Compromised cardiovascular function in aged rats corresponds with increased expression and activity of calcium/calmodulin dependent protein kinase IIδ in aortic endothelium.

Ageing is the greatest risk factor for cardiovascular disease. Calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) plays a fundamental role in the pathology of heart disease yet a potential role for CaMKIIδ 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 CaMKIIδ 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 CaMKIIδ expression and CaMKII activation occur in parallel with the pathological progression associated with ageing of the heart and vasculature. Specifically, CaMKIIδ expression is significantly increased and activated in the endothelium of aged aorta. As such, CaMKIIδ 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.

Cobalt Administration Causes Reduced Contractility with Parallel Increases in TRPC6 and TRPM7 Transporter Protein Expression in Adult Rat Hearts.

Exposure to circulating cobalt (Co2+) 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 Co2+ on the heart in vivo and specifically on cardiac fibroblasts in vitro. Adult male rats were treated with CoCl2 (1 mg/kg) for either 7 days or 28 days. Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure Co2+ uptake into various organs of the body. Co2+ accumulated in the heart over time with significant levels evident after only 7 days of treatment. There was no evidence of cardiac remodelling following Co2+ 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 Co2+ 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 Co2+ specifically into rat cardiac fibroblasts was measured over 72 h and was shown to dramatically increase with increasing concentrations of applied CoCl2. Expression levels of TRPC6 and TRPM7 proteins were both significantly elevated in these cells following Co2+ treatment. In conclusion, Co2+ 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 Co2+ treatment and this may contribute to the Co2+-induced cardiotoxicity observed over time.

CaMKIIδ interacts directly with IKKß and modulates NF-κB signalling in adult cardiac fibroblasts.

Calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) acts as a molecular switch regulating cardiovascular Ca2+ 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 IKKß but not IKKα. SPR analysis further supported a direct interaction between CaMKIIδ and IKKß 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 IKKß was evident following Proximity Ligation Assays in adult cardiac fibroblasts. This study provides new evidence supporting direct interaction between CaMKIIδ and IKKß in pro-inflammatory signalling in cardiac fibroblasts and could represent a feature that may be exploited for therapeutic benefit.