R-CEPIA1er as a new tool to directly measure sarcoplasmic reticulum [Ca] in ventricular myocytes

Bovo, Elisa; Martin, J.L.; Tyryfter, Jollyn; Tombe, Pieter P. de; Zima, Aleksey V.

doi:10.1152/ajpheart.00175.2016

Cited by 23 publications

(23 citation statements)

References 31 publications

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“…In this study, only indirect and global ROS measurements were used although development of local ROS measurements could provide further insights. For local [Ca 2+ ], targeted probes are available but cell culture 60–62 complicates the application as culture affects TT ultrastructure.…”

Section: Discussionmentioning

confidence: 99%

Hyperactive ryanodine receptors in human heart failure and ischaemic cardiomyopathy reside outside of couplons

Dries

Santiago

Gilbert

et al. 2018

Cardiovascular Research

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AimsIn ventricular myocytes from humans and large mammals, the transverse and axial tubular system (TATS) network is less extensive than in rodents with consequently a greater proportion of ryanodine receptors (RyRs) not coupled to this membrane system. TATS remodelling in heart failure (HF) and after myocardial infarction (MI) increases the fraction of non-coupled RyRs. Here we investigate whether this remodelling alters the activity of coupled and non-coupled RyR sub-populations through changes in local signalling. We study myocytes from patients with end-stage HF, compared with non-failing (non-HF), and myocytes from pigs with MI and reduced left ventricular (LV) function, compared with sham intervention (SHAM).Methods and resultsSingle LV myocytes for functional studies were isolated according to standard protocols. Immunofluorescent staining visualized organization of TATS and RyRs. Ca2+ was measured by confocal imaging (fluo-4 as indicator) and using whole-cell patch-clamp (37°C). Spontaneous Ca2+ release events, Ca2+ sparks, as a readout for RyR activity were recorded during a 15 s period following conditioning stimulation at 2 Hz. Sparks were assigned to cell regions categorized as coupled or non-coupled sites according to a previously developed method. Human HF myocytes had more non-coupled sites and these had more spontaneous activity than in non-HF. Hyperactivity of these non-coupled RyRs was reduced by Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition. Myocytes from MI pigs had similar changes compared with SHAM controls as seen in human HF myocytes. As well as by CaMKII inhibition, in MI, the increased activity of non-coupled sites was inhibited by mitochondrial reactive oxygen species (mito-ROS) scavenging. Under adrenergic stimulation, Ca2+ waves were more frequent and originated at non-coupled sites, generating larger Na+/Ca2+ exchange currents in MI than in SHAM. Inhibition of CaMKII or mito-ROS scavenging reduced spontaneous Ca2+ waves, and improved excitation–contraction coupling.ConclusionsIn HF and after MI, RyR microdomain re-organization enhances spontaneous Ca2+ release at non-coupled sites in a manner dependent on CaMKII activation and mito-ROS production. This specific modulation generates a substrate for arrhythmia that appears to be responsive to selective pharmacologic modulation.

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Section: Discussionmentioning

confidence: 99%

Hyperactive ryanodine receptors in human heart failure and ischaemic cardiomyopathy reside outside of couplons

Dries

Santiago

Gilbert

et al. 2018

Cardiovascular Research

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“…Once caffeine was washed out, the RyR inhibitor ruthenium red (10 M) was applied to stop Ca 2ϩ release and allow measurement of the rate of luminal [Ca 2ϩ ] ER recovery. At the end of each experiment, the R-CEPIA1er signal was calibrated by addition of the Ca 2ϩ ionophore ionomycin (Iono) (21). ER Ca 2ϩ recovery, monitored by [Ca 2ϩ ] ER accumulation, was analyzed to determine the maximum ER Ca 2ϩ uptake rate (i.e.…”

Section: Structural and Cellular Determinants Of Serca Conformationmentioning

confidence: 99%

“…where F is the R-CEPIA1er fluorescence; F max and F min are the fluorescence level at 10 mM Ca 2ϩ /Iono before and after depletion of ER Ca 2ϩ with caffeine (10 mM), respectively. The R-CEPIA1er Ca 2ϩ dissociation constant (K d ) is 390 M based on in situ calibrations (21). At the end of each experiment, the R-CEPIA1er signal (F max ) was calibrated with addition of Iono (100 M).…”

Section: Measuring Er Ca 2؉ Uptake In Permeabilized Hek-293 Cellsmentioning

confidence: 99%

Redistribution of SERCA calcium pump conformers during intracellular calcium signaling

Raguimova

Smolin

Bovo

et al. 2018

Journal of Biological Chemistry

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The conformational changes of a calcium transport ATPase were investigated with molecular dynamics (MD) simulations as well as fluorescence resonance energy transfer (FRET) measurements to determine the significance of a discrete structural element for regulation of the conformational dynamics of the transport cycle. Previous MD simulations indicated that a loop in the cytosolic domain of the SERCA calcium transporter facilitates an open-to-closed structural transition. To investigate the significance of this structural element, we performed additional MD simulations and new biophysical measurements of SERCA structure and function. Rationally designed mutations of three acidic residues of the loop decreased SERCA domain-domain contacts and increased domain-domain separation distances. Principal component analysis of MD simulations suggested decreased sampling of compact conformations upon N-loop mutagenesis. Deficits in headpiece structural dynamics were also detected by measuring intramolecular FRET of a Cer-YFP-SERCA construct (2-color SERCA). Compared with WT, the mutated 2-color SERCA shows a partial FRET response to calcium, whereas retaining full responsiveness to the inhibitor thapsigargin. Functional measurements showed that the mutated transporter still hydrolyzes ATP and transports calcium, but that maximal enzyme activity is reduced while maintaining similar calcium affinity. In live cells, calcium elevations resulted in concomitant FRET changes as the population of WT 2-color SERCA molecules redistributed among intermediates of the transport cycle. Our results provide novel insights on how the population of SERCA pumps responds to dynamic changes in intracellular calcium.

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“…[Ca 2+ ]ER measurements. [Ca 2+ ]ER was recorded as changes in fluorescence intensity of the genetically encoded ER-targeted Ca 2+ sensor R-CEPIA1er (29). R-CEPIA1er was excited with a 514 nm line of the argon laser and signal was collected at >560 nm.…”

Section: In-cell Calcium Uptake and Spontaneous Calcium Release Methodsmentioning

confidence: 99%

Dwarf open reading frame (DWORF) peptide is a direct activator of the sarcoplasmic reticulum calcium pump SERCA

Fisher

Bovo

Cho

et al. 2020

Preprint

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The cardiac sarcoplasmic reticulum calcium pump, SERCA, sequesters calcium in the sarco-endoplasmic reticulum (SR/ER) and plays a critical role in the contraction-relaxation cycle of the heart. A well-known regulator of SERCA in cardiac muscle is phospholamban (PLN), which interacts with the pump and reduces its apparent calcium affinity. A newly discovered SERCA regulatory subunit in cardiac muscle, dwarf open reading frame (DWORF), has added a new level of SERCA regulation. In this report, we modeled the structure of DWORF and evaluated it using molecular dynamics simulations. DWORF structure was modeled as a discontinuous helix with an unwound region at Pro15. This model orients an N-terminal amphipathic helix along the membrane surface and leaves a relatively short C-terminal transmembrane helix. We determined the functional regulation of SERCA by DWORF using a membrane reconstitution system. Surprisingly, we observed that DWORF directly activated SERCA by increasing its turnover rate. Furthermore, in-cell imaging of calcium dynamics demonstrated that DWORF increased SERCA-dependent ER calcium load, calcium reuptake rate, and spontaneous calcium release. Together, these functional assays suggest opposing effects of DWORF and PLN on SERCA function. The results agree with fluorescence resonance energy transfer experiments, which revealed changes in the affinity of DWORF for SERCA at low versus high cytosolic calcium concentrations. We found that DWORF has a higher affinity for SERCA in the presence of calcium, while PLN had the opposite behavior, a higher affinity for SERCA in low calcium. We propose a new mechanism for DWORF regulation of cardiac calcium handling in which DWORF directly enhances SERCA turnover by stabilizing the conformations of SERCA that predominate during elevated cytosolic calcium.

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R-CEPIA1er as a new tool to directly measure sarcoplasmic reticulum [Ca] in ventricular myocytes

Cited by 23 publications

References 31 publications

Hyperactive ryanodine receptors in human heart failure and ischaemic cardiomyopathy reside outside of couplons

Hyperactive ryanodine receptors in human heart failure and ischaemic cardiomyopathy reside outside of couplons

Redistribution of SERCA calcium pump conformers during intracellular calcium signaling

Dwarf open reading frame (DWORF) peptide is a direct activator of the sarcoplasmic reticulum calcium pump SERCA

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