Nicolas Deguines

Karine Princé

Anne-Caroline Prévot

Benoît Fontaine

Assessing the emergence of pro-biodiversity practices in citizen scientists of a backyard butterfly survey

Keywords: Citizen scientists, Lepidoptera, Pro-environmental behaviors, Nature-based monitoring program, Urban green spaces

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Evil Quartet causing extinctions [START_REF] Diamond | Overview of recent extinctions[END_REF], habitat change remains among the most important threats to biodiversity worldwide [START_REF] Godet | What conservation does[END_REF]IPBES, 2019). In Europe, urbanization is the prime driver of land use change [START_REF] Eea | The European environment state and outlook 2010: Synthesis[END_REF]. The suite of environmental degradation associated with urbanization (e.g., increase in impervious surfaces, air and soil pollution) generally leads to a reduction in the diversity of a wide range of taxa [START_REF] Marzluff | Worldwide urbanization and its effects on birds[END_REF][START_REF] Mckinney | Urbanization as a major cause of biotic homogenization[END_REF] and a biotic homogenization at large geographical scales [START_REF] Deguines | Functional homogenization of flower visitor communities with urbanization[END_REF]La Sorte et al., 2007;[START_REF] Mckinney | Urbanization as a major cause of biotic homogenization[END_REF]. Improving the suitability of urban environments for wild species thus is a conservation issue [START_REF] Hall | The city as a refuge for insect pollinators[END_REF].

Within cities, backyards may constitute 16-47% of urban green space in Europe (based on estimates from the UK and France), and as much as 86% as found in León, Nicaragua [START_REF] Baldock | A systems approach reveals urban pollinator hotspots and conservation opportunities[END_REF][START_REF] Goddard | Scaling up from gardens: biodiversity conservation in urban environments[END_REF][START_REF] Mimet | Contribution of private gardens to habitat availability, connectivity and conservation of the common pipistrelle in Paris[END_REF]. Urban backyards can act as refuges for biodiversity [START_REF] Goddard | Scaling up from gardens: biodiversity conservation in urban environments[END_REF][START_REF] Levé | Domestic gardens as favorable pollinator habitats in impervious landscapes[END_REF][START_REF] Sperling | The importance of urban backgardens on plant and invertebrate recruitment: a field microcosm experiment[END_REF], and as corridors connecting green spaces [START_REF] Mimet | Contribution of private gardens to habitat availability, connectivity and conservation of the common pipistrelle in Paris[END_REF][START_REF] Rudd | Importance of backyard habitat in a comprehensive biodiversity conservation strategy: a connectivity analysis of urban green spaces[END_REF]. Yet, to fulfil this potential, there is a need to improve backyard suitability for biodiversity [START_REF] Daniels | Does variation in garden characteristics influence the conservation of birds in suburbia?[END_REF][START_REF] Fontaine | Impact of urbanization and gardening practices on common butterfly communities in France[END_REF][START_REF] Pardee | Native plants are the be predictors of bee richness and abundance in backyard gardens[END_REF].

Citizen science (CS), defined as a method of integrating public outreach and scientific data collection [START_REF] Cooper | Citizen science as a tool for conservation in residential ecosystems[END_REF] through the involvement of volunteers in research [START_REF] Dickinson | Citizen science as an ecological research tool: challenges and benefits[END_REF], could help change the management of these private spaces for greater biodiversity benefits. The success of nature-based CS programs in advancing the field of ecology is well established [START_REF] Mckinley | Citizen science can improve conservation science, natural resource management, and environmental protection[END_REF]. From a conservation perspective, Couvet and colleagues (2008) highlighted the greater social legitimacy of CS biodiversity indicators, generated from public-collected data, which may help bridge the gap between research findings and policy implementation (Arlettaz et al., 2010;[START_REF] Toomey | Navigating the space between research and implementation in conservation[END_REF].

Participating in CS programs may also increase the biodiversity knowledge of volunteers [START_REF] Deguines | Fostering close encounters of the entomological kind[END_REF][START_REF] Silvertown | Crowdsourcing the identification of organisms: A case-study of iSpot[END_REF], and these programs are further hoped to commit volunteers into heightened pro-environmental behaviors [START_REF] Chase | Citizen Science: Exploring the Potential of Natural Resource Monitoring Programs to Influence Environmental Attitudes and Behaviors[END_REF][START_REF] Toomey | Can citizen science lead to positive conservation attitudes and behaviors?[END_REF]. To date, however, evidence on whether pro-environmental behaviors are adopted by volunteers is scarce and based on qualitative information from a limited number of volunteers and/or a short period of time [START_REF] Cosquer | Observations of everyday biodiversity: A new perspective for conservation[END_REF][START_REF] Crall | The impacts of an invasive species citizen science training program on participant attitudes, behavior, and science literacy[END_REF][START_REF] Jordan | floras within and across continents: biogeographical consequences of human-mediated biotic interchange[END_REF][START_REF] Lewandowski | Butterfly citizen scientists in the United States increase their engagement in conservation[END_REF][START_REF] Sharma | From citizen science to citizen action: analysing the potential for a digital platform to cultivate attachments to nature[END_REF]. A temporal and quantitative assessment is lacking to assess this issue.

In this study, we investigated whether backyard owners joining the French Butterfly citizen science project (BCSP) adopt pro-environmental behaviors in their backyards.

Butterflies forage on nectar from flowers and the amount of nectar resources is a strong driver of butterfly abundance and richness in anthropogenic landscapes [START_REF] Luppi | Local and landscape drivers of butterfly richness and abundance in a human-dominated area[END_REF].

Conversely, butterflies are negatively affected by pesticides [START_REF] Forister | Increasing neonicotinoid use and the declining butterfly fauna of lowland California[END_REF][START_REF] Gilburn | Are neonicotinoid insecticides driving declines of widespread butterflies?[END_REF]. Provisioning nectariferous plants and reducing the use of pesticides are two conservation actions with demonstrated benefits for butterflies in backyards [START_REF] Fontaine | Impact of urbanization and gardening practices on common butterfly communities in France[END_REF]. Based on data from 2362 BCSP volunteers who participated two to eight years between 2006 and 2013, we assessed how participation may foster pro-environmental behaviors. We focused on two behaviors that have a direct impact on butterflies and biodiversity, i.e., the provision of nectar resources and the use of pesticides in backyards.

Research in environmental psychology showed that the simple provision of information is not enough, on its own, to induce behavioral changes [START_REF] Byerly | Nudging pro-environmental behavior: evidence and opportunities[END_REF][START_REF] Osbaldiston | Environmental sustainability and behavioral science[END_REF][START_REF] Schultz | Conservation means behavior[END_REF]. However, more than simply receiving general information about surrounding biodiversity and how to support it, volunteers in CS programs live so-experiences of nature [START_REF] Clayton | Transformation of experience: toward a new relationship with nature[END_REF], with explicit attention to biodiversity, that could facilitate pro-biodiversity practices [START_REF] Prévot | Routine experiences of nature in cities can increase personal commitment toward biodiversity conservation[END_REF]. We therefore expected that participation would encourage volunteers to shift towards butterfly-friendly gardening practices. Specifically, we hypothesized that sustained participation for multiple years can lead to adopting the two studied pro-biodiversity practices (i.e., provisioning more nectar resources and decreasing pesticide use). Additionally, within-year degree of participation during the period of sustained engagement (Ponciano and Brasileiro, 2014) may be seen as a quantitative measure of motivation for monitoring butterflies and we expected it to be associated with higher provision of nectar resources and lower use of pesticides.

However, individual behavioral changes are constrained by a set of interacting factors, such as attitudes, habits, personal capabilities, social norms, and context [START_REF] Stern | Toward a coherent theory of environmentally significant behavior[END_REF]. In s the identity of its owners (e.g., interests and activities such as recreation, eating, growing fruits or vegetables, connecting to nature; [START_REF] Clayton | Domesticated nature: motivations for gardening and perceptions of environmental impact[END_REF]. There is also evidence that personal experience and social norms can influence practices [START_REF] Ajzen | From publications to public actions: when conservation biologists bridge the gap between research and implementation[END_REF][START_REF] Goddard | Why garden for wildlife? Social and ecological drivers, motivations and barriers for biodiversity management in residential landscapes[END_REF][START_REF] Uren | Exploring social and cultural to ecologically sensitive residential garden design[END_REF]. For example, gardeners from rural origins or inhabiting rural areas may use more pesticides in their backyards compared with urban counterparts [START_REF] Barrault | Les pratiques de jardinage face aux risques sanitaires et environnementaux des pesticides. Les approches différenciées de la France et du Québec[END_REF][START_REF] Coppin | Is Pesticide Use Socially Acceptable? A Comparison between Urban and Rural Settings[END_REF]. Finally, backyard management is also influenced by its size [START_REF] Barrault | Les pratiques de jardinage face aux risques sanitaires et environnementaux des pesticides. Les approches différenciées de la France et du Québec[END_REF][START_REF] Clayton | Domesticated nature: motivations for gardening and perceptions of environmental impact[END_REF]Freeman et al., 2012;[START_REF] Riboulot-Chetrit | Making space for disorder in the garden: developing biophilia to conciliate aesthetics and biodiversity[END_REF]. Owners of large garden with a vegetable garden and fruit trees may be particularly prone to using pesticides [START_REF] Barrault | Les pratiques de jardinage face aux risques sanitaires et environnementaux des pesticides. Les approches différenciées de la France et du Québec[END_REF]. In our analyses, we thus accounted for the role of backyard size and its position along an urbanization gradient, as well as the presence of a vegetable garden or fruit trees in the backyard in determining pro-biodiversity practices of volunteers. Specifically, we tested whether the latter four variables could mediate the effect of sustained participation on the provisioning of nectar resources or the use of pesticides by volunteers in their backyard.

Lastly, general attention towards environmental and biodiversity issues have gained momentum in European countries (European Commission, 2013). These variations in collective norms could be linked with potential changes in gardening practices. In this regard, our study assesses across seven cohorts of volunteers (i.e., joining the BCSP program in seven consecutive years) whether behavioral changes are associated to being involved in this naturefocused CS program, strengthening our confidence that any observed pattern may not be confounded with temporal changes occurring in the overall French population.

MATERIALS and METHODS

Data collection and localization

The Opération Papillons -Vigieproject database for France; Noé -Muséum national d'Histoire naturelle, Paris, France; https://www.sciences-participatives-au-jardin.org/) is a citizen science program in which volunteers record butterflies in their backyard following a simple protocol [START_REF] Fontaine | Impact of urbanization and gardening practices on common butterfly communities in France[END_REF]. Upon registration, volunteers give their consent that the data they provide can be used for scientific studies. The program is open to the general public with no entomological skills required, as butterfly identification is based on a closed list of 28 species/group of species.

Each year from March to October, volunteers identify and count butterflies in their backyard and are invited to upload monthly lists of butterfly species abundance. Within a month, no minimum amount of time of observations is required (but participants qualitatively report their frequency of observations). In average, volunteers participated (i.e., uploaded butterfly counts) 4.98 months annually (SE = 0.04 months, min. = 1, max. = 8). To motivate volunteers, a monthly newsletter reported on overall participation, highlighted a Butterfly of the month , and shared results of the project; additionally, a tip of the month and a plant of the month sections could suggest pro-environmental behaviors (e.g., traditional crop varieties may better tolerate pests and reduce the need for pesticides).

We used data from the first eight years of the program (2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013). We reduced our dataset to volunteers who uploaded butterfly counts (i.e., participated) for several consecutive years, the minimum being two years (regardless the number of months of participation per year). Some volunteers interrupted their participation for one or several years. Because we wanted to assess potential effects of sustained (i.e., continuous) involvement into the BCSP program, we further restrained our dataset to volunteers with no annual break in participation.

Upon registration, participants provided the size of their backyard as well as its localization (the municipality -smallest administrative district in France). Backyard size ranged from 20 m² to 6000 m² (median = 1000 m², Q1 = 600 m², Q3 = 2000 m²). We characterized urbanization context of each backyard by computing the percentage of urban land use in the municipality (using Artificial surfaces from the first level of the Corine Land Cover 2006 database; [START_REF] Bossard | CORINE Land Cover[END_REF]; it ranged from 0% to 100% (median = 7%, Q1 = 2%, Q3 = 26%). While backyard size and urbanization context were slightly correlated tau = -0.33, P<0.001), there was no worrisome collinearity that would prevent their inclusion in the same statistical model (see 2.3 and computations of variance inflation factors). Participants also declared the presence of a vegetable garden or of fruit trees.

pro-biodiversity gardening practices

Participants were annually asked to fill out a questionnaire regarding the presence of some specific backyard features and plants, from a closed list, as well as their use of Regularly . After 2009, this question was split into 5 more-detailed questions: 1) Are you using insecticides? , 2) herbicides? , 3) fungicides? , 4) slug pellets? , and 5)

Bordeaux mixture? (the latter is a fungicide authorized in organic agriculture); again,

Never Occasionally

Regularly each of these questions.

Regularly we converted responses as binary variables (0 Never , 1 Occasionally Regularly ). To obtain for the whole 2006-2013 period a consistent index of pesticide use within backyards, we lumped responses to the 5 questions asked after 2009 into a single one: 0

Occasionally Regularly Some volunteers did not fill this questionnaire every year, in which case one or both indices could not be computed for a given year. We therefore further restricted our dataset to volunteers that provided backyard information for at least two years, including the first year of their participation to serve as a baseline against which changes in garden practices following sustained participation could be assessed. Our final dataset included 2362 volunteers distributed across all mainland France (Fig. 1).

Statistical analyses

As there were multiple observations per volunteer, we relied on mixed-effects individual gardening practices (provision of nectar resources and pesticide use) changed over time since the start of participation. All statistical analyses were performed with R version 3.5.2 (R Core Team, 2018) and, in particular, R package lme4 [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF]. Volunteers were structured in seven cohorts corresponding to their first year of participation (Fig. Additionally, to account for potential annual unmeasured variations (e.g., climate effects on we also included year as a random effect on the intercept. We computed variance inflation factors [START_REF] Zuur | Mixed effects models and extensions in ecology with R, Statistics for Biology and Health[END_REF] of all explanatory variables and found no evidence of collinearity (all VIF values < 1.5). Assumptions of homoscedasticity and normality of residuals of the model were met. Spatial independence of model residuals was confirmed graphically by computing a variogram [START_REF] Zuur | Mixed effects models and extensions in ecology with R, Statistics for Biology and Health[END_REF]. Two-way interactions which had no significant effects (P > 0.05) were removed from the models to better interpret single effects. This mixed-effect model was based on a sample size of 9009 observations from 2362 volunteers.

To investigate variations of pesticide use by volunteers in their backyard, we performed a generalized linear mixed-effects model with a binomial family and a logit link.

The response variable was binary, corresponding to using pesticides (1) or not (0). We accounted for the change in how information regarding pesticide use within backyards was gathered and treated (see above -biodiversity gardening practices) by including the type of recorded information regarding pesticide use as a fixed effect (two levels: single question and five questions). Other fixed effects were the same as in the linear mixed-effects model presented above. Specifying the same random-effect structure as above led to a singular model fit. To resolve this issue, we followed Bates and colleagues (2018) and simplified the random effect structure by removing the effect of year on the intercept. There was no collinearity among our explanatory variables (VIF values < 1.5) and spatial independence of model residuals was confirmed with a variogram. Two-way interactions which had no significant effects (P > 0.05) were removed from the model to better interpret single effects. This mixed-effect model was based on a sample size of 8636 observations from 2362 volunteers; observations number differs from the nectar resources linear mixed-effects model because volunteers were allowed to only partially fill in the backyard information annual questionnaire. We carried out post-hoc analyses to further interpret how significant effects of two sustained participation x vegetable garden sustained participation x fruit trees ) affected pesticide use. Specifically, we ran separate generalized linear mixed-effects models for volunteers with or without a vegetable garden (regardless the presence of fruit trees) and with or without fruit trees (regardless the presence of a vegetable garden).

Duration of sustained participation ranged from two to eight years and was unbalanced (765 and 166 volunteers participated during two and eight years respectively; the median sustained participation duration was three years). To ensure this would not lead to biased estimates of the relationships between explanatory variables and gardening practices, we performed a randomization procedure [START_REF] Manly | Randomization, bootstrap and Monte Carlo methods in biology[END_REF]. First, we randomly sampled (with replacement) 166 volunteers from each participation duration to generate a random dataset.

Second, we ran the mixed-effects models to this randomly sampled dataset, and repeated this procedure over 1000 iterations. We then compared the observed estimates (from the observed whole dataset) with the distribution expected with constant number of participants (166) per sustained participation duration (obtained from the 1000 iterations). We concluded from this procedure that results obtained from models using the observed (i.e., whole) dataset can be trusted (Supporting Information).

RESULTS

Both indices of gardening practices significantly changed with sustained participation (i.e., the time in years since entering the Butterfly citizen science project), and a set of other explanatory variables had effects on their own or mediated participation effects (Table 1).

Nectar resources in backyards

The index of nectar resources significantly increased with sustained participation (Fig. 2a), and we found no evidence that this effect was mediated by other backyard variables (size, urbanization context, presence of a vegetable garden or fruit trees; Table 1). In average, after eight years of participation, the index of nectar resources increased by 13.7%. Withinyear participation was also significantly positively associated with backyard nectar resources (Fig. 2b), but its effect was relatively weak: for every additional month of participation, nectar resources increased by 1%. Backyard size was strongly and positively correlated with nectar resources (Fig. 2c), with the index of nectar resources increasing by 33% from 100 m² to 1000 m² and then heading toward a plateau. Increasing urbanization context was significantly correlated with lower nectar resources in backyards (Fig. 2d). Finally, backyards that included a vegetable garden or fruit trees were associated to higher provisioning of nectar resources (Fig. 2e-f).

Pesticide use in backyards

The use of pesticides in backyards was significantly correlated with sustained participation; however, this relationship depended on whether or not volunteers had a vegetable garden or fruit trees in their backyard (Table 1).

Sustained participation was associated to lower use of pesticide by volunteers who did not have a vegetable garden in their backyard, but this relationship was weaker for volunteers tending a vegetable garden (Fig. 3a). Yet, post-hoc analyses detected significant effects of sustained participation on pesticide use in volunteers without or with a vegetable garden (P = 0.005 and P < 0.001 based on a sample size of 3052 and 5584 observations respectively).

After eight years of participation, the probability of pesticide use decreased by 78% and 23%

in volunteers without or with a vegetable garden respectively.

Similarly, sustained participation was associated with lower pesticide use by volunteers who did not have fruit trees in their backyard, but this relationship was weaker in volunteers having fruit trees (Fig. 3b). Post-hoc analyses revealed a significant relationship between sustained participation and pesticide use by volunteers having fruit trees in their backyard or not (P < 0.001 and P = 0.010 based on a sample size of 6824 and 1812 observations respectively). After eight years of participation, the probability of pesticide use in volunteers without or with a vegetable garden decreased by 73% and 37% respectively.

Additionally, backyard size was associated to increased probability of using pesticides (Fig. 3c). From a 100 m² to a 1000 m² backyard, probability of using pesticides increased by 28%.

DISCUSSION

Using temporal data on gardening practices from a nature-based citizen science program, we provided strong evidence that shifts towards biodiversity-friendly gardening practices may occur through CS volunteering. To our knowledge, this is the first time that an assessment of whether participating in a nature-based CS programs is associated to the implementation of pro-biodiversity actions is based on such a large number of volunteers, surveyed annually over multiple years.

We found positive correlations between sustained participation and level of probiodiversity practices, i.e. growing nectar-rich flowering plants and decreasing pesticide use. This is consistent with previous results from Cosquer and colleagues (2012), who carried out interviews of 30 volunteers from the same CS program. Similarly, a recent study reported that 95% of 139 volunteers from different butterfly CS programs across the United States declared participating more in conservation actions since engaging in one of their program [START_REF] Lewandowski | Butterfly citizen scientists in the United States increase their engagement in conservation[END_REF]. However, as these authors noted, volunteers could have increased their involvement regardless of joining a CS program, following potential changes in social norms regarding environmental and biodiversity issues. In this regard, an additional strength of the evidence presented here relies in our dataset including seven cohorts of volunteers joining the BCSP program in consecutive years (Fig. 1), and observed changes in gardening practices can be attributed with greater confidence to joining this nature-based CS program. Our quantitative and large-scale approach thus complements the existing body of qualitative evidence [START_REF] Cosquer | Observations of everyday biodiversity: A new perspective for conservation[END_REF][START_REF] Lewandowski | Butterfly citizen scientists in the United States increase their engagement in conservation[END_REF], and allows emphasizing that, beyond the acknowledged value for research in ecology, nature-based CS can also directly enhance local conservation measures at potentially broad geographical scale.

Our analyses also highlighted the importance of other variables than participation in affecting levels of pro-biodiversity practices. In particular, backyard size was the strongest predictor of nectar resources provisioning and a substantial one of pesticide use. Interestingly, backyard size had contrasting effects as larger backyards had higher nectar resources (i.e., a pro-biodiversity practice), but owners used more pesticides (i.e., a detrimental practice for biodiversity). Among the eight groups of plants used to calculate the nectar index, only two are spontaneous, while the presence of the others depends on the gardener decision to plant them. Such decision is most likely influenced by the physical constraints imposed by the size of the backyard, limiting the space that can be dedicated to different activities. The reasons for owners of larger backyards to harbor greater nectar resources cannot be determined from our dataset, and the aesthetic value of flowers may be the prime motivation, more than promoting biodiversity [START_REF] Clayton | Domesticated nature: motivations for gardening and perceptions of environmental impact[END_REF]. In line with this, greater pesticide use in large backyards appeared to be mostly due to greater application of herbicides and Bordeaux mixture (2010-2013 data from detailed pesticide use by volunteers), suggesting the will to maintain safety and order by controlling unwanted vegetation [START_REF] Clayton | Domesticated nature: motivations for gardening and perceptions of environmental impact[END_REF][START_REF] Riboulot-Chetrit | Making space for disorder in the garden: developing biophilia to conciliate aesthetics and biodiversity[END_REF].

Whether a backyard was used to grow food had multiple effects on pro-biodiversity practices implemented by volunteers. Greater amount of nectar resources was found in backyards where a vegetable garden or fruit trees were present. This could be interpreted as a way for gardeners to attract pollinators required for crop pollination [see for example [START_REF] Torres | Specificities of French community gardens as environmental stewardships[END_REF] in the context of community gardening], but it could simply be that gardeners tending a vegetable garden or fruits trees enjoy growing plants and thus are more likely to spend time planting different species; additional data would be needed to investigate this and other motives that volunteers may have in the present case. Most importantly, the presence of a vegetable garden or fruit trees in backyards weakened but did not prevent the reduction in pesticide use associated to sustained participation. Greater use of pesticides by backyard owners growing food had been found previously [START_REF] Barrault | Les pratiques de jardinage face aux risques sanitaires et environnementaux des pesticides. Les approches différenciées de la France et du Québec[END_REF]; therefore, the fact that participation to nature-based CS was able to prompt a reduction in using these chemicals in such context is very promising. Indeed, while the ban on the domestic use of some pesticides enforced in France since January 2019 should improve backyard quality for biodiversity, routine-experience of nature as proposed by nature-based CS programs may help prevent shifts towards pesticides considered as less harmful but that can still have detrimental environmental effects (e.g., the Bordeaux mixture, used in organic agriculture and remaining allowed for domestic use; Bourdais, 1999).

Studies based on self-reported data may be prone to the two following limits. First, researchers may obtain more responses from a subset of highly motivated persons. In the present study, we maximized the number of volunteers that we could consider in the analyses, including every volunteer of the BCSP program participating for at least two consecutive years and from whom we had received backyard information in at least the first year of participation and another year. Additionally, we ran a randomization procedure to check that the reduced number of long-term volunteers did not bias results from our mixed-effects modeling Information). Second, respondents may be biased in their reporting, being influenced by what is thought of as socially desirable. In our case, the primary use of understand the influence of gardening practices on butterflies. This clearly advertised biodiversity-focused objective may have prevented biased reporting due to social desirability.

Indeed, the reported data were used by Fontaine and colleagues (2016) who successfully detected positive and negative effects of the index of nectar resources and pesticide use on butterflies, respectively, as expected from the literature [START_REF] Forister | Increasing neonicotinoid use and the declining butterfly fauna of lowland California[END_REF][START_REF] Gilburn | Are neonicotinoid insecticides driving declines of widespread butterflies?[END_REF][START_REF] Luppi | Local and landscape drivers of butterfly richness and abundance in a human-dominated area[END_REF]. Thus, while our dataset may not be exempt of bias, it likely well describes practices in volunteers of the BCSP program.

Our findings confirmed that participation to nature-based CS program can prompt probiodiversity practices in volunteers, with direct local benefits for conservation. An analysis of interviews of 30 volunteers of the BCSP suggested that the development of awareness of butterflies and understanding of their ecological needs led to the intentional implementation of pro-conservation actions [START_REF] Cosquer | Observations of everyday biodiversity: A new perspective for conservation[END_REF]. Regular attentive observations of butterflies for the program constituted routine experiences of nature that may have primed volunteers towards adopting pro-biodiversity practices [START_REF] Prévot | Routine experiences of nature in cities can increase personal commitment toward biodiversity conservation[END_REF][START_REF] Prévot | Vigie-Lea[END_REF]. As recommended elsewhere [START_REF] Lewandowski | Butterfly citizen scientists in the United States increase their engagement in conservation[END_REF], we encouraged the adoption of biodiversity-friendly gardening practices (through newsletters): this may have been particularly effective in spurring changes in backyard management, because volunteers were helping biodiversity conservation their main reasons for participating [START_REF] Cosquer | Observations of everyday biodiversity: A new perspective for conservation[END_REF][START_REF] Prévot | Vigie-Lea[END_REF]. It is also possible that belonging to a community of observers (e.g., receiving newsletters, engaging in a program led by the National Museum of Natural History and Noé, an environmental NGO) has favored changes in attitudes and social norms towards greater acceptance of backyards features benefitting butterflies. Last but not least, our results may be particularly expected a citizen science program engaging backyard owners. Indeed, volunteers managed their backyard the way they chose, and perceived control to meet a particular outcome (i.e., perception of self-efficacy) was found to be positively associated with the probability to engage into pro-environmental behaviors [START_REF] Ajzen | From publications to public actions: when conservation biologists bridge the gap between research and implementation[END_REF][START_REF] Hines | Analysis and Synthesis of Research on Responsible Environmental Behavior: A Meta-Analysis[END_REF]. Sustained participation to a nature-based CS program may allow experiencing the causality between practices and biodiversity outcomes [START_REF] Cosquer | Observations of everyday biodiversity: A new perspective for conservation[END_REF], and the shift towards probiodiversity behaviors would then be likely, thanks to high perceived control. Complementary data would be required to understand h or available time may influence behavioral changes in the context of participation in citizen science.

pollinator hotspots [START_REF] Baldock | A systems approach reveals urban pollinator hotspots and conservation opportunities[END_REF][START_REF] Levé | Domestic gardens as favorable pollinator habitats in impervious landscapes[END_REF], and are thus of paramount importance for urban conservation strategies of butterflies and the wider flower visitor fauna. Yet, improving their quality through biodiversity-friendly management (e.g., planting nectar-rich or host plants, reducing mowing frequency) will require wishful personal involvement from the owners. We highlighted the roles of different factors in determining adoption of pro-biodiversity practices by citizen scientists. This calls for collaborations between biologists and social scientists if we are to succeed in further changing behaviors towards conservation goals [START_REF] Schultz | Conservation means behavior[END_REF]. Different tools exist to favour pro-environmental changes, but uncertainties remain regarding their efficiency under various conditions and for different behaviors [START_REF] Byerly | Nudging pro-environmental behavior: evidence and opportunities[END_REF][START_REF] Schultz | Strategies for promoting proenvironmental behavior[END_REF]. Beyond provisioning information and encouraging volunteers to engage in conservation, biologists involved in nature-based CS programs should embrace collaborations with psychological scientists to design and test interventions for enhancing adoption of probiodiversity behaviors [START_REF] Clayton | Psychological science, conservation, and environmental sustainability[END_REF]. For example, by designing experimental emails or newsletters, we could test the effectiveness of different strategies such as Messenger effect, Norms, or Salience [START_REF] Byerly | Nudging pro-environmental behavior: evidence and opportunities[END_REF] spurring behavioral changes in volunteers.

Given the tens of thousands of citizen scientists monitoring biodiversity in their backyards in Europe and North America [START_REF] Cannon | Trends in the use of private gardens by wild birds in Great Britain 1995-2002[END_REF][START_REF] Lorrillière | Winter bird survey in French gardens, two complementary schemes for a wide array of questions[END_REF][START_REF] Princé | Climate change in our backyards: the reshuffling of North 585[END_REF], this exciting avenue of interdisciplinary research represents critical stakes for biodiversity conservation in cities.

SUPPORTING INFORMATION

Methodological details and results of the randomization procedure are available online (Appendix S1).

  1): 2006 (n = 767), 2007 (n = 722), 2008 (n = 355), 2009 (n = 97), 2010 (n = 130), 2011 (n = 144), or 2012 (n = 147).The index of nectar resources displayed an approximately Gaussian distribution; thus, although it could only take integer values between 0 and 19, we decided to include it as the response variable of a linear-mixed effect model. Explanatory variables included the time (in years) since a volunteer started participating (sustained participation), the mean number of months of participation per year for each volunteer (within-year participation), the size of the backyard (backyard size; log transformed to improve residuals behavior), the percentage of urban land use in the s municipality (urbanization context), the presence of a vegetable garden in the backyard (vegetable garden), and the presence of fruit trees in the backyard (fruit trees). We further tested whether these four latter variables mediated the effect of sustained participation on the index of nectar resources by including each in a two-way interaction sustained participation . We included the longitude and latitude (its centroid) to account for potential spatial auto-correlation in our dataset. There were multiple observations per volunteer (from two to eight), and volunteers were clustered in seven cohorts (Fig.1). We accounted for these dependences in our dataset by including a random effect. Intercept and slope of sustained participation was allowed to vary among volunteers within cohorts [coded as (1 + sustained participation | cohort_ID : volunteer_ID)].

  pesticides. None of the backyard features, plants, or level of pesticide use was a requirement to participate. Based on this information, we computed the two following indices: nectar resources, and pesticide use.

	We used Bergerot	(2010) ranking of plant attractiveness for butterflies
	(related to nectar production) to compute the index of nectar resources in the backyard as
	following: the presence of butterfly bushes (Buddleja spp.), knapweeds (Centaurea spp.),
	lavenders (Lavandula spp.) or brambles (Rubus spp.) was scored 3 for each taxon; the
	presence of valerians (Valeriana spp.), clovers (Trifolium spp.) or aromatic plants (e.g.,

Rosmarinus officinalis/spp., Thymus spp.) was scored 2 for each; the presence of geraniums (Pelargonium spp.) was scored 1. The final index was computed by summing all these scores (range: 0-19). All plants are common backyard species across the bioclimatic regions of France.

The questions regarding pesticide use in the backyard varied in the period of the study.

From 2006 to 2009,

Is your garden treated with pesticides (e.g., insecticides, antkillers, aphid-killers or fungicides)? volunteers could answer Never , Occasionally , or

ACKNOWLEDGMENTS

We thank the thousands of volunteers of the Opération Papillons -Vigie Nature (2019) program for their effort and time monitoring butterflies. We are grateful to the NGO Noé and to the Vigie-Nature team for managing the citizen science program, to A-C Maurice and members of the CESCO for fruitful methodological discussions, and to two anonymous reviewers for helpful suggestions. ND was funded by the BiodivERsA3-2015-104 (BIOVEINS) grant, and KP was funded by the ANR-17-CE04-0012 (VGI4BIO). Funding sources were not involved in any step of this study.

AUTHORS CONTRIBUTIONS

Nicolas Deguines: Conceptualization, Methodology, Formal Analysis, Visualization, Writing -Original Draft, Writing -Reviewing and Editing. Karine Princé: Conceptualization, Methodology, Writing -Reviewing and Editing. Anne-Caroline Prévot: Conceptualization, Writing -Reviewing and Editing. Benoît Fontaine: Conceptualization, Data Curation, Methodology, Writing -Reviewing and Editing, Monitoring Scheme Management.

e shown for the linear mixed-effects model (Nectar resources) and the generalized linear mixed-effects model (Pesticide use), along with their associated 95% confidence intervals, and P-values. Sust. participation, Urban. cont., Pres. veg. garden, and Pres. fruit trees stand for sustained participation, urbanization context, of vegetable garden within the (vs absence), and presence of fruit trees within the backyard (vs absence) respectively. Two-way interactions associated to a P-value > 0.05 were sequentially removed from the complete models (see 2.3).


Zeineb Haj Slimane

Ibrahim Bedioune

Patrick Lechêne

Audrey Varin

Florence Lefebvre

Philippe Mateo

Valérie Domergue-Dupont

Matthias Dewenter

Wito Richter

Marco Conti

Zeineb Haj Slimane

Ali El-Armouche

Jin Zhang

Rodolphe Fischmeister

Grégoire Vandecasteele
email: gregoire.vandecasteele@u-psud.fr

Haj Slimane

Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes

Keywords: cAMP-dependent protein kinase, compartmentation, 3'-5' cyclic nucleotide phosphodiesterases, Ser/Thr protein phosphatases, nucleus

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.

Introduction

The cAMP-dependent protein kinase (PKA) is critically involved in the regulation of cardiac function by catecholamines acting on β-adrenoceptors (β-ARs) as well as several other hormonal and circulating factors acting through other Gs protein-coupled receptors (GsPCRs).

In the absence of cAMP, the PKA holoenzyme is a heterotetramer composed of two catalytic (C) subunits that are bound and inhibited by a dimer of regulatory (R) subunits. GsPCR occupancy leads to activation of adenylyl cyclases (AC) and increase in intracellular [cAMP].

Cooperative binding of cAMP to the R subunits leads to dissociation and activation of the C subunits which phosphorylate multiple protein targets in various subcellular compartments. [START_REF] Tasken | Localized effects of cAMP mediated by distinct routes of protein kinase A[END_REF] In cardiac myocytes, the best described PKA targets are proteins involved in excitationcontraction coupling (ECC) including the sarcolemmal L-type Ca 2+ channel, the ryanodine receptor (RyR2) and phospholamban (PLB), as well as the myofilament proteins cardiac myosin-binding protein C (cMyBP-C) and troponin I. [START_REF] Bers | Cardiac excitation-contraction coupling[END_REF] The velocity and specificity of β-AR regulation is determined by the localization of the PKA holoenzyme to its targets by A-kinase anchoring proteins (AKAPs) [START_REF] Scott | A-kinase anchoring proteins: getting to the heart of the matter[END_REF] and by the spatiotemporal pattern of cAMP signaling, which results from the interplay between cAMP production by ACs and cAMP degradation by cyclic nucleotide phosphodiesterases (PDEs). [START_REF] Mika | PDEs create local domains of cAMP signaling[END_REF] In addition, PKA activity is counterbalanced by Ser/Thr protein phosphatases (PPs). [START_REF] Herzig | Effects of serine/threonine protein phosphatases on ion channels in excitable membranes[END_REF] In cardiac myocytes, the PDEs that degrade cAMP belong to 5 major families (PDE1-4 and PDE8) [START_REF] Mika | PDEs create local domains of cAMP signaling[END_REF] , whereas the major cardiac PPs are PP1, PP2A and PP2B. [START_REF] Herzig | Effects of serine/threonine protein phosphatases on ion channels in excitable membranes[END_REF] The development of genetically encoded indicators of cAMP levels and PKA activity has been instrumental in defining the spatiotemporal characteristics of cAMP signals and PKA activity elicited by GsPCRs in living cardiac myocytes. The use of targeted sensors to various intracellular compartments revealed subcellular compartments with distinct cAMP responses and PKA phosphorylation gradients upon β-AR stimulation. [START_REF] Leroy | Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF][START_REF] Saucerman | Systems analysis of PKA-mediated phosphorylation gradients in live cardiac myocytes[END_REF] These studies also identified the cAMP-specific PDE4 family as a major negative regulator of cAMP generated by β-ARs. [START_REF] Leroy | Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF] Three genes encoding PDE4 (Pde4a, Pde4b and Pde4d) are expressed in cardiac tissue, and recent studies have emphasized the importance of PDE4B and PDE4D for β-AR regulation of cardiac ECC (reviewed in [START_REF] Mika | PDEs create local domains of cAMP signaling[END_REF] ).

PKA regulates numerous other effectors in cardiac myocytes, notably the transcription factors belonging to the cAMP-response element binding protein (CREB) family and class II histone deacetylases (HDACs) 4 and 5 in the nucleus. [START_REF] Muller | Transcriptional regulation by cAMP in the heart[END_REF][START_REF] Ha | PKA phosphorylates histone deacetylase 5 and prevents its nuclear export, leading to the inhibition of gene transcription and cardiomyocyte hypertrophy[END_REF][START_REF] Backs | Selective repression of MEF2 activity by PKA-dependent proteolysis of HDAC4[END_REF][START_REF] Chang | Acute beta-Adrenergic Activation Triggers Nuclear Import of Histone Deacetylase 5 and Delays Gq-induced Transcriptional Activation[END_REF] According to the classical view of nuclear PKA signaling, cAMP binds to PKA outside of the nucleus and, after dissociation from the R subunits, the C subunits cross the nuclear envelope by passive diffusion, which is a slow process. [START_REF] Harootunian | Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion[END_REF] However, recent evidence indicates that in HEK293 cells, a nuclear resident pool of PKA exists but is isolated from cAMP generated at the plasma membrane by AKAP-anchored PDE4. [START_REF] Sample | Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP[END_REF] In cardiac myocytes, it was reported that muscle A-kinase anchoring protein (mAKAP) targets PKA to multiple subcellular compartments including the nucleus [START_REF] Yang | A-kinase anchoring protein 100 (AKAP100) is localized in multiple subcellular compartments in the adult rat heart[END_REF] and the nuclear membrane. [START_REF] Kapiloff | mAKAP and the ryanodine receptor are part of a multi-component signaling complex on the cardiomyocyte nuclear envelope[END_REF] Moreover, mAKAP binds PDE4D3, a specific PDE4 isoform, which may control cAMP levels in this compartment and thereby the release of PKA C subunits into the nucleus. [START_REF] Dodge | mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module[END_REF][START_REF] Dodge-Kafka | The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways[END_REF] However, the modalities of nuclear PKA regulation in adult cardiomyocytes remain unknown. This issue is important given the role of the mAKAP complex and class II HDACs in the control of pathological cardiac hypertrophy [START_REF] Dodge-Kafka | The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways[END_REF][START_REF] Haberland | The many roles of histone deacetylases in development and physiology: implications for disease and therapy[END_REF] and of the CREB family of transcription factors in multiple aspects of cardiac function, including hypertrophy and apoptosis [START_REF] Tomita | Inducible cAMP early repressor (ICER) is a negative-feedback regulator of cardiac hypertrophy and an important mediator of cardiac myocyte apoptosis in response to beta-adrenergic receptor stimulation[END_REF] as well as the deleterious effects of chronic β1-AR overexpression in the heart. [START_REF] Lewin | Critical role of transcription factor cyclic AMP response element modulator in beta1-adrenoceptormediated cardiac dysfunction[END_REF] In this study, we compared the spatiotemporal dynamics of cytoplasmic and nuclear cAMP and PKA activity in adult cardiac myocytes using recombinant FRET probes targeted to these compartments. We found that upon β-AR stimulation, cAMP increases with similar fast kinetics in both compartments, whereas PKA activation is considerably delayed in the nuclei compared to the cytoplasm. Our results reveal for the first time the respective roles of PDE3 and PDE4 and of PP1, PP2A and PP2B in the differential integration of cytoplasmic and nuclear PKA responses to β-AR stimulation in cardiac myocytes.

Methods

All experiments performed conform to the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J no. L358, 18 December 1986), the local ethics committee (CREEA Ile-de-France Sud) guidelines, and the French decree n o 87-848 of October 19, 1987 (J Off République Française, 20 October 1987, pp 12245-12248). Authorizations to perform animal experiments according to this decree were obtained from the French Ministère de l'Agriculture, de la Pêche et de l'Alimentation (nº D 92-283, December 13, 2012). A total of 72 rats and 7 mice were used for this study.

FRET-Based Sensors of cAMP and PKA Activity

Cytoplasmic cAMP was measured using Indicator of cAMP using Epac 3 (ICUE3), a fluorescence resonance energy transfer (FRET)-based sensor. [START_REF] Dipilato | The role of membrane microdomains in shaping beta2-adrenergic receptor-mediated cAMP dynamics[END_REF] Nuclear localization of ICUE3 was achieved by adding a C-terminal nuclear localization signal (ICUE3-NLS). [START_REF] Sample | Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP[END_REF] PKA activity was measured with the FRET-based A-kinase activity reporter 3 (AKAR3). [START_REF] Allen | Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters[END_REF] Because untargeted AKAR3 is localized both in the cytoplasm and the nucleus, a specific cytoplasmic localization of AKAR3 was achieved by adding a C-terminal nuclear export signal to AKAR3 (AKAR3-NES). A specific nuclear localization was obtained by adding a C-terminal nuclear localization signal (AKAR3-NLS). [START_REF] Allen | Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters[END_REF] Adenoviruses encoding Ad.AKAR3-NES and Ad.AKAR3-NLS were generated using the ViraPower Adenoviral Expression System (Invitrogen) according to the manufacturer's protocol. The adenovirus encoding ICUE3 was kindly provided by Dr. Yang K. Xiang (University of California, Davis). The adenovirus encoding ICUE3-NLS was generated by Welgen Inc.

Isolation of Adult Rat Ventricular Myocytes (ARVMs)

Male Wistar rats (200-250 g) were subjected to anaesthesia by intraperitoneal injection of pentothal (0.1 mg/g). Individual ventricular myocytes were obtained by retrograde perfusion of the hearts as previously described. [START_REF] Rochais | Negative feedback exerted by cAMP-dependent protein kinase and cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes: an in vivo study using adenovirus-mediated expression of CNG channels[END_REF] See Data Supplement for details.

Adenoviral Infection

The medium was replaced by 300 µL of FBS-free MEM containing one of the following adenoviruses: Ad-AKAR3-NES, Ad-AKAR3-NLS, Ad-ICUE3 or Ad-ICUE3-NLS. In some experiments, PKA was blocked by co-infecting the cells with adenoviruses encoding a rabbit muscle PKA inhibitor (Ad.PKI) [START_REF] Lum | Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction[END_REF] . All experiments were done 24-48 h after cell isolation.

FRET Measurements of Cytoplasmic and Nuclear cAMP and PKA Activity

Cells were maintained at room temperature (20-25°C) in the same Ringer solution as described above. Images were captured every 5 s using the 40x oil immersion objective of an inverted microscope (Nikon) connected to a Cool SNAP HQ2 camera (Photometrics) controlled by the Metafluor software (Molecular Devices). CFP was excited during 300 ms by a Xenon lamp (Nikon) using a 440/20BP filter and a 455LP dichroic mirror. Dual emission imaging of CFP and YFP was performed using a Dual view emission splitter equipped with a 510 LP dichroic mirror and BP filters 480/30 nm and 535/25 nm, respectively.

Western Blot Studies

ARVMs were lysed in cold HNTG buffer, proteins were resolved by SDS-PAGE, and transferred to nitrocellulose membranes. Total and phosphospecific antibodies against CREB and cMyBP-C and a calsequestrin antibody were used as detailed in the Data supplement.

Data Analysis

For FRET measurements with AKAR3-NES and ICUE3, average fluorescence intensity was measured in the entire cell or in a portion of the cytoplasm with identical results. For AKAR3-NLS and ICUE3-NLS, average fluorescence intensity was measured in a region of interest inside the nuclei. Background was subtracted and YFP intensity was corrected for CFP bleedthrough before calculating the ratio. Ratio images were obtained with ImageJ software.

Average time course of the ratio represents the mean of all cells measured in a given experimental condition. The data were normalized to the ratio measured before the stimulus and expressed as percent change over basal. Student's t-test was used to compare two groups.

Western blot data were compared with Mann-Whitney test. CRC were compared by Fisher test. p-value <0.05 was considered statistically significant. Densitometric analyses of Western blots were performed using Quantity one software (Bio-Rad).

Results

Real Time Measurements of Cytoplasmic and Nuclear PKA Activity in Adult Rat

Ventricular Myocytes

Adenoviruses were generated to express the FRET-based A-kinase activity reporter 3 targeted to the cytoplasm (AKAR3-NES) and the nucleus (AKAR3-NLS) in ARVMs. YFP emission images were acquired by confocal microscopy to examine their subcellular localization. As shown in Figure 1A, myocytes infected at a multiplicity of infection (MOI) of 1,000 active viral particles per cell showed AKAR3-NES expression in the cytoplasm but not in the nuclei 24 h after infection. AKAR3-NLS expression could be detected slightly earlier (18 h) in the nuclei and was excluded from the cytoplasm (Figure 1B). However, this specific localization was dependent on the time of expression and was completely lost at 48 h, unless the MOI was reduced to 200 (Supplemental Figure 1). In order to test the functionality of AKAR3 in each compartment, cells infected with Ad.AKAR3-NES or Ad.AKAR3-NLS at MOI 1,000 for 24 h were stimulated with the β-AR agonist isoprenaline (Iso, 1 µM) and the CFP and YFP fluorescence were measured simultaneously in the entire cell for AKAR3-NES and in the two nuclei for AKAR3-NLS (Figure 1C and1D). As shown on the graphs and the corresponding pseudocolor images, Iso strongly increased the YFP/CFP ratio in the cytoplasm as well as in both nuclei. The average change was significantly larger in the cytoplasm (+38.6 ± 2.1%) than in the nuclei (+20.4 ± 2.1%, p<0.001) and both were abolished by application of the PKA inhibitor H89 (10 µM) or by co-expression of the PKA inhibitor peptide PKI (Figure 1E and1F). In the absence of Iso, neither H89 nor PKI had any effect on the basal YFP/CFP ratio in the cytoplasm or in the nuclei (Supplemental Figure 2). To further characterize PKA activation by β-AR stimulation in the two compartments, concentration-response curves (CRC) to Iso were generated. Supplemental Figure 3A shows a typical experiment in a myocyte expressing AKAR3-NES and exposed to increasing concentrations of Iso between 0.1 nM and 1 µM. The YFP/CFP emission ratio increased in a concentration-dependent manner until 30 nM Iso, and the effect was fully reversible upon washout. Supplemental Figure 3B compares the average CRC obtained in the cytoplasm (black squares) and in the nucleus (white squares). Hill fit of the data yielded apparent maximal effects (Emax) of +46.1  2.1% and +27.6  6.2%, and halfmaximal activation values (EC50) of 1.8  0.3 nM and 4.2  1.0 nM in the cytoplasm and the nuclei, respectively. The two curves were statistically different as indicated by Fisher test (p<0.001). When CRC to Iso were repeated in ARVMs infected with Ad.AKAR3-NES or Ad.AKAR3-NLS at MOI 1,000 during 48h and the fluorescence was measured in the entire cell, these differences were no longer observed (Supplemental Figure 4). Thus, the decreased efficiency and potency of Iso in the nucleus compared to the cytoplasm cannot be attributed to different properties of the targeted probes.

Kinetics of cAMP and PKA Activation in the Cytoplasm and in the Nucleus of Cardiac

Myocytes

In addition to these steady state differences, the kinetics of PKA activation was much faster in the cytoplasm than in the nuclei upon β-AR stimulation (Figure 2A andB). Indeed, half maximal PKA activation (t1/2on) was reached ~30 s after addition of Iso in the cytoplasm, but required ~3 min in the nuclei (Figure 2B). This difference was also observed upon direct AC activation with the hydrosoluble forskolin analogue L-858051 (L-85, 30 µM, Figure 2C and 2D) or upon global PDE blockade with IBMX (300 µM, Figure 2E and2F). In both compartments the response to Iso was faster than the response to L-85 and to IBMX, and in the case of L-85, the maximal nuclear PKA activation (+38.8 ± 1.5%) was significantly higher than that obtained with Iso (+21.4 ± 2.4%, p<0.001 versus L-85) or IBMX (+22.1 ± 3.1%, p<0.01 versus L-85). To test whether restricted cAMP diffusion could be involved in the slow phosphorylation of AKAR3-NLS upon β-AR stimulation, we compared the kinetics of cAMP in the cytoplasm and the nuclei using the untargeted cAMP sensor ICUE3 [START_REF] Dipilato | The role of membrane microdomains in shaping beta2-adrenergic receptor-mediated cAMP dynamics[END_REF] and a nuclear version of the sensor, ICUE3-NLS. [START_REF] Sample | Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP[END_REF] Myocytes infected with Ad.ICUE3 and Ad.ICUE3-NLS expressed the probe in the cytoplasm and nuclei, respectively (Figure 3A). β-AR stimulation with Iso (1 µM) produced a fast increase in cAMP with similar onset kinetics (t1/2on ~20 s) in both compartments (Figure 3B and3C).

In a next series of experiments, we compared the effect of a transient β-AR stimulation (Iso, 100 nM, 15 s) similar to those elicited by a startle response on cytoplasmic and nuclear PKA activities. As shown in Figure 4A, this induced a maximal activation of PKA in the cytoplasm (+49.7 ± 2.0%, P<0.001), but a marginal increase in the nuclei (+3.2 ± 0.9%, P<0.01). Similar differences were observed when comparing the PKA phosphorylation of myofilament protein cMyBP-C at Ser282 with that of the nuclear transcription factor CREB at Ser133. cMyBP-C was significantly phosphorylated upon a short (15 s) Iso stimulation (Figure 4C) while CREB was not (Figure 4D). Prolonging the Iso application to 15 min did not further increase cMyBP-C phosphorylation (Figure 4C), but significantly increased CREB phosphorylation (Figure 4D). In parallel experiments, we verified that Iso stimulation (100 nM, 15 s and 15 min) did not modify total cMyBP-C and CREB protein levels (Supplemental Figure 5). These results suggest that the PKA-dependent regulation of contractility can be dissociated from regulation of gene expression during short β-AR stimulation.

Regulation of Cytoplasmic and Nuclear PKA Activity in Cardiac Myocytes

We next sought to characterize the regulatory mechanisms governing PKA activation in the two compartments. We have shown previously that PDE3 and PDE4 account for the majority of the cAMP-hydrolyzing activity in ARVMs and that PDE4 is predominant in the degradation of cAMP generated by β-ARs at the plasma membrane and in the cytoplasm. [START_REF] Leroy | Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF] Therefore, we examined the respective contribution of these PDEs to the regulation of PKA activity in the cytoplasm and in the nuclei using the specific PDE3 inhibitor cilostamide (Cil) and the specific PDE4 inhibitor Ro 20-1724 (Ro). Neither Cil (1µM) alone nor Ro (10 µM) alone had any effect on the basal YFP/CFP ratio (Supplemental Figure 6). ARVMs overexpressing AKAR3-NES and AKAR3-NLS were briefly exposed to Iso (100 nM, 15 s) in the presence of Cil or Ro. Each inhibitor was applied during 3 minutes before the Iso pulse and continuously thereafter. As illustrated in Figure 5, PDE3 inhibition with Cil did not alter cytoplasmic or nuclear PKA activation by Iso (Figure 5A and 5B), whereas Ro greatly prolonged PKA activation in the cytoplasm (t1/2 off = 3.4 ± 0.2 min, versus 1.7 ± 0.1 min for Iso alone, p<0.001) and in the nuclei (Figure 5C and5D). Because in cardiac myocytes PDE4D is enriched at the nuclear envelope, [START_REF] Dodge | mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module[END_REF][START_REF] Dodge-Kafka | The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways[END_REF][START_REF] Lugnier | Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei[END_REF] we examined β-AR regulation of cytoplasmic and nuclear PKA activity in adult mouse ventricular myocytes (AMVMs) from Pde4d-deficient (Pde4d -/-) mice. [START_REF] Jin | Generation of PDE4 knockout mice by gene targeting[END_REF] In these myocytes, the response to Iso pulse stimulation in the cytoplasm was slightly but significantly prolonged compared to WT myocytes (t1/2 off = 2.4 ± 0.3 min in Pde4d -/-versus 1.5 ± 0.2 min, in WT, p<0.05, Figure 5E). In the nuclei, similarly to what we observed in ARVMs, Iso pulse stimulation had virtually no effect on PKA activity in WT AMVMs (+1.0 ± 0.5%, p<0.01, Figure 5F). However, in Pde4d -/-myocytes, Iso stimulation increased nuclear PKA activity by 4-fold (+3.8 ± 0.9% in Pde4d -/-versus WT, p<0.05, Figure 5F). These results are consistent with PDE4D being an important regulator of nuclear cAMP/PKA signalling.

Contribution of Ser/Thr Phosphatases to the Regulation of Cytoplasmic and Nuclear

PKA Responses in ARVMs

In cardiac myocytes, PP1 and PP2A are the major Ser/Thr protein phosphatases that counterbalance PKA activity. To assess the contribution of these PPs to the modulation of the cytoplasmic and nuclear PKA responses, FRET experiments were performed in ARVMs expressing AKAR3-NES or AKAR3-NLS exposed to Iso pulse (100 nM, 15 s) in the presence of Calyculin A (CalyA, 100 nM), which inhibits both PP1 and PP2A. As shown in Figure 6A, CalyA markedly slowed down the dephosphorylation of AKAR3 by Iso in the cytoplasm (t1/2 off =7.0 ± 1.6 min versus 1.5 ± 0.1 min for Iso alone, p<0.05), and unmasked a strong PKA response in the nucleus, which remained sustained for at least 8 min (Figure 6B). To determine the participation of PP2A to these effects, okadaïc acid (OA) was used at a concentration of 100 nM, at which it preferentially inhibits this phosphatase. [START_REF] Bialojan | Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics[END_REF] As shown in Figure 6C, OA also slowed down the dephosphorylation of AKAR3 in the cytoplasm (t1/2 off =3.0 ± 0.3 min versus 1.5 ± 0.1 min for Iso alone, p<0.001), although to a lesser extent than CalyA (p<0.001). In contrast, OA had no effect on the nuclear PKA response to Iso pulse stimulation (Figure 6D).

In another series of experiments, a potential role for PP2B (calcineurin) was investigated using the PP2B-selective inhibitor cyclosporin A (CsA, 5 µM). As shown in Figures 6E and6F, CsA did not modify the PKA response to Iso, neither in the cytoplasm or the nucleus. Together, these results indicate that upon β-AR stimulation, PP1 and PP2A contribute to the AKAR3 dephosphorylation in the cytoplasm, whereas PP1 plays a major role in the nucleus.

Discussion

Although it is known that PKA can regulate cardiac gene expression through nuclear transcription factors such as CREB and specific class II HDACs, the modalities of nuclear PKA regulation have remained elusive in cardiac myocytes. Here, we show the dynamics of this regulation in response to β-AR stimulation in living adult cardiomyocytes. Our results indicate that β-AR stimulation of nuclear PKA activity is temporally dissociated from that of nuclear cAMP elevation and from that of cytoplasmic PKA activation. This is consistent with activation of the PKA holoenzyme in the cytoplasm and translocation of the C subunits to the nucleus.

We show that PDE4 is an upstream negative regulator of this process, with PDE4D playing an important role to control nuclear PKA activity, whereas PP1 and PP2A are downstream negative regulators. Importantly, the contribution of PP1 and PP2A to the termination of the β-AR PKA response differs between these two compartments, with PP1 being predominant in the nucleus.

One major finding was that independently of the mechanism of cAMP elevation, the kinetics of PKA activation were much slower in the nuclei than in the cytoplasm, as previously reported in model cell lines [START_REF] Sample | Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP[END_REF][START_REF] Allen | Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters[END_REF] and primary neurons. [START_REF] Gervasi | Dynamics of protein kinase A signaling at the membrane, in the cytosol, and in the nucleus of neurons in mouse brain slices[END_REF] Importantly, when measuring cytoplasmic and nuclear cAMP upon β-AR stimulation, no such delay was observed (Figure 3) indicating that limited cAMP diffusion was not the cause of the slow increase in nuclear PKA activity. This was consistent with activation of PKA holoenzyme in the cytoplasm and slow translocation of the C subunits into the nuclei by passive diffusion. [START_REF] Harootunian | Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion[END_REF] As a result, a short β-AR stimulation marginally activated PKA and CREB phosphorylation in the nuclei while it maximally activated PKA and cMyBP-C phosphorylation in the cytoplasm (Figure 4). Such temporal dissociation of PKA action in the two compartments may have important implications for cardiac physiology and pathology. The fast kinetics of cytoplasmic PKA activation observed here closely mimic the kinetics of L-type Ca 2+ current stimulation [START_REF] Leroy | Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF] and emphasize the importance of fast PKA activation for the velocity and efficiency of the fight-or-flight response.

The modest consequences on nuclear PKA activity and CREB phosphorylation suggest that PKA regulation of cardiac contractility can be dissociated from regulation of gene expression during short term sympathetic stimulation. However, cAMP may also activate other effectors such as Epac, which was shown to be localized not only at the plasma membrane but also at the nuclear/perinuclear area in cardiomyocytes. [START_REF] Dodge-Kafka | The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways[END_REF][START_REF] Pereira | Epac enhances excitation-transcription coupling in cardiac myocytes[END_REF] In adult rat ventricular myocytes, Epac activation was shown to contribute to the prohypertrophic effects of β-AR stimulation [START_REF] Metrich | Epac mediates beta-adrenergic receptor-induced cardiomyocyte hypertrophy[END_REF] in part by increasing nuclear [Ca 2+ ] and CaMKII-dependent nuclear export of HDAC5, with consequent derepression of the transcription factor MEF2. [START_REF] Pereira | Epac enhances excitation-transcription coupling in cardiac myocytes[END_REF] Our results also show that sustained β-AR activation, as may occur during intense and prolonged physical exercise or following myocardium injury, increases nuclear PKA activity and CREB phosphorylation.

While the CREB/CREM family of transcription factors may contribute to β-AR-dependent maladaptive remodelling, [START_REF] Tomita | Inducible cAMP early repressor (ICER) is a negative-feedback regulator of cardiac hypertrophy and an important mediator of cardiac myocyte apoptosis in response to beta-adrenergic receptor stimulation[END_REF][START_REF] Lewin | Critical role of transcription factor cyclic AMP response element modulator in beta1-adrenoceptormediated cardiac dysfunction[END_REF] PKA can also phosphorylate HDAC5 in the nucleus. [START_REF] Ha | PKA phosphorylates histone deacetylase 5 and prevents its nuclear export, leading to the inhibition of gene transcription and cardiomyocyte hypertrophy[END_REF][START_REF] Chang | Acute beta-Adrenergic Activation Triggers Nuclear Import of Histone Deacetylase 5 and Delays Gq-induced Transcriptional Activation[END_REF] This, in contrast to phosphorylation by CaMKII and PKD, [START_REF] Mckinsey | Activation of the myocyte enhancer factor-2 transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14-3-3 to histone deacetylase 5[END_REF][START_REF] Vega | Protein kinases C and D mediate agonist-dependent cardiac hypertrophy through nuclear export of histone deacetylase 5[END_REF][START_REF] Wu | Local InsP3dependent perinuclear Ca2+ signaling in cardiac myocyte excitation-transcription coupling[END_REF] favours its nuclear retention, and the repression of the hypertrophic gene program. [START_REF] Ha | PKA phosphorylates histone deacetylase 5 and prevents its nuclear export, leading to the inhibition of gene transcription and cardiomyocyte hypertrophy[END_REF][START_REF] Chang | Acute beta-Adrenergic Activation Triggers Nuclear Import of Histone Deacetylase 5 and Delays Gq-induced Transcriptional Activation[END_REF] Altogether these results illustrate the dynamic and versatile nature of nuclear cAMP signalling, which may exert both beneficial and detrimental influences on the adult heart depending on the respective balance between the different target effectors.

Our finding that PDE4 inhibition strongly prolonged β-AR stimulation of PKA activity in the cytoplasm, whereas PDE3 had no effect, is consistent with the relative contribution of these two PDE families to cAMP degradation in the cytoplasm of ARVMs following a short β-AR stimulation. [START_REF] Leroy | Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF] Analysis of cardiomyocytes from Pde4d -/-mice further show the participation of this specific isoform to termination of β-AR responses in ARVMs, which is consistent with the reported role of PDE4D in potentiating β-AR-stimulated contraction rate in neonatal cardiomyocytes from Pde4d -/-34 and the reported roles of PDE4D variants in β-AR signalling and ECC in heart. [START_REF] Mika | PDEs create local domains of cAMP signaling[END_REF][START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF][START_REF] Beca | Phosphodiesterase 4D regulates baseline sarcoplasmic reticulum Ca2+ release and cardiac contractility, independently of L-type Ca2+ current[END_REF] PDE3 and PDE4 represent the major PDE activities in cardiac nuclei and are primarily localised at the nuclear envelope. [START_REF] Lugnier | Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei[END_REF] Here we show that following short β-AR stimulation PDE4 predominates to control nuclear PKA activation. Results in cardiomyocytes from Pde4d -/-mice further indicate that the lack of PDE4D enhanced nuclear PKA responses to β-AR stimulation, which is consistent with the presence this isoform at the nuclear membrane. [START_REF] Dodge | mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module[END_REF][START_REF] Dodge-Kafka | The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways[END_REF][START_REF] Lugnier | Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei[END_REF] Because at the nuclear envelope, PDE4D is integrated in a macromolecular complex including mAKAP and is involved in the regulation of cardiomyocyte hypertrophy, [START_REF] Dodge-Kafka | The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways[END_REF][START_REF] Pare | The mAKAP complex participates in the induction of cardiac myocyte hypertrophy by adrenergic receptor signaling[END_REF] our results suggest that dysregulation of nuclear PKA activity following the loss of PDE4D may participate in the late onset dilated cardiomyopathy observed in Pde4d -/- mice. [START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF] In HEK293 cells, PDE4 inhibition dramatically accelerated the nuclear PKA response to a maintained application of forskolin, implying that PDE4 inhibition uncovered a nuclear pool of PKA in these cells. [START_REF] Sample | Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP[END_REF] Although the pulse stimulation used here did not allow unambiguous determination of the onset kinetics, our data do not support a similar acceleration of nuclear PKA activation by PDE4 inhibition in ARVMs (Figure 5). Rather, a different arrangement of components may exist in cardiac myocytes, with PDE4 being located at the nuclear envelope [START_REF] Dodge | mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module[END_REF][START_REF] Lugnier | Characterization of cyclic nucleotide phosphodiesterase isoforms associated to isolated cardiac nuclei[END_REF] where it controls the extent of C subunits released upon β-AR stimulation and transferred into the nucleus from an extranuclear pool of PKA holoenzyme.

Our study shows that Ser/Thr PPs PP1 and PP2A contribute differently to AKAR3 dephosphorylation between the cytoplasm and the nucleus. The balanced contribution of PP1 and PP2A in the cytoplasm is consistent with these two PPases playing a major role in controlling the phosphorylation status of ECC proteins and in counteracting the β-AR/cAMP/PKA-mediated functional effects in cardiac myocytes. [START_REF] Herzig | Effects of serine/threonine protein phosphatases on ion channels in excitable membranes[END_REF] In contrast, PP2B inhibition had no effect, suggesting a minor participation of calcineurin in AKAR3 dephosphorylation in the bulk cytoplasm and the nuclei when PP1 and PP2A are active. A more important participation of PP2B might occur in pathological contexts such as cardiac pressure-overload hypertrophy where PP2B is activated. [START_REF] Lim | Calcineurin expression, activation, and function in cardiac pressure-overload hypertrophy[END_REF] In agreement with this hypothesis, it was reported that PP2B blunts β-AR phosphorylation of PLB in spontaneously hypertensive rats. [START_REF] Macdonnell | Calcineurin inhibition normalizes beta-adrenergic responsiveness in the spontaneously hypertensive rat[END_REF] Moreover, whereas PP2B is not detected in the nuclei of normal heart, it is clearly localized in this compartment in the diseased myocardium. [START_REF] Hallhuber | Inhibition of nuclear import of calcineurin prevents myocardial hypertrophy[END_REF] While our work was in revision, Yang et al. published an independent report in neonatal cardiomyocytes where they found similar kinetics differences in cytoplasmic and nuclear PKA activation which supports activation of the PKA holoenzyme in the cytoplasm and translocation of the C subunits to the nucleus in neonatal myocytes. [START_REF] Yang | PKA catalytic subunit compartmentation regulates contractile and hypertrophic responses to beta-adrenergic signaling[END_REF] Yang et al. however did not investigate the role of PDE families in β-AR response as done here. Their modelling approach predicts a determinant role of phosphatase activity on the amplitude of nuclear PKA response, which is consistent with what we found here. PP1 has been shown to have crucial functions in the nucleus of other cell types. [START_REF] Bollen | Signaling by protein phosphatases in the nucleus[END_REF] Our data suggest that a nuclear PP1 efficiently suppresses PKA activation in the nucleus, and this could provide new opportunities to selectively manipulate nuclear β-AR signalling independently of ECC regulation. As stated above, it has been proposed recently that PKA favours nuclear accumulation of HDAC5, thereby inhibiting cardiomyocyte hypertrophy. [START_REF] Ha | PKA phosphorylates histone deacetylase 5 and prevents its nuclear export, leading to the inhibition of gene transcription and cardiomyocyte hypertrophy[END_REF][START_REF] Chang | Acute beta-Adrenergic Activation Triggers Nuclear Import of Histone Deacetylase 5 and Delays Gq-induced Transcriptional Activation[END_REF] Thus, nuclear PP1 inhibition may enhance this cardioprotective effect of β-AR stimulation without increasing the phosphorylation of PLB and RyR2 as observed upon global PP1 inhibition, which -although beneficial on the short termis associated with an increased risk of ventricular arrhythmia and the development of a progressive cardiomyopathy with aging. [START_REF] Wittkopper | Constitutively active phosphatase inhibitor-1 improves cardiac contractility in young mice but is deleterious after catecholaminergic stress and with aging[END_REF] In summary, our results demonstrate for the first time that β-AR stimulated PKA activity is differentially regulated in the cytoplasm and the nuclei of adult rat ventricular myocytes, and delineate the respective role of PDE3 and PDE4 and of PPs in this process. Our results unveil PP1 as a critical negative regulator of nuclear PKA in response to β-AR stimulation and this should have important functional consequences for the control of nuclear PKA targets such as the CREB family of transcription factors and class II HDACs, which are involved in pathological cardiac remodelling and in the adverse effects of chronic β-AR stimulation.

Cell stimulation for Western Blot studies

ARVMs were incubated in FBS-free MEM containing 100 nM isoprenaline (Iso) for 15 seconds or 15 minutes. Stimulation was stopped by fast exchange of the Iso-containing medium with 2 mL of cold PBS (PAA Laboratories).

Western Blot studies

ARVMs were lysed in cold HNTG lysis buffer containing (in mM): Hepes (pH 7.5) 50; NaCl 400; Na-pyrophosphate 10; MgCl2 1.5; EGTA 1; Glycerol 10%, Triton X100 1% supplemented with phosphatase inhibitor cocktail (Roche) and a protease inhibitor cocktail (Roche). The lysates were centrifuged at 13,000g and proteins were resolved by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were saturated with 3% BSA and incubated overnight at 4°C with phosphospecific antibodies against CREB (Ser133, Cell Signaling Technology) and cMyBP-C (Ser282, Alexis). Immunoreactive proteins were revealed using anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz). The membranes were stripped with Reblot plus Strong solution (Millipore) and reprobed with a

Supplemental Figures

Supplemental Figure 1

Figure 1 .

 1 Figure 1. Measurements of cytoplasmic and nuclear PKA activity in ARVMs. A and B,

Figure 2 .

 2 Figure 2. Comparative kinetics of cytoplasmic and nuclear PKA activation in response to

Figure 3 .

 3 Figure 3. Comparative measurements of cytoplasmic and nuclear cAMP responses to a

Figure 4 .

 4 Figure 4. Comparison of cytoplasmic and nuclear PKA responses upon brief β-AR stimulation

Figure 5 .

 5 Figure 5. Role of PDE3 and PDE4 in the regulation of cytoplasmic and nuclear PKA activity.

Figure 6 .

 6 Figure 6. Contribution of Ser/Thr phophatases to the apparent PKA activity in the cytoplasm

Supplemental Figure 2 :

 2 : Subcellular localization of AKAR3-NES and AKAR3-NLS in ARVMs depends on expression level. YFP images of ARVMs infected with Ad.AKAR3-NES and Ad.AKAR3-NLS at MOI 200 or MOI 1000 for 48h obtained by confocal microscopy. Scale bars represent 10 µm. PKA inhibition with H89 or PKI has no significant effect on the basal FRET ratio measured with AKAR-NES in the cytoplasm and AKAR-NLS in the nucleus. Mean variation (±S.E.M.) of the basal YFP/CFP ratio induced by H89 (10 µM) in ARVMs expressing AKAR-NES (A) or AKAR-NLS (B). PKI was co-expressed with the FRET sensors by adenoviral infection, and the mean variation (±S.E.M.) in YFP/CFP ratio were compared to cells expressing AKAR-NES alone or AKAR-NLS alone (n=7 cells each). For AKAR-NES, the numbers next to the bars indicate the number of cells. For AKAR-NLS, the first number below the bars indicates the number of cells and the second indicates the number of nuclei. Data are from 5 rats.

Supplemental Figure 3 .Supplemental Figure 6 :

 36 Cumulative concentration-response curves (CRCs) of cytoplasmic and nuclear PKA activity in response to Iso in ARVMs. A, Time course of the normalized YFP/CFP ratio in response to increasing concentrations of Iso in a myocyte expressing AKAR3-NES. Pseudo-color images depict the variations of the YFP/CFP emission ratio at each Iso concentration. B, Comparison of the average effect of Iso on cytoplasmic (black squares) and nuclear (white squares) PKA activity. N refers to the number of cells, n refers to the number of nuclei. Data are from 6 rats. The curves were fitted with the Hill equation. Values are means ± S.E.M. Statistical significance is indicated as ***, p<0.001. Inhibition of PDE3 or inhibition of PDE4 have no significant effect on the basal FRET ratio measured with AKAR-NES in the cytoplasm and AKAR-NLS in the nucleus. Mean variation (±S.E.M.) of the basal YFP/CFP ratio induced by the PDE3 inhibitor cilostamide (Cil) and the PDE4 inhibitor Ro 20-1724 (Ro) in ARVMs expressing AKAR-NES (A) or AKAR-NLS (B). For AKAR-NES, the numbers next to the bars indicate the number of cells tested. For AKAR-NLS, the first number indicates the number of cells and the second indicates the number of nuclei. Data are from 2 rats.

Acknowledgements

We thank Pierre Bobin, Dr. Jérôme Leroy and Dr. Martine Pomérance for helpful discussion of the data. We thank Valérie Nicolas (imaging core facility, University Paris-Sud, IFR 141) for assistance with confocal microscopy. We are also grateful to Dr. Hazel Lum (University of Illinois) for providing the adenovirus encoding PKI and to Dr. Yang K. Xiang (University of California Davis) for providing the adenovirus encoding ICUE3.

ZHS was a recipient of doctoral grants from the CORDDIM program of Région Ile-de-France and from the Fondation pour la Recherche Médicale. This work was supported by ANR grant 2010 BLAN 1139-01 (to GV), the Fondation Leducq for the Transatlantic Network of Excellence cycAMP grant 06CVD02 (to RF) and NIH grant R21HL107960 (to WR).

Funding

Haj Slimane et al.

Data Supplement

Conflict of interest: none declared

Supplemental Methods

Reagents

Isoprenaline (Iso), H-89 dihydrochloride hydrate (H89), 3-isobutyl-1-methylxantine (IBMX) and Cyclosporin A (CsA) were from Sigma, L-858051 (L-85), Ro 20-1724 (Ro), Okadaic Acid (OA) and Calyculin A (CalyA) were from Calbiochem, Cilostamide was from Tocris Bioscience.

Isolation of adult rat ventricular myocytes

Male Wistar rats (200-250 g) were subjected to anaesthesia by intraperitoneal injection of pentothal (0.1 mg/g), and hearts were quickly excised. Freshly isolated cells were suspended in minimal essential medium (M 4780, Sigma) containing 2.5% fetal bovine serum (FBS, PAA Laboratories), 1% penicillin-streptomycin and 2% HEPES (pH 7.6) and plated on 35 mm, laminin-coated glass bottom culture dishes for FRET experiments (10 4 cells/dish) or on 60 mm, laminin-coated culture dishes for western blot experiments (2x10 5 cells/dish).

Myocytes were left to adhere for 2 h in a 95% O2, 5% CO2 incubator at 37 °C.

Isolation of adult mouse ventricular myocytes

Generation of Pde4d-deficient (Pde4d -/-) mice has been described previously. [START_REF] Tasken | Localized effects of cAMP mediated by distinct routes of protein kinase A[END_REF] Three to six month-old wild type (WT) and Pde4d -/-males with a mixed 129/Ola (25%) and C57BL/6 (75%) background were anesthetized by intraperitoneal injection of pentothal (0.15 mg/g), and hearts were quickly removed. Ventricular myocytes were obtained by retrograde Langendorff perfusion of the heart as previously described. [START_REF] Bers | Cardiac excitation-contraction coupling[END_REF] Freshly isolated ventricular myocytes were suspended in MEM (Gibco 51200) supplemented with 5% FBS, 2% penicillin-streptomycin, 0.1% BSA, 2 mM L-glutamine, 10 mM BDM and Insulin Transferin Haj Slimane et al.

calsequestrin antibody (Affinity BioReagents) used as a loading control or MyBPC3 (K-16) against total cMyBP-C (Santa Cruz). Total CREB was detected with 48H2 antibody (Cell Signaling Techonology).

Supplemental References