Tiotropium bromide, a long acting muscarinic receptor antagonist triggers intracellular calcium signalling in the heart

Abstract

Background and purpose: Tiotropium bromide (TB) is a long acting muscarinic receptor antagonist used to manage chronic obstructive pulmonary disease (COPD). Recent meta-analyses suggest an increased risk of cardiovascular events with TB. Ca²⁺/calmodulin dependent kinase II (CaMKII) and L-type Ca²⁺ channels regulate Ca²⁺ concentrations allowing management of Ca²⁺ across membranes. Pathological increases in Ca²⁺ are initially slow and progressive, however once the cytosolic concentration rises >1-3 μM from ~100 nM, calcium overload occurs and can lead to cell death. Ipratropium bromide, a short acting muscarinic receptor antagonist has previously been found to induce Ca²⁺ mediated eryptosis. The aim of this study was to investigate the role of Ca²⁺ in Tiotropium bromide mediated cardiotoxicity.

Experimental approach: Isolated Sprague-Dawley rat hearts were perfused with TB (10-0.1 nM) ± KN-93 (400 nM) or nifedipine (1 nM). Hearts were stained to determine infarct size (%) using triphenyltetrazolium chloride (TTC), or snap frozen to determine p-CaMKII (Thr₂₈₆) expression. Cardiomyocytes were isolated using a modified Langendorff perfusion and enzymatic dissociation before preparation for Fluo 3-AM staining and flow cytometric analysis.

Key results: TB increased infarct size compared to controls by 6.91-8.41%, with no effect on haemodynamic function. KN-93/nifedipine with TB showed a 5.90/7.38% decrease in infarct size compared to TB alone, the combined use of KN-93 with TB also showed a significant increase in left ventricular developed pressure whilst nifedipine with TB showed a significant decrease in coronary flow. TB showed a 42.73% increase in p-CaMKII (Thr₂₈₆) versus control, and increased Ca²⁺ fluorescence by 30.63% in cardiomyocytes.

Conclusions and implications: To our knowledge, this is the first pre-clinical study to show that Tiotropium bromide induces Ca²⁺ signalling via CaMKII and L-type Ca²⁺ channels to result in cell damage. This has significant clinical impact due to long term use of TB in COPD patients, and warrants assessment of cardiac drug safety.

Keywords: Ca(2+)/Calmodulin Kinase II (CaMKII); Calcium; Cardiotoxicity; Long acting muscarinic receptor antagonist; Tiotropium bromide.