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

George W. P. Madders, Marion Barthe, Flora Lefebvre, Emilie Langlois, Florence Lefebvre, Patrick Lechene, Xavier Iturrioz, Maya Dia, Catherine Llorens-Cortes, Tâp Ha-Duong, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

George W. P. Madders, Marion Barthe, Flora Lefebvre, Emilie Langlois, Florence Lefebvre, et al. Distinct functions of cardiac β-adrenergic receptors in the T-tubule vs. outer surface membrane. eLife, 2025, 14, pp.e84243. hal-05271952

HAL Id: hal-05271952

https://hal.science/hal-05271952v1

Submitted on 22 Sep 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Distinct functions of cardiac β-adrenergic receptors in the T-tubule vs. outer surface membrane

George W. P. Madders<fnref n="1" />,*, Marion Barthé<fnref n="1" />,*, Flora Lefebvre<fnref n="2" />, Emilie Langlois<fnref n="2" />, Florence

Lefebvre<fnref n="1" />, Patrick Lechêne<fnref n="1" />, Maya Dia<fnref n="1" />, Xavier Iturrioz<fnref n="3" />,<fnref n="4" />, Catherine Llorens-Cortes<fnref n="3" />,<fnref n="4" />, Tâp Ha-Duong<fnref n="5" />, Laurence Moine<fnref n="2" />, Nicolas Tsapis<fnref n="2" />, Rodolphe Fischmeister<fnref n="1" />,#

<footnote n="1">¹Université Paris-Saclay, Inserm, UMR-S 1180, Orsay, France.</footnote>

<footnote n="2">²Université Paris-Saclay, CNRS, Institut Galien Paris-Saclay, Orsay, France.</footnote>

<footnote n="3">³Collège de France, CIRB, INSERM U1050/CNRS UMR 7241, Paris, France.</footnote>

<footnote n="4">⁴Université Paris-Saclay, CEA, Département Médicaments et Technologies pour la Santé, SIMoS, Gif-sur-Yvette, France.</footnote>

<footnote n="5">⁵Université Paris-Saclay, CNRS, BioCIS, Orsay, France.</footnote>

<footnote>*Authors with equal contribution</footnote>

<footnote>#Corresponding author: Rodolphe Fischmeister, PhD</footnote>

<footnote>E-mail: rodolphe.fischmeister@inserm.fr</footnote>

<footnote>**Competing interests:** The authors declare no competing interests.</footnote>

<footnote>Classification: Biological Sciences/Physiology</footnote>

<footnote>**Keywords:** T-tubules • membrane compartmentation • size exclusion • β-adrenergic receptors • PEGylation • excitation-contraction coupling • calcium channels • cyclic AMP • cAMP-dependent protein kinase • physiology</footnote>
**Abstract**

β-adrenoceptors (β-ARs) regulate cardiac function during sympathetic nerve stimulation. β-ARs are present in both the cardiac T-tubule (TTM) and outer surface membrane (OSM), but how their location impacts 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 covalently 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 T-tubule network. The affinity of PEG-Iso and Iso on β₁- and β₂-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 of adult rat ventricular myocytes (ARVMs) due to the presence of the extracellular glycocalyx. 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 cAMP and PKA than Iso. Thus, OSM β-ARs in ARVMs control mainly cytosolic cAMP/PKA pathway and contractility, while TTM β-ARs control mainly nuclear cAMP, PKA and consequent 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 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 (PEG-Iso) 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 cAMP/PKA and nuclear protein
phosphorylation.
Introduction

The β-adrenergic receptor (β-AR) is a key player in the regulation of cardiac function during sympathetic nerve stimulation. The classical pathway for β-AR receptor signalling is activation of adenylyl cyclases (AC) via Gαs, resulting in increased intracellular cAMP levels ([cAMP]i) [1]. Three types of β-ARs are expressed in the heart, respectively β₁-, β₂- and β₃-ARs. β₁- and β₂-ARs are both positively coupled to the AC/cAMP cascade and involved in cardiac performance [2] while β₃-ARs may act via either cAMP or cGMP depending on the cell type (atrial vs. ventricular myocytes) [3, 4]. The primary target of cAMP is the cAMP-dependent protein kinase (PKA) that in turn phosphorylates several key proteins involved in the excitation-contraction (EC) coupling, such as the L-type Ca²⁺ channel (LTCC or CaV1.2), phospholamban, troponin I, etc. [5]. The phosphorylation of CaV1.2 (or its regulatory protein Rad [6, 7]) leads to enhanced LTCC current (Ica,L) and sarcoplasmic reticulum (SR) Ca²⁺ release via the ryanodine type 2 receptor (RyR2), contributing to enhanced Ca²⁺ transients and contraction [8, 9]. Phosphorylation of phospholamban increases Ca²⁺ uptake into the SR which accelerates Ca²⁺ transient decay and, together with troponin I phosphorylation, speeds up relaxation. Whereas short term stimulation of β-AR/cAMP is beneficial for the heart, chronic activation of this pathway results in altered Ca²⁺ signalling, cardiac hypertrophy and fibrosis, leading to ventricular dysfunction [10] and cardiac arrhythmias [11-15].

The cell membrane of cardiomyocytes is characterized by invaginations of the surface membrane, occurring primarily perpendicular to myocyte longitudinal edges, at intervals of ~1.8–2 µm, that form a complex interconnected tubular network penetrating deep into the cell interior [16]. This network is called the transverse (T-) tubules (TT) system, although the invaginations may often bifurcate in the axial direction or form branches [16]. These tubular
structures are found mostly in adult ventricular myocytes, where they represent about 30% of the total cell membrane [17, 18], and occur near the sarcomeric z-discs [19], forming functional junctions with the SR called dyads. They are critical for EC coupling by concentrating LTCCs and positioning them at close proximity of RyR2 clusters at the junction of SR to form Ca²⁺ release units [20, 21]. During an action potential, TTs propagate the cell-membrane depolarization inside the cell allowing Ca²⁺ entry to trigger successive Ca²⁺ release thus promoting synchronicity as well as ensuring efficiency of the Ca²⁺-induced Ca²⁺ release (CICR) phenomenon [22]. Their enrichment with ion channels and the presence of signaling pathways components such as β-ARs [23] and ACs [24] make these structures essential for cardiomyocyte function and regulation.

Biochemical assays following membrane fractionation have provided indirect evidence that membrane proteins may have different properties whether they are located in TTM or OSM. Other indirect evidence came from experiments in cardiomyocytes in which the TTM was uncoupled from OSM (detubulation) using a hyperosmotic shock with molar concentrations of formamide [17, 25-33]. However, none of these approaches allowed to investigate in an intact cardiomyocyte whether the function of a given membrane receptor differs if the receptor is located in OSM or TTM. To do so requires being able to separately activate or inhibit the receptor in OSM or TTM which has not been feasible so far. Here, we tackled this challenge by developing a size exclusion strategy using the PolyEthylene-Glycol (PEG)ylation technology. By covalently linking isoprenaline (Iso) to a PEG chain, we increased the size of the β-AR agonist to prevent it from accessing the TT network. Our working hypothesis is that PEGylated isoprenaline (PEG-Iso) activates only β-ARs present in OSM while free Iso activates β-ARs present in both OSM and TTM. We thus characterized the
pharmacological properties of PEG-Iso and compared its functional effects in adult rat ventricular myocytes (ARVMs) with those of Iso.

## Results

### PEGylation constrains molecules outside the TT network

The rationale for using ligand PEGylation as a tool to impede ligand diffusion into T-tubules came from the observation that fluorescent PEG<sub>5000</sub> molecules were unable to access to TTs. A typical representative experiment (out of 15) using confocal microscopy is shown in Figure 1. When ARVMs were exposed during 15 min to 100 µM of PEG<sub>5000</sub> molecules functionalized with Fluorescein Isothiocyanate (FITC), no fluorescence was seen throughout the cell (Figure 1A, B). On the contrary, when the cells were exposed during 15 min to 100 µM fluorescein, fluorescence was seen with clear staining of the TTs (Figure 1C, D). We suspected that the lack of access of PEG-FITC in the TTs was due to steric hindrance due to the presence of glycocalyx matrix. To test this hypothesis, ARVMs were first exposed during 1 hr to a solution containing 0.25 U/mL neuraminidase, an enzyme that degrades sialic acid in the glycocalyx matrix [34].

As shown in Figure 1E, F, this allowed PEG-FITC to enter the TTs as evidenced by the striated profile now observed. Summary data (Figure 1G) reflects these observations where fluorescence intensity is reduced from clearly stained TTs in FITC to almost 0 fluorescence in PEG-FITC. Degradation of the glycocalyx by neuraminidase restores fluorescence intensity to the same level as that seen in free FITC.
PEG-Isoprenaline binds to β₁- and β₂-adrenergic receptors

The above experiments demonstrate that PEG<sub>5000</sub> molecules are unable to access the TTs in our experimental conditions. We thus synthesized PEGylated isoprenaline (PEG-Iso) by covalently linking isoprenaline to PEG<sub>5000</sub> molecules (Figure 1 - supplement 1). Our rationale was that since PEG-Iso does not access TTs, it will not reach β-ARs located in the TTM. But the question remained whether PEG-Iso would be able to bind to β-ARs located in the OSM. To address this question, we performed radioligand binding studies in purified membranes from Chinese Hamster Ovary (CHO) cells overexpressing either human β<sub>1</sub>- or β<sub>2</sub>-ARs. Competition curves between [<sup>125</sup>Iodo]cyanopindolol and Iso or PEG-Iso were used to measure K<sub>i</sub> values of both ligands for the β<sub>1</sub>- and β<sub>2</sub>-ARs. As shown in Figure 2A and B, PEG-Iso binds to both β<sub>1</sub>- and β<sub>2</sub>-ARs, but with an affinity which is ~2-orders of magnitude lower than Iso. Functional experiments using homogeneous time-resolved fluorescence (HTRF) technology on HEK 293 cells, that stably expressed either the β<sub>1</sub>- or β<sub>2</sub>-ARs showed that the EC<sub>50</sub> for cAMP stimulation induced by Iso was 7.4 ± 3.6 pM vs. 39.4 ± 24.2 nM (n=4) for PEG-Iso in β<sub>1</sub>-AR expressing cells and 3.4 ± 7.7 pM vs. 20.2 ± 69.8 nM (n=4) for PEG-Iso in β<sub>2</sub>-AR expressing cells. Thus, the ability of PEG-Iso to activate β<sub>1</sub>- and β<sub>2</sub>-ARs was reduced by the same order of magnitude for both receptors which should not bias the following cellular experiments.

We hypothesized that the decreased affinity of PEG-Iso could be due to wrapping of
the PEG chain around Iso moiety reducing its exposure to solvent (water). To test this, we used
molecular dynamics simulations to evaluate the solvent-accessible surface area (SASA) of Iso
moiety in PEG-Iso compared to free Iso. Figure 2C shows that free Iso SASA remains steady
over the whole studied time range (black line). In contrast, the Iso moiety in PEG-Iso (blue line)
has a SASA which largely fluctuates and on multiple instances reaches values close to zero,
indicating its frequent shielding by the PEG chain. We calculated that only 1.2% of PEG-Iso
conformations have an Iso SASA larger than 90% of the free Iso average value. This means that over a time lapse of 100 s, the Iso moiety is only unwrapped from the PEG chain and able to bind to a receptor for ≈1 s of the experiment.

## Comparison of the effects of PEG-Iso and Iso on cytosolic cAMP in ARVMs

The next series of experiments were designed to test whether PEG-Iso was able to produce a functional β-AR response in ARVMs. First, cytosolic cAMP ([cAMP]ᵢ) was monitored in isolated ARVMs expressing the FRET-based sensor Epac-S<sup>H187</sup> [35]. As seen in Figure 3, both Iso (Figure 3A, B) and PEG-Iso (Figure 3C, D) produced a concentration-dependent increase in [cAMP]ᵢ. However, as highlighted in Figure 3E, there were two major differences: 1) the concentration-response curve to PEG-Iso was shifted ~100-fold towards larger concentrations as compared to Iso; 2) the maximal efficacy of PEG-Iso was significantly lower by ~30% than that of Iso. The former was anticipated based on the lower binding affinity of PEG-Iso to β-ARs shown above. The latter can be explained by the fact that PEG-Iso only activates β-ARs in OSM while Iso activates β-ARs in both OSM and TTM, thus producing a larger [cAMP]ᵢ elevation.

## Comparison of the effects of PEG-Iso and Iso on I<sub>Ca,L</sub>

Next, the ability of Iso and PEG-Iso to stimulate the L-type Ca²⁺ current, I<sub>Ca,L</sub> was compared at a single concentration of each, 10 nM Iso and 1 µM PEG-Iso, shown above to produce an equivalent response on [cAMP]ᵢ (Figure 3). As shown in the individual experiments in Figure 4A and B, both Iso and PEG-Iso produced a similar increase in the amplitude of I<sub>Ca,L</sub> (Figure 4C).

## Comparison of the effects of PEG-Iso and Iso on excitation-contraction coupling
To investigate the impact of stimulating TTM and OSM β-ARs on EC coupling, Ca²⁺ transients and sarcomere shortening measurements were simultaneously recorded in Fura-2-loaded ARVMs and their response to PEG-Iso and Iso evaluated. Here, two concentrations of PEG-Iso were used, 100 and 300 nM, and compared to two affinity-adjusted concentrations of Iso that produce similar elevations of [cAMP]ᵢ, 1 and 3 nM. As shown in Figure 5 and 6, 100 and 300 nM PEG-Iso increased contractility (Figure 5B), Ca²⁺ transients (Figure 6B) and accelerated their relaxation kinetics (Figure 5D and 6D). These effects were not different from those produced by 1 and 3 nM Iso, respectively, for either sarcomere shortening (Figure 5A, C), Ca²⁺ transient amplitude (Figure 6A, C) or relaxation kinetics (Figure 5C, D; Figure 6C, D).

## Comparison of the effects of PEG-Iso and Iso on cytosolic and nuclear PKA activity

The observation that equipotent concentrations of PEG-Iso and Iso on [cAMP]ᵢ produced equivalent effects on *I*<sub>Ca,L</sub> and EC coupling, may suggest that OSM β-ARs are not functionally different from their TTM homologs as long as their degree of activation leads to similar [cAMP]ᵢ responses. However, the above experiments do not exclude possible differences in the compartmentalization of intracellular cAMP cascade when cAMP is synthesized upon OSM or TTM β-AR stimulation. To obtain further insight, we compared the effect of PEG-Iso and Iso on PKA activity in the bulk cytoplasm and in the nucleus. For that, we used genetically encoded A-kinase activity FRET-based reporters (AKAR3) targeted to these compartments by the addition of a nuclear export sequence (NES), and a nuclear localizing sequence (NLS), respectively [36]. As shown previously [37, 38], adenoviral transfer allowed robust and compartment-specific expression of these biosensors after 24 h in ARVMs. In the experiments shown in Figure 7, four increasing concentrations of Iso (0.3, 1, 3 and 10 nM: Figure 7A, C) and PEG-Iso (10, 30, 100 nM and 1 µM: Figure 7B, D) were successively applied to ARVMs infected
with AKAR3-NES probe and cytosolic PKA activation was measured. Both compounds produced a clear concentration-dependent increase in cytosolic PKA activity, with a similar efficacy (maximal cytosolic PKA response of 59.7 ± 1.9% for Iso compared to 55.1 ± 3.9% for PEG-Iso; P=0.33) but a ~100-fold lower potency for the effect of PEG-Iso as compared to Iso. However, when both nuclear cAMP (Figure 8) and nuclear PKA (Figure 9) activity was measured, clear differences between the effects of Iso and PEG-Iso were observed: While PEG-Iso and Iso still produced a concentration-dependent increase in nuclear cAMP/PKA activity, the largest response to Iso (Figure 8C and 9C) was ≈2-fold greater compared to the largest response to PEG-Iso (Figure 8D and 9D; P=0.002 (cAMP) and P=0.033 (PKA), respectively). The same difference is observed when measuring the total nuclear protein phosphorylation by nuclear PKA (Figure 9–figure supplement 1) after stimulation with 10 nM Iso or 1 µM PEG-Iso by western blot.

Figure 9 – figure supplement 2 shows a plot of cytosolic and nuclear PKA activity as a function of cytosolic cAMP from the average data in the experiments in Figure 3, 7 and 9 for three concentrations of PEG-Iso (10 and 100 nM and 1 µM) and Iso (0.3, 1 and 10 nM). It shows that for any given measured increase in [cAMP]ᵢ, PEG-Iso is more efficient than Iso to increase PKA activity in the cytosol (Figure 9 – figure supplement 2A), while on the contrary Iso is more efficient than PEG-Iso to increase PKA activity in the nucleus (Figure 9 – figure supplement 2B).

Discussion

The main function of the TT system is to provide proximity between LTCCs in the TTM and RyR2 in the SR membrane [21, 39]. However, TTM also contains many other membrane proteins, such as receptors, enzymes, ion channels and transporters. These include plasma
membrane Ca²⁺-ATPase [40], Na⁺-Ca²⁺ exchanger [41, 42], Na⁺ channels [41], Na⁺/K⁺-ATPase [43], ACs [24, 44] and β-ARs [23, 45]. Density of these membrane proteins is usually found to be higher in the TTM than in the external membrane. Hence, the TT network plays a determinant role in rapid activation and synchronous Ca²⁺ release, cellular signalling and, consequently, cardiac contraction in both human and animal models [46-48].

Any given membrane protein is likely to serve different functions and be regulated in a different manner whether it is located in TTM or in OSM. Attempts to address this important question have so far been based on the elimination of the TT network (detubulation) using a hyperosmotic shock with molar concentrations of formamide [17, 25-33]. This technique, first used in skeletal muscle [49], was introduced in the cardiac field by Kawai *et al.* [33] and provided an excellent experimental tool for numerous T-tubular studies. However, the potential role of shock-induced detubulation in physiologically and pathophysologically relevant conditions is essentially unknown. The method is very harsh on the cells with fewer than 10% of the cells surviving the procedure, which raises issues about whether the "survivors" are truly representative cells [18]. Furthermore, osmotic shock produces a sealing of the TTs which remain present as vesicles inside the cell [25] and may still respond to hormonal or pharmacological challenges and signalling cascades even if they are electrically disconnected from the surface membrane [50, 51].

To overcome these limitations, we introduce here a completely different approach based on size exclusion. We proposed to enlarge the size of a ligand molecule by attaching it to a 5 kDa chain of PEG so that it does not access TTM but remains active on OSM. PEG is a hydrophilic, flexible and rather inert polymer. The covalent linking of one or several PEG chains to a therapeutic molecule, called PEGylation [52], is commonly used in several products on
the market [53]. PEGylated molecules exhibit improved biodistribution and pharmacokinetics, better stability and solubility, reduced immunogenicity and longer plasma half-life due to both reduced renal filtration and proteolysis, when compared to non-PEGylated analogues [54, 55]. In our study, we diverted the same technology towards an entirely different objective: Instead of using PEGylated molecules to improve drug formulation, pharmacokinetics and efficacy, we propose to use PEGylated drugs as *key holders* to prevent drug (*key*) access in the TT network and thus limit its access to the outer surface of the cell. We provide a proof of concept that this approach works using PEG$_{5000}$ functionalized with non-permeant FITC (PEG-FITC): When ARVMs were exposed to PEG-FITC, fluorescence was only seen on the periphery of the cell while when the non-PEGylated free diffusible dye was used, fluorescence was seen in the TT compartment (Figure 1). Thus, large molecular weight PEGs are prevented to diffuse within TTs whereas small molecular weight fluorophore can easily enter.

This finding was surprising, though, since longitudinal TT diameter from healthy control ARVMs varies from 50 to 350 nm (~200 nm on average [56]) [47, 56], which is significantly larger than the size of a PEG$_{5000}$ molecule, which is ≈5 nm. However, a number of recent studies have shown that solute movement in the TT network is strongly restricted even though the diameter of TTs is far larger than the molecular dimensions of typical solutes [57-61]. Impediment of solute movement in the TT network was proposed to be caused by the presence of the glycocalyx [62] at the cell surface, reducing the effective diameter of a TT [34, 58, 59]. This was supported by two findings. Firstly, that pre-treatment of isolated ARVMs with neuraminidase, an enzyme which cleaves sialic acid from oligosaccharide chains in the glycoprotein matrix on the external cell surface, allowed PEG-FITC to enter the TT compartment (Figure 1). Secondly, when ARVMs pre-treated with neuraminidase were stimulated with PEG-Iso the FRET derived responses were much larger than in the non-treated
cells, indicative of increased access to the β-ARs in the TTM, similar to free Iso (Figure 9 – figure supplement 3). The cytosolic cAMP response increased by ≈ 100% (Figure 9 – figure supplement 3A), whilst nuclear cAMP (Figure 9 – figure supplement 3C) and nuclear PKA responses (Figure 9 – figure supplement 3E and 3G) were ≈ 50% greater. Controls using Iso confirmed this to be an effect of the glycocalyx being degraded rather than a direct effect of neuraminidase on the β-ARs (Figure 9 – figure supplement 3B, D and F). Thus, when the protocol used for cell isolation is sufficiently gentle to preserve the integrity of the extracellular matrix in the TT, which must be the case in our experimental conditions, it is possible to prevent a ligand from accessing the TTM if the size of the ligand molecule is sufficiently enlarged. PEG molecules of 5000 Da MW appear to provide a good compromise between solubility issues and size exclusion capability.

Several studies have shown localization of β-ARs (particularly β₂-ARs [63, 64]) in caveolae. Our hypothesis is that, unlike what is occurring within TT, caveolae are not filled with a dense extracellular matrix, possibly because of a better access to enzymatic digestion during cell isolation. Future experiments will test this hypothesis by disrupting caveolae with methyl-beta-cyclodextrin [65] to see how this affects the response to PEG-Iso.

PEG-Iso was thus synthetized by linking Iso to PEG$_{5000}$ molecules. Characterization of its binding properties showed that PEG-Iso binds to β₁- and β₂-ARs but with ~2-orders of magnitude lower affinity than Iso (Figure 2). Since PEG was grafted on the lateral amino group of Iso (Figure 1 – figure supplement 1), a position shown to maintain its affinity for β-ARs [66] and away from the active pharmacophore for β-AR binding, it was unlikely that the chemical modification *per se* was the reason for the decreased affinity. We confirmed that this was the case by detubulating cells with 300 µM imipramine [18]. Without the TT network only the
OSM β-ARs were available for stimulation. Under these conditions, affinity-adjusted Iso and PEG-Iso had identical effects on cytosolic and nuclear cAMP/PKA stimulation (Figure 9 – figure supplement 4). We also addressed the possibility that PEG could change the membrane permeability of Iso and differentially stimulate intracellular β-ARs, the existence of which has been proposed by other groups [67]. We found that irrespective of whether we used a membrane permeable (propranolol) or membrane impermeable (sotalol) β-AR antagonist, the cAMP stimulatory effects of Iso could be fully inhibited (Figure 9 – figure supplement 5), and so under our conditions the stimulatory effects of Iso are confined to the cell membrane. Therefore, the only difference between Iso and PEG-Iso is the ability to bind the β-ARs and not an effect of PEG itself.

We thus explored a possible involvement of the PEG conformation in this phenomenon using molecular dynamics simulations. Because of its hydrophilicity and flexibility, PEG is very dynamic in solution and constantly changes conformation. Consequently, the Iso moiety in PEG-Iso is rarely fully accessible to solvent and spends most of the time with the PEG chain wrapped around it (Figure 2). Based on the calculations, only ~1% of PEG-Iso conformations are able to bind β-ARs as compared to 100% for free Iso, which is in the order of magnitude of the measured difference in their respective binding affinity on β-ARs.

When applied on ARVMs, PEG-Iso produced stimulatory effects on [cAMP]ᵢ, PKA activity, *I*<sub>Ca,L</sub>, sarcomere shortening and Ca²⁺ transients which all had the hallmarks of a β-AR stimulation (Figure 3 to 9). Because of the reduced binding affinity towards β-ARs, PEG-Iso produced its effect at ~2-orders of magnitude larger concentrations than Iso. But there were two other striking differences between the effects of PEG-Iso and Iso which strongly suggest that the two ligands did not act on the exact same populations of receptors.
First, PEG-Iso produced a stimulation of [cAMP]ᵢ with a much lower efficacy than Iso (Figure 3). The simplest explanation for this result is that PEG-Iso activates only β-ARs located in the OSM while Iso activates β-ARs located in both OSM and TTM. Since both populations of β-ARs are likely coupled to ACs [24, 44], one would expect an activation of OSM β-ARs with PEG-Iso to lead to a smaller maximal increase in [cAMP]ᵢ than an activation of all β-ARs with Iso. This diminution is in line with decreased cAMP production sites by detubulation [18]. Cyclic AMP must emanate mainly from sarcolemmal β₁-ARs, which produce highly diffusible signals throughout the cell in contrast to β₂-ARs [68]. However, there are conflicting results regarding the distribution of β-ARs on the cardiac cell membrane. Immunohistochemical data have suggested that β₁- and β₂-ARs are present in OSM and TTM in mouse heart [69]. Using radioligand binding, β₁-AR density was found to be almost 2-fold more concentrated in OSM that TTM, whereas β₂-AR density was evenly distributed across the entire cell surface in dog heart [70]. Using nanoscale live-cell scanning ion conductance and fluorescence resonance energy transfer microscopy techniques in healthy ARVMs, Nikolaev *et al.* found that β₁-ARs are distributed across the entire cell surface while β₂-ARs are localized exclusively in TTM [23]. While the distribution of β₁-ARs across the entire cell surface was confirmed in ARVMs in another study using formamide detubulation, β₂-ARs were found to be only present in OSM [26]. Additional experiments using PEGylated selective β₁- and β₂-AR agonists and antagonists are needed to solve this issue and to precisely characterize the role of each β-AR subtype in the different membrane compartments.

Second, PEG-Iso produced a much lower stimulation of nuclear PKA activity than Iso (Figure 9) even though both ligands were equally efficient in stimulating cytosolic PKA activity (Figure 7). Also, when cytosolic and nuclear PKA activity measured during PEG-Iso or Iso stimulation were plotted as a function of [cAMP]ᵢ measured under the same conditions, PEG-
Iso was found to be more efficient than Iso to increase PKA activity in the cytosol, while on the contrary Iso is more efficient than PEG-Iso to increase PKA activity in the nucleus (Figure 9 – figure supplement 2). A likely interpretation of these results is that β-ARs located in TTM (or a β-AR subtype more concentrated in TTM) are more efficiently coupled to nuclear PKA than those located in OSM, while β-ARs located in OSM (or a β-AR subtype more concentrated in OSM) are more efficiently coupled to cytosolic PKA. In a recent study, we showed that β₂-ARs, unlike β₁-ARs, are inefficient in activating nuclear PKA activity in ARVMs [37]. Thus, if the ratio of β₂/β₁ receptors is larger in OSM than in TTM, as discussed above [26], then this would explain the lower efficacy of PEG-Iso to activate nuclear PKA activity as compared to Iso. Recent studies have shown that perinuclear PKA has the ability to activate without detachment of the catalytic subunits, and thus to phosphorylate its targets in close proximity [71, 72]. According to these studies, only supra-physiological concentrations of cAMP could lead to dissociation of the catalytic subunits and their translocation into the nucleus. Perinuclear PKA is anchored to the nuclear membrane via its interaction with mAKAP and its dynamics are finely regulated by AC5 and PDE4D3 [73], AC5 being concentrated in TTM [24] and PDE4D3 being linked to mAKAP [74]. The location of AC5 may therefore be crucial for nuclear signalling and explain why β-ARs present in TTM are necessary for activation of nuclear PKA. One major limitation of our data is that there is a reduction in TTs over the ≈36-hour infection period needed for robust FRET sensor expression (Figure 9 – figure supplement 6). Therefore, the differences between OSM and TTM may be even greater than we have observed with respect to nuclear PKA activation.

Another important finding from this study concerns the contribution of OSM β-ARs to the regulation of EC coupling. Activation of β-ARs only in OSM with PEG-Iso produced a similar increase in I<sub>Ca,L</sub> amplitude, sarcomere shortening and Ca<sup>2+</sup> transients as Iso, when both ligands
were used at concentrations producing an equivalent elevation of [cAMP]i. Since OSM
contributes to ~60% of total cell membrane in ARVMs [18], 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. It would also suggest that small
GTPase Rab proteins mediated increases in Cav1.2 trafficking to the TTM that occur in
response to β-AR activation [75] are sufficiently activated by OSM cAMP alone for a similar
increase in Ica,L amplitude to have been observed. 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. A recent study showed that under basal conditions, cAMP
is bound to proteins and its diffusion in the cytosol is slow; but when cAMP increases in the
cytosol, e.g. as a result of β-AR stimulation, the cAMP binding sites become saturated and
cAMP diffuses freely [76].

While this study provides new insight on the differential function of OSM vs. TTM β-ARs in healthy cardiomyocytes, it raises obvious questions on how their role is altered in cardiac pathology. A reduction in the density and a disorganization of the TT network is frequently observed in pathological conditions [20, 56, 77], with a substantial impact on both EC coupling and contractile function [21]. A previous study showed a redistribution of β₂-ARs from TTM to OSM in failing ARVMs [23]. As β-ARs are not simply bystanders but also participate in the remodeling during pathological hypertrophy and failure, it is important to know how the OSM vs. TTM subpopulations of β-AR subtypes contribute to this process. The PEGylation technology should enable elucidation of the changes that occur in OSM vs. TTM distribution of β-ARs in ventricular cells isolated from failing hearts, and to determine how this impacts on cellular function.In conclusion, this study provides a novel approach to distinguish the function of a membrane protein depending on its location on the cell membrane. Whereas
we focused here on the function of β-ARs, the size exclusion strategy provided by ligand PEGylation can be extended to other ligands, such as selective agonists or antagonists of β-AR subtypes or other G-protein coupled receptors, dihydropyridine agonists or antagonists of LTCCs, etc. More generally, it should pave the road to exploring and comparing the function of any membrane receptor, channel, transporter or enzyme in TTM and OSM compartments in intact cardiomyocytes.

## Materials and Methods

**Animals.** All animal care and experimental procedures were performed in accordance with the ARRIVE guidelines and conform to the European Community guiding principles in the Care and Use of Animals (Directive 2010/63/EU of the European Parliament), the local Ethics Committee (CREEA Ile-de France Sud) guidelines, and the French decree no. 2013-118 of February 1st, 2013 on the protection of animals used for scientific purposes (JORF no. 0032, February 7th, 2013, p2199, text no. 24). Animal experiments according were carried out according to the European Community guiding principles in the care and use of animals (2010/63/UE), the local Ethics Committee (CREEA Ile-de-France Sud) guidelines and the French decree n° 2013-118 on the protection of animals used for scientific purposes. Adult male rats (Rattus norvegicus) belonging to the Wistar strain (Janvier, Le Genest St Isle, France) have been used. This is a versatile strain of albino rats, selected by Donaldson in 1906 at the Wistar Institute (USA).

**Synthesis of PEG-lso.** Isoprenaline was grafted into the reactive end of 5000 Da PEG (PEG$_{5000}$). A carbodiimide reaction was carried out between the carboxylic acid of PEG and the amine
function of Iso (Figure 1 – figure supplement 1). In a 25 mL amber vial, 41.3 mg of DCC (0.2 mmol; 2eq), 23 mg of NHS (0.2 mmol; 2eq) and 500 mg of OH-PEG$_{5000}$-COOH (0.1 mmol; 1eq) were solubilized in 10 mL of dichloromethane. 1 mL of triethylamine (TEA) was added to the syringe. The solution was stirred for 5 hr at RT. 21.1 mg isoprenaline (0.1 mmol; 1 eq) were added to the reaction mixture. The mixture was left to stir overnight. The solution was then filtered through silica gel and precipitated twice in cold diethyl ether. After evaporation of the ether, the final powder was solubilised in H$_{2}$O.

**Purification of PEG-Iso.** A purification step was introduced to obtain a pure PEG-Iso product and to eliminate the various by-products obtained, potential impurities and especially traces of ungrafted Iso which could interfere in the biological experiments. A HPLC 1290 Infinity II - (Agilent Technologies) consisting of a 4-channel binary pump, UV/visible detector (PDA diode array), 1260 Infinity DEDL light scattering detector and C18 XBridge column, 4.6 x 150 mm, 5 µm (17 mL/min flow rate) was used. The mobile phase consisted of a mixture of water + 0.1% formic acid and acetonitrile. This phase was pushed according to a gradient from 1 to 100% in 15 min into the stationary phase for the purification of PEG-Iso. Detection was carried out in the UV at 254 and 280 nm. PEG-Iso was solubilized in water. Several successive injections of 300 µL each were performed. The retention time of PEG-Iso was 11 min. The fractions containing PEG-Iso were recovered, evaporated and then frozen and freeze-dried. Finally, the powder obtained was stored at -20°C and protected from light.

**Cardiomyocyte isolation and culture.** Adult male Wistar rats (250–300 g) were anesthetized by intraperitoneal injection of pentobarbital (0.1 mg/g) and hearts were excised rapidly. Individual adult rat ventricular cardiomyocytes were obtained by retrograde perfusion of the
heart as previously described [78]. For enzymatic dissociation, the hearts were perfused
during 5 min at a constant flow of 6 mL/min at 37°C with a Ca²⁺-free Ringer solution containing
(in mM): NaCl 117, KCl 5.7, NaHCO₃ 4.4, KH₂PO₄ 1.5, MgCl₂ 1.7, D-glucose 11.7, Na₂-
phosphocreatine 10, taurine 20, and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
(HEPES) 21, pH 7.1. This was followed by a perfusion at 4 mL/min for 40 min with the same
solution containing 1 mg/mL of collagenase A (Roche Diagnostics GmbH, Mannheim,
Germany) plus 300 µM ethylene glycol tetraacetic acid (EGTA) and CaCl₂ to adjust free Ca²⁺
concentration to 20 µM. The ventricles were then separated, finely chopped and gently
agitated to dissociate individual cells. The resulting cell suspension was filtered on gauze and
the cells were allowed to settle down. The supernatant was discarded and cells resuspended
four more times with Ringer solution at increasing [Ca²⁺] from 20 to 300 µM. Freshly isolated
cells were suspended in minimal essential medium (MEM: M 4780; Sigma, St Louis, MO USA)
containing 1.2 mM [Ca²⁺] supplemented with 2.5% foetal bovine serum (FBS, Invitrogen,
Cergy-Pontoise, France), 1% penicillin-streptomycin (P-S), 20 mM HEPES (pH 7.6), and plated
on 35 mm, laminin-coated (10 mg/mL) culture dishes at a density of 10⁴ cells per dish and kept
at 37°C (5% CO₂). After 1 hr, the medium was replaced by 300 µL of FBS-free MEM. To perform
FRET imaging, the medium was replaced by 300 µL of FCS-free MEM or transduced with an
adenovirus encoding either an Epac-SH187 FRET-based sensor[35], or an AKAR3 FRET-based
sensor[36] addressed to the cytosol or the nucleus, at a multiplicity of infection (MOI) of 1000
pfu/cell. Cells were cultivated for 36 h prior to the experiments. Patch-clamp and IonOptix
experiments were performed on cells 24 h after dissociation

**Confocal imaging.** Ventricular cardiomyocytes from adult rats were plated on 35 mm glass bottom Petri dishes. Two hours after plating, the control cells were stained with commercial
fluorescent PEG$_{5000}$ functionalized with FITC (Fluorescein Isothiocyanate) (Nanocs) or free Fluorescein (Sigma-Aldrich). Other cells were treated with neuraminidase (Sigma-Aldrich) for 1 hr at 37°C at a concentration of 0.25 U/mL. After which they were washed and re-incubated in physiological buffer solution, cells were then stained with PEG-FITC. All molecules were dissolved in Ringer containing (in mM): NaCl 121.6, KCl 5.4, MgCl$_2$ 1.8, CaCl$_2$ 1.8, NaHCO$_3$ 4, NaH$_2$PO$_4$ 0.8, D-glucose 5, sodium pyruvate 5, HEPES 10, adjusted to pH 7.4. The acquisitions were made with a Leica TCS SP5 confocal microscope using a white light laser and an X40 oil immersion objective. The cells were excited at 495 nm and fluorescence was recovered at wavelengths >510 nm.

**Fluorescence intensity quantification.** FITC or PEG-FITC that enters the T-tubules (TT) should flag the TTs, similar to Di4Anepps. Thus, we used the same established method used to measure the density of TT in cells. The centre fluorescence image from each cell was selected for a thresholding analysis using an ImageJ plugin described previously [79] and TT quantification was performed using the ImageJ free software. On each image, a brightness/contrast correction was applied to correct acquisition settings differences. Images were filtered with a despeckle filter and a median filter. The image was then binarized using an auto local threshold (Autothreshold plug-in). The threshold method and parameters were visually adjusted to ensure the coherence of the binarized image with the original one. A region of interest was defined inside the cell excluding the plasma membrane then a percent area was calculated. This value corresponds to the ratio of the number of white pixels over the size of the region multiplied by 100 and thus, constitutes an index of the TT density where the fluorophore is visible.
**Detubulation.** The TT network was chemically removed from a subset of control cells by a process termed detubulation as previously described [18]. Briefly, cells were exposed to 300 µM imipramine for 15 min at room temperature, after which they were washed with physiological Ringer's solution and used for FRET. Detubulation was performed ≈ 1 hr before use in functional experiments. Detubulation was confirmed through confocal microscopy with 4 µM Di4Anepps. Cells were excited at 495 nm and fluorescence was recovered at wavelengths >510 nm.

**Molecular dynamics simulations.** Molecular dynamics simulations of free Iso and PEG Iso were carried out to predict their conformations in aqueous solution and to assess the solvent-accessibility of Iso moiety once grafted to the PEG. A 3000 Da PEG molecule was used in these simulations. The 2D chemical structure of each of the two molecules was first converted into a three-dimensional structure using ChemAxon's MarvinSketch chemical editing software. The topology and GAFF force field parameters [80] used in this study were then automatically generated using the ACPYPE program [81]. Initial three-dimensional structure was then placed in the centre of 13.4 nm side cubic simulation. These dimensions allow the solute not to interact with its virtually created images due to the periodic boundary conditions used to simulate an infinitely duplicated mesh. The box was then filled with water (model TIP3P [82]) and 150 mM NaCl. The system was then subjected to 2 short simulations (1 ns each) to equilibrate first the temperature around 300 K and then the pressure around 1 bar. The Nosé-Hoover [83, 84] (coupling time τT = 0.5 ps) and Parrinello-Rahman [85] (τP = 2.5 ps) algorithms were used for maintaining constant temperature and pressure, respectively. Finally, each of the two molecules was subjected to a production run of 150 ns. All simulations were performed using the GROMACS software version 2016 [86]. To analyse the trajectories, we
used the GROMACS internal *gmx sasa* tool to calculate the solvent-accessible surface area (SASA) for Iso moiety in both free Iso or PEG-Iso molecules. To compare the solvent-accessibility of Iso moiety in free Iso and PEG-Iso, the percentage of PEG-Iso conformations with a SASA<sub>PEG-Iso</sub> ≥ 0.9 SASA<sub>free-Iso</sub> was calculated.

**Binding experiments.** Binding assays were carried out in a final volume of 100 µL, containing a membrane suspension of CHO cells overexpressing human β₁- or β₂-adrenergic receptors, 145 pM of the radioligand [¹²⁵Iodo]cyanopindolol and non-radioactive ligands at concentrations ranging from 100 pM to 1 mM. Incubation was carried out for 2 hr at room temperature and terminated by addition of 4 mL PBS. Then, rapid filtration was performed through Whatman GF/C glass fiber filters previously soaked in PBS containing 0.33% polyethyleneimine using a compact cell harvester (Millipore® 1225 Sampling Vacuum Manifold). Binding reaction was transferred to the filters and washed 3 times with Wash Buffer. Filter-bound radioactivity was measured in a gamma counter. All determinations were performed in triplicate at least.

**HTRF quantification of cAMP production.** cAMP was quantified using the HTRF cAMP Gs dynamic detection kit (Revvity, Codolet, France) based on homogeneous time-resolved fluorescence (HTRF) technology. The stimulation was done in the stimulation buffer (stimulation buffer 1X (Revvity) containing 1 mM 3-isobutyl-methylxanthine, IBMX). Briefly, HEK 293 cells stably expressing the human β₁- or β₂-ARs were resuspended in stimulation buffer and dispensed into 384-well plate (1,000 cells per well (5 µl)) and stimulated with increasing concentrations (0.1 pM to 100 µM) of Iso diluted in stimulation buffer (5 µl/well) for 30 min at 20°C. Cells were then lysed, and cAMP levels were determined following
manufacturer instructions. HTRF signal was read with the microplate reader Envision Excite (Perkin Elmer, Turku, Finland).

**FRET imaging.** FRET experiments were performed at room temperature 36 hr after cell plating. Cells were maintained in a Ringer solution containing (in mM): NaCl 121.6, KCl 5.4, MgCl₂ 1.8; CaCl₂ 1.8; NaHCO₃ 4, NaH₂PO₄ 0.8, D-glucose 5, sodium pyruvate 5, HEPES 10, adjusted to pH 7.4. Images were captured every 5 s using the 40x oil immersion objective of a Nikon TE 300 inverted microscope connected to a software-controlled (Metafluor, Molecular Devices, Sunnyvale, CA, USA) cooled charge coupled (CCD) camera (Sensicam PE, PCO, Kelheim, Germany). Cells were excited for 150-300 ms by a Xenon lamp (100 W, Nikon, Champigny-sur-Marne, France) using a 440/20BP filter and a 455LP dichroic mirror. Dual emission imaging was performed using an Optosplit II emission splitter (Cairn Research, Faversham, UK) equipped with a 495LP dichroic mirror and BP filters 470/30 (CFP) and 535/30 (YFP), respectively. Spectral bleed-through into the YFP channel was subtracted using the formula: $YFP_{corr}=YFP-0.6xCFP$.

**Electrophysiological experiments.** The whole cell configuration of the patch-clamp technique was used to record $I_{Ca,L}$. Patch electrodes had resistance of 0.5–1.5 MΩ when filled with internal solution containing (in mM): CsCl 118, EGTA 5, MgCl₂ 4, Na₂-phosphocreatine 5, Na₂ATP 3.1, Na₂GTP 0.42, CaCl₂ 0.062, Hepes 10, adjusted to pH 7.3 with CsOH. External Cs⁺-Ringer solution contained (in mM): NaCl 107.1, CsCl 20, NaHCO₃ 4, NaH₂PO₄ 0.8, D-glucose 5, sodium pyruvate 5, Hepes 10, MgCl₂ 1.8, CaCl₂ 1.8, adjusted to pH 7.4 with NaOH. The cells were depolarized every 8 s from –50 mV to 0 mV for 400 ms. The liquid junction potential was adjusted to zero current between pipette and bath solution before the cells were attached to
obtain a tight gigaseal (>1GΩ). The use of -50mV as holding potential allowed the inactivation
of voltage-dependent sodium currents. Currents were not compensated for capacitance and
leak currents. The amplitude of I<sub>Ca,L</sub> was measured as the difference between the peak inward
current and the current at the end of the depolarization pulses. The cells were voltage-
clamped a patch-clamp amplifier (either RK-400 (Bio-Logic, Claix, France) or Axopatch 200B
(Axon Instruments, Inc., UnionCity, CA, USA)). Currents were analog filtered at 5 kHz and
digitally sampled at 10 kHz using a 16-bit analog to digital converter (DT321; Data translation,
Marlboro, MA, USA or a Digidata1440A, Axon Instruments) connected to a PC (Dell, Austin,
TX, USA).

**Measurements of Ca²⁺ transients and sarcomere shortening.** All experiments were performed at room temperature. Isolated ARVMs were loaded with 1 µM Fura-2 AM (Thermo Fischer Scientific) and Pluronic acid (0.012%, Thermo Fischer Scientific) for 15 min in a Ringer solution containing (in mM): KCl 5.4, NaCl 121.6, sodium pyruvate 5, NaHCO₃ 4.013, NaH₂PO₄ 0.8, CaCl₂ 1.8, MgCl₂ 1.8, glucose 5, Hepes 10, pH 7.4 with NaOH. Sarcomere shortening and Fura-2 ratio (measured at 512 nm upon excitation at 340 and 380 nm) were simultaneously recorded in Ringer solution, using a double excitation spectro fluorimeter coupled with a video detection system (IonOptix, Milton, MA, USA). Myocytes were electrically stimulated, with biphasic field pulses (5 V, 4 ms) at a frequency of 1 Hz. Ca²⁺ transient amplitude was measured by dividing the twitch amplitude (difference between the end-diastolic and the peak systolic ratios) by the end-diastolic ratio, thus corresponding to the percentage of variation in the Fura-2 ratio. Similarly, sarcomere shortening was assessed by its percentage variation, which was obtained by dividing the twitch amplitude (difference between the end-diastolic and the peak systolic sarcomere length) by the end-diastolic sarcomere length. Relaxation kinetics
were estimated by a non-linear fit of the decaying part of the Ca²⁺ transient and sarcomere shortening traces with the following equation: $X(t) = A \cdot \exp(-(t-t_0)/\tau)+A_0$, where $t_0$ is zero time, $A_0$ the asymptote of the exponential, $A$ the relative amplitude of the exponential and $\tau$ the time constant of relaxation. The maximum first derivative of transients during the deflexion allowed determination of the rising velocities of the signals. All parameters were calculated offline with dedicated software (IonWizard 6x, IonOptix R).

**Protein expression analysis.** Cardiomyocytes were treated with or without isoprenaline 10 nM or PEG-Iso 1 µM (for 15 min) and the nuclei were enriched using a purification kit (ProteoExtract® Subcellular Proteome, Millipore). Nuclear proteins were examined by immunoblot for phosphorylation of PKA substrates using Phospho-(Ser/Thr) PKA Substrate antibody (Cell Signaling, Cat# 9621, RRID: AB_330304) and total protein per lane. The ratio of phospho-PKA expression was assessed by densiometric scanning of immunoblots.

**Statistical analysis.** All results are expressed as mean ± SEM. Statistical analysis was performed using GraphPad software (GraphPad Software V.10.4.1, Inc., La Jolla, CA, USA). Normal distribution was tested by the Shapiro-Wilk normality test. For simple two-group comparison that satisfied parametric assumptions, we used an unpaired Student's t test. Differences between multiple concentrations of the same drug were analysed using an ordinary 1-way ANOVA with Tukey post hoc test. A P value<0.05 was considered statistically significant.

**Funding and Acknowledgements**

MB was supported by doctoral grant from the Laboratory of Excellence LERMIT supported by the French National Research Agency (ANR-10-LABX-33) under the program "Investissements
d'Avenir" ANR-11-IDEX-0003-01. She also received a doctoral grant from the Fondation pour la Recherche Médicale. This work was also funded by grants ANR-15-CE14-0014-01 and ANR-23-CE14-0027-01 'CARDIOPEG' to RF which was used to support GM, alongside a postdoctoral research award grant from the Fondation Lefoulon-Delalande. Authors would like to thank K. Leblanc (BioCIS) for help with preparative HPLC and Dr R. Jockers (Institut Cochin, France), who kindly provided the HEK 293 cells expressing β-adrenergic receptors.
References

1. Leroy, J., G. Vandecasteele, and R. Fischmeister, Cyclic AMP signaling in cardiac myocytes. Curr Op Physiol, 2018. **1**: p. 161-171.

2. Xiang, Y.K., Compartmentalization of β-adrenergic signals in cardiomyocytes. Circ Res, 2011. **109**(2): p. 231-44.

3. Schobesberger, S., et al., β₃-Adrenoceptor redistribution impairs NO/cGMP/PDE2 signalling in failing cardiomyocytes. Elife, 2020. **9**: p. e52221.

4. Skeberdis, V.A., et al., β₃-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca²⁺ current. J Clin Invest, 2008. **118**: p. 3219-3227.

5. Bers, D.M., Cardiac excitation-contraction coupling. Nature, 2002. **415**(6868): p. 198-205.

6. Leroy, J. and R. Fischmeister, β-adrenergic regulation of the L-type Ca²⁺ current: The missing link eventually discovered. Med Sci (Paris), 2020. **36**(6-7): p. 569-572.

7. Liu, G., et al., Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics. Nature, 2020. **577**(7792): p. 695-700.

8. Bers, D.M., Calcium cycling and signaling in cardiac myocytes. Ann Rev Physiol, 2008. **70**: p. 23-49.

9. Eisner, D.A., et al., Calcium and excitation-contraction coupling in the heart. Circ Res, 2017. **121**(2): p. 181-195.

10. Osadchii, O.E., Myocardial phosphodiesterases and regulation of cardiac contractility in health and cardiac disease. Cardiovasc Drugs Ther, 2007. **21**: p. 171-94.

11. Boluyt, M.O., et al., Alterations in cardiac gene expression during the transition from stable hypertrophy to heart failure -Marked upregulation of genes encoding extracellular matrix components. Circ Res, 1994. **75**(1): p. 23-32.

12. Morisco, C., et al., β-Adrenergic cardiac hypertrophy is mediated primarily by the β₁-subtype in the rat heart. J Mol Cell Cardiol, 2001. **33**(3): p. 561-573.

13. Engelhardt, S., et al., Progressive hypertrophy and heart failure in β₁-adrenergic receptor transgenic mice. Proc Natl Acad Sci USA, 1999. **96**(12): p. 7059-7064.

14. Antos, C.L., et al., Dilated cardiomyopathy and sudden death resulting from constitutive activation of protein kinase A. Circ Res, 2001. **89**(11): p. 997-1004.

15. Karam, S., et al., Cardiac overexpression of PDE4B blunts β-adrenergic response and maladaptive remodeling in heart failure. Circulation, 2020. **142**(2): p. 161-174.

16. Hong, T. and R.M. Shaw, Cardiac T-tubule microanatomy and function. Physiol Rev, 2017. **97**(1): p. 227-252.

17. Brette, F., L. Salle, and C.H. Orchard, Quantification of calcium entry at the T-tubules and surface membrane in rat ventricular myocytes. Biophys J, 2006. **90**(1): p. 381-389.

18. Bourcier, A., et al., Imipramine as an alternative to formamide to detubulate rat ventricular cardiomyocytes. Exp Physiol, 2019. **104**(8): p. 1237-1249.

19. Caldwell, J.L., et al., Dependence of cardiac transverse tubules on the BAR domain protein amphiphysin II (BIN-1). Circ Res, 2014. **115**: p. 986-96.

20. Guo, A., et al., Emerging mechanisms of T-tubule remodelling in heart failure. Cardiovasc Res, 2013. **98**(2): p. 204-15.

21. Sipido, K.R. and H. Cheng, T-tubules and ryanodine receptor microdomains: on the road to translation. Cardiovasc Res, 2013. **98**(2): p. 159-61.
22. Fu, Y., et al., Isoproterenol promotes rapid ryanodine receptor movement to BIN1 organized dyads. Circulation, 2016. **133**(4): p. 388-97.

23. Nikolaev, V.O., et al., β₂-Adrenergic receptor redistribution in heart failure changes cAMP compartmentation. Science, 2010. **327**(5973): p. 1653-7.

24. Timofeyev, V., et al., Adenylyl cyclase subtype-specific compartmentalization: Differential regulation of L-type Ca²⁺ current in ventricular myocytes. Circ Res, 2013. **112**(12): p. 1567-76.

25. Moench, I., et al., Resolution of hyposmotic stress in isolated mouse ventricular myocytes causes sealing of t-tubules. Exp Physiol, 2013. **98**: p. 1164-77.

26. Cros, C. and F. Brette, Functional subcellular distribution of β₁- and β₂-adrenergic receptors in rat ventricular cardiac myocytes. Physiol Rep, 2013. **1**(3): p. e00038.

27. Brette, F., et al., Spatiotemporal characteristics of SR Ca²⁺ uptake and release in detubulated rat ventricular myocytes. J Mol Cell Cardiol, 2005. **39**: p. 804-12.

28. Fowler, M.R., et al., Functional consequences of detubulation of isolated rat ventricular myocytes. Cardiovasc Res, 2004. **62**(3): p. 529-537.

29. Brette, F., L. Salle, and C.H. Orchard, Differential modulation of L-type Ca²⁺ current by SR Ca²⁺ release at the T-tubules and surface membrane of rat ventricular myocytes. Circ Res, 2004. **95**(1): p. e1-7.

30. Thomas, M.J., et al., Localization and function of the Na⁺/Ca²⁺-exchanger in normal and detubulated rat cardiomyocytes. J Mol Cell Cardiol, 2003. **35**(11): p. 1325-37.

31. Despa, S., et al., Na/Ca exchange and Na/K-ATPase function are equally concentrated in transverse tubules of rat ventricular myocytes. Biophys J, 2003. **85**(5): p. 3388-3396.

32. Brette, F., K. Komukai, and C.H. Orchard, Validation of formamide as a detubulation agent in isolated rat cardiac cells. Am J Physiol Heart Circ Physiol, 2002. **283**(4): p. H1720-H1728.

33. Kawai, M., M. Hussain, and C.H. Orchard, Excitation-contraction coupling in rat ventricular myocytes after formamide-induced detubulation. Am J Physiol, 1999. **277**(2 Pt 2): p. H603-9.

34. Parfenov, A.S., et al., Aqueous diffusion pathways as a part of the ventricular cell ultrastructure. Biophys J, 2006. **90**(3): p. 1107-19.

35. Klarenbeek, J., et al., Fourth-generation EPAC-based fret sensors for cAMP feature exceptional brightness, photostability and dynamic range: Characterization of dedicated sensors for flim, for ratiometry and with high affinity. PLoS One, 2015. **10**: e0122513.

36. Allen, M.D. and J. Zhang, Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem Biophys Res Commun, 2006. **348**(2): p. 716-21.

37. Bedioune, I., et al., PDE4 and mAKAPβ are nodal organizers of β₂-AR nuclear PKA signaling in cardiac myocytes. Cardiovasc Res, 2018. **114**(11): p. 1499-1511.

38. Haj Slimane, Z., et al., Control of cytoplasmic and nuclear protein kinase A activity by phosphodiesterases and phosphatases in cardiac myocytes. Cardiovasc Res, 2014. **102**(1): p. 97-106.

39. Carl, S.L., et al., Immunolocalization of sarcolemmal dihydropyridine receptor and sarcoplasmic reticular triadin and ryanodine receptor in rabbit ventricle and atrium. J Cell Biol, 1995. **129**(3): p. 673-682.

40. Brette, F. and C. Orchard, T-tubule function in mammalian cardiac myocytes. Circ Res, 2003. **92**(11): p. 1182-92.
41. Scriven, D.R.L., P. Dan, and E.D.W. Moore, Distribution of proteins implicated in excitation-contraction coupling in rat ventricular myocytes. Biophys J, 2000. **79**(5): p. 2682-2691.

42. Yang, Z., et al., Na⁺-Ca²⁺ exchange activity is localized in the T-tubules of rat ventricular myocytes. Circ Res, 2002. **91**(4): p. 315-322.

43. Berry, R.G., et al., Differential distribution and regulation of mouse cardiac Na⁺/K⁺-ATPase α₁ and α₂ subunits in T-tubule and surface sarcolemmal membranes. Cardiovasc Res, 2007. **73**(1): p. 92-100.

44. Gao, T.Y., et al., Identification and subcellular localization of the subunits of L-type calcium channels and adenylyl cyclase in cardiac myocytes. J Biol Chem, 1997. **272**(31): p. 19401-19407.

45. Gorelik, J., et al., Spatial control of the βAR system in heart failure: the transverse tubule and beyond. Cardiovasc Res, 2013. **98**(2): p. 216-24.

46. Song, L.S., et al., Orphaned ryanodine receptors in the failing heart. Proc Natl Acad Sci USA, 2006. **103**(11): p. 4305-10.

47. Soeller, C. and M.B. Cannell, Examination of the transverse tubular system in living cardiac rat myocytes by 2-photon microscopy and digital image-processing techniques. Circ Res, 1999. **84**(3): p. 266-275.

48. Hatano, A., et al., Critical role of cardiac t-tubule system for the maintenance of contractile function revealed by a 3D integrated model of cardiomyocytes. J Biomech, 2012. **45**: p. 815-23.

49. Argiro, V., Excitation-contraction uncoupling of striated muscle fibres by formamide treatment: evidence of detubulation. J Muscle Res Cell Motil, 1981. **2**(3): p. 283-94.

50. Moench, I. and A.N. Lopatin, Ca²⁺ homeostasis in sealed t-tubules of mouse ventricular myocytes. J Mol Cell Cardiol, 2014. **72**: p. 374-83.

51. Scardigli, M., et al., Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes. J Physiol, 2024. **602**(5): p. 791-808.

52. Veronese, F.M. and J.M. Harris, Introduction and overview of peptide and protein pegylation. Adv Drug Deliv Rev, 2002. **54**(4): p. 453-6.

53. Pasut, G. and F.M. Veronese, State of the art in PEGylation: the great versatility achieved after forty years of research. J Control Release, 2012. **161**(2): p. 461-72.

54. Abuchowski, A., et al., Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. J Biol Chem, 1977. **252**(11): p. 3578-81.

55. Harris, J.M. and R.B. Chess, Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov, 2003. **2**(3): p. 214-21.

56. Wagner, E., et al., Stimulated emission depletion live-cell super-resolution imaging shows proliferative remodeling of T-tubule membrane structures after myocardial infarction. Circ Res, 2012. **111**(4): p. 402-14.

57. Entcheva, E., Uncovering an electrically heterogeneous cardiomyocyte by FRAP-quantified diffusion in the T-tubules. Proc Natl Acad Sci USA, 2018. **115**(4): p. E560-E561.

58. Hong, T., et al., Cardiac BIN1 folds T-tubule membrane, controlling ion flux and limiting arrhythmia. Nat Med, 2014. **20**: p. 624-32.

59. Kong, C.H.T., et al., Solute movement in the t-tubule system of rabbit and mouse cardiomyocytes. Proc Natl Acad Sci USA, 2018. **115**(30): p. E7073-E7080.
60. Scardigli, M., et al., *Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy*. Proc Natl Acad Sci USA, 2017. **114**(22): p. 5737-5742.

61. Uchida, K. and A.N. Lopatin, *Diffusional and electrical properties of T-tubules are governed by their constrictions and dilations*. Biophys J, 2018. **114**(2): p. 437-449.

62. Langer, G.A., J.S. Frank, and K.D. Philipson, *Ultrastructure and calcium exchange of the sarcolemma, sarcoplasmic reticulum and mitochondria of the myocardium*. Pharmacol Ther, 1982. **16**(3): p. 331-76.

63. Wright, P.T. et al., *Caveolin-3 regulates compartmentation of cardiomyocyte β₂-adrenergic receptor-mediated cAMP signaling*. J Mol Cell Cardiol, 2014. **67**: p. 38-48.

64. Wright, P.T. et al., *Cardiomyocyte membrane structure and cAMP compartmentation produce anatomical variation in β₂AR-cAMP responsiveness in murine hearts*. Cell Rep, 2018. **23**:459-469.

65. Calaghan, S. and E. White, E. *Caveolae modulate excitation-contraction coupling and β₂-adrenergic signalling in adult rat ventricular myocytes*. Cardiovasc Res, 2006. **69**:816-824.

66. Ruffolo, R.R., W. Bondinell, and J.P. Hieble, *α- and β-Adrenoceptors: From the gene to the clinic. 2. Structure-activity relationships and therapeutic applications*. J Med Chem, 1995. **38**(19): p. 3681-3716.

67. Wang, Y., et al., *Intracellular beta(1)-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility*. Circ Res, 2021. **128**(2): p. 246-261.

68. Nikolaev, V.O., et al., *Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching β₁-adrenergic but locally confined β₂-adrenergic receptor-mediated signaling*. Circ Res, 2006. **99**(10): p. 1084-1091.

69. Zhou, Y.Y., et al., *Spontaneous activation of β₂-but not β₁-adrenoceptors expressed in cardiac myocytes from β₁β₂ double knockout mice*. Mol Pharmacol, 2000. **58**(5): p. 887-894.

70. He, J.Q., et al., *Crosstalk of β-adrenergic receptor subtypes through Gᵢ blunts beta-adrenergic stimulation of L-type Ca²⁺ channels in canine heart failure*. Circ Res, 2005. **97**(6): p. 566-573.

71. Smith, F.D., et al., *Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation*. Elife, 2013. **2**: p. e01319.

72. Smith, F.D., et al., *Local protein kinase A action proceeds through intact holoenzymes*. Science, 2017. **356**(6344): p. 1288-1293.

73. Diviani, D., et al., *A-kinase anchoring proteins: Scaffolding proteins in the heart*. Am J Physiol Heart Circ Physiol, 2011. **301**(5): p. H1742-H1753.

74. Dodge-Kafka, K.L., et al., *The protein kinase A anchoring protein mAKAP co-ordinates two integrated cAMP effector pathways*. Nature, 2005. **437**: p. 574-578.

75. Del Villar, S.G., et al., *beta-Adrenergic control of sarcolemmal Ca(V)1.2 abundance by small GTPase Rab proteins*. Proc Natl Acad Sci U S A, 2021. **118**(7):e2017937118.

76. Bock, A., et al., *Optical mapping of cAMP signaling at the nanometer scale*. Cell, 2020. **182**(6): p. 1519-1530.

77. Lawless, M., et al., *Phosphodiesterase 5 inhibition improves contractile function and restores transverse tubule loss and catecholamine responsiveness in heart failure*. Sci Rep, 2019. **9**(1): p. 6801.
78. Mika, D., et al., *Differential regulation of cardiac excitation-contraction coupling by cAMP phosphodiesterase subtypes*. Cardiovasc Res, 2013. **100**(2): p. 336-46.

79. Heinzel, F.R., et al., *Spatial and temporal inhomogeneities during Ca2+ release from the sarcoplasmic reticulum in pig ventricular myocytes*. Circ Res, 2002. **91**(11): p. 1023-30.

80. Wang, J., et al., *Development and testing of a general amber force field*. J Comput Chem, 2004. **25**(9): p. 1157-74.

81. Sousa da Silva, A.W. and W.F. Vranken, *ACPYPE - AnteChamber PYthon Parser interfacE*. BMC Res Notes, 2012. **5**: p. 367.

82. Jorgensen, W.L., et al., *Comparison of simple potential functions for simulating liquid water*. J Chemical Physics, 1983. **79**(2): p. 926-935.

83. Hoover, W.G., *Canonical dynamics: Equilibrium phase-space distributions*. Phys Rev A Gen Phys, 1985. **31**(3): p. 1695-1697.

84. Nosé, S., *A unified formulation of the constant temperature molecular dynamics methods*. J Chemical Physics, 1984. **81**(1): p. 511-519.

85. Parrinello, M. and A. Rahman, *Polymorphic transitions in single crystals: A new molecular dynamics method*. J Applied Physics, 1981. **52**(12): p. 7182-7190

86. Abraham, M.J., et al., *GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers*. SoftwareX, 2015. **1-2**: p. 19-25.
<figure><img src="image_3.png" /><figcaption>**Figure 1. Localization of PEG-FITC in freshly isolated adult rat ventricular cardiomyocytes.**</figcaption></figure>

Typical confocal images (A, C, E) and plot profiles representing the fluorescence intensity measured across the cell (B, D, F) of ARVMs incubated during 15 min with either 100 µM PEG<sub>5000</sub>-FITC (A, B), 100 µM free fluorescein (C, D), or 100 µM PEG<sub>5000</sub>-FITC after a 1h treatment with 0.25 U/mL neuraminidase (+nmd) (E, F). Summary fluorescence intensity for cells stained with FITC and PEG-FITC either in control (-, blue) conditions or after treatment with neuraminidase (+, green) (G). Bars represent the mean ± S.E.M. n/N = 8-18/2-4. Kruskall-Wallis test with multiple comparisons was used: **** p<0.0001; ns, non-significant.

The following figure supplements are available for figure 1:
**Figure supplement 1.** PEG-Isoprenaline synthesis.
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

**Figure 2.** Binding properties of Iso and PEG-Iso on β-ARs. β₁- or β₂-AR CHO membrane preparations (2.5 µg/triplicate) were incubated for 2hr with 0.25 nM [¹²⁵Iodo]cyanopindolol and increasing concentrations of unlabeled Iso or PEG-Iso. (A) Example of a typical competition binding of Iso and PEG-Iso on β₁-ARs. (B) Example of a typical competition binding of Iso and PEG-Iso on β₂-ARs. Kᵢ values were, respectively, 2.0 ± 0.7 µM and 1.4 ± 0.7 mM for Iso and PEG-Iso on β₁-ARs; 2.1 ± 0.4 µM and 0.4 ± 1.0 mM for Iso and PEG-Iso on β₂-ARs (N=5). (C) Time evolution of solvent-accessible surface area (SASA) of Iso (black trace) and PEG-Iso (blue trace) using molecular dynamics simulations. Two representative conformations of PEG-Iso with large or small SASA are shown.
<figure><img src="image_9.png" /><figcaption>**Figure 3.** Effects of Iso and PEG-Iso on cytosolic cAMP in adult rat ventricular cardiomyocytes. Freshly isolated ARVMs were infected with an adenovirus encoding the Epac-SH187 FRET-based cytosolic cAMP sensor for 48 hr at 37°C at a multiplicity of infection of 1000 pfu/cell. (A) Typical experiment showing the time course of CFP/YFP ratio during successive applications of four increasing Iso concentrations: 0.3, 1, 10 and 100 nM. (B) Similar experiment showing the time course of CFP/YFP ratio during successive applications of four increasing PEG-Iso concentrations: 10 and 100 nM, 1 and 10 µM. Pseudo-color images shown above the main graphs were taken at times indicated by the corresponding letters in the graphs. (C, D) Summary data from several similar experiments as in (A) and (B), respectively. The bars show the mean ± S.E.M. of the data shown by symbols. 16 cells from 3 rats were used in (C); 32 cells from 4 rats in (D). One-way ANOVA and Tukey's multiple comparisons post hoc test were used: ** p<0.01; *** p<0.001; ns, non significant. (E) Comparison of the concentration-response curves in C and D. A fit of the data to the Michaelis-Menten equation allowed to estimate Emax and EC50 values for the effects of Iso and PEG-Iso, as well as 95% confidence intervals (CI 95%) for each parameter: Emax=168.9% (CI 95% 141.1, 196.8) and EC50=4.6 nM (CI 95% 1.3, 7.9) for Iso; Emax=107.3% (CI 95% 98.1, 116.5) and EC50=297.5 nM (CI 95% 174.1, 420.9) for PEG-Iso. The two curves are statistically different at p<0.05 (*) since there was no overlap of CI 95% for each parameter.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Figure 4.** Comparison of the effects of PEG-Iso and Iso on *I*<sub>Ca,L</sub>. The whole-cell patch-clamp technique was applied to ARVMs after 24 hr culture. (A) Typical experiment showing the time course of peak *I*<sub>Ca,L</sub> current density upon application of 10 nM Iso and during washout. (B) Similar experiment showing the response of peak *I*<sub>Ca,L</sub> current density to 1 µM PEG-Iso. Individual current traces shown above the graphs were taken at times indicated by the corresponding letters on the graphs. (C) Mean ± S.E.M. increase in peak *I*<sub>Ca,L</sub> in response to 10 nM Iso or 1 µM PEG-Iso stimulation. Student's unpaired t-test: ns, nonsignificant, n=11-12.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Figure 5.** Comparison of the effects of PEG-Iso and Iso on sarcomere shortening.
Representative traces of sarcomere shortening recorded in ARVMs paced at 0.5 Hz and loaded with Fura-2 AM (1 µM) showing the effects of Iso (1 and 3 nM, **A**) and PEG-Iso (100 and 300 nM, **B**). The bars in (**A**) and (**B**) show the mean ± S.E.M. of the data shown by symbols. (**C**, **D**) Average time-to-50% relaxation of sarcomere shortening from experiments shown in (**A**) and (**B**), respectively. 30 cells from 4 rats were used in (**A**) and (**C**); 34 cells from 4 rats in (**B**) and (**D**). One-way ANOVA and Tukey's post hoc test: \* p<0.05; \*\*\* p<0.001; ns, non-significant.</figcaption></figure>
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>**Figure 6.** Comparison of the effects of PEG-Iso and Iso on Ca²⁺ transients. Representative traces of Ca²⁺ transients recorded in ARVMs paced at 0.5 Hz and loaded with Fura-2 AM (1 µM) showing the effect of Iso (1 and 3 nM, A) and PEG-Iso (100 and 300 nM, B). The bars in (A) and (B) show the mean ± S.E.M. with individual data points denoted by symbols. (C, D) Exponential time constant (*Tau*) of relaxation of Ca²⁺ transients from experiments shown in (A) and (B), respectively. 30 cells from 4 rats were used in (A) and (C); 34 cells from 4 rats in (B) and (D). One-way ANOVA and Tukey's post hoc test: * p<0.05; ** p<0.01; *** p<0.001; ns, non-significant.</figcaption></figure>
<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

**Figure 7. Comparison of the effects of PEG-Iso and Iso on cytosolic PKA activity.** Freshly isolated ARVMs were infected with an adenovirus encoding the AKAR3-NES FRET-based PKA sensor for 48 hr at 37°C at a multiplicity of infection of 1000 pfu/cell. (A) Typical experiment showing the time course of YFP/CFP ratio during successive applications of three increasing Iso concentrations: 0.3, 1 and 3 nM. (B) Similar experiment showing the time course of YFP/CFP ratio during successive applications of three increasing PEG-Iso concentrations: 10, 30 and 100 nM. Pseudo-color images shown above the main graphs were taken at times indicated by the corresponding letters in the graphs. (C, D) Summary data from several similar experiments as in (A) and (B), respectively. The bars show the mean ± S.E.M. of the data shown by symbols. 3 rats and 8-10 cells were used in C; 3-5 rats and 13-18 cells in (D). One-way ANOVA and Tukey's multiple comparisons post hoc test: ** p<0.01; *** p<0.001.
<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

**Figure 8. Comparison of the effects of PEG-Iso and Iso on nuclear cAMP activity.** Freshly isolated ARVMs were infected with an adenovirus encoding the nuclear Epac-SH187-3NLS FRET-based nuclear cAMP sensor for 48h at 37°C at a multiplicity of infection of 1000 pfu/cell. (A) Typical experiment showing the time course of CFP/YFP ratio during successive applications of increasing Iso concentrations and (B) a dose-response of increasing PEG-Iso concentrations. Pseudo-color images shown above the main graphs were taken at times indicated by the corresponding letters in the graphs. (C, D) Summary data from several similar experiments as in (A) and (B), respectively. The bars show the mean ± S.E.M. of the data shown by symbols. 2 rats and 6 cells were used in (C) and 6 cells from 3 rats in (D). One-way ANOVA and Tukey's multiple comparisons post hoc test were used: * p<0.05; ** p<0.01.
<figure><img src="image_28.png" /></figure>

**Figure 9. Comparison of the effects of PEG-Iso and Iso on nuclear PKA activity.** Freshly isolated ARVMs were infected with an adenovirus encoding the AKAR3-NLS FRET-based PKA sensor for 48h at 37°C at a multiplicity of infection of 1000 pfu/cell. (A) Typical experiment showing the time course of YFP/CFP ratio during successive applications of three increasing Iso concentrations: 0.3, 1 and 3 nM. (B) Similar experiment showing the time course of YFP/CFP ratio during successive applications of three increasing PEG-Iso concentrations: 10, 30 and 100 nM. Pseudo-color images shown above the main graphs were taken at times indicated by the corresponding letters in the graphs. (C, D) Summary data from several similar experiments as in (A) and (B), respectively. The bars show the mean ± S.E.M of the data shown by symbols. 3 rats and 10-12 cells were used in C; 3-5 rats and 10-17 cells in (D). One-way ANOVA and Tukey's multiple comparisons post hoc test: * p<0.05; ** p<0.01; *** p<0.001. The following figure supplements are available for figure 9:

**Figure supplement 1.** Comparison of the effects of PEG-Iso and Iso on nuclear proteins phosphorylation levels by PKA.

**Figure supplement 2.** Plot of cytosolic and nuclear PKA activity as a function of cytosolic cAMP in response to Iso and PEG-Iso.

**Figure supplement 3.** PEG-Iso cytosolic cAMP and nuclear cAMP/ PKA responses after treatment of cells with neuraminidase.

**Figure supplement 4.** Detubulation normalises the effect of Iso and PEG-Iso.

**Figure supplement 5.** Effect of permeant (propranolol) and impermeant (sotalol) β-blockers on nuclear PKA response to β-AR stimulation by Iso.

**Figure supplement 6.** T-tubule network is still robust up to 48 hours post-isolation.
Supplemental Figures

<figure><img src="image_29.png" /><figcaption>**Figure 1** – figure supplement 1. PEG-Isoprenaline synthesis. **Top**, Concept of chemical grafting between PEG<sub>5000</sub> and Iso; **Bottom**, chemical coupling between PEG and Iso using carbodiimide reaction.</figcaption></figure>
<figure><img src="image_30.png" /><figcaption>**Figure 9** – figure supplement 1. Comparison of the effects of PEG-Iso and Iso on nuclear proteins phosphorylation levels by PKA. Freshly isolated ARVMs were treated with or without Iso 10 nM or PEG-Iso 1 µM during 15 min. Nuclear proteins were extracted, and proteins phosphorylated by PKA were revealed using a phospho-PKA substrate antibody. (A) Total proteins labelling as a loading control; (B) Immunoblot for phosphorylation of PKA substrates using a specific phospho-PKA antibody; (C) Quantification of phospho-PKA levels normalized on total protein, N=3. One-way ANOVA and Tukey's post hoc test: \* p<0.05; \*\* p<0.01. (see also Supplemental Figure 2—source data 1). The bars show the mean ± S.E.M. of the data.</figcaption></figure>
A

<figure><img src="image_31.png" /></figure>

B

<figure><img src="image_32.png" /></figure>

Figure 9 – figure supplement 2. Plot of cytosolic and nuclear PKA activity as a function of cytosolic cAMP in response to Iso and PEG-Iso. For three concentrations of Iso (0.3, 1 and 10 nM) and PEG-Iso (10 nM, 100 nM and 1 µM), the average percent increase (± S.E.M.) in YFP/CFP ratio for cytosolic PKA activity (A, taken from Figure 7) and nuclear PKA activity (B, taken from Figure 8) are plotted as a function of the average percent increase (± S.E.M.) in CFP/YFP ratio for cytosolic cAMP (taken from Figure 3).
<figure><img src="image_33.png" /><figcaption>**Figure 9** – figure supplement 3. PEG-Iso cytosolic cAMP and nuclear cAMP/ PKA responses after treatment of cells with neuraminidase. Freshly isolated ARVMs were infected with an adenovirus encoding the (A, B) the Epac-S<sup>H187</sup> FRET-based cytosolic cAMP sensor, (C, D) Epac-SH187-3NLS FRET-based nuclear cAMP or the (E, F) AKAR-NLS FRET-based nuclear PKA sensor for 48h at 37°C at a multiplicity of infection of 1000 pfu/cell. On the day of experimentation, a subset of cells was pretreated with 0.25 U/mL neuraminidase (+nmd, green) for 1h at 37°C. All cells were washed in physiological solution before use and responses to 10 µM PEG-Iso (blue) with and without neuraminidase were recorded. An equivalent Iso concentration of 100 nM Iso (black) was used as a negative control for the effects of neuraminidase. (F) A representative increase in nuclear PKA response to PEG-Iso ± neuraminidase. Symbols are from individual cells together with the mean ± S.E.M. Unpaired T-test was performed in each condition: \* p<0.05; \*\* p<0.01. n=4-13.</figcaption></figure>
<figure><img src="image_34.png" /><figcaption>Figure 9 – figure supplement 4. Detubulation normalises the effect of Iso and PEG-Iso. T-tubules were chemically removed by incubating the cells with 300 µM imipramine for 15 minutes. Loading the cells with Di4Anepps on a 10x spinning disk confocal showed that control cells had a dense (a) intracellular T-tubule network, whilst this had been completely detubulated (b) in imipramine treated cells. FRET experiments showed that without T-tubules and only OSM β-ARs the response to stimulation with Iso (black) and PEG-Iso (blue) elicited the same cytosolic cAMP (c), cytosolic PKA (d) and nuclear PKA (e) responses. Data points are of the mean ± S.E.M. ns, not significant. Tested by two-way ANOVA. Confocal images were checked for detubulation in N=3 isolations. FRET n = 2-12.</figcaption></figure>
<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

Figure 9 – figure supplement 5. Effect of permeant (propranolol) and impermeant (sotalol) β-blockers on nuclear PKA response to β-AR stimulation by Iso. Freshly isolated ARVMs were infected with an adenovirus encoding the AKAR-NLS FRET-based nuclear PKA sensor for 48h at 37°C at a multiplicity of infection of 1000 pfu/cell. On the day of experimentation, cells were pretreated with either vehicle, propranolol 1 µM (red) or sotalol 25 µM (orange) for 15 mins prior to stimulation with Iso 10 nM (black). (a) Representative traces of typical responses to Iso stimulation alone or with β-blockers being preincubated for 15 mins and added with Iso. (b) and (c) Baseline and percentage of YFP/CFP increase after Iso with or without β-blockers, respectively. The bars present mean ± S.E.M. with N= 3 rats, n=8-13 cells with 15-26 nuclei. One-way ANOVA and Tukey's multiple comparisons post hoc test were performed: *** p<0.001.
<figure><img src="image_38.png" /><figcaption>**Figure 9** – figure supplement 6. T-tubule network is still robust up to 48 hours post-isolation.
The density of T-tubules was quantified in adult rat ventricular myocytes after 0 (a), 24 (b) and 48 hours (c) post-isolation using 2 µM Di4Anepps on a 10x spinning disk confocal. Summary quantification supports a decrease in the T-tubule network over 48 hours but still a robust network at the end of the experimental time frame (d). One-way ANOVA with Tukey's post-hoc test performed; n=13-31. *** p<0.001; **** p<0.0001. The bars show the mean ± S.E.M. of the data.</figcaption></figure>