C1. Calcium sparks- brief

Question 1

Headings pneumonic

Answer 1

IWTFCDPAAAC

Question 2

Headings (list)

Answer 2

(Intro)
Initiation and termination
Waves
Transients
Function
Calcium clocks
Disputing calcium clocks
Phosphorylation and luminal calcium sensing sites
Atrial vs ventricular myocytes and IP3Rs
Arrhythmogenesis
Artial fibrillation
CPVT and heart failure
(Conc)

Question 3

Introduction

Answer 3

Calcium sparks are localized and spontaneous calcium fluxes through RyR2 receptors in cardiac myocytes.

They are key physiological events but are also linked to arrhythmias, especially through their role in calcium waves.

Question 4

Initiation and termination subheadings (list)

Answer 4

Cheng 1993

Termination

Question 5

(Initiation and termination) Cheng 1993

Answer 5

Cheng et al. first identified calcium sparks in resting rat cardiomyocytes using confocal microscopy and Fluo-3 dye.

Their experiments confirmed that these events were intrinsic to the SR and not due to external calcium influx.

Question 6

(Initiation and termination) Termination

Answer 6

Calcium sparks rapidly terminate due to a combination of SR calcium depletion and feedback control on RyR2 gating.

However, mechanisms like RyR2 adaptation and structural models suggest spark termination is multifactorial and not fully understood.

Question 7

Waves subheadings (list)

Answer 7

Propagation

Cheng 1996

Buffering within the cytoplasm

Venkataraman 2012

Question 8

(Waves) Propagation

Answer 8

Under high SR calcium conditions, calcium sparks can activate adjacent CRUs, resulting in propagating calcium waves.

Normally, spatial separation of dyads limits wave formation, keeping calcium release events localized.

Question 9

(Waves) Cheng 1996

Answer 9

Increasing extracellular calcium concentration significantly elevated calcium wave frequency in myocytes.

The majority of wave events were preceded by calcium sparks, suggesting sparks are the initiating event.

Question 10

(Waves) Buffering within the cytoplasm

Answer 10

Enhanced cytoplasmic calcium buffering reduces calcium diffusion and wave propagation.

This mechanism is disrupted during ischemia-reperfusion injury, which increases calcium overload and wave frequency.

Question 11

(Waves) Venkataraman 2012

Answer 11

Temporary ischemia in isolated rat hearts led to increased calcium wave activity below the point of ligation.

Reperfusion reversed this effect, highlighting how ischemia-induced calcium overload drives spontaneous wave generation.

Question 12

Transients subheadings (list)

Answer 12

L- type calcium channels

Barencas-Ruiz 1987

Calcium-binding proteins excitation contraction-coupling

Question 13

(Transients) L-type calcium channels

Answer 13

L-type calcium channels open in response to depolarization, allowing calcium influx into the dyadic space.

This influx initiates a much larger release of calcium from the SR through RyR2 in a process known as CICR.

Question 14

(Transients) Barencas-Ruiz 1987

Answer 14

Voltage-clamp experiments in isolated myocytes showed that calcium transients depend on L-type channels and RyR2.

Calcium dye imaging confirmed that blocking either component diminished the transients, establishing their functional link.

Question 15

(Transients) Calcium-binding proteins excitation contraction-coupling

Answer 15

During a calcium transient, calcium binds to proteins like troponin and calmodulin to trigger contraction and signaling.

Over 90% of transient calcium is buffered by these proteins, modulating the strength and duration of contraction.

Question 16

Function subheadings (list)

Answer 16

SERCA and calcium leak

Excitation- contraction coupling

Question 17

(Function) SERCA and calcium leak

Answer 17

SERCA pumps actively resequester calcium into the SR to maintain intracellular homeostasis.

To balance SERCA activity, a continuous calcium leak, potentially in the form of spontaneous sparks, is present.

Question 18

(Function) Excitation- contraction coupling

Answer 18

Calcium sparks act as elementary units that summate to produce a full cellular calcium transient.

During a heartbeat, thousands of sparks synchronize to generate a global rise in intracellular calcium concentration.

Question 19

Calcium clocks subheadings (list)

Answer 19

Sparks and SR in pacemaking

Rigg & Terrar 1996

Model- spontaneous release triggers oscillations

Capel & Terrar 2015

Question 20

(Calcium clocks) Sparks and SR in pacemaking

Answer 20

In pacemaker cells, calcium sparks contribute to rhythmic firing by triggering NCX-mediated depolarization.

These spontaneous events arise from the small but persistent open probability of RyR2 channels.

Question 21

(Calcium clocks) Rigg & Terrar 1996

Answer 21

Inhibition of RyR2 or SERCA in atrial cells led to a measurable reduction in heart rate.

This demonstrated that spontaneous SR calcium release is integral to pacemaker activity.

Question 22

(Calcium clocks) Model- spontaneous release triggers oscillations

Answer 22

The calcium clock model suggests that SR calcium release initiates rhythmic depolarizations via NCX activation.

Calcium is recycled back into the SR by SERCA, sustaining the oscillatory behavior of pacemaker cells.

Question 23

(Calcium clocks) Capel & Terrar 2015

Answer 23

SAN myocytes treated with calcium chelators lost their rhythmic firing ability.

Confocal imaging confirmed that calcium sparks and transients ceased, supporting the calcium clock hypothesis.

Question 24

Disputing calcium clocks subheadings (list)

Answer 24

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 25

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

Answer 25

Calcium handling protein isoforms vary between SAN and ventricular cells, allowing for specialized function. This regional difference helps explain how spontaneous activity is localized and not arrhythmogenic in other areas.

Question 26

(Disputing) Superfusion of Li+

Answer 26

Blocking NCX with lithium suppressed spontaneous action potentials in pacemaker cells. This highlights the essential role of electrogenic calcium extrusion in rhythm generation.

Question 27

(Disputing) Rigg 2000 and PKA- dependant mechanisms

Answer 27

Beta-adrenergic stimulation increased heart rate via SR calcium release, as shown by isoprenaline experiments. Inhibition of RyR2 shifted the response curve, indicating dependence on calcium release from the SR.

Question 28

(Disputing) PKA/exercise and arrhythmias

Answer 28

Calcium spark frequency increases during exercise via β-adrenergic stimulation, helping regulate the calcium clock and pacemaking activity. While this regulation is essential for physiological responses, its disruption can heighten RyR2 sensitivity to Ca²⁺, contributing to pathological arrhythmias.

Question 29

(Disputing) Vinogradova 2002

Answer 29

Beta-adrenergic agonists increased calcium release events and pacemaker firing in SAN cells. Blocking RyR2 reversed these effects, confirming a link between sympathetic tone and calcium spark activity.

Question 30

Phosphorylation and luminal calcium sensing sites subheadings (list)

Answer 30

RyR2 associated accessory proteins van Oort 2010 Györke 1998

Question 31

(Phosphorylation) RyR2 associated accessory proteins

Answer 31

Accessory proteins like FKBP12.6 and CASQ2 modulate RyR2 gating via structural changes. Their effects are mediated through reversible phosphorylation by kinases such as PKA and CaMKII.

Question 32

(Phosphorylation) van Oort 2010

Answer 32

van Oort et al. (2010) showed that phosphorylation at RyR2 residue S2814 increased calcium spark frequency in a knock-in mouse model. However, inconsistent findings across studies suggest that the effect of phosphorylation may depend on the specific RyR2 residue targeted.

Question 33

(Phosphorylation) Györke 1998

Answer 33

Györke et al. (1998) showed that higher luminal calcium increases RyR2 open probability, using canine SR microsomes in a planar lipid bilayer system. Despite limitations in physiological mimicry, the findings highlight how SR calcium overload can enhance spark activity and contribute to arrhythmogenesis in heart failure.

Question 34

Atrial vs ventricular myocytes and IP3Rs subheadings (list)

Answer 34

Atrial myocytes Ventricular myocytes Lipp 2000

Question 35

(Myocytes) Atrial myocytes

Answer 35

Atrial myocytes lack transverse tubules and show larger, longer-lasting calcium sparks, especially at the cell periphery. They have a higher expression of IP3 receptors (IP3Rs), which contribute to more frequent and spatially widespread sparks.

Question 36

(Myocytes) Ventricular myocytes

Answer 36

These myocytes contain well-organized dyadic junctions between L-type calcium channels and RyR2 receptors, allowing uniform calcium spark distribution. They rely less on IP3Rs, in contrast to atrial cells, and maintain tightly regulated calcium release through their structural configuration.

Question 37

(Myocytes) Lipp 2000

Answer 37

Western blotting and microscopy revealed IP3R expression is significantly higher in atrial vs. ventricular myocytes. IP3 stimulation increased calcium spark frequency in atrial and SAN cells, but rodent models used may limit translational relevance.

Question 38

Arrhythmogenesis subheadings (list)

Answer 38

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

Question 39

(Arrhythmogenesis) Transient inward current and EADs/ DADs

Answer 39

Spontaneous calcium sparks can activate NCX, causing transient inward currents leading to early (EADs) or delayed afterdepolarizations (DADs). These afterdepolarizations can trigger ectopic action potentials and contribute to arrhythmia initiation.

Question 40

(Arrhythmogenesis) Fowler 2020 and NCX exchange

Answer 40

In a rabbit heart failure model, increased late calcium release events were linked to prolonged action potentials. This supports the role of spontaneous sparks in promoting EADs via NCX activity during cardiac dysfunction.

Question 41

(Arrhythmogenesis) Bögelholz 2016 and NCX mediated calcium extrusion

Answer 41

Overexpression of NCX in mice enhanced spontaneous action potentials following DADs during atrial pacing. This suggests elevated NCX activity can drive arrhythmogenesis even without increasing DAD frequency itself.

Question 42

Atrial fibrillation subheadings (list)

Answer 42

Hove- Madsen 2004 Voigt 2012 Maintenance of AF

Question 43

(AF) Hove- Madsen 2004

Answer 43

Right atrial cells from AF patients had significantly more calcium sparks than those without AF. This was attributed to RyR2 hyperphosphorylation and contributed to increased DADs and ectopic activity.

Question 44

(AF) Voigt et al in 2012

Answer 44

In chronic AF patients, SR calcium leak and RyR2 open probability were increased, elevating spontaneous spark activity. Although atrial samples were from the right atrium, results support a role for DADs in AF pathophysiology.

Question 45

(AF) Maintenance of AF

Answer 45

While calcium sparks can initiate atrial fibrillation, sustained AF requires structural changes like fibrosis. Electrical remodeling and impaired conduction pathways play a greater role in perpetuating the arrhythmia.

Question 46

CPVT and heart failure subheadings (list)

Answer 46

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

Question 47

(CPVT and HF) Mutations to RYRs apparatus including calsequestrin and triadin

Answer 47

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by mutations in components of the RyR2 complex. These mutations increase spark frequency, with RyR2 mutations being the most common form of CPVT1.

Question 48

(CPVT and HF) Jiang 2004

Answer 48

HEK293 cells transfected with mutant CPVT RyR2 channels exhibited a higher spark rate compared to wild-type. Findings have since been validated in human and mouse cardiomyocytes, showing clinical relevance.

Question 49

(CPVT and HF) RyR2 mutations and flecainide

Answer 49

CPVT-associated RyR2 mutations increase sensitivity to cytosolic calcium, triggering pathological sparks during stress. Flecainide reduces arrhythmic events by blocking RyR2-mediated calcium release, especially when combined with beta-blockers.

Question 50

(CPVT and HF) Hilliard 2010 and flecainide

Answer 50

Flecainide reduced calcium spark amplitude and frequency in CPVT mouse models, independent of sodium channel activity. These findings suggest that RyR2 inhibition is its primary anti-arrhythmic mechanism.

Question 51

(CPVT and HF) Shan 2010 and ryanodine receptor in chronic heart failure

Answer 51

In a chronic PKA-hyperphosphorylation model, RyR2 channels had increased open probability and caused arrhythmias. Following myocardial infarction, these hyperphosphorylated channels led to more frequent ventricular tachycardia and sudden death.

Question 52

Conclusion

Answer 52

Calcium sparks and waves are spontaneous SR calcium release events crucial for cardiac excitation-contraction coupling. Their dysregulation contributes to arrhythmias, and therapies targeting RyR2, such as flecainide, show clinical promise.