Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.
The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.
Biomechanical forces, such as hemodynamic shear stress, are intimately involved in cardiac morphogenesis. In response to hemodynamic shear forces, myocardial ridges and grooves develop in a wave-like trabecular network in alignment with the direction of the shear stress across the atrioventricular (AV) valve1. Cardiac trabeculation is necessary to increase contractile function and myocardial mass2. Mutations in Notch signaling pathways result in congenital heart defects in humans and other vertebrates3. For example, gata1a4 and tnnt2a5 morpholino oligonucleotides (MO) have been shown to reduce erythropoiesis, while erythropoietin mRNA (EPO)6 and Isoproterenol (ISO)7 increase red blood cells and heart rate respectively, and therefore wall shear stress (WSS). Furthermore, ErbB2 signaling, downstream of Notch, promotes cardiomyocyte proliferation and differentiation to generate contractile force, which in turn activates Notch signaling8,9. It is suggested that shear stress governs Notch signaling driven trabeculation for ventricular development. Currently, there are many studies that attempt to further understand the genetic programming events leading to congenital heart defects (CHD)10,11,12, but very little are investigating how mechanical forces influence the forming heart.
In order to investigate the mechanical forces acting on the endocardium, close observation during the developmental period needs to be implemented. However, it is challenging to obtain good quality images of in vivo beating samples due to the inherence of traditional microscopy13. In order to observe the development over time within a sample, physical sectioning and staining, therefore, need to occur13,14,15. Although confocal microscopy is widely used to image the 3-D structure of samples14,16, these imaging systems' acquisition is still limited by slow scanning speeds.
Light-sheet fluorescence microscopy (LSFM) is a unique imaging technique that allows the visualization of in vivo dynamic events with long working distance13. This technique uses a light-sheet fluorescent microscopy to optically section a sample17. Due to illumination of only a thin sheet of light on the sample, there is a reduction in photo-bleaching and photo toxicity13,18. The large field of view and long working distance allows for large samples to stay intact as they are imaged13,14,17. The low magnification allows for a larger area to be imaged, while the long working distance allows for thicker samples to be imaged without compromising the signal-to-noise ratio. Many groups have used LSFM to image entire embryos17, brains14,18, muscles and hearts19 among other tissues, showing the diverse types of samples that can be imaged.
Although previous research demonstrated reduced hemodynamic shear force by occluding the inflow or outflow tracks of the zebrafish heart, the information is solely qualitative. It results in an abnormal third chamber, diminished cardiac looping, and impaired valve formation20. The 4-D LSFM images give a new perspective into the way the hemodynamic shear forces affect the development of the cardiac tissue. These mechanical forces may activate force-sensitive signaling molecules and induce the formation of the trabecular ridges. Because of the added time aspect of 4-D imaging, one is able to track changes in development in real time, which could lead to new revelations that had gone unnoticed previously. The zebrafish is an ideal model for imaging because scientists can observe an entire vertebrate animal versus only cell-cell interactions. Oxygen can also diffuse through the entire embryo, which allows development to occur without depending on the vascular system, unlike in mammalian development. Even though the zebrafish heart lacks the pulmonary organs, which require a four-chambered heart, there is a large number of cardiac genes that are conserved between zebrafish and humans21.
In this manuscript, we describe how to use light-sheet fluorescence microscopy to image the developing trabeculae in zebrafish hearts under various circumstances. First, injection of gata1a4 or tnnt2a5 MOs were used to lower the blood viscosity, and therefore WSS. The morphology of the heart was then recorded. In a separate group of fish, we increased the WSS by administering EPO mRNA6 or isoproterenol7 and observed the results. We also conducted a cell study with different pulsatile or oscillatory flow rates. After imaging each group, we found that WSS sensed by the endocardium via Notch signaling initiates trabeculation.
The following methods were performed in compliance with UTA and UCLA IACUC protocols. These experimental groups were used with transgenic Tg(cmlc2:gfp), the wea (weak atrium) or clo (cloche) mutants: (a) wild-type (WT) control, (b) gata1a MO, and (c) tnnt2a MO injections (Table 1).
Model | Name | Modified Genes | Phenotype | Reference |
Control | Wild type | None | N/A | |
Tg(cmlc2:gfp) | Cardiac myosin light chain | Green flourescence specifically expressed in the myocardium | 34 | |
Decreased Wall Shear Stress | Weak atrium (wea) mutant | Atrium-specific myosin heacy chain | Atrium cannot contract, compact ventricle, thick myocardial wall, narrow lumen, dialated atrium, | 37 |
Cloche (clo) mutant | N/A | Complete removal of endocardium, unaturally large atrium with small ventricle, nonexistent cardiac cushins | 41 | |
gata1a MO | gata1a | Anemia from lack of red blood cells | 4 | |
tnnt2a MO | tnnt2a | 39 | ||
Increased Wall Shear Stress | EPO mRNA | None | Severe polycythemia, increase in number of circulating blood cells, increased viscosity | 6 |
Isoprotenerol | None | Increased cardiac rate | 7 |
Table 1: Zebrafish descriptions. Definitions and descriptions of Zebrafish used in experiments.
1. Study Setup
2. Imaging Techniques
3. qRT-PCR analysis
4. In vitro Human Aortic Endothelial Cell (HAEC) Experiments
5. Rescue and over-expression of Notch signaling
6. Quantification of Fractional Shortening and volume change over time
LSFM was used in this manuscript in order to acquire high-resolution 2-D and 3-D pictures. As seen in Figure 1A and 1B, the illumination lens directs the light sheet at the sample. Because of the thinness of the light sheet, only a single plane is illuminated. The detection lens is positioned perpendicular to the illumination lens and is focused on the illuminated plane (Figure 1B). The light sheet from the illumination lens then scans the sample from left to right (Figure 1C). The scanning allows less photo-bleaching, photo-toxicity, and has a lower signal-to-noise ratio.
LSFM was applied to Tg(cmlc2a:gfp) embryos to visualize the entire cardiac structure34 by rapid scanning (> 30 s) with a voxel resolution of 0.65 x 0.65 x 1 µm. Due to a single plane illumination, background noise was significantly reduced (Figure 1). Trabecular ridges protruded into the ventricular lumen at 75 hpf, and a trabecular network clearly was shown at 100 hpf (Figure 2A, B). Gata1a MO micro-injection reduced hematopoiesis and viscosity by 90%4,35. This decreased viscosity attenuated hemodynamic shear stress, resulting in a delayed initiation and density of trabecular network at 75 hpf and 100 hpf when compared to control group (Figure 2C, D). Furthermore, Notch ligands (Dll4, Jag1, and Jag2), receptor (Notch1b), and downstream signaling components (Nrg1 and ErbB2) were down-regulated in response to gata1a MO injection (p < 0.05, n=5) (Figure 2K)2,8,36. Co-injection of gata1a MO with 5 μM of Nrg1 mRNA up-regulated Notch signaling-related gene expression and rescued trabecular formation at 75 hpf and 100 hpf (Figure 2E, F, K). Thus, gata1a MO reduced hemodynamic forces leading to down-regulation of Notch signaling, whereas Nrg1 mRNA rescue up-regulated Notch-related genes and re-initiated trabeculation.
In order to further prove our hypothesis that changing hemodynamic forces and thus trabeculation via Notch signaling, we introduced the wea mutant that develops a non-contractile atrium37. In the absence of ventricular inflow and hemodynamic forces during atrial systole, wea mutants express lower cardiac mRNA levels of Notch signaling component genes and target genes compared to controls, and harbor non-trabeculated, small and weakly contracting ventricles (Figure 2G, L). However, Nrg1 mRNA micro-injection into the wea mutants up-regulated the Notch signaling pathway, accompanied by the partial rescue of trabecular ridges, leading to a more pronounced contractile ventricle despite a non-contractile atrium (Figure 2H, L)2. In this context, the wea mutants corroborated the relation between the non-contractile atrium and a non-trabeculated ventricle, underscoring the hemodynamic modulation of trabeculation via Notch signaling.
Tnnt2a MO was delivered to stop cardiac contraction by inhibiting cardiac troponin T38,39. The tnnt2a MO-injected fish were completely devoid of circulation and ventricular WSS due to significant down-regulated Notch signaling, leading to a smooth ventricular surface and thin wall when compared to control group (Figure 2I, M). To investigate whether WSS on the endocardium was a requirement for trabeculation, we also have cloche (clo) mutants for which endocardium and the endothelial lining of the heart do not develop40,41. The clo mutant failed to develop cardiac trabeculae and showed smaller sized ventricle and incomplete cardiac looping (Figure 2J). Thus, the endocardial lining is necessary to sense hemodynamic shear stress to activate Notch signaling (Figure 2M). However, when the hemodynamic shear stress was increased by increasing hematopoiesis or treatment of isoproterenol to increase contractility, the expression levels of Notch-related genes did not increase, and the trabecular network morphology did not change (Figure 3).
To further demonstrate the relationship between shear stress and Notch signaling, HAECs were used for in vitro tests. A pulsatile flow was exerted on confluent cells to simulate the hemodynamic shear stress observed by endothelial cells. It is shown that compared to static culture, pulsatile culture increases expression of Notch-related genes (Figure 4). Furthermore, treatment with the ADAM10 inhibitor significantly reduced Notch signaling (Figure 4).
Next, we calculated ventricular fractional shortening, and ventricular cavity change in volume over time for the wild-type, gata1a MO, AG1478, and those rescued by Nrg1 injection groups at 50 hpf, 75 hpf, and 100 hpf (Figure 5A-C). Treatment with ErbB signaling inhibitor, AG1478, significantly delayed and reduced fractional shortening during ventricular diastole at 100 hpf (Figure 5A). Both gata1a MO and AG1478 treatment reduced ventricular chamber size during contraction, as assessed by the changes in 4-D LSFM (Figure 5E, F). Both groups developed an increased end-systolic and -diastolic volume when compared to the wild-type. Co-injection of Nrg1 mRNA with gata1a MO restored both fractional shortening, and Ejection Fraction toward those of the wild type.
Figure 1: Fluorescent light sheet overview. (A) Representative LSFM system where the sample is placed in the intersection of the light sheet from the illumination lens (IL) and the detection path of the detection lens (DL). (B) The cylindrical lens behind the IL creates the micron-sized light sheet (purple) within the sample, which excites a thin sheet of the sample. The detection lens records the emitted fluorescent signal. (C) A schematic of the thin laser sheet (purple) optically sectioning the zebrafish embryo which moves right to left. This figure has been modified from Lee et al17. Please click here to view a larger version of this figure.
Figure 2: Ventricular morphologies of genetically manipulated Tg(cmlc:gfp) zebrafish with Notch Ligand, receptor, and target gene expression. (A) The trabecular network is shown at 75 hpf of control zebrafish. (B) The trabecular network at 100 hpf of control zebrafish. (C) Injection of gata1a MO at the 1- to 4-cell stage showed little to no trabeculation at 75 hpf. (D) Injection of gata1a MO into zebrafish had delayed trabecular network formation. (E) Co-injection of gata1a MO and human Nrg1 mRNA partially restored trabeculation at 75 hpf. (F) Co-injection of gata1a MO and human Nrg1 mRNA almost completely restored the trabecular network at 100 hpf. (G) wea mutant at 100 hpf shows no trabeculation and a smooth ventricular wall. (H) Injection of human Nrg1 into wea mutant zebrafish partially restored trabeculation at 100 hpf. (I) Injection of tnnt2a MO showed no trabeculation at 75 hpf (not shown) and 100 hpf. (J)clo mutant showed no trabeculation at 100 hpf. (K) Injection of the gata1a MO significantly reduced mRNA expression of Notch1, Nrg1, and Jag2 (t-test, *P < 0.05, n=5 vs. control). Co-injection of gata1a MO and human Nrg1 mRNA significantly increased the expression of Notch1b, Nrg1, ErbB, and Jag1 compared to control. (L) The wea mutant had the significantly lower expression of Notch-related genes (t-test, *P < 0.05, n=5 vs. control). Injection of human Nrg1 increased the expression of Notch-related genes; Jag1 and Jag2 were significantly higher than control. (M) The tnnt2a MO injection and clo mutant showed a significantly lower expression of Notch-related genes (t-test, *P < 0.05, n=5 vs. control). Scale bars: 50 μm. Data are shown as the mean standard deviation. This figure has been modified from Lee et al17. Please click here to view a larger version of this figure.
Figure 3: Increasing WSS in manipulated Tg(cmlc:gfp) zebrafish with Notch ligand, receptor and target gene expression in vivo. Increasing the WSS via over-expression of hematopoiesis with the injection of EPO(A) or addition of ISO (B) via increasing heart rate did not increase trabeculation and had a trabecular network similar to that of the control. (C) The injection of EPO mRNA or Isoproterenol did not change the expression of Notch ligand, receptor or target genes (t-test, *P < 0.05, n=5 vs. control). This figure has been modified from Lee et al17. Please click here to view a larger version of this figure.
Figure 4: Endothelial cells subject to oscillatory or pulsatile shear stress in vitro. HAECs subject to the pulsatile shear stress of τaverage = 30 x 10-5 N/s at 1 Hz had upregulated Notch-related gene expressions and downregulated of Notch-related gene expressions when the ADAM10 inhibitor was applied (t-test, *P < 0.05, n=5 vs. control). This figure has been modified from Lee et al17. Please click here to view a larger version of this figure.
Figure 5: Effect of trabeculation of fractional shortening and ejection fraction. (A-C) Addition of AG1478 and gata1a MO significantly decreased the fractional shortening at 100 hpf, but co-injection of human Nrg1 and gata1a MO improved it. (D) gata1a injection and AG1478 significantly decreased the fractional shortening at 100 hpf (t-test, *P < 0.05, n=5 vs. control). Co-injection of gata1a and Nrg1 mRNA improved the fractional shortening. (E-F) Integrating the 4-D synchronization algorithm with LSFM showed that AG1478 and gata1a MO had increased end systolic and diastolic volumes at 75 hpf (E) and 100 hpf (F). Data are shown as the mean standard deviation. This figure has been modified from Lee et al17. Please click here to view a larger version of this figure.
Zebrafish Jag1 | Forward | CCGCGTATGTTTGAAGGAGTATCAGTCG |
Backward | CAGCACGATCCGGGTTTTGTCG | |
Zebrafish Jag2 | Forward | AGCCCTAGCAAAACGAGCGACG |
Backward | GCGTGAATGTGCCGTTCGATCAA | |
Zebrafish Dll4 | Forward | CAAAGTGGGAAGCAGACAGAGCTAAGG |
Backward | CGGTCATCCCTGGGTGTGCATT | |
Zebrafish BMP10 | Forward | GCATCAAGGGGCCACTCGTGTAGA |
Backward | TCGTCTCACTCCACTAGGTCCCATACTG | |
Zebrafish ErBb2 | Forward | GATCAGGACTGCCAAACATTGACGTCT |
Backward | AGCAGCACACTGAACATGGCAGCA | |
Zebrafish Nrg-1 | Forward | GTGTGTTTGTCCCTGTGGACGCGT |
Backward | CCTCCTGGAGCTTCCCCTCAAACA | |
Zebrafish Notch1b | Forward | CAGAGAGTGGAGGCACAGTGCAATCC |
Backward | GCCGTCCCATTCACACTCTGCATT |
Table 3: Primers used for Zebrafish screening.
In this protocol, we have shown that 4-D imaging can be used to track the development of a trabecular network in response to changes in biomechanical forces. In particular, the shear stress experienced by endothelial cells initiates the Notch signaling cascade, which in turn promotes trabeculation. In this manuscript, we have shown that (1) gata1a MO injection decreased hematopoiesis and therefore it reduced wall shear stress, (2) tnnt2a MO injection inhibited ventricular contractile function to reduce wall shear stress, and (3) wea mutants lacked atrial contraction, which then decreased the hemodynamic force applied to the ventricle. By reducing the shear stress on the cardiac walls, we quantified the Notch signaling via qRT-PCR and found that with reduced WSS, there was reduced Notch signaling. To further prove our hypothesis, clo mutants that lacked endocardium did not form trabeculae, and subjecting endothelial cells to pulsatile shear stress with ADAM10 present showed that shear stress activates Notch signaling.
The other imaging techniques such as confocal microscopy can be used to image samples in 3-D14. However, this technique and others have many drawbacks. For example, the confocal imaging cannot penetrate deeply into samples, has the low axial resolution, and slow scanning speeds19,42. Because of the penetration depth limit in confocal imaging, small samples, such as zebrafish embryos, can only be imaged in its entirety. Two-photon confocal microscopy improves the penetration depth, but the resolution, slow scanning speed, and high cost make this technique undesirable13. There have been studies that combine two-photon microscopy and LSFM successfully18, however, the drawbacks are still prominent42.
LSFM, on the other hand, has the least photo-bleaching and damage13,18,42, fast acquisition speed, and similar penetration depth13. Additionally, because of the dual lenses and excitation of a single plane (Figure 1), the background noise is substantially reduced43. However, the samples need to be embedded into gels to immobilize the sample, which makes it difficult to apply to in vivo models other than zebrafish14,17,19. Also, utilization of the fluorescent light limits the depth penetration to a few millimeters. Even though LSFM is a great method for visualization, it is critical that it is supplemented with quantitative data. Because LSFM is only qualitative, it can be subject to various interpretations. Using qRT-PCR, we could confirm that the LFSM 4-D images were a correct representation of the events occurring in the live samples. Occasionally in larger samples, stripes and shadows can form in the images. However, dual-sided illumination or mSPIM can be utilized to reduce these artifacts42. Because LSFM is so versatile, its capability of capturing high resolution and overall better-quality images makes the future of LSFM promising. Mentioned previously, LSFM can be combined with other imaging systems for successful imaging18,42.
We demonstrated previously that applying a pulsatile shear stress of 23 x 10-5 N at 1 Hz for 24 h substantially upregulates Notch signaling in vitro(Figure 4)17. We further demonstrated that shear forces activate Notch signaling by genetic manipulation of hematopoiesis (gata1a MO)4, cardiac contraction (tnnt1a MO)5, atrial contraction (wea mutant), endocardial deletion (clo mutant), and localization of Notch signaling (Tg[flk:mCherry;tp1:gfp]). We describe here that by lowering the wall shear stress in the ventricle, that Notch-related genes were down-regulated (Figure 2K, L, M). Furthermore, we were able to demonstrate that Nrg1 mRNA was able to rescue trabeculation, restore Notch signaling, and improve ventricular fractional shortening and ejection fraction (Figure 2E, F, H, K-M, Figure 5). The restoration of cardiac function was shown by the increase in contractile function-mediated hemodynamic forces that activated Notch signaling (Figure 2K-M).
We also determined that the shear stress-activated Notch signaling is endocardial dependent. By using gata1a MO, tnnt2a MO, wea mutant or AG1478 treatment, trabeculation was inhibited and Notch signaling was down-regulated (Figure 2K-M). However, Nrg1 rescued trabeculation and Notch signaling in wea mutants and when co-injected with gata1a MO (Figure 2D, H, K, L). When wall shear stress (EPO injection) or Notch signaling (10 pg/nL Nrg1 injection) was increased, there was no change in the EPO group in terms of morphology, but abnormal ventricular morphogenesis was present in the Nrg1 increased dose (Figure 3).
In conclusion, we have shown that the mechanotransduction of shear stress activates the Notch signaling pathway. By applying 4-D LSFM and genetically engineered zebrafish, we developed a new model system that shows how critical blood flow is during cardiac development to its morphology, contractility, and overall function.
The authors have nothing to disclose.
The authors would like to express gratitude to William Talbot from Stanford University for providing the Human Nrg1 cDNA and to Deborah Yelon from UCSD for providing the wea mutants. The authors would also like to thank Cynthia Chen for helping with image acquisition. This study was supported by grants NIH HL118650 (to T.K. Hsiai), HL083015 (to T.K. Hsiai), HD069305 (to N.C. Chi and T.K. Hsiai.), HL111437 (to T.K. Hsiai and N.C. Chi), HL129727 (to T.K. Hsiai), T32HL007895 (to R.R. Sevag Packard), HL 134613 (to V. Messerschmidt) and University of Texas System STARS funding (to J. Lee).
Clontech Hifi PCR pre-mix | Takara | 639298 | PCR mastermix 1.1.1.1, 1.1.1.2, 1.1.1.5, 1.1.2.1, 3.1.4 |
Human Nrg1 cDNA | Gift from William Talbot, Stanford University, Stanford, California, USA | N/A | Used for trabeculation rescue 1.1.1.3, 1.1.1.4, 1.1.2.1 |
CFX Connect™ Real-Time PCR Detection System | Bio-Rad | 1855201 | PCR Machine 1.1.1.5, 1.1.1.6, 1.1.2.2, 1.1.3.2 |
pCS2+ | GE Health | Plasmid used to synthesize mRNA 1.1.2.1, 1.1.2.4, 1.1.2.5 |
|
Nucleospin purification kit | Clontech | 740609.25 | DNA Purification 1.1.2.3, 1.1.2.4, 1.1.6.2 |
T4 DNA ligase | Clontech | 2011A | PCR Ligation solution 1.1.2.5 |
Stellar competent cells | Clontech | 636763 | E. coli cells used for transformation 1.1.2.6, 1.1.3.1, 1.1.3.2 |
Lipofectamine 2000 transfection reagent | Life Technologies | 11668027 | Transfection reagent 1.1.4 |
mMessage SP6 kit | Invitrogen | AM1340 | Kit used to synthesize mRNA 1.1.6.3 |
Aurum Total RNA Mini Kit | Bio-Rad | 7326820 | Purifies RNA 1.1.6.4, 3.1.2 |
GeneTools 4.3.8 | GeneTools | N/A | Software for primer design 1.2.1, 3.1.3 |
EPO cDNA | Creative Biogene | CDFH006026 | Increases WSS 1.2.2, 1.2.3, 1.1.7 |
AG1478 | Sigma-Aldrich | T4182 | ErbB inhibitor 1.3.1 |
E3 medium | To grow embryos 1.3.1, 1.3.2, 5.1.5 |
||
DAPT | Sigma-Aldrich | D5942 | γ-secretase inhibitor 1.3.2 |
Agarose | Sigma-Aldrich | A9539 | Used for mounting embryos 2.1.1.1 |
ORCA-Flash4.0 LT Digital CMOS camera | Hamamatsu Photonics | C11440-42U | Used to capture Images 2.1.1.2, 2.1.1.3 |
Amira Software | FEI Software | N/A | Visualized and Analysed images into 3D, and 4D 2.1.5.1.1-2.1.5.2.8 |
Tricaone mesylate | Sigma-Aldrich | 886-86-2 | Used to humanely sedated or sacrifice embryos 3.1.1 |
iScript cDNA Synthesis Kit | Bio-Rad | 1708890 | Synthesizes cDNA 3.1.2 |
Eppendorf 5424 microcentrifuge | Eppendorf | 05-400-005 | Microcentrifuge 4.1.1.3 |
GI254023X | Sigma-Aldrich | 260264-93-5 | ADAM10 inhibitor 4.1.2, 4.1.3 |
Isoprenaline hydrochloride | Sigma-Aldrich | I5627 | Isoproterenol increases WSS 5.1.5 |
MATLAB | Mathworks | N/A | Cardiac mechanics analysis |