The role of ca2+/calmodulin kinase II in tiotropium bromide mediated cardiotoxicity

S. Cassambai, S. Dean, K. L. Harvey, C. J. Mee, A. Hussain. Health and Life Sciences, Coventry University, Coventry, UNITED KINGDOM.

Introduction

Recent meta-analyses have highlighted adverse cardiac effects associated with the long acting muscarinic receptor antagonist (LAMA), tiotropium bromide(1). LAMAs are used to inhibit cholinergic-induced bronchoconstriction in chronic obstructive pulmonary disease (COPD). Clinical studies have correlated an increase in stroke, myocardial infarction and cardiovascular related mortality, with the use of LAMAs(2). Calcium concentrations are tightly regulated and drive cardiac contractility; however, pathological increases lead to cell death(3). A key regulator in calcium signalling is the Ca2+/calmodulin-dependent kinase II (CaMKII), which is also involved in Akt activation(2), although Akt promotes cell survival, constitutive activation of Akt can itself result in cell death(5). This study assessed infarct/risk ratio and p-Akt expression in tiotropium-administered hearts with or without the CaMKII inhibitor, KN-93.

Methods

Isolated perfused hearts from male Sprague-Dawley rats (300 ± 50g) were subjected to stabilisation (20-minutes), followed by perfusion (155-minutes) ± tiotropium bromide (10nM - 0.1nM) or Tiotropium bromide (1-nM) ± the specific CaMKII inhibitor, KN-93 (400nM). All drugs were dissolved in DMSO. Following this, hearts were stained to determine infarct/risk ratio (%) using triphenyl-tetrazolium chloride (TTC), or snap frozen for Western blotting to determine p-Akt (Ser473) expression. Data were analysed using one-way ANOVA and LSD, presented as mean ± SEM.

Results

Tiotropium (10nM - 0.1nM) administration during perfusion, significantly increased infarct/risk ratio compared with control. Administration of KN-93 (400nM) showed no significant difference with respect to control (9.5 ± 0.9% vs. 10.3 ± 1.9%), however co-administration of KN-93 with tiotropium (1nM) attenuated infarct development (12.8 ± 2.8% vs. 18.7 ± 1.8%, p<0.02, n=3-5). Western blot analysis showed a significant increase in p-Akt (Ser473) expression in tiotropium treated groups compared to time-matched controls (88.7 ± 6.0% vs. 36.2 ± 5.1%, p<0.0001 at 1nM, n=3), which was abrogated with co-administration of KN-93 (88.7 ± 6.0% vs. 60.5 ± 4.9%, p<0.01).

Conclusion

This is the first pre-clinical study to suggest that tiotropium increases infarct/risk ratio in an isolated perfused heart model via CaMKII, and upregulation of p-Akt. This may account for the adverse cardiac side-effects seen clinically.

References

(1) Singh S et al. (2011) BMJ (Clinical Research Ed.) 342, d3215

(2) Cazzola M et al. (2015) The Lancet Respiratory Medicine 3 (15), 593-595

(3) Orrenius S et al. (2015) Biochemical and Biophysical Research Communications 460 (1), 72-81

(4) Rokhlin O et al. (2007) Cancer Biology & Therapy 6 (5), 732-742

(5) Castino R et al. (2008) Journal of Neuroendocrinology 20 (10), 1165-1175