Rapid myocardial and cardiac relaxation is essential for normal physiology. Mechanical relaxation mechanisms are now known to be dependent on strain rate. This protocol provides an overview of the acquisition and analysis of experiments to further study the mechanical control of relaxation.
Diastolic dysfunction is a common phenotype across cardiovascular disease presentations. In addition to elevated cardiac stiffness (elevated left ventricular end-diastolic pressure), impaired cardiac relaxation is a key diagnostic indicator of diastolic dysfunction. While relaxation requires the removal of cytosolic calcium and deactivation of sarcomeric thin filaments, targeting such mechanisms has yet to provide effective treatments. Mechanical mechanisms, such as blood pressure (i.e., afterload), have been theorized to modify relaxation. Recently, we showed that modifying the strain rate of a stretch, not afterload, was both necessary and sufficient to modify the subsequent relaxation rate of myocardial tissue. The strain rate dependence of relaxation, called the mechanical control of relaxation (MCR), can be assessed using intact cardiac trabeculae. This protocol describes the preparation of a small animal model, experimental system and chamber, isolation of the heart and subsequent isolation of a trabecula, preparation of the experimental chamber, and experimental and analysis protocols. Evidence for lengthening strains in the intact heart suggests that MCR might provide new arenas for better characterization of pharmacological treatments, along with a method to assess myofilament kinetics in intact muscles. Therefore, studying the MCR may elucidate a path to novel approaches and new frontiers in the treatment of heart failure.
Cardiac relaxation is impaired in nearly all forms of heart failure (including heart failure with reduced ejection fraction) and in many cardiovascular diseases. In addition to numerous methods to evaluate cardiac function in permeabilized muscles, the evaluation of intact cardiac muscles is gaining interest. Such tissues are assessed unloaded (ends free to contract) or loaded (length or force controlled). Historically, intact isolated myocytes have been evaluated in an unloaded condition, where the cell body is free to shorten during contraction. Intact cardiac trabeculae are often evaluated in isometric conditions, where the length is not allowed to change, but stress (force per cross-sectional area) is generated. Both intact myocyte and trabeculae methods are beginning to converge with modifications of load1,2.
Protocols for load-clamping a muscle (i.e., controlling a muscle's developed stress at a specified value that simulates physiologic afterloads) have been developed over several decades3,4,5. In intact cardiac tissues, load-clamps allow researchers to more closely mimic the in vivo cardiac cycle using isotonic or Windkessel-like afterloads6,7,8,9. The goal of this protocol is to obtain data used to quantify the MCR (i.e., the strain rate dependence of the relaxation rate)8,9.
While the MCR protocol has been adapted from prior work, the focus of this protocol (compared to similar protocols utilizing intact cardiac tissues) is on biomechanical mechanisms that modify relaxation. There are several protocols utilizing load-clamping3,4,5,7,10 and protocols focusing on Windkessel models1,2,11, but this protocol describes specifically how stretch prior to relaxation modifies the relaxation rate. We have shown that this control occurs during a proto-diastolic period8, a phase originally described by Wiggers12. In normal healthy hearts, the myocardium undergoes a lengthening strain during ejection prior to aortic valve closure (i.e., prior to isovolumic relaxation)13. This is mimicked by prolonging the duration of afterload control until the muscle begins to stretch. Clinical evidence suggests that this lengthening may be attenuated or lost in disease states14, and the implications and mechanisms of altered end-systolic strain rates have not been fully elucidated. Given the sparse treatment options for diastolic diseases and heart failure with a preserved ejection fraction, we posit that MCR may provide insight into novel mechanisms underlying impaired relaxation.
While the gross dissection described here focuses on rodents, trabecula isolation can be performed from any intact heart, and has previously been used with a human cardiac trabecula8. Similarly, the data acquisition and analysis can also be applied to cardiomyocytes or other isolated muscle types1,10. The discussion includes commentary on possible alterations and adaptations to the method, along with limitations, such as caution against utilizing papillary muscles because of the mechanical properties of the chordae9.
The following protocol has been approved by the Institutional Animal Care and Use Committee of Wayne State University. The protocol here describes steps for utilizing rodent experimental subjects, but can be adapted for use in other model organisms.
1. Preparation
2. Gross dissection and cannulation
3. Isolation and equilibration of trabecula
4. Data acquisition
5. Data analysis
A representative data set is shown in Figure 4, and additional results can be found in prior publications8,9. Briefly, the strain rate is calculated from the derivative of the strain, just prior to isometric relaxation. Strain is the length as a function of time divided by the length of the muscle at optimal length. Relaxation rate is calculated as 1/τ, where τ is the exponential time constant16. Multiple strain rates and their resultant relaxation rates are required to determine the mechanical control of relaxation (MCR). These data are plotted on a relaxation rate versus strain rate graph. The slope of the line provides the MCR index.
Note that end systolic and diastolic strain rates are not likely to exceed 1 s-1. Therefore, the slope should only include strain rates < 1 s-1. The relaxation rate at low strain rates can be confounded by changes in the minimum time derivative of stress (dStress/dtmin), and in these cases, data from stretches less than approximately 0.15 s-1 may be ignored.
Figure 1: Setup of gross dissection and heart cannulation area. From right to left: a. The anesthesia induction chamber for the animal is located near the dissection area. b. A snorkel for optional volatile gas scavenger. c. A dissection pad, where the animal will be placed supine, is flanked by (clockwise) d. a nosecone, to provide continued anesthesia to the rodent, e. hemostats, f. surgical scissors, g. curved fine scissors placed for easy access with the dominant (cutting) hand, h. curved iris forceps placed for easy access with the non-dominant hand. Upper limbs of the rodent may be affixed to the dissection pad using tape, and i. a dissection dish (or small beaker) with perfusion solution should be placed nearby to rinse the heart. j. An area staged for cannulation must be placed nearby. A syringe with an appropriate cannula is mounted on a ring stand. k. (Inset) A closer image of a 16 G cannula, with 1 mm of PE205 tubing attached and a suture loosely knotted. Please click here to view a larger version of this figure.
Figure 2: Experimental area and tools. (A) Experimental area. The experimental chamber and data acquisition system are prepared on a nearby inverted microscope (left). Trabecula isolation and mounting occurs under a stereoscope (right). (B) Tips of two forceps, large and small Vannas scissors, and a custom glass probe are prepared by soaking in 10% BSA solution. (C) Additional dissection tools. A silicone elastomer-plated dish allows for mounting the heart during fine dissection. A 7 mL transfer pipette with the end cut is shown below the dish and glass probe. The cut tip of the transfer pipette is discarded, and an enlarged bore is used to transfer the muscle, with minimal risk of stretching or dehydration. (D) Magnified image of the end of a glass pipette, which is approximately 2 mm long and 0.25-0.5 mm in diameter. Please click here to view a larger version of this figure.
Figure 3: Guide to dissection. (A) Gross dissection should begin with a transverse cut (1. green) through the skin into the peritoneal cavity below the xyphoid process (lower ribs and xyphoid are indicated by a thin gray line). Parasagittal cuts from the peritoneal cavity up the ribcage should follow (2. teal and 3. blue lines), after which the diaphragm should be cut. A hemostat can then be used to clamp the xyphoid process and elevate the chest wall toward the head. (B) An intact rat heart pinned to a silicon-elastomer dish, oriented with a view of the right ventricular outflow tract (RVOT), right atrium (RA), aorta (Ao), and left atrium (LA). The first set of cuts should be from the RVOT to the apex along the septum (1. Yellow line). A second set of cuts should be from the RVOT along the base of the heart then across the right atrium (2. orange line). (C) The RVOT can be carefully pulled away from the aorta to open the heart and pin it back. Yellow and orange lines correspond to cuts described in B. Free standing trabeculae are often found near the base of the RV free wall and near the septum, but can occur anywhere (red lines indicate common locations). (D) Magnified view of the glass probe under a trabecula (yellow arrow indicates intersection of trabecula and probe). (E) A trabecula (red arrow) is mounted to the experimental system between a force transducer (left) and motor (center-right), and is flanked by two pacing leads (horizontal above and below the trabecula). Please click here to view a larger version of this figure.
Figure 4: Representative traces and results. (A) A single cardiac trabecula illuminated using a gooseneck LED light at a steep (~75°) angle from the axis of the microscope lens, which enhances contrast of the trabecula. In this example, two images are tiled to show the full length of the trabecula. (B) Stress-time (top) and strain-time (bottom) curves for the same trabecula. An isometric twitch, along with three load-clamp twitches at increasing end systolic strain rates are shown. (C) Representative MCR calculation. MCR is defined as the slope of the line between the relaxation rate and strain rate. As noted in the discussion, strain rates may be restricted to provide a quantitative, reproducible MCR. Please click here to view a larger version of this figure.
Table 1: Solutions. Please click here to download this Table.
Mechanical control of relaxation (MCR) quantifies the dependence of the myocardial relaxation rate on the strain rate of the muscle proceeding relaxation8,9. Strain rate, rather than afterload, is both necessary and sufficient to modify the relaxation rate8. As interventions to modify the calcium rate have not been proven to substantially improve cardiac relaxation, mechanical intervention may provide new insights into the mechanism and provide a new treatment for diastolic dysfunction.
The protocol to modify the myocardial strain rate described here utilizes an isotonic load-clamp8,9. A strength of the isotonic load-clamp is the quantitative control of the afterload stress. Windkessel-like protocols could be used to further probe changes in afterload, preload, and cardiac work2,6,7. A ramp not controlled by the load-clamp could also be used to better isolate the change in strain from strain rate. Regardless, the afterload itself does not appear to be a strong modifier of the relaxation rate8.
The protocol may also be adapted to approach more physiological conditions for temperature and pacing rate. The current protocol details were used to show the presence of the MCR. Conducting experiments in physiological conditions is generally recommended, depending on the experimental question. However, experiments performed at 37 °C, or at high pacing rates, can more rapidly induce rundown (damage) to the muscle. A solution with improved oxygen carrying capacity may be needed. Further, the data acquisition must be able to sample the length and force fast enough to resolve the rapid twitches and provide feedback control.
The current protocol does not describe the measurement of calcium or the measurement and control of sarcomere lengths. Calcium measurements have been addressed in other protocols11, while sarcomere length measurement can be added with appropriate equipment. Sarcomere length control is not utilized in current MCR studies, because muscle length is the most correlative parameter to the clinical condition19. Further sarcomere length control (vs. muscle length control) would provide specific answers to kinetic questions, but is unlikely to add to translational knowledge due to inter-sarcomere variation and the minimum understanding of sarcomere length changes in vivo.
Three experimental considerations are highlighted here to increase the reproducibility of the data.
First, free-standing cardiac trabeculae may be difficult to find in some animals (unpublished results and communications). While twitching muscles can be found in most rats, a reasonable success rate for obtaining data from trabecula in rats is one in three. Trabecula success may be higher with Brown Norway x Lewis F1 rats, which have also been used historically20 and reported to have more trabeculae (unpublished communications). For mice, success rates are likely to be lower, with less than one in 10 expected for mice from a BL/6 background; however, a higher rate is expected for mice from a FVBN background (unpublished communications and observations).
Second, damage to muscles can reduce output. If the developed forces are less than 10 mN mm-2 at 25 °C and 0.5 Hz pacing, investigators may need to perform troubleshooting to assess if inadvertent stretching or contact between metal forceps and muscle is occurring, if solutions are not properly prepared, or if pacing or experimental equipment is functioning properly. Other protocols utilizing intact trabecula have suggested using Luer-lock syringes as transfer vessels11. While this is possible, especially if the user controls a very slow flow rate or smaller muscle segment, the current protocol utilizes a much larger bore transfer pipette to minimize possible damage. Another step where ischemic damage may occur is during dissection. The aorta should be cannulated and flushed with perfusion solution within 3 min of the first abdominal cut (rat) or cervical dislocation (mouse), similar to limits listed in cardiomyocyte isolation protocols21,22. This minimizes the time when the cardiac tissue is not exposed to the cardioplegia-like perfusion solution. Further, dissections lasting more than 30 min generally do not produce twitching trabecula. Thus, operators should practice rapid but careful dissection to minimize damage. A cross-sectional area above 0.2 mm2 (2 x 10-7 m2) may suffer from core ischemia20.
Third, the way in which muscles are attached to the motor and force transducer should be considered. This protocol currently focuses on hooks and free-standing trabeculae. The sometimes rapid strain rate of the stretch prior to relaxation can cause a muscle to slide if not affixed properly, which is why the current protocol does not use "baskets" to hold the trabecula23,24. Alternate mounting methods (adhesives, clips, etc25,26) can also be considered and validated. The protocol described here utilizes trabeculae and not papillary muscles. The chordae of the papillary muscle induce a series elasticity that can inhibit changes to the MCR9. However, the exact placement of the attachments in the muscle is unlikely to impact the measures, because the trabecula length (and diameter) vary substantially.
A limitation of piercing the muscle ends with hooks is that the mounting point itself can also be damaged. Possible tearing of the affixed muscle tissue with frequent contractions (depending on their strength) may change the length or series elasticity. This rate of tearing is difficult to control. Similarly, damage to the tissue and the hook can be exacerbated during stretching, also potentially causing problems. Visual inspection, and the developed force values remaining >80% of the equilibrated isometric force, should be used to assess whether the preparation is damaged and should be excluded.
Another limitation or consideration affects what experimental questions can be answered by the method. For example, consider the use of 2,3-butanedione monoxime (BDM) in the perfusion solution. BDM is a phosphatase, which may alter the function of the muscle. In addition, the long period of unloading and lack of pacing mean that the latent phosphorylation state has likely changed. Thus, caution should be used if trying to directly assess the muscle contractility of an animal (vs. differences between genotypes or treatments), as the contractile state has likely changed. However, the impact of phosphorylation may be assessed pharmacologically by adding an agonist or antagonist of the pathway.
In summary, MCR provides insight into how relaxation is regulated by muscle movement (strain rate). MCR may help to provide better insight into the diagnosis and monitoring of diastolic disease, along with targets for pharmacological intervention, such as modifying myosin kinetics. The protocol and advice outlined here lay out knowledge developed over several years of trials, and should be applicable to other systems and models of cardiac disease.
The authors have nothing to disclose.
This work is supported by the National Institutes of Health (1R01HL151738) and the American Heart Association (18TPA34170169).
18 or 16 gauge blunted needle/canula | for cannulation of rat aorta, use 1mm of PE160 or PE205 tubing as stop | ||
2,3-Butanedione Monoxime | Sigma-Aldrich | B0753-25G | |
23 gauge blunted needle/canula | for cannulation of mouse aorta, use 1mm of PE50 tubing as stop | ||
5 mL syringe | BD Luer-Lock | 309646 | |
95% Oxygen/5% CO2 | AirGas | Z02OX9522000043 | |
Anethesia system | EZ Systems | EZ-SA800 | Can use any appropriate anethesia method/system |
Bovine Serum Albumin | Fisher BioReagents | BP-1600 | to coat tips of fine forcepts, scissors |
Calcium Chloride Dihydrate | Fisher Chemical | C79-500 | |
Containers/dissection dishes | FisherBrand | 08-732-113 | Weigh dishes for creating dissection plates |
Crile Hemostat | Fine Science Tools | 13005-14 | for mouse gross dissection |
D-(+)-Glucose | Sigma-Aldrich | G8270-1KG | |
Data acquisition software | SLControl | ||
Data acquisition system | MicrostarLabs | DAP5216a | Can use any DAQ. This is a PCI based data acqusition for use with SLControl; must have a PC with a PCI slot |
Data analysis software | Mathworks | Matlab | Custom Script |
Dumont #3 Forceps | Fine Science Tools | 11231-30 | 2x for cannulation of aorta |
Dumont #5 Forceps | Fine Science Tools | 11254-20 | 2x for trabecula isolation |
Experimental system | Aurora Scientific | 801C | Can use any appropriate experimental chamber with force and length control |
Fine Scissors, curved | Fine Science Tools | 14061-09 | for removal of heart |
Gooseneck Piggyback Illuminator | AmScope | LED-6WA | |
HEPES | Sigma-Aldrich | H3375-250G | |
Imaging software | IrfanView | ||
Iris Forceps | World Precision Instruments | 15915 | for removal of heart |
Isoflurane | VetOne | 502017 | |
Magnesium Chloride Hexahydrate | Sigma-Aldrich | M2670-100G | |
Magnesium Sulfate | Sigma-Aldrich | M7506-500G | |
Mayo Scissors | Fine Science Tools | 14110-15 | for rat gross dissection |
Metzenbaum Scissors | Fine Science Tools | 14116-14 | for mouse gross dissection |
Microscope connected camera | Flir | BFS-U3-27S5M-C | Includes acquisition software |
Microscope/digital imaging system | Olympus | IX-73 | Can use any appropriate microscope. Needed to measure muscle length, cross sectional area |
Mounting Pin/Needle | BD PrecisionGlide | 305136 | For holding heart to dish. 27 G x 1-1/4 |
Mounting Pin/Needle | Fine Science Tools | 26000-40 | For holding heart to dish. 0.4mm diameter insect pin (Alt to 27G needle) |
Oxygen (O2) | AirGas | OX USP300 | |
Peristaltic Pump | Rainin | Rabbit | Can be any means to create flow in experimental chamber |
pH and Oxygen sensor | Mettler Toledo | SevenGo pH and DO | |
Potassium Bicarbonate | Sigma-Aldrich | 237205-100G | |
Potassium Chloride | Fisher Chemical | P217-500 | |
Potassium Phosphate Monobasic | Sigma-Aldrich | 795488-500G | |
Rochester-pean Hemostat | World Precision Instruments | 501708 | for rat gross dissection |
Silk Suture, Size: 4/0 | Fine Science Tools | 18020-40 | cut to ~1.5 inch pieces, soaked in water |
Sodium Bicarbonate | Sigma-Aldrich | S6297-250G | |
Sodium Chloride | Sigma-Aldrich | S9888-1KG | |
Sodium Hydroxide | Sigma-Aldrich | S8045-500G | |
Sodium Phosphate Dibasic | Sigma-Aldrich | S7907-100G | |
Stereomicroscope | AmScope | SM-1TX | |
Student Vannas Spring Scissors | Fine Science Tools | 91500-09 | for opening of the RV |
SYLGARD 184 Silicone Elastomer Base | Dow Corning | 3097358-1004 | For creating dissection plates |
Syringe Holder | Harbor Frieght | Helping Hands 60501 | Can be used as alternate for ring stand |
Taurine | Sigma-Aldrich | T0625-1KG | |
Transfer Pipette | FisherBrand | 13-711-7M | cut ~1" from tip to widen bore |
Vannas Spring Scissors | Fine Science Tools | 15000-00 | for trabecula isolation |