Methods for isolating atrial cells from large mammals and humans

Voigt, Niels; Pearman, Charles M.; Dobrev, Dobromir; Dibb, Katharine M.

doi:10.1016/j.yjmcc.2015.07.006

Cited by 35 publications

(27 citation statements)

References 114 publications

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“…Cardiomyocyte isolation was performed according to previously established protocols ( Llach et al, 2011 ; Molina et al, 2012 ; Voigt et al, 2015 ). Briefly, enzymatic digestion was carried out in a Ca 2+ -free Tyrode solution containing 0.5 mg/ml collagenase (Worthington type 2, 300 u/mg), 0.25 mg/ml proteinase (Sigma type XXIV, 11 u/mg solid) and 5% bovine fatty acid-free albumin.…”

Section: Methodsmentioning

confidence: 99%

Profibrotic, Electrical, and Calcium-Handling Remodeling of the Atria in Heart Failure Patients With and Without Atrial Fibrillation

et al. 2018

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Atrial fibrillation (AF) and heart failure (HF) are common cardiovascular diseases that often co-exist. Animal models have suggested complex AF-promoting atrial structural, electrical, and Ca2+-handling remodeling in the setting of HF, but data in human samples are scarce, particularly regarding Ca2+-handling remodeling. Here, we evaluated atrial remodeling in patients with severe left ventricular (LV) dysfunction (HFrEF), long-standing persistent (‘chronic’) AF (cAF) or both (HFrEF-cAF), and sinus rhythm controls with normal LV function (Ctl) using western blot in right-atrial tissue, sharp-electrode action potential (AP) measurements in atrial trabeculae and voltage-clamp experiments in isolated right-atrial cardiomyocytes. Compared to Ctl, expression of profibrotic markers (collagen-1a, fibronectin, periostin) was higher in HFrEF and HFrEF-cAF patients, indicative of structural remodeling. Connexin-43 expression was reduced in HFrEF patients, but not HFrEF-cAF patients. AP characteristics were unchanged in HFrEF, but showed classical indices of electrical remodeling in cAF and HFrEF-cAF (prolonged AP duration at 20% and shorter AP duration at 50% and 90% repolarization). L-type Ca2+ current (ICa,L) was significantly reduced in HFrEF, cAF and HFrEF-cAF, without changes in voltage-dependence. Potentially proarrhythmic spontaneous transient-inward currents were significantly more frequent in HFrEF and HFrEF-cAF compared to Ctl, likely resulting from increased sarcoplasmic reticulum (SR) Ca2+ load (integrated caffeine-induced current) in HFrEF and increased ryanodine-receptor (RyR2) single-channel open probability in HFrEF and HFrEF-cAF. Although expression and phosphorylation of the SR Ca2+-ATPase type-2a (SERCA2a) regulator phospholamban were unchanged in HFrEF and HFrEF-cAF patients, protein levels of SERCA2a were increased in HFrEF-cAF and sarcolipin expression was decreased in both HFrEF and HFrEF-cAF, likely increasing SR Ca2+ uptake and load. RyR2 protein levels were decreased in HFrEF and HFrEF-cAF patients, but junctin levels were higher in HFrEF and relative Ser2814-RyR2 phosphorylation levels were increased in HFrEF-cAF, both potentially contributing to the greater RyR2 open probability. These novel insights into the molecular substrate for atrial arrhythmias in HF-patients position Ca2+-handling abnormalities as a likely trigger of AF in HF patients, which subsequently produces electrical remodeling that promotes the maintenance of the arrhythmia. Our new findings may have important implications for the development of novel treatment options for AF in the context of HF.

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Section: Methodsmentioning

confidence: 99%

Profibrotic, Electrical, and Calcium-Handling Remodeling of the Atria in Heart Failure Patients With and Without Atrial Fibrillation

et al. 2018

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“…Human atrial cardiomyocyte isolation and electrophysiological recordings. Right atrial appendages were obtained from 12 patients with SR. Atrial cardiomyocytes were isolated using a standard protocol (Voigt et al, 2015), suspended in storage solution (mM: KCl 20, KH 2 PO 4 10, glucose 10, K-glutamate 70, β-hydroxybutyrate 10, taurine 10, EGTA 10, albumin 1, pH = 7.4) and investigated within 6 h. Only well-striated, rod-shaped myocytes were used for current recordings.…”

Section: Dogsmentioning

confidence: 99%

The inward rectifier current inhibitor PA‐6 terminates atrial fibrillation and does not cause ventricular arrhythmias in goat and dog models

Yuan

Varkevisser

Opačić

et al. 2017

British J Pharmacology

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Background and PurposeThe density of the inward rectifier current (IK1) increases in atrial fibrillation (AF), shortening effective refractory period and thus promoting atrial re‐entry. The synthetic compound pentamidine analogue 6 (PA‐6) is a selective and potent IK1 inhibitor. We tested PA‐6 for anti‐AF efficacy and potential proarrhythmia, using established models in large animals.Experimental ApproachPA‐6 was applied i.v. in anaesthetized goats with rapid pacing‐induced AF and anaesthetized dogs with chronic atrio‐ventricular (AV) block. Electrophysiological and pharmacological parameters were determined.Key ResultsPA‐6 (2.5 mg·kg−1·10 min−1) induced cardioversion to sinus rhythm (SR) in 5/6 goats and prolonged AF cycle length. AF complexity decreased significantly before cardioversion. PA‐6 accumulated in cardiac tissue with ratios between skeletal muscle : atrial muscle : ventricular muscle of approximately 1:8:21. In SR dogs, PA‐6 peak plasma levels 10 min post infusion were 5.5 ± 0.9 μM, PA‐6 did not induce significant prolongation of QTc and did not affect heart rate, PQ or QRS duration. In dogs with chronic AV block, PA‐6 did not affect QRS but lengthened QTc during the experiment, but not chronically. PA‐6 did not induce TdP arrhythmias in nine animals (0/9) in contrast to dofetilide (5/9). PA‐6 (200 nM) inhibited IK1, but not IK,ACh, in human isolated atrial cardiomyocytes.Conclusion and ImplicationsPA‐6 restored SR in goats with persistent AF and, in dogs with chronic AV block, prolonged QT intervals, without inducing TdP arrhythmias. Our results demonstrate cardiac safety and good anti‐AF properties for PA‐6.

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“…Single myocytes were isolated using an enzymatic digestion technique, as described previously Voigt et al 2015). Animals were killed by I.V.…”

Section: Myocyte Isolationmentioning

confidence: 99%

Increased Ca buffering underpins remodelling of Ca²⁺ handling in old sheep atrial myocytes

Clarke

Caldwell

Pearman

et al. 2017

The Journal of Physiology

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Key points Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart.Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur.We report alterations to the atrial systolic Ca2+ transient that have implications for the function of the atrial in the elderly.We describe a novel mechanism by which increased Ca buffering can account for changes to systolic Ca2+ in the old atria.The present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops. AbstractMany cardiovascular diseases, including those affecting the atria, are associated with advancing age. Arrhythmias, including those in the atria, can arise as a result of electrical remodelling or alterations in Ca2+ homeostasis. In the atria, age‐associated changes in the action potential have been documented. However, little is known about remodelling of intracellular Ca2+ homeostasis in the healthy aged atria. Using single atrial myocytes from young and old Welsh Mountain sheep, we show the free Ca2+ transient amplitude and rate of decay of systolic Ca2+ decrease with age, whereas sarcoplasmic reticulum (SR) Ca content increases. An increase in intracellular Ca buffering explains both the decrease in Ca2+ transient amplitude and decay kinetics in the absence of any change in sarcoendoplasmic reticulum calcium transport ATPase function. Ageing maintained the integrated Ca2+ influx via I Ca‐L but decreased peak I Ca‐L. Decreased peak I Ca‐L was found to be responsible for the age‐associated increase in SR Ca content but not the decrease in Ca2+ transient amplitude. Instead, decreased peak I Ca‐L offsets increased SR load such that Ca2+ release from the SR was maintained during ageing. The results of the present study highlight a novel mechanism by which increased Ca buffering decreases systolic Ca2+ in old atria. Furthermore, for the first time, we have shown that SR Ca content is increased in old atrial myocytes.

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Methods for isolating atrial cells from large mammals and humans

Cited by 35 publications

References 114 publications

Profibrotic, Electrical, and Calcium-Handling Remodeling of the Atria in Heart Failure Patients With and Without Atrial Fibrillation

Profibrotic, Electrical, and Calcium-Handling Remodeling of the Atria in Heart Failure Patients With and Without Atrial Fibrillation

The inward rectifier current inhibitor PA‐6 terminates atrial fibrillation and does not cause ventricular arrhythmias in goat and dog models

Increased Ca buffering underpins remodelling of Ca²⁺ handling in old sheep atrial myocytes

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Methods for isolating atrial cells from large mammals and humans

Cited by 35 publications

References 114 publications

Profibrotic, Electrical, and Calcium-Handling Remodeling of the Atria in Heart Failure Patients With and Without Atrial Fibrillation

Profibrotic, Electrical, and Calcium-Handling Remodeling of the Atria in Heart Failure Patients With and Without Atrial Fibrillation

The inward rectifier current inhibitor PA‐6 terminates atrial fibrillation and does not cause ventricular arrhythmias in goat and dog models

Increased Ca buffering underpins remodelling of Ca2+ handling in old sheep atrial myocytes

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Increased Ca buffering underpins remodelling of Ca²⁺ handling in old sheep atrial myocytes