Name: magnetic adjustment of afterload in engineered heart tissues
Uploaded: 2020-05-05T13:30:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Magnetic Adjustment of Afterload in Engineered Heart Tissues at JoVE.com

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.

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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. 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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>&lt;strong&gt;MMC-100-01000&lt;/strong&gt;</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. Dit systeem kan worden gebruikt om gemanipuleerde hartweefsels bloot te stellen aan verschillende regimes van afterload, om de effecten van deze stimuli op weefselkrachtontwikkeling, remodellering en rijping te bestuderen. Onze techniek maakt het mogelijk om nauwkeurig te onderwerpen kloppen weefsels om een breed scala van aanpasbare afterload routines over langdurige perioden van cultuur zonder zelfs de noodzaak om de celkweek plaat te openen. Onze methode kan worden aangepast om na belastin...

Video: Magnetic Adjustment of Afterload in Engineered Heart Tissues

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Bioengineering

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Medicine

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

In Vivo Mechanotransduction Study Using Magnetic Beads in the Zebrafish Heart

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

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

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

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

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

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents

To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, mice undergo RR upon removal of the aortic constriction. In this paper, we describe a step-by-step procedure to perform a minimally invasive surgical aortic debanding in mice. Echocardiography was subsequently used to assess the degree of cardiac hypertrophy and dysfunction during LV remodeling and RR and to determine the best timing for aortic debanding. At the end of the protocol, terminal hemodynamic evaluation of the cardiac function was conducted, and samples were collected for histological studies. We showed that debanding is associated with surgical survival rates of 70-80%. Moreover, two weeks after debanding, the significant reduction of ventricular afterload triggers the regression of ventricular hypertrophy (~20%) and fibrosis (~26%), recovery of diastolic dysfunction as assessed by the normalization of left ventricular filling and end-diastolic pressures (E/e&#39;&#160;and LVEDP). Aortic debanding is a useful experimental model to study LV RR in rodents. The extent of myocardial recovery is variable between subjects, therefore, mimicking the diversity of RR that occurs in the clinical context, such as aortic valve replacement. We conclude that the aortic banding/debanding model represents a valuable tool to unravel novel insights into the mechanisms of RR, namely the regression of cardiac hypertrophy and the recovery of diastolic dysfunction.

Here we describe a step-by-step protocol of surgical aorta debanding in the well-established mice model of aortic-constriction. This procedure not only allows studying the mechanisms underlying the left ventricular reverse remodeling and regression of hypertrophy but also to test novel therapeutic options that might accelerate myocardial recovery.

To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, ...

Quantification of Biventricular Function and Morphology by Cardiac Magnetic Resonance Imaging in Mice with Pulmonary Artery Banding

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. Assessment of RV function and morphology is complex, partly due to the complex shape of the RV. Currently, cardiac magnetic resonance (CMR) imaging is the golden standard for noninvasive assessment of RV function and morphology. The current protocol describes CMR imaging in a mouse model of RV pressure load induced by pulmonary artery banding (PAB). PAB is performed by placing a 6-0 suture around the pulmonary artery over a 23 G needle. The PAB gradient is determined using echocardiography at 2 and 6 weeks. At 6 weeks, the right and left ventricular morphology and function is assessed by measuring both end-systolic and end-diastolic volumes and mass by ten to eleven cine slices 1 mm thick using a 9.4 T magnetic resonance imaging scanner equipped with a 1,500 mT/m gradient. Representative results show that PAB induces a significant increase in RV pressure load, with significant effects on biventricular morphology and RV function. It is also shown that at 6 weeks of RV pressure load, cardiac output is maintained. Presented here is a reproducible protocol for the quantification of biventricular morphology and function in a mouse model of RV pressure load and may serve as a method for experiments exploring determinants of RV remodeling and dysfunction.

To understand the pathophysiology of right ventricular (RV) adaptation to abnormal loading, experimental models are crucial. However, assessment of RV dimensions and function is complex and challenging. This protocol provides a method to perform cardiac magnetic resonance imaging (CMR) as a noninvasive benchmark procedure in mice subjected to RV pressure load.

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. ...

Cardiac Response to &#946;-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of specific treatments on the heart. Due to the feasibility of genetic manipulations the mouse has become the most common mammalian animal model to study cardiac function and to search for new potential therapeutic targets. Here we describe a protocol to determine cardiac function&#160;in vivo using pressure-volume loop measurements and analysis during basal conditions and under &#946;-adrenergic stimulation by intravenous infusion of increasing concentrations of isoproterenol. We provide a refined protocol including ventilation support taking into account the positive end-expiratory pressure to ameliorate negative effects during open-chest measurements, and potent analgesia (Buprenorphine) to avoid uncontrollable myocardial stress evoked by pain during the procedure. All together the detailed description of the procedure and discussion about possible pitfalls enables highly standardized and reproducible pressure-volume loop analysis, reducing the exclusion of animals from the experimental cohort by preventing possible methodological bias.

Here we describe a cardiac pressure-volume loop analysis under increasing doses of intravenously infused isoproterenol to determine the intrinsic cardiac function and the &#946;-adrenergic reserve in mice. We use a modified open-chest approach for the pressure-volume loop measurements, in which we include ventilation with positive end-expiratory pressure.

Determination of the cardiac function is a robust endpoint analysis in animal models of cardiovascular diseases in order to characterize effects of ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Magnetic Adjustment of Afterload in Engineered Heart Tissues

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