyoblast cells (H9c2) were cultured under standard conditions and seeded at 40x10³ cells/spheroid in ultra-low adherence plates with 25 mM glucose DMEM, 10% FBS, 1% pen/strep, whilst human ventricular cells (AC16) were cultured in DMEM/F12 media, 12.5% FBS and 1% pen/strep, and seeded at 30x10³ cells/well in media with 2.5% FBS. Both spheroid types were treated with 100 nM insulin and a combination of fatty acids. The H9c2 spheroids were harvested 96 hours after seeding, whilst the AC16 spheroids were cultured for 14 days. Messenger RNA expression levels (real-time PCR) and protein levels (Western blot) for metabolism, mitochondrial dynamics, function and autophagy were measured, whilst scanning and transmission electron microscopy (SEM and TEM) was used to image surface and cellular ultra-structure.

This study established reproducible spheroids for both cell types. Spheroid formation promoted filament development in both models, irrespective of treatment. The metabolic manipulation of H9c2 spheroids decreased the size and impacted the extra-cellular matrix (ECM) morphology. At a cellular level, metabolic manipulation significantly decreased insulin-stimulated phosphorylation of mTOR (Ser2481) and Akt (Ser472), increased mitochondrial biogenesis (PGC1-α) and fragmentation (phospho-Drp Ser616), and significantly increased mitochondrial β-oxidation. Treatment did not change G6PD, PFK2 or Cpt1b mRNA levels. The same treatment conditions decreased mRNA levels of Opa1, MAP1LC3B, ATP5A, CoQ7 and CoQ10B relative to the untreated control in the AC16 spheroid model, but did not affect the protein levels of ATM, mTOR, p62, or Opa1, nor ECM formation.

This study characterized two cardiovascular spheroid models that are stable, require little infrastructure to set up, and respond to metabolic stimuli. Whilst the H9c2 cardiac model mimic insulin resistance, the human ventricular spheroid model showed dysregulation at the transcriptional but not the protein level. Importantly, the long-term viability and metabolic responsiveness of the novel AC16 cardiovascular model provide a simplified, high-throughput platform to investigate mitochondrial response in metabolic disease development and drug screening.