Introduction:

Heart Failure with preserved Ejection Fraction [HFpEF] is a pandemic associated with hypertension and diabetes amongst other co-morbidities. It has been established that fibrosis plays a major role in this form of heart failure. However, the functional mechanisms of cardiac fibroblasts (CFs) in HFpEF and more importantly, how these cells interact with cardiomyocytes (CMs) and impact calcium (Ca²⁺) signalling is limited. This limitation is majorly due to a lack of physiologically relevant cellular models.

 

Aims/Objectives:

To study the CF-CM interaction in an in vitro model of HFpEF, this study aimed to evaluate the Ca²⁺ response of CFs in diabetic, hypertensive, and HFpEF conditions. Furthermore, the electrophysiology of CMs co-cultured with CFs, under these pathological conditions was assessed.

 

Methods:

Adult human male CFs (HcFb) (Promocell) at Passage 3 were seeded at a density of 1×10⁴ cells per well in a 96-well plate and grown for 24 hours in fibroblast growth medium 3 (FGM3) supplemented with 10% Foetal Calf Serum (FCS) and 1% Penicillin/Streptomycin (P/S). Cells were serum starved for 24 hours prior to treatment. For CFs Ca²⁺ signalling, fibroblasts were treated for 72 hours with high glucose (22mM) to simulate diabetes, high Angiotensin-II (Ang-II) (200nM) to simulate hypertension, or both conditions to simulate HFpEF. Cells were stained with 2µM Cal520 AM calcium indicator and the Ca²⁺ response induced by the addition of 20nM Endothelin-1 was measured by confocal microscopy. For co-culture, adult male rabbit CMs were freshly isolated following all ethical requirements by the UK Home Office Licence. CMs were cultured with(out) CFs in either Medium 199 (M199) or FGM3, using treatment conditions similar to the above. Following treatment, CMs were harvested, stained, and electrically stimulated at 1Hz to measure single cell Ca²⁺ transients.

 

Results:

The Ca²⁺ transient amplitude of CMs was significantly reduced when cultured in FGM3 or co-cultured with HcFb in M199, compared to M199 (M199: 0.766±0.112 versus FGM3: 0.145±0.0266; co-cultured myocytes in M199: 0.347±0.0385, N=1 rabbit and n≥5 cells per group; p< 0.001, one-way ANOVA). Endothelin-1-induced Ca²⁺ response in CFs was significantly enhanced with high glucose when compared to control, hypertensive and HFpEF conditions (Control: 3.67±1.32 versus Diabetic: 13.7±1.34; Hypertensive: 6.29±1.04; HFpEF: 7.14±1.27, N=2 biological replicates with n=3 technical replicates each; p< 0.05, one-way ANOVA). Using the same treatment conditions, similar effects on Ca²⁺ transient amplitude were observed in electrically stimulated co-cultured CMs. (Control: 0.294±0.0164 versus Diabetic: 0.716±0.108; Hypertensive: 0.611±0.0935; HFpEF: 0.546±0.0460, N=1 rabbit and n≥5 cells per group; p< 0.05, one-way ANOVA). These results are contrary to existing data that show no significant change in Ca²⁺ transient amplitude of CMs treated with high glucose and increased amplitude with high Ang-II treatment.

 

Conclusions:

Ultimately, these findings suggest different regulation of Ca²⁺ by CFs during hypertension and HFpEF when compared with that seen during diabetes. Furthermore, data indicate a notable influence of CFs on CM Ca²⁺ transient amplitude. Ongoing work is exploring the role and mode of intercellular communication by both cell types in cardiomyopathy.