G L Lippi

How to organize the preparation of a scientific paper

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.

1 To whom this note is addressed This note is a contribution which aims at offering some advice to Ph.D. students and Young Scientists1 in the Natural Sciences (and Mathematics) who are confronted with the writing of a scientific publication. As always, advice depends strongly on people: what works well for someone does not for another person. Thus, the best way of profiting from it is to compare various sources (which exist in abundance on this topic) and to take what is most suited to one's needs and style. In addition, different disciplines have separate requirements2 , thus it is a good idea to also ask around for more information in one's field.

The scope of this contribution is help palliating difficulties which at times arise in preparing a scientific manuscript, which may end up into a writer block or fear of the blank page. Two outcomes are common in these situations: either the page remains blank, or it is filled -at times lavishly -with sentences which, upon later reading, convey little or unintelligible information. Both lead to a sense of frustration: the young writer feels inadequate to the task; the more experienced colleague (e.g., the Thesis Advisor) faces the daunting task of rearranging confused, poorly written information. Indeed, one of the natural reactions to feeling overwhelmed by the task is to start writing long circumlocutions whose sense is clear (at best) only to the writer. This is particularly true for non-native speakers who have to face the additional difficulty of more limited vocabulary and syntactical choices.

The unfortunate consequence is a negative impact on the young colleague's writing and productivity; it is therefore important to aim at palliating the problem before it arises. I hope that the suggestions of the present contribution will result in a happier and more productive experience for all involved.

The writing of a paper can be decomposed into three main parts:

A. Organization of the writing task and structuring the manuscript. This is the main objective of this note and will be detailed in the following.

B. Writing style. Since English is the international scientific language (at least in the sciences targeted by this note) and most writers are native speakers of another language, there is a double hurdle to be overcome: linguistic proficiency (for grammatical and syntactic correctness), and effective communication.

B.1 No specific advice will be given here for English language help for non-native speakers.

Everyone working in the sciences is nowadays sufficiently proficient to communicate in English, but writing may raise additional difficulties. It is possible to find online help for grammar and syntax 3 .

B.2 Scientific writing advice: this may vary somewhat from one discipline to the other, but there are some basic rules that hold for all, such as conciseness and precision. There is no point in repeating excellent advice that has already been given 4 , but my general recommendation is to look for contributions on the web.

B.3 I personally recommend the use of free synonym online dictionaries -numerous and overall good -which help avoiding disturbing repetitions: the less proficient the writer with the English language, the more frequent the word recurrence. You will quickly identify, by trial and error, your preferred web sites. Bilingual dictionaries, numerous on the web, can also be useful but my recommendation is to compare the offered translation to a range of synonyms. The proposed translation may not be the most suitable to your needs. Google Translate is a nice tool, but my advice is to use it with moderation when you write a scientific text. Its translations are of fairly good quality but, in my opinion, they do not stand up to the desired level for a scientific paper.

Finally, I would like to transmit one piece of advice I have heard multiple times from native English speakers and that ought to be treasured. An often needed and (mis-)used verb in scientific writing is "to allow": unless you are a native English speaker (or exceptionally proficient in that language) ban it from your vocabulary and replace it with a synonym. The cause of its misuse is its need for a preposition, unnatural to the non-native speaker.

C. Technical writing style deals with presentation: figure preparation, recommended font size, formatting, numbering, insertion and punctuation with equations, etc. Each journal has its own guidelines, which ought to be checked before getting down to truly writing the paper. Some of the guidance, however, is broader and covers a whole discipline. Thus, it is always good for the newcomer to familiarize oneself with the general usage by consulting the technical advice (often) offered by Scientific Societies and/or by their non-profit journals 5 .

To summarize this introduction, adapt the various suggestions (preceding and following) to your needs and supplement them by searching the Web for resources. The amount of advice freely available is very abundant and, by careful selection, you can find practically anything you need. A useful summary on how to write a scientific paper can be found at: https://www.grammarly.com/blog/how-to-write-a-research-paper/ 2 Choice of journal Each discipline and each journal have their own characteristics. Choosing the right journal is in itself a crucial step that needs to be discussed with your Thesis Advisor. In fact, the preparation of the manuscript will depend, in its more advanced stages, on the journal choice, since the text has to 3 A well-advertised, free site, is the following: https://www.grammarly.com/ There are certainly others, but I haven't checked any of them. Interested people are invited to search the web.

4 General scientific writing style tips are effectively conveyed by Barbara Goss Levi, a Senior Editor at Physics Today -they can be downloaded at: https://www.aps.org/publications/apsnews/199806/rules.cfm K. Friedman, at Reviews of Modern Physics, offers a detailed list of advice on scientific writing.

Although some parts are somewhat specific, anyone can benefit from reading the text, available online at: https://d22izw7byeupn1.cloudfront.net/files/rmpguapa.pdf 5 A good technical style and notation guide for physics is freely available online at: https://cdn.journals.aps.org/files/styleguide-pr.pdf match the goals of the chosen journal. In spite of the differences among branches of science, two basic kinds of publications can be identified in the bibliography: Letters and Archival papers (finer details may vary with discipline and publisher).

In broad terms, a Letter is a short report on original research which focusses on an outstanding finding. The emphasis on the indeterminate article signifies that there must be one central point (with a couple of side points which may be needed to corroborate it) of sufficient importance to justify the communication, rather than an ensemble of results. Often the Publisher will require the Letter to have a sufficiently broad scope to be useful to an audience beyond a strict specialty. A Letter may also be useful to give advance notice of an important result to the research community. As such, it should concentrate on the main message, giving the strictly necessary details for supporting the statements. In this case, it is expected that the Letter be followed by an Archival publication, which will contain the full details of the investigation and will also dwell on side issues which are important for the specialized community, but unnecessary for the broader audience.

An Archival publication, instead, presents original research, with a complete overview of the general context and existing literature (relevant to the specific work -a Review paper instead will summarize and discuss a large amount of literature covering a given field). The problem to be solved is explicitly stated, together with the work methodology, the conceptualization of a solution, and the theoretical (or experimental) background which complements the investigation. The data are presented, together with the (experimental) acquisition or (theoretical) computational methodology, their analysis, discussion and conclusions. If a Letter has preceded the Archival publication, new findings that may have appeared -e.g. following discussions and debates, or simply as a consequence of additional work -will be explicitly added and discussed in the archival presentation.

These are just generic guidelines which classify the two main kinds of publications used in the natural sciences for the dissemination of original research. The main burden of journal selection falls on the more experienced researchers; however, once the journal has been chosen it is good practice for the younger collaborator to go and read the Publisher's description on the targeted journal's scope and read the advice on the expected paper content and structure.

Your manuscript

Before starting with a technical point-by-point description of an organization strategy, we are going to review the points which need to be addressed in the preparation of a paper (this list should be supplemented, at least, by the one given at the end of Section 1 -cf. grammarly site). The first point you need to have in mind is the message that you want to disseminate. Not only do you need to know what the message is, but it has to be clear in your mind, both as a concept and as a sequence of the steps needed for its presentation. Keep in mind that you may think the ideas obvious, but discover that the process needed for their explanation may be a lot more complex than expected. Discuss it with your Thesis Advisor and/or Coworkers to see whether: 1. your arguments are well-structured, clear and convincing; and 2. you are correctly targeting the Readership you want to address.

The Readership choice has been crucial in determining the journal. As already mentioned in section 2 this choice conditions the form of presentation and, in turn, influences the organization of the paper in terms of arguments (general and broad for a Letter, more technical and specialized for an Archival publication) and of level of detail.

Having a clear idea in mind of how and for whom you want to pitch your contribution will help you defining the organization of the manuscript. As a reviewer for journals, I have encountered situations where the targeted audience and the style did not match: for instance a very detailed and pedantic presentation for a paper destined to attract interest from a wider research community. If one wants to attract non-specialists to a new field, the paper must give the broad lines and stress the novelty and importance of the results, inciting others to seek for similar features in other systems. In such a case, writing several pages to discuss all the little details of their particular implementation is neither going to attract Readers outside the specialty, nor is it going to render the paper understandable, as non-specialists will get lost in unnecessary details.

The definition of scope and target, thus the presentation strategy, may take a few attemps before reaching a satisfactory level, and discussions with Colleagues and Thesis Advisors are certainly a very useful way of meeting the challenge, thus highly recommended. The investment will be well-justified, since it will later make you gain time by avoiding lengthy (even multiple) text rewritings before the correct harmony is found.

The following practical advice for organizing the writing of a paper may appear a bit long and painstaking. It is purposefully detailed and broken up in many steps. If you feel comfortable with the preparation of your paper you can compact some of the proposed steps together, reducing their number. The reason for presenting them separately is to offer the most detailed break-down, from which one can more easily construct personalized procedures. In addition, there are always situations where the task becomes harder (for instance, writing a more complex paper) for which the more parcelled version of the planning may become useful. Finally, and generally true, the perfect advice does not exist; there is only advice from which you can select what works best for you. Thus, this is just a proposal from which anyone can shape their own procedure. In the end, with growing experience, anyone develops one's personal way of working. This scheme becomes, therefore, the fall-back, basic plan.

Detailed plan

Practically, we can break down the organization process into a few steps, each with substeps.

Step 1: Determining the paper's message

• Preliminary step

Prepare a list of ideas/results you want to present in your paper Checkpoint No. 1

1.1 Check with your Thesis Advisor (and/or other experienced Coworkers, if appropriate) which parts of the content you suggest are suitable for a scientific publication. As a young researcher, your perspective is often very narrowly focussed on your work. Thus, you risk emphasizing details which are not so important (or are already known) while you might overlook aspects which would hold weight with the research community. This is not a shortcoming of yours, but simply the consequence of being a beginner in the field; experienced researchers who change field would do the same, consulting with colleagues who are recognized experts before embarking on projects and publications.

1.2 Decide with your Thesis Advisor the form of the paper (Letter or Archival) and determine the journal you want to submit to -the latter may not be a final decision, as there may be the choice left between two or three suitable journals. At this stage this suffices, as you don't need yet to closely follow the formatting instructions of any journal (it will come later).

Step 2: Organizing the manuscript's structure Step 3: Inserting content into the section structure

• The task is now to start putting content into the structure you have planned.

At this stage you are writing bullet points, not full text. Writing full sentences (and good English) may take the focus away from content. Thus, concentrate on ideas and substance.

Collect the thoughts and ideas which belong to each of the sections you have outlined in the previous step; each idea will become a subsection

For each section, describe each idea in a couple of lines, one idea per subsection. This subdivision is artificial, but useful to categorize and prioritize the rank that each thought or point has at this stage (you will most likely remove most of the subsections, at the end).

For those ideas that need further development, introduce additional subsections each containing a single argument that needs to be made. You can repeat the process as in a chain (which eventually will end) if the idea requires multiple levels of discussion. Make sure you identify the level in the sequence of subsections to which each argument belongs. This chain-like structure allows you to identify the sequence of steps needed to prove your point and can be invaluable in gaining time and preparing a much clearer discussion.

Remember that subsections may contain equations, figures or any other means of explanation. All these tools are part of the presentation and should be seamlessly integrated in your exposition.

Remember to insert, at this stage, the arguments from the literature which back up your statements (or those papers to which you want to compare your work). Make a note of the paper(s), book(s) or whatever support you will need for the statement; the reference must be clearly identifiable, even if the full citation is not developed. This is a work in progress, but the literature should appear now.

Checkpoint No. 3

3.a Show the content you have prepared to your Thesis Advisor. There should be enough detail to be understandable; however, it is not impossible that the formulations are understandable only to you. Thus plan for a live discussion to present your ideas point by point.

3.b Record the feedback throughout the discussion. It is very likely that, while discussing the various points you have prepared, necessary changes may surface, such as modifications in priorities, order of presentation, changes in the arguments, etc. Note them down in real time (on paper or directly in an electronic file) and make sure you have enough detail to reconstruct the arguments and the changes. Also, verify that you bibliography is suitable; if additional references are needed, make sure that you have an unambiguous way of retrieving it and that the points to be made in its respect are clear to you.

3.c

In the course of the discussion, the need for some additional checks/work may emerge, required to better support your findings. It is a good timing for it and you should neither be surprised nor worried. Typically, it is a matter of additions which do not required a large amount of work, but which can bring substantial benefits.

3.d Discuss with your Thesis

Advisor whether the journal choice can be finalized, now that the paper structure and content are well established.

3.e You should be leaving the meeting (or written exchange) in a condition to start writing out the text (and, if needed, carry out the additional checks).

Remark: it shouldn't have escaped your attention that so far you have not written, and not even planned three of the required parts of the manuscript: abstract, introduction and conclusions. You may also have only a very preliminary title -just to have an idea of where you are going. This is quite alright, since these sections have to be adapted to the content of your paper and until the latter is well-defined you would be waisting your time working on them.

Step 4: Writing the first version of your main sections This step is optional and can be run in parallel with step 4

Step 4A: Preparing Supplementary Material

• This step, numbered 4A, is (nearly) independent of the writing of the main paper, as it concerns the preparation of additional material. It is not a mandatory step. It concerns various forms of complementary information that the Authors may wish to make available to Readers, through the electronic posting of specialized sections (often called Supplementary Material) with technical information providing useful, but not essential, support. Instances may include lengthy mathematical derivations, computer codes, additional figures, construction details for an experiment, technological points, etc. Depending on the Publisher, it may also be possible to publish data, animations, high resolution and/or interactive figures, videos, etc.

The position of this step in the sequence of tasks is somewhat arbitrary. This is the first place where it makes sense to start considering it. Any later time is also possible (of course, before submission!).

After verification of the Publisher's offer for posting Supplementary Material [check: some Journals do not accept it!], discuss with your Thesis Advisor -in one of your meetings or by email -whether it would be beneficial to your manuscript. Remember that this section cannot be used to squeeze into a limited number of pages a paper which would require several more -the Supplementary Material is only an optional complement; the paper must remain fully understandable without it.

Evaluate and discuss with your Advisor which form of Supplementary Material would be useful for your paper: additional section, video, etc.

Since this Material focusses on technical issues, it will be easier to write. You just need to present/explain the content.

Although the optional nature of this section may (apparently) lessen its importance, nonetheless, it should be carefully written. A poorly prepared Supplementary Material will induce a negative opinion on the work5 .

It is a good idea to search for articles similar to yours in your targeted Journal to find Supplementary Material sections. From them (possibly a few) you can get an idea of the typical style for this section.

In order to prepare the Supplementary Material, follow the same steps that you have used for your main sections.

Checkpoint No. 4A

4A You can choose to treat the task separately, sending materials to your Thesis Advisor for her/his revision on the Supplementary Material. Most of the exchanges should be doable by email, as the preparation of this section is more straightforward: it only contains technical information and can be rather dry in style. It is crucial, however, to be complete, clear and to avoid mistakes (grammar, syntax, typos, etc.). In-person discussions on this task can probably be embedded into another scheduled meeting.

The following step [5] can be run in parallel with step 4. At variance with step 4A, it is not optional and is of crucial importance. Whether you want to prepare it in parallel with point 4 or not will depend both on your preferences and on the availability of your Thesis Advisor or collaborating Co-Author. If it is difficult to get an appointment, then you may want to go ahead with point 5 at the same time as 4, since the procedure is that of steps 2 and 3. If your Thesis Advisor is easily available, then you can treat this task as a separate one. In any case, you should be aware of the fact that both 4 and 5 are substantial steps which require a considerable time investment: they shouldn't be taken lightly.

Step 5: Planning Conclusions, Introduction, Abstract (and Title)

• Since the organization of the main body of the paper (i.e., your original work) is now completed, and since you may have already written a good first draft for those sections, it is now time to start considering the trimmings -i.e., what follows and precedes the core of your presentation.

• Remember: although these parts are accessories to your core message, their role is of paramount importance in attracting (Title and Abstract), leading and engaging (Introduction) the Reader, as well as in making sure that the central points have been duly understood and retained (Conclusions). It is therefore crucial to pay as close an attention to these sections as you have done with your core work. The reason for delaying their preparation is that they have to be adapted to your original contribution (and not vice-versa). Advancing their writing would most likely result in a waste of time and effort, because they would later have to be reshaped to match your message.

Start working from the Conclusions section and apply the previous Steps 2 and 3 -with their corresponding checkpoints.

This time, you want to make a list of the points that your contribution is making. Prioritize them and break them down in sections and subsections, as you have done to present your original work. Remember to concentrate only on the main points and refrain from the temptation of including every single detail of your paper: only the most important results should stand out here. As for the main text, you should write the text only after a check with your Thesis Advisor.

Remember the acknowledgments section (check with your Thesis Advisor and Coworkers, as well) both to recognize help you may have received, as well as support (financial or otherwise).

Now apply

Steps 2 and 3 to the Introduction. The latter is probably the most difficult part to write, as it must a give critical overview of the literature relevant to your contibution, placing it into the general context. Prepare your work well and expect the need for detailed discussions with your Thesis Advisor and Coworkers. Remember to apply the advice previously seen at Steps 2 and 3.

Repeat the procedure with the Abstract. Check the journal's rules (often there is a length limit). Read abstracts from the journal in which you are going to publish to match the style. Make a bullet list of the twothree main points that you are making in your manuscript. Write the abstract if very concise form. Even though the abstract is very short, you will probably need to discuss it with your Thesis Advisor more than once, given its importance: the abstract is at times the only piece of information available to potential Readers, who will decide, on its basis, whether they are interested in your work

The Title is the ultimate advertisement for your paper and its appearence as last item shows that it has to be a one-line, eye-catching summary of your paper. It has to be carefully thought, given its necessary briefness and importance. Use the same criteria as for the abstract and plan for repeated exchanges with your Thesis Advisor. It may be a good idea to conceive more than one version of the title to explore various possibilities.

Checkpoint No. 5

5 Since the organization exercise for these sections is the same as what has been done for Steps 2 and 3, you should expect to check with your Advisor on the structure of your proposed Conclusion, Introduction, Abstract sections and Title. The procedure will the same as already practiced for the body of the paper. Make sure you include in your meeting a discussion of the title (cf. last point of the list).

Step 6: Last iterations

• The main body of the paper is now at a stage where it is being polished, both for details and English presentation.

Polish the writing, adding all the details left out so far (if any) and make use -if needed -of online writing help.

Make sure to follow the style guidelines of the selected journal for the presentation of figures, equations, etc.

• The Conclusions, Introduction and Abstract are one step behind (equivalent of step 4 for the main paper).

Prepare a text -preliminary or advanced -for the three sections. It is likely that the Conclusions will advance more readily than the Introduction. For the latter, feedback and perhaps some direct writing from your Thesis Advisor or Coworker(s) will be beneficial (or needed).

Checkpoint No. 6

6 By now, you have gained enough experience and, by comparing to the previous checkpoints, you know what to do in your discussions.

6.1 The main body of the text has to be checked over (perhaps a final time).

6.2 Verify with your Thesis Advisor the progress on Conclusions, Introduction and Abstract.

Iterate the writing and the discussions as needed, to converge towards a finalized text also for these parts.

At this point, you are in good shape for completing the paper, as you have gone through most of the organizational steps and hurdles. As a repeat of what previously stated: the proposed scheme is meant as a suggestion on how to organize the writing. You will obtain best results by comparing with other schemes (which certainly exist online) and by extracting from it something that is most suited to you and to your collaborators.

Practice is the answer to all challenges. There is no teaching that can replace that. In fact, "to teach" is a verb that ought to be banned from everyone's vocabulary (not only non-English speakers). Even though "teaching" is half of my job, I think that this is a poor description of the task -learning is the only true activity and this can be achieved only through practice. What "teachers" can do is only support the learning process by showing known techniques, guiding the learner (and we are all learners, especially in research) and offering some advice; however, knowing full well that what is excellent help for one person may be absolutely useless for another. Thus, I hope that you will treat this advice for what it is, and take charge of your learning process in writing your papers.

Step 7: Final checks and submission

• The writing is practically completed and it is time for the last checks (including misspellings, typos, etc.).

Plan for your final reading, checking with the Journal's guidelines on all the technical points (figures, formatting, etc.) to make sure that the paper is ready for submission. Your Thesis Advisor may (correctly) delegate to you this step and you need to ensure that everything is in order to avoid unnecessary and annoying delays coming from the non-respect of basic rules.

Verify that all the material is ready for the submission. For the record, this step is often done in writing (email), since the submitting Co-Author certifies all Authors' agreement.

7.2 One Author is chosen to be in charge of the submission with the Journal. Her/his duties reside in: 1. uploading the manuscript onto the Journal's website (nowadays this is typically done in electronic form); 2. keeping in touch with the Journal; 3. answering Journal's questions in relation to the submission; 4. serving as communication channel between Journal and all the Authors (thus, all information has to be circulated as soon as it arrives).

7.3 Of course, as part of the previous point the Referee reports -which come to the submitting Author -have to be immediately circulated to all Co-Authors. Typically, the submitting Author also coordinates the preparation of the Authors' answer and of the possible modifications of the manuscript for resubmission. The follow-up work (new submission with all the needed material) is part of the submitting Author's duties.

You may be chosen as the Co-Author in charge of the submission.

If you have been designated as the Author in charge of the submission to the Journal, you need to make sure that you are in possession of all of the following material before you endevour to accomplish the task. Notice that some of the following points may need to be checked by your Thesis Advisor because of the nature of the required information. This may take some time, thus you ought to plan for that. You should also expect, and be prepared for the possibility of having to suspend the submission process (hopefully saving the part of the work you have already done) to double check for information or parts of text that may be required by the web page (cf. below for examples). Each Journal has its own peculiar requirements, thus it is impossible to provide an exhaustive list here.

Step S: Submitting the paper on the Journal's website

• This part can be rather time consuming -more than you would expect -and requires preparation. The list below is a contribution towards being ready. However, it cannot substitute a careful reading of all the guidelines and actually going through the process, since different Journal may have different requirements. I am trying to collect the most commonly required information, presented here in an order which will not match the one of your Journal.

S.1 Make sure that you have a submission letter addressed to the Editor, answering all the required points (to be checked on the guidelines). This letter has to be double-checked with your Thesis Advisor.

S.2 If you are submitting online (most likely nowadays) make sure that you have access to the site: you will probably need to register for an account, in first place. In unlucky cases this may take a day or two.

S.3 Make sure that you have all the information asked by the web page (hard to know ahead!), which is likely to include, among others, the affiliations of all Authors and their email addresses.

S.4 A list of keywords -often needed also on the manuscript -is likely to be asked by the web page. At times, it has to be chosen from the Journal's database: you may have to discover the allowed keywords and check possible choices with your Thesis Advisor or a designated Co-Author. Keep in mind that these choices are not always obvious, even for experienced researchers.

S.5 It is not uncommon for the web page to ask you to specify funding sources and to seek their identification online. This is at times a sticky step which results in considerable time waste, since you may have to work around ways of identifying the relevant match.

S.6 It is common for Journals to ask the Authors to suggest names of Referees -typically at least 3. The Journal will not necessarily contact them, but will use the suggestions to look through their database to more easily identify suitable experts. It is therefore important to make very judicious choices on this point and you absolutely need the input of your Thesis Advisor and experienced Colleagues. On the other hand, the Authors may choose not to suggest Referees (allowed).

S.7 It is also customary for the Journal to allow the Authors to reject some potential Referees because of conflict of interest (or any other reasonjustifications are not normally required). Again, you need to check with your Thesis Advisor and Co-Authors whether some name should be put on this list.

S.8 One of the Co-Authors has to be designated as Corresponding Author, i.e., the Author who will receive correspondence, requests for information, questions, etc. Make sure you have cleared with your Thesis Advisor who the Corresponding Author is going to be before embarking on the submission.

S.9 If the paper is in Letter format (or in any case has length restrictions) it is common for the web page to run some check to verify its potential length in print. This is sometimes a sticky point because your estimates may not match those of the web page and you may need to go back to your Co-Authors to discuss trimming the text a bit.

•

  Now that you have agreed on the main content of the paper and its format (short or long paper, i.e., Letter or Archival) go ahead with the following steps Organize your paper by determining what are the needed sections (e.g., experimental setup, model description, mathematical background, etc.) Make sure you have a section for your results Make sure you have a section for their discussion Choose provisional section titles more appropriate than the generic categories suggested above. This helps you focus on the message contained in each section. This advice does not hold for those journals and research fields which, unfortunately, stifle the creative process of result dissemination by imposing a set structure (and title sections). In spite of this, even in those cases this step remains a useful practice. The main purpose for dividing your work into sections is to help structuring the communication. If your paper is a letter, which is typically a brief communication devoid of sections, you will eventually remove all your section headers and transform your manuscript into a single section. Get feedback, making sure that you fully understand the suggestions and note down their implementation. It is alright to ask questions and request clarifications, as it is crucial to fully understand the instructions. A good job at this stage can save you precious time and frustration at a later time.

If possible, choose now the text editing mode (Word or LaTeX) which will allow you to make the submission. This amounts to a gain in time, as you won't have to convert later to a different tool. If you by chance already know to which journal the paper will be submitted, then you can even download the template and write your notes directly into it. Otherwise, for those disciplines in which there is a general-purpose format accepted by several journals (LaTeX has some basic, standard formats, which can be later adapted) it is a good idea to select it.

Checkpoint

No. 2 2.a Show your plan to your Thesis Advisor or any other Designated Collaborator (depending on the organizational structure of your group) 2.b

•

  The aim of this step is to detail the material that you have laid out and start writing the text of your main sections, using the outcome of the last exchange with your Thesis Advisor

	Develop the points that you have made, first subsection by subsection,
	then working your way up to each section. Your text may not yet flow
	entirely, but you should have full sentences that clearly develop the con-
	cepts.
	Remember to insert the literature citations in the appropriate spots while
	you are writing the text.
	You may go through the text in more than one pass if this makes the
	writing easier. Don't be afraid of completing a subsection half-way down.
	If your organization is good, you should be able to write the different
	parts semi-independently -you will only need to make connections in a
	later global reading, if the text does not flow.
	Checkpoint No. 4
	4 This step will most likely require a serious reading, thus you should first hand-in (or send-in
	electronically) your text. You should be receiving written comments from your Thesis Advisor
	(or designated Coworker). If your text was not yet written in full English form, you will have
	received a return on the skeleton, otherwise the comments can even contain a first round of
	corrections. After you have read the comments, you can ask for an in-person meeting to clarify
	any (likely) outstanding point that you don't understand or on which you disagree.

Remember that text composition can be best done in stages. Write a first version, then set it aside. When you come back to it, you can better see what is unclear and needs amending. You can iterate as many times as needed.

When writing don't forget the opportunity offered by online writing help (I previously mentioned the grammarly site, but there are others); this may lighten your work by checking for mistakes and fluidifying the presentation.

The choice of journal should have been finalized in your last meeting (checkpoint 3.d). If this is still not the case, bring this point up either in writing (e.g. email) or in the next meeting. But don't delay this step.

Together with the journal choice, it is now time to go and read the guidelines. If the journal offers a template (whether LaTeX or Word style) it is now time to convert your draft into that format (unless you have already done this).

  This step requires matching the list of what you have available with the Journal's guidelines After a final, global check, verify with your Thesis Advisor and your Co-Authors the last version of the paper. Everyone has to agree to the submission of the manuscript in the now agreed final form, thus you need to send the last version by email, explicitly asking for last-minute changes or for approval. If changes are made, then the step has to be re-iterated.

	Checkpoint No. 7
	7.1

In the following, I will be referring only to "Thesis Advisors" for simplicity. Since this note is addressed also to young colleagues, possibly beyond their Ph.D. Thesis, read in place of Thesis Advisor an Experienced Collaborator (e.g., the head of the research group).

My advice may be more suitable to physicists, since this is my field.

It is unfortunately not uncommon, at least at the reviewing stage, to read Supplementary Material sections that haven't been properly drafted. In this case, you compromise your reputation with Reviewers and Journal Editor.

Step 6: Submitting the paper on the Journal's website -continued S.10 Make sure that you have checked beforehand what is required by the Journal at submission. At times, one single file with the complete paper is to be uploaded. This is the case in which the Journal wants to simplify procedures until the Reviewers recommend publication. Additional information (e.g., high quality figures) will be requested only once a positive recommendation for publication is received. In other cases, you will have to upload from the start the individual figures in the suitable format (to be checked in the Journal's guidelines).

S.11 Make sure you have the Supplementary Material file, animations, movies, etc. ready at hand for submission.

S.12 A statement on the paper's relevance and novelty may be required, often asking for a justification for the Journal's choice. There is typically a limited number of words allowed for this section, which has to be filled into an online box as "text only". Some submissions sites do not ask for this piece of information.


Aniella Abi-Gerges

Liliana Castro

Jérôme Leroy

Valérie Domergue

Rodolphe Fischmeister

Dr Grégoire Vandecasteele
email: gregoire.vandecasteele@universite-paris-saclay.fr

Selective Changes in Cytosolic β-adrenergic cAMP Signals and L-Type Calcium Channel Regulation by Phosphodiesterases during Cardiac Hypertrophy

Background

In cardiomyocytes, phosphodiesterases (PDEs) type 3 and 4 are the predominant enzymes that degrade cAMP generated by β-adrenergic receptors (β-ARs), impacting notably the regulation of the L-type Ca 2+ current (ICa,L). Cardiac hypertrophy (CH) is accompanied by a reduction in PDE3 and PDE4, however, whether this affects the dynamic regulation of cytosolic cAMP and ICa,L is not known.

Methods and Results

CH was induced in rats by thoracic aortic banding over a time period of five weeks and was confirmed by anatomical measurements. Left ventricular myocytes (LVMs) were isolated from CH and sham-operated (SHAM) rats and transduced with an adenovirus encoding a Förster resonance energy transfer (FRET)-based cAMP biosensor or subjected to the whole-cell configuration of the patch-clamp technique to measure ICa,L. Aortic stenosis resulted in a 46% increase in heart weight to body weight ratio in CH compared to SHAM. In SHAM and CH LVMs, a short isoprenaline stimulation (Iso, 100 nM, 15 s) elicited a similar transient increase in cAMP with a half decay time (t1/2off) of ~50 s. In both groups, PDE4 inhibition with Ro-201724 (10 µM) markedly potentiated the amplitude and slowed the decline of the cAMP transient, this latter effect being more pronounced in SHAM (t1/2off ~ 250 s) than in CH (t1/2off ~ 150 s, P<0.01). In contrast, PDE3 inhibition with cilostamide (1 µM) had no effect on the amplitude of the cAMP transient and a minimal effect on its recovery in SHAM, whereas it potentiated the amplitude and slowed the decay in CH (t1/2off ~ 80 s). Iso pulse stimulation also elicited a similar transient increase in ICa,L in SHAM and CH, although the duration of the rising phase was delayed in CH. Inhibition of PDE3 or PDE4 potentiated ICa,L amplitude in SHAM but not in CH. Besides, while only PDE4 inhibition slowed down the decline of ICa,L in SHAM, both PDE3 and PDE4 contributed in CH. 3 Conclusion These results identify selective alterations in cytosolic cAMP and ICa,L regulation by PDE3 and PDE4 in CH, and show that the balance between PDE3 and PDE4 for the regulation of β-AR responses is shifted toward PDE3 during CH.

Introduction

The intracellular second messenger cAMP modulates a myriad of processes in the cardiovascular system, from short-term regulation of contraction and relaxation to long-term effects on growth and survival. In the heart, an elevation of cAMP, particularly through βadrenergic receptor (β-AR) stimulation, exerts positive inotropic and lusitropic effects by activating the cAMP-dependent protein kinase (PKA) which regulates the main components of the excitation-contraction coupling (ECC) process. These include sarcolemmal L-type Ca 2+ channels (LTCCs) which underlie the L-type Ca 2+ current (ICa,L), ryanodine receptors (RyR2) responsible for Ca 2+ release from the sarcoplasmic reticulum (SR), the SR Ca 2+ -ATPase (SERCA2) inhibitor phospholamban (PLB), and the myofilament proteins troponin I and myosin-binding protein C [START_REF] Bers | Calcium cycling and signaling in cardiac myocytes[END_REF].

The intracellular cAMP levels result from the equilibrium between its synthesis by adenylyl cyclases (AC) and its degradation by cyclic nucleotide phosphodiesterases (PDEs). Five PDE families can degrade cAMP in the heart: PDE1, which is activated by Ca 2+ -calmodulin; PDE2, which is stimulated by cGMP; PDE3, which is inhibited by cGMP; PDE4 which is activated by PKA; and PDE8. In cardiomyocytes, the PDE3 and PDE4 families prevail to hydrolyze cAMP and regulate ECC. The respective contribution of these two families varies depending on the species, the concentration of cAMP, and the subcellular compartment under investigation [START_REF] Osadchii | Myocardial phosphodiesterases and regulation of cardiac contractility in health and cardiac disease[END_REF]. In rats and mice, PDE4 predominates to hydrolyze cAMP generated by β-AR stimulation in the cytosol and at the plasma membrane and to regulate ICa,L [START_REF] Mongillo | Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases[END_REF][START_REF] Nikolaev | Novel single chain cAMP sensors for receptor-induced signal propagation[END_REF][START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF][START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias in mice[END_REF]. However, when PDE4 is inhibited, PDE3 becomes predominant to degrade cAMP, and to regulate ICa,L [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF]. Further studies identified the association of specific isoforms of these two PDE families with the main actors of cardiac ECC: PDE3A1 and PDE4D with SERCA2/PLB [START_REF] Beca | PDE3A Regulates Basal Myocardial Contractility Through Interacting with SERCA2a-Signaling Complexes in Mouse Heart[END_REF][START_REF] Beca | Phosphodiesterase 4D regulates baseline sarcoplasmic reticulum Ca 2+ release and cardiac contractility, independently of L-type Ca 2+ current[END_REF][START_REF] Ahmad | Regulation of sarcoplasmic reticulum Ca 2+ ATPase 2 (SERCA2) activity by phosphodiesterase 3A (PDE3A) in human myocardium: phosphorylation-dependent interaction of PDE3A1 with SERCA2[END_REF], PDE4D with RyR2 [START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF], and PDE4B with LTCC [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias in mice[END_REF].

Impairment of cAMP-mediated signaling is a hallmark of pathological cardiac hypertrophy (CH) and heart failure (HF). The main changes include a decreased β1-AR density, an uncoupling of β-ARs from Gs proteins, an increased Gi expression and an increased G-protein-coupled receptor kinase (GRK) activity [START_REF] Lohse | What is the role of β-adrenergic signaling in heart failure?[END_REF]. In addition, β2-ARs were reported to be redistributed from Ttubules to peripheral sarcolemma in HF [START_REF] Nikolaev | β2-adrenergic receptor redistribution in heart failure changes cAMP compartmentation[END_REF]. Downstream of cAMP synthesis, contrasted results were obtained regarding PDE modifications in human and in various animal models of CH and HF [START_REF] Osadchii | Myocardial phosphodiesterases and regulation of cardiac contractility in health and cardiac disease[END_REF]. Although a majority of studies found a reduction in PDE3 [START_REF] Smith | Development of decompensated dilated cardiomyopathy is associated with decreased gene expression and activity of the milrinone-sensitive cAMP phosphodiesterase PDE3A[END_REF][START_REF] Sato | Mechanisms of desensitization to a PDE inhibitor (milrinone) in conscious dogs with heart failure[END_REF][START_REF] Ding | Functional role of phosphodiesterase 3 in cardiomyocyte apoptosis: implication in heart failure[END_REF][START_REF] Ding | A positive feedback loop of phosphodiesterase 3 (PDE3) and inducible cAMP early repressor (ICER) leads to cardiomyocyte apoptosis[END_REF] and in PDE4 [START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF][START_REF] Richter | Conserved expression and functions of PDE4 in rodent and human heart[END_REF] some studies found no change in PDE3 [START_REF] Movsesian | Sarcoplasmic reticulum-associated cyclic adenosine 5'-monophosphate phosphodiesterase activity in normal and failing human hearts[END_REF][START_REF] Der Leyen | Mechanism underlying the reduced positive inotropic effects of the phosphodiesterase III inhibitors pimobendan, adibendan and saterinone in failing as compared to nonfailing human cardiac muscle preparations[END_REF][START_REF] Chiu | Milrinone, a cyclic AMP-phosphodiesterase inhibitor, has differential effects on regional myocardial work and oxygen consumption in experimental left ventricular hypertrophy[END_REF] or even an increase in PDE3 and/or PDE4 [START_REF] Takahashi | Enhanced activities and gene expression of phosphodiesterase types 3 and 4 in pressure-induced congestive heart failure[END_REF][START_REF] Mokni | Concerted regulation of cGMP and cAMP phosphodiesterases in early cardiac hypertrophy induced by angiotensin II[END_REF][START_REF] Mika | Synergic PDE3 and PDE4 control intracellular cAMP and cardiac excitation-contraction coupling in a porcine model[END_REF].

In the case of PDE3, some of this heterogeneity could be linked to the existence of a polymorphism in the promoter region of the PDE3A gene that regulates transcription by cAMP, for example in response to treatment with PDE3 inhibitors [START_REF] Sucharov | A PDE3A Promoter Polymorphism Regulates cAMP-Induced Transcriptional Activity in Failing Human Myocardium[END_REF]. Deregulation of cAMP in specific microdomains due to genetic or pharmacological impairment of PDEs can cause arrhythmias and HF [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias in mice[END_REF][START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF][START_REF] Mika | Synergic PDE3 and PDE4 control intracellular cAMP and cardiac excitation-contraction coupling in a porcine model[END_REF][START_REF] Ghigo | PI3Kgamma Protects against Catecholamine-Induced Ventricular Arrhythmia through PKA-mediated Regulation of Distinct Phosphodiesterases[END_REF][START_REF] Bobin | Calmodulin kinase II inhibition limits the pro-arrhythmic Ca 2+ waves induced by cAMP-phosphodiesterase inhibitors[END_REF]. Therefore, an obvious question is whether CH and HF are associated with specific disturbances in local cAMP microdomains differentially regulated by PDEs. Recent studies using transgenic mice expressing cAMP biosensors targeted to the plasma membrane and the SR have suggested that CH is accompanied by a spatial redistribution of PDEs leading to disturbed cAMP compartmentation [START_REF] Sprenger | In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease[END_REF][START_REF] Perera | Microdomain Switch of cGMP-Regulated Phosphodiesterases Leads to ANP-Induced Augmentation of β-Adrenoceptor-Stimulated Contractility in Early Cardiac Hypertrophy[END_REF][START_REF] Bastug-Ozel | Heart failure leads to altered β2-adrenoceptor/cyclic adenosine monophosphate dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain[END_REF].

In a rat model of compensated CH induced by aortic stenosis, we have shown previously that the expression and the activity of PDE3 and PDE4 were decreased, and that this was associated with a less stringent control of β-AR cAMP signals by these PDEs at the plasma membrane [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on βadrenergic cAMP signals[END_REF]. However, whether these modifications affect β-AR regulation of cAMP levels in the cytosol and the local regulation of ICa,L was not investigated. Thus, this study was designed to characterize the dynamic regulation of cytosolic cAMP and macroscopic ICa,L by PDE3 and PDE4 in left ventricular myocytes (LVMs) from sham-operated (SHAM) and hypertrophied (CH) rats, using a FRETbased cAMP sensor and the whole cell configuration of the patch-clamp technique. We show that balance between PDE3 and PDE4 in regulating the β-AR stimulation of cytosolic cAMP and ICa,L is shifted from PDE4 in healthy cardiomyocytes to PDE3 in hypertrophied cells.

Methods

All experiments were carried out according to the European Community guiding principles in the care and use of animals (2010/63/UE, 22 September 2010), the local Ethics committee (CEEA26 CAPSud) guidelines and the French decree n°2013-118, 1st February 2013 on the protection of animals used for scientific purposes (JORF n°0032, 7 February 2013 p2199, text n°24). Animal experiments were approved by the French Ministry of Agriculture (prefectural agreement N°2016-108 and agreement to our animal facility N° C 92-019-01).

Induction of cardiac hypertrophy (CH) in Rats

Fourty male Wistar rats were used in this study. CH was induced at three weeks of age (body weight<60g). The animals were anesthetized (pentobarbital 60 mg/kg) and a stainless steel hemoclip of 0.6 mm ID was placed on the ascending aorta via thoracic incision. Age-matched control rats (SHAM) underwent the same procedure without placement of the clip. Five weeks after surgery, rats were sacrificed and body, heart, lung, liver and kidney were weighted. Tibia length was measured using a vernier caliper (Table 1). Rats that exhibited signs of congestive HF (i.e. ascite, serous cavity effusion, or necrotic lungs, kidney or liver) were excluded from the study.

Cardiomyocyte isolation, culture and infection

Hearts were mounted on a Langendorff apparatus for retrograde perfusion of the aorta and coronaries with collagenase A (Roche, Meylan, France) as described previously [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on βadrenergic cAMP signals[END_REF]. Digestion time was 50 min for hearts from SHAM animals and varied between 90 and 120 min in the aortic stenosis group. At the end of the enzymatic digestion, the left ventricles were excised, chopped finely, and agitated manually to dissociate individual myocytes. Freshly isolated cells were suspended in minimal essential medium (MEM: M 4780; Sigma, St Louis, MO USA) containing 1.2 mM Ca 2+ , 2.5% fetal bovine serum (FBS, Invitrogen, Cergy-Pontoise, France), 1% penicillinstreptomycin and 2% HEPES (pH 7.6) and plated on 35 mm, laminin-coated culture dishes (10 µg/mL laminin, 2 h) at a density of 10 4 cells per dish. After 1h the medium was replaced by 300 µL of FBS-free MEM. A portion of the freshly isolated cells was incubated with an adenovirus encoding the cAMP sensor Epac2-camps [START_REF] Nikolaev | Novel single chain cAMP sensors for receptor-induced signal propagation[END_REF] (MOI=1000 pfu/cell) for measurement of cytosolic cAMP as described below. The remaining non-infected cells were used for ICa,L measurements.

ICa,L and cAMP measurements were performed at room temperature 2h and 48h after cell plating, respectively.

Electrophysiological experiments

Cell membrane capacitance and ICa,L were measured using the patch-clamp technique in the wholecell configuration as described previously [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF]. Voltage-clamp protocols were generated by a challenger/09-VM programmable function generator (Kinetic Software, Atlanta, GA, USA). The cells were voltage-clamped using a patch-clamp amplifier (model RK-400; Bio-Logic, Claix, France). Currents were analogue-filtered at 3 KHz and digitally sampled at 10 KHz using a 12-bit analogue-to-digital converter (DT2827; Data translation, Marlboro, MA, USA) connected to a compatible PC (386/33 Systempro; Compaq Computer Corp., Houston, TX, USA). Cell membrane capacitance was determined as the current elicited by a voltage ramp at 1 V/s between -90 mV and -70 mV. For ICa,L measurement, the freshly isolated cells were depolarized every 8 s to 0 mV during 400 ms. Fast sodium current was inactivated by maintaining the cells at a holding potential of -50 mV and potassium currents were blocked by replacing all K + ions with external and internal

Live cell imaging

Epac2-camps was used to measure cytosolic cAMP signals in SHAM and CH cardiomyocytes, as previously described [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF]. Cells were maintained in a K + -Ringer solution containing (in mM): NaCl 121.6, KCl 5.4, MgCl2 1.8; CaCl2 1.8; NaHCO3 4, NaH2PO4 0.8, D-glucose 5, sodium pyruvate 5, HEPES 10, adjusted to pH 7.4. Images were captured every 5 s using the 40x oil immersion objective of a Nikon TE 300 inverted epifluorescence microscope connected to a softwarecontrolled (Metafluor, Molecular Devices, Sunnyvale, CA, USA) cooled charge coupled (CCD) camera (Sensicam PE; PCO, Kelheim, Germany). Pixel size was 6.45 µm and a binning of 2 was used thus image resolution was (6.45x2)/40 ~ 0.32µm. CFP was excited during 150-300 ms by a Xenon lamp (100 W, Nikon, Champigny-sur-Marne, France) using a 440/20BP filter and a 455LP dichroic mirror. Dual emission imaging of CFP and YFP was performed using an Optosplit II emission splitter (Cairn Research, Faversham, UK) equipped with a 495LP dichroic mirror and BP filters 470/30 and 535/30, respectively.

Data analysis

Cellular hypertrophy was evaluated by measuring cell membrane capacitance (Table 1). The maximal amplitude of ICa,L was measured as the difference between the peak inward current and the current at the end of the 400-ms duration pulse at 0 mV. Currents were not compensated for capacitance and leak currents. Basal ICa,L density (dICa,L) was calculated for each experiment as the ratio of current amplitude to cell capacitance. In imaging experiments, average fluorescence intensity was measured in a region of interest comprising the entire cell or a significant part of the cell. Background was subtracted and YFP intensity was corrected for CFP spillover into the 535 nm channel before calculating the CFP/YFP ratio. Ratio images were obtained with ImageJ software (National Institutes of Health). Kinetic parameters (t1/2on and t1/2off) were determined using Microsoft Excel software. The time of the last baseline point before the signal first increased was defined as t0. tpeak represents the time required to reach the maximum of the signal from t0. t1/2on value is calculated as the time required for half maximal increase from t0 while t1/2off is calculated as the time required for half maximal decay from tpeak. Data are expressed as mean ± S.E.M. Statistical analysis was performed with GraphPad Prism software. Normal distribution was tested by the Shapiro-Wilk normality test. For simple two-group comparison, we used an unpaired Student t-test or a Mann-Whitney test when the data did not follow a normal distribution. Two-way, mixed-effects ANOVA with Sidak's post-hoc test was used to compare the time courses of cAMP and ICa,L in SHAM and CH myocytes. Two-way ANOVA followed by Tukey's or Sidak's post-hoc test was used to compare maximal amplitude and kinetic parameters of cAMP and ICa,L in SHAM and CH myocytes. Statistical difference was considered significant when P<0.05.

Drugs

Isoprenaline (Iso) was from Sigma (Saint Louis, MO USA). Cilostamide was from Tocris Bioscience (Ellisville, MI USA): It blocks PDE3 with an IC50 of about 0.042 µM [START_REF] Stoclet | Cyclic nucleotide phosphodiesterases as therapeutic targets in cardiovascular diseases[END_REF] and was used here at a 1 µM concentration. Ro 20-1724 was kindly provided by Hoffman-La-Roche (Basel, Switzerland): It blocks PDE4 with an IC50 value around 1 µM [START_REF] Rose | Cyclic AMP-mediated regulation of vascular smooth muscle cell cyclic AMP phosphodiesterase activity[END_REF][START_REF] Rich | In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors[END_REF] and was used here at 10 µM. At these concentrations, Cil and Ro were shown to be selective for PDE3 and PDE4 respectively (Supplemental Table 1).

Results

Induction and characterization of cardiac hypertrophy in rats

In order to study the β-AR regulation of cytosolic cAMP and ICa,L during CH, we used a model of pressure overload which we characterized in a previous study [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on βadrenergic cAMP signals[END_REF]. CH was generated by banding of the ascending aorta in male Wistar rats over a time period of 5 weeks as described in Methods.

Anatomical data summarized in Table 1 indicate that heart weight (HW) of CH rats was increased by 46% compared to sham-operated (SHAM) rats, whereas body weight (BW) and tibia length (TL) were similar in both groups. The weight of lung, liver, kidney, and right ventricle were also unchanged (data not shown) indicating the absence of congestive HF. From these data, we can classify this model as compensated CH (Table 1).

Regulation of cytosolic cAMP by transient β-AR stimulation in SHAM and CH cardiomyocytes

To determine whether CH impacts on the dynamics of cytosolic cAMP, LVMs from SHAM and CH rats were transduced with an adenovirus encoding the FRET-based sensor, Epac2-camps [START_REF] Nikolaev | Novel single chain cAMP sensors for receptor-induced signal propagation[END_REF]. This sensor can detect rapid changes in cAMP and is distributed throughout the cytosol [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF]. Figure 1A shows pseudo color images of the CFP/YFP ratio in a SHAM and a CH myocyte before Iso stimulation (baseline), at the maximum of the Iso pulse (100 nM, 15 s) and after washout of the β-AR agonist (recovery). In these examples, the CFP/YFP ratio was homogeneous in the cytosol, although in certain SHAM and CH myocytes a striated pattern was observed. However, no obvious difference in cAMP distribution within the cell was observed between SHAM and CH myocytes.

The average time course of several experiments measuring cytosolic cAMP in response to a 15 s pulse of Iso is presented in Figure 1B. In both SHAM and CH myocytes, Iso induced a rapid and transient increase in the CFP/YFP ratio presenting similar maximal amplitude (Figure 1C), equivalent rising phase kinetics (t1/2on, Figure 1D) and comparable decay kinetics (t1/2off, Figure 1E).

Modulation of β-AR cAMP signals by PDEs in SHAM and CH cardiomyocytes

In healthy cardiomyocytes, cAMP generated by β-ARs is tightly controlled by PDE3 and PDE4 [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF][START_REF] Nikolaev | Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching β1-adrenergic but locally confined β2adrenergic receptor-mediated signaling[END_REF]. We tested whether this was also the case in CH myocytes. In the absence of Iso, selective inhibition of PDE3 with cilostamide (Cil, 1 µM) or PDE4 with Ro 20-1724 (Ro, 10 µM) had no effect on the FRET ratio in either SHAM or CH cells (data not shown). Since PDE4 plays a prominent role in the termination of β-AR cAMP signals in healthy rat cardiomyocytes [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF] we first compared the effect of Ro on the cytosolic cAMP transient elicited by a 15 s pulse of Iso (100 nM) in SHAM and CH myocytes. As shown by the average time courses of Figure 2, Ro strongly increased the maximal amplitude and prolonged the duration of the cAMP response in both SHAM (Figure 2A) and CH myocytes (Figure 2B). The effects of Ro on the maximal amplitude (Figure 2C) and on the duration of the rising phase (Figure 2D) were not different between SHAM and CH. In contrast, as shown in Figure 2E, the effect of Ro on the decay kinetics of the cAMP transient was significantly more pronounced in SHAM than in CH (in the presence of Ro, t1/2off was 273.3 ± 25.4 s in SHAM versus 176.3 ± 15.9 s in CH, P<0.001). These results indicate that PDE4 plays a more prominent role to degrade cytosolic cAMP generated by β-AR in SHAM than in CH myocytes.

In a next series of experiments, we compared the effect of PDE3 inhibition on the cytosolic cAMP transient in SHAM and CH myocytes (Figure 3). As shown in Figure 3A and Figure 3B, Cil (1 µM) had a minimal effect in SHAM myocytes, whereas it potentiated the cAMP response to a pulse stimulation with Iso (100 nM) in CH myocytes. Cil increased the amplitude of the response by ~35% (from +8.2 ± 0.9%, n=16 to +11.0 ± 0.5%, n=19, P<0.05) in CH whereas it had no effect in SHAM (Figure 3C). In both groups, PDE3 inhibition did not modify the onset kinetics of the cAMP response (Figure 3D). Cil also induced a significant prolongation of the cAMP response to Iso in CH LVMs (t1/2off was 52.6 ± 3.5 s with Iso alone and 79.1 ± 6.2 s in the presence of Cil) in contrast to SHAM LVMs (Figure 3E) These results indicate that while PDE3 plays a minor role in the regulation of β-AR cAMP signals in SHAM myocytes, its contribution becomes more important in CH myocytes. Taken together, these results suggest that in SHAM myocytes, PDE4 is major to control the amplitude and the duration of cytosolic cAMP signals elicited by a short β-AR stimulation whereas in CH myocytes, both PDE3 and PDE4 contribute to modulate the amplitude and the decay phase of the cAMP transient generated by a brief β-AR stimulation.

Regulation of ICa,L by transient β-AR stimulation in SHAM and CH cardiomyocytes

In order to determine whether the regulation of ICa,L by a brief β-AR stimulation is altered in CH, the whole-cell configuration of the patch clamp technique was used. Consistent with cardiomyocyte hypertrophy, cell membrane capacitance, which is proportional to the surface of the plasma membrane, was significantly increased by ~31% in CH versus SHAM LVMs. ICa,L amplitude was also significantly larger (+38%) in CH compared to SHAM LVMs, so that basal ICa,L density, which is the ratio of ICa,L current to membrane capacitance, was unchanged between the two groups (Table 1). As illustrated by the individual ICa,L current traces of Figure 4A and by the average time course of ICa,L amplitude in Figure 4B, a brief application of Iso (100 nM, 15 s) elicited an apparently similar transient increase in ICa,L in both groups. Indeed, both the maximal increase in ICa,L and recovery rate were unchanged (Figure 4C and4E). However, the kinetics of ICa,L augmentation were significantly slower in CH compared to SHAM LVMs (t1/2on=30.1±1.8 s, n=17 in CH versus 21.9±0.9 s, n=27 in SHAM, P<0.001, Figure 4D).

Regulation of ICa,L by PDEs in SHAM and CH cardiomyocytes

In order to determine whether PDE regulation of ICa,L is modified in CH, we first applied Cil (1 µM) or Ro (10 µM) in the absence of β-AR stimulation in SHAM and CH cells. However, neither inhibitor alone had an effect on basal ICa,L (Supplemental Figure 1). We next tested the effect of PDE4 inhibition with Ro on top of a transient β-AR stimulation with Iso (100 nM, 15 s). As shown in Figure 5A and 5B, in both SHAM and CH LVMs, Ro potentiated the Iso-stimulated ICa,L In SHAM, PDE4 inhibition increased the maximal ICa,L amplitude by ~50% (Figure 5C), had no effect on the rising kinetics (Figure 5D) and nearly doubled ICa,L recovery (Figure 5E, t1/2off was 128.4 ± 11.7 s, n=16 with Iso alone and 219.1 ± 24.8 s, n=9 in the presence of Ro, P<0.01). However in CH myocytes, Ro did not significantly increase the maximum of ICa,L response to Iso (Figure 5C) and appeared less effective in prolonging its decay phase (Figure 5E, t1/2off was 166.0 ± 23.2 s, n=10 with Iso alone and 249.3 ± 24.8 s, n=9 in the presence of Ro, P<0.05). These data suggest an altered regulation of ICa,L by PDE4 in CH myocytes.

In a second series of experiments, the effect of Cil (1 µM) was tested on the β-AR regulation of ICa,L (Figure 6). In SHAM LVMs, Cil showed a strong tendency to potentiate the maximal increase of ICa,L in response to Iso pulse stimulation (Figure 6A and 6C) whereas there was no potentiating effect of Cil on ICa,L maximal amplitude in CH LVMs (Figure 6B and6C). PDE3 inhibition did not modify the rising kinetics of ICa,L in either group (Figure 6D), whereas it slowed down ICa,L recovery in CH but not in SHAM LVMs (Figure 6E, t1/2off was 166.7±18.5 s, n=15 with Iso alone and 238.1±27.2 s, n=17 in the presence of Cil, P<0.05). These results highlight two main differences in the regulation of Ica,L by PDE3/4 between SHAM and CH cells: First, whereas both PDE3 and PDE4 appear to limit the maximal ICa,L amplitude upon a transient -AR stimulation in SHAM myocytes, this effect is lost in CH myocytes. Second, whereas PDE4 is predominant to control the termination of Iso-stimulated ICa,L and is able to mask the contribution of PDE3 in SHAM myocytes as shown in a previous study [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF], it was no longer the case in CH myocytes.

Selective alterations in cytosolic cAMP and ICa,L compartments in cardiac hypertrophy

In Figure 7, the kinetics of cytosolic cAMP and ICa,L transients induced by Iso alone or in the presence of PDE inhibitors are summarized and compared between SHAM and CH myocytes.

Additionally, kinetics of CFP/YFP ratio are compared to those of ICa,L within the same group. This latter comparison highlights the faster onset and decay kinetics of cAMP compared to ICa,L, in SHAM and CH myocytes, as observed previously in normal rat cardiomyocytes [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF], which indicates that molecular events occurring downstream of cAMP, i.e. phosphorylation and dephosphorylation of LTCC or a closely associated protein, are rate-limiting for both the rise and the decay of ICa,L. When both PDEs are functional, the only difference between SHAM and CH cells is a slower rising phase of ICa,L in CH cells (Figures 7A and7B). This difference is no longer observed when either PDE3 or PDE4 are blocked (Figures 7C and7E). Blockade of PDE4 slows the decay phase of cAMP and ICa,L transients compared to Iso in both groups, underlining the major role played by PDE4 in controlling the duration of cAMP-triggered events. In the presence of Ro, the FRET signal returns more rapidly to baseline in CH than in SHAM myocytes, in contrast to the decrease rates of ICa,L which remain similar between SHAM and CH cells (Figure 7D). These results suggest that in CH compared to SHAM, there is more PDE activity remaining in the cytosol (probably PDE3), whereas it is unchanged nearby L-type Ca 2+ channels. In contrast, when PDE3 is blocked the duration of the decay phase of the cAMP transient is similar in both groups, whereas ICa,L decay becomes significantly slower in CH cells compared to SHAM (Figure 7F), which is consistent with altered regulation of ICa,L by PDE4 in CH cells.

Discussion

The major goal of this study was to determine how CH induced by pressure overload modifies the dynamic regulation of cytosolic cAMP and ICa,L by PDE3 and PDE4 upon a brief β-AR stimulation, which mimics the sympathetic nerve discharge elicited during a startle response. While the amplitude and the duration of the cytosolic cAMP and ICa,L responses were preserved in CH, our results reveal a specific rearrangement of PDE3 and PDE4 in the bulk cytosol and in the vicinity of LTCCs that maintains the homeostasis of the β-AR response in diseased myocytes (Figure 8). Indeed, whereas PDE4 was the main family modulating cytosolic β-AR cAMP transients in SHAM, both PDE3 and PDE4 controlled the amplitude and the recovery of these cAMP signals in CH. The subcellular reorganization of PDEs in CH LVMs also modified the β-AR regulation of Ica,L by inducing i) a slower rate of ICa,L increase upon Iso stimulation; ii) an abrogated modulation by PDE3 and PDE4 of Iso-augmented maximal ICa,L amplitude; and iii) a rearrangement in the PDEs governing the recovery of ICa,L from β-AR stimulation. This molecular remodeling likely prevents abnormal prolongation of Ca 2+ influx after β-AR stimulation and consequent deleterious Ca 2+ overload in CH.

The conclusions drawn in the present paper rely on the comparison of cardiomyocytes isolated from sham-operated rats or rats with aortic banding. As indicated in the Methods section, the digestion time had to be increased to isolate CH myocytes, most likely due to the increased cardiac mass and fibrosis in banded animals [START_REF] Creemers | Molecular mechanisms that control interstitial fibrosis in the pressure-overloaded heart[END_REF]. Although this was not associated with readily observable damages of CH myocytes, this constitutes a limitation since we cannot exclude that it contributed to some of the differences observed between normal and CH myocytes. In addition, all experiments of this study were performed at room temperature. Although experimental conditions of cAMP and ICa,L measurements were rigorously similar between normal and CH myocytes, we cannot exclude a potential impact of temperature on some of the results reported here. In this study, cAMP was measured with an untargeted FRET sensor in the bulk cytosol. As indicated in the result section, the CFP/YFP ratio was homogeneous in most cells, but a striated pattern was observed in some cells. Based on our previous observations of similar striations upon injection of the FRETbased cAMP sensor FlCRhR, or upon injection of a 70 kDa fluorescein-dextran molecule into frog cardiomyocytes [START_REF] Goaillard | Simultaneous measurements of intracellular cAMP and L-type Ca 2+ current in single frog ventricular myocytes[END_REF], we think that these striations reflect accumulation of the probe in less crowded regions of the myocyte. In the case of the genetically-encoded Epac2-camps used in the present study, this phenomenon is probably influenced by the expression level. For a given multiplicity of infection, there is a cell-to-cell variability in the amount of sensor which is expressed in different myocytes, which probably explains the occurrence of striations in some cells and not in others. Importantly however, these striations could be observed both in sham and hypertrophied myocytes so that the two cell populations did not differ in this respect. While the modulation of β-ARs cAMP signals by PDE3 and PDE4 in the bulk cytosol have been largely documented in normal cardiomyocytes, much less is known in heart disease [START_REF] Sprenger | In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease[END_REF]. However, due to the compartmentalized nature of cAMP, it should be kept in mind that the changes reported in this study concern global cAMP levels and may not apply to specific microdomains in cardiomyocytes.

In CH myocytes, the cytosolic cAMP transient generated by a brief β-AR stimulation was not different from that observed in SHAM myocytes. Given that β-AR desensitization is a hallmark of HF [START_REF] Lohse | What is the role of β-adrenergic signaling in heart failure?[END_REF] and is also observed in CH induced by aortic banding [START_REF] Chevalier | β-adrenergic system is modified in compensatory pressure cardiac overload in rats: physiological and biochemical evidence[END_REF], one could have expected the cytosolic cAMP transient to be reduced. However, along with β-AR desensitization, the total cAMP-hydrolytic activity is decreased in this model of CH [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on βadrenergic cAMP signals[END_REF]. Therefore, these results suggest that during CH, a reduction in cytosolic cAMP-PDE activity compensates for β-AR desensitization, resulting in unchanged cAMP transient in this compartment. At variance with this result, a decreased cAMP response to sustained β-AR stimulation was observed in CH myocytes when cAMP was measured with CNG channels at the sarcolemma [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on βadrenergic cAMP signals[END_REF]. This suggests that compartment-specific cAMP alterations occur during CH, where local rather than global PDE activity determines the response [START_REF] Ghigo | cAMP/PKA signaling compartmentalization in cardiomyocytes: Lessons from FRET-based biosensors[END_REF]. Alternatively, a difference linked to the stimulation protocol (i.e. pulse stimulation used in the present study versus cumulative Iso application in the previous one) cannot be excluded. Indeed, during cumulative concentration response experiments, prolonged agonist application should favour β-AR desensitization compared to a 15 s pulse stimulation.

The next question was whether the control of β-AR cAMP signals by PDE3 and PDE4 differ in SHAM versus CH myocytes. Our results confirm previous studies showing that PDE4 is the main PDE degrading cytosolic cAMP in healthy adult cardiomyocytes from rats and mice [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF][START_REF] Nikolaev | Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching β1-adrenergic but locally confined β2adrenergic receptor-mediated signaling[END_REF].

Besides, PDE4 is able to compensate for PDE3 inhibition in the cytosol of normal cells, a property which is likely facilitated by PKA-dependent activation of PDE4 [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF]. In contrast, in CH cells both PDE3 and PDE4 limit the amplitude and duration of the cAMP transient. The decreased contribution of PDE4 in the bulk cytosol of CH cells is consistent with the decreased PDE4 activity observed in this model [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on βadrenergic cAMP signals[END_REF] and with a previous study in cardiomyocytes from mice with transaortic constriction (TAC) [START_REF] Sprenger | In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease[END_REF]. Interestingly, when PDE4 was inhibited, cAMP decay was faster in CH compared to SHAM, implying that the remaining cytosolic cAMP-PDE activity is higher in CH than in SHAM (Figure 7D). Importantly, this difference was not observed when PDE3 was inhibited (Figure 7F). These observations, together with the fact that PDE3 is largely predominant to terminate β-AR cAMP responses when PDE4 is inhibited [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF], strongly suggest that the relative contribution of PDE3 to cAMP degradation in the cytosol is increased in CH myocytes. This interpretation may seem at odds with the decreased total PDE3 activity in this model [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on βadrenergic cAMP signals[END_REF].

However, because PDE4 decreases even more than PDE3, the PDE3/PDE4 activity ratio is actually increased in CH compared to SHAM [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on βadrenergic cAMP signals[END_REF]. In addition, it is likely that the global PDE3 activity measured in vitro, at a fixed substrate concentration, differs from the PDE3 activity in intact cardiomyocytes challenged with Iso and Ro 20-1724. In particular, PDE3 regulation by cGMP is not taken into account in such enzymatic assay. cGMP effectively inhibits the cAMP-hydrolytic activity of PDE3 with Ki value of about 0.06-0.6 μM [START_REF] Osadchii | Myocardial phosphodiesterases and regulation of cardiac contractility in health and cardiac disease[END_REF] and its concentration is decreased in pathological cardiac hypertrophy [START_REF] Tsai | Cyclic GMP signaling in cardiovascular pathophysiology and therapeutics[END_REF]. Thus, decreased cGMP levels in CH cells could be involved in PDE3 activation, as occurs in aortic rings with endothelial dysfunction due to heart failure in rats [START_REF] Hubert | Alteration of vascular reactivity in heart failure: role of phosphodiesterases 3 and 4[END_REF]. In addition, recent studies have documented a decreased localization of PDE3 in specific microdomains of the plasma membrane in CH [START_REF] Perera | Microdomain Switch of cGMP-Regulated Phosphodiesterases Leads to ANP-Induced Augmentation of β-Adrenoceptor-Stimulated Contractility in Early Cardiac Hypertrophy[END_REF] and HF [START_REF] Bastug-Ozel | Heart failure leads to altered β2-adrenoceptor/cyclic adenosine monophosphate dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain[END_REF] which could result in the relocalization of PDE3 in the cytosol. Alternatively, we cannot exclude that other PDEs participate in this difference. Possible candidates include PDE1 and PDE2 which are increased in animal models and human HF, albeit not in CH [START_REF] Knight | PDE1C deficiency antagonizes pathological cardiac remodeling and dysfunction[END_REF][START_REF] Hashimoto | Acute Enhancement of Cardiac Function by Phosphodiesterase Type 1 Inhibition[END_REF][START_REF] Mehel | Phosphodiesterase-2 Is Up-Regulated in Human Failing Hearts and Blunts β-Adrenergic Responses in Cardiomyocytes[END_REF] and PDE8, which was reported in mouse cardiomyocytes [START_REF] Patrucco | Phosphodiesterase 8A (PDE8A) regulates excitation-contraction coupling in ventricular myocytes[END_REF]. Although we did not test directly the potential contribution of these PDEs to cAMP-degradation in CH cardiomyocytes, it should be emphasized that they represent minor contributors to cAMP hydrolysis compared to PDE3 and PDE4, not only in SHAM but also in CH ventricular myocytes [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on βadrenergic cAMP signals[END_REF].

ICa,L is a critical determinant of Ca 2+ -induced Ca 2+ release which constitutes a canonical example of local regulation by β-ARs and PDEs [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF][START_REF] Jurevicius | cAMP compartmentation is responsible for a local activation of cardiac Ca 2+ channels by beta-adrenergic agonists[END_REF][START_REF] Timofeyev | Adenylyl cyclase subtype-specific compartmentalization: differential regulation of L-type Ca 2+ current in ventricular myocytes[END_REF]. Thus, using the same protocol of brief β-AR stimulation as for cytosolic cAMP, we compared the dynamic stimulation of ICa,L in SHAM and CH, and the influence of PDE3 and PDE4 in this process. In the absence of β-AR stimulation, basal ICa,L density was similar between SHAM and CH myocytes, which is consistent with a number of previous studies [START_REF] Scamps | Calcium current in single cells isolated from normal and hypertrophied rat heart. Effects of β-adrenergic stimulation[END_REF][START_REF] Mukherjee | L-type calcium channel abundance and function with cardiac hypertrophy and failure: a review[END_REF][START_REF] Xu | Intermolecular failure of L-type Ca 2+ channel and ryanodine receptor signaling in hypertrophy[END_REF]. While most analyses point to a maintenance of whole-cell ICa,L density in CH and HF, an increased open probability has been evidenced at the single channel level [START_REF] Schroder | Increased availability and open probability of single L-type calcium channels from failing compared with nonfailing human ventricle[END_REF][START_REF] Sanchez-Alonso | Microdomain-Specific Modulation of L-Type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure[END_REF]. A possible explanation is that diseased myocytes have fewer but more active channels [START_REF] Zhang | Calcium currents in postinfarction rat cardiac myocytes[END_REF][START_REF] He | Reduction in density of transverse tubules and L-type Ca 2+ channels in canine tachycardia-induced heart failure[END_REF][START_REF] Chen | L-type Ca 2+ channel density and regulation are altered in failing human ventricular myocytes and recover after support with mechanical assist devices[END_REF].

Upon pulse β-AR stimulation, ICa,L augmented more slowly in CH than in SHAM, although the onset kinetics of cAMP in the cytosol were not modified. These results are not necessarily contradictory if one considers that submembrane and cytosolic cAMP signals display different kinetics and represent distinct microdomains separated by restricted diffusion [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF]. In addition, mechanisms downstream of cAMP may explain this difference. Indeed, pathological CH and HF are associated with altered PKA expression [START_REF] Zakhary | Protein kinase A (PKA)-dependent troponin-I phosphorylation and PKA regulatory subunits are decreased in human dilated cardiomyopathy[END_REF][START_REF] Han | Human heart failure is accompanied by altered protein kinase A subunit expression and post-translational state[END_REF], reorganized association with AKAPs [START_REF] Zakhary | Regulation of PKA binding to AKAPs in the heart: alterations in human heart failure[END_REF][START_REF] Aye | Reorganized PKA-AKAP associations in the failing human heart[END_REF], and increased phosphatase activity [START_REF] Neumann | Increased expression of cardiac phosphatases in patients with end-stage heart failure[END_REF][START_REF] Yamada | Inhibition of protein phosphatase 1 by inhibitor-2 gene delivery ameliorates heart failure progression in genetic cardiomyopathy[END_REF], all of which could contribute to a slower phosphorylation of ICa,L in CH LVMs. Although upon β-AR stimulation ICa,L increased slower in CH than in SHAM, the peak stimulation was similar. This contrasts with previous studies showing that the maximal response to Iso is depressed in CH and HF [START_REF] Scamps | Calcium current in single cells isolated from normal and hypertrophied rat heart. Effects of β-adrenergic stimulation[END_REF][START_REF] Chen | L-type Ca 2+ channel density and regulation are altered in failing human ventricular myocytes and recover after support with mechanical assist devices[END_REF]. As indicated previously for cAMP, this might be explained by the different protocols used in the different studies, i.e. sustained versus pulse stimulation. In SHAM, application of 100 nM Iso during 15 s was not maximal since ICa,L could be further potentiated by inhibition of PDE3 or PDE4. Interestingly, this was not the case in CH. ICa,L amplitude is a crucial determinant of systolic Ca 2+ concentration and therefore contraction. Hence, these results are consistent with an attenuated contractile response to PDE3 and PDE4 inhibitors in rats with cardiac hypertrophy [START_REF] Osadchii | Contractile responses to selective phosphodiesterase inhibitors following chronic β-adrenoreceptor activation[END_REF] and with the decreased inotropic effect of PDE3 inhibitors in failing human hearts [START_REF] Der Leyen | Mechanism underlying the reduced positive inotropic effects of the phosphodiesterase III inhibitors pimobendan, adibendan and saterinone in failing as compared to nonfailing human cardiac muscle preparations[END_REF] or in dogs with HF [START_REF] Sato | Mechanisms of desensitization to a PDE inhibitor (milrinone) in conscious dogs with heart failure[END_REF]. Mechanistically, the blunted effect of PDE inhibitors could be due to impaired regulation of subsarcolemmal cAMP by PDE3 and PDE4, as shown previously [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on βadrenergic cAMP signals[END_REF]. In the case of PDE3, this is corroborated by recent independent studies showing decreased contribution of this PDE to cAMP hydrolysis in specific microdomains of the plasma membrane. [START_REF] Perera | Microdomain Switch of cGMP-Regulated Phosphodiesterases Leads to ANP-Induced Augmentation of β-Adrenoceptor-Stimulated Contractility in Early Cardiac Hypertrophy[END_REF][START_REF] Bastug-Ozel | Heart failure leads to altered β2-adrenoceptor/cyclic adenosine monophosphate dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain[END_REF] The blunted effect of PDE3/4 inhibitors on maximal ICa,L amplitude could also be related to a larger fraction of LTCC being activated already in basal conditions in CH myocytes, as indicated by a number of previous reports in failing myocytes. [START_REF] Schroder | Increased availability and open probability of single L-type calcium channels from failing compared with nonfailing human ventricle[END_REF][START_REF] Sanchez-Alonso | Microdomain-Specific Modulation of L-Type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure[END_REF][START_REF] Zhang | Calcium currents in postinfarction rat cardiac myocytes[END_REF][START_REF] He | Reduction in density of transverse tubules and L-type Ca 2+ channels in canine tachycardia-induced heart failure[END_REF][START_REF] Chen | L-type Ca 2+ channel density and regulation are altered in failing human ventricular myocytes and recover after support with mechanical assist devices[END_REF][START_REF] Bryant | Altered distribution of ICa impairs Ca release at the t-tubules of ventricular myocytes from failing hearts[END_REF] Hence, similar to what was reported for PDE4 and RyR2 in HF [START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF], a decrease in PDE activity nearby Ca 2+ channels might contribute to increased basal activity of LTCC and therefore to a lack of the potentiating effect of PDE inhibitors on maximal ICa,L in CH myocytes.

We have shown previously in rat myocytes that ICa,L decay following a transient Iso stimulation is not modified by PDE3 inhibition, as shown here in SHAM (Figure 4E). However, concomitant PDE3 and PDE4 inhibition considerably slowed ICa,L recovery in a similar way as IBMX, indicating a critical role of these two PDE families for ICa,L recovery in normal myocytes [START_REF] Leroy | Spatiotemporal dynamics of β-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF]. Although concomitant inhibition of PDE3 and PDE4 in SHAM and CH would have been useful to ensure that the situation is the same especially in hypertrophied myocytes, analysis of the recovery kinetics of ICa,L following β-AR stimulation suggests that both PDE3 and PDE4 are required to terminate the response in CH and further support the notion that PDE4 regulation of ICa,L is impaired in CH. Indeed, when PDE3 was blocked, ICa,L decay was significantly slower in CH, whereas it was unchanged when PDE4 was inhibited (Figure 7F and7D). These results strongly argue for altered regulation of ICa,L by PDE4 during CH. Among the various PDE4 isoforms expressed in cardiomyocytes, we showed that PDE4B is predominant for β-AR regulation of ICa,L [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias in mice[END_REF] and is decreased in CH [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on βadrenergic cAMP signals[END_REF]. In normal cardiomyocytes, PDE4B co-localizes with LTCCs in the T-tubules [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias in mice[END_REF] and during HF, LTCCs are redistributed from T-tubules to the peripheral sarcolemma. [START_REF] Sanchez-Alonso | Microdomain-Specific Modulation of L-Type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure[END_REF][START_REF] Bryant | Altered distribution of ICa impairs Ca release at the t-tubules of ventricular myocytes from failing hearts[END_REF] Thus, decreased PDE4B expression and/or a rearrangement away from LTCCs could contribute to abnormal regulation of ICa,L by PDE4 in CH.

The complete loss of PDE4B in mice potentiates β-AR stimulation of ICa,L and the occurrence of spontaneous Ca 2+ release events, as well as the susceptibility to ventricular tachycardia [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias in mice[END_REF]. In CH, our results indicate that the contribution of PDE3 to ICa,L recovery after β-AR stimulation compensates for impaired PDE4 regulation, and may therefore constitute a safety mechanism that preserves hypertrophic cardiomyocytes from Ca 2+ overload and arrhythmias. Although PDE3 is classically considered as the major PDE regulating cardiac contractility in large mammals [START_REF] Eschenhagen | PDE4 in the human heart -major player or little helper?[END_REF],

there is increasing evidence that PDE4 contributes to β-AR regulation of cAMP and ECC in dog [START_REF] Molina | Interventricular differences in β-adrenergic responses in the canine heart: role of phosphodiesterases[END_REF], pig [START_REF] Mika | Synergic PDE3 and PDE4 control intracellular cAMP and cardiac excitation-contraction coupling in a porcine model[END_REF] and human [START_REF] Molina | Cyclic adenosine monophosphate phosphodiesterase type 4 protects against atrial arrhythmias[END_REF]. Thus, hypothesizing that PDE4 regulation of ICa,L is impaired in human HF, this could contribute to the increased risk of arrhythmias observed in this condition, especially in the context of chronic treatment with PDE3 inhibitors, which were shown to increase cardiac arrhythmias and mortality in severe HF [START_REF] Holmes | Milrinone in congestive heart failure: observations on ambulatory ventricular arrhythmias[END_REF][START_REF] Packer | Effect of oral milrinone on mortality in severe chronic heart failure. The PROMISE Study Research Group[END_REF][START_REF] Teerlink | Ambulatory ventricular arrhythmias in patients with heart failure do not specifically predict an increased risk of sudden death. PROMISE (Prospective Randomized Milrinone Survival Evaluation) Investigators[END_REF]. indicates the first of the statistically significant difference between Iso and Iso + Ro on the graph.

ns: not significant (P>0.05). Two-way, mixed-effects ANOVA followed by Sidak's post-hoc test.

In C-E, *P<005, **P<0.01, ***P<0.001 indicates statistically significant difference between Iso and Iso + Ro; $$$ P<0.001 indicates statistically significant difference between SHAM and CH LVMs. Two-way ANOVA with Tukey's post-hoc test.

  Cs + . Extracellular solution contained (in mM): NaCl 107.1, CsCl 20, NaHCO3 4, NaH2PO4 0.8, D-Glucose 5, sodium pyruvate 5, HEPES 10, CaCl2 1.8, MgCl2 1.8 and was adjusted to pH 7.4 with NaOH. Control and drug-containing solutions were applied to the exterior of the cell by placing the cell at the opening of 250 µm inner diameter capillary tubing. Patch electrodes (0.5-1 M) were filled with control internal solution containing (in mM): CsCl 118, EGTA 5, MgCl2 4, sodium phosphocreatine 5, Na2ATP 3.1, Na2GTP 0.42, CaCl2 0.062 (pCa 8.5), HEPES 10, adjusted to pH 7.3 with CsOH. All the experiments were done at room temperature, and the temperature did not vary by more than 1°C in a given experiment.

Figure 1 .

 1 Figure 1. Cytosolic cAMP response to transient β-AR stimulation in SHAM and CH

Figure 2 .

 2 Figure 2. Effect of PDE4 inhibition on cytosolic cAMP upon a β-AR stimulation in LVMs

Figure 3 .

 3 Figure 3. Effect of PDE3 inhibition on cytosolic cAMP upon a β-AR stimulation in LVMs

Figure 4 .

 4 Figure 4. Regulation of ICa,L by β-AR stimulation in SHAM and CH cardiomyocytes.

Figure 5 .

 5 Figure 5. Effect of PDE4 inhibition on ICa,L upon transient β-AR stimulation in SHAM and

Figure 6 .

 6 Figure 6. Effect of PDE3 inhibition on ICa,L upon transient β-AR stimulation in SHAM and

Figure 7 .

 7 Figure 7. Comparative kinetics of cytosolic cAMP and ICa,L responses to β-AR stimulation

Figure 8 .

 8 Figure 8. Proposed contribution of PDE3 and PDE4 to the termination of β-AR responses in

Table 1 : Cardiac phenotype of sham-operated (SHAM) and aortic stenosis (CH) rats

 1

	Anatomy	SHAM	N	CH	N	P-value
	BW (g)	325 ± 10	23	324 ± 17	17	n.s
	TL (cm)	3.51 ± 0.06	23	3.52 ± 0.05	17	n.s
	HW (g)	1.47 ± 0.03	23	2.15 ± 0.08	17	P < 0.001
	HW/BW (mg/g)	4.60 ± 0.13	23	6.93 ± 0.47	17	P < 0.001
	HW/TL (mg/cm)	422 ± 9	23	614 ± 28	17	P < 0.001
	Cell parameters	SHAM	n	CH	n	P-value
	Capacitance (pF)	209 ± 8	53	274 ± 18	35	P < 0.01
	ICa,L amplitude (pA)	1395 ± 77	53	1926 ± 77	35	P < 0.05
	ICa,L density (pA/pF)	6,9 ± 0.4	53	7.3 ± 0.7	35	n.s.

All data are expressed as mean ± S.E.M. Abbreviations used: BW, body weight; TL, tibia length; HW, heart weight; N, number of animals in each group; n number of cells in each group. Statistically significant differences between aortic stenosis (CH) and sham-operated (SHAM) rats are indicated with P value; n.s.: not significantly different (Student t-test).

Acknowledgments

We thank Florence Lefebvre and Patrick Lechène for skillful technical assistance. We are also grateful to Dr. Viacheslav Nikolaev (University of Hamburg), Dr. Stefan Engelhardt (University of Munich) and Dr. Martin Lohse (University of Würzburg) for the adenovirus encoding Epac2camps.

Funding

This work was supported by a joint fellowship from the French and Lebanese governments, Partenariat Hubert Curien, CEDRE N° 42338SA to A.A.G and G.V. UMR-S1180 is a member of the Laboratory of Excellence LERMIT supported by a grant from the French National Research Agency (ANR-10-LABX-33) under the program "Investissements d'Avenir" ANR-11-IDEX-0003-01. This work was also funded by grant ANR R19163LL to GV, ANR-16-ECVD-0007-01 (PDE4HEART) to RF and the Fondation de France (to G.V.).

Disclosure

None.