Prolonged β-adrenergic stimulation disperses ryanodine receptor clusters in cardiomyocytes
Abstract
Ryanodine Receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that 'dispersion' of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged β-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion, and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hour triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of CaMKII reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of β-adrenergic stimulation over the longer term, and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology.
Data availability
Custom codes used in this study were written in Python, and is available at the public repository https://gitlab.com/louch-group/ryr-tt-correlative-analsyis.
Article and author information
Author details
Funding
Norwegian Research Council
- Xin Shen
- William Edward Louch
European Research Council
- Xin Shen
- Terje R Kolstad
- William Edward Louch
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Christopher L-H Huang, University of Cambridge, United Kingdom
Ethics
Animal experimentation: All animal experiments were performed in accordance with the Norwegian Animal Welfare Act and NIH Guidelines, and were approved by the Ethics Committee of the University of Oslo and the Norwegian animal welfare committee (FOTS ID 20208). The majority of the experiments were performed on adult male Wistar rats (250-350 g) purchased from Janvier Labs (Le Genest-Saint-Isle, France). Rats were group housed at 22{degree sign}C on a 12 h:12 h light-dark cycle, with free access to food and water. Cardiomyocytes isolated from transgenic RyR2-S2814D and RyR2-S2814A mice were provided by the laboratory of Xander Wehren (Baylor College of Medicine, Texas, United States) where experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011) and approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. A total of 64 rats and 6 mice were used in this study.
Version history
- Received: February 15, 2022
- Preprint posted: February 19, 2022 (view preprint)
- Accepted: July 31, 2022
- Accepted Manuscript published: August 1, 2022 (version 1)
- Version of Record published: August 25, 2022 (version 2)
Copyright
© 2022, Shen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,253
- Page views
-
- 364
- Downloads
-
- 10
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cell Biology
- Neuroscience
Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2α is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2α have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.
-
- Cell Biology
Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here, we report that deletion of tricalbins (Tcb1, Tcb2, and Tcb3), tethering proteins at endoplasmic reticulum (ER)–plasma membrane (PM) and ER–Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine (PHS) accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus–vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous PHS and Tcb3-deleted cells, supporting that PHS transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.