Prolonged β-adrenergic stimulation disperses ryanodine receptor clusters in cardiomyocytes and has implications for heart failure

  1. Xin Shen
  2. Jonas van den Brink
  3. Anna Bergan-Dahl
  4. Terje R Kolstad
  5. Einar S Norden
  6. Yufeng Hou
  7. Martin Laasmaa
  8. Yuriana Aguilar-Sanchez
  9. Ann P Quick
  10. Emil KS Espe
  11. Ivar Sjaastad
  12. Xander HT Wehrens
  13. Andrew G Edwards
  14. Christian Soeller
  15. William E Louch

  • Curated by eLife

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    Evaluation Summary:

    This study applies super-resolution imaging to the distribution of Ca2+ release channels before and after adrenergic stimulation. They make comparisons between healthy and failing cardiomyocytes. The results are specifically applicable to the understanding of contractile function in cardiac failure.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Ryanodine receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that ‘dispersion’ of RyR clusters disrupts Ca 2+ 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 hr triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of Ca 2+ /calmodulin-dependent protein kinase II (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 Ca 2+ spark fidelity and magnitude, and slowed release kinetics from Ca 2+ 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 Ca 2+ waves. However, during HF, RyR dispersion was linked to impaired Ca 2+ 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.

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  1. Version published to 10.7554/elife.77725 on eLife
  2. eLife

    Evaluation Summary:

    This study applies super-resolution imaging to the distribution of Ca2+ release channels before and after adrenergic stimulation. They make comparisons between healthy and failing cardiomyocytes. The results are specifically applicable to the understanding of contractile function in cardiac failure.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    In this study, Xin Shen et al. aim to establish a link between prolonged β-Andrenergic receptor (β-AR) stimulation and the fragmentation of Calcium Release Units (CRUs) in healthy cardiomyocytes and demonstrate that phosphorylation of ryanodine receptor (RyR) by downstream effectors of β-AR (PKA and CaMKII) is the driver of this fragmentation. They then aim to measure the effects of prolonged β-AR stimulation on the measurable properties of calcium-induced calcium release (CICR) and deduce the consequences for CICR efficacy. Finally, the authors seek to infer the role of β-AR induced CRU fragmentation in heart failure by comparing the results obtained from healthy β-AR stimulated cardiomyocytes with results from failing cardiomyocytes.

    This work is a logical progression from a previous study demonstrating RyR dispersion in failing cardiomyocytes (Kolstad et al. eLife 2018;7:e39427) and the synergy of super-resolved RyR imaging, functional Ca2+ imaging, and Ca2+ spark simulation is the paper's strength. This work also takes full advantage of the previously published extension of the super-resolved imaging of RyR clustering to three dimensions (Shen et al. J Physiol 597.2 (2019) pp 399-418) in order to obtain more accurate reconstructions of CRUs and to also enable the reconstruction of correlated t-tubule/RyR images. Correspondingly, the supporting spark simulations are now generated using spatial models that are a more realistic 3D representation of the range of dyadic CRU organisation. A potential weakness in their approach lies with the use of indirect immuno-labelling of receptors which will introduce larger linkage errors to the RyR localisation data, compounding the localisation error. That said the linkage error appears to have been reduced somewhat by the judicious selection of an Alexa-647 labelled Fab secondary. Plus intuitively, greater positional uncertainty is likely to result in a small systematic underestimation of CRU fragmentation that would not have a major impact on the relative results reported in this study or their interpretation.

    Generally, the experiments in this paper are well thought out, with appropriate controls, and achieve the aims of the authors. Their 3D dSTORM data indicates that after 60 minutes of β-AR stimulation there is a significant disruption of RyR clustering in dyadic CRUs. Both PKA and CaMKII activity is convincingly implicated by control experiments that partially reverse the cluster dispersion when the activity of either effector is inhibited. Non-phosphorylatable or phosphomimetic RyR mutants are offered as direct evidence that the phosphorylation of RyR by CaMKII indeed drives the observed RyR dispersion, but it is notable that the same controls are not available for the site of RyR phosphorylation by PKA. The properties of Ca2+ sparks, transients, waves, and spark-mediated Ca2+ leak are thoroughly quantified before and after prolonged β-AR stimulation. The measured disruption to CICR is then shown to be reversible by inhibition of CaMKII and PKA. Simulations of sparks using a mathematical model are required to predict the effects of β-AR stimulation on spark fidelity and silent Ca2+ leak, in order to complete the picture. It is reassuring that the simulation results do not contradict experimental results for spark time course variables that are reported by both and the authors are careful to distinguish between observations made in vivo and in silico. 3D dSTORM and Ca2+ imaging results obtained from post-infarction cardiomyocytes were found to mimic important aspects of the results β-AR stimulation of healthy cells including their reversibility by CaMKII or PKA inhibition. The totality of this wealth of data is consistent with RyR disruption during heart failure being caused by the downstream effects of prolonged β-AR stimulation.

    Interestingly, their results suggest that the downstream effects of prolonged β-AR stimulation have different functional consequences in healthy and failing cells. This has implications for why β-AR blockade can have a beneficial effect on failing cardiomyocytes and also if control of RyR dispersion is to be considered as a potential therapeutic target. Additionally, with this work as an example, the correlative 3D localisation microscopy-confocal reconstruction technique is likely to find applications in the study of other cellular processes.

  4. eLife

    Reviewer #2 (Public Review):

    This work reports several interesting and novel results that tie RyR2 dispersion during prolonged β-Adrenergic stimulation with isoproterenol to receptor phosphorylation by CaMKII and PKA. The potential clinical relevance is highlighted with the comparison and correlation to the RyR2 dispersion that occurs during HF, building and substantially expanding upon previous work by this group. Indeed, this group has recognized strengths in nanoscale analyses of RyR2 and here uses their own impressive 3D correlative reconstruction approach to provide a striking visual demonstration of the distribution of RyR2 in relation to the nearby t-tubule membrane. The group implements a broad array of techniques to investigate the functional implications of the RyR2 dispersion that they elegantly demonstrate with super-resolution microscopy, including in-depth analyses of Ca2+ spark and transient dimensions, and kinetics. The imaging techniques are well-executed and beautifully presented and support the novel conclusion that prolonged ISO stimulation leads to RyR2 dispersion. It also raises interesting discussion points that will stimulate future studies including evoking Vatner and Lederer's idea that non-uniform SR Ca2+ depletion may create "fire-breaks" that prevent Ca2+ wave propagation. Altogether, this is an exciting and timely study and will have an impact in the field that is currently only beginning to understand and explore the functional implications of nanoscale changes in RyR2 and other EC-coupling proteins.

    The conclusions are mostly well supported by data, but a few aspects of image acquisition and data analysis need to be clarified and extended.

    - A critical control is missing to demonstrate the effects of 2 hrs ISO treatment on RyR2 arrangements. This will strengthen the conclusions from the CaMKII and PKA inhibitor RYR2 dispersion reversal experiments which utilize a protocol that includes a 1 hr ISO stimulation followed by a 1Hr ISO + inhibitor treatment.

    - The mathematical modeling results would also benefit from more explanation of how they complement, align, and expand on the experimental studies.

  5. Version published to 10.1101/2022.02.18.481024v1 on bioRxiv