RWOP3 Cardiac fibroblasts modulate Ca²⁺signalling in cardiac myocytes under hyperglycaemic and hypertensive conditions

Heart Failure with preserved Ejection Fraction (HFpEF), is a multifactorial disease associated with hypertension, diabetes and obesity, and is predominantly observed in females. HFpEF results in a slowly relaxing, fibrotic myocardium, and modifications in cardiomyocyte (CM) Ca²⁺-handling-proteins are observed in animal models in vivo. However, the role of cardiac fibroblasts (CFs) in the HFpEF phenotype remains unclear. This study aims to develop a HFpEF-like cellular model to explore abnormal Ca²⁺-signalling of CFs and CMs in diabetic-hypertensive conditions. Adult human male and female CFs (Promocell) and primary left ventricular CMs isolated from adult NZW male rabbits, were cultured independently or co-cultured using media containing 22 mM Glucose, 200 nM Angiotensin-II or a combination of both (to mimic diabetes, hypertension or HFpEF, respectively), for a duration of 24h. Both cell types were then loaded with 2µM Cal520AM calcium indicator. CFs were chemically stimulated using 20 nM Endothelin-1 while CMs were electrically field-stimulated at 1Hz to generate Ca²⁺-responses. The alterations in intracellular Ca²⁺ were measured by confocal microscopy. The Ca²⁺-transient-amplitude (ΔF/F₀) in CFs was enhanced with 22 mM Glucose [Control: 0.158±0.015; Diabetes: 0.273±0.014; n=3 humans with 3 technical replicates each; p< 0.001]. The Ca²⁺-transient-amplitude in CMs increased with Angiotensin-II [Control: 0.750±0.101; Hypertension: 2.02±0.32; N=3 rabbits; p< 0.0001]. In myocytes co-cultured with female CFs, the Ca²⁺-transient-amplitude was sustained with Glucose and Angiotensin-II [Control: 0.346±0.064; Diabetes: 0.359±0.049; Hypertension: 0.296±0.037; N=3 rabbits]. However, Ca²⁺-spark-frequency (sparks.µm².s⁻¹) in CMs was elevated after co-culture with female CFs, across all pathological conditions [Control: 3.56±0.71; Diabetes: 13.0±1.1; Hypertension: 16.5±1.1; HFpEF: 22.9±1.2; N=3 rabbits; p< 0.001]. These findings show remodelling of Ca²⁺-signalling in CFs and CMs, in an in vitro model of HFpEF. A sex disparity observed in female co-culture mimics that seen in human HFpEF. Further work is needed to understand how this in vitro model correlates to an in vivo model of HFpEF.