Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C77
Integrated assessment of cardiac contractility and calcium/calmodulin protein kinase II delta expression and activity following acute and chronic isoprenaline administration
L. Mooney1, S. J. Coker1, M. Skinner2, S. Currie1
1. Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom. 2. Safety Assessment, AstraZeneca, Macclesfield, United Kingdom.
Calcium/calmodulin protein kinase II delta (CaMKIIδ) plays a central role in normal cardiac calcium handling and contractility and has also been identified as a molecular switch, triggering contractile dysfunction in cardiomyopathy. As such, it may be an important intracellular target for drugs that alter cardiac performance and could be a useful parameter to measure in cardiac pharmacological safety studies. Here we present data showing the effects of isoprenaline on cardiac contractility and CaMKIIδ expression and activation in male Dunkin-Hartley guinea pigs (GPs) following acute and chronic administration. In acute experiments, contractility was assessed by measurement of left ventricular (LV) dP/dtmax. GPs (430-640g) were anaesthetised with fentanyl (50 µg kg-1 s.c.) followed by sodium pentobarbital (50-60 mg kg-1 i.p.) and a Millar Tip catheter was placed in the lumen of the LV. Cumulative doses of isoprenaline (Iso; 0.1, 0.3, 1.0 nmol kg-1 min-1) or vehicle (saline, Sal) were infused i.v. for 15 min each dose (n=4). LV dP/dtmax was increased by Iso (3325±288 to 8500±591 mmHg s-1, p<0.05, one way ANOVA plus Dunnett’s test) while Sal had no effect (3415±487 to 3536±155 mmHg s-1). In chronic experiments, contractility was assessed by echocardiography. GPs (450-650g) were anaesthetised with 6 mL kg-1 Hypnorm/Hypnovel. Iso (1.5 µmoles kg-1 day-1, n=4) or vehicle (acidified saline, AS; n = 2) was delivered via a minipump for 6 days. AS did not alter LV diastolic diameter (LVDD) and fractional shortening (FS) (6.4±0.6 to 6.6±0.3 mm and 68±5 to 75±7 %, respectively) while LV systolic diameter (LVSD) decreased (2.0±0.5 to 1.6±0.4 mm, p<0.05, paired t-test). Iso increased both LVDD and LVSD (5.8±0.6 to 7.1±0.3 mm and 2.0±0.4 to 3.8±0.2 mm, respectively, p<0.05) while FS decreased (67±4 to 46±2 %, p<0.05). Quantitative immunoblotting was performed to assess total CaMKIIδ protein levels in GP LV homogenates. Following acute Iso no changes were seen in expression, however, chronic Iso increased CaMKIIδ expression to 1.01±0.03 vs 0.63±0.07 in AS (p<0.05, unpaired t-test). CaMKII activity was assessed by incorporation of γ-32P into a CaMKII peptide substrate, autocamtide. Acute Iso increased CaMKII activity to 2.5±0.32 compared with 1.9±0.13 in Sal (p<0.05, unpaired t-test), while chronic Iso increased CaMKII activity to 1.89±0.10 vs 1.11±0.09 in AS (p<0.05). In conclusion, both acute and chronic Iso treatment in vivo altered cardiac contractility and CaMKII activity and/or expression. Future work will explore possible correlations to determine whether drugs that exhibit cardiotoxic or therapeutic effects in vivo may work via actions on CaMKIIδ, thus identifying CaMKIIδ as a useful marker of cardiac safety.
Where applicable, experiments conform with Society ethical requirements