Inositol trisphosphate receptor contribution to ventricular cardiac fibroblast function in healthy and hypertrophied mouse hearts

Currie, Susan and Martin, Tamara Patricia and Salleh, Hanif and McCluskey, Claire and Bushell, Trevor John (2016) Inositol trisphosphate receptor contribution to ventricular cardiac fibroblast function in healthy and hypertrophied mouse hearts. FASEB Journal, 30 (1 Supp). ISSN 0892-6638

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Abstract

Cardiac Fibroblasts play a critical role in pathological cardiac remodelling. Using a minimally invasive transverse aortic banding (MTAB) mouse model of cardiac hypertrophy, we investigated whether Inositol trisphosphate receptor type 2 (IP3R2) expression and activity contributes not only to cardiac contractile dysfunction, but also to changes in cardiac fibroblast function. Cardiac contractility was assessed in vivo using echocardiography and in vitro using stimulation of isolated tissue preparations in organ baths. Cardiac fibroblasts were isolated from sham-operated and MTAB hearts under sterile conditions using chunk digestion and maintained in short-term culture. Quantitative immunoblot assessment of whole ventricular homogenates showed that IP3R2 expression was significantly increased in MTAB hearts (IP3R2:GAPDH ratio: 0.378±0.06 vs 0.130±0.03, MTAB vs sham respectively, n=7, p<0.001). Further individual analysis of samples comparing in vivo contractile function with corresponding IP3R protein expression showed a clear distinction between the sham and MTAB groups. MTAB animals with <40% Fractional Shortening showed a clear correlation with higher expression of IP3R2 (Spearman r=−0.79, p<0.05, n=15). The contribution of IP3R activity to contractile function was further assessed in vitro using isolated ventricular preparations stimulated with 1uM isoprenaline in the presence and absence of the Ca2+ release modulator 2-aminoethyl diphenylborinate (2-APB) (10uM). Preparations from both MTAB and sham-operated hearts showed reduced contractile activity in the presence of 2-APB (~35% and 25% reduction respectively, n=2). Analysis of intracellular Ca2+ release channel expression in isolated cardiac fibroblasts revealed a lack of any ryanodine receptor (RyR) expression but significant expression of IP3R2. In fibroblasts isolated from MTAB hearts, significantly higher expression of IP3R2 was evident (IP3R2:GAPDH ratio: 0.252±0.02 vs 0.101±0.008, MTAB vs sham respectively, n=3, p<0.001) along with higher phosphorylation of IP3R at Ser1756. Fibroblasts from MTAB hearts also exhibited hyperproliferative growth characteristics as assessed following 1uM Angiotensin II stimulation of cells over a period of 48h (average cell number per area, 59.6±2.5 vs 41.8±2.3, MTAB vs sham, n=3, p=0.006). Treatment with 2-APB significantly reduced both proliferation and Angiotensin II-mediated Ca2+ release in these cells. In conclusion and for the first time, we have shown increased IP3R2 expression and IP3R phosphorylation specifically in ventricular cardiac fibroblasts isolated from hypertrophied hearts. Expression of IP3R2 shows a strong correlation with cardiac contractility, where increased IP3R2 expression not only parallels decreased contractile performance but may also influence fibroblast hyperproliferation. This highlights the possibility that altered Ca2+ handling at the level of the cardiac fibroblast could be mechanistically important in pathological remodelling of the heart.