Introduction
Heart Failure with preserved Ejection Fraction (HFpEF), is a multifactorial disease associated with hypertension, diabetes and obesity, and is predominantly observed in females1. HFpEF results in a slowly relaxing, fibrotic myocardium, and modifications in cardiomyocyte (CM) Ca2+-handling-proteins are observed in animal models in vivo2. However, how patient comorbidities may influence cardiac fibroblasts (CFs) and how this may then influence myocytes in the HFpEF phenotype remains unclear. This study aims to develop an in vitro model -which includes aspects of HFpEF to explore abnormal Ca2+-signalling of CFs and CMs in hyperglycaemic-hypertensive conditions.
Methods
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 22mM Glucose (HG), 200nM Angiotensin-II (AngII) 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 20nM Endothelin-1 while CMs were electrically field-stimulated at 1Hz to generate Ca2+-responses. The alterations in intracellular Ca2+ were measured by confocal microscopy.
Results
The Ca2+ transient amplitude (ΔF/F0) in CFs was enhanced with 22mM Glucose (HG) [Control: 0.158±0.015; HG: 0.273±0.014; n=3 humans with 3 technical replicates each; p< 0.001]. The Ca2+-transient-amplitude in CMs increased with AngII [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 HG and AngII [Control: 0.346±0.064; HG: 0.359±0.049; Ang: 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].
Conclusions
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.