CaMKII augments ROS and Ca2+ entry processes in female cardiac fibroblasts in hyperglycemic and hypertensive conditions

Introduction Heart Failure with preserved Ejection Fraction (HFpEF) affects over 50% of patients with HF, majority of whom are females with conditions such as hypertension, obesity and diabetes. Our in vivo animal models of these conditions show increased fibrosis and oxidized Calcium-calmodulin dependent protein kinase II (CaMKII) activity. At the cellular level, mitochondrial health is known to be implicated in HF, and our in vitro models have signified the impact of female cardiac fibroblasts (CFs) on altered calcium (Ca2+) signalling in cardiac myocytes (CMs), during co-culture. However, knowledge on the characteristics of CFs in HFpEF still remains obscure. Here, we investigated altered Ca2+ signalling, mitochondrial Reactive Oxygen Species (ROS) production, and the therapeutic potential of inhibiting CaMKII activity in CFs, during hyperglycaemia and hypertension.

Methods Adult human CFs (Promocell) sourced from both female and male donors were cultured under hyperglycaemic (22 mM Glucose), hypertensive (200nM Angiotensin II) or HFpEF-like (hyperglycaemic plus hypertensive) conditions, in the absence or presence of a CaMKII inhibitor (5µM KN93), for 48 hours. Following pathological conditioning of CFs, cells were loaded with Cal520AM calcium indicator or MitoSOX red mitochondrial superoxide indicator. Live cell fluorescence imaging was utilised to assess Ca2+ activity and mitochondrial ROS production in CFs.

Results Female CFs treated under hyperglycaemic conditions showed a greater Endothelin-1-induced Ca2+ transient amplitude (ΔF/F0) relative to control and hypertensive conditioning [Control: 0.184±0.008; Diabetes: 0.305±0.026; Hypertension: 0.208±0.030; HFpEF: 0.241±0.008, n=4passages]. Mitochondrial superoxide (AU) levels were elevated only in female CFs, in both hyperglycaemic and hypertensive conditions [Control: 18.0±2.0; Diabetes: 36.7±3.4; Hypertension: 29.5±4.5; HFpEF: 22.7±2.0, n=4passages]. These alterations in Ca2+ transient amplitude and superoxide production were impeded by the presence of KN93 [Ca2+ Transient Amplitude- Control: 0.197±0.020; Diabetes: 0.167±0.022; Hypertension: 0.199±0.037; HFpEF: 0.205±0.023, Mitochondrial superoxide- Control: 16.6±2.4; Diabetes: 14.7±2.6; Hypertension: 13.3±1.1; HFpEF: 18.1±2.6, [n=4passages].

Conclusions These results indicate CaMKII is important in mitochondrial oxidative stress in female CFs, in hyperglycaemic and hypertensive conditions. Further work is needed to investigate the importance of these processes in the development of fibrosis in HFpEF.