Introduction: Myocardial ischemia leads to contractile dysfunction and myocardial damage. We established a hypoxic living myocardial slice (LMS) model to study the myocardium's molecular and cellular alterations occurring during hypoxia. We aimed to identify novel protective therapies using an integrative multi-omics method. LMS are precision-cut thin tissue sections of myocardial tissue and aim to bridge the gap between in vivo and in vitro models with multicellular context and functional readouts.

Purpose: This study aims to investigate the contractile response and alterations in gene and protein expressions of LMS in hypoxic conditions and to determine molecular candidates that can modulate contractile impairment in hypoxia.

Methods and Results: LMS were generated from adult rat, pig and human end-stage heart failure hearts and were subjected to 9% O₂ concentration or kept in normoxic conditions. Hypoxia and normoxia-LMS were cultivated for 24 hours in biomimetic cultivation chambers with continuous force recordings, or force-length relationships were measured upon acute decrease in oxygen to 9% vs control-LMS kept at 18% O₂. Morphological studies were done to quantify cardiomyocyte cross-sectional area and sarcomere length. Additionally, the expressions of marker genes for hypoxia and genes involved in electromechanical coupling were measured by RT-qPCR. RNAseq and protein/metabolites mass spectrometry-base profiling was done from porcine normoxic and hypoxic LMS. Multi/panomics analysis was performed using mixOmics DIABOLO and MibiOmics WGNCA, combining information about LMS force and RNA/proteins/metabolites expressions.

LMS of three species were cultured at 18% or 9% O₂, and contractile function was recorded continuously for 24h. The force after 24 hours in LMS significantly decreased for all three species. We also measured the acute functional effects of hypoxia development in fresh rat LMS. The LMS were stretched to 200% of the original length, and the force was decreased in hypoxic LMS. To assess morphological changes, cross-sections of PFA-fixated rat LMS were performed and stained with Hoechst33342 and WGA. The cardiomyocyte cross-sectional area was significantly smaller in hypoxic rat LMS. The sarcomere length variability of the slices was significantly larger in the 9% O₂ LMS group. We also observed changes in marker gene expressions for hypoxia and expressional changes of genes coding ion channels, calcium handling genes and stress responses.

Further, we analyzed parts of the same porcine LMS by RNAseq or mass spectrometry-based proteomics/metabolomics. A multi-omics model determined a differentiating signature of hypoxic samples using selected genes. Each sample had its active force annotated for integrated analysis to produce a panomics dataset. MixOmics analysis revealed BAG3 as an important, negatively correlated protein to the amplitude.

Conclusion: Based on contractile, structural, and multi/pan-OMICs analyses, we report a novel multi-species ex-vivo model of cardiac hypoxia in the acute and cultured LMS. We found that BAG3, a known player in heart disease, was deregulated in our model. These findings suggest that targeting BAG3 regulation and expression in our model is a potential strategy for finding a treatment for ischemic heart disease.