Calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) acts as a molecular switch regulating cardiovascular Ca handling and contractility in health and disease. Activation of CaMKIIδ is also known to regulate cardiovascular inflammation and is reported to be required for pro-inflammatory NF-κB signalling. In this study the aim was to characterise how CaMKIIδ interacts with and modulates NF-κB signalling and whether this interaction exists in non-contractile cells of the heart. Recombinant or purified CaMKIIδ and the individual inhibitory -κB kinase (IKK) proteins of the NF-κB signalling pathway were used in autoradiography and Surface Plasmon Resonance (SPR) to explore potential interactions between both components. Primary adult rat cardiac fibroblasts were then used to study the effects of selective CaMKII inhibition on pharmacologically-induced NF-κB activation as well as interaction between CaMKII and specific IKK isoforms in a cardiac cellular setting. Autoradiography analysis suggested that CaMKIIδ phosphorylated IKKβ but not IKKα. SPR analysis further supported a direct interaction between CaMKIIδ and IKKβ but not between CaMKIIδ and IKKα or IKKγ. CaMKIIδ regulation of IκΒα degradation was explored in adult cardiac fibroblasts exposed to pharmacological stimulation. Cells were stimulated with agonist in the presence or absence of a CaMKII inhibitor, autocamtide inhibitory peptide (AIP). Selective inhibition of CaMKII resulted in reduced NF-κB activation, as measured by agonist-stimulated IκBα degradation. Importantly, and in agreement with the recombinant protein work, an interaction between CaMKII and IKKβ was evident following Proximity Ligation Assays in adult cardiac fibroblasts. This study provides new evidence supporting direct interaction between CaMKIIδ and IKKβ in pro-inflammatory signalling in cardiac fibroblasts and could represent a feature that may be exploited for therapeutic benefit.

Exposure to circulating cobalt (Co 2+ ) in patients with metal-on-metal orthopaedic hip implants has been linked to cardiotoxicity but the underlying mechanism(s) remain undefined. The aim of the current study was to examine the effects of Co 2+ on the heart in vivo and specifically on cardiac fibroblasts in vitro. Adult male rats were treated with CoCl 2 (1 mg/kg) for either 7 days or 28 days. Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure Co 2+ uptake into various organs of the body. Co 2+ accumulated in the heart over time with significant levels evident after only 7 days of treatment. There was no evidence of cardiac remodelling following Co 2+ treatment as assessed by heart weight:body weight and left ventricular weight:body weight. However, a decrease in fractional shortening, as measured using echocardiography, was observed after 28 days of Co 2+ treatment. This was accompanied by increased protein expression of the ion transient receptor potential (TRP) channels TRPC6 and TRPM7 as assessed by quantitative immunoblotting of whole cardiac homogenates. Uptake of Co 2+ specifically into rat cardiac fibroblasts was measured over 72 h and was shown to dramatically increase with increasing concentrations of applied CoCl 2 . Expression levels of TRPC6 and TRPM7 proteins were both significantly elevated in these cells following Co 2+ treatment. In conclusion, Co 2+ rapidly accumulates to significant levels in the heart causing compromised contractility in the absence of any overt cardiac remodelling. TRPC6 and TRPM7 expression levels are significantly altered in the heart following Co 2+ treatment and this may contribute to the Co 2+ -induced cardiotoxicity observed over time.

Ageing is the greatest risk factor for cardiovascular disease. Calcium/calmodulin dependent protein kinase II (CaMKII) plays a fundamental role in the pathology of heart disease yet a potential role for CaMKII in cardiovascular pathology associated with ageing remains unclear. Taking a combined in vivo and in vitro approach, we have for the first time investigated whether CaMKII expression and CaMKII activity may be altered following age-related cardiovascular deterioration. Both cardiac contractility and aortic blood flow are compromised in aged rats and we have shown that this occurs in parallel with increased inflammation and crucially, autonomous activation of CaMKII. Endothelial cells isolated from young and aged aortae exhibit differences in cell phenotype and physiology. In line with observations in aortic tissue, aged aortic endothelial cells also show increased basal levels of pro-inflammatory markers and oxidative stress with concurrent increased basal activation of CaMKII. These results are the first to demonstrate that elevated CaMKIIexpression andCaMKII activation occur in parallel with the pathological progression associated with ageing of the heart and vasculature. Specifically, CaMKIIexpression is significantly increased and activated in the endothelium of aged aorta. As such, CaMKII could serve as an important marker of endothelial dysfunction that accompanies the ageing process and may be an appropriate candidate for investigating targeted therapeutic intervention.

Nuclear factor-kappa beta (NF-kβ) pro-inflammatory signalling is important in modulating endothelial dysfunction and may be important in vascular dysfunction associated with the ageing process. Recent studies in the heart have highlighted Calcium/calmodulin-dependent protein kinase II (CaMKII) as a novel regulator of NF-kβ signalling. However nothing is known of the role this interaction could play in regulating dysfunction of the vasculature during ageing. Here we (i) characterise NF-kβ signalling in vascular endothelial cells and examine the potential for CaMKII modulation and (ii) determine whether CaMKIIδ expression is altered in ageing.Using human umbilical vein endothelial cells (HUVECs) as an in vitro model system, initial experiments have established that pro-inflammatory NF-kB signalling is activated in response to both tumour necrosis factor α (TNF-α) and interleukin-1β (IL-1β) stimulation. This was shown by a significant reduction in IkBα expression (1.18 ± 0.16 vs. 0.48 ± 0.13, control vs. TNF-α 15 min stimulation , p = 0.008, n = 3); (1.53 ± 0.22 vs. 0.16 ± 0.09 control vs. IL-1β 15 min stimulation ,p = 0.004, n = 3) and increased phosphorylation of the p65 subunit (0.69 ± 0.1 vs. 1.22 ± 0.12 ,control vs. TNF-α 5 min stimulation, p = 0.006, n = 3); (0.54 ± 0.07 vs. 1.86 ± 0.21, control vs. IL-1β 5 min stimulation , p = 0.005, n = 3). We have identified CaMKIIδ as the predominant isoform expressed in HUVECs and shown CaMKII is also activated in response to TNF-α (1.3-fold increase at 10 min stimulation, p = 0.01, n = 3) and IL-1β. Further work has assessed expression of CaMKIIδ in freshly isolated rat aorta. Tissue lysate preparations have compared CaMKIIδ expression in young and aged aortae. Parallel studies in hearts from the same animals have shown CaMKIIδ expression is increased in whole ventricular homogenates prepared from aged versus young hearts (2.3-fold increase, n = 3). These results reflect similar alterations in CaMKIIδ aged hearts as previously observed in models of cardiovascular disease. This highlights the possibility that CaMKII could be pivotal in modulating cardiovascular changes that occur during ageing.Future work will involve selective inhibition of CaMKIIδ in the HUVEC model system by siRNA transfection prior to cytokine stimulation. This will establish involvement of CaMKIIδ in modulation of NF-kB signalling in a vascular endothelial cell line. Studies can then be performed in primary aortic endothelial cells from both young and aged animals to further investigate this interaction and the potential importance in modulating endothelial dysfunction during ageing.