The authors thank Jutta Starbatty for her support in tissue culture work, Axel Kirchhof for photography, Alice Casagrande Cesconetto for editing work, and a special thanks to B&#252;lent Aksehirlioglu for technical support in the development of this device. B.B. was supported by a DZHK (German Centre for Cardiovascular Research) Scholar Grant, M.L.R. by a Whitaker International Postdoctoral Scholar Grant and M.N.H. by funds from the DZHK.

<ol>
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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=EURObservational+Research+Programme:+regional+differences+and+1-year+follow-up+results+of+the+Heart+Failure+Pilot+Survey+(ESC-HF+Pilot).">EURObservational Research Programme: regional differences and 1-year follow-up results of the Heart Failure Pilot Survey (ESC-HF Pilot).</a> European Journal of Heart Failure. 15 (7), 808-817 (2013).</li><li>Mozaffarian, D., 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=Heart+disease+and+stroke+statistics--2015+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2015 update: a report from the American Heart Association.</a> Circulation. 131 (4), 29 (2015).</li><li>Klautz, R. J., Teitel, D. F., Steendijk, P., van Bel, F., Baan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+afterload+and+contractility+in+the+newborn+heart:+evidence+of+homeometric+autoregulation+in+the+intact+circulation.">Interaction between afterload and contractility in the newborn heart: evidence of homeometric autoregulation in the intact circulation.</a> Journal of the American College of Cardiology. 25 (6), 1428-1435 (1995).</li><li>Liedtke, A. J., Pasternac, A., Sonnenblick, E. H., Gorlin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+canine+ventricular+dimensions+with+acute+changes+in+preload+and+afterload.">Changes in canine ventricular dimensions with acute changes in preload and afterload.</a> The American Journal of Physiology. 223 (4), 820-827 (1972).</li><li>Toischer, K., 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=Differential+cardiac+remodeling+in+preload+versus+afterload.">Differential cardiac remodeling in preload versus afterload.</a> Circulation. 122 (10), 993-1003 (2010).</li><li>Zhang, 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=Cellular+Hypertrophy+and+Increased+Susceptibility+to+Spontaneous+Calcium-Release+of+Rat+Left+Atrial+Myocytes+Due+to+Elevated+Afterload.">Cellular Hypertrophy and Increased Susceptibility to Spontaneous Calcium-Release of Rat Left Atrial Myocytes Due to Elevated Afterload.</a> PloS one. 10 (12), 0144309 (2015).</li><li>Hori, M., 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=Loading+sequence+is+a+major+determinant+of+afterload-dependent+relaxation+in+intact+canine+heart.">Loading sequence is a major determinant of afterload-dependent relaxation in intact canine heart.</a> The American Journal of Physiology. 249, 747-754 (1985).</li><li>Schotola, 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=The+contractile+adaption+to+preload+depends+on+the+amount+of+afterload.">The contractile adaption to preload depends on the amount of afterload.</a> ESC Heart Failure. 4 (4), 468-478 (2017).</li><li>Sonnenblick, E. H., Downing, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Afterload+as+a+primary+determinat+of+ventricular+performance.">Afterload as a primary determinat of ventricular performance.</a> The American Journal of Physiology. 204, 604-610 (1963).</li><li>Hirt, M. N., 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=Increased+afterload+induces+pathological+cardiac+hypertrophy:+a+new+in+vitro+model.">Increased afterload induces pathological cardiac hypertrophy: a new in vitro model.</a> Basic Research in Cardiology. 107 (6), 307 (2012).</li><li>Hirt, M. N., 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=Deciphering+the+microRNA+signature+of+pathological+cardiac+hypertrophy+by+engineered+heart+tissue-+and+sequencing-technology.">Deciphering the microRNA signature of pathological cardiac hypertrophy by engineered heart tissue- and sequencing-technology.</a> Journal of Molecular and Cellular Cardiology. 81, 1-9 (2015).</li><li>Dorn, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fuzzy+logic+of+physiological+cardiac+hypertrophy.">The fuzzy logic of physiological cardiac hypertrophy.</a> Hypertension. 49 (5), 962-970 (2007).</li><li>Hill, J. A., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+plasticity.">Cardiac plasticity.</a> The New England Journal of Medicine. 358 (13), 1370-1380 (2008).</li><li>Maillet, M., van Berlo, J. H., Molkentin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+physiological+heart+growth:+fundamental+concepts+and+new+players.">Molecular basis of physiological heart growth: fundamental concepts and new players.</a> Nature Reviews Molecular Cell Biology. 14 (1), 38-48 (2013).</li><li>Stenzig, 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=DNA+methylation+in+an+engineered+heart+tissue+model+of+cardiac+hypertrophy:+common+signatures+and+effects+of+DNA+methylation+inhibitors.">DNA methylation in an engineered heart tissue model of cardiac hypertrophy: common signatures and effects of DNA methylation inhibitors.</a> Basic Research in Cardiology. 111 (1), 9 (2016).</li><li>Werner, T. R., Kunze, A. C., Stenzig, J., Eschenhagen, T., Hirt, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+miR-140-3p+prevents+functional+deterioration+in+afterload-enhanced+engineered+heart+tissue.">Blockade of miR-140-3p prevents functional deterioration in afterload-enhanced engineered heart tissue.</a> Scientific Reports. 9 (1), 11494 (2019).</li><li>Leonard, 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=Afterload+promotes+maturation+of+human+induced+pluripotent+stem+cell+derived+cardiomyocytes+in+engineered+heart+tissues.">Afterload promotes maturation of human induced pluripotent stem cell derived cardiomyocytes in engineered heart tissues.</a> Journal of Molecular and Cellular Cardiology. 118, 147-158 (2018).</li><li>Rodriguez, M. L., Werner, T. R., Becker, B., Eschenhagen, T., Hirt, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetics-Based+Approach+for+Fine-Tuning+Afterload+in+Engineered+Heart+Tissues.">Magnetics-Based Approach for Fine-Tuning Afterload in Engineered Heart Tissues.</a> ACS Biomaterials Science &amp; Engineering. 5 (7), 3663-3675 (2019).</li><li>Mannhardt, 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=Automated+Contraction+Analysis+of+Human+Engineered+Heart+Tissue+for+Cardiac+Drug+Safety+Screening.">Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening.</a> Journal of Visualized Experiments: JoVE. (122), e55461 (2017).</li></ol>

TE and MNH are co-founders of EHT Technologies GmbH. All other authors have nothing to disclose.

The protocol outlined herein describes a new technique for magnetically altering afterload in engineered heart tissues. This technique relies upon the use of a piezoelectric stage to translate a plate of strong magnets towards and away from magnetically responsive racks of silicone posts. The closer the two sets of magnets, the stronger the afterload experienced by the EHTs cultured on them.
There are several steps that are critical to the successful production and use of this system. While fabricating the magnetically responsive silicone racks, it is crucially important to ensure that all of the magnets within the posts are oriented with the same polarity. If a magnet is placed in the reverse orientation, it will serve to weaken rather than augment the strength of the magnetic field. Similarly, this polarity should match that of all the magnets within the magnet plate, else the two sets of magnets will repel, rather than attract one another. Additionally, where possible, refrain from using magnetic materials in the construction of the afterload adjustment device, as they can interfere with the magnetic field. Aluminum is suggested as a primary construction material for this reason. Similarly, if using a piezoelectric stage other than the one listed in the Table of Materials, ensure that it is resistant to magnetic fields and standard cell culture conditions (e.g., 37 &#176;C, 100% humidity, and high CO2 and O2-concentrations). Lastly, keep in mind that most piezoelectric linear stages are meant to be mounted horizontally, as they tend to have a low load capacity. As such, if the weight of the magnet plate exceeds this load capacity, a counterweight should be used to unload the motor.
Despite best practices, it is quite difficult to keep the encoder surface pristine. When this occurs, the stage will stop moving before reaching the target position when running in closed-loop mode. The motion controller&#8217;s red LED will flash, additionally the motion controller software will display a &#8220;No encoder detected&#8221;-error message. To remedy this, the user should clean the surface of the encoder with a lint-free piece of cloth soaked in isopropyl alcohol and let it air dry.
This protocol demonstrates the steps taken by our lab to produce and implement this system. However, several of these steps could be achieved by different means. For example, one could use a force transducer, rather than optical means, to confirm the relationship between magnet spacing and post stiffness. Additionally, custom posts could be designed and fabricated with embedded magnets and braces. However, we have found that precise positioning of these objects is easier to accomplish manually. To fine-tune the range of afterloads applicable by this system, these posts can be manufactured with differing basal stiffnesses or with a different number of magnets. Though, using too many magnets will impede post bending. Alternatively, this can also be achieved by adjusting the size and strength of the magnets within the magnet plate. Larger and stronger magnets will yield higher afterloads.
There are several limitations to this method that could be improved upon in future versions of this system. Namely, the range of applicable afterloads is physically limited by the maximum and minimum spacing between the post magnets and plate magnets. Ideally, the posts used for this system would place the tissues as close to the base of the tissue culture dish as possible, without directly allowing the tissue to touch the bottom of the plate. However, the posts used in these studies were commercially made prior to the development of this system, so the lengths of the posts were not optimized for this platform. Similarly, since the EHT contractility analysis system was constructed prior to this system, it was not designed to allow for electrical cords in or out of the measurement space. As such, the presence of these cords resulted in a small air gap, which allowed for gases to slowly leak out of the inner chamber. This could be ameliorated by adjusting gas flow rates accordingly. However, ideally, a future embodiment of this system would have insulated exit and entry points for these cords. Should one wish to perform these experiments in the absence of the EHT contractility analysis system, the afterload tuning platform can instead be placed within an incubator. Though, the system in its entirety will only fit within a standard incubator if one or more of the shelves are removed, rendering this space unavailable for other cell and or tissue culture purposes. To optically observe the tissues in either environment, lights will be necessary. The LED lights used for this system were found to give off a substantial amount of heat. If left on for long periods, this heat could potentially damage the tissues. As such, for these studies, the lights were only used for short periods while assessing the contractility of the tissues. However, should one desire to consistently observe the tissues, the lighting system will have to be optimized for these purposes.
Afterload has been previously studied in EHT models16,23. However, these works presented techniques that were only able to achieve a singular static increase in load. Alternatively, this work demonstrated how a magnetics-based platform can be used to fine-tune and temporally regulate afterload in EHTs. The results from two separate sets of experiments were used to exemplify the wide range of afterload regimens that can be applied to EHTs using this device. Intended future applications of this system include studies on the effect of the applied afterload regimen (dose and duration) on both tissue maturation and pathological remodeling.

Afterload is the systolic load on the ventricle after it has begun to eject blood1. During cardiac development, an appropriate afterload is of critical importance for cardiomyocyte maturation2. In adulthood, low levels of ventricular afterload (e.g., in bedridden patients with high-level spinal cord injury3 or in very special cases like spaceflight4) can result in hypotrophy of the heart. Conversely, high afterload can lead to cardiac hypertrophy5. While cardiac hypertrophy in endurance athletes or pregnant women is considered beneficial and physiological, hypertrophy associated with long-term arterial hypertension or severe aortic valve stenosis is detrimental as it predisposes one to cardiac arrhythmias and heart failure6. Although the 5-year mortality rate for heart failure patients has reduced from ~70% in the 1980s6 to 40&#8211;50%7 presently, there is still a great need for new therapeutic treatment options for this highly prevalent condition (currently 2.2% of the population in the Western world)8.
In order to investigate the molecular mechanisms of pathological cardiac hypertrophy and to test preventive or therapeutic strategies for treating this disease, in vivo models of afterload have been developed9,10,11,12. While these models have offered beneficial insights into the effects of afterload on ventricular performance, they do not allow for fine control over afterload magnitude. Alternatively, in vitro studies of afterload performed on excised hearts and muscle preparations allow for finer control over tissue loading, but these models are not conducive to longitudinal studies13,14,15.
To overcome these issues, we developed an in vitro model of elevated afterload in engineered heart tissues (EHTs)16,17. This model is a 3-dimensional culture format for rat heart cells embedded in a fibrin matrix suspended between flexible hollow silicone posts. These tissues beat spontaneously (against the resistance of the silicone posts) and perform auxotonic work. We have increased afterload applied to EHTs by a factor of 12 in previous experiments by the insertion of rigid metal braces into the hollow silicone posts for one week. This led to a multitude of changes, characteristic of pathological cardiac hypertrophy18,19,20: cardiomyocyte hypertrophy, partial necroptosis, a decline in contractile force, the impairment of tissue relaxation, reactivation of the fetal gene program, a metabolic shift from fatty acid oxidation to anaerobic glycolysis, and an increase in fibrosis. Though this procedure has been successfully employed in several studies17,21,22, it has some disadvantages. There are only two states, low or very high (12-fold) afterload, and the procedure requires manual handling of the EHTs, which limits its temporal flexibility and poses the risk of contamination.
Recently, Leonard et al. used a similar technique to modulate afterload in EHTs cultured on silicone posts23. Braces of varying lengths were placed around the outside of the posts to restrict their bending motion. The authors of this study reported that a singular small-to-medium increase in load enhanced force development and maturation of human iPS-derived EHTs, while higher loads resulted in a pathological state. However, similar to our own system, this technique only allows for singular increases in afterload, the magnitude of which is dictated by the length of the braces. As such, fine alterations in afterload, modifications in afterload over time, and precise loading regimens are not possible with these techniques.
Here, we provide the protocol for a system that can be used to modulate post-resistance, i.e., afterload of EHTs magnetically24. This platform facilitates the fine-tuning of afterload, enables user-defined afterload regimens, and ensures EHT sterility.

<table>
	<tbody>
		<tr>
			<td>Cylindrical plate magnets</td>
			<td>HKCM</td>
			<td>9962-55184</td>
			<td>h = 14 mm, d = 13 mm</td>
		</tr>
		<tr>
			<td>Cylindrical post magnets</td>
			<td>HKCM</td>
			<td>9962-63571</td>
			<td>h = 2 mm, d = 0.5 mm</td>
		</tr>
		<tr>
			<td>Dental wire</td>
			<td>Ormco</td>
			<td>266-1316</td>
			<td>d = 0.016 inches (0.406 mm)</td>
		</tr>
		<tr>
			<td>GraphPad</td>
			<td>GraphPad Software, La Jolla, California, USA</td>
			<td>version 6.00 for Windows</td>
			<td></td>
		</tr>
		<tr>
			<td>Motion control software for piezo motor</td>
			<td>Micronix USA</td>
			<td></td>
			<td>free download on manufacturer homepage</td>
		</tr>
		<tr>
			<td>Motion controller for piezo motor</td>
			<td>Micronix USA</td>
			<td>MMC-100-01000</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical contractility analysis platform</td>
			<td>EHT technologies</td>
			<td>A0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezoelectric linear motor</td>
			<td>Micronix USA</td>
			<td>PPS-20-15206</td>
			<td>fitted with linear optical encoder, incubator-environment compatible</td>
		</tr>
		<tr>
			<td>Styrene Rod</td>
			<td>Plastruct</td>
			<td>MR-15</td>
			<td>d= 0.015 inches (0.381 mm)</td>
		</tr>
		<tr>
			<td>USB camera</td>
			<td>Reichelt Elektronik</td>
			<td>REFLECTA 66142</td>
			<td></td>
		</tr>
	</tbody>
</table>

magnetic adjustment of afterload in engineered heart tissues

Afterload is known to drive the development of both physiological and pathological cardiac states. As such, studying the outcomes of altered afterload states could yield important insights into the mechanisms controlling these critical processes. However, an experimental technique for precisely fine-tuning afterload in heart tissue over time is currently lacking. Here, a newly developed magnetics-based technique for achieving this control in engineered heart tissues (EHTs) is described. In order to produce magnetically responsive EHTs (MR-EHTs), the tissues are mounted on hollow silicone posts, some of which contain small permanent magnets. A second set of permanent magnets is press-fit into an acrylic plate such that they are oriented with the same polarity and are axially-aligned with the post magnets. To adjust afterload, this plate of magnets is translated toward (higher afterload) or away (lower afterload) from the post magnets using a piezoelectric stage fitted with an encoder. The motion control software used to adjust stage positioning allows for the development of user-defined afterload regimens while the encoder ensures that the stage corrects for any inconsistencies in its location. This work describes the fabrication, calibration, and implementation of this system to enable the development of similar platforms in other labs around the world. Representative results from two separate experiments are included to exemplify the range of different studies that can be performed using this system.

Magnet post stiffness quantification 
A horizontally oriented magnetically responsive silicone post was mounted in a fixed position, and an axially aligned calibration magnet was placed at several defined distances (&#8220;magnet spacings&#8221;) from this post. Test loads of known weight were suspended from the end of the silicone post, causing the post to bend. This deflection was quantified optically. A linear relationship between the gravitational force of the test load and resulting post deflection was observed at all magnet spacings (Figure 6A). The values of stiffness derived from these linear relationships followed a negative exponential trend with increasing magnet spacing (Figure 6B).
Stepwise afterload increase 
Control and magnetically responsive EHTs (MR-EHTs) produced from rat hearts were cultured in the absence of magnetic afterload (0.6 mN/mm for control tissues and 0.91 mN/mm for MR-EHTs) until a plateau in contractile force was reached. On this day (24 days following EHT casting), MR-EHTs and control EHTs had similar mean forces (0.29 mN versus 0.22 mN). Over the next week, the afterload exerted on MR-EHTs was incrementally increased from 0.91 to 6.85 mN/mm, while afterload for control EHTs remained constant. Mean contractile force increased with increasing afterload up to 0.95 mN, which marks more than a 3-fold increase in force compared to the average value (0.29 mN) measured for control EHTs (Figure 7A). Post deflection, on the other hand, decreased compared to control tissues. On the last day of culture, the mean deflection measured for MR-EHTs was only 0.11 mm compared to 0.48 mm for control EHTs (Figure 7B). From day 27 on, force production rate and force decay rate were higher in MR-EHTs than in control EHTs while there was only a transient increase in work over days 25&#8211;28 (Supplementary Figure 2).
Interval afterload regimen 
Rat EHTs on magnetically responsive silicone posts (MR-EHTs) were cultured at a minimal afterload of 0.91 mN/mm until a plateau in contractile force was reached. From this day (17 days following EHT casting) onward, MR-EHTs underwent a 7-day afterload regimen which exposed the EHTs to cycles of afterloads alternating between 0.91 and 6.85 mN/mm (Figure 8A). The afterload of control EHTs was kept constant at 0.60 mN/mm over the entire duration of culture. Following this intervention, average forces for MR-EHTs increased by 12.0% compared to day 17, while those measured for control EHTs only increased by 1.5% over the same time frame (Figure 8B). However, these differences were not statistically significant. Moreover, no significant differences in force production rate, force decay rate and contractile work were measured (Supplementary Figure 3). This implies that the selected afterload regimen was not an efficient means of increasing EHT contractility.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60811/60811fig01.jpg" /> 
Figure 1: Assembled magnetically responsive silicone racks.&#160;(A) Orthogonal view and (B) sectional view of assembled magnetically responsive silicone racks containing five magnets. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60811/60811fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60811/60811fig02.jpg" /> 
Figure 2: Magnet plate.&#160;Photograph of the magnet plate and its attachment bracket. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60811/60811fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60811/60811fig03.jpg" /> 
Figure 3: Mechanical drive system.&#160;Photograph showing the system of mechanical drives used to adjust the horizontal position of the 24-well plate with respect to the magnet plate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60811/60811fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60811/60811fig04.jpg" /> 
Figure 4: LED plate.&#160;Photograph of the LED plate used to illuminate EHTs for optical contractility analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60811/60811fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60811/60811fig05.jpg" /> 
Figure 5: Fully assembled afterload tuning device.&#160;Photograph of the fully assembled afterload tuning device including the LED plate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60811/60811fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60811/60811fig06.jpg" /> 
Figure 6: Optical determination of post stiffness.&#160;(A) The deflection of a magnetically responsive silicone post in the presence of an external calibration magnet and under the influence of five test weights was assessed at nine determined magnet spacings (five shown as examples). (B) Determined relationship between magnet spacing and post stiffness. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60811/60811fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60811/60811fig07.jpg" /> 
Figure 7: Contractile response of EHTs to a stepwise increase in afterload.&#160;Contractile measurements of control EHTs (black line) and EHTs cultured on magnetically responsive posts (MR-EHTs; blue line) over a culture period of 31 days. (A) MR-EHTs had slightly higher average contractile forces than control EHTs under baseline conditions. From day 25 on, however, this difference was amplified with increasing afterload. (B) Post deflection was similar between both groups until day 27. Past afterload values of 3.5 mN/mm, post deflection for MR-EHTs dropped substantially. Here, n = 10 MR-EHTs and n = 10 control EHTs were analyzed by fitting a mixed model (REML = restricted maximum likelihood) and Sidak&#8217;s multiple comparison test. The error bars in graphs represent standard error of the mean, ** p &#60; 0.01, *** p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60811/60811fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60811/60811fig08.jpg" /> 
Figure 8: Contractile response of EHTs to an interval afterload protocol.&#160;The contractile behavior of magnetically responsive EHTs under the influence of a fluctuating regimen of afterload was observed. (A) The afterload regimen, which was initiated on day 17 (d17), exposed the MR-EHTs to 40 s intervals of minimal afterload (0.91 mM/mm) followed by 40 s intervals of maximal afterload (6.85 mN/mm) for 7 days. (B) MR-EHTs (blue bars) showed a trend towards increasing forces during the interval afterload protocol, while the forces measured for control EHTs (black bars) remained relatively unchanged. For these experiments, n = 10 EHTs were analyzed per group and this data was statistically compared using a 2-way ANOVA and Sidak&#8217;s multiple comparison test. Error bars in graphs represent standard error of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60811/60811fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 Rat EHT</td>
			<td>24 Rat EHTs (+10%)</td>
			<td>1 hiPSC-CM EHT</td>
			<td>24 hiPSC-CM EHTs (+10%)</td>
			<td colspan="2">Component</td>
		</tr>
		<tr>
			<td>5 x 105</td>
			<td>1.3 x 107</td>
			<td>1 x 106</td>
			<td>2.6 x 107</td>
			<td colspan="2">Cells (either neonatal rat heart ventricular cells or hiPSC-derived cardiomyocytes)</td>
		</tr>
		<tr>
			<td>5.57 &#956;L</td>
			<td>147 &#956;L</td>
			<td>5.57 &#956;L</td>
			<td>147 &#956;L</td>
			<td colspan="2">2x DMEM: 20% heat-inactivated horse serum, 20% 10x DMEM, 2% penicillin/streptomycin, 58% aqua ad iniectabilia</td>
		</tr>
		<tr>
			<td>2.53 &#956;L</td>
			<td>66.8 &#956;L</td>
			<td>2.53 &#956;L</td>
			<td>66.8 &#956;L</td>
			<td colspan="2">Fibrinogen: 200 mg/mL Fibrinogen dissolved in 0.9% NaCl</td>
		</tr>
		<tr>
			<td>-</td>
			<td>-</td>
			<td>0.1 &#956;L</td>
			<td>2.64 &#956;L</td>
			<td colspan="2">Y-27632</td>
		</tr>
		<tr>
			<td>ad 100 &#956;L</td>
			<td>ad 2640 &#956;L</td>
			<td>ad 100 &#956;L</td>
			<td>ad 2640 &#956;L</td>
			<td colspan="2">EHT-casting medium: 88% DMEM, 10% heat-inactivated fetal calf serum, 1 % penicillin/streptomycin, 1% L-glutamine</td>
		</tr>
	</tbody>
</table>
Table 1: Reconstitution mix for generating EHTs.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Command name</td>
			<td>Syntax</td>
			<td colspan="2">Description</td>
		</tr>
		<tr>
			<td>Move absolute</td>
			<td>1MVA[x]</td>
			<td colspan="2">Stage moves to position [x] in mm</td>
		</tr>
		<tr>
			<td>Set velocity</td>
			<td>1VEL[x]</td>
			<td colspan="2">Stage movement velocity set to [x] in mm/s</td>
		</tr>
		<tr>
			<td>Emergency stop</td>
			<td>1EST</td>
			<td colspan="2">Stops any movement</td>
		</tr>
		<tr>
			<td>Wait for stop</td>
			<td>1WST</td>
			<td colspan="2">Only during program recording; Waits for completion of previous movement command before executing next command</td>
		</tr>
		<tr>
			<td>Wait for time period</td>
			<td>1WTM[x]</td>
			<td colspan="2">Only during program recording; Waits for time period [x] in ms</td>
		</tr>
		<tr>
			<td>Beginn program recording</td>
			<td>1PGM[x]</td>
			<td colspan="2">Begin program recording in slot [x]; Note: Slot [x] needs to be free</td>
		</tr>
		<tr>
			<td>End program recording</td>
			<td>1END</td>
			<td colspan="2">End program recording and save program</td>
		</tr>
		<tr>
			<td>Erase program</td>
			<td>1ERA[x]</td>
			<td colspan="2">Erase program saved in slot [x]</td>
		</tr>
		<tr>
			<td>Execute program</td>
			<td>1EXC[x]</td>
			<td colspan="2">Execute program saved in slot [x]</td>
		</tr>
		<tr>
			<td>Loop program</td>
			<td>1PGL[x]</td>
			<td colspan="2">[x]=1 Program loop mode ON [x]=0 Program loop mode turned off</td>
		</tr>
		<tr>
			<td>Read and clear errors</td>
			<td>1ERR?</td>
			<td colspan="2">Request error report</td>
		</tr>
	</tbody>
</table>
Table 2: Useful commands for afterload tuning experiments.
Supplementary Figure 1: Dimensions of silicone racks. (A) Top view, (B) sectional side view, and (C) detailed post view of the silicone racks used for these studies. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60811/60811supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 2: Additional contractile parameters for stepwise afterload increase. Contractile measurements of control EHTs (black line) and EHTs cultured on magnetically responsive posts (MR-EHTs; blue line) over a culture period of 31 days. (A) MR-EHTs had a significantly higher force production rate than control EHTs from day 27 on. (B) The rate of force decay was also significantly greater in MR-EHTs than in control from day 27 onward. (C) While contractile work measured in control EHTs gradually increased over the entire period of culture, the contractile work produced by MR-EHTs peaked on day 26 and dropped thereafter to levels below control. Yet, work in MR-EHTs was never significantly higher than that in control EHTs. Here, n = 10 MR-EHTs and n = 10 control EHTs were analyzed by fitting a mixed model (REML = restricted maximum likelihood) and Sidak&#8217;s multiple comparison test. The error bars in graphs represent standard error of the mean, * p &#60; 0.05, *** p &#60; 0.001. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60811/60811supfig02.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 3: Additional contractile parameters for interval afterload protocol. The contractile behavior of magnetically responsive EHTs (MR-EHTs) under the influence of a fluctuating regimen of afterload was observed. (A) During the interval afterload protocol, MR EHTs (blue bars) initially showed a trend towards higher force production rates compared to control EHTs (black bars), but these differences were not significant and diminished towards the end of the experiment. (B) Force decay rates measured in MR-EHTs and control EHTs were statistically similar throughout the interval afterload protocol. (C) Contractile work measured for MR-EHTs increased during the first days of the afterload interval protocol but decreased on the last day. The contractile work measured for control EHTs did not noticeably change during this period of time. Work in MR-EHTs was never significantly higher than work in control EHTs. For these experiments, n = 10 EHTs per group were analyzed by 2-way ANOVA and Sidak&#8217;s multiple comparison test. Error bars in graphs represent standard error of the mean. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60811/60811supfig03.jpg" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about Magnetic Adjustment of Afterload in Engineered Heart Tissues at JoVE.com

Magnetic Adjustment of Afterload in Engineered Heart Tissues

This protocol provides detailed methods describing the fabrication and implementation of a magnetics-based afterload tuning platform for engineered heart tissues.

Afterload is known to drive the development of both physiological and pathological cardiac states. As such, studying the outcomes of altered afterload ...

magnetic-adjustment-of-afterload-in-engineered-heart-tissues

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

5.8K Views.  University Medical Center Hamburg-Eppendorf. This protocol provides detailed methods describing the fabrication and implementation of a magnetics-based afterload tuning platform for engineered heart tissues.

Video: Magnetic Adjustment of Afterload in Engineered Heart Tissues

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls are composed of cellulose and non-cellulosic polysaccharides together with proteins, phenolic compounds and water. 90% of the cell wall is composed of&#160;polysaccharides (e.g., pectins) and arabinogalactan proteins (AGPs). The fluorescent immunolocalization of specific glycan epitopes in plant histological sections remains a key tool to uncover remodeling of wall polysaccharide networks, structure and components.
Here, we report an optimized fluorescent immunolocalization procedure to detect glycan epitopes from AGPs and pectins in plant tissues. Paraformaldehyde/glutaraldehyde fixation was used along with LR-White embedding of the plant samples, allowing for a better preservation of the tissue structure and composition. Thin sections of the embedded samples obtained with an ultra-microtome were used for immunolocalization with specific antibodies. This technique offers great resolution, high specificity, and the chance to detect multiple glycan epitopes in the same sample. This technique allows subcellular localization of glycans and detects their level of accumulation in the cell wall. It also permits the determination of spatio-temporal patterns of AGP and pectin distribution during developmental processes. The use of this tool may ultimately guide research directions and link glycans to specific functions in plants. Furthermore, the information obtained can complement biochemical and gene expression studies.

This protocol describes in detail how the plant material for immunolocalization of Arabinogalactan proteins and pectins is fixed, embedded in a hydrophilic acrylic resin, sectioned and mounted on glass slides. We show cell wall related epitopes will be detected with specific antibodies.

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls ...