Distinct functions of cardiac β-adrenergic receptors in the T-tubule vs. outer surface membrane

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β-adrenoceptors (β-ARs) regulate cardiac function during sympathetic nerve stimulation. β-ARs are present in both cardiac T-tubule (TTM) and outer surface membrane (OSM), but how their location impacts on their function is unknown. Here, we developed a technology based on size exclusion to explore the function of β-ARs located in the OSM. We synthetized a PEG-Iso molecule by covalent linking isoprenaline (Iso) to a 5000 Da PolyEthylene-Glycol (PEG) chain to increase the size of the β-AR agonist and prevent it from accessing the TT network. The affinity of PEG-Iso and Iso on β 1 - and β 2 -ARs was measured using radioligand binding. Molecular dynamics simulation was used to assess PEG-Iso conformation and visualise the accessibility of the Iso moiety to water. Using confocal microscopy, we show that PEGylation constrains molecules outside the T-tubule network due to the presence of the extracellular matrix. β-AR activation in OSM with PEG-Iso produced a lower stimulation of [cAMP] i than Iso but a larger stimulation of cytosolic PKA at equivalent levels of [cAMP] I and similar effects on excitation-contraction coupling parameters. However, PEG-Iso produced a much lower stimulation of nuclear PKA than Iso. Thus, OSM β-ARs control mainly cytosolic cAMP/PKA pathway and contractility, while TTM β-ARs control mainly nuclear PKA and nuclear protein phosphorylation. Size exclusion strategy using ligand PEGylation provides a unique approach to evaluate the respective contribution of T-tubule vs. outer surface membrane proteins in cardiac cells.

Significance Statement

β-adrenoceptors (β-ARs) regulate cardiac function during sympathetic nerve stimulation. They are present in both cardiac T-tubule and outer surface membranes, but how their location impacts on their function is unknown. By linking the β-AR agonist isoprenaline (Iso) to a PolyEthylene-Glycol (PEG) chain, we increased the size of the agonist to prevent it from entering the T-tubules. Thus, PEG-Iso is only able to activate β-ARs in the outer surface membrane. With this size exclusion strategy, we show that β-ARs located in the outer surface membrane control mainly cytosolic cAMP/PKA pathway and contractility, while those located in the T-tubule membrane control mainly nuclear PKA and nuclear protein phosphorylation.

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  1. eLife assessment

    This study describes an interesting approach using PEGylated isoprenaline to selectively activate beta-adrenergic receptors in the surface sarcolemma of ventricular myocytes. While the concept is compelling, and the core of an interesting and impactful study is presented, the results are preliminary and incomplete at this stage, and would benefit from more rigorous validation of the approach. The work will be of interest to cardiac cell biologists and pharmacologists.

  2. Reviewer #1 (Public Review):

    In this study, Barthe et al. developed an approach to selectively activate beta-adrenergic receptors in the sarcolemma of ventricular myocytes. The approach involved the linking of a 5Kd PEG chain to the beat agonist isoprenaline. This prevents the agonist from entering transverse tubules. Using this approach, the authors find that activation of beta-adrenergic receptors in the surface sarcolemma of ventricular myocytes leads to lower cytosolic cAMP levels but longer-lasting effects on EC coupling than when TT receptors were activated.

    Strengths of the study:

    1. The PEG-ISO, size exclusion approach is very interesting and useful.
    2. The observation that activation of beta-adrenergic receptors in the surface sarcolemma of ventricular myocytes leads to lower cytosolic cAMP levels, but longer-lasting effects on EC coupling than when TT receptors were activated is interesting.
    3. The observation that beta-adrenergic receptors in the TT lead to stronger nuclear activation of nuclear cAMP/PKA signaling is interesting.

    Weaknesses of the study:

    1. There seems to be a paucity of mechanistic insights into the study.
    2. It is unclear what would be the ideal control for these experiments. Would the addition of the PEG chain, by itself, alter the binding of and activation of beta-adrenergic receptors regardless of their location?
    3. The novelty of the findings is unclear, as other studies have suggested differential effects of beta-adrenergic receptors in membrane compartments.

    Impact on the field:

    1. PEG-ISO may become a useful strategy to selectively activate surface sarcolemmal beta-adrenergic receptors.
  3. Reviewer #2 (Public Review):

    Barthé et al. present a manuscript examining membrane-domain specific signaling by βAR stimulation in cardiomyocytes. Specifically, the authors seek to use a size exclusion approach using PEGylated-isoproterenol to allow only surface sarcolemmal βAR receptor stimulation without T-tubule βAR stimulation. This innovative approach was advanced using confocal microscopy to determine the accessibility of the PEGylated substrates to the T-tubule network. The authors show comparable responses of L-type Ca channels, Ca transients, and contraction using equipotent doses of PEG-Iso and Iso, but differences in nuclear and cytoplasmic cAMP responses based on FRET reporters.

    1. The size exclusion strategy using PEGylation technology is well rationalized and well supported by the physicochemical characterization of PEGylated Iso. This represents a novel strategy to decipher cardiomyocyte cell surface signaling from T-tubule network signaling resulting from the stimulation of β-adrenergic receptors. This approach can be used to study the compartmentalization of various signaling pathways in cardiomyocytes as well as in other cell types that exhibit complex cytoarchitecture. The authors use multiple cAMP FRET sensors as well as assay a number of relevant physiological cellular responses to assess the effect of Iso vs. PEGylated Iso which are informative.

    1. The authors' evidence that PEG-FITC does not penetrate the TT network is not convincing as presented in Figure 1. A single confocal image from one cell showing a lack of fluorescence (Figure 1A) could be due to an outlier cell or lack of penetration to more central regions of the cell where images are taken from. More convincing would be a confocal Z-scan series comparing PEG-FITC and FITC in ARVM. Some form of quantification of T-tubule network density from multiple cells would provide even more robust evidence, similar to the many studies that have done this characterization in models of dilated cardiomyopathy showing a loss of TT network. This exclusion of PEG-FITC provides the critical foundation for the paper and it is somewhat unanticipated given the large dimensions of the t-tubules relative PEG-Iso, so strong data here are particularly important.

    2. The conclusion on line 160 that 'the maximal efficacy of PEG-Iso was significantly lower by 30% than that of Iso,' may be overstated. What approach was used to conclude significantly differently as this implies a statistical comparison? Were the concentration-response curves fit to determine maximal responses? In the examples given, the responses are continuing to increase at the highest concentrations tested, so it is difficult to simply compare the responses to the highest doses tested.

    3. For experiments using adenovirus delivery of FRET-based sensor, the culture of ARVM is required which may impact the biology. Such culture is known to result in changes in cell structure and physiology with loss of the TT network over time. It is essential for the authors to demonstrate that under the conditions of their FRET experiments, the cells continue to exhibit a robust TT network.

    4. As pointed out by the authors, the interpretation of OSM/TTM adrenergic receptor functions in this study is limited by the fact that the relative contributions of β-adrenergic receptor subtypes had not been assessed. This particularly complicates the interpretation of their results in that the authors demonstrate in Figure 2 that PEGylation increases the Ki for Iso for β1 receptors by 700-fold whereas the increase for β2 receptors is about 200-fold. Thus, the relative contribution of β1 and β2 receptors to a 'comparable' dose of Iso and PEGylated Iso will potentially be different. Could that difference in relative β1/β2 receptors be the cause of the different 'efficacy of nuclear and cytoplasmic' cAMP changes between the two tested ligands in Figure 8 and supplemental Figure 3? This would fundamentally alter the conclusions of the paper.

    5. The equipotent doses of Iso and PEG-Iso were initially defined based on their ability to elevate global [cAMP]i. The authors then further demonstrated that such equipotent doses of Iso and PEG-Iso also had equal effects on ICa,L amplitude, Ca2+ transient parameters, and cellular contractility (shortening), presumably because they raised global [cAMP]i to the same levels. These findings seem to defy the importance of nanodomain organization and local [cAMP]i in the regulation of LTCCs, Ca2+ cycling proteins, and contractile machinery. The authors argued that "Since OSM contributes to ~60% of total cell membrane in ARVMs, either β-ARs and ACs are more concentrated in OSM than TTM, or they are in large excess over what is needed to activate PKA phosphorylation of proteins involved in EC coupling. Also, cAMP produced at OSM must diffuse rapidly in the cytosol in order to activate PKA phosphorylation of substrates located deep inside the cell, such as LTCCs in TTM" (lines 336-341). Although this argument may be valid at high concentrations of Iso and PEG-Iso when PKA activation is saturated, it also implies that discrepancy could be detectable at lower (non-saturating) doses of Iso and PEG-Iso. Thus, additional experiments using lower Iso and PEG-Iso doses are required to support this notion.

    6. The size excluded compartment for PEG-Iso proposed by the authors is the TT network, but this ignores other forms of sarcolemmal nanodomains such as caveolae, which include β2 receptors and AC, and may exhibit similar if not great sensitivity to the size exclusion approaches pioneered by the authors.

  4. Reviewer #3 (Public Review):

    The manuscript by Barthe et al compares the effects derived from the application of isoprenaline (Iso) or isoprenaline covalently linked to PEG (PEG-Iso) on adult rat ventricular myocytes (ARVM). Iso is a well-characterized β-AR agonist and the authors work under the assumption that PEGylation of Iso prevents it from accessing the T-tubules. Therefore, due to its larger size, PEG-Iso is only able to activate β-ARs located on the outer surface membrane (OSM), and any additional effect observed by Iso stimulation is attributed to the activation of β-ARs located in T-tubules. First, the authors determined that the affinity of PEG-Iso for β-ARs is about 100 times lower than the one of Iso. Then, they analyze the effects of Iso (10 nM) and PEG-Iso (1 µM) on calcium channel currents, contractility, calcium transients, and cytosolic and nuclear PKA activity. They only found a stronger effect of Iso on nuclear pKA activity. Therefore they conclude that, while OSM β-ARs stimulation mainly results in positive inotropy and lusitropy, T-tubules ARs stimulation mainly results in increased nuclear pKA activity.

    Overall the manuscript is well written and the findings are biologically important from the perspective of understanding the mechanism of β-AR stimulation as well as in assigning the functional contribution of β-ARs in the OSM and in the T-tubules. However, the major conclusion is not strongly supported by the data. The interpretation of the results is all based on the assumption that PEG-Iso is excluded by the T-tubules, but no experiment presented here rigorously demonstrates this.

    1. The only indication that PEG-Iso may be excluded by the T-tubules is one confocal image in which FITC or PEG-FITC were applied on ARVM. No experiment has been performed to assess if PEG-Iso is indeed not able to enter the T-tubules.
    The treatment of ARVM with neuraminidase made the T-tubules accessible to PEG-FITC. If the authors could demonstrate that neuraminidase treatment followed by PEG-Iso would result in similar nuclear pKA activity as Iso, this would strengthen their conclusion.
    2. The fact that PEG-Iso treatment resulted in a lower increase of intracellular cAMP (Figure 3) could also be due to the activation of a smaller fraction of β-ARs, independent of their localization.