This protocol describes how to measure contractility in adult human primary cardiomyocytes from donor hearts with the MyoBLAZER system, a reliable platform for assessing drug-induced changes in contractility during preclinical development.
The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This paper describes a protocol for assessing changes in contractility in adult human primary ventricular cardiomyocytes utilizing the MyoBLAZER, a non-invasive optical method that preserves the normal physiology and pharmacology of the cells. This optical recording method continuously measures contractility transients from multiple cells in parallel, providing both medium-throughput and valuable information for each individual cell in the field of view, enabling the real-time tracking of drug effects. The cardiomyocyte contractions are induced by paced electrical field stimulation, and the acquired bright field images are fed to an image-processing software that measures the sarcomere shortening across multiple cardiomyocytes. This method rapidly generates different endpoints related to the kinetics of contraction and relaxation phases, and the resulting data can then be interpreted in relation to different concentrations of a test article. This method is also employed in the late stages of preclinical development to perform follow-up mechanistic studies to support ongoing clinical studies. Thus, the adult human primary cardiomyocyte-based model combined with the optical system for continuous contractility monitoring has the potential to contribute to a new era of in vitro cardiac data translatability in preclinical medical therapy development.
Myocardial contractility (inotropy), which represents the natural capacity of the heart muscle to contract, is a key property of cardiac function and depends on the dynamics of the electro-mechanical coupling. Drug-induced changes in myocardial contractility are desired to treat heart disease (e.g., heart failure) and unsought in the context of cardiotoxicity (e.g., reduction in left ventricular ejection fraction). Therefore, preclinical contractility models must be associated with accurate predictivity to make sure novel drugs can succeed during clinical development. However, current preclinical strategies, which rely on reductionist artificial cellular models (e.g., genetically engineered immortalized cell lines that overexpress specific cardiac targets of interest) and non-human animal models, have shown significant limitations and have been found to be associated with high drug attrition rates (i.e., a high rate of false signals)1,2,3,4. Accordingly, it is imperative to establish new and reliable human cellular heart contractility models that are associated with high power (i.e., high rate of true signals) to predict drug outcomes in humans and, hence, help to accelerate the launch of new therapies5.
The groundbreaking methods recently established for the recovery of human donor hearts for research6,7,8,9,10 and in cardiomyocyte isolation techniques11,12,13,14,15 have provided a unique opportunity for conducting human-based studies during preclinical development. To this end, adult human primary cardiomyocytes have already shown utility in assessing drug-induced changes in human heart contractility11,12,13,14. The current article details the protocol for investigating the contractility effects of novel compounds in adult human cardiomyocytes.
All methods were carried out in accordance with relevant guidelines and regulations. All human hearts used for the studies were non-transplantable and ethically obtained by informed legal consent (first person or next-of-kin) from cadaveric organ donors in the United States (US). The recovery protocols and in vitro experimentation were pre-approved by Institutional Review Boards (IRBs ) at transplant centers within the US Organ Procurement Transplant Network (OPTN). Furthermore, all transfers of the donor hearts are fully traceable and periodically reviewed by US federal authorities.
NOTE: Apply all necessary safety procedures during the execution of this protocol, including wearing appropriate personal protective equipment (e.g., laboratory coats, safety glasses, gloves).
1. Isolation of cardiomyocytes (1 day before measuring contractility)
2. Laminin coating preparation (1 day before measuring contractility)
3. Preparation of Ca2+-tolerant cardiomyocytes (on the day of measuring contractility)
4. Preparation of the recording system (on the day of measuring contractility)
NOTE: The recording system includes a temperature control box, stimulator, computer-controlled pressure-driven perfusion, pressure-driven perfusion bottles, in-house acquisition software permitting the selection of regions of interest (ROIs) and display of contractility transients, inverted microscope, cell chamber, and camera (Figure 1).
5. Preparation of test compounds (on the day of measuring contractility)
6. Plating of cardiomyocytes (on the day of measuring contractility)
7. Recording of cardiomyocyte contractility
8. Turning off the optical contractility recording system
9. Analysis of contractility data
Described in this protocol is a procedure for measuring contractility in isolated adult human primary cardiomyocytes from organ donors and evaluating acute changes in contractility parameters induced by a test compound. The measurement of the contractility transient is accomplished using a video-based cell geometry system, the MyoBLAZER, that measures sarcomere dynamics, and the adult heart condition is emulated by inducing contractility with electrical stimulation at the physiological pacing frequency (1 Hz). The functional viability of the cardiomyocytes is confirmed by assessing their excitation-contraction coupling (i.e., cellular processing linking electrical excitation [the sarcolemmal action potential] with contraction). There are no spontaneous contractility transients in human cardiomyocytes, and the cardiomyocytes respond to external electrical stimulation with contractility/relaxation cycles (Figure 2C, E), as well as to isoproterenol, a β-adrenergic agonist (Figure 2F,G). Isoproterenol causes a concentration-dependent increase in contractility (Figure 2G), and its effects on the kinetics of contractility transient can also be characterized (Figure 2D)12.
Figure 1: Experimental setup of the optical contractility recording system. Bright-field contractility recordings are made from adult human primary cardiomyocytes as previously described11,12,13,14. The setup includes a (A) temperature control box, (B) field stimulator, (C,D) pressure-driven perfusion system, (E) computer pressure-driven program, (F) pressure plus bottles, (G) acquisition software, (H) selection of ROIs with acquisition software, (I) display of contractility transients with acquisition software, (J) inverted microscope, (K) microscope chamber, and (L) camera. All features of the equipment are given in the Table of Materials. Please click here to view a larger version of this figure.
Figure 2: Validation of human cardiomyocyte contractility measurement and the effect of β-adrenergic stimulation. (A) Phase contrast microscopy image of a representative adult human primary cardiomyocyte, (B) user-defined regions of interest (ROI) placed over the images of healthy cardiomyocytes and parallel to the axis of contraction, (C) display of contractility transients obtained from the same ROIs in (B) with the acquisition software, (D) parameters related to the contractility transient measured with the acquisition software: contractility amplitude, time to peak, Decay 30, Decay 90, TR90. Additional parameters can also be measured: baseline sarcomere length, time to 50% peak, peak height, sarcomere length at peak contractility, maximum contraction velocity, and maximum relaxation velocity. (E) Typical contractility transients recorded from an adult human primary ventricular myocyte at a pacing frequency of 1 Hz in the presence of vehicle control (F) and after exposure to 30 nM isoproterenol, a non-selective β-adrenergic agonist. The contractility transients demonstrated in panels E and F were collected from the same cardiomyocyte. Isoproterenol from human cardiomyocytes paced at 1 Hz pacing frequency was used to generate the potency information. (G) Typical cumulative concentration-response curve generated by human cardiomyocyte contractility measurement with isoproterenol (n = 8 cells). Please click here to view a larger version of this figure.
Perfusion Sequence | Vehicle solution | Concentration 1 (µM) | Concentration 2 (µM) | Concentration 3 (µM) | Concentration 4 (µM) | Wash |
Treatment Time | 120 s | 300 s | 300 s | 300 s | 300 s | 300 s |
Stimulation Frequency | 1 Hz | 1 Hz | 1 Hz | 1 Hz | 1 Hz | 1 Hz |
Table 1: Stimulation protocol and test compound application sequence. The stability of contractility transients is assessed by continuous recording for 120 s in solution C, establishing the vehicle control (typically in 0.1% DMSO). Subsequently, the test article concentration is applied for a minimum of 300 s or until a steady-state effect is attained. Four ascending test article concentrations are used, providing cumulative concentration-effect (C-E) curves. At the end of the cumulative additions of the test article, a 300-s washout period can be implemented.
This manuscript provides a detailed protocol for the adult human cardiomyocyte contractility-based optical system for a simplified medium-throughput method that enables the testing of the acute efficacy and cardiotoxicity of novel compounds. This optical contractility recording system is easy to use, allows recordings from multiple cells in parallel, enables the simultaneous assessment of cell health, physiology, and pharmacology, comes with automated and rapid data analysis (a run of multiple cells is analyzed in 5 s), and permits rapid data collection (concentration-response curve every 30 min/compound/device). Taking into consideration these attributes, the recording system can be used not only to detect the effects of drugs on cardiomyocyte contractility but also to provide structure-activity relationship data for supporting medicinal chemistry efforts during the early phases of drug discovery16. Since tens of millions of cells can be obtained from a cardiomyocyte isolation protocol, the application of the optical contractility recording system-cardiomyocyte platform is currently being explored to achieve increased testing capacity (with the use of well-based plates) with reduced cost. Moreover, the assessment of drug effects on the systolic and relaxation parameters measured with the recording system can provide multiparametric mechanistic profiling of inotropic drugs12. Additionally, cardiomyocyte contractility data can be used to rank novel drugs from most to least cardiotoxic (e.g., safety margin) and from least to most efficacious (e.g., potency margin). Follow-up cardiomyocyte contractility studies can also be conducted to support development programs that have been associated with a clinical decrease in myocardial contractility12.
Another significant advantage of using the human cardiomyocyte contractility optical recording system is its alignment with the 3Rs concept (replacement, reduction, and refinement)17 since it can be considered as an alternative method that avoids or replaces the use of animals for data generation within the pharmaceutical industry. This 3Rs benefit can also be extended to academic cardiac research. The entirety of current knowledge of cardiomyocyte physiology and pharmacology comes from academic research studies conducted with cells isolated from animal hearts18. Thus, the human cardiomyocyte optical contractility model opens the possibility for critical translational studies to be performed. To perform these studies, protocols for the preservation and shipment of human adult cardiomyocytes must be developed (currently under evaluation in AnaBios' laboratory), and the contractility system must have the ability to record changes in sarcomere length from non-human cardiomyocytes (this is the case with the optical contractility recording system since sarcomeres are well conserved among species).
The human cardiomyocyte contractility system can emulate several physiological conditions (e.g., electromechanical coupling, pacing frequency mimicking heart rate, body temperature, the integration of all human cardiac targets) and has demonstrated translational value as a key component in drug discovery11,12,13,14, although it cannot mimic the changes in mechanical load and shear stress seen during the cardiac contractile cycle. The structure and function of cardiac extracellular matrices are now better understood19, the development of such matrices can potentially help overcome the mechanical load limitation, and matrices with different heart-like stiffnesses are currently being evaluated in AnaBios' laboratory. Another limitation of the human cardiomyocyte optical contractility system is the absence of the network of nerves that supplies the heart (e.g., sympathetic and parasympathetic fibers)20. This neuro-cardiac contact can be re-established with the co-application of neurotransmitters (e.g., isoproterenol, an agonist of β-adrenoceptor receptors; acetylcholine, an agonist of M2 muscarinic receptors), with the compound being assessed for its potential effects on cardiomyocyte contractility. Furthermore, the contractility transients are recorded with no simultaneous measurements of action potentials and Ca2+ transients, which are also essential when evaluating drug effects on the electrocardiogram and Ca2+ handling. Although this omission can be considered a limitation of the system, it is not too critical to have since the recordings of action potential signals (with the current-clamp method or voltage-sensitive dyes) and Ca2+ transients (with Ca2+ indicators/dyes) can be associated with cytotoxicity. Such cytotoxic effects can impact the assessment of novel drugs to modulate heart contractility. On the contrary, the use of a non-invasive optical method that preserves the health, physiology, and pharmacology of the cardiomyocytes, like the recording system described in this protocol, would not only ensure that the highest quality of contractility data is obtained but also provide data that can predict well the contractile effects of novel drugs in humans.
The authors have nothing to disclose.
This work was supported by the AnaBios Corporation and an NIH Small Business Innovation Research (SBIR) grant (1R44TR003162-01).
100–1000 µL Filtered, Wide Orifice, Sterile tips | Pipette | UF-1000W | |
100 mL, Duran pressure plus bottles | DWK Life Sciences | 218102406 | |
1 L, 0.22 µm Vacuum Filter system | VWR | 567-0020 | |
290 mmol/kg Osmolarity Standard | Wescor | OA-029 | |
Benchtop pH Meter | Mettler Toledo | https://www.mt.com/us/en/home/products/Laboratory_Analytics_Browse/pH-meter/pH-meters.html | |
Calcium Chloride dihydrate (CaCl2) | Sigma-Aldrich | C3881 | |
Camera | Optronis GmbH | Cyclone-25-150-M | https://optronis.com/en/products/cyclone-25-150/ |
Corning 25 mm x25 mm Square #1 Cover Glass | Corning | 2845-25 | |
Cyclone-25-150 | Optronis | https://optronis.com/en/products/cyclone-25-150/ | |
D-(+)-Glucose | Sigma-Aldrich | G8270 | |
Digital Timer/Stopwatch | Fisher Scientific | 14-649-17 | |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D8418 | |
Eight-well rectangular polystyrene sterile culture plate | Thermo Fisher Scientific | 73521-426 | https://us.vwr.com/store/product/4679368/nunclontm-delta-rectangular-dishes-polystyrene-sterile-thermo-scientific |
FHD Microscope Chamber System | IonOptix | ||
Flow EZ, Modular pressure-based flow controller with a computer driven program version 1.1.0.0. | Fluigent OxyGEN | ||
Heavy Duty Vacuum Bottles | VWR | 16211-080 | |
HEPES | Sigma-Aldrich | H3375 | |
Human Recombinant Laminin 521 | BioLamina | LM521-05 | |
Idex Chromatography Tubing, Natural FEP, 1/16" OD x 0.030" ID | Cole-Palmer | 1520L | |
Kimberly-Clark Professional Kimtech Science Kimwipes | Fisher Scientific | 06-666 | |
L-(-)-Malic acid | Sigma-Aldrich | 112577 | |
Lactobionic acid | Sigma-Aldrich | 153516 | |
L-Glutamic acid | Sigma-Aldrich | 49449 | |
L-Histidine | Sigma-Aldrich | H8000 | |
Magnesium Chloride hexahydrate (MgCl2) | Sigma-Aldrich | M9272 | |
Microscope Temperature Control Stage Warmer | AmScope | TCS-100 | |
MyoPacer Field Stimulator | IonOptix | ||
Nunc Rectangular Dishes | Thermo Scientific | 267062 | |
Olympus IX83P1ZF Ixplore Standard microscope | Olympus | https://www.olympus-lifescience.com/en/microscopes/inverted/ixplore-standard/?campaignid=657680540&adgroupid =116963199831&keyword=ix73%20 microscope&gclid=EAIaIQobChMIl qjyiMWP-AIVVx-tBh2JoQ85EAA YASAAEgLp3fD_BwE |
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pH 4.01, 7.00, and 10.01 Standards | Oakton | WD-05942-10 | |
Potassium Chloride (KCl) | Sigma-Aldrich | 746436 | |
Potassium Hydroxide (KOH) | Sigma-Aldrich | P4494 | |
Potassium phosphate monobasic (KH2PO4) | Sigma-Aldrich | 795488 | |
Prism Software | GraphPad Software – Dotmatics | https://www.graphpad.com/ | |
RBS 25 Liquid Detergent | Sigma-Aldrich | 83460 | |
Sharps Container | Uline | S-15307 | |
SigmaPlot analysis software | Systat Software Inc. | https://systatsoftware.com/ | |
Sodium Chloride (NaCl) | Sigma-Aldrich | S3014 | |
Sodium Hydroxide (NaOH) | Sigma-Aldrich | 221465 | |
Student Dumont #5 Forceps | Fine Science Tools | 91150-20 | |
Sucrose | Sigma-Aldrich | S7903 | |
Taurine | Sigma-Aldrich | T0625 | |
Temperature Control Box | Warner Insturments | TC-324C | |
Vapor Pressure Osmometer | ELITechGroup | Model 5600 | |
Wheaton 20 mL Vials | DWK Life Sciences | 225288 |