β2‐adrenergic stimulation potentiates spontaneous calcium release by increasing signal mass and co‐activation of ryanodine receptor clusters

Arrhythmogenesis has been increasingly linked to cardiac ryanodine receptor (RyR) dysfunction. However, the mechanistic relationship between abnormal RyR function and arrhythmogenesis in the heart is not clear. We hypothesize that, under abnormal RyR conditions, triggered activity will be caused by spontaneous calcium release (SCR) events that depend on transmural heterogeneities of calcium handling. We performed high-resolution optical mapping of intracellular calcium and transmembrane potential in the canine left ventricular wedge preparation ( n = 28). Rapid pacing was used to initiate triggered activity under normal and abnormal RyR conditions induced by FKBP12.6 dissociation and β-adrenergic stimulation (20–150 μM rapamycin, 0.2 μM isoproterenol). Under abnormal RyR conditions, almost all preparations experienced SCRs and triggered activity, in contrast to control, rapamycin, or isoproterenol conditions alone. Furthermore, under abnormal RyR conditions, complex arrhythmias (monomorphic and polymorphic tachycardia) were commonly observed. After washout of rapamycin and isoproterenol, no triggered activity was observed. Surprisingly, triggered activity and SCRs occurred preferentially near the epicardium but not the endocardium ( P < 0.01). Interestingly, the occurrence of triggered activity and SCR events could not be explained by cytoplasmic calcium levels, but rather by fast calcium reuptake kinetics. These data suggest that, under abnormal RyR conditions, triggered activity is caused by multiple SCR events that depend on the faster calcium reuptake kinetics near the epicardium. Furthermore, multiple regions of SCR may be a mechanism for multifocal arrhythmias associated with RyR dysfunction.

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Faculty Opinions recommendation of Deviant ryanodine receptor-mediated calcium release resets synaptic homeostasis in presymptomatic 3xTg-AD mice.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1164013.624669 ◽

2009 ◽

Author(s):

Jane Sullivan

Keyword(s):

Ryanodine Receptor ◽

Calcium Release ◽

Synaptic Homeostasis

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Loss of dysferlin or myoferlin results in differential defects in excitation–contraction coupling in mouse skeletal muscle

Scientific Reports ◽

10.1038/s41598-021-95378-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

David Y. Barefield ◽

Jordan J. Sell ◽

Ibrahim Tahtah ◽

Samuel D. Kearns ◽

Elizabeth M. McNally ◽

...

Keyword(s):

Ryanodine Receptor ◽

Calcium Binding ◽

Physiological Role ◽

Muscle Disease ◽

Calcium Handling ◽

Membrane Repair ◽

Tubule Formation ◽

Ec Coupling ◽

Associated Proteins ◽

Receptor Clusters

AbstractMuscular dystrophies are disorders characterized by progressive muscle loss and weakness that are both genotypically and phenotypically heterogenous. Progression of muscle disease arises from impaired regeneration, plasma membrane instability, defective membrane repair, and calcium mishandling. The ferlin protein family, including dysferlin and myoferlin, are calcium-binding, membrane-associated proteins that regulate membrane fusion, trafficking, and tubule formation. Mice lacking dysferlin (Dysf), myoferlin (Myof), and both dysferlin and myoferlin (Fer) on an isogenic inbred 129 background were previously demonstrated that loss of both dysferlin and myoferlin resulted in more severe muscle disease than loss of either gene alone. Furthermore, Fer mice had disordered triad organization with visibly malformed transverse tubules and sarcoplasmic reticulum, suggesting distinct roles of dysferlin and myoferlin. To assess the physiological role of disorganized triads, we now assessed excitation contraction (EC) coupling in these models. We identified differential abnormalities in EC coupling and ryanodine receptor disruption in flexor digitorum brevis myofibers isolated from ferlin mutant mice. We found that loss of dysferlin alone preserved sensitivity for EC coupling and was associated with larger ryanodine receptor clusters compared to wildtype myofibers. Loss of myoferlin alone or together with a loss of dysferlin reduced sensitivity for EC coupling, and produced disorganized and smaller ryanodine receptor cluster size compared to wildtype myofibers. These data reveal impaired EC coupling in Myof and Fer myofibers and slightly potentiated EC coupling in Dysf myofibers. Despite high homology, dysferlin and myoferlin have differential roles in regulating sarcotubular formation and maintenance resulting in unique impairments in calcium handling properties.

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Analysis of Dihydropyridine and Ryanodine Receptor Clusters in Healthy and Failing Human Cardiac Myocytes

Heart Lung and Circulation ◽

10.1016/j.hlc.2008.05.579 ◽

2008 ◽

Vol 17 ◽

pp. S232

Author(s):

David Crossman ◽

Christian Soeller ◽

Peter Ruygrok ◽

Mark Cannell

Keyword(s):

Cardiac Myocytes ◽

Ryanodine Receptor ◽

Receptor Clusters

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Spontaneous calcium release in tissue from the failing canine heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01320.2008 ◽

2009 ◽

Vol 297 (4) ◽

pp. H1235-H1242 ◽

Cited By ~ 36

Author(s):

Gregory S. Hoeker ◽

Rodolphe P. Katra ◽

Lance D. Wilson ◽

Bradley N. Plummer ◽

Kenneth R. Laurita

Keyword(s):

Sarcoplasmic Reticulum ◽

Calcium Release ◽

Tissue Level ◽

Calcium Handling ◽

Canine Heart ◽

Myocardial Cells ◽

Spontaneous Release ◽

Adrenergic Stimulation ◽

Electrical Instability ◽

Delayed Afterdepolarization

Abnormalities in calcium handling have been implicated as a significant source of electrical instability in heart failure (HF). While these abnormalities have been investigated extensively in isolated myocytes, how they manifest at the tissue level and trigger arrhythmias is not clear. We hypothesize that in HF, triggered activity (TA) is due to spontaneous calcium release from the sarcoplasmic reticulum that occurs in an aggregate of myocardial cells (an SRC) and that peak SCR amplitude is what determines whether TA will occur. Calcium and voltage optical mapping was performed in ventricular wedge preparations from canines with and without tachycardia-induced HF. In HF, steady-state calcium transients have reduced amplitude [135 vs. 170 ratiometric units (RU), P < 0.05] and increased duration (252 vs. 229 s, P < 0.05) compared with those of normal. Under control conditions and during β-adrenergic stimulation, TA was more frequent in HF (53% and 93%, respectively) compared with normal (0% and 55%, respectively, P < 0.025). The mechanism of arrhythmias was SCRs, leading to delayed afterdepolarization-mediated triggered beats. Interestingly, the rate of SCR rise was greater for events that triggered a beat (0.41 RU/ms) compared with those that did not (0.18 RU/ms, P < 0.001). In contrast, there was no difference in SCR amplitude between the two groups. In conclusion, TA in HF tissue is associated with abnormal calcium regulation and mediated by the spontaneous release of calcium from the sarcoplasmic reticulum in aggregates of myocardial cells (i.e., an SCR), but importantly, it is the rate of SCR rise rather than amplitude that was associated with TA.

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Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling

The Journal of General Physiology ◽

10.1085/jgp.200709790 ◽

2007 ◽

Vol 130 (4) ◽

pp. 365-378 ◽

Cited By ~ 57

Author(s):

Sanjeewa A. Goonasekera ◽

Nicole A. Beard ◽

Linda Groom ◽

Takashi Kimura ◽

Alla D. Lyfenko ◽

...

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Calcium Release ◽

Striated Muscle ◽

Single Channel ◽

Triple Mutant ◽

Double Mutation ◽

Excitation Contraction Coupling ◽

Contraction Coupling ◽

Functional Studies

Ca2+ release from intracellular stores is controlled by complex interactions between multiple proteins. Triadin is a transmembrane glycoprotein of the junctional sarcoplasmic reticulum of striated muscle that interacts with both calsequestrin and the type 1 ryanodine receptor (RyR1) to communicate changes in luminal Ca2+ to the release machinery. However, the potential impact of the triadin association with RyR1 in skeletal muscle excitation–contraction coupling remains elusive. Here we show that triadin binding to RyR1 is critically important for rapid Ca2+ release during excitation–contraction coupling. To assess the functional impact of the triadin-RyR1 interaction, we expressed RyR1 mutants in which one or more of three negatively charged residues (D4878, D4907, and E4908) in the terminal RyR1 intraluminal loop were mutated to alanines in RyR1-null (dyspedic) myotubes. Coimmunoprecipitation revealed that triadin, but not junctin, binding to RyR1 was abolished in the triple (D4878A/D4907A/E4908A) mutant and one of the double (D4907A/E4908A) mutants, partially reduced in the D4878A/D4907A double mutant, but not affected by either individual (D4878A, D4907A, E4908A) mutations or the D4878A/E4908A double mutation. Functional studies revealed that the rate of voltage- and ligand-gated SR Ca2+ release were reduced in proportion to the degree of interruption in triadin binding. Ryanodine binding, single channel recording, and calcium release experiments conducted on WT and triple mutant channels in the absence of triadin demonstrated that the luminal loop mutations do not directly alter RyR1 function. These findings demonstrate that junctin and triadin bind to different sites on RyR1 and that triadin plays an important role in ensuring rapid Ca2+ release during excitation–contraction coupling in skeletal muscle.

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Protein Kinase A Phosphorylation of the Cardiac Calcium Release Channel (Ryanodine Receptor) in Normal and Failing Hearts

Journal of Biological Chemistry ◽

10.1074/jbc.m207028200 ◽

2002 ◽

Vol 278 (1) ◽

pp. 444-453 ◽

Cited By ~ 158

Author(s):

Steven Reiken ◽

Marta Gaburjakova ◽

Silvia Guatimosim ◽

Ana M. Gomez ◽

Jeanine D'Armiento ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Ryanodine Receptor ◽

Calcium Release ◽

Calcium Release Channel ◽

Release Channel ◽

Kinase A

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Phosphorylation of the ryanodine receptor 2 at serine 2030 is required for a complete β-adrenergic response

The Journal of General Physiology ◽

10.1085/jgp.201812155 ◽

2018 ◽

Vol 151 (2) ◽

pp. 131-145 ◽

Cited By ~ 10

Author(s):

Duilio M. Potenza ◽

Radoslav Janicek ◽

Miguel Fernandez-Tenorio ◽

Emmanuel Camors ◽

Roberto Ramos-Mondragón ◽

...

Keyword(s):

Ryanodine Receptor ◽

Cardiac Contractility ◽

Phosphorylation Site ◽

Adrenergic Stimulation ◽

Permeabilized Cells ◽

Whole Cell Patch Clamp ◽

Adrenergic Response ◽

Pump Activity ◽

Ryr2 Channel ◽

A Site

During physical exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in protein kinase A (PKA)–mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent “hyperphosphorylation” of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca2+ signaling and excitation–contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A+/+). We assessed ECC gain by using whole-cell patch–clamp recordings and confocal Ca2+ imaging during β-ARs stimulation with isoproterenol (Iso) and consistent SR Ca2+ loading and L-type Ca2+ current (ICa) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca2+ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca2+ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A+/+ myocytes. Using Ca2+ spark recovery analysis, we found that mutant RyR Ca2+ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca2+ waves and increases latency to the first spontaneous Ca2+ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during β-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs.

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