C2. Pacemaking- full Flashcards

Question

(Disputing) PKA/exercise and arrhythmias

Answer 1

● 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 2

● 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 3

Baruscotti 2011 and the necessity of HCN isoforms for pacemaker activity Hoesl 2008 and importance of If Himeno 2011

Answer 4

● There is still debate as to the necessity of HCN isoforms for pacemaker activity. ● This is exemplified in two opposing studies by Baruscotti et al in 2011 and Hoesl et al in 2008. ● Baruscotti et al developed a floxed-HCN4 mouse, expressing inducible Cre-recombinase under a cardiac-specific alpha-MHC promoter. ● Tamoxifen administration in these mice caused severe bradycardia, shown by in vivo ECG. ● Furthermore, isolated cells from these mice had decreased DD slope under current-clamp conditions.

Answer 5

● However, these findings were not replicated by Hoesl et al, who generated SAN-specific HCN4 knockout mice by expressing Cre-recombinase under the HCN4 promoter itself. ● In vivo ECGs showed no change in heart rate, despite a reduction in funny current in isolated cells exposed to hyperpolarising pulses. ● Differences between these studies may be explained by the more generalised knockout employed by Baruscotti et al, and thus the SAN-specific knockout provides stronger evidence against the necessity of If for pacemaker activity. ● Proponents of the membrane clock hypothesis argue that the importance of If is shown by clinical trials reporting bradycardia in response to the HCN inhibitor, ivabradine.

Answer 6

● Critics of the calcium clock model often reference a study performed by Himeno et al in 2011, in which BAPTA-AM chelation of voltage clamped SAN myocytes did not reduce pacemaker activity, observed in current clamp mode. ● However, the Lakatta group has argued that the methodology behind these experiments is flawed, and that the failure to measure intracellular calcium in these myocytes was a severe limitation of this study.

Answer 7

Rigg 2003 Models and mechanisms for cooperation

Answer 8

● One of the major advances in the field of pacemaker activity was made by Rigg et al in 2003, who observed the relationship between intracellular calcium and the membrane clock. ● The authors used voltage-clamped SAN myocytes and loaded cells with the rapid calcium chelator BAPTA. ● When hyperpolarising stimuli were administered to the myocytes, there was less If in cells under BAPTA-chelation. ● The authors applied a degree of hyperpolarising stimuli. ● They determined that the maximal intensity of funny current was not affected, but the activation voltages for If were shifted to more negative membrane potentials. ● The authors then showed that W-7, a calmodulin antagonist was able to mimic the effects of calcium-chelation. ● However, these calmodulin antagonists are capable of multiple off-target effects on other calcium-binding proteins.

Answer 9

● Nonetheless, these findings contributed to the inception of the calcium clock model by the Lakatta group. ● This model states that the membrane clock and calcium clock act cooperatively to modulate and entrain one another thus maintaining normal sinus rhythm. ● Figure 1 The diagram in Figure 2 describes some of the further proposed mechanisms for cooperation between intracellular calcium and membrane channels. ● CaMKII is also capable of acting directly on ryanodine receptors along with SERCA2 to have both positive chronotropic and lusitropic effects respectively. ● Figure 2

Answer 10

Yanic 2014 Mutations and sick sinus syndrome D’Souza 2014 RyR2 mutations

Answer 11

● Yaniv et al in 2014 have shown the importance of both the membrane and calcium clocks in maintaining beating intervals in isolated rabbit sinoatrial node cells. ● The authors treated these cells with ivabradine to inhibit funny current, or inhibited SERCA2 with cyclopiazonic acid to disrupt the membrane clock. ● The authors voltage clamped these isolated SAN cells, and were able to show that the frequency of action potential firing was decreased, as was the variability of the action potential intervals. ● Decoupling of the pacemaker mechanisms within the SAN is associated with aberrant electrical activity.

Answer 12

● Mutations in both membrane and calcium-handling proteins can have deleterious effects on SAN pacemaking. ● For instance, mutations in HCN proteins are commonly associated with sick sinus syndrome. ● Patients with this condition typically present with varied atrial arrhythmias, syncope, and fatigue.

Answer 13

● Exercise is another situation in which the coupled clock may become dysregulated, and one clock may proceed to dominate. ● D’Souza et al in 2014 evaluated this possibility by first demonstrating that exercise in rats by training with uphill running was sufficient to generate bradycardia ● The authors concluded this was attributable to electrophysiological remodelling of SAN cells as the bradycardia persisted in the presence of autonomic nervous blockade with propranolol and atropine in vivo. ● The authors then patch-clamped isolated rat cells from trained and untrained animals in the whole cell configuration. ● Administration of rapid hyperpolarising pulses showed that there was a reduction in whole-cell If conductance in trained mice. ● Therefore, the authors examined expression of the HCN4 mRNA in sinus node cells using qPCR. ● It was observed there was a reduction in HCN4 expression in trained rats compared with sedentary, and the authors showed that this correlated with reduced levels of Tbx3 and increased expression of miR1, which have all previously been shown to affect HCN4 expression in other studies. ● These findings show that the membrane clock dominates in causing bradycardia after training. ● Although it is important to note that the authors did not examine calcium release in trained SAN cells, which may have also been important in the response to training.

Answer 14

● As well as HCN mutations, calcium clock mutations, such as RyR2 mutations in CPVT can cause SAN dysfunction. ● Patients with CPVT often present with paradoxical atrial bradycardia, which results from calcium-depletion in the SR. ● This further emphasises the importance of correct membrane and calcium clock functioning for physiological SAN pacemaker activity.

Answer 15

Theories Dobrzynski 2003 and secondary pacemaker sites Ventricular tissue vs pacemaking tissue Mattick 2007 and constitutively active phosphodiesterase enzymes Coppen 1999 and Cx45 vs Cx43 AVN and ventricular conduction system

Answer 16

● There are a number of theories as to why pacemaker activity is typically localised to the SAN, and not the ventricles. ● Proponents of the membrane clock hypothesis argue this is to do with absence of the funny current from ventricular cells, whilst researchers that believe in the calcium clock argue that there are subtle differences in calcium handling machinery.

Answer 17

● One particular paper by Dobrzynski et al in 2003 has attempted to define the molecular correlates of AVN pathology. ● The authors used an excitation contraction uncoupler to enable easier study of atrioventricular preparations, the authors used the voltage sensitive dye di-4-ANEPPS to determine areas of spontaneous electrical activity in the AVN. ● The authors applied caesium to these preparations and observed the cessation of electrical activity under electrodes. ● The authors then demonstrated that the AVN cells responsible for the pacemaker activity selectively expressed Cx45 immunostaining, but not Cx43, similar to the expression profile in the SAN. ● Furthermore, the junctional pacemaker site in the AVN expressed the If channel protein HCN4. ● However, the authors recognised that the combination of dissection trauma and di-4-ANNEPS administration may have distorted the location of pacemaker activity in the AVN. ● Given the importance of secondary pacemaker sites after damage to the SAN, the area of secondary pacemaker research may perhaps become a greater area of study in future. ● This may also help with an understanding of ectopic pacemaker sites in the pathophysiology of arrhythmias such as atrial fibrillation.

Answer 18

● The majority of the calcium handling machinery within the cell is expressed in ventricular tissue. ● Indeed, proponents of the membrane clock theory have used this fact to argue against the role of calcium oscillations in pacemaking. ● Although SANC, like ventricular myocytes, express high levels of Ca2+-inhibited AC types 5 and 6, the discoveries of Ca2+-activated AC types, such as AC1 and AC8 in rabbit and guinea-pig SANC, provide a clear difference between the ventricular tissue and pacemaking tissue.

Answer 19

● This was first noted by Mattick et al in 2007, who showed expression of AC1 in the SAN, but not ventricles, at both protein and mRNA levels using immunohistochemistry and RT-PCR respectively. ● Another potential difference between ventricular tissue and sinoatrial node tissue is the expression of constitutively active phosphodiesterase enzymes, reducing the potential for aberrant cAMP signalling inducing tachycardias. ● In the heart, a key feature that distinguishes pacemaker tissue from surrounding atrial muscle is the absence of the stabilizing influence of IK1. ● The presence of IK1 channels in atrial and ventricular myocytes is responsible for the ~−90 mV resting membrane potential in these cells, dominated by the equilibrium potential for potassium ions in physiological solutions. ● In the absence of IK1 the SAN membrane potential is not forced to rest at this potential, and the tissue is more excitable.

Answer 20

● Other important characteristics are the presence of the low-conductance connexin protein Cx45 as opposed to the high-conductance Cx43. ● Coppen et al in 1999 showed this using immunocytochemistry for Cx45, Cx40 and Cx43 in the SA nodal regions and the surrounding crista terminalis. ● The authors showed expression of Cx45 and Cx40 in the SA node, but not the crista terminalis. ● In contrast, Cx43 was not expressed in the SA node. ● These results were verified by Western blotting. ● In future, it would be interesting to see if an inducible knockout of Cx45 in the SAN was sufficient to attenuate spontaneous activity, and if knock-in in the ventricle was sufficient to produce spontaneous activity.

Answer 21

● There are regions of the heart capable of secondary pacemaker activity. ● The AVN and ventricular conduction system act as secondary (accessory) pacemakers to secure repetitive ventricular contraction in conditions of SAN failure or AV block. ● In addition, the lower rim of the atrial myocardium, just above the AV junction, and the myocardium surrounding the systemic venous return contain nodal-like cells, which, under pathological conditions, can acquire ectopic pacemaker activity.

Answer 22

● There has been considerable effort devoted to understanding the molecular basis for pace-making. ● Two prominent groups have contested the relative importance of the membrane and calcium clocks. ● Both groups provide compelling evidence from mouse-models, electrophysiology, and pharmacology to support their hypotheses. ● However, it is now commonly accepted that these pacemaker mechanisms act cooperatively to prevent aberrant impulse generation. ● The necessity of both clocks is exemplified by mutations to HCN4 and RyR2, which result in clock uncoupling and SAN arrhythmias. ● These clocks may be important in different situations, including over different time scales in the response to various stimuli.

C2. Pacemaking- full Flashcards

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