C1. Calcium sparks- full Flashcards

Question

(Disputing) Lakatta group and different isoforms of SERCA and Ca2+-activated AC

Answer 1

● Critics of the calcium clock also question the ubiquitous expression of calcium handling machinery throughout the heart. ● They argue that this would predispose individuals to ectopic firing and arrhythmias. ● Although, the Lakatta group argues to the contrary, by demonstrating that different isoforms of SERCA and Ca2+-activated adenylate cyclase are expressed in the SAN compared to the ventricles. ● Furthermore, those critical of the calcium clock argue that when cells are isolated from the SAN, they’re calcium homeostasis is dysregulated, and thus this leads to potentially erroneous results regarding the importance of the calcium clock.

Answer 2

● Rapid superfusion of cells with Li+ blocks the NCX transporter, which attenuated the inward current in response to ramp depolarisation. ● When action potentials were observed in current-clamp mode, superfusion of Li+ inhibited action potential generation. ● The rate and amplitude of SR Ca2+ cycling is proposed to be controlled by the amount of free Ca2+ in the system, the SR Ca2+ pumping rate and the number of activated RyR’s. ● The LCR period and amplitude determine the time and amplitude of the late exponential phase of the DD, respectively, and thus determine whether the membrane achieves its excitation threshold to generate the next rhythmic APs via activation of INCX.

Answer 3

● Rigg et al in 2000 showed a potential mechanism for the regulation of heart rate by SR calcium release. ● Ryanodine administration shifted the dose-response curve for isoprenaline to the right, with the response being percentage change in heart rate, recorded by extracellular electrodes in isolated hearts. ● These findings indicate that beta adrenergic stimulation increases heart rate by an SR calcium-dependent mechanism. ● Isoprenaline administration increased calcium transients as measured by Indo-1 AM addition. ● However, the frequency of pacemaking was reduced in Indo-1 loaded cells, which the authors concluded was a result of the inherent buffering capacity of the calcium dye. ● Furthermore, ryanodine was sufficient to attenuate isoprenaline-induced calcium transients. ● It would have been interesting if these experiments were conducted in the presence of nifedipine to determine that the calcium transients were completely intracellular in origin. ● Basal PKA-dependent phospholamban phosphorylation modulates kinetics of SR Ca2+ pumping, and phosphorylation of RyRs alters threshold of spontaneous activation of RyRs by Ca2+ within the SR. ● PKA-dependent mechanisms also regulate function of surface electrogenic proteins and couple M and Ca2+ clocks.

Answer 4

● Method; Used confocal imaging and fluo-3AM to measure CaT in isolated rabbit SANCs ● Results; Demonstrated that 0.1micromol/L isoproterenol induces 3-fold increase in no of Ca release events during diastolic depolarisation [CRDDs] per cycle, a shift to higher CRDD amplitudes and increase in spatial width ● This resulted in acceleration of diastolic depolarisation rate and increased SANC firing rate ● However, application of ryanodine = able to abolish effect of beta-AR stimulation ● Conclusion: Suggests that beta-AR stimulation increases firing rate in rabbit SANCs via recruiting additional local RyR Ca release and synchronising its occurrence ● These Ca sparks can then activate NCX, producing an inward current that can contribute to decay of the pacemaker potential – this = Ca clock hypothesis [Maltsev and Lakatta, 2008] ● Limitation; Did not provide mechanistic insight as to how beta-AR stimulation achieves this – now know that this = through activation of the kinase PKA, which phosphorylates RyR2 tetrameric monomers, LTCCs and phospholamban, resulting in dissociation of RyR2 accessory proteins [e.g. FKBP12.6] thus increasing intracellular Ca release

Answer 5

● Ca spark frequency = increased during physiological states such as exercise through beta-adrenergic stimulation ● Then enables modulation of Ca clock system and opportunity to regulate pacemaking ● Whilst regulation of Ca release events = important during physiological events such as exercise, disruption of regulation can increase RyR sensitivity to Ca2+ -> pathological arrhythmias

Answer 6

RyR2 associated accessory proteins van Oort 2010 Györke 1998

Answer 7

● As highlighted above, Ca sparks in atria and SAN can be regulated by IP3 ● Other methods by which Ca spark frequency can be modulated = through RyR2 associated accessory proteins, including FKBP12.6, calmodulin [CaM] and calsequesterin [CASQ2], which induce RyR2 domain rearrangement and conformational changes that alter open/close probability of RyR2, and SERCA accessory proteins [e.g. phospholamban] ● Binding of accessory proteins in turn = regulated by reversible phosphorylation of RyR2 tetrameric monomers ● This is controlled by kinases such as PKA and CaMKII, as well as phosphatases ● Oxidation and X-ROS signalling also plays a role

Answer 8

● Phosphorylation of the RyR2 receptor is also thought to increase the frequency of ryanodine sparks. ● van Oort et al in 2010 generated a knock-in mice model with the constitutively phosphorylated S2814D variant of the RyR2 protein. ● After the mice were killed, the frequency of calcium sparks was compared using fluorescent calcium indicators and confocal microscopy. ● It was found that phosphorylation of this specific residue increased the prevalence of the calcium sparks. ● However, other studies have found that phosphorylation of RyR2 does not increase calcium sparks. ● Differences in results are likely due to studying the phosphorylation of different amino acid residues in the RyR2 protein.

Answer 9

● The incidence of calcium sparks can be increased by luminal calcium sensing sites on the ryanodine receptor apparatus. ● This was shown by Györke et al in 1998 who incorporated SR microsomes from canine models into a planar lipid bilayer structure. ● The authors varied the calcium concentration on the cis (cytosolic) and trans (SR luminal) side of the planar lipid bilayer. ● Single channel recordings using voltage clamp showed the open probability was increased by higher luminal [Ca2+]. ● However, the experiments did not hold the potential difference across the bilayer at a representative physiological value for myocytes. ● Understanding the mechanism of SR calcium sensing is relevant to arrhythmias as SR calcium overload has been reported in heart failure patients, who are predisposed to arrhythmogenesis.

Answer 10

Atrial myocytes Ventricular myocytes Lipp 2000

Answer 11

● Largely lack TTs and contain both peripheral junctional and central non-junctional CRUs. ● Spontaneous Ca2+ sparks are larger and longer lasting than their ventricular counterparts (300,000 Ca2+ in 12 ms versus 100,000 Ca2+ in 7 ms). ● High prevalence at the periphery.

Answer 12

● The microdomain of calcium release is a dyad subspace formed by a cluster of RyR2 (30-100 channels) on the terminal cisternae side and a smaller cluster of LCC on the T-tubular wall, in the case of cardiac myocyte. ● The dimension of this subspace is at the range of 10-15 nm height and 200-400 nm width. ● As described above, calcium sparks in the sino-atrial node can contribute to the calcium clock which influences the pacemaker potential. ● However, whilst ventricular myocytes = dyadic junctions throughout the volume of the cell facilitating Ca sparks throughout, atrial myocytes = higher expression of IP3R2s, which can facilitate Ca spark generation.

Answer 13

● Method: Used Western blotting, PCR & IP3 binding analysis to demonstrate that expression of IP3Rs = six-fold higher in atrial myocytes in comparison to ventricular myocytes. ● Furthermore, using Fluo-4 & confocal microscopy, demonstrated that stimulation of quiescent atrial myocytes with IP3-BM (cell permeant form) increased no & frequency of spontaneous Ca2+ sparks. ● Using same techniques, Ju et al (2011) demonstrated that IP32Rs = also found within SAN, & that addition of IP3-BM increased frequency of Ca2+ sparks at edge of SAN (as seen with atrial myocytes). ● Conclusion: Overall, suggests that IP3Rs = role in modulating Ca2+ sparks, perhaps playing greater role in atria & SAN than ventricles. ● Limitations: Use of rodent models in these studies limits applicability to humans due to sig diff electrical cycles. ● Rodents = much more rapid heart rate, shorter Ca2+ influx period, different K+ dynamics & significantly lower contribution of NCX to outward Ca2+ current. ● To improve, use higher species e.g. rabbits, which have slower heart rates and more similar rate determining currents to improve translation to humans.

Answer 14

Transient inward current and EADs/ DADs Fowler 2020 and NCX exchange Bögelholz 2016 and NCX mediated calcium extrusion

Answer 15

● Some of the first evidence that intracellular calcium transients such as those described were capable of causing arrhythmias, came prior to our knowledge of calcium sparks themselves. ● Early researchers noted the occurrence of transient inward current in response to arrhytmogenic stimuli. ● The transient inward current described is capable of either augmenting early afterdepolarisations (EADs) or generating de novo delayed afterdepolarisations (DADs). ● EADs are produced due to the interaction between calcium currents and repolarising potassium currents. ● Both the EADs and DADs are sufficient to precipitate further aberrant electrical activity and arrhythmias.

Answer 16

● Fowler et al in 2020, where they used myocytes isolated from rabbit models of heart failure with reduced ejection. ● The heart failure myocytes had increased occurrence of these late calcium events. ● Current clamp recordings showed that there was a prolongation of the cardiac action potential relative to control rabbits. ● Whilst these results would appear to show that late calcium sparks during the cardiac action potential contribute to EADs, it is important to note that the coronary ligation model used in the rabbits is not representative of the broad spectrum of clinical heart failure. ● Spontaneous Ca2+ release from the SR elevates intracellular Ca2+ concentration which can activate Na+-Ca2+ exchange. ● The activation of Na+-Ca2+ exchange can cause afterdepolarisations, due to the electrogenic nature of the protein, if this occurs in late systole this can result in EADs.

Answer 17

● The potential for NCX to initiate these DADs relevant to arrhythmias was described by Bögelholz et al in 2016 who artificially over-expressed the NCX protein in mice and subjected the atrial cardiomyocytes to atrial pacing designed to induce fibrillation. ● Patch clamp experiments in the current clamp mode showed no significant increase in DADs. ● However, the number of spontaneous action potentials triggered by DADs was increased nearly 20-fold compared to wild-type mice. ● Whilst these results show that NCX mediated calcium extrusion may be sufficient to cause arrhythmogenesis, it is important to note that the model of overexpression is unlikely to represent normal physiology. ● Further experiments could test whether cardiospecific knockouts of NCX in atrial myocytes prevented any DADs. ● Figure 1

Answer 18

Hove- Madsen 2004 Voigt et al in 2012 Maintenance of AF

Answer 19

● Atrial fibrillation is the most common arrhythmia in patients. ● Dysfunctional calcium homeostasis is accepted to play a role in the initiation of ectopic electrical activity in AF. ● Hove-Madsen et al in 2004 isolated cells from excised right-atrial appendages in patients with or without AF. ● Using fluo3-AM and confocal microscopy, they were able to show that the frequency of calcium sparks was significantly higher in patients with AF. ● These findings may be caused by increased phosphorylation of the RyR2 channel in AF. ● Furthermore, the aforementioned DADs caused by NCX activity secondary to SR calcium sparks may play a role in the pathophysiology of AF.

Answer 20

● Voigt et al in 2012 took right atrial samples from 69 chronic AF patients and matched controls. ● They observed that the calcium leak during diastole was significantly increased in cAF patients compared with controls. ● This correlated with an increased open-probability of RyR2 receptors on the SR, and also observation of calcium sparks using Fluo-4AM and confocal microscopy. ● The incidence of spontaneous calcium events during diastole, and the corresponding DADs was shown using current clamp methods. ● This provides evidence for the role of DADs in inducing the arrhythmias seen in heart failure. ● However, the right atrial samples may not have been the most appropriate sample to take in the case of AF. ● Ectopic electrical activity that initiates AF is typically seen in pulmonary veins and the interatrial septum, not necessarily the right atrium.

Answer 21

● However, it is important to note that whilst aberrant calcium events are capable of initiating atrial fibrillation, they are not sufficient for the maintenance of the disease. ● Instead, this typically requires significant atrial remodelling and fibrosis. ● Electrical conductance abnormalities typically help propagate the disease.

Answer 22

Mutations to RYRs apparatus including calsequestrin and triadin Jiang 2004 RyR2 mutations and flecainide Hilliard 2010 and flecainide Shan 2010 and ryanodine receptor in chronic heart failure

Answer 23

● CPTV is an inherited condition that causes cardiac arrhythmias. ● The disease is caused by a variety of different mutations in the ryanodine receptor apparatus, including calsequestrin and triadin. ● However, the most common mutation that causes CPVT1 is to the RyR2 channel itself.

Answer 24

● Jiang et al in 2004 showed that these RyR2 mutations were gain of function. ● The authors transfected either wild-type or CPVT-mutant RyR2s into HEK293 cell lines and loaded them with fluo3-AM. ● Under confocal line-scan microscopy, the occurrence of Ca2+ sparks was significantly higher in HEK cells transfected with CPVT-mutant RyR2 channels. ● However, the use of the embryonic kidney line raises questions as to the validity of these results in myocytes. ● Nonetheless, these findings have been supported by more recent studies in isolated ventricular myocytes from both humans and mouse models.

Answer 25

● RyR2 mutations can increase the sensitivity of the ryanodine receptor to calcium concentrations on the cytosolic side of the SR membrane. ● The unifying feature of CPVT mutations is that their deleterious effects are only present upon sympathetic nervous system activation, often during exercise, and resulting in sudden cardiac death. ● One of the most promising new drugs in the treatment of CPVT is the class 1c anti-dysrhythmic, flecainide. ● Several clinical trials have shown that combination therapy of flecainide with beta blockers is effective at reducing the recurrence of tachycardias, particularly during exercise.

Answer 26

● The mechanism of action for flecainide was initially controversial, given that the drug is traditionally thought to blockade voltage gated sodium channels. ● However, Hilliard et al in 2010 identified another potential mechanism of action by comparing Wistar rats with Casq2-/- models of CPVT. ● Flecainide reduced the amplitude and intensity of the calcium sparks in the CPVT model. ● This was sufficient to prevent arrhythmogenic calcium waves. ● These findings were supported by Kryshtal et al in 2021 who used a similar experimental design in the presence of the voltage-gated sodium channel (VGSC) blocker tetrodotoxin (TTX). ● Flecainide administration had the same effect on calcium sparks in the presence or absence of TTX. ● Furthermore, using a modified version of flecainide (NM-FL) that was unable to block RyR2 channels did not improve symptoms in the CPVT mice model. ● These results together suggest that RyR2 blockade and inhibition of Ca2+ is the primary mechanism of action for flecainide. ● However, this must be treated with caution given that the two studies described, used only one mouse model of one of the many mutations capable of causing CPVT.

Answer 27

● The ryanodine receptor is not just affected by genetic aberrations in CPVT, instead there can be modulation by intracellular kinases. ● This can occur particularly in prolonged conditions such as chronic heart failure. ● Shan et al in 2010 used a mice model of a constitutively hyperphosphorylated RyR2. ● The authors developed a RyR2 S2808D knockin mice model. ● The authors found that mimicking chronic PKA phosphorylation with isoproterenol caused cardiomyopathy with significantly reduced ejection fraction in the knockin mice. ● When these channels were isolated and recorded in the presence of 150nM calcium, the single channel patch clamp recordings in lipid bilayers showed that these channels had a greater open probability. ● After myocardial infarction, these mice were more pre-disposed to ventricular tachycardia and death following the LAD ligation model of MI. ● Together, these findings demonstrate that the chronic hyperphosphorylation of RyR2 in heart failure predisposes to the development of arrhythmia due to increased open probability of the RyR2 channel. ● It would have been interesting for the authors to study calcium transients with calcium dyes in myocytes isolated from the canine models, to determine the frequency of arrhythmogenic events.

Answer 28

● Calcium sparks and waves are spontaneous events caused by the opening of RyR2 channels in CRUs. ● There is now strong evidence to suggest that there is an increased frequency of Ca2+ sparks in both patients and animal models of arrhythmias. ● When these calcium events occur in the late stages of the cardiac action potential they can cause EADs. ● In diastole, aberrant calcium sparks result in increased NCX activity which drives DADs. ● Flecainide, a drug which targets RyR2 and thus inhibits calcium sparks, has already shown promise in the treatment of the inherited arrhythmia CPVT, paving the way for future investigation into other Ca2+ spark inhibitors.

C1. Calcium sparks- full Flashcards

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