C2. Pacemaking- detailed

Question 1

Headings pneumonic

Answer 1

ICMCDDCDL

Question 2

Headings (list)

Answer 2

Introduction

Clinical context/ future therapies

Membrane clock

Calcium clocks

Disputing calcium clocks

Debate between hypotheses

Coupled clock

Dysregulation of coupled clock

Localisation of pacemaker activity to the SAN

Question 3

Introduction subheadings (list)

Answer 3

Spontaneous action potentials and diastolic depolarisation

Membrane clock vs calcium clock

Question 4

(Introduction) Spontaneous action potentials and diastolic depolarisation

Answer 4

The SAN is the primary site of spontaneous electrical activity in the heart.

This automaticity arises partly due to the absence of inwardly-rectifying K+ channels.

Each action potential is preceded by diastolic depolarisation, essential for SAN firing.

Understanding this process is vital for treating SAN arrhythmias and developing biological pacemakers.

Question 5

(Introduction) Membrane clock vs calcium clock

Answer 5

The DiFrancesco group promotes the funny current-driven “membrane clock” theory of pacemaking.

The Lakatta group proposes that spontaneous calcium release forms a “calcium clock.”

Both models face experimental limitations and may not fully reflect human physiology.

A hybrid “coupled-clock” theory integrates both mechanisms to explain pacemaker redundancy.

Question 6

Clinical context/ future therapies subheadings (list)

Answer 6

Keith & Flack 1907

Pacemakers

Protze 2017 and stem cells

HCN channels, stem cells and connexin-43 proteins

Qu 2003

Question 7

(Clinal/ future) Keith & Flack 1907

Answer 7

● Keith & Flack in 1907 reported on the presence of a region in the sino-auricular junction responsible for the heart’s automaticity.

● They described the passage of electricity from this fibrous tissue to the interauricular septum.

● These findings validated early physiological studies that showed the heart’s rhythm began in the neighbourhood of the great veins.

Question 8

(Clinal/ future) Pacemakers

Answer 8

Artificial pacemakers are used to regulate abnormal heart rhythms caused by AV block or other conditions.

Devices may be temporary or permanent, involving a pulse generator and pacing leads.

Limitations include procedural risks, hardware longevity, and complications.

These drawbacks motivate research into biological pacemaker development.

Question 9

(Clinal/ future) Protze 2017 and stem cells

Answer 9

Protze et al. 2017 developed pacemaker-like cells from human pluripotent stem cells (hPSCs).

These cells showed SAN gene expression and pacemaking ability in vitro and in vivo.

BMP4 and TGF-β inhibition improved culture efficiency, with further enhancement using right atrial tissue.

Limited survival post-transplant remains a barrier to clinical application.

Question 10

(Clinal/ future) HCN channels, stem cells and connexin-43 proteins

Answer 10

● Despite conflicting reports as to the necessity of HCN channels for pacemaking activity, these ion channels have been described as a potential therapeutic option for delivery of a biological pacemaker.

● Other biological pacemaker avenues of research have focused on transplantation of pacemaker-like cells developed from embryonic stem cells into the SAN.

● Furthermore, it may not be essential for cells to express all pacemaker currents, if these cells can be made to couple with existing pacemaker cells with connexin-43 proteins.

Question 11

(Clinal/ future) Qu 2003

Answer 11

Qu et al. 2003 used gene therapy to express HCN2 channels in canine atria.

Vagal stimulation suppressed native SAN activity while ECG tracked induced pacing.

Patch clamp confirmed cAMP-sensitive funny current in transduced cells.

Results were limited to short-term evaluation (3–4 days), leaving long-term effects unknown.

Question 12

Membrane clock subheadings (list)

Answer 12

Funny current

DiFrancesco & Tortora 1991

Shi 1999

Question 13

(Membrane clock) Funny current

Answer 13

● If channels spontaneously open at the maximum diastolic potential, enabling inward flow of current carried by predominantly sodium ions.

● This depolarisation increases the open probability of voltage gated CaT channels in late DD, causing sufficient depolarisation to open L-type voltage gated calcium channels (VGCCs), triggering the upstroke of the SAN action potential.

● The funny current was proposed to be the target of autonomic modulation of heart rate.

● As such, research was directed into a number of factors that may influence the intensity of funny current in sinoatrial node cardiomyocytes.

Question 14

(Membrane clock) DiFrancesco & Tortora 1991

Answer 14

DiFrancesco & Tortora showed that cAMP enhances funny current in excised rabbit SAN patches.

PKA alone did not mimic this effect, suggesting a specific cAMP-dependent mechanism.

cAMP shifted the If activation curve to more positive voltages, supporting its role in pacemaking.

The study excluded parasympathetic influence and lacked in situ cellular context.

Question 15

(Membrane clock) Shi 1999

Answer 15

● Shi et al in 1999 ​used RT-PCR to verify that HCN1 & HCN4 (as they were later named), were selectively expressed in the SAN.

● These findings have been used to argue that If is fundamental to pace-making, as the expression of the channels facilitating If is localised to the SAN and other neurons that exhibit pacemaker-like activity.

● The funny current channels are not the only membrane channels involved in automaticity in the sinoatrial node.

● Indeed, there has been some suggestion that both L-type and T-type voltage gated calcium channels may contribute to the diastolic depolarisation.

Question 16

Calcium clocks subheadings (list)

Answer 16

Sparks and SR in pacemaking

Rigg & Terrar 1996

Model- spontaneous release triggers oscillations

Capel & Terrar 2015

Question 17

(Calcium clocks) Sparks and SR in pacemaking

Answer 17

● The release of Ca2+ also occurs spontaneously under the form of sparks or openings of single RyR2 sensitive Ca2+ channels.

● This is the result of the small, but non-zero open probability of these ryanodine receptor channels.

● The importance of sarcoplasmic reticulum in pacemaking was first noticed by Rigg & Terrar in the late 1990s.

Question 18

(Calcium clocks) Rigg & Terrar 1996

Answer 18

Rigg & Terrar found that ryanodine and SERCA inhibition reduced heart rate in guinea pig atrium.

SAN action potential frequency and diastolic depolarisation slope were reduced in single-cell recordings.

Results imply SR calcium release plays a critical role in pacemaker activity.

Funny current contribution was not directly assessed but may have been involved.

Question 19

(Calcium clocks) Model- spontaneous release triggers oscillations

Answer 19

● These findings formed the basis of the calcium clock model.

● This model argues that spontaneous calcium release from the SR, due to the non-zero open probability of RyR2 channels, triggers oscillations in local calcium release.

● This calcium release is proposed to activate the electrogenic NCX protein, which drives depolarisation and spontaneous electrical activity.

● Rhythmic depolarisations are maintained by calcium reuptake into the SR by SERCA.

Question 20

(Calcium clocks) Capel & Terrar 2015

Answer 20

● Capel & Terrar in 2015, using isolated guinea-pig SAN myocytes, found that addition of BAPTA to the pipette solution in whole-cell voltage clamp conditions resulted in rapid cessation of rhythmic cellular pacemaking activity, which was not seen in control myocytes.

● Concurrently loading the cells with Fluo5F and imaging under confocal microscopy showed that these oscillatory calcium transients proceeded until BAPTA addition, thus supporting the calcium clock hypothesis.

Question 21

Disputing calcium clocks subheadings (list)

Answer 21

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

Superfusion of Li+

Rigg 2000 and PKA-dependent mechanisms

PKA/exercise and arrhythmias

Vinogradova 2002

Question 22

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

Answer 22

● 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.

Question 23

(Disputing) Superfusion of Li+

Answer 23

● 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.

Question 24

(Disputing) Rigg 2000 and PKA-dependent mechanisms

Answer 24

Rigg et al. showed that ryanodine reduces the heart rate response to isoprenaline.

Calcium transients were enhanced by isoprenaline and attenuated by Indo-1 and ryanodine.

Suggests SR calcium and RyR2 sensitivity are involved in β-adrenergic modulation.

PKA phosphorylation affects both SR dynamics and surface ion channels.

Question 25

(Disputing) PKA/exercise and arrhythmias

Answer 25

● 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

Question 26

(Disputing) Vinogradova 2002

Answer 26

Vinogradova et al. found that β-adrenergic stimulation increases local calcium release events in rabbit SAN. Ryanodine blocked this effect, linking Ca2+ release to pacing acceleration. Sparks activated NCX current, supporting the calcium clock hypothesis. PKA-mediated phosphorylation of RyR2 and associated proteins was implicated.

Question 27

Debate between hypotheses subheadings (list)

Answer 27

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

Question 28

(Debate) Baruscotti 2011 ​and the necessity of HCN isoforms for pacemaker activity

Answer 28

Baruscotti et al. used cardiac-specific inducible knockout mice to study HCN4. Tamoxifen-induced HCN4 deletion caused bradycardia on ECG and reduced SAN DD slope. This supports a role for HCN4 in automaticity. Contrasting results from Hoesl et al. fuel ongoing debate about HCN necessity.

Question 29

(Debate) Hoesl 2008 and importance of If

Answer 29

● 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.

Question 30

(Debate) Himeno 2011

Answer 30

● 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.

Question 31

Coupled clock subheadings (list)

Answer 31

Rigg 2003 Models and mechanisms for cooperation

Question 32

(Coupled clock) Rigg 2003

Answer 32

Rigg et al. used calcium chelators and SAN myocyte voltage clamp to assess Ca-If interaction. BAPTA reduced If current amplitude and shifted activation to more negative potentials. W-7, a calmodulin blocker, mimicked calcium chelation effects. Findings support calcium’s role in modulating funny current gating.

Question 33

(Coupled clock) Models and mechanisms for cooperation

Answer 33

● 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

Question 34

Dysregulation of coupled clock subheadings (list)

Answer 34

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

Question 35

(Dysregulation) Yanic 2014

Answer 35

● 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.

Question 36

(Dysregulation) Mutations and sick sinus syndrome

Answer 36

● 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.

Question 37

(Dysregulation) D’Souza 2014

Answer 37

D’Souza et al. showed that exercise-induced bradycardia in rats persists despite autonomic blockade. Trained SAN cells had reduced If conductance and HCN4 expression. Altered transcriptional regulators (Tbx3, miR1) correlated with reduced HCN4. Results suggest membrane clock dominance in exercise adaptation, though calcium contribution was not studied.

Question 38

(Dysregulation) RyR2 mutations

Answer 38

● 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.

Question 39

Localisation of pacemaker activity to the SAN subheadings (list)

Answer 39

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

Question 40

(Localisation) Theories

Answer 40

● 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.

Question 41

(Localisation) Dobrzynski 2003 and secondary pacemaker sites

Answer 41

Dobrzynski et al. mapped pacemaker sites in AVN using optical voltage-sensitive dyes. AVN pacemaker cells expressed Cx45 and HCN4, similar to SAN. Study highlighted secondary pacemakers’ role following SAN dysfunction. Imaging limitations and trauma may have influenced pacemaker localisation.

Question 42

(Localisation) Ventricular tissue vs pacemaking tissue

Answer 42

● 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.

Question 43

(Localisation) Mattick 2007 and constitutively active phosphodiesterase enzymes

Answer 43

Mattick et al. showed SAN-specific expression of AC1 and reduced cAMP responsiveness. Constitutively active phosphodiesterases help limit inappropriate SAN activation. Absence of IK1 channels allows SAN excitability and less negative resting potential. This functional architecture distinguishes SAN from atrial or ventricular tissue.

Question 44

(Localisation) Coppen 1999 and Cx45 vs Cx43

Answer 44

Coppen et al. found SAN expresses low-conductance Cx45, while adjacent regions express Cx43. Cx40 was also present in the SAN but not Cx43, confirming nodal specificity. Western blotting validated these findings. Future studies may assess how Cx45 knockouts affect pacemaker activity.

Question 45

(Localisation) AVN and ventricular conduction system

Answer 45

● 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.

Question 46

Conclusion

Answer 46

Both membrane and calcium clocks contribute to pacemaker activity. Experimental evidence supports the coupled-clock model. Genetic mutations (e.g., HCN4, RyR2) disrupt pacemaking and highlight interdependence. Understanding their synergy is key for advancing arrhythmia therapies.