Development of human precision cut heart slices as a novel platform for target identification and validation in the heart

Abstract

Cardiovascular disease is a term used to broadly describe a range of devastating conditions, each with poor prognosis and limited treatment options. Cardiac fibrosis is one of the primary mechanisms attributed to heart failure and is characterised by cardiomyocyte loss, accumulation of extracellular matrix and increased stiffness of the cardiac muscle, culminating in impaired cardiac function. Widely employed in vitro and in vivo models used to study mechanisms of cardiac fibrosis have significant limitations and fail to account for the complexity of human heart tissue. Therefore these mechanisms need to be interrogated in more physiologically relevant human models to identify novel therapeutic targets. Here we describe the development of a novel physiologically relevant ex vivo human precision cut heart (PCHS) model for investigating mechanisms of cardiac fibrosis and determining the efficacy of novel therapeutics. Left ventricular tissue was processed to create PCHS and cultured in conventional static culture or using a novel bioreactor system. Differences in PCHS viability and metabolic activity demonstrated the beneficial effects of the bioreactor system on PCHS culture. To investigate inflammation and fibrosis, PCHS were challenged with recombinant human TGF-β1, CTGF, IL-11, Angiotensin II or Galectin-3. Fibrotic and inflammatory responses were assessed through quantification of chemokines, cytokines, ECM and matrix remodelling proteins. Next, unbiased mass-based spectrometry and bulk RNA sequencing was utilised to assess changes in gene and protein expression. Using these methods we discovered that Galectin-3 induced a robust inflammatory response in PCHS, whilst TGF-β1 produced a potent fibrotic response. Ingenuity Pathway Analysis (IPA) identified upstream regulators of inflammation and fibrosis and 32 candidate compounds were selected for assessment as potential novel therapeutics. Of these compounds, n=11 exhibited promising anti-inflammatory effects whilst n=9 exhibited robust anti-fibrotic effects in PCHS. Finally through the use of four distinct cutting-edge molecular biology approaches, we identified number of soluble factors present in the circulation of a well-characterised cohort of patients with a spectrum of severity of cardiac disease. These included 32 differentially expressed proteins, identified by proteomic analysis and 8 markers identified using multi-plex ELISA. We have optimised and developed a medium throughput culture system of 3D human cardiac tissue. The PCHS are highly reproducible and viable enabling us to interrogate mechanisms regulating inflammation and fibrosis in intact human tissue. We are able to modulate inflammation and fibrosis through the application of novel therapeutic compounds to determine their efficacy in human cardiac tissue. Whilst a small scale study of a well characterised group of female DMD carriers with a range in severity of cardiac involvement has identified a number of potential blood based biomarkers of cardiac fibrosis.