The authors would like to express gratitude to William Talbot from Stanford University for providing the Human&#160;Nrg1&#160;cDNA and to Deborah Yelon from UCSD for providing the&#160;wea&#160;mutants. The authors would also like to thank Cynthia Chen for helping with image acquisition. This study was supported by grants NIH HL118650 (to T.K. Hsiai), HL083015 (to T.K. Hsiai), HD069305 (to N.C. Chi and T.K. Hsiai.), HL111437 (to T.K. Hsiai and N.C. Chi), HL129727 (to T.K. Hsiai), T32HL007895 (to R.R. Sevag Packard), HL 134613 (to V. Messerschmidt) and University of Texas System STARS funding (to J. Lee).

The following methods were performed in compliance with UTA and UCLA IACUC protocols. These experimental groups were used with transgenic Tg(cmlc2:gfp), the wea (weak atrium) or clo (cloche) mutants: (a) wild-type (WT) control, (b) gata1a MO, and (c) tnnt2a MO injections (Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Model</td>
			<td>Name</td>
			<td>Modified Genes</td>
			<td>Phenotype</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>Wild type</td>
			<td>None</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Tg(cmlc2:gfp)</td>
			<td>Cardiac myosin light chain&#160;</td>
			<td>Green flourescence specifically expressed in the myocardium</td>
			<td>34</td>
		</tr>
		<tr>
			<td rowspan="4">Decreased Wall Shear Stress</td>
			<td>Weak atrium (wea) mutant</td>
			<td>Atrium-specific myosin heacy chain</td>
			<td>Atrium cannot contract, compact ventricle, thick myocardial wall, narrow lumen, dialated atrium,&#160;</td>
			<td>37</td>
		</tr>
		<tr>
			<td>Cloche (clo) mutant</td>
			<td>N/A</td>
			<td>Complete removal of endocardium, unaturally large atrium with small ventricle, nonexistent cardiac cushins</td>
			<td>41</td>
		</tr>
		<tr>
			<td>gata1a MO</td>
			<td>gata1a&#160;</td>
			<td>Anemia from lack of red blood cells</td>
			<td>4</td>
		</tr>
		<tr>
			<td>tnnt2a MO</td>
			<td>tnnt2a</td>
			<td></td>
			<td>39</td>
		</tr>
		<tr>
			<td rowspan="2">Increased Wall Shear Stress</td>
			<td>EPO mRNA</td>
			<td>None</td>
			<td>Severe polycythemia, increase in number of circulating blood cells, increased viscosity</td>
			<td>6</td>
		</tr>
		<tr>
			<td>Isoprotenerol</td>
			<td>None</td>
			<td>Increased cardiac rate</td>
			<td>7</td>
		</tr>
	</tbody>
</table>
Table 1: Zebrafish descriptions. Definitions and descriptions of Zebrafish used in experiments.
1. Study Setup
<ol>
	<li>Prepare Nrg1 and EPO mRNA for trabeculation rescue.
	<ol>
		<li>Obtain Human Nrg1 cDNA and amplify from a donor plasmid. 
		Note: The Human Nrg1 cDNA was gifted from William Talbot from Stanford University.
		<ol>
			<li>Take out necessary reagents and materials, namely PCR mastermix (stored in -20 &#176;C), primers (See Table 2) (stored in -20 &#176;C), a PCR plate (stored at room temperature), and a 20 &#956;L pipette (stored at room temperature). See Table of Materials for example products. 
			<img alt="Table 2" src="/files/ftp_upload/57763/57763tbl2v2.jpg" /> 
			Table 2: Primers for human Nrg1 cloning.</li>
			<li>Prepare the mastermix for each primer set in triplicate using the 20 &#956;L pipette. For 1 set of triplicates, use 37.5 &#956;L of the mastermix, 3 &#956;L of DNA-free water, 4.5 &#956;L of primer. For more triplicates, simply multiply by the number of samples.</li>
			<li>Dilute the working solution Human Nrg1 cDNA (20 ng/&#956;g concentration) in the primer strip with DNA-free water so that there is 10 &#956;L total per well per sample. 
			Note: Ensure that each solution under step 1.1.1 is evenly distributed. Do this by mixing each solution by pipetting up and down. To prevent cross-contamination, use one pipette tip for only one sample.</li>
			<li>Aliquot the Nrg1 cDNA with the 10 &#956;L multi-channel pipette to desired wells in the PCR plate.</li>
			<li>Add 14.5 &#956;L of the mastermix to the cDNA in the wells. Apply a sticky cover to the PCR plate to seal the wells, and place into the PCR machine.</li>
			<li>Start the PCR machine and ensure that the cycle reaches appropriate temperatures according to the enzymes and primers used.</li>
		</ol>
		</li>
		<li>Clone the Nrg1 cDNA into the plasmid pCS2+ at the BamHI and EcoRI sites using excess enzymes BamHI and EcoRI to ensure cDNA is ligated in all plasmids.
		<ol>
			<li>Mix the Nrg1 cDNA with the donor plasmid pCS2+, commercially available master mix solution and primers (Table 2). 
			Note: The total reaction volume should be 50 &#956;L with 25 &#956;L of master mix, 3 &#956;L of primers and 1 &#956;L of 20 ng/&#956;L donor plasmid with Nrg1 cDNA.</li>
			<li>Place the mix from step 1.1.2.1 in the PCR machine and set the parameters to meet the following specifications: 98 &#176;C for 10 s, 55 &#176;C for 5 or 15 s, and 72 &#176;C for 5 s/kb.</li>
			<li>Purify the PCR product using a purification kit following the manufacturer&#39;s instructions. Elute the product into 50 &#956;L of elution buffer.</li>
			<li>Digest the purified PCR product and 5 &#956;g of pCS2+ with BamHI and EcoRI separately at 37 &#176;C for 2 h in a 200 &#956;L reaction volume. Purify the resulting digestion with a commercial kit and elute in 20 &#956;L and 100 &#956;L of Tris-HCl (pH 8.5), respectively.</li>
			<li>Ligate 4 &#956;L of Nrg1 cDNA and 2 &#956;L of the digested pCS2+ plasmid in a 25 &#956;L reaction with 1 &#956;L of a ligase catalyst at 16 &#176;C overnight. 
			Note: The procedure can be stopped here for overnight incubation.</li>
			<li>Use 2 &#956;L of the ligation solution from step 1.1.2.5 and transform into 50 &#956;L of E. coli bacteria cells suitable for cloning (see Table of Materials).</li>
		</ol>
		</li>
		<li>Confirm that the transformation took place by screening the Nrg1 cDNA clones using PCR. Choose clones at random and add to a solution of specific primers, polymerase, and deoxyribonucleotide triphosphates (dNTPs). 
		Note: The controls were the vector (pCS2+) with and without the insert (human cDNA). Do not pick too big of a colony because excessive bacteria can inhibit PCR.
		<ol>
			<li>Pick 8 colonies from the transformation and screen pCS2+-Nrg1 clones using the PCR primers from Table 2.</li>
			<li>Culture one clone with Nrg1 cDNA to isolate the pCS2+-Nrg1 plasmid DNA. Inoculate with 100 &#956;L of E. coli bacteria with pCS2+-Nrg1 plasmid into 100 mL of LB media, and culture by shaking (160 - 225 RPM) at 37 &#176;C.</li>
		</ol>
		</li>
		<li>Transfect purified pCS2+-Nrg1 plasmid into HEK-293 cells in a 6-well plate using a commercial transfection reagent following the manufacturer&#39;s instructions.</li>
		<li>24 h after transfection, lyse the cells using a lysis buffer and run through an SDS-PAGE gel, transfer to a gel membrane, and then stain with anti-Nrg1 antibody22. Verify Nrg1 protein expression using a Western Blot. 
		Note: The control is an empty pCS2+ plasmid. Can be paused here if gel membrane transfer occurs overnight.</li>
		<li>Synthesize Nrg1 mRNA using a commercial product similar to that in the Table of Materials and follow the manufacturer&#39;s instructions.
		<ol>
			<li>Take 5 &#956;L of the pCS2+-Nrg1 DNA from the bacteria isolated culture, and digest with NotI in a 200 &#956;L reaction for 2 h at 37 &#176;C to linearize the plasmid.</li>
			<li>Purify the linearized plasmid with a commercial kit and elute in 20 &#956;L of Tris-HCl (pH 8.5).</li>
			<li>Conduct in vitro transcription with a commercial RNA isolation kit in a 20 &#956;L reaction using 5 &#956;L of the linearized plasmid following the manufacturer&#39;s instructions.</li>
			<li>Add the in vitro transcribed Nrg1 to 350 &#956;L of RNA lysis buffer and then purify with an RNA isolation kit. Elute the RNA into 60 &#956;L of Tris-HCl (pH 8.5).</li>
			<li>Measure the RNA concentration and store at -80 &#176;C for future use.</li>
		</ol>
		</li>
		<li>Follow steps 1.1.6.1 - 1.1.6.5 above for the EPO cDNA and mRNA preparation but clone the cDNA into the pCS2+ plasmid at EcoRI and XhoI sites instead.</li>
	</ol>
	</li>
	<li>Inject Morpholino into zebrafish
	<ol>
		<li>Design23 the MO injections using an online tool (Table of Materials) against the ATG sequence of gata1a4 (5&#8242;-CTGCAAGTGTAGTATTGAAGATGTC-3&#8242;) and tnnt2a5 (5&#8242;-CATGTTTGCTCTGATCTGACACGCA-3&#8242;).</li>
		<li>Add gata1a and tnnt2a MO separately to the nuclease-free water to make the final concentrations of 8 ng/nL and 4 ng/nL, respectively. Repeat with EPO mRNA with a final concentration of 20 pg/nL. Ensure that the final volume is 1 mL.</li>
		<li>Inject 1 nL of the 8 ng/nL of gata1a MO and 1 nL of the 4 ng/nL of tnnt2a MO into separate zebrafish embryos at the 1 to the 4-cell stage to reduce ventricular wall shear stress (WSS)24. To increase WSS, inject 1 nL of 20 pg/nL of EPO mRNA6 into the zebrafish embryos at the 1- to 4- cell stage.</li>
	</ol>
	</li>
	<li>Chemically treat zebrafish to inhibit trabeculation
	<ol>
		<li>Using a 20 mL pipette, dilute the 10 mg/mL AG1478 in 1% DMSO in E3 medium to a final concentration of 5 &#956;M at 30 hpf into a 15 mL tube. As a control, treat each fish with 1% DMSO only.</li>
		<li>Add N-[N-(3,5-Difluorophenactyl)-L-Alanyl]-S-Phenylglycine t-butyl ester (DAPT) in 1% DMSO (100 &#956;M) to E3 medium in a 15 mL tube to inhibit Notch signaling at 40 hpf in a similar fashion. As a control, treat with only 1% DMSO.</li>
	</ol>
	</li>
</ol>
2. Imaging Techniques
<ol>
	<li>4-D cardiac LSFM Imaging with synchronization algorithm
	<ol>
		<li>Take 2-D images of the entire fish embryo over multiple cardiac beating cycles using the steps below (Figure 1) . Ensure that the light-sheet thickness is approximately 5 &#956;m. Take 500 x-y frames with an exposure time of 10 ms. Set the z-scanning to 1 &#956;m for lossless digital sampling according to the Nyquist sampling principle25.
		<ol>
			<li>Create a 1% (w/v) agarose gel by adding 1 g of agarose to 100 mL and heating it until all agarose is dissolved. Load a small plastic tube with the embryo and agarose using a 20 &#956;L pipette and secure it on the stage.</li>
			<li>Open the image viewing software and click Live to see the live view of the sample. Adjust the objective using the knob so that the sample is in focus. Similarly, move the stage so that the live view of the sample shows the top of the embryo.</li>
			<li>Take images 500 times for 5 s at 10X magnification using the microscope&#39;s software26.</li>
			<li>Move the stage using the motor to a new layer (1 &#956;m in the z-axis) and repeat the imaging procedure.</li>
			<li>Repeat above 2 steps until entire heart is fully imaged.</li>
		</ol>
		</li>
		<li>Ensure data integrity by disregarding the first and last cardiac cycle images. Find the period of the cardiac cycle by matching images that were taken at the same time point in the cardiac cycle, but in different periods. Use the following equation that was developed to find the period: 
		<img alt="Equation 1" src="/files/ftp_upload/57763/57763eq1.jpg" /> (1) 
		where D is the cost function used for fitting a period hypothesis, Im the captured image, &#964;&#39; the time the image was captured, zk the z-direction index of the image, xm is the vector of the pixel index, and T&#39; is the periodic hypothesis.</li>
		<li>Use the following equation to find the relative shift between two pictures in similar stages: 
		<img alt="Equation 2" src="/files/ftp_upload/57763/57763eq2.jpg" /> (2) 
		where Qk&#39;,k denotes a cost function for the relative shift, R is the possible spatial neighborhood, L is the total time of capturing the image, x is the pixel index, zk and zk&#39; are the first and second z-direction slices, t is time, and s is the relative shift hypothesis.</li>
		<li>Convert the relative shift to absolute shift with respect to the first image and solve the linear equation using the pseudo-inverse approach, to find the absolute relation to align the next section of images as previously described27.</li>
		<li>The final 4-D images were processed using image processing software (Table of Materials).
		<ol>
			<li>Stacking 2-D images into 3-D
			<ol>
				<li>Open a commercial image processing software.</li>
				<li>Click Open Data.</li>
				<li>Select all the images to be stacked into 3-D &#62; Click Load.</li>
				<li>Add in the voxel size of 0.65 x 0.65 x 1 &#62; Click OK.</li>
				<li>Click the Multi-Planar View box to visualize the 3-D image.</li>
				<li>Right-click the blue slice_1.tiff box &#62; Select Display &#62; Select Volren.</li>
				<li>Click Edit &#62; Select Options &#62; Select Edit Colormap.</li>
				<li>Adjust the color using the boxes outlined in red &#62; Click OK.</li>
			</ol>
			</li>
			<li>Converting 3-D to 4-D
			<ol>
				<li>Click &#34;File &#62; Open Time Series Data.</li>
				<li>Select the 3-D tiffs that were just made &#62; Click Load.</li>
				<li>Enter the same voxel size as before.</li>
				<li>Right click the blue slice_1.tiff box &#62; Select &#34;Display&#34; &#62; Select Volren.</li>
				<li>Click Edit &#62; Select Options &#62; Select Edit Colormap. Adjust the color using the boxes &#62; Click OK.</li>
				<li>Right click Time Series Control &#62; Click Movie maker &#62; Press the Play button in order to watch the movie.</li>
				<li>Add a file name, frame size, frame rate, quality equal to 1, type Monoscopic &#62; Click Apply</li>
				<li>Export the video. Open the video in an appropriate software.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. qRT-PCR analysis
<ol>
	<li>Zebrafish heart RNA Isolation to quantify Notch ligands, receptors, and target genes&#39; expressions
	<ol>
		<li>Sacrifice the zebrafish by subjecting them to an overdose of tricaine methylate28,29. Using a previously described protocol30, the zebrafish hearts were excised and prepared for RNA isolation.</li>
		<li>Isolate the total RNA, and synthesize the cDNA using the cDNA synthesis kit following the manufacturer&#39;s instructions.</li>
		<li>Design the PCR primers for the Notch ligands Jag1, Jag2, Dll4, receptor Notch1b, and signaling related genes Nrg1 and ErbB2. See Table 3 for the primers we used.</li>
		<li>Add the primers from step 3.1.3, the isolated mRNA from step 3.1.2, and a commercial mastermix to the PCR plate. Run the qRT-PCR at the appropriate temperatures and times according to the enzymes used. Normalize the results to zebrafish &#945;-actin. 
		Note: This will calculate the expression levels for each of the ligands, receptors, and genes.</li>
	</ol>
	</li>
</ol>
4. In vitro Human Aortic Endothelial Cell (HAEC) Experiments
<ol>
	<li>Dynamic Shear Stress Model
	<ol>
		<li>Culture HAEC cells with HAEC culture media. Once fully confluent, expose the cells to laminar flow and pulsatile flow of 23 x 10-5 N at the rate of 1 Hz for 24 h.
		<ol>
			<li>Warm up EC media/DMEM 10% FBS in a water bath for 20 min.</li>
			<li>Retrieve HAEC cells from liquid nitrogen tank and place the vial into the water bath until melted.</li>
			<li>Using a 1,000 &#956;L pipette, remove the thawed cells and put them in a 15 mL tube with 3-5 mL of media. Centrifuge the cell solution at 53 x g for 3 min.</li>
			<li>Aspirate the solution off and add enough media for a total volume of 10 mL. Add the cell solution to a sterile T75 flask, cap the flask, and distribute the cells evenly on the bottom of the plate.</li>
			<li>Mark the flask with initials, date, cell type, and passage number. Incubate the flask at 37 &#176;C, 5% CO2, until cells are 80% confluent. Remember to change the media every 2-3 days.</li>
			<li>Using a commercial pump, attach the tubing to the flask, and set the parameters mentioned above in step 4.1.131,32,33.</li>
		</ol>
		</li>
		<li>Add GI254023X to 50 mL of HAEC medium at a final concentration of 5 &#956;M for 30 min.</li>
		<li>With the pre-mixed GI254023X, an ADAM10 inhibitor which suppresses Notch signaling, conduct the same laminar flow or pulsatile flow experiments as in 4.1.1.</li>
		<li>Quantify Notch ligands (Jag1, Jag2, and Dll4) and Notch target genes (hairy and enhancer of split [Hes]) using qRT-PCR for four groups of HAEC samples (laminar flow, laminar flow + GI254023X, pulsatile flow, pulsatile flow + GI254023X) described previously32.</li>
	</ol>
	</li>
</ol>
5. Rescue and over-expression of Notch signaling
<ol>
	<li>Inject 1 nL of the prepared Nrg1 mRNA at a concentration of 5 pg/nL (from step 1.1.6) at the 1- to 4-cell stage with the gata1a MO in order to overexpress Notch target genes24.</li>
	<li>Inject 1 nL of the Nrg1 mRNA into wea mutant in a similar way24.</li>
	<li>Double the concentration of Nrg1 mRNA (10 pg/nL) and inject24 1 nL into zebrafish to observe the ventricular morphology.</li>
	<li>Inject 1 nL of the 20 pg/nL of EPO mRNA6 into the zebrafish embryos at the 1- to 4- cell stage to augment hematopoiesis to increase ventricular WSS as previously shown24.</li>
	<li>Inject 1 nL of the 50 &#956;M Isoproterenol7, which increases the rate of contractility, to the E3 medium for 24 h while zebrafish are cultured as previously shown24.</li>
	<li>Perform 4-D LSFM imaging for each group. Follow the instructions in steps 2.1.1-2.1.5.2.8.</li>
</ol>
6. Quantification of Fractional Shortening and volume change over time
<ol>
	<li>Perform 4-D LSFM imaging for each group at 50, 75, and 100 hpf. Follow instructions in steps 2.1.1-2.1.5.2.8. Divide each image so each has 600 nodes for replication of the cardiac wall motion.</li>
	<li>Measure the change in diameter of the ventricle during diastole and systole using the following formula: 
	<img alt="Equation 3" src="/files/ftp_upload/57763/57763eq3.jpg" />&#160; (3) 
	Load 3-D ventricular images at every 0.1 s with visualization software and measure the volume.</li>
	<li>Plot the volume change over time for each experimental group at 75 hpf and 100 hpf.</li>
</ol>

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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>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> Cardiovascular Research. 91 (2), 279-288 (2011).</li><li>BioRad. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> General Protocol for Western Blotting. 6376, (2018).</li><li>GeneTools. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gene Tools Oligo Design Website. , (2018).</li><li>Mullins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish Course. , (2013).</li><li>Grenander, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Probability and Statistics: The Harald Cram&#233;r Volume. , (1959).</li><li>Fei, P., 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+Light-Sheet+Fluorescent+Microscopy+for+Multi-Scale+and+Rapid+Imaging+of+Architecture+and+Function.">Cardiac Light-Sheet Fluorescent Microscopy for Multi-Scale and Rapid Imaging of Architecture and Function.</a> Scientific Reports. 6, 22489 (2016).</li><li>Liebling, M., Forouhar , A. S., Gharib, M., Fraser, S. E., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-dimensional+cardiac+imaging+in+living+embryos+via+postacquisition+synchronization+of+nongated+slice+sequences.">Four-dimensional cardiac imaging in living embryos via postacquisition synchronization of nongated slice sequences.</a> Journal of Biomedical Optics. 10 (5), (2005).</li><li>Adeoye, A. A., 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=Combined+effects+of+exogenous+enzymes+and+probiotic+on+Nile+tilapia+(Oreochromis+niloticus)+growth,+intestinal+morphology+and+microbiome.">Combined effects of exogenous enzymes and probiotic on Nile tilapia (Oreochromis niloticus) growth, intestinal morphology and microbiome.</a> Aquaculture. 463, 61-70 (2016).</li><li>Matthews, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+Euthanasia+in+Zebrafish.">Anesthesia and Euthanasia in Zebrafish.</a> ILAR Journal. 53 (2), 192-204 (2012).</li><li>Singleman, C., Holtzman, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Dissection+in+Larval,+Juvenile+and+Adult+Zebrafish,+Danio+rerio.">Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio.</a> Journal of Visualized Experiments : JoVE. (55), e3165 (2011).</li><li>Li, 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=Disturbed+Flow+Induces+Autophagy,+but+Impairs+Autophagic+Flux+to+Perturb+Mitochondrial+Homeostasis.">Disturbed Flow Induces Autophagy, but Impairs Autophagic Flux to Perturb Mitochondrial Homeostasis.</a> Antioxidants &amp; Redox Signaling. 23 (15), 1207-1219 (2015).</li><li>Li, 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=Shear+Stress-Activated+Wnt-Angiopoietin-2+Signaling+Recapitulated+Vascular+Repair+in+Zebrafish+Embryos.">Shear Stress-Activated Wnt-Angiopoietin-2 Signaling Recapitulated Vascular Repair in Zebrafish Embryos.</a> Arteriosclerosis, thrombosis, and vascular biology. 34 (10), 2268-2275 (2014).</li><li>Baek, K. I., 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=Flow-Responsive+Vascular+Endothelial+Growth+Factor+Receptor-Protein+Kinase+C+Isoform+Epsilon+Signaling+Mediates+Glycolytic+Metabolites+for+Vascular+Repair.">Flow-Responsive Vascular Endothelial Growth Factor Receptor-Protein Kinase C Isoform Epsilon Signaling Mediates Glycolytic Metabolites for Vascular Repair.</a> Antioxidants &amp; Redox Signaling. 28 (1), 31-43 (2018).</li><li>Huang, C. J., Tu, C. T., Hsiao, C. D., Hsieh, F. J., Tsai, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+a+myocardium-specific+GFP+transgene+reveals+critical+regulatory+elements+in+the+cardiac+myosin+light+chain+2+promoter+of+zebrafish.">Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.</a> Developmental Dynamics. 228 (1), 30-40 (2003).</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), (2009).</li><li>Grego-Bessa, 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=Notch+signaling+is+essential+for+ventricular+chamber+development.">Notch signaling is essential for ventricular chamber development.</a> Dev Cell. 12 (3), 415-429 (2007).</li><li>Berdougo, E., Coleman, H., Lee, D. H., Stainier, D. Y. R., Yelon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+weak+atrium/atrial+myosin+heavy+chain+disrupts+atrial+function+and+influences+ventricular+morphogenesis+in+zebrafish.">Mutation of weak atrium/atrial myosin heavy chain disrupts atrial function and influences ventricular morphogenesis in zebrafish.</a> Development. 130 (24), 6121 (2003).</li><li>Arnaout, 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=Zebrafish+model+for+human+long+QT+syndrome.">Zebrafish model for human long QT syndrome.</a> Proc Natl Acad Sci U S A. 104 (27), 11316-11321 (2007).</li><li>Chi, N. C., 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=Genetic+and+physiologic+dissection+of+the+vertebrate+cardiac+conduction+system.">Genetic and physiologic dissection of the vertebrate cardiac conduction system.</a> PLoS Biol. 6 (5), e109 (2008).</li><li>Liao, 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=The+zebrafish+gene+cloche+acts+upstream+of+a+flk-1+homologue+to+regulate+endothelial+cell+differentiation.">The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation.</a> Development. 124 (2), 381-389 (1997).</li><li>Stainier, D. Y., Weinstein, B. M., Detrich, H. W., Zon, L. I., Fishman, M. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+enhancement+in+a+light-sheet-based+microscope+(SPIM).">Resolution enhancement in a light-sheet-based microscope (SPIM).</a> Optics Letters. 31 (10), 1477-1479 (2006).</li></ol>

The authors have nothing to disclose.&#160;

In this protocol, we have shown that 4-D imaging can be used to track the development of a trabecular network in response to changes in biomechanical forces. In particular, the shear stress experienced by endothelial cells initiates the Notch signaling cascade, which in turn promotes trabeculation. In this manuscript, we have shown that (1) gata1a MO injection decreased hematopoiesis and therefore it reduced wall shear stress, (2) tnnt2a MO injection inhibited ventricular contractile function to reduce ...

An erratum was issued for:&#160;<a href="https://www.jove.com/video/57763/light-sheet-fluorescence-microscopy-to-capture-4-dimensional-images">Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart</a>.&#160;Additional author names were added, and an author affiliation was updated.
The author list was updated from:
Victoria Messerschmidt*1, Zachary Bailey*1, Kyung In Baek2, Richard Bryant1, Rongsong Li2, Tzung K. Hsiai2, Juhyun Lee1
to:
Victoria Messerschmidt*1, Zachary Bailey*1, Kyung In Baek2, Yichen Ding2, Jeffrey J. Hsu2, Richard Bryant1, Rongsong Li2, Tzung K. Hsiai2, Juhyun Lee1
The author affiliation for Rongsong Li was updated from:
Department of Medicine (Cardiology) and Bioengineering, UCLA
to:
College of Health Science and Environmental Engineering, Shenzhen Technology University

An erratum was issued for: <a href="https://www.jove.com/video/57763/light-sheet-fluorescence-microscopy-to-capture-4-dimensional-images">Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart</a>. Additional&#160;author names were&#160;added, and an author affiliation was updated.

Erratum: Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

Biomechanical forces, such as hemodynamic shear stress, are intimately involved in cardiac morphogenesis. In response to hemodynamic shear forces, myocardial ridges and grooves develop in a wave-like trabecular network in alignment with the direction of the shear stress across the atrioventricular (AV) valve1. Cardiac trabeculation is necessary to increase contractile function and myocardial mass2. Mutations in Notch signaling pathways result in congenital heart defects in humans and other vertebrates3. For example, gata1a4 and tnnt2a5 morpholino oligonucleotides (MO) have been shown to reduce erythropoiesis, while erythropoietin mRNA (EPO)6 and Isoproterenol (ISO)7 increase red blood cells and heart rate respectively, and therefore wall shear stress (WSS). Furthermore, ErbB2 signaling, downstream of Notch, promotes cardiomyocyte proliferation and differentiation to generate contractile force, which in turn activates Notch signaling8,9. It is suggested that shear stress governs Notch signaling driven trabeculation for ventricular development. Currently, there are many studies that attempt to further understand the genetic programming events leading to congenital heart defects (CHD)10,11,12, but very little are investigating how mechanical forces influence the forming heart.
In order to investigate the mechanical forces acting on the endocardium, close observation during the developmental period needs to be implemented. However, it is challenging to obtain good quality images of in vivo beating samples due to the inherence of traditional microscopy13. In order to observe the development over time within a sample, physical sectioning and staining, therefore, need to occur13,14,15. Although confocal microscopy is widely used to image the 3-D structure of samples14,16, these imaging systems&#39; acquisition is still limited by slow scanning speeds.
Light-sheet fluorescence microscopy (LSFM) is a unique imaging technique that allows the visualization of in vivo dynamic events with long working distance13. This technique uses a light-sheet fluorescent microscopy to optically section a sample17. Due to illumination of only a thin sheet of light on the sample, there is a reduction in photo-bleaching and photo toxicity13,18. The large field of view and long working distance allows for large samples to stay intact as they are imaged13,14,17. The low magnification allows for a larger area to be imaged, while the long working distance allows for thicker samples to be imaged without compromising the signal-to-noise ratio. Many groups have used LSFM to image entire embryos17, brains14,18, muscles and hearts19 among other tissues, showing the diverse types of samples that can be imaged.
Although previous research demonstrated reduced hemodynamic shear force by occluding the inflow or outflow tracks of the zebrafish heart, the information is solely qualitative. It results in an abnormal third chamber, diminished cardiac looping, and impaired valve formation20. The 4-D LSFM images give a new perspective into the way the hemodynamic shear forces affect the development of the cardiac tissue. These mechanical forces may activate force-sensitive signaling molecules and induce the formation of the trabecular ridges. Because of the added time aspect of 4-D imaging, one is able to track changes in development in real time, which could lead to new revelations that had gone unnoticed previously. The zebrafish is an ideal model for imaging because scientists can observe an entire vertebrate animal versus only cell-cell interactions. Oxygen can also diffuse through the entire embryo, which allows development to occur without depending on the vascular system, unlike in mammalian development. Even though the zebrafish heart lacks the pulmonary organs, which require a four-chambered heart, there is a large number of cardiac genes that are conserved between zebrafish and humans21.
In this manuscript, we describe how to use light-sheet fluorescence microscopy to image the developing trabeculae in zebrafish hearts under various circumstances. First, injection of gata1a4 or tnnt2a5 MOs were used to lower the blood viscosity, and therefore WSS. The morphology of the heart was then recorded. In a separate group of fish, we increased the WSS by administering EPO mRNA6 or isoproterenol7 and observed the results. We also conducted a cell study with different pulsatile or oscillatory flow rates. After imaging each group, we found that WSS sensed by the endocardium via Notch signaling initiates trabeculation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Clontech Hifi PCR pre-mix&#160;</td>
			<td>Takara&#160;</td>
			<td>639298</td>
			<td>PCR mastermix 
			1.1.1.1, 1.1.1.2, 1.1.1.5, 1.1.2.1, 3.1.4</td>
		</tr>
		<tr>
			<td>Human Nrg1 cDNA</td>
			<td>Gift from William Talbot, Stanford University, Stanford, California, USA</td>
			<td>N/A</td>
			<td>Used for trabeculation rescue 
			1.1.1.3, 1.1.1.4, 1.1.2.1</td>
		</tr>
		<tr>
			<td>CFX Connect&#8482; Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>1855201</td>
			<td>PCR Machine 
			1.1.1.5, 1.1.1.6, 1.1.2.2, 1.1.3.2</td>
		</tr>
		<tr>
			<td>pCS2+</td>
			<td>GE Health</td>
			<td></td>
			<td>Plasmid used to synthesize mRNA 
			1.1.2.1, 1.1.2.4, 1.1.2.5</td>
		</tr>
		<tr>
			<td>Nucleospin purification kit&#160;&#160;</td>
			<td>Clontech</td>
			<td>740609.25</td>
			<td>DNA Purification 
			1.1.2.3, 1.1.2.4, 1.1.6.2</td>
		</tr>
		<tr>
			<td>T4 DNA ligase&#160;</td>
			<td>Clontech</td>
			<td>2011A</td>
			<td>PCR Ligation solution 
			1.1.2.5</td>
		</tr>
		<tr>
			<td>Stellar competent cells</td>
			<td>Clontech</td>
			<td>636763</td>
			<td>E. coli cells used for transformation 
			1.1.2.6, 1.1.3.1, 1.1.3.2</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 transfection reagent</td>
			<td>Life Technologies</td>
			<td>11668027</td>
			<td>Transfection reagent 
			1.1.4</td>
		</tr>
		<tr>
			<td>mMessage SP6 kit</td>
			<td>Invitrogen</td>
			<td>AM1340</td>
			<td>Kit used to synthesize mRNA 
			1.1.6.3</td>
		</tr>
		<tr>
			<td>Aurum Total RNA Mini Kit</td>
			<td>Bio-Rad</td>
			<td>7326820</td>
			<td>Purifies RNA&#160; 
			1.1.6.4, 3.1.2</td>
		</tr>
		<tr>
			<td>GeneTools 4.3.8</td>
			<td>GeneTools</td>
			<td>N/A</td>
			<td>Software for primer design 
			1.2.1, 3.1.3</td>
		</tr>
		<tr>
			<td>EPO cDNA</td>
			<td>Creative Biogene</td>
			<td>CDFH006026</td>
			<td>Increases WSS 
			1.2.2, 1.2.3, 1.1.7</td>
		</tr>
		<tr>
			<td>AG1478</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T4182</td>
			<td>ErbB inhibitor 
			1.3.1</td>
		</tr>
		<tr>
			<td>E3 medium</td>
			<td></td>
			<td></td>
			<td>To grow embryos 
			1.3.1, 1.3.2, 5.1.5</td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D5942</td>
			<td>&#947;-secretase inhibitor 
			1.3.2</td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A9539</td>
			<td>Used for mounting embryos 
			2.1.1.1</td>
		</tr>
		<tr>
			<td>ORCA-Flash4.0 LT&#160;Digital CMOS camera</td>
			<td>Hamamatsu Photonics</td>
			<td>C11440-42U</td>
			<td>Used to capture Images 
			2.1.1.2, 2.1.1.3</td>
		</tr>
		<tr>
			<td>Amira Software</td>
			<td>FEI Software</td>
			<td>N/A</td>
			<td>Visualized and Analysed images into 3D, and 4D 
			2.1.5.1.1-2.1.5.2.8</td>
		</tr>
		<tr>
			<td>Tricaone mesylate&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>886-86-2</td>
			<td>Used to humanely sedated or sacrifice embryos 
			3.1.1</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>1708890</td>
			<td>Synthesizes cDNA 
			3.1.2</td>
		</tr>
		<tr>
			<td>Eppendorf 5424 microcentrifuge</td>
			<td>Eppendorf</td>
			<td>05-400-005</td>
			<td>Microcentrifuge 
			4.1.1.3</td>
		</tr>
		<tr>
			<td>GI254023X</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>260264-93-5</td>
			<td>ADAM10 inhibitor 
			4.1.2, 4.1.3&#160;</td>
		</tr>
		<tr>
			<td>Isoprenaline hydrochloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>I5627</td>
			<td>Isoproterenol increases WSS 
			5.1.5</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>N/A</td>
			<td>Cardiac mechanics analysis</td>
		</tr>
	</tbody>
</table>

light-sheet fluorescence microscopy to capture 4-dimensional images of the effects of modulating shear stress on the developing zebrafish heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

LSFM was used in this manuscript in order to acquire high-resolution 2-D and 3-D pictures. As seen in Figure 1A and 1B, the illumination lens directs the light sheet at the sample. Because of the thinness of the light sheet, only a single plane is illuminated. The detection lens is positioned perpendicular to the illumination lens and is focused on the illuminated plane (Figure 1B). The light sheet from the illum...

Watch this Scientific Journal Video about Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart at JoVE.com

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

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Research

JoVE Journal

Bioengineering

8.2K Views.  The University of Texas at Arlington. Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

Video: Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via semiautomatic segmentation, from an in vivo computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 &deg;. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.

A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...