Abstract
Long acting muscarinic receptor antagonists (LAMAs) are utilised for the management of chronic obstructive pulmonary disease (COPD), to alleviate parasympathetic mediated bronchoconstriction. Amongst COPD patients, cardiovascular comorbidities are increasingly common, such as ischaemic heart disease, and account for the greatest proportion of deaths. Clinical studies have highlighted a possible link between the use of LAMAs such as Tiotropium bromide and adverse cardiovascular effects. Muscarinic receptors are present in the heart, and therefore all LAMAs possess potential cardiovascular risk. The current study investigated the effect of LAMA administration in whole heart Langendorff models of normoxic conditions and myocardial ischaemia/reperfusion injury using 3 month old Sprague Dawley rats. The Langendorff model was used to assess the effect of LAMAs on haemodynamic function; tissue from the models were assessed for infarct size analysis using triphenyl tetrazolium chloride (TTC) staining or were prepared for western blot analysis of Akt and CaMKII. Tissue from the models was also used for qPCR quantification of genes associated with myocardial infarction and oxidative stress. Cardiomyocytes were isolated from 3 month old rats using a modified Langendorff set up, with flow cytometric analysis of caspase-3, apoptotic cell death, oxidative stress and calcium release.Aclidinium, Tiotropium and Umeclidinium bromide but not Glycopyyronium bromide exacerbated myocardial ischaemia/reperfusion injury, assessed by infarct size to risk ratio when administered at the onset of reperfusion without significantly affecting haemodynamic function. Tiotropium bromide has been on the market the longest and is the most widely prescribed long acting muscarinic receptor antagonist. This was further tested in normoxic cardiac models, and was also found to induce damage, assessed by infarct size analysis. The use of the natural muscarinic agonist, acetylcholine inhibited Tiotropium bromide induced infarct size in normoxic and ischaemia/reperfusion models, suggesting that direct muscarinic signalling was involved in Tiotropium bromide mediated cardiac damage. Inhibitors and adjunctive agents including cyclosporin A (CsA), wortmannin, KN-93, Z-DEVD-FMK, nifedipine and resveratrol were used to determine the mechanisms of cardiotoxicity following administration of Tiotropium bromide; as well as determining the effect of Tiotropium bromide on Akt and CaMKII phosphorylation, reactive oxygen species generation and intracellular Ca2+ release and caspase-3 activation.
The study found that Tiotropium bromide mediated infarct size is attenuated following inhibition of pathways known to be involved in cardiotoxicity, such as mPTP opening, reactive oxygen species (ROS) generation, Ca2+ overload and caspase-3 activation. The study determined that Tiotropium bromide in cardiomyocytes exerts a greater effect on Ca2+ mediated signalling than on the generation of ROS. Additionally, caspase-3 is significantly activated, indicating a mechanism of cell death, however with no significant effect on apoptosis or necrosis as a whole. The study also showed that Tiotropium bromide affects the regulation of some genes such as caspase-3 involved in myocardial infarction through upregulation, and downregulates certain genes such as Akt1, peroxiredoxin 4 and superoxide dismutase 2 involved in cellular protection against oxidative stress.
In conclusion, the effect of LAMAs on myocardial ischaemia/reperfusion injury reflects the observation of increased cardiovascular complications amongst COPD patients, independently of other risk factors such as smoking. The effect of Tiotropium bromide in normoxic conditions suggests that muscarinic receptor antagonism in otherwise healthy heart exerts a Ca2+ driven mechanism of cardiotoxicity, involving abhorrent activation of Akt and CaMKII. These detrimental effects can be attenuated with the use of CsA, L-type Ca2+ channel blockers and antioxidants as well as acetylcholine. This study identifies the potential risks of LAMA use on the heart and urges for the development of robust preclinical tests which can identify compounds that may cause adverse cardiovascular complications. The observation that haemodynamic function was not adversely affected despite an increase in myocardial infarction, show that the dependence on functional parameters alone can not sufficiently identify cardiovascular risk.
| Date of Award | Nov 2018 |
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| Original language | English |
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| Supervisor | Afthab Hussain (Supervisor), Christopher Mee (Supervisor) & Sadie Dean (Supervisor) |