scholarly journals Ryanodine receptor dispersion disrupts Ca2+ release in failing cardiac myocytes

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Terje R Kolstad ◽  
Jonas van den Brink ◽  
Niall MacQuaide ◽  
Per Kristian Lunde ◽  
Michael Frisk ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Myocytes ◽  
Ryanodine Receptor ◽  
Release Kinetics ◽  
Ryanodine Receptors ◽  
Cardiac Contractility ◽  
Super Resolution ◽  
Slow Kinetics ◽  
Spark Generation ◽  
Resolution Imaging

Reduced cardiac contractility during heart failure (HF) is linked to impaired Ca2+ release from Ryanodine Receptors (RyRs). We investigated whether this deficit can be traced to nanoscale RyR reorganization. Using super-resolution imaging, we observed dispersion of RyR clusters in cardiomyocytes from post-infarction HF rats, resulting in more numerous, smaller clusters. Functional groupings of RyR clusters which produce Ca2+ sparks (Ca2+ release units, CRUs) also became less solid. An increased fraction of small CRUs in HF was linked to augmented ‘silent’ Ca2+ leak, not visible as sparks. Larger multi-cluster CRUs common in HF also exhibited low fidelity spark generation. When successfully triggered, sparks in failing cells displayed slow kinetics as Ca2+ spread across dispersed CRUs. During the action potential, these slow sparks protracted and desynchronized the overall Ca2+ transient. Thus, nanoscale RyR reorganization during HF augments Ca2+ leak and slows Ca2+ release kinetics, leading to weakened contraction in this disease.

scholarly journals Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes

2009 ◽  
Vol 106 (52) ◽  
pp. 22275-22280 ◽  
Author(s):  
David Baddeley ◽  
Isuru D. Jayasinghe ◽  
Leo Lam ◽  
Sabrina Rossberger ◽  
Mark B. Cannell ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Ryanodine Receptor ◽  
Single Channel ◽  
Resolution Imaging ◽  
Rat Cardiac Myocytes ◽  
Receptor Distribution

scholarly journals Pachymic Acid Attenuated Doxorubicin-Induced Heart Failure by Suppressing miR-24 and Preserving Cardiac Junctophilin-2 in Rats

2021 ◽  
Vol 22 (19) ◽  
pp. 10710
Author(s):  
Nahla N. Younis ◽  
Alaa Salama ◽  
Mohamed A. Shaheen ◽  
Rana G. Eissa
Keyword(s):  
Electron Microscopy ◽  
Heart Failure ◽  
Fiber Diameter ◽  
Ryanodine Receptors ◽  
Cardiac Contractility ◽  
Histological Assessment ◽  
Contraction Coupling ◽  
Pachymic Acid ◽  
T Tubules ◽  
Mitochondrial Number

Defects in cardiac contractility and heart failure (HF) are common following doxorubicin (DOX) administration. Different miRs play a role in HF, and their targeting was suggested as a promising therapy. We aimed to target miR-24, a suppressor upstream of junctophilin-2 (JP-2), which is required to affix the sarcoplasmic reticulum to T-tubules, and hence the release of Ca2+ in excitation–contraction coupling using pachymic acid (PA) and/or losartan (LN). HF was induced with DOX (3.5 mg/kg, i.p six doses, twice weekly) in 24 rats. PA and LN (10 mg/kg, daily) were administered orally for four weeks starting the next day of the last DOX dose. Echocardiography, left ventricle (LV) biochemical and histological assessment and electron microscopy were conducted. DOX increased serum BNP, HW/TL, HW/BW, mitochondrial number/size and LV expression of miR-24 but decreased EF, cardiomyocyte fiber diameter, LV content of JP-2 and ryanodine receptors-2 (RyR2). Treatment with either PA or LN reversed these changes. Combined PA + LN attained better results than monotherapies. In conclusion, HF progression following DOX administration can be prevented or even delayed by targeting miR-24 and its downstream JP-2. Our results, therefore, suggest the possibility of using PA alone or as an adjuvant therapy with LN to attain better management of HF patients, especially those who developed tolerance toward LN.

scholarly journals Mitochondrial calcium overload is a key determinant in heart failure

2015 ◽  
Vol 112 (36) ◽  
pp. 11389-11394 ◽  
Author(s):  
Gaetano Santulli ◽  
Wenjun Xie ◽  
Steven R. Reiken ◽  
Andrew R. Marks
Keyword(s):  
Heart Failure ◽  
Mitochondrial Function ◽  
Posttranslational Modifications ◽  
Ryanodine Receptors ◽  
Cardiac Contractility ◽  
Major Effect ◽  
Genetic Enhancement ◽  
Macromolecular Complex ◽  
Atp Production

Calcium (Ca2+) released from the sarcoplasmic reticulum (SR) is crucial for excitation–contraction (E–C) coupling. Mitochondria, the major source of energy, in the form of ATP, required for cardiac contractility, are closely interconnected with the SR, and Ca2+ is essential for optimal function of these organelles. However, Ca2+ accumulation can impair mitochondrial function, leading to reduced ATP production and increased release of reactive oxygen species (ROS). Oxidative stress contributes to heart failure (HF), but whether mitochondrial Ca2+ plays a mechanistic role in HF remains unresolved. Here, we show for the first time, to our knowledge, that diastolic SR Ca2+ leak causes mitochondrial Ca2+ overload and dysfunction in a murine model of postmyocardial infarction HF. There are two forms of Ca2+ release channels on cardiac SR: type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s). Using murine models harboring RyR2 mutations that either cause or inhibit SR Ca2+ leak, we found that leaky RyR2 channels result in mitochondrial Ca2+ overload, dysmorphology, and malfunction. In contrast, cardiac-specific deletion of IP3R2 had no major effect on mitochondrial fitness in HF. Moreover, genetic enhancement of mitochondrial antioxidant activity improved mitochondrial function and reduced posttranslational modifications of RyR2 macromolecular complex. Our data demonstrate that leaky RyR2, but not IP3R2, channels cause mitochondrial Ca2+ overload and dysfunction in HF.

scholarly journals Live Cell Super-Resolution Imaging of Transverse Membrane Tubules in Heart Failure

2012 ◽  
Vol 102 (3) ◽  
pp. 223a-224a
Author(s):  
Eva Wagner ◽  
Marcel Lauterbach ◽  
Tobias Kohl ◽  
Volker Westphal ◽  
George S.B. Williams ◽  
...  
Keyword(s):  
Heart Failure ◽  
Super Resolution ◽  
Live Cell ◽  
Resolution Imaging ◽  
Membrane Tubules

scholarly journals Intracellular β1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility

2020 ◽  
Author(s):  
Ying Wang ◽  
Qian Shi ◽  
Minhui Li ◽  
Meimi Zhao ◽  
Gopireddy Raghavender Reddy ◽  
...  
Keyword(s):  
Single Molecule ◽  
Organic Cation ◽  
Cardiac Contractility ◽  
Super Resolution ◽  
Organic Cation Transporter ◽  
Proximity Ligation ◽  
Resolution Imaging ◽  
Organic Cation Transporter 3 ◽  
And Function ◽  
Phospholamban Phosphorylation

Rationale: β 1 -adrenoceptors (β 1 ARs) exist at intracellular membranes and Organic Cation Transporter 3 (OCT3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β 1 AR in cardiac contractility remains to be elucidated. Objective: Test localization and function of intracellular β 1 AR on cardiac contractility. Methods and Results: Membrane fractionation, super-resolution imaging, proximity ligation, co-immunoprecipitation and single-molecule pulldown demonstrated a pool of β 1 ARs in mouse hearts that was associated with sarco/endoplasmic reticulum Ca 2+ -ATPase at the sarcoplasmic reticulum (SR). Local protein kinase A (PKA) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared to wild type (WT), myocytes lacking OCT3 (OCT3KO) responded identically to the membrane-permeant βAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3KO selectively suppressed calcium transients and contraction responses to norepinephrine, but not isoproterenol. Furthermore, sotalol, a membrane-impermeant βAR-blocker suppressed isoproterenol-induced PKA activation at the PM, but permitted PKA activation at the SR, phospholamban phosphorylation and contractility. Moreover, pretreatment with sotatol in OCT3KO myocytes prevented norepinephrine induced PKA activation at both PM and the SR and contractility. Conclusions: Functional β 1 ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular β 1 ARs requires catecholamine transport via OCT3.

scholarly journals Caspofungin Modulates Ryanodine Receptor-Mediated Calcium Release in Human Cardiac Myocytes

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Christian Koch ◽  
Jennifer Jersch ◽  
Emmanuel Schneck ◽  
Fabian Edinger ◽  
Hagen Maxeiner ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Critically Ill ◽  
Ryanodine Receptor ◽  
Critically Ill Patients ◽  
Calcium Release ◽  
Ryanodine Receptors ◽  
Intracellular Ca ◽  
Hemodynamic Impairment ◽  
Dose Dependent Increase ◽  
Dose Dependent

ABSTRACT Recent studies showed that critically ill patients might be at risk for hemodynamic impairment during caspofungin (CAS) therapy. The aim of our present study was to examine the mechanisms behind CAS-induced cardiac alterations. We revealed a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) after CAS treatment. Ca2+ ions were found to be released from intracellular caffeine-sensitive stores, most probably via the activation of ryanodine receptors.

scholarly journals Super-Resolution Imaging Using a Novel High-Fidelity Antibody Reveals Close Association of the Neuronal Sodium Channel NaV1.6 with Ryanodine Receptors in Cardiac Muscle

2020 ◽  
Vol 26 (1) ◽  
pp. 157-165
Author(s):  
Heather L. Struckman ◽  
Stephen Baine ◽  
Justin Thomas ◽  
Louisa Mezache ◽  
Kirk Mykytyn ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Cardiac Muscle ◽  
Cardiac Myocytes ◽  
Ryanodine Receptor ◽  
Affinity Purification ◽  
Super Resolution ◽  
Proximity Ligation Assay ◽  
High Fidelity ◽  
Voltage Gated Sodium Channel ◽  
Voltage Gated

AbstractThe voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (NaV1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for NaV1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal NaV1.6 antibody and undertook super-resolution microscopy studies of NaV1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (NaV1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of NaV1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel NaV1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the NaV1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.

scholarly journals Live Cell Palm Techniques for Super Resolution Imaging of Murine Cardiac Myocytes

2018 ◽  
Vol 114 (3) ◽  
pp. 549a ◽  
Author(s):  
Yufeng Hou ◽  
Ornella Manfra ◽  
Jia Li ◽  
Xin Shen ◽  
William E. Louch
Keyword(s):  
Cardiac Myocytes ◽  
Super Resolution ◽  
Live Cell ◽  
Resolution Imaging

scholarly journals Peptide ligands of the cardiac ryanodine receptor as super-resolution imaging probes

2020 ◽  
Author(s):  
Thomas M. D. Sheard ◽  
Luke Howlett ◽  
Hannah Kirton ◽  
Zhaokang Yang ◽  
Georgina Gurrola ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Ryanodine Receptor ◽  
Peptide Binding ◽  
Super Resolution ◽  
Fluorescent Imaging ◽  
Confocal Imaging ◽  
Structural Basis ◽  
Functional Changes ◽  
Imaging Probes ◽  
Resolution Imaging

AbstractTo study the structural basis of pathological remodelling and altered calcium channel functional states in the heart, we sought to re-purpose high-affinity ligands of the cardiac calcium channel, the ryanodine receptor (RyR2), into super-resolution imaging probes. Imperacalcin (IpCa), a scorpion toxin peptide which induces channel sub-conduction states, and DPc10, a synthetic peptide corresponding to a sequence of the RyR2, which replicates arrhythmogenic CPVT functional changes, were used in fluorescent imaging experiments.Isolated adult rat ventricular cardiomyocytes were saponin-permeabilised and incubated with each peptide. IpCa-A546 became sequestered into the mitochondria. This was prevented by treatment of the permeabilised cells with the ionophore FCCP, revealing a striated staining pattern in confocal imaging which had weak colocalisation with RyR2 clusters. Poor specificity (as an RyR2 imaging probe) was confirmed at higher resolution with expansion microscopy (proExM) (~70 nm).DPc10-FITC labelled a striated pattern, which had moderate colocalisation with RyR2 cluster labelling in confocal and proExM. There was also widespread non-target labelling of the Z-discs, intercalated discs, and nuclei, which was unaffected by incubation times or 10 mM caffeine. The inactive peptide mut-DPc10-FITC (which causes no functional effects) displayed a similar labelling pattern.Significant labelling of structures unrelated to RyR2 by both peptide conjugates makes their use as highly specific imaging probes of RyR2 in living isolated cardiomyocytes highly challenging.We investigated the native DPc10 sequence within the RyR2 structure to understand the domain interactions and proposed mechanism of peptide binding. The native DPc10 sequence does not directly interact with another domain, and but is downstream of one such domain interface. The rabbit Arg2475 (equivalent to human Arg2474, mutated in CPVT) in the native sequence is the most accessible portion and most likely location for peptide disturbance, suggesting FITC placement does not impact peptide binding.

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