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

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4 George W. P. Madders¹,*, Marion Barthé¹,*, Flora Lefebvre², Emilie Langlois², Florence

5 Lefebvre¹, Patrick Lechêne¹, Maya Dia¹, Xavier Iturrioz³,⁴, Catherine Llorens-Cortes³,⁴, Tâp Ha-
6 Duong⁵, Laurence Moine², Nicolas Tsapis², Rodolphe Fischmeister¹,#

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⁹Université Paris-Saclay, Inserm, UMR-S 1180, Orsay, France.

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²Université Paris-Saclay, CNRS, Institut Galien Paris-Saclay, Orsay, France.

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³Collège de France, CIRB, INSERM U1050/CNRS UMR 7241, Paris, France.

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⁴Université Paris Saclay, CEA, Département Médicaments et Technologies pour la Santé, SIMoS, Gif-sur-Yvette, France.

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⁵Université Paris-Saclay, CNRS, BioCIS, Orsay, France.

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*Authors with equal contribution

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#Corresponding author: Rodolphe Fischmeister, PhD

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**E-mail:** [rodolphe.fischmeister@inserm.fr](mailto:rodolphe.fischmeister@inserm.fr)

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**Competing interests:** The authors declare no competing interests.

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Classification: Biological Sciences/Physiology

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**Keywords:** T-tubules • membrane compartmentation • size exclusion • β-adrenergic

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receptors • PEGylation • excitation-contraction coupling • calcium channels • cyclic AMP •

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cAMP-dependent protein kinase • physiology

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31 **Abstract**

32 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 33 how their location impacts their function is unknown. Here, we developed a technology 34 based on size exclusion to explore the function of β-ARs located in the OSM. We synthetized 35 a PEG-Iso molecule by covalently linking isoprenaline (Iso) to a 5000 Da PolyEthylene-Glycol 36 (PEG) chain to increase the size of the β-AR agonist and prevent it from accessing the T-37 tubule network. The affinity of PEG-Iso and Iso on β₁- and β₂-ARs was measured using 38 radioligand binding. Molecular dynamics simulation was used to assess PEG-Iso 39 conformation and visualise the accessibility of the Iso moiety to water. Using confocal 40 microscopy, we show that PEGylation constrains molecules outside the T-tubule network of 41 adult rat ventricular myocytes (ARVMs) due to the presence of the extracellular glycocalyx. 42

43 β-AR activation in OSM with PEG-Iso produced a lower stimulation of [cAMP]ᵢ than Iso but a 44 larger stimulation of cytosolic PKA at equivalent levels of [cAMP]ᵢ and similar effects on 45 excitation-contraction coupling parameters. However, PEG-Iso produced a much lower 46 stimulation of nuclear cAMP and PKA than Iso. Thus, OSM β-ARs in ARVMs control mainly 47 cytosolic cAMP/PKA pathway and contractility, while TTM β-ARs control mainly nuclear 48 cAMP, PKA and consequent nuclear protein phosphorylation. Size exclusion strategy using 49 ligand PEGylation provides a unique approach to evaluate the respective contribution of T-50 tubule vs. outer surface membrane proteins in cardiac cells. 51

52 **Significance Statement**

53 β-adrenoceptors (β-ARs) regulate cardiac function during sympathetic nerve stimulation.
54 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.

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64 **Introduction**

65 The β-adrenergic receptor (β-AR) is a key player in the regulation of cardiac function during
66 sympathetic nerve stimulation. The classical pathway for β-AR receptor signalling is
67 activation of adenylyl cyclases (AC) via Gαs, resulting in increased intracellular cAMP levels
68 ([cAMP]ᵢ) [1]. Three types of β-ARs are expressed in the heart, respectively β₁-, β₂- and β₃-
69 ARs. β₁- and β₂-ARs are both positively coupled to the AC/cAMP cascade and involved in
70 cardiac performance [2] while β₃-ARs may act via either cAMP or cGMP depending on the
71 cell type (atrial vs. ventricular myocytes) [3, 4]. The primary target of cAMP is the cAMP-
72 dependent protein kinase (PKA) that in turn phosphorylates several key proteins involved in
73 the excitation-contraction (EC) coupling, such as the L-type Ca²⁺ channel (LTCC or Ca_v1.2),
74 phospholamban, troponin I, etc. [5]. The phosphorylation of Ca_v1.2 (or its regulatory protein
75 Rad [6, 7]) leads to enhanced LTCC current (I<sub>Ca,L</sub>) and sarcoplasmic reticulum (SR) Ca²⁺ release
76 via the ryanodine type 2 receptor (RyR2), contributing to enhanced Ca²⁺ transients and
77 contraction [8, 9]. Phosphorylation of phospholamban increases Ca²⁺ uptake into the SR
78 which accelerates Ca²⁺ transient decay and, together with troponin I phosphorylation,
79 speeds up relaxation. Whereas short term stimulation of β-AR/cAMP is beneficial for the
80 heart, chronic activation of this pathway results in altered Ca²⁺ signalling, cardiac
81 hypertrophy and fibrosis, leading to ventricular dysfunction [10] and cardiac arrhythmias
82 [11-15].

83 The cell membrane of cardiomyocytes is characterized by invaginations of the surface
84 membrane, occurring primarily perpendicular to myocyte longitudinal edges, at intervals of
85 ~1.8–2 µm, that form a complex interconnected tubular network penetrating deep into the
86 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
111 pharmacological properties of PEG-Iso and compared its functional effects in adult rat
112 ventricular myocytes (ARVMs) with those of Iso.

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114 **Results**

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116 **PEGylation constrains molecules outside the TT network**

117 The rationale for using ligand PEGylation as a tool to impede ligand diffusion into T-tubules
118 came from the observation that fluorescent PEG<sub>5000</sub> molecules were unable to access to TTs.

119 A typical representative experiment (out of 15) using confocal microscopy is shown in Figure
120 1. When ARVMs were exposed during 15 min to 100 µM of PEG<sub>5000</sub> molecules functionalized
121 with Fluorescein Isothiocyanate (FITC), no fluorescence was seen throughout the cell (Figure
122 1A, B). On the contrary, when the cells were exposed during 15 min to 100 µM fluorescein,
123 fluorescence was seen with clear staining of the TTs (Figure 1C, D). We suspected that the
124 lack of access of PEG-FITC in the TTs was due to steric hindrance due to the presence of
125 glycocalyx matrix. To test this hypothesis, ARVMs were first exposed during 1 hr to a solution
126 containing 0.25 U/mL neuraminidase, an enzyme that degrades sialic acid in the glycocalyx
127 matrix [34]. As shown in Figure 1E, F, this allowed PEG-FITC to enter the TTs as evidenced by
128 the striated profile now observed. Summary data (Figure 1G) reflects these observations
129 where fluorescence intensity is reduced from clearly stained TTs in FITC to almost 0
130 fluorescence in PEG-FITC. Degradation of the glycocalyx by neuraminidase restores
131 fluorescence intensity to the same level as that seen in free FITC.

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134 PEG-Isoprenaline binds to β₁- and β₂-adrenergic receptors

135 The above experiments demonstrate that PEG₅₀₀₀ molecules are unable to access the TTs in
136 our experimental conditions. We thus synthetized PEGylated isoprenaline (PEG-Iso) by
137 covalently linking isoprenaline to PEG₅₀₀₀ molecules (Figure 1 – supplement 1). Our rationale
138 was that since PEG-Iso does not access TTs, it will not reach β-ARs located in the TTM. But
139 the question remained whether PEG-Iso would be able to bind to β-ARs located in the OSM.
140 To address this question, we performed radioligand binding studies in purified membranes
141 from Chinese Hamster Ovary (CHO) cells overexpressing either human β₁- or β₂-ARs.
142 Competition curves between [¹²⁵Iodo]cyanopindolol and Iso or PEG-Iso were used to
143 measure Kᵢ values of both ligands for the β₁- and β₂-ARs. As shown in Figure 2A and B, PEG-
144 Iso binds to both β₁- and β₂-ARs, but with an affinity which is ~2-orders of magnitude lower
145 than Iso. Functional experiments using homogeneous time-resolved fluorescence (HTRF)
146 technology on HEK 293 cells, that stably expressed either the β₁- or β₂-ARs showed that the
147 EC₅₀ for cAMP stimulation induced by Iso was 7.4 ± 3.6 pM vs. 39.4 ± 24.2 nM (n=4) for PEG-
148 Iso in β₁-AR expressing cells and 3.4 ± 7.7 pM vs. 20.2 ± 69.8 nM (n=4) for PEG-Iso in β₂-AR
149 expressing cells. Thus, the ability of PEG-Iso to activate β₁- and β₂-ARs was reduced by the
150 same order of magnitude for both receptors which should not bias the following cellular
151 experiments.

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

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

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

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

177 Next, the ability of Iso and PEG-Iso to stimulate the L-type Ca²⁺ current, I<sub>Ca,L</sub> was compared at
178 a single concentration of each, 10 nM Iso and 1 µM PEG-Iso, shown above to produce an
179 equivalent response on [cAMP]<sub>i</sub> (Figure 3). As shown in the individual experiments in Figure
180 4A and B, both Iso and PEG-Iso produced a similar increase in the amplitude of I<sub>Ca,L</sub> (Figure
181 4C).
182

183 **Comparison of the effects of PEG-Iso and Iso on excitation-contraction coupling**

184 To investigate the impact of stimulating TTM and OSM β-ARs on EC coupling, Ca²⁺ transients
185 and sarcomere shortening measurements were simultaneously recorded in Fura-2-loaded
186 ARVMs and their response to PEG-Iso and Iso evaluated. Here, two concentrations of PEG-
187 Iso were used, 100 and 300 nM, and compared to two affinity-adjusted concentrations of Iso
188 that produce similar elevations of [cAMP]ᵢ, 1 and 3 nM. As shown in Figure 5 and 6, 100 and
189 300 nM PEG-Iso increased contractility (Figure 5B), Ca²⁺ transients (Figure 6B) and
190 accelerated their relaxation kinetics (Figure 5D and 6D). These effects were not different
191 from those produced by 1 and 3 nM Iso, respectively, for either sarcomere shortening
192 (Figure 5A, C), Ca²⁺ transient amplitude (Figure 6A, C) or relaxation kinetics (Figure 5C, D;
193 Figure 6C, D).

194

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

196 The observation that equipotent concentrations of PEG-Iso and Iso on [cAMP]ᵢ produced
197 equivalent effects on I<sub>Ca,L</sub> and EC coupling, may suggest that OSM β-ARs are not functionally
198 different from their TTM homologs as long as their degree of activation leads to similar
199 [cAMP]ᵢ responses. However, the above experiments do not exclude possible differences in
200 the compartmentalization of intracellular cAMP cascade when cAMP is synthesized upon
201 OSM or TTM β-AR stimulation. To obtain further insight, we compared the effect of PEG-Iso
202 and Iso on PKA activity in the bulk cytoplasm and in the nucleus. For that, we used
203 genetically encoded A-kinase activity FRET-based reporters (AKAR3) targeted to these
204 compartments by the addition of a nuclear export sequence (NES), and a nuclear localizing
205 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).

228

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

315 We thus explored a possible involvement of the PEG conformation in this
316 phenomenon using molecular dynamics simulations. Because of its hydrophilicity and
317 flexibility, PEG is very dynamic in solution and constantly changes conformation.
318 Consequently, the Iso moiety in PEG-Iso is rarely fully accessible to solvent and spends most
319 of the time with the PEG chain wrapped around it (Figure 2). Based on the calculations, only
320 ~1% of PEG-Iso conformations are able to bind β-ARs as compared to 100% for free Iso,
321 which is in the order of magnitude of the measured difference in their respective binding
322 affinity on β-ARs.

323 When applied on ARVMs, PEG-Iso produced stimulatory effects on [cAMP]ᵢ, PKA
324 activity, I<sub>Ca,L</sub>, sarcomere shortening and Ca<sup>2+</sup> transients which all had the hallmarks of a β-AR
325 stimulation (Figure 3 to 9). Because of the reduced binding affinity towards β-ARs, PEG-Iso
326 produced its effect at ~2-orders of magnitude larger concentrations than Iso. But there were
327 two other striking differences between the effects of PEG-Iso and Iso which strongly suggest
328 that the two ligands did not act on the exact same populations of receptors.

329 First, PEG-Iso produced a stimulation of [cAMP]ᵢ with a much lower efficacy than Iso
330 (Figure 3). The simplest explanation for this result is that PEG-Iso activates only β-ARs
331 located in the OSM while Iso activates β-ARs located in both OSM and TTM. Since both
332 populations of β-ARs are likely coupled to ACs [24, 44], one would expect an activation of
333 OSM β-ARs with PEG-Iso to lead to a smaller maximal increase in [cAMP]ᵢ than an activation
334 of all β-ARs with Iso. This diminution is in line with decreased cAMP production sites by
335 detubulation [18]. Cyclic AMP must emanate mainly from sarcolemmal β₁-ARs, which
336 produce highly diffusible signals throughout the cell in contrast to β₂-ARs [68]. However,
337 there are conflicting results regarding the distribution of β-ARs on the cardiac cell
338 membrane. Immunohistochemical data have suggested that β₁- and β₂-ARs are present in
339 OSM and TTM in mouse heart [69]. Using radioligand binding, β₁-AR density was found to be
340 almost 2-fold more concentrated in OSM that TTM, whereas β₂-AR density was evenly
341 distributed across the entire cell surface in dog heart [70]. Using nanoscale live-cell scanning
342 ion conductance and fluorescence resonance energy transfer microscopy techniques in
343 healthy ARVMs, Nikolaev *et al.* found that β₁-ARs are distributed across the entire cell
344 surface while β₂-ARs are localized exclusively in TTM [23]. While the distribution of β₁-ARs
345 across the entire cell surface was confirmed in ARVMs in another study using formamide
346 detubulation, β₂-ARs were found to be only present in OSM [26]. Additional experiments
347 using PEGylated selective β₁- and β₂-AR agonists and antagonists are needed to solve this
348 issue and to precisely characterize the role of each β-AR subtype in the different membrane
349 compartments.

350 Second, PEG-Iso produced a much lower stimulation of nuclear PKA activity than Iso
351 (Figure 9) even though both ligands were equally efficient in stimulating cytosolic PKA
352 activity (Figure 7). Also, when cytosolic and nuclear PKA activity measured during PEG-Iso or
353 Iso stimulation were plotted as a function of [cAMP]ᵢ measured under the same conditions,
354 PEG-Iso was found to be more efficient than Iso to increase PKA activity in the cytosol, while
355 on the contrary Iso is more efficient than PEG-Iso to increase PKA activity in the nucleus
356 (Figure 9 – figure supplement 2). A likely interpretation of these results is that β-ARs located
357 in TTM (or a β-AR subtype more concentrated in TTM) are more efficiently coupled to
358 nuclear PKA than those located in OSM, while β-ARs located in OSM (or a β-AR subtype more
359 concentrated in OSM) are more efficiently coupled to cytosolic PKA. In a recent study, we
360 showed that β₂-ARs, unlike β₁-ARs, are inefficient in activating nuclear PKA activity in ARVMs
361 [37]. Thus, if the ratio of β₂/β₁ receptors is larger in OSM than in TTM, as discussed above
362 [26], then this would explain the lower efficacy of PEG-Iso to activate nuclear PKA activity as
363 compared to Iso. Recent studies have shown that perinuclear PKA has the ability to activate
364 without detachment of the catalytic subunits, and thus to phosphorylate its targets in close
365 proximity [71, 72]. According to these studies, only supra-physiological concentrations of
366 cAMP could lead to dissociation of the catalytic subunits and their translocation into the
367 nucleus. Perinuclear PKA is anchored to the nuclear membrane via its interaction with
368 mAKAP and its dynamics are finely regulated by AC5 and PDE4D3 [73], AC5 being
369 concentrated in TTM [24] and PDE4D3 being linked to mAKAP [74]. The location of AC5 may
370 therefore be crucial for nuclear signalling and explain why β-ARs present in TTM are
371 necessary for activation of nuclear PKA. One major limitation of our data is that there is a
372 reduction in TTs over the ≈36-hour infection period needed for robust FRET sensor
373 expression (Figure 9 – figure supplement 6). Therefore, the differences between OSM and
374 TTM may be even greater than we have observed with respect to nuclear PKA activation.

375 Another important finding from this study concerns the contribution of OSM β-ARs to
376 the regulation of EC coupling. Activation of β-ARs only in OSM with PEG-Iso produced a
377 similar increase in I<sub>Ca,L</sub> amplitude, sarcomere shortening and Ca<sup>2+</sup> transients as Iso, when
378 both ligands were used at concentrations producing an equivalent elevation of [cAMP]<sub>i</sub>.
379 Since OSM contributes to ~60% of total cell membrane in ARVMs [18], either β-ARs and ACs
380 are more concentrated in OSM than TTM, or they are in large excess over what is needed to
381 activate PKA phosphorylation of proteins involved in EC coupling. It would also suggest that
382 small GTPase Rab proteins mediated increases in Ca<sub>v</sub>1.2 trafficking to the TTM that occur in
383 response to β-AR activation [75] are sufficiently activated by OSM cAMP alone for a similar
384 increase in I<sub>Ca,L</sub> amplitude to have been observed. Also, cAMP produced at OSM must diffuse
385 rapidly in the cytosol in order to activate PKA phosphorylation of substrates located deep
386 inside the cell, such as LTCCs in TTM. A recent study showed that under basal conditions,
387 cAMP is bound to proteins and its diffusion in the cytosol is slow; but when cAMP increases
388 in the cytosol, e.g. as a result of β-AR stimulation, the cAMP binding sites become saturated
389 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.

408

## 409 **Materials and Methods**

410

**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
414 Committee (CREEA Ile-de France Sud) guidelines, and the French decree no. 2013-118 of
415 February 1st, 2013 on the protection of animals used for scientific purposes (JORF no. 0032,
416 February 7th, 2013, p2199, text no. 24). Animal experiments according were carried out
417 according to the European Community guiding principles in the care and use of animals
418 (2010/63/UE), the local Ethics Committee (CREEA Ile-de-France Sud) guidelines and the
419 French decree n° 2013-118 on the protection of animals used for scientific purposes. Adult
420 male rats (Rattus norvegicus) belonging to the Wistar strain (Janvier, Le Genest St Isle,
421 France) have been used. This is a versatile strain of albino rats, selected by Donaldson in
422 1906 at the Wistar Institute (USA).

423

424 **Synthesis of PEG-Iso.** Isoprenaline was grafted into the reactive end of 5000 Da PEG
425 (PEG<sub>5000</sub>). A carbodiimide reaction was carried out between the carboxylic acid of PEG and
426 the amine function of Iso (Figure 1 – figure supplement 1). In a 25 mL amber vial, 41.3 mg of
427 DCC (0.2 mmol; 2eq), 23 mg of NHS (0.2 mmol; 2eq) and 500 mg of OH-PEG<sub>5000</sub>-COOH (0.1
428 mmol; 1eq) were solubilized in 10 mL of dichloromethane. 1 mL of triethylamine (TEA) was
429 added to the syringe. The solution was stirred for 5 hr at RT. 21.1 mg isoprenaline (0.1 mmol;
430 1 eq) were added to the reaction mixture. The mixture was left to stir overnight. The
431 solution was then filtered through silica gel and precipitated twice in cold diethyl ether. After
432 evaporation of the ether, the final powder was solubilised in H<sub>2</sub>O.

433

434 **Purification of PEG-Iso.** A purification step was introduced to obtain a pure PEG-Iso product
435 and to eliminate the various by-products obtained, potential impurities and especially traces
436 of ungrafted Iso which could interfere in the biological experiments. A HPLC 1290 Infinity II -
437 (Agilent Technologies) consisting of a 4-channel binary pump, UV/visible detector (PDA
438 diode array), 1260 Infinity DEDL light scattering detector and C18 XBridge column, 4.6 x 150
439 mm, 5 µm (17 mL/min flow rate) was used. The mobile phase consisted of a mixture of
440 water + 0.1% formic acid and acetonitrile. This phase was pushed according to a gradient
441 from 1 to 100% in 15 min into the stationary phase for the purification of PEG-Iso. Detection
442 was carried out in the UV at 254 and 280 nm. PEG-Iso was solubilized in water. Several
443 successive injections of 300 µL each were performed. The retention time of PEG-Iso was 11
444 min. The fractions containing PEG-Iso were recovered, evaporated and then frozen and
445 freeze-dried. Finally, the powder obtained was stored at -20°C and protected from light.
446

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

471
472 **Confocal imaging.** Ventricular cardiomyocytes from adult rats were plated on 35 mm glass
473 bottom Petri dishes. Two hours after plating, the control cells were stained with commercial
474 fluorescent PEG₅₀₀₀ functionalized with FITC (Fluorescein Isothiocyanate) (Nanocs) or free
475 Fluorescein (Sigma-Aldrich). Other cells were treated with neuraminidase (Sigma-Aldrich) for
476 1 hr at 37°C at a concentration of 0.25 U/mL. After which they were washed and re-
477 incubated in physiological buffer solution, cells were then stained with PEG-FITC. All
478 molecules were dissolved in Ringer containing (in mM): NaCl 121.6, KCl 5.4, MgCl₂ 1.8, CaCl₂
479 1.8, NaHCO₃ 4, NaH₂PO₄ 0.8, D-glucose 5, sodium pyruvate 5, HEPES 10, adjusted to pH 7.4.
480 The acquisitions were made with a Leica TCS SP5 confocal microscope using a white light
481 laser and an X40 oil immersion objective. The cells were excited at 495 nm and fluorescence
482 was recovered at wavelengths >510 nm.

483
484 **Fluorescence intensity quantification.** FITC or PEG-FITC that enters the T-tubules (TT) should
485 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.

497

**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.

505

**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 $SASA_{PEG-Iso} \ge 0.9 SASA_{free-Iso}$ 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 $\beta_1$- or $\beta_2$-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
534 Manifold). Binding reaction was transferred to the filters and washed 3 times with Wash
535 Buffer. Filter-bound radioactivity was measured in a gamma counter. All determinations
536 were performed in triplicate at least.

537
538 HTRF quantification of cAMP production. cAMP was quantified using the HTRF cAMP Gs
539 dynamic detection kit (Revvity, Codolet, France) based on homogeneous time-resolved
540 fluorescence (HTRF) technology. The stimulation was done in the stimulation buffer
541 (stimulation buffer 1X (Revvity) containing 1 mM 3-isobutyl-methylxanthine, IBMX). Briefly,
542 HEK 293 cells stably expressing the human β₁- or β₂-ARs were resuspended in stimulation
543 buffer and dispensed into 384-well plate (1,000 cells per well (5 µl)) and stimulated with
544 increasing concentrations (0.1 pM to 100 µM) of Iso diluted in stimulation buffer (5 µl/well)
545 for 30 min at 20°C. Cells were then lysed, and cAMP levels were determined following
546 manufacturer instructions. HTRF signal was read with the microplate reader Envision Excite
547 (Perkin Elmer, Turku, Finland).

548
549 FRET imaging. FRET experiments were performed at room temperature 36 hr after cell
550 plating. Cells were maintained in a Ringer solution containing (in mM): NaCl 121.6, KCl 5.4,
551 MgCl2 1.8; CaCl2 1.8; NaHCO3 4, NaH2PO4 0.8, D-glucose 5, sodium pyruvate 5, HEPES 10,
552 adjusted to pH 7.4. Images were captured every 5 s using the 40x oil immersion objective of
553 a Nikon TE 300 inverted microscope connected to a software-controlled (Metafluor,
554 Molecular Devices, Sunnyvale, CA, USA) cooled charge coupled (CCD) camera (Sensicam PE,
555 PCO, Kelheim, Germany). Cells were excited for 150-300 ms by a Xenon lamp (100 W, Nikon,
556 Champigny-sur-Marne, France) using a 440/20BP filter and a 455LP dichroic mirror. Dual
557 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<sub>corr</sub>=YFP-0.6xCFP.

**Electrophysiological experiments.** The whole cell configuration of the patch-clamp technique was used to record *I*<sub>Ca,L</sub>. Patch electrodes had resistance of 0.5–1.5 MΩ when filled with internal solution containing (in mM): CsCl 118, EGTA 5, MgCl<sub>2</sub> 4, Na<sub>2</sub>-phosphocreatine 5, Na<sub>2</sub>ATP 3.1, Na<sub>2</sub>GTP 0.42, CaCl<sub>2</sub> 0.062, Hepes 10, adjusted to pH 7.3 with CsOH. External Cs<sup>+</sup>-Ringer solution contained (in mM): NaCl 107.1, CsCl 20, NaHCO<sub>3</sub> 4, NaH<sub>2</sub>PO<sub>4</sub> 0.8, D-glucose 5, sodium pyruvate 5, Hepes 10, MgCl<sub>2</sub> 1.8, CaCl<sub>2</sub> 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 Ca<sup>2+</sup> 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.exp(-(t-t₀)/τ)+A₀, where t₀ is zero time, A₀ the asymptote of the exponential, A the relative amplitude of the exponential and τ 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
606 antibody (Cell Signaling, Cat# 9621, RRID: AB_330304) and total protein per lane. The ratio of
607 phospho-PKA expression was assessed by densiometric scanning of immunoblots.

608
609 **Statistical analysis.** All results are expressed as mean ± SEM. Statistical analysis was
610 performed using GraphPad software (GraphPad Software V.10.4.1, Inc., La Jolla, CA, USA).

611 Normal distribution was tested by the Shapiro-Wilk normality test. For simple two-group
612 comparison that satisfied parametric assumptions, we used an unpaired Student's t test.

613 Differences between multiple concentrations of the same drug were analysed using an
614 ordinary 1-way ANOVA with Tukey post hoc test. A P value<0.05 was considered statistically
615 significant.

616
617 **Funding and Acknowledgements**

618 MB was supported by doctoral grant from the Laboratory of Excellence LERMIT supported by
619 the French National Research Agency (ANR-10-LABX-33) under the program
620 "Investissements d'Avenir" ANR-11-IDEX-0003-01. She also received a doctoral grant from
621 the Fondation pour la Recherche Médicale. This work was also funded by grants ANR-15-
622 CE14-0014-01 and ANR-23-CE14-0027-01 'CARDIOPEG' to RF which was used to support GM,
623 alongside a postdoctoral research award grant from the Fondation Lefoulon-Delalande.

624 Authors would like to thank K. Leblanc (BioCIS) for help with preparative HPLC and Dr R.
625 Jockers (Institut Cochin, France), who kindly provided the HEK 293 cells expressing β-
626 adrenergic receptors.

627
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833
834 **Figure Legends**

835

836 **Figure 1. Localization of PEG-FITC in freshly isolated adult rat ventricular cardiomyocytes.**

837 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.

844

845 The following figure supplements are available for figure 1:

846 **Figure supplement 1. PEG-Isoprenaline synthesis.**

847

848 **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. *K*ᵢ 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.

857

858 **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-S<sup>H187</sup> FRET-based cytosolic cAMP sensor for 48 hr at 37°C at a multiplicity of infection of 860 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)

859

861

862
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.

876

**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.

885
886 **Figure 5.** Comparison of the effects of PEG-Iso and Iso on sarcomere shortening.
887 Representative traces of sarcomere shortening recorded in ARVMs paced at 0.5 Hz and
888 loaded with Fura-2 AM (1 µM) showing the effects of Iso (1 and 3 nM, **A**) and PEG-Iso (100
889 and 300 nM, **B**). The bars in (**A**) and (**B**) show the mean ± S.E.M. of the data shown by
890 symbols. (C, D) Average time-to-50% relaxation of sarcomere shortening from experiments
891 shown in (A) and (B), respectively. 30 cells from 4 rats were used in (A) and (C); 34 cells from
892 4 rats in (B) and (D). One-way ANOVA and Tukey's post hoc test: * p<0.05; *** p<0.001; ns,
893 non-significant.

894
895 **Figure 6.** Comparison of the effects of PEG-Iso and Iso on Ca²⁺ transients. Representative
896 traces of Ca²⁺ transients recorded in ARVMs paced at 0.5 Hz and loaded with Fura-2 AM (1
897 µM) showing the effect of Iso (1 and 3 nM, **A**) and PEG-Iso (100 and 300 nM, **B**). The bars in
898 (A) and (B) show the mean ± S.E.M. with individual data points denoted by symbols. (C, D)
899 Exponential time constant (Tau) of relaxation of Ca²⁺ transients from experiments shown in
900 (A) and (B), respectively. 30 cells from 4 rats were used in (A) and (C); 34 cells from 4 rats in
901 (B) and (D). One-way ANOVA and Tukey's post hoc test: * p<0.05; ** p<0.01; *** p<0.001;
902 ns, non-significant.

903
904 **Figure 7.** Comparison of the effects of PEG-Iso and Iso on cytosolic PKA activity. Freshly
905 isolated ARVMs were infected with an adenovirus encoding the AKAR3-NES FRET-based PKA
906 sensor for 48 hr at 37°C at a multiplicity of infection of 1000 pfu/cell. (A) Typical experiment
907 showing the time course of YFP/CFP ratio during successive applications of three increasing
908 Iso concentrations: 0.3, 1 and 3 nM. (B) Similar experiment showing the time course of
909 YFP/CFP ratio during successive applications of three increasing PEG-Iso concentrations: 10,
910 30 and 100 nM. Pseudo-color images shown above the main graphs were taken at times
911 indicated by the corresponding letters in the graphs. (C, D) Summary data from several
912 similar experiments as in (A) and (B), respectively. The bars show the mean ± S.E.M. of the
913 data shown by symbols. 3 rats and 8-10 cells were used in C; 3-5 rats and 13-18 cells in (D).
914 One-way ANOVA and Tukey's multiple comparisons post hoc test: ** p<0.01; *** p<0.001.

915
916 **Figure 8. Comparison of the effects of PEG-Iso and Iso on nuclear cAMP activity.** Freshly
917 isolated ARVMs were infected with an adenovirus encoding the nuclear Epac-SH187-3NLS
918 FRET-based nuclear cAMP sensor for 48h at 37°C at a multiplicity of infection of 1000
919 pfu/cell. (A) Typical experiment showing the time course of CFP/YFP ratio during successive
920 applications of increasing Iso concentrations and (B) a dose-response of increasing PEG-Iso
921 concentrations. Pseudo-color images shown above the main graphs were taken at times
922 indicated by the corresponding letters in the graphs. (C, D) Summary data from several
923 similar experiments as in (A) and (B), respectively. The bars show the mean ± S.E.M. of the
924 data shown by symbols. 2 rats and 6 cells were used in (C) and 6 cells from 3 rats in (D). One-
925 way ANOVA and Tukey's multiple comparisons post hoc test were used: * p<0.05; ** p<0.01.

926
927 **Figure 9. Comparison of the effects of PEG-Iso and Iso on nuclear PKA activity.** Freshly
928 isolated ARVMs were infected with an adenovirus encoding the AKAR3-NLS FRET-based PKA
929 sensor for 48h at 37°C at a multiplicity of infection of 1000 pfu/cell. (A) Typical experiment
930 showing the time course of YFP/CFP ratio during successive applications of three increasing
931 Iso concentrations: 0.3, 1 and 3 nM. (B) Similar experiment showing the time course of
932 YFP/CFP ratio during successive applications of three increasing PEG-Iso concentrations: 10,
933 30 and 100 nM. Pseudo-color images shown above the main graphs were taken at times
934 indicated by the corresponding letters in the graphs. (C, D) Summary data from several
935 similar experiments as in (A) and (B), respectively. The bars show the mean ± S.E.M of the
936 data shown by symbols. 3 rats and 10-12 cells were used in C; 3-5 rats and 10-17 cells in (D).
937 One-way ANOVA and Tukey's multiple comparisons post hoc test: * p<0.05; ** p<0.01; ***
938 p<0.001.

939 The following figure supplements are available for figure 9:

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

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

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

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

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

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

950

951 **Supplemental Figures**

952

953 **Figure 1 – figure supplement 1.** PEG-Isoprenaline synthesis. Top, Concept of chemical
954 grafting between PEG<sub>5000</sub> and Iso; Bottom, chemical coupling between PEG and Iso using
955 carbodiimide reaction.

956

957 **Figure 9 – figure supplement 1.** Comparison of the effects of PEG-Iso and Iso on nuclear
958 proteins phosphorylation levels by PKA. Freshly isolated ARVMs were treated with or
959 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.

966

967 **Figure 9 - figure supplement 1 - source data 1**

TIF files containing original western blots for Figure 9 - figure supplement 1A and B.

969

970 **Figure 9 - figure supplement 1 - source data 2**

PRISM, PDF and Excel files for the western blot analysis in Figure 9 - figure supplement 1A-C.

972

973 **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).

979

980 **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.

980

981

982

983

984

985

986

987

988

989

990

991
992
993 **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.

1002
1003 **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.

1014
1015 **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.
<figure><img src="image_1.png" /></figure>

B

Fluorescence intensity (A.U)

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

<figure><img src="image_3.png" /></figure>

D

Fluorescence intensity (A.U)

<figure><img src="image_4.png" /></figure>

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

F

Fluorescence intensity (A.U)

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

G

Visible T-tubule density (A.U.)

<figure><img src="image_7.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure 4</figcaption></figure>
A

<figure><img src="image_11.png" /></figure>

B

<figure><img src="image_12.png" /></figure>

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

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

C

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

D

<figure><img src="image_16.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>Figure 6</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>Figure 7</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>Figure 8</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>Figure 9</figcaption></figure>
<figure><img src="image_21.png" /><figcaption>PEG$_{5000}$ with reactive subunit in
blue</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>Iso</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>PEG$_{5000}$-Iso</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>HO-PEG$_{5000}$-COOH</figcaption></figure>

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

<figure><img src="image_26.png" /><figcaption>Isoprenaline, R= 2(-CH$_{3}$)</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>HO-PEG$_{5000}$-Isoprenaline, R= 2(-CH$_{3}$)</figcaption></figure>

Figure 1 – figure supplement 1
A
Loading control (total proteins)

<figure><img src="image_28.png" /></figure>

B
pPKA substrate

<figure><img src="image_29.png" /></figure>

C

<figure><img src="image_30.png" /></figure>

Figure 9 - figure supplement 1
A

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

B

<figure><img src="image_32.png" /><figcaption>Figure 9 – figure supplement 2</figcaption></figure>
<figure><img src="image_33.png" /><figcaption>Figure 9 – figure supplement 3</figcaption></figure>
<figure><img src="image_34.png" /><figcaption>Figure 9 – figure supplement 4</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
<figure><img src="image_38.png" /></figure>

Figure 9 – figure supplement 6