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

Shabana Cassambai; Christopher J. Mee; Derek Renshaw; Afthab Hussain

doi:10.1016/j.taap.2019.114778

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

Shabana Cassambai, Christopher J. Mee, Derek Renshaw, Afthab Hussain

University of Leicester

Research output: Contribution to journal › Article › peer-review

6 Citations (Scopus)

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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.

Original language	English
Article number	114778
Journal	Toxicology and Applied Pharmacology
Volume	384
Early online date	13 Oct 2019
DOIs	https://doi.org/10.1016/j.taap.2019.114778
Publication status	Published - 1 Dec 2019

Bibliographical note

NOTICE: this is the author’s version of a work that was accepted for publication in Toxicology and Applied Pharmacology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Toxicology and Applied Pharmacology, 384, (2019)DOI: 10.1016/j.taap.2019.114778

© 2019, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/10.1016/j.taap.2019.114778

Keywords

Long acting muscarinic receptor antagonist
Cardiotoxicity
Tiotropium bromide
Calcium
Ca2+/Calmodulin Kinase II (CaMKII)

ASJC Scopus subject areas

Toxicology
Pharmacology

Access to Document

10.1016/j.taap.2019.114778

Post-PrintAccepted author manuscript, 1.17 MBLicence: CC BY-NC-ND

Cite this

@article{e67ecb85d7454bab8fe25fd5f349b4a4,

title = "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 = "Long acting muscarinic receptor antagonist, Cardiotoxicity, Tiotropium bromide, Calcium, Ca2+/Calmodulin Kinase II (CaMKII)",

author = "Shabana Cassambai and Mee, {Christopher J.} and Derek Renshaw and Afthab Hussain",

note = "NOTICE: this is the author{\textquoteright}s version of a work that was accepted for publication in Toxicology and Applied Pharmacology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Toxicology and Applied Pharmacology, 384, (2019)DOI: 10.1016/j.taap.2019.114778 {\textcopyright} 2019, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/10.1016/j.taap.2019.114778 ",

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language = "English",

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T1 - Tiotropium bromide, a long acting muscarinic receptor antagonist triggers intracellular calcium signalling in the heart

AU - Cassambai, Shabana

AU - Mee, Christopher J.

AU - Renshaw, Derek

AU - Hussain, Afthab

N1 - NOTICE: this is the author’s version of a work that was accepted for publication in Toxicology and Applied Pharmacology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Toxicology and Applied Pharmacology, 384, (2019)DOI: 10.1016/j.taap.2019.114778 © 2019, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/10.1016/j.taap.2019.114778

PY - 2019/12/1

Y1 - 2019/12/1

N2 - 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.

AB - 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.

KW - Long acting muscarinic receptor antagonist

KW - Cardiotoxicity

KW - Tiotropium bromide

KW - Calcium

KW - Ca2+/Calmodulin Kinase II (CaMKII)

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

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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Derek Renshaw, Centre Director

Cite this

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

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Profiles

Derek Renshaw, Centre Director

Cite this