We thank the members of the Vermot lab for discussions and comments on the protocol. We are grateful to all the staff members of the Imperial College London fish facility. This project has received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program: GA N&#176;682939, Additional Ventures (award number 1019496), the MRC (MR/X019837/1) and the BBSRC (BB/Y00566X/1). CV-P was supported by a Bioengineering Departmental Scholarship (Imperial College London). HF was supported by the JSPS KAKENHI (23H04726 and 24K02207), the JST FOREST program (23719210), the Uehara Memorial Foundation, the Cell Science Research Foundation, the Takeda Medical Research Foundation, and the Novartis Research Foundation.

The procedures for working with zebrafish embryos described in this protocol adhere to the European directive 2010/63/EU and Home Office guidelines under the project licence PP6020928.
1. Obtaining zebrafish embryos for bead grafting
<ol>
	<li>Cross the relevant zebrafish line and grow the embryos in Danieau&#39;s medium at 28.5 &#176;C. For more detailed instructions on crossing zebrafish lines, refer to the JoVE Science Education Database27.</li>
	<...

In Vivo Mechanotransduction Study Using Magnetic Beads in the Zebrafish Heart

<ol>
	<li>Streisinger, G., Walker, C., Dower, N., Knauber, D., Singer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291 (5813), 293-296 (1981).</li><li>Tu, S., Chi, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+in+cardiac+development+and+congenital+heart+birth+defects.">Zebrafish models in cardiac development and congenital heart birth defects.</a> Differentiation. 84 (1), 4-16 (2012).</li><li>Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+genetics+and+vertebrate+heart+formation.">Zebrafish genetics and vertebrate heart formation.</a> Nat Rev Genet. 2 (1), 39-48 (2001).</li><li>Brown, D. R., Samsa, L. A., Qian, L., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+study+of+heart+development+and+disease+using+zebrafish.">Advances in the study of heart development and disease using zebrafish.</a> J Cardiovasc Dev Dis. 3 (2), 13 (2016).</li><li>Hoo, J. Y., Kumari, Y., Shaikh, M. F., Hue, S. M., Goh, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+A+versatile+animal+model+for+fertility+research.">Zebrafish: A versatile animal model for fertility research.</a> Biomed Res Int. 2016 (2016), 9732780 (2016).</li><li>Bakkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+to+study+cardiac+development+and+human+cardiac+disease.">Zebrafish as a model to study cardiac development and human cardiac disease.</a> Cardiovasc Res. 91 (2), 279-288 (2011).</li><li>Briggs, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish:+a+new+model+organism+for+integrative+physiology.">The zebrafish: a new model organism for integrative physiology.</a> Am J Physiol Regul Integr Comp Physiol. 282 (1), R3-R9 (2002).</li><li>Veldman, M. B., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+developmental+model+organism+for+pediatric+research.">Zebrafish as a developmental model organism for pediatric research.</a> Pediatr Res. 64 (5), 470-476 (2008).</li><li>Duchemin, A. L., Vignes, H., Vermot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanically+activated+piezo+channels+modulate+outflow+tract+valve+development+through+the+Yap1+and+Klf2-Notch+signaling+axis.">Mechanically activated piezo channels modulate outflow tract valve development through the Yap1 and Klf2-Notch signaling axis.</a> Elife. 8, e44706 (2019).</li><li>Vignes, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+mechanical+forces+drive+endocardial+cell+volume+decrease+during+zebrafish+cardiac+valve+morphogenesis.">Extracellular mechanical forces drive endocardial cell volume decrease during zebrafish cardiac valve morphogenesis.</a> Dev Cell. 57 (5), 598-609.e595 (2022).</li><li>Steed, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=klf2a+couples+mechanotransduction+and+zebrafish+valve+morphogenesis+through+fibronectin+synthesis.">klf2a couples mechanotransduction and zebrafish valve morphogenesis through fibronectin synthesis.</a> Nat Commun. 7, 11646 (2016).</li><li>Chow, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+forces+regulate+zebrafish+heart+valve+delamination+by+modulating+Nfat+signaling.">Cardiac forces regulate zebrafish heart valve delamination by modulating Nfat signaling.</a> PLoS Biol. 20 (1), e3001505 (2022).</li><li>Fukui, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioelectric+signaling+and+the+control+of+cardiac+cell+identity+in+response+to+mechanical+forces.">Bioelectric signaling and the control of cardiac cell identity in response to mechanical forces.</a> Science. 374 (6565), 351-354 (2021).</li><li>Vignes, H., Vagena-Pantoula, C., Vermot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+control+of+tissue+shape:+Cell-extrinsic+and+-intrinsic+mechanisms+join+forces+to+regulate+morphogenesis.">Mechanical control of tissue shape: Cell-extrinsic and -intrinsic mechanisms join forces to regulate morphogenesis.</a> Semin Cell Dev Biol. 130, 45-55 (2022).</li><li>Juan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pkd+and+piezo+gene+family+members+are+required+for+atrioventricular+valve+formation.">Multiple pkd and piezo gene family members are required for atrioventricular valve formation.</a> Nat Commun. 14 (1), 214 (2023).</li><li>Juan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cardiac+contractions+using+Cre-lox+and+degron+strategies+in+zebrafish.">Control of cardiac contractions using Cre-lox and degron strategies in zebrafish.</a> Proc Natl Acad Sci U S A. 121 (3), e2309842121 (2024).</li><li>Duchemin, A. L., Vignes, H., Vermot, J., Chow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+in+cardiovascular+morphogenesis+and+tissue+engineering.">Mechanotransduction in cardiovascular morphogenesis and tissue engineering.</a> Curr Opin Genet Dev. 57, 106-116 (2019).</li><li>Kalogirou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracardiac+flow+dynamics+regulate+atrioventricular+valve+morphogenesis.">Intracardiac flow dynamics regulate atrioventricular valve morphogenesis.</a> Cardiovasc Res. 104 (1), 49-60 (2014).</li><li>da Silva, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=egr3+is+a+mechanosensitive+transcription+factor+gene+required+for+cardiac+valve+morphogenesis.">egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis.</a> Sci Adv. 10 (20), eadl0633 (2024).</li><li>Bartman, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+myocardial+function+affects+endocardial+cushion+development+in+zebrafish.">Early myocardial function affects endocardial cushion development in zebrafish.</a> PLoS Biol. 2 (5), E129 (2004).</li><li>Steed, E., Boselli, F., Vermot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+driven+cardiac+valve+morphogenesis.">Hemodynamics driven cardiac valve morphogenesis.</a> Biochim Biophys Acta. 1863 (7 Pt B), 1760-1766 (2016).</li><li>Vermot, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversing+blood+flows+act+through+klf2a+to+ensure+normal+valvulogenesis+in+the+developing+heart.">Reversing blood flows act through klf2a to ensure normal valvulogenesis in the developing heart.</a> PLoS Biol. 7 (11), e1000246 (2009).</li><li>B&#228;ck, M., Gasser, T. C., Michel, J. B., Caligiuri, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+factors+in+the+biology+of+aortic+wall+and+aortic+valve+diseases.">Biomechanical factors in the biology of aortic wall and aortic valve diseases.</a> Cardiovasc Res. 99 (2), 232-241 (2013).</li><li>Hahn, C., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+in+vascular+physiology+and+atherogenesis.">Mechanotransduction in vascular physiology and atherogenesis.</a> Nat Rev Mol Cell Biol. 10 (1), 53-62 (2009).</li><li>Boselli, F., Steed, E., Freund, J. B., Vermot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+shear+stress+patterns+predict+the+orientation+of+convergent+tissue+movements+in+the+embryonic+heart.">Anisotropic shear stress patterns predict the orientation of convergent tissue movements in the embryonic heart.</a> Development. 144 (23), 4322-4327 (2017).</li><li>Hove, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracardiac+fluid+forces+are+an+essential+epigenetic+factor+for+embryonic+cardiogenesis.">Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis.</a> Nature. 421 (6919), 172-177 (2003).</li><li>JoVE Science Education database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+II:+Mouse,+zebrafish,+and+chick.+Zebrafish+breeding+and+embryo+handling.">Biology II: Mouse, zebrafish, and chick. Zebrafish breeding and embryo handling.</a> JoVE. , (2023).</li><li>Arrenberg, A. B., Stainier, D. Y., Baier, H., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+cardiac+function.">Optogenetic control of cardiac function.</a> Science. 330 (6006), 971-974 (2010).</li></ol>

Critical steps in the protocol and troubleshooting
Mounting of zebrafish embryos
The quantity of agarose used to mount the embryos is important. The dome formed should not be excessively large, as this can hinder the manipulation of the bead from the surface to the embryos. Conversely, it should not be too small; having multiple beads atop the agarose, positioned close to the embryos and their yolk, can cause confusion. A v...

Since its introduction in the late 1970s1, the zebrafish (Danio rerio) has emerged as a powerful model system for studying the intricacies of cardiac development and congenital heart disorders. Unlike most vertebrates, including mouse and chick embryos, which rely on a functional cardiovascular system and cannot survive early heart defects, zebrafish provide a unique advantage by enabling the investigation of severe heart phenotypes. This is due to their small size, which facilitates sufficient oxygen supply through passive diffusion, allowing survival even in the absence of heart contraction and active blood circulation2,3,4. Furthermore, among the many significant features of zebrafish is the optical transparency of their embryos, which enables non-invasive monitoring of the developing heart5,6,7,8.
Mechanical forces continuously provide feedback to the heart valve morphogenetic programs9,10,11,12,13,14,15,16,17,18,19,20,21,22, and aberrant blood flow is widely acknowledged as a shared factor in various cardiovascular disorders23,24. In zebrafish, cardiac valve development relies on heart contraction and mechanical forces generated by the beating heart. Multiple zebrafish mutants have demonstrated the significance of heart-generated mechanical stimuli in valvulogenesis. Remarkably, the complete absence of heart contraction and, consequently, blood flow due to mutations of cardiac troponin T (tnnt2) in silent heart (sih) mutants results in the absence of tissue convergence and endocardial cell (EdC) clustering during early morphogenetic stages25.
Intracardiac hemodynamics and mechanical forces generated by the blood flow emerge as fundamental epigenetic components shaping the development of the zebrafish embryonic heart. Numerous studies suggest that proper cardiac morphogenesis in zebrafish requires distinct flow stimuli, and deviations from these physiological patterns lead to heart valve defects10,13,14,22,26. Here, we describe an effective method, adapted from Fukui et al.13, to manipulate mechanical forces in vivo by grafting a 30 &#181;m to 60 &#181;m diameter magnetic bead within the developing zebrafish beating heart. The technique involves microsurgical insertion of a bead into the cardiac lumen of anesthetized larvae without perturbing heart function. The presence of the bead leads to the amplification of the mechanical forces experienced by EdCs, directly triggering mechanical stimulus-dependent calcium influx13. This approach enables the investigation of mechanotransduction pathways that regulate heart morphogenesis and offers a means to deepen our understanding of the role of mechanical forces in valve formation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Essential equipment for zebrafish raising, breeding, and embryo collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-bottom dish (35 mm x 15&#160;mm)</td>
			<td>VWR International</td>
			<td>734-2905</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Eppendorf</td>
			<td>EP5382000031</td>
			<td>Eppendorf ThermoMixer C</td>
		</tr>
		<tr>
			<td>Jewelers forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F6521-1EA</td>
			<td>Dumont No. 5, L 4 1/4 in., Inox alloy</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 2 mL</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 mg/mL tricaine stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Danieau&#39;s medium (60x stock solution)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PureCube Glutathione MagBeads</td>
			<td>Cube Biotech</td>
			<td>32201</td>
			<td></td>
		</tr>
		<tr>
			<td>PTU (1-phenyl-2-thiourea)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure low melting point agarose</td>
			<td>Invitrogen</td>
			<td>16520-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Danieau&#39;s medium (60x stock solution)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>34.8 g NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>1.6 g KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>5.8 g CaCl2&#183;2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>9.78 g MgCl2&#183;6H2O&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>442611-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissolve the ingredients in H2O to a final volume of 2 L. Adjust the pH to 7.2 using NaOH, then autoclave.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 mg/mL tricaine stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>400 mg of tricaine powder (Ethyl 3-aminobenzoate methanesulfonate salt)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>97.9 mL double-distilled H2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.1 mL 1 M Tris (pH 9)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adjust the pH to 7, then aliquot and store at -20 &#176;C.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

manipulating mechanical forces in the developing zebrafish heart using magnetic beads

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction and physical stimuli generated by the beating heart. Intracardiac hemodynamics, driven by blood flow, emerge as fundamental information shaping the development of the embryonic heart. Here, we describe an effective method to manipulate mechanical forces in vivo by grafting a 30 &#181;m to 60 &#181;m diameter magnetic bead in the cardiac lumen. The insertion of the bead is conducted through microsurgery in anesthetized larvae without perturbing heart function and enables artificial alteration of the boundary conditions, thereby modifying flow forces in the system. As a result, the presence of the bead amplifies the mechanical forces experienced by endocardial cells and can directly trigger mechanical stimulus-dependent calcium influx. This approach facilitates the investigation of mechanotransduction pathways that govern heart development and can provide insights into the role of mechanical forces in cardiac valve morphogenesis.

Author Spotlight: Understanding Mechanical Forces Involved in Shaping the Zebrafish Heart

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

We focus on understanding how mechanical forces shape the zebrafish heart using advanced imaging techniques. By combining our knowledge of biology, physics, microscopy, and computing, we develop tools for optical imaging and image analysis to study how mechanical stimuli influence the way the cardiovascular system develops. A beating heart generates several type of forces, such as pressure for the shear and contractor force.Although each of these forces has a function in vitro culture systems, it is difficult to separate these parameters in the in vivo heart. By developing our approach, we aim to tackle this challenging task. We have established a novel approach to assess, adapt biological output caused by external force stimulation, and we verified our force spin and constant signal in crosscutting the cardiac cells.As a result this approach enables us to impetrate other aspects of tissue neurogenesis in the world of mechanical biology. This method enables the study of heart function in zebrafish embryo via targeted mechanical stimulation. Unlike genetic, pharmacological, or optogenetic approaches, pit grafting offers a more direct way to influence heart physiology through mechanical means.Pit grafting is viable for examining the roles of mechanical forces and calcium influx in cardiac biological processes. It provides means to deepen our understanding of the mechanical transduction pathways involved in heart development and function.

Examples of successful bead grafting are shown in Figure 3, Video 3, and Video 4. The magnetic bead was correctly positioned within the atrium of the zebrafish heart, allowing for unobstructed blood flow and no observed hemorrhage. Additionally, the heart walls maintained their structural integrity without collapsing (Figure 3 and Video 3). After 24 h, the embryo showed no signs of pericardial edema, further con...

This protocol outlines a method for grafting a magnetic bead into the developing zebrafish heart through microsurgery, enabling the manipulation of mechanical forces in vivo and triggering mechanical stimulus-dependent calcium influx in endocardial cells.

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction ...

manipulating-mechanical-forces-developing-zebrafish-heart-using

Research

JoVE Journal

Developmental Biology

610 visualizações.  Imperial College London. Nós nos concentramos em entender como as forças mecânicas moldam o coração do peixe-zebra usando técnicas avançadas de imagem. Ao combinar nosso conhecimento de biologia, física, microscopia e computação, desenvolvemos ferramentas para imagens ópticas e análise de imagens para estudar como os estímulos mecânicos influenciam a maneira como o sistema cardiovascular se desenvolve. Um coração batendo gera vários tipos de forças, como pressão pa...