Patent Application
20220196636
Improvements in methodologies for assessing drug cardiotoxicity during preclinical testing would remove dangerous drugs early on from the pipeline and retain effective drugs, thereby providing cost savings for pharmaceutical companies. The general approach to cardiotoxicity testing has been to evaluate a drug's ability to delay action potential repolarization by measuring its inhibition of hERG channels and the degree to which it causes QT prolongation, a marker for risk of tissue-level arrhythmias. This approach, however, does not capture the biological complexity of cardiac cells and can result in inaccurate assessment of a drug's arrhythmogenic potential. In the last decade, new tools for preclinical cardiac testing have emerged that address this shortcoming by relying on human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), which represent large numbers of patient-specific cardiac cells. This new field has expanded to include more complex, tissue-like preparations of hPSC-CMs and new tools for functional evaluation of the cells, based on fluorescent reporters, automated patch clamp, and impedance measurements, in an effort to improve drug testing accuracy. Most approaches, however, have been restricted to spontaneous activity of cardiac preparations and fail to control for the rate-dependence of the various ion currents, which can influence the response to drugs.
In some aspects, described herein is a method for evaluating a sample comprising cardiac cells. The method comprises providing at least one sample comprising one or more cardiac cells; pacing the one or more cardiac cells at two or more fixed rates; and measuring a fixed response of the one or more cardiac cells at each of the two or more fixed rates.
In some aspects, described herein is a method for evaluating the cardiac effect of a compound. The method comprises providing at least one sample comprising one or more cardiac cells; contacting the at least one sample with the compound; pacing the one or more cardiac cells at two or more fixed rates; and measuring a fixed response of the one or more cardiac cells to the compound at each of the two or more fixed rates. In some aspects, a plurality of samples is provided, wherein each of the samples comprises at least one cardiac cell.
In some aspects, the method further comprises measuring a spontaneous response of the one or more cardiac cells to the compound prior to pacing the one or more cardiac cells at the two or more fixed rates. In certain aspects, the two or more fixed rates have a range from about 0.5 Hz to about 2 Hz.
Measuring the spontaneous response and/or fixed response of the one or more cardiac cells may comprise measuring one or more of pace-capture, repolarization time, contraction strength, and conduction velocity. In some aspects, the method further comprises reporting the measured spontaneous response and/or fixed response of the one or more cardiac cells on a binary classification scale. The reported spontaneous response and/or fixed response of each cardiac cell may be compiled into a single visual representation. In some aspects, the visual representation is color coded.
In certain aspects, the sample comprises a single cell, a cell monolayer, a cell cluster, engineered cardiac tissue, cardiac organoid, cardiac tissue explant, or whole heart.
Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:
The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the presently disclosed subject matter are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.
Drug development is a long and costly process characterized by a high degree of attrition in potential drug candidates. For example, an average of 12 years and $2.6 billion in research and development is required to bring a drug to market. Only 13.8% of drugs make it from Phase I to approval. Cardiac effect is the most common reason for withdrawing candidate drugs from the development pipeline, with 30% of drugs withdrawn during the clinical development phase (most because of adverse cardiovascular effects, Arrowsmith and Miller, 2013) and a high number of withdrawn in the post-marketing phase (Magdy et al., 2018).
As such, a need exists for accurate methods of screening drugs for cardiac effect early in the development pipeline. Disclosed herein are new analytical tools for assessing cardiac responses to candidate drugs. In particular, disclosed herein are new methods for evaluating the cardiac effect of compounds that involve measuring the response of one or more cardiac cells to a given compound at different pacing rates, thus allowing for evaluation of the rate-dependence of various ion currents that can be affected by cardiac drugs as well as the cardiac cell response over the entire dynamic range of normal and abnormal heart rates.
The advancement in cardiac differentiation strategies for human pluripotent stem cells (hPSCs) (Burridge et al., 2012) has opened up opportunities for new in vitro studies of human cardiomyocytes (CMs). Widespread and reliable use of hPSC-CMs, however, requires the development of preparations that can recapitulate essential features of myocardial structure and function: e.g., elongated CMs in arrays that mimic myofiber bundles, coordinated contraction, fast and uniform conduction of action potentials (APs), and appropriate sensitivity to cardioactive drugs. To this end, a variety of strategies have been employed to make tissue-like constructs, including casting hPSC-CMs in hydrogels (Tzatzalos et al., 2015), seeding them onto synthetic matrices (Ma et al., 2014), and fabricating cell sheets (Matsuura et al., 2012). These efforts have resulted in structurally organized, multicellular preparations that promote more mature states of cardiac gene expression, contraction, calcium handling, and conduction. Their ultimate utility for in vitro studies, however, may be hampered by the inability to maintain functionality during long-term culture and the absence of instructive cues typically present in the adult myocardium.
An emerging strategy is to use decellularized myocardial matrix as a source of biochemical, topographical, and biomechanical cues present in the heart to direct differentiation and maturation of PSC-CMs. Decellularized myocardial matrix decreases stem cell pluripotency and induces differentiation in iPSCs (Carvalho, 2012) and early cardiac progenitor cells (Lu et al., 2013). The idea that multi-component extracellular matrix (ECM) can enhance cardiac differentiation has been demonstrated with hydrogels composed of solubilized acellular porcine matrix (Duan et al., 2011) or solubilized basement membrane Matrigel preparations (Zhang et al., 2012). Cell culture coatings made from these solubilized, acellular matrix sources or from decellularized sheets of supporting cells enhance the structural organization of CMs (Baharvand et al., 2005) and temporally advance the expression of cardiac genes and proteins in cardiac progenitor cells (French et al., 2012). Acellular matrix can improve the response of single hiPSC-CMs to cardiac drugs so that they more closely resemble that of adult CMs (Feaster et al., 2015). While these findings indicate that decellularized matrix may be suited to guide cellular organization, promote CM lineage commitment, accelerate maturation, and promote better physiological responses to cardiac drugs, reseeding decellularized myocardium with hPSCs and differentiating these cells into a dense tissue-like network of CMs has proven difficult. Furthermore, an important step toward the creation of a truly tissue-like preparation of human CMs would be the demonstration of a high degree of electrophysiological and contractile function in preparations generated on decellularized matrices.
While decellularized myocardium provides biological cues that direct cardiomyocytes organization and function, repopulating it to create a highly functional electrical and mechanical syncytium has proven challenging. The presently disclosed subject matter provides, in part, engineered heart slices from thin decellularized sheets and heart cells derived from reprogrammed human skin cells, blood cells and other somatic cells that can be used for long-term electrophysiological and contractile studies and drug testing. In the presently disclosed subject matter, decellularized myocardial slices were repopulated with hiPSC-CMs to make EHS. The EHS manufactured in this way exhibited coordinated contractions and anisotropic electrical conduction; could be cultured and retained electrical and contractile function for >200 days; and had different sensitivities to ion-channel drugs than cell monolayers. As provided herein, this EHS can be used to evaluate cardiac effect of a candidate drug compound.
More particularly, described herein are methods for evaluating a sample comprising cardiac cells. The methods described herein may assess the ability of a sample comprising cardiac cells to recapitulate essential features of myocardial structure and function. For example, the methods described herein may be used to measure any number of responses that are indicative of electrophysiological maturity, physiological state and/or long-term health of a sample comprising cardiac cells. The methods involve providing at least one sample comprising one or more cardiac cells, pacing the one or more cardiac cells at two or more fixed rates; and measuring a fixed response of the one or more cardiac cells at each of the two or more fixed rates. In some embodiments, the method comprises measuring a spontaneous response of the one or more cardiac cells before pacing the cells at the two or more fixed rates.
Also described herein are methods for evaluating the cardiac effect of a compound. The method for evaluating the cardiac effect of a compound comprises obtaining at least one sample comprising one or more cardiac cells, administering the compound to each sample, pacing the one or more cardiac cells at two or more fixed rates, and measuring a fixed response of the one or more cardiac cells to the compound at each of the two or more fixed rates. The cardiac cell may be paced at the first of the two or more fixed rates before or after administration of the compound. In some embodiments, the method further comprises measuring a spontaneous response of the one or more cardiac cells to the compound prior to pacing the one or more cardiac cells at the two or more fixed rates.
For any of the methods described herein a plurality of samples may be provided, each sample comprising one or more cardiac cells. Alternatively, a single sample may be provided.
Any suitable method may be used to pace the cardiac cell at the two or more fixed rates. Pacing can be applied in multiple ways as known in the art, including through electrical and optogenetic systems.
The cardiac cell may be paced at any suitable rate. In general, the pacing rates may span the physiological and pathophysiological range appropriate for the intended species. Accordingly, the pace rates used may vary based upon the species from which the cardiac cell is derived. For example, the heart rate of smaller mammals, such as a mouse, tends to be faster than the heart rate of larger mammals, such as a human. Accordingly, selection of the appropriate pace rates will be dependent on the species of cardiac cell used. In some embodiments, the cardiac cell may be paced at two or more fixed rates, wherein each rate ranges from about 0.3 Hz (18 bpm) to about 10 Hz (600 bpm). For example, the cardiac cell may be paced at about 0.1 Hz to about 10 Hz, about 0.5 Hz to about 7.5 Hz, about 1 Hz to about 5 Hz, about 2 Hz to about 4 Hz, or about 3 Hz. For example, the cardiac cell may be paced at two or more of about 0.5 Hz, about 1 Hz, about 1.5 Hz, or about 2 Hz.
In particular embodiments, the cardiac cell may be paced about 30 beats per minute (bpm) to about 600 bpm. For example, the cardiac cell may be paced at about 30-600 bpm, about 40-500 bpm, about 50-400 bpm, about 60-300 bpm, about 70-200 bpm, about 80-190 bpm, about 90-180 bpm, about 100-170 bpm, about 110-160 bpm, about 120-150 bpm, or about 130-140 bpm. For example, the cardiac cell may be paced at about 30 bpm, about 40 bpm, about 50 bpm, about 60 bpm, about 70 bpm, about 80 bpm, about 90 bpm, about 100 bpm, about 110 bpm, about 120 bpm, about 130 bpm, about 140 bpm, about 150 bpm, about 160 bpm, about 170 bpm, about 180 bpm, about 190 bpm, about 200 bpm, about 210 bpm, about 220 bpm, about 230 bpm, about 240 bpm, or about 250 bpm. In some embodiments, each fixed rate is unique (i.e., each rate is different from each other rate). For example, in some embodiments the first fixed rate is different from the second fixed rate. In some embodiments, the cardiac cell may be paced at two or more sequentially increasing rates. In other embodiments, the cardiac cell may be paced at two or more sequentially decreasing rates.
The cardiac cell may be paced at any suitable number of fixed rates. In general, the number of fixed rates may be selected according to the needs of the user and may be dependent on the sample type used. In some embodiments, the cardiac cell may be paced at 2-20 fixed rates. For example, the cardiac cell may be paced at two fixed rates, three fixed rates, four fixed rates, five fixed rates, six fixed rates, seven fixed rates, eight fixed rates, nine fixed rates, ten fixed rates, 11 fixed rates, 12 fixed rates, 13 fixed rates, 14 fixed rates, 15 fixed rates, 16 fixed rates, 17 fixed rates, 18 fixed rates, 19 fixed rates, 20 fixed rates, or more than 20 fixed rates.
The disclosed methods involve measuring the response of the cardiac cell. In some embodiments, the disclosed methods involve measuring the response of the cardiac cell to the compound. As used herein, the term “response” indicates “spontaneous response” and/or “fixed response” unless otherwise specified. The term “spontaneous response” may refer to the response of the cardiac cell while the cell is not being paced at a fixed rate. For example, the term “spontaneous response” may refer to the response of the cardiac cell prior to pacing the cell at the first fixed rate. For example, the term “spontaneous response” may refer to the response of the cardiac cell after administration of the compound but prior to pacing the cell at the first fixed rate. The term “spontaneous response” may also refer to the response of the cardiac cell after administration of the compound after pacing has been turned off. The term “fixed response” refers to the response of the cardiac cell while the cell is being paced at a given fixed rate. For example, the term “fixed response” may refer to the response of the cardiac cell after administration of the compound and while pacing the cell at each fixed rate. In this effect, multiple fixed responses may be measured, wherein each response may be measured at each fixed rate. For example, a first fixed response may be measured while pacing at the first fixed rate, a second fixed response may be measured while pacing at the second fixed rate, a third fixed response may be measured while pacing at the third fixed rate, and so forth.
Measuring the response of the cardiac cell comprises measuring any suitable indication of cardiac excitability, automaticity, refractoriness, contraction, and/or conduction. For example, measuring the response of the cardiac cell may comprise obtaining any suitable electrophysiological recording of the cardiac cell. Suitable electrophysiological recordings include measurements of transmembrane voltage, intracellular calcium, action potential parameters, calcium transient parameters, and the like. For example, electrophysiological recordings may comprise recording of action potentials. Measuring the response of the cardiac cell may be performed using any suitable method known in the art, including multielectrode recordings, automated patch clamp, impedance measurements, voltage mapping, calcium mapping, time-lapse imaging, and the like. In some embodiments, contraction traces may be recorded. Contraction recordings may be measured using imaging, force transducers, optical indicators, and the like.
In some embodiments, measuring the response comprises measuring one or more of pace-capture, repolarization time, contraction strength, and conduction velocity. For example, pace-capture measurements may be obtained to indicate whether the cardiac cell lost beats, maintained the same number of beats, or generated extra beats compared to the number of beats at the fixed pace rate. For example, action potentials or contraction traces may be recorded to measure pace-capture of the cardiac cell. Alternatively or in combination, action potentials could be recorded to measure action potential repolarization time and/or action potential conduction velocity. Alternatively or in combination, contraction strength may be measured. Any suitable measurement or combination of measurements may be used to evaluate the sample.
Any of the methods described herein may further comprise reporting the measured response of the one or more cardiac cells on an arbitrary classification scale. In some embodiments, the methods may further comprise reporting the measured response on a binary classification scale. For example, fractions of lost and extra beats are designated in the example in
Any of the methods described herein may further comprise compiling the reported spontaneous response and/or fixed response of each cardiac cell into a single visual representation. This visual representation is also interchangeably referred to herein as “GRIDS,” “GRIDS map,” or “grid of responses indicating drug sensitivity.” For example, the spontaneous response and the fixed response at each of the two or more fixed pace rates for each cardiac cell may be compiled into a single visual representation. In some embodiments, the visual representation is color coded. For example, responses below the given threshold can be coded blue and responses above the given threshold can be coded as red. In some embodiments, responses above and below the given threshold can be color coded on a continuous color gradient. These colors are only intended to be nonlimiting examples, other colors could be substituted according to individual preference.
In some embodiments, the methods further comprise assessing the cardiac effect of the compound based upon the measured response of the one or more cardiac cells to the compound. The term “cardiac effect” as used herein refers to the impact of the compound on cardiac function. For example, cardiac effect may indicate an alteration in cardiac function or no change in cardiac function. Alterations in cardiac function may be impaired cardiac function or enhanced cardiac function. In some instances, impaired cardiac function may indicate cardiotoxicity of a drug. For example, in some instances it would be undesirable to have a drug that causes cardiac dysfunction or damage, which may be manifested as extra beats leading to elevated arrhythmia risk or lost beats leading to conduction block. In other instances, it may be desirable to identify a drug that causes changes in cardiac function. For example, it may be desirable to identify a drug that causes lost beats for use in pathophysiological conditions that cause abnormally high pacing rates. As another example, it may be desirable to identify a drug that produces extra beats for use in pathophysiological conditions that cause very low pacing rates or as a candidate for patients with elevated arrhythmia risk.
In some embodiments, cardiac effect may be indicated by one or more electrophysiological differences following addition of the compound. For example, cardiac effect of the compound may be indicated by differences in one or more of cardiac excitability, automaticity, refractoriness, and/or conduction following addition of the compound. For example, cardiac effect of the compound may be indicated by abnormal pace-capture measurements (i.e. lost beats or extra beats), changes in repolarization time, changes in contraction strength, and/or changes in conduction velocity.
In particular, the disclosed methods allow for evaluation of compounds at fixed pacing rates, thus more effectively mimicking daily or day-to-day physiological conditions in which the heart rate is not constant. For example, heart rate may vary from one day to the next, during sleep, wakefulness, the stress response, exercise conditions, and the like. Accordingly, the disclosed methods enable a more realistic evaluation of the cardiac effect of compounds compared to methods currently used in the art.
In some embodiments, the disclosed methods may be used for testing of a single compound. In other embodiments, the disclosed methods may be used for testing multiple compounds. For example, the method may be used to test the effects of multiple compounds, wherein the multiple compounds are added to the sample simultaneously. Alternatively, multiple compounds may be added to the sample sequentially. Such embodiments may be useful in conditions where compounds are intended for simultaneous or sequential use in a subject. In some embodiments, the disclosed methods may be adapted for use in high-throughput drug screens.
The methods described herein may be used on any suitable sample. Any type of cardiac cell may be used in the disclosed methods. Suitable cardiac cells include human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), embryonic stem cell-derived cardiac cells, directly reprogrammed cells (from fibroblasts or other somatic cells), natural cardiac cells isolated from native tissue (both human and animal), and various cardiac cell lines. In some embodiments, a computer model of a cardiac cell may be used. The sample may be a single cell, a cell monolayer, a cell cluster, engineered cardiac tissue, cardiac organoid, cardiac tissue explant, or whole heart. In particular embodiments, the sample may comprise engineered heart slices (EHS), as described in U.S. Pat. No. 10,183,097 for Engineered Cardiac Derived Compositions and Methods of Use, to Tung and Blazeski, issued Jan. 22, 2019, the entire contents of which are incorporated herein by reference.
Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.
Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.
Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ±100% in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.
The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The following Examples are offered by way of illustration and not by way of limitation.
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for cardiac studies, but their structural and functional immaturity precludes their use as faithful models of adult myocardium. The presently disclosed subject matter provides engineered heart slices (EHS), preparations of decellularized porcine myocardium repopulated with hiPSC-CMs that exhibit structural and functional improvements over standard culture. EHS exhibited multicellular, aligned bundles of elongated CMs with organized sarcomeres, positive inotropic responses to isoproterenol, anisotropic conduction of action potentials, and electrophysiological functionality for more than 200 days.
In some embodiments, the presently disclosed subject matter provides a drug assay, referred to herein as “GRIDS,” that serves as a “fingerprint” of cardiac drug sensitivity for a range of pacing rates and drug concentrations. GRIDS maps characterize differences in drug sensitivity between EHS and monolayers more clearly than changes in action potential durations or conduction velocities. EHS represent a tissue-like model for long term culture, structural and functional improvement, and higher fidelity drug response of hiPSC-CMs.
Slices of porcine myocardium 12 mm in diameter and 300 μm in thickness were sectioned and decellularized as previously described (Blazeski et al., 2015). The dECM slices were spread on plastic 12-mm diameter coverslips, with the perimeter of each slice wrapped around the edges of the coverslip. Coverslips with slices were placed in wells of standard 24-well culture plates and kept in PBS with antibiotics for up to 2 weeks prior to reseeding.
hiPSC Differentiation and Culture
Wild type (WT) and LQT2 hiPSC lines with a heterozygous A422T mutation in the hERG channel (Spencer et al., 2014) were gifts from Dr. Bruce Conklin. Both hiPSC lines were differentiated using a monolayer-based protocol (Boheler et al., 2014; Wang et al., 2015). The age of the EHS (d26 to d201) is given as the time in days from the start of hiPSC-CM differentiation (d0). On d9, the medium was switched to RPMI 1640 containing B-27 with insulin, and this medium was used for the remainder of time both prior to and after seeding on the dECM slices.
On d10-12, hiPSC-CM monolayers were dissociated using 0.05% Trypsin-EDTA and plated on dECM slices affixed to coverslips at a density of 0.8 to 1.3 million cells/cm2. EHS were maintained in culture for 16 to 191 days prior to evaluation by optical mapping or contraction measurements.
Standard fixation and immunostaining techniques were applied to slices before and after decellularization to label F-actin, nuclei, collagen I, collagen III, and laminin. Unstained dECM slices were also imaged by second harmonic generation (SHG). EHS were fixed and stained using standard techniques for cardiac troponin I (cTnI), α-actinin, connexin 43 (Cx43), vimentin, F-actin, and nuclei (DAPI). All samples were imaged by confocal microscopy. Nuclear elongation and orientation were analyzed in confocal images of EHS and monolayers using custom MATLAB scripts. Cellular structures in EHS were imaged by transmission electron microscopy.
WT d24-78 EHS were placed in a 35-mm tissue culture mm dish filled with Tyrode's solution and maintained at 31±0.1° C. for the duration of the experiment. A section of each EHS was detached from the edge of the coverslip so that it could move freely. Each EHS was paced at 1 Hz, 1.5 Hz, and 2 Hz while the free region was imaged with a CCD camera. A custom MATLAB script was used to segment the image and calculate the change in EHS area over time, which was used as a measure of contraction.
Each EHS was placed in Tyrode's solution and stained with 10 μM di-4-ANEPPS for 10 min at 37° C. The EHS was rinsed several times in a dish with warm Tyrode's solution, and then immersed in Tyrode's solution containing 10 μM blebbistatin to suppress contraction. The dish was placed on a 37° C. heated stage for the duration of the experiment. At least 5 min after adding blebbistatin, the EHS was stimulated with a point electrode and optically mapped using a CMOS camera (MiCAM Ultima-L, SciMedia). The EHS was paced by 5 ms monophasic rectangular pulses at stepwise increasing rates starting at 0.5 Hz. For some samples, E-4031, chromanol 293B, nifedipine, Bay K 8644, BaCl2, or cromakalim was added for 7 min prior to mapping. Mapping data were analyzed using custom MATLAB scripts (details provided in Supplemental Information). AP durations at 30% and 80% repolarization (APD30 and APD80) were calculated from the optical voltage signal. For drug studies, APD and conduction velocity (CV) measurements at each concentration were plotted as a percentage of APD and CV measured at baseline, with no drug present.
All data are presented as mean±SD. A Wilcoxon Rank-Sum test was used to determine statistical significance between control and drug groups for WT EHS contraction experiments, and between WT and LQT2 EHS. Paired, unequal variance, two-tailed t-tests were used for statistical tests of significance between experimental groups in all other drug studies, and unpaired, unequal variance, two-tailed t-tests were performed to determine the statistical significance between experimental measurements of d62-82 and d201 EHS. Differences were considered statistically significant at p<0.05.
Thin Slices of Decellularized Myocardium Promote Growth and Global Alignment of hiPSCCMs.
12-mm diameter plugs of left ventricular myocardium from porcine hearts were sectioned into 300-μm thick slices using a vibratome (
Decellularization left intact the ECM components of collagen I (
Differentiated progeny from hiPSCs were seeded onto the dECM slices at d10-12 (
Comparisons were then made between d55 hiPSC-CMs seeded at low density to make EHS and those on Geltrex-coated cell culture dishes to evaluate the effect of the matrix on cellular shape and organization. CMs grown on Geltrex were cobblestone-like and had randomly-oriented sarcomeres (
For example, when hiPSC-CMs were maintained for a prolonged period of time (139 days) as EHS and subsequently dissociated and replated under standard 2D culture systems, the cultivated cells retained an elongated morphology and exhibited highly organized sarcomeres (
Spontaneous and asynchronous contractions were apparent in EHS within 24 hours of cell seeding, but this transitioned to synchronous contractions in about one week's time. At d28 and d74, we evaluated contraction as the change in EHS area in a region where the slice edge was freed from the coverslip. The contracting hiPSC-CMs deformed the ECM, permitting the change in area to be monitored as an approximation of the degree of contraction. When stimulated at a pacing cycle length (PCL) of 666 ms (1.5 Hz, n=8), the EHS area in the field of view decreased, on average, by 2.0±1.3% from its value at rest (
Increasing the pacing rate from 1.5 Hz (666 ms PCL) to 2 Hz (500 ms PCL) resulted in an area change of 0.7±0.1 (negative force-frequency relationship) that was statistically significant, whereas decreasing the pacing rate from 1.5 Hz to 1 Hz (1000 ms PCL) resulted in an area change (1.1±0.1) that was not statistically significant (
EHS at d54-58 could be pace-captured starting from a PCL of 1000 ms down to 425±51 ms (n=6) and exhibited propagation of APs throughout the entire preparation (
The high degree of electrophysiological function in EHS was applicable to other hiPSC lines. This was demonstrated through the analyses of EHS made with hiPSC-CMs derived from a patient with confirmed LQT2. LQT2 is a cardiac disorder characterized by prolonged ventricular repolarization arising from mutations in the rapid delayed potassium channel (hERG) that increases the incidence of arrhythmias (Tester and Ackerman, 2014). As with WT EHS, LQT2 EHS cells were aligned and organized into multicellular strands (
In addition to having structurally elongated and organized CMs (
EHS Differ from Standard Monolayer Cultures in their Response to Ion Channel Modulating Drugs, when Evaluated Using GRIDS Analysis.
Based on the improved adult-like cellular morphology, more ventricular-like APs (
EHS and monolayers were superfused with the rapidly activating potassium current (IKr) blocker, E-4031, at concentrations ranging from 1 nM to 10 μM (
The slow delayed rectifier K+ current (IKs) was then blocked with chromanol 293B to test its effects on EHS and monolayer cultures (
Therefore, chromanol 293B had a limited effect on either preparation, suggesting that IKs is poorly expressed or not functionally active in both monolayers and EHS. Aside from differences in potassium channels, whether L-type calcium channels might also be differentially expressed in EHS and in monolayers was tested, so the channel blocker nifedipine was applied (
In addition to the GRIDS analysis, the occurrence of drug-induced prolongations of repolarization, which is an index of liability for acquired long-QT syndrome (Wood and Roden, 2004), as well as conduction slowing, which can be an early sign of conduction block was evaluated. At a PCL of 1500 ms, E-4031 prolonged APD80 (
GRIDS analysis was applied to contraction data collected from monolayers of hiPSC-CMs. Each monolayer (cultured on a plastic coverslip) was placed in a 35-mm dish filled with cell culture medium and set on the stage of a microscope (LS720, Etaluma) fitted within a cell culture incubator. Monolayers were superfused with increasing concentrations of E-4031 (
When treated with E-4031 (
In this study, dECM slices were repopulated with hiPSC-CMs to make EHS with coordinated and syncytial contractile and electrophysiological function. On EHS, reseeded hiPSC-CMs organize in a similar manner to the ordered arrays of fibers found in the native myocardium (Veeraraghavan et al., 2014), becoming elongated and aligned in bundles, with elongated and oriented nuclei, and well-defined, aligned sarcomeres. This is an improvement from the morphology seen in standard cultures, where hiPSC-CMs appear more cobblestone-like and have randomly-oriented sarcomeres. Gap junction staining for Cx43, however, was observed around the periphery of cells in the EHS and does not localize at intercalated discs, reflecting a level of structural immaturity that has also been found in other studies of hPSC-CMs (Zhang et al., 2013). Nevertheless, the presently disclosed EHS are a well-connected and reproducible functional syncytium of CMs that exhibits uniform conduction and coordinated contraction over an area of about 1 cm2. These characteristics overcome the problem of variable and patchy conduction that occurs in other preparations using decellularized myocardium caused by non-uniform cell seeding (Guyette et al., 2016; Lu et al., 2013; Oberwallner et al., 2014).
The cellular organization of EHS and the resultant anisotropic conduction of APs is guided by the retention of the native oriented matrix in the dECM slice. This strategy to utilize the topographical cues of the ECM differs from those used in other studies to align CMs, including microcontact printing (Wang et al., 2014), hydrogel compaction in the presence of non-myocytes (Liau et al., 2011) and fabricated microgrooved (Rao et al., 2013), nanogrooved (Macadangdang et al., 2015), electrospun (Wanjare et al., 2017), and wrinkled (Wang et al., 2013) substrates. The use of decellularized matrix may confer benefits not present in the other approaches, as there is mounting evidence that the complex chemistry of the matrix can promote stem cell differentiation (Ng et al., 2011) and electrophysiological maturation of CMs (Herron et al., 2016). The presently disclosed study of EHS demonstrates the suitability of this platform to study electrophysiological function over long-term culture.
The presently disclosed experimental approach involves the routine production of batches of 10 to 20 thin tissue slices from ventricular plugs that are subsequently decellularized in parallel. This decellularization method leaves behind a scaffold that retains an organized and aligned structure, made up of multiple ECM components. While various decellularization methods have been developed (Badylak et al., 2010), the method of Ott and colleagues was selected because it preserves non-collagen proteins (particularly, fibrillin, heparin sulfate, and laminin (Guyette et al., 2016), with a trade-off of decreased retention of collagen (Akhyari et al., 2011). These noncollagen components may be particularly beneficial for promoting the differentiation and maintenance of hPSC-CMs (Nakayama et al., 2014). However, because the composition and mechanical properties of the ECM change during development of the heart from the post-natal to adult stage (Gershlak et al., 2013), further work needs to be done to identify the developmental stage that will yield ECM best suited for growth and maintenance of hPSC-CMs.
The presently disclosed method for decellularizing slices is amenable to the use of native myocardium from a variety of sources and can be used to compare the effect of different species, chamber locations, and developmental states of the ECM source on CM electrophysiology and contraction. Automation of the process of anchoring the dECM slice to a support will be necessary if large numbers of ECM scaffolds are needed to make EHS for drug discovery and screening.
While EHS exhibited a positive inotropic response to isoproterenol, their fractional shortening (around 2%) was much lower than the 30% fractional shortening reported in adult hearts (Colan et al., 1984), and they also exhibited a negative force-frequency relationship.
Further improvements, such as increasingly rapid pacing during culture, can be used to improve contractility and achieve the positive force-frequency relationship found in adult myocardium (Ronaldson-Bouchard et al., 2018). EHS also exhibited rate-dependent decreases of APD and CV, as well as anisotropic conduction, as is found in adult human heart (Yue et al., 2005).
However, CV in EHS was less than half of that measured in the adult ventricle (Durrer et al., 1970), and the anisotropy ratio of conduction was substantially less than that in the adult ventricle (Peters and Wit, 1998). The loss of CM alignment in layers of cells farther from the matrix surface likely contributed to a diminished anisotropy ratio. Amenability to long-term culture is a powerful feature of EHS, because prolonged culture advances the structural organization, cardiac gene expression, and contractile and electrophysiological function of hPSC-CMs (Lundy et al., 2013). Maintaining multi-layers of hiPSC-CMs in standard culture plates for periods of weeks is difficult, as they can detach from the underlying substrate, although individual hiPSC-CMs have been maintained for up to 120 days (Lundy et al., 2013), and multicellular embryoid bodies of hiPSC-CMs have been maintained for up to 360 days (Kamakura et al., 2013). In the presently disclosed study, EHS allowed for stable, long-term culture of a functional syncytium of hiPSC-CMs. Electrophysiological functionality was maintained for more than 200 days—EHS could be pace-captured at PCLs as short as 400 ms, and APs continued to propagate as before throughout the entirety of the preparation, although with some loss of CV that may occur as non-myocytes proliferate over time in EHS culture.
Remarkably, hiPSC-CMs cultured long-term within EHS retained their elongated morphology and aligned sarcomeres even after removal from the dECM slice, suggesting that the ECM may have durable effects on cell phenotype. Aside from cues from the ECM, additional steps may be required to optimize the structural organization and function of EHS in long-term culture.
The EHS preparation holds promise for preclinical cardiac effect testing, where accurate prediction of arrhythmia risk is essential to remove hazardous drugs from the development pipeline. Currently, drugs are tested for their ability to inhibit hERG and cause QT prolongation, a marker for risk of developing Torsades de Pointes (TdP), a tissue-level arrhythmia (Farkas and Nattel, 2010). Most studies of this kind are performed on heterologous expression systems which lack the full complement of cardiac ion channels (Fermini et al., 2016). Such assays also do not account for offsetting mechanisms from non-hERG ion channels that may render a drug safe (Redfern et al., 2003). The EHS preparation addresses these shortcomings as a functional syncytium of human CMs and allows for a multitude of mechanisms by which drugs can affect excitability, including effects on ion currents and electrical coupling.
The GRIDS assay described herein provides a new tool for evaluating drug sensitivity in the context of cellular automaticity and excitability. Changes in spontaneous beating rates are often used to evaluate drug sensitivity of hiPSC-CMs (Gilchrist et al., 2015) and can be altered by drugs that act on ICa,L or IKr (Blazeski et al., 2012). Electrophysiological measurements at variable spontaneous beat rates, however, fail to control for the rate-dependence of the various ion currents. On the other hand, GRIDS evaluates the effect of drugs during electrical pacing at different fixed rates. These periodic stimuli introduce controlled, dynamic changes into the system, and the resulting beating patterns are an integrated effect of automaticity, excitability, and refractoriness. The GRIDS map for a given drug is comprised of pace-capture responses across multiple dosing and electrical pacing regimes, and it can serve as a “fingerprint” of the drug sensitivity. The lowermost row of the GRIDS map reflects the effect of drug on spontaneous rate in the absence of electrical stimulation, while the remaining rows delineate the range of pace-capture across drug dosages. The leftmost column of the map delineates the range of pace-capture under drug-free conditions, while the remaining columns are at different drug dosages. The localization of red blocks in the upper right of the maps for E-4031 and nifedipine (monolayer only) indicates loss of capture of paced beats in the presence of high concentrations of the drug and short PCLs. Localization of blue blocks in the lower right of the maps for BaCl2 indicates a higher fraction of samples with spontaneous activity as drug concentration increased, and the large area of green for chromanol 293B and nifedipine (EHS only) indicates very little response to the drug. Further, across the four drugs tested, in the GRIDS maps the regions of red or blue blocks tended to be smaller, and the region of green tended to be larger, for EHS than for monolayers, revealing that EHS are comparatively less sensitive to these drugs when evaluated for effects on their excitability and ability to capture during pacing. This suggests that EHS express relatively more of the repolarizing currents IKr and IK1, which is responsible for maintaining the resting potential in adult ventricular cells (Doss et al., 2012), than do monolayers. On the other hand, the absence of a chromanol 293B effect supports the notion that both EHS and monolayers have low levels of IKs, as has been previously described for hPSC-CMs (Ma et al., 2011). The GRIDS maps also indicate that EHS remain excitable at all PCLs and concentrations of nifedipine, whereas monolayers are unable to be pace-captured for every beat for some combinations of PCL and nifedipine concentration. Increased ICa,L in EHS would explain why for the same level of ICa,L block at a given concentration of nifedipine, EHS would retain enough residual ICa,L to remain excitable while monolayers would not. Alternatively, the excitability of EHS may be governed more by INa than by ICa,L (as in more mature ventricular tissue) compared with that of monolayers (either due to differences in ion channels or because hiPSC-CMs in EHS are less depolarized), so that block of ICa,L does not decrease excitability.
The presently disclosed GRIDS maps were able to differentiate the responses of EHS and monolayers to the panel of drugs tested even though measurements of a single electrophysiological parameter, APD prolongation, did not provide a clear snapshot of relative drug sensitivity. In both EHS and monolayers, E-4031 and BaCl2 prolonged APD, chromanol 293B did not change APD, and nifedipine shortened APD. Further, differences in drug responses between EHS and monolayers were not detected with respect to APD for E-4031 (
Differences in drug responses of EHS, which tend to be less sensitive but more robust in their ability to be electrically paced over a wider range of rates and drug concentrations when compared to monolayers, can be attributed to a variety of factors. One possibility is that cells in EHS are a more densely-packed, thicker syncytium that experiences a lower effective drug concentration than cells in monolayers where diffusion is not limited. Also, differences in the mechanical and biochemical environment in EHS compared to monolayers can result in differences in cell phenotype and AP morphology seen in EHS (more elongated cells with organized sarcomeres), which can affect drug responses. Additionally, EHS experience an increase in non-myocytes over time, and this modulation of cell-cell interactions in the preparation could also impact drug responses. Multicellular preparations with large areas, like EHS, will be needed in future studies to assess the risk for reentrant arrhythmias, which require room for circuitous wavefront propagation to occur. Further, EHS are tissue-like models that can be used in studies aimed at treatment discovery and at creating clinically-relevant disease models.
EHS take advantage of the complex biochemical and structural cues of the myocardial ECM to guide the alignment of CMs. Seeded hiPSC-CMs organize as multicellular, anisotropic bundles that contract the EHS and propagate APs uniformly throughout the preparation. EHS can be used for long-term culture of hiPSC-CMs to interrogate processes of cell maturation and response to drugs over time. Drug sensitivity can be evaluated for a range of concentrations and under different pacing rates using the GRIDS assay, which has revealed differences in drug sensitivity between EHS and cell monolayers. In summary, EHS are tissue-like models that can be used in long-term electrophysiology and drug studies.
Unless Otherwise Stated, Reagents were Acquired from Thermo Fisher Scientific, Waltham, Mass.
Hearts obtained from slaughterhouse pigs were rinsed in distilled and deionized water to remove blood and stored overnight at −20° C. The following day, the hearts were allowed to thaw at room temperature for 1 hour. A metal 12-mm diameter punch was sterilized using 70% ethanol and used to punch out plugs of tissue from the left ventricle. Plugs were trimmed to fit into 35-mm culture dishes and stored at −80° C. until slicing, a minimum of 16 hours. Plugs were allowed to thaw in room temperature distilled water supplemented with 100 U/mL Penicillin-Streptomycin and 0.1× antibiotic-antimycotic. After thawing, plugs were blotted dry, placed in a 35-mm culture dish with the epicardium pressed against the bottom of the dish, and embedded in 4% w/v low gelling temperature agarose (Sigma-Aldrich Corp., St. Louis, Mo.) dissolved into distilled water with penicillin-streptomycin and antibiotic-antimycotic. The agarose was allowed to solidify at 4° C. for 15 minutes, and then the agarose disc containing the plug was removed from the culture dish and attached to the cutting stage of a vibratome (7000smz, Campden Instruments, Lafayette, Ind.) using cyanoacrylate glue (3M, Maplewood, Minn.) with the epicardium positioned at the top of the plug. The plug was sectioned into 300 μm-thick slices parallel to the epicardium using a ceramic blade oscillating at a frequency of 100 Hz with an amplitude of 1 mm and advancing at a speed of 0.01 to 0.03 mm/second. The cutting solution in which the plug was immersed (phosphate buffered saline (PBS) supplemented with antibiotics) was kept at 4-8° C. during slicing. Slices were stored in PBS supplemented with antibiotics at 4° C. overnight.
Slices were decellularized using a procedure modified from Ott et al., 2008. All detergents for decellularization were diluted in distilled water supplemented with antibiotics. PBS also was supplemented with the same antibiotics. Slices were each placed in a well of a 12-well plate, and 1 mL of each of the decellularization solutions was added to each well. The plate was placed on a rotator (Orbit 1000 Digital Shaker, Labnet International Inc., Edison, N.J.) and agitated 180 rpm in the presence of the following solutions: 1% sodium dodecyl sulfate (SDS) for 3 hours (replaced with fresh solution after 1.5 hours), water for 15 minutes, 1% Triton-X 100 (Sigma-Aldrich Corp.) for 7 minutes, and PBS for 45 minutes (replaced with fresh solution every 15 minutes). Samples were left in PBS on rotator at 160 rpm overnight to rinse out any remaining detergents.
Plastic 12-mm coverslips were immersed in 70% ethanol and wiped dry. After an overnight wash in PBS, slices were carefully handled with forceps, spread over the coverslips and wrapped around the edges of the coverslips. Slices attached to the coverslips were placed into wells of a 24-well plate, immersed in PBS with antibiotics, and stored at 4° C. until seeding (up to 2 weeks).
hiPSC Differentiation and Culture
Wild type and LQT2 hiPSCs were plated into wells of 6-well plates coated with 1:200 Geltrex:DMEM/F-12. For the first 22 hours, hiPSCs were maintained in Essential 8 medium (E8) with 10 μM Y-27632 dihydrochloride (Tocris Bioscience, Bristol, UK). Afterwards, hiPSCs were rinsed with DMEM/F-12 and fed with E8 medium every day. On the fourth day, when cells had reached about 80% confluence, the medium was replaced with RPMI 1640 supplemented with B-27, minus insulin and 6 μM CHIR-99021 (Selleck Chemicals, Houston, Tex.) to initiate differentiation (d0 of differentiation). Over the course of the next week, medium was changed as follows: RPMI 1640 with B-27 without insulin (B-27 minus) on d2, B-27 minus and 5 μM IWR-1 (Sigma-Aldrich Corp.) on d3, B-27 minus on d5 and d7, and RPMI 1640 with B-27 with insulin (B-27 plus) on d9 and every other day afterwards. Spontaneous beating in the monolayers was observed starting at d7 to d10. On d10 to d12, the hiPSC-CM monolayers were washed with 0.5 mM EDTA (Mediatech, Inc., Manassas, Va.) and then incubated in EDTA for 5 minutes at 37° C. Afterwards, the EDTA was aspirated off, and 0.05% Trypsin-EDTA was added for 3 minutes at 37° C. Cells were triturated before Defined Trypsin Inhibitor was added to stop the digestion. The resultant suspension was centrifuged at 200 g for 5 minutes (Centrifuge 5702, Eppendorf AG, Hamburg, Germany). After aspirating off the supernatant, the cell pellet was resuspended in B-27 plus. The PBS was removed from dECM slices, and the suspension of hiPSC-CMs was pipetted on top of the slices at a density of 0.8 to 1.3 million cells per cm2. Slices were maintained in culture for 16 to 190 days, and the B-27 plus medium was replaced every other day for the duration of culture.
dECM slices or EHS were fixed in 4% paraformaldehyde solution (Affymetrix, Inc., Cleveland, Ohio) for 10 minutes and rinsed twice with PBS. Samples were stored in PBS at 4° C. until immunostaining. To immunostain for ECM proteins, slices were immersed in Target Retrieval Solution (Dako North America, Inc., Carpinteria, Calif.) for 20 minutes in a steamer.
Samples were subsequently rinsed in distilled, deionized water for 5 minutes, blocked with 10% peroxide solution in water (Sigma-Aldrich Corp.) for 10 minutes, and rinsed twice for 5 minutes each time with Dulbecco's Phosphate Buffered Saline (DPBS). Samples were incubated in primary antibodies against collagen I (C2456, Sigma-Aldrich Corp.), collagen III (ab7778, Abcam, Cambridge, Mass.), and laminin (L9393, Sigma-Aldrich Corp.). The next day, slices were washed three times for 5 minutes each with TBS-T (0.05% Tween 20, Sigma-Aldrich Corp.) in Tris Buffered Saline (TBS, Quality Biological, Gaithersburg, Md.) and incubated in secondary antibodies (Invitrogen, Waltham, Mass.) for 45 minutes at room temperature. EHS were shielded from exposure to light and washed in three rounds of TBS-T for 5 minutes each round.
Afterwards, samples were mounted onto slides, a drop of Prolong Gold Antifade Mountant was added onto the slices, and a glass slide was placed on top. Samples were left to dry for a minimum of 24 hours before acquiring images using a confocal microscope (LSM 510 Meta, Zeiss, Oberkochen, Germany). Fibrillar collagen in unstained dECM slices was also imaged by second harmonic generation (SHG) using a multiphoton microscope (710NLO, Zeiss) with excitation at 880 nm, and emission acceptance at 415-450 nm.
To immunostain for cellular proteins, EHS were permeabilized with cold 0.5% Triton-X100 (Sigma-Aldrich Corp.) in PBS for 20 minutes, followed by blocking with 10% goat serum (Life Technologies, Carlsbad, Calif.) for 1 hour at room temperature. Primary antibodies against cardiac troponin I (T8665-13F, United States Biological, Pittsburgh, Pa.), α-actinin (A7811,Sigma-Aldrich Corp.), connexin 43 (C6219, Sigma-Aldrich Corp.), or vimentin (M0725, Dako North America, Inc.) in antibody diluent (Dako North America, Inc.) were added overnight at 4° C. The next day, EHS were washed with TBS-T as described above. Afterwards, samples were stained with 4′,6-Diamidino-2 Phenylindole, Dihydrochloride (DAPI) for 25 minutes at room temperature. Samples were subjected to three more rounds of TBS-T washing before the addition of a drop of Prolong Gold Antifade Mountant and a glass slide on top of each sample.
Alternatively, some samples were permeabilized as previously described and stained with Alexa Fluor 488 Phalloidin and DAPI at room temperature for 25 minutes. Samples were washed in TBS-T and mounted for imaging as described above. Images of stained samples were acquired using a confocal microscope (LSM 510 Meta, Zeiss). To prepare samples for transmission electron microscopy, EHS were fixed in 2.5% glutaraldehyde and 3 mM MgCl2 in 0.1 M sodium cacodylate buffer (pH 7.2) overnight at 4° C. Samples were subsequently rinsed three times (15 minutes per rinse) in a 0.1 M sodium cacodylate buffer supplemented with 3 mM MgCl2 and 3% sucrose, shielded from light, and left for one hour in a solution of 1% osmium tetroxide in 3 mM MgCl2 and 0.1 M sodium cacodylate buffer. After undergoing two rounds of rinsing with water for 5 minutes each, the samples were stained in 2% aqueous uranyl acetate in the dark for 1 hour. Samples were then dehydrated in a graded series of ethanol washes (30%, 50%, 70%, 90%, and three washes of 100% ethanol), followed by two washes with propylene oxide (for 5 minutes each) and an overnight incubation in 1:1 propylene oxide:epon. The next day, samples were incubated in epon with catalyst three times for 2 hours each at room temperature and for 2 days at 60° C. Samples were examined using a transmission electron microscope (Phillips/FEI BioTwin CM120 TEM, Hillsboro, Oreg.).
SHG images were acquired of three types of decellularized slices (dECM slices) after decellularizing: fresh slices (native slices from sections of fresh plugs prepared right after the hearts were acquired that were decellularized), frozen slices (fresh slices that were stored at −80° C. for over 48 hours), and frozen plus (fresh plugs that were stored at −80° C. for over 48 hours, thawed, and subsequently sectioned). Both fresh and frozen plugs were acquired from the same hearts, and fresh and frozen slices were derived from adjacent sections of the same plug. SHG z-stack images had an area of 1.875 mm2 and were processed in MATLAB to quantify fiber orientation and fiber degree of alignment. After compressing z-stack images in ImageJ using a maximum intensity z-axis projection, each compressed image was converted to an RGB image using ImageJ and segmented in MATLAB. SHG images were then segmented into 6889 subregions. The sectioning frame originated at the top left corner of the image and continuously shifted to the right or downwards by 0.0133 mm to create a new subregion with an area of 0.071 mm2. The absolute logarithmic magnitude of the 2D Fast-Fourier transform (2D-FFT) was calculated for each subregion in the segmented image. The direct current frequency was shifted to the center of the 2D-FFT plot, and the magnitude of the 2D-FFT was normalized so that it had a magnitude of 1. Since the 2D-FFT is symmetric, only half of it was used in the analysis. The angle of orientation from the horizontal axis of each coordinate in the 2D-FFT was calculated by converting from Cartesian to polar coordinates.
The radial sum of the 2D-FFT, normalized to the number of pixels along each radius, was plotted as a function of the orientation for each subregion. The orientation of the maximum 2DFFT sum determined the angle perpendicular to the fiber orientation in that subregion. The mean and standard deviation of the fiber orientation were calculated for all subregions in each image. Mean values of fiber orientation were plotted for 8 slices within each group. The fiber degree of alignment was calculated as the maximum 2D-FFT sum divided by the total sum of the 2D-FFTs of all subregions within an image.
Time-matched EHS and monolayers were immunostained with DAPI and imaged using a confocal microscope, as described above. Images of nuclei were thresholded above a baseline noise level and segmented, removing overlapping areas as necessary, by using a previously described method (Plissiti et al., 2014) with some modifications. A recursive search for concavity points was performed; when a concavity point was found, the outline between it and the convex hull was searched for more concavities. Further, the case where non-adjoining sections of an outline are part of the same nucleus was permitted. An ellipse was fitted to each nucleus using a method previously described (Fitzgibbon et al., 1999). The nuclear elongation ratio, mean angular orientation, and standard deviation of the angular orientations were calculated, as previously described (Bray et al., 2010).
Flow cytometry analysis was performed on d17 (prior to monolayer seeding), and on monolayers and EHS that had been seeded at d17 and cultured for 61 days (d68). Monolayers and d17 cells were washed with 5 mM EDTA and subsequently incubated in 5 mM EDTA at 37° C. for 5 minutes. The EDTA solution was aspirated off and replaced with 0.05% Trypsin-EDTA for 4-6 minutes at 37° C., until the cells easily detached from the bottom of the wells when the culture dish was agitated. The Trypsin-EDTA solution containing cells was neutralized using Defined Trypsin Inhibitor, and the cells were centrifuged at 200 g for 5 minutes before fixation.
EHS were unhooked from their underlying plastic coverslip, transferred to a 12-well plate and rinsed with DPBS containing calcium and magnesium (DPBS +/+). After removing the DPBS, EHS were incubated in 10 mg/mL of collagenase IV with 10% fetal bovine serum in DPBS +/+ and 50 μg/mL of DNAse I in 0.15 M NaCl (Sigma-Aldrich Corp.) for 30 minutes on a shaker at 37° C. The collagenase and DNAse solution were subsequently removed, and the EHS were rinsed twice with PBS (without calcium or magnesium). After removing the PBS, EHS were incubated in 0.05% Trypsin-EDTA for 5 minutes on a shaker at 37° C. and broken up by pipetting up and down. The Trypsin-EDTA solution containing the recovered cells was neutralized and centrifuged as described above prior to fixation.
Dissociated cells were fixed in 4% paraformaldehyde (Affymetrix) for 10 min at room temperature. Cells were then simultaneously and permeabilized and blocked in PBS containing 0.1% BSA (Sigma-Aldrich Corp.), 5% goat serum, and 0.1% Triton-X 100 (Sigma-Aldrich Corp.) for 30 min. Cells were incubated with mouse anti-cTnT antibody diluted 1:200 in FACS buffer (PBS with 0.1% BSA and 0.1% Triton-X 100) for 1 hr at 4° C., washed three times with FACS buffer, and incubated with anti-mouse Alexa Fluor® 488 antibody in FACS buffer (1:200, Invitrogen) for 30 min at 22° C. in the dark. After washing and re-suspending in PBS with 0.1% BSA, cells were strained through a 30 μm filter and run on a FACSCalibur cytometer (BD Biosciences, Woburn, Mass.). Secondary controls consisted of cells incubated only with antimouse Alexa Fluor® 488 secondary antibody. Single cells were identified and gated based on their forward and side scatter, and cardiomyocytes were gated based on their cTnT expression.
Data were analyzed using FlowJo X Software. Histogram counts were extracted by gating on unstained cells and cells stained only with secondary antibody control (to remove autofluorescence and background nonspecific secondary staining).
Tyrode's solution was prepared by combining 1.8 mM CaCl2, 5 mM glucose, 5 mM HEPES, 1 mM MgCl2, 5.4 mM KCl, 135 mM NaCl, and 0.33 mM NaH2PO4 in ddH2O and adding NaOH to raise the pH to 7.4 (all chemicals from Sigma-Aldrich Corp.). EHS were placed in a 35-mm dish filled with Tyrode's solution and set on a stage heated to 31±0.1° C. (Warner Instruments, Hamden, Conn.). A section of each EHS was unhooked and allowed to move freely throughout the duration of the experiment. The EHS were allowed to equilibrate in Tyrode's for 5 minutes before the start of pacing with a point electrode. Each sample was paced for 1 minute at each of 3 cycle lengths (1000 ms, 666 ms, 500 ms), while the freely moving region was kept approximately vertical in the field of view and imaged at 4× magnification using a CCD camera (Swiftcam, Swift, Schertz, Tex.) at a rate of 14-17 fps with 320×256 pixel resolution. Samples also were imaged during pacing at 666 ms after the application of 1 μM isoproterenol (Sigma-Aldrich Corp.) for 2 minutes and after washout of the drug for 2 minutes. A pacing cycle length shorter than 1000 ms was applied in the presence of isoproterenol to overcome the increase in spontaneous rate in response to the drug. Custom MATLAB scripts were used to segment the images by applying a user-defined threshold to assign each pixel in the image as either belonging to the EHS, which was darker, or the background, which was lighter. From this, the area of EHS was calculated in each image. The change in area from a fully relaxed state (reference frame, designated as 100% EHS area) was determined for each frame in the time series. The minimum area (maximum change in area) was averaged over time (multiple cycles of contraction) and relaxation for each pacing condition. The change in area in the presence of isoproterenol and after washout was compared to the change in area prior to the application of drug for each EHS.
EHS were stained with 10 μM of the voltage-sensitive dye di-4-ANEPPS (Sigma-Aldrich Corp.) in Tyrode's solution for 10 minutes at 37° C. Afterwards, EHS were rinsed twice with Tyrode's and placed in a 35-mm dish filled with Tyrode's and 10 μM of the contraction inhibitor blebbistatin (Sigma-Aldrich Corp.). This dish was set on a stage heated to 37° C. and allowed to equilibrate for at least 5 minutes. Samples were point paced with at least 30 stimulus pulses at a range of cycle lengths, starting from 2000 ms and decreasing until the they lost capture. EHS were optically mapped during pacing using a 100×100 pixel CMOS camera (MiCAM Ultima-L, SciMedia, Costa Mesa, Calif.).
During drug studies, recordings at a range of pacing rates were taken at baseline (prior to the addition of drug) before replacing the solution in the dish with Tyrode's supplemented with blebbistatin and the lowest dose of drug studied. The sample was paced at the same range of rates and mapped 7 minutes after the addition of drug. Afterwards, the bath solution was replaced with Tyrode's with blebbistatin and the next lowest drug dose and the procedure repeated. This method was applied for all doses of each drug and, except where noted in the figures, only one drug was tested per sample. In these studies, we superfused EHS or monolayers with the following drugs: E-4031 (Tocris Bioscience), BaCl2 (Sigma-Aldrich Corp.), Chromanol 293B (Tocris Bioscience), Nifedipine (Tocris Bioscience), Bay K 8644 (Tocris Bioscience), and Cromakalim (Sigma-Aldrich Corp.).
Optical mapping data was analyzed using custom MATLAB scripts. Recordings at each pixel were de-noised using a previously described method (Little and Jones, 2010) to regulate total signal variance and convolved with a 5×5 spatial Gaussian filter. Activation times were defined as the maximum of the derivative of membrane potential (dV/dt), which was calculated as previously described (Chartrand, 2011). Histograms of local conduction velocities for each EHS were fitted to a Gaussian curve and the mean of the curve was defined as the average conduction velocity (CV). To determine longitudinal and transverse CVs, a bimodal Gaussian curve was fitted to the local CVs. Action potential durations at 30 and 80 percent repolarization (APD30 and APD80) were determined for all local traces over the recording region for each EHS and fit with Gaussian curves to determine the mean value for each EHS, as described for CV measurements. For drug studies, average APD and CV measurements for each dose were normalized by average APD and CV measurements at baseline (without the drug).
mRNA was isolated from EHS (d69) and monolayers (d22) using the following procedure: incubation in TRIzol Reagent for 5 minutes at room temperature, incubation in chloroform for 3 minutes, centrifugation at 12,000 g for 15 minutes at 4° C., collection of colorless phase that separated at the top of the centrifuged sample, addition of isopropyl alcohol and incubation at room temperature for 10 minutes, centrifugation at 12,000 g for 10 minutes at 4° C., solubilization of RNA pellet in 75% ethanol, centrifugation at 7,500 g for 5 minutes at 4° C., air drying of sample, and resuspension of the RNA pellet in DEPC-treated water. mRNA from two EHS were combined for each EHS replicate.
Reverse transcription was performed to create cDNA with the PCR Master Mix kit, using the MyGo Mini PCR system (IT-IS Life Science Ltd., Republic of Ireland). RT-PCR was performed on each target in triplicate, using the following primers:
The PCR program run for each sample consisted of 120 seconds hold at 95° C., 40 cycles of amplification that alternated between 90° C. and 65° C., 10 seconds pre-melt hold at 95° C., and a melting step that increased from 60° C. to 97° C. at 0.1° C./second. CT values were obtained using MyGo Mini PCR Software (IT-IS Life Science Ltd.) and CT was calculated for each gene of interest (GOI) in EHS to determine fold change over transcript levels expressed monolayers according to the formula:
ΔΔCT=CEHST,GOI−CEHST,ACTN2−CMnT,GOI+CMnT,ACTN2
where CEHST,GOI refers to CT of EHS for the GOI, CEHST,ACTN2 refers to CT of EHS for ACTN2 (α-actinin, which was used as a normalizing gene), CMnT,GOI refers to CT of monolayers for the GOI, and CMnT,ACTN2 refers to CT of monolayers for ACTN2.
All data are presented as mean±SD. Measurements of nuclear elongation were log transformed and reported as the interval of the log-transformed mean±SD after inverse transformation into linear space (Bland and Altman, 1996). For contraction experiments, a one-tailed Wilcoxon test was used to determine statistically significant differences from 1 for isoproterenol-treated and washout groups. For studies on rate-dependence of contraction, a two-tailed Wilcoxon test was used to determine statistically significant differences from 1 for groups paced at 500 ms and 1000 ms cycle lengths. A two-tailed Wilcoxon test was also used for statistical significance between APD measurements for WT and LQT2 EHS. Paired, unequal variance, two-tailed t-tests were performed for all other drug studies and unpaired, unequal variance, two-tailed t-tests were performed for optical mapping studies not involving drugs, studies comparing d62-82 EHS to d201 EHS, and for orientation analysis of fresh slices, frozen slices, and frozen plugs.
All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references (e.g., websites, databases, etc.) mentioned in the specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.
Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims
This application claims priority to U.S. Provisional Patent Application No. 62/826,030, filed Mar. 29, 2019, which is incorporated herein by reference in its entirety.
This invention was made with government support under HL120959 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/25709 | 3/30/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62826030 | Mar 2019 | US |
