Human tissue model of ejection fraction preserved heart failure (HFPEF) for discovery of therapeutic agents and therapeutic targets

Abstract

The present disclosure provides methods for identifying therapeutic agents for treating ejection fraction preserved heart failure. The method may comprise: contacting a cardiac tissue or cardiac organoid with an effective amount of transforming growth factor-pi and an effective amount of endothelin-1 (ET-1) to induce ejection fraction preserved heart failure in the cardiac tissue; exposing the cardiac tissue or cardiac organoid exhibiting heart failure with preserved ejection fraction to a candidate therapeutic agent; and if the cardiac tissue or cardiac organoid induces a reduction in any one or more of the following characteristics of the cardiac tissue or cardiac organoid if the candidate therapeutic agent induces a reduction in any one or more of the following characteristics of the cardiac tissue or cardiac organoid, if yes, identifying the candidate therapeutic agent as a therapeutic agent for ejection fraction preserved heart failure: passive strain, stiffness, myocardial cell hypertrophy, or fibrosis. In some embodiments, the candidate therapeutic agent identified as a therapeutic agent for treating ejection fraction preserved heart failure induces an increase in contraction kinetics or relaxation kinetics. Additional embodiments of the disclosed methods identify therapeutic agents that induce any combination of the above altered characteristics of cardiac tissue and / or cardiac organoids.

Description

Technical Field

[0001] The present disclosure generally relates to the fields of medicine and healthcare, and more particularly to the field of cardiovascular healthcare. Background Art

[0002] Heart failure with preserved ejection fraction (HFpEF) is becoming the most prevalent cause of heart failure [1-3]. HFpEF accounts for more than 50% of all heart failure cases. Compared with many other cardiovascular diseases, the prevalence of HFpEF is increasing, and the increasing incidence has led to a significant increase in morbidity, mortality, and social costs [1-4]. HFpEF affects the elderly in an age-dependent manner, and its prevalence is increasing in Europe and the United States as the population ages. The mortality rate from HFpEF is high and comparable to that of heart failure with reduced ejection fraction (HFrEF). Unfortunately, many of the therapies that reduce mortality and morbidity in HFrEF have little effect on HFpEF. Patients with HFpEF do not respond to the standard care for HFrEF because the biology and clinical courses of these two diseases are very different. The main clinical manifestations of patients with HFpEF are exercise intolerance and dyspnea on exertion. Such dyspnea can be severe, can occur at very low levels of exertion, and can thus limit physical activity, leading to a downward spiral of inactivity and dysregulation, exacerbating the underlying heart disease. Physiologically, HFpEF is characterized by 1) delayed and incomplete left ventricular (LV) relaxation, 2) reduced LV compliance, 3) atrioventricular mismatch (AV coupling mismatch), 4) abnormal ventriculo-aortic coupling, and 5) chronotropic incompetence. However, the exact mechanisms underlying this basic pathological symptom are not clear.

[0003] A number of animal experimental models, including mice, rats, and pigs, have been established and reported to simulate the disease heart failure with preserved ejection fraction (HFpEF).

[0004] There are several problems / defects in the existing technical working methods. Existing HFpEF models are not of human origin and thus cannot reproduce all the key characteristic clinical manifestations caused by species specificity and other inherent differences (for example, the mouse heart is significantly smaller in size, has a heart rate 10 times higher, the pig has a heart anatomy similar to that of humans, but has a significantly different response to pharmacological challenges).

[0005] Heart failure with preserved ejection fraction (HFpEF) accounts for more than 50% of all heart failure cases. Compared with many other cardiovascular diseases, the prevalence of HFpEF is increasing, resulting in significant morbidity, mortality, and social costs. HFpEF affects the elderly in an age-dependent manner, and its prevalence is increasing in Europe and the United States as the population ages. The mortality from HFpEF is high and comparable to that of heart failure with reduced ejection fraction (HFrEF). Unfortunately, many of the therapies that reduce mortality and morbidity in HFrEF have little effect on HFpEF. Patients with HFpEF do not respond to the standard care procedures for HFrEF because the biology and clinical course of these two diseases are very different.

[0006] Two pathological features have been shown to be significantly prevalent in patients with HFpEF: cardiomyocyte hypertrophy and cardiac fibrosis. In large prospective myocardial tissue analyses of HFpEF, myocardial fibrosis and hypertrophy are common features [11,12]. During the past decade, a great deal of effort has focused on elucidating the complex responses of cardiomyocytes to various hypertrophic stimuli and the signaling pathways that progress from cardiac hypertrophy to heart failure [13 - 21]. Endothelin-1 has been shown to be an effective hypertrophic stimulus for cardiomyocytes and has been shown to be elevated in patients with HFpEF

[22] . Cardiac fibrosis (CF) is highly associated with heart failure (HF), especially with HFpEF. Although CF has traditionally been considered a secondary phenomenon, it has recently been proposed that it plays a major role in the progression of HFpEF

[23] . The clinical outcomes of patients with severe aortic stenosis who undergo aortic valve replacement are related to the severity of CF

[24] . Based on this finding, CF is associated with the mortality of patients with HFpEF

[25] . Activation of the neurohumoral pathway directly stimulates fibroblasts. Members of the transforming growth factor-β family secreted in the cardiac interstitium play specific roles in activating specific aspects of the fibrotic response. Secreted fibrogenic mediators and matrix cell proteins bind to cell surface receptors in fibroblasts and induce the synthesis, processing, and metabolism of the extracellular matrix.

[0007] There is an urgent need to better understand the underlying cellular and molecular processes of HFpEF in order to develop targeted therapies for this largely untreated patient population. Although several animal experimental models have been established and reported to mimic human diseases, these animal experimental models are not of human origin and therefore cannot reproduce all the key characteristic clinical manifestations caused by species specificity and other inherent differences (e.g., the mouse heart is significantly smaller in size, has a heart rate 10 times higher, pigs have a heart anatomy similar to that of humans, but have significantly different responses to pharmacological agents).

[0008] Accordingly, there is still a need in the art for methods of treating heart failure with preserved ejection fraction, and thus, there is still a need in the art for therapeutic agents and therapeutic targets for treating the condition, as well as robust and accurate models for use in screening methods for identifying such therapeutic agents and targets. Summary of the Invention

[0009] A first in vitro human HFpEF heart model has been reported by using a combination of state-of-the-art human pluripotent stem cells (hPSCs) in a cardiac tissue engineering approach, where the hPSCs are directed to differentiate into human ventricular cardiomyocytes (hVCMs) for assembly into engineered tissue: the method involves human ventricular cardiac tissue strips (hvCTSs) and three-dimensional (3D) mechanoelectrical coupling, fluid jet micro-human ventricular-like cardiac organoid chambers (hvCOCs), which faithfully reproduce pathological features that have been shown to be significantly prevalent in patients with HFpEF, namely cardiomyocyte hypertrophy and cardiac fibrosis. The disclosed materials, methods, and systems can be used as tools for drug discovery and preclinical in vitro testing of drug and therapy candidates.

[0010] Three-dimensional bioengineered tissue constructs have been developed using cardiomyocytes derived from human pluripotent stem cells. It allows for the simulation of different cardiovascular disease states and the testing of drugs and biologics in human tissue. The in vitro human HFpEF heart model disclosed herein has been extensively validated using a series of phenotypic assessments. Human PSCs (HES2: human embryonic stem cells; ESI, NIH code ES02 and L-EdV: human induced pluripotent stem cells) are directed to differentiate into human ventricular cardiomyocytes (hvCMs) for assembly into engineered tissue in the form of human ventricular cardiac tissue strips (hvCTSs) and three-dimensional (3D) mechanoelectrical coupling, fluid jet micro-human ventricular-like cardiac organoid chambers (hvCOCs) [6-9].

[0011] The present disclosure provides materials and methods that exhibit new and unique features. Compared with the prior art, the present disclosure provides the use of biologics for inducing disease without the need for patient-specific human induced pluripotent stem cells. Additionally, the disclosed model is suitable for complex phenotypic measurements and is capable of reproducing the characteristic phenotypes seen in patients. In some exemplary embodiments, although the HFpEF phenotype (i.e., reversing HFpEF symptoms) is rescued in engineered tissue using adeno-associated virus gene therapy AAV1-SERCA, the disclosed model is suitable for evaluating the ability of any agent or candidate agent to rescue or improve HFpEF symptoms.

[0012] One aspect of the present disclosure relates to a method for identifying a therapeutic agent for treating heart failure with preserved ejection fraction, the method comprising: (a) contacting cardiac tissue with an effective amount of transforming growth factor-β1 and an effective amount of endothelin-1 (ET-1) to induce heart failure with preserved ejection fraction in the cardiac tissue; (b) exposing the cardiac tissue exhibiting heart failure with preserved ejection fraction to a candidate therapeutic agent, and (c) if, compared to the absence of the candidate therapeutic agent, in the presence of the candidate therapeutic agent, (i) the passive strain of the cardiac tissue, (ii) the cardiac tissue stiffness, (iii) the cardiomyocyte hypertrophy, or (iv) the level of cardiac fibrosis is reduced, or (v) the contractile kinetics or (vi) the relaxation kinetics is increased, or any combination thereof, then identifying the candidate therapeutic agent as a therapeutic agent for heart failure with preserved ejection fraction. In some embodiments, the cardiac tissue is formed into cardiac tissue strips. In some embodiments, the cardiac tissue strips are formed between two posts, and the reduction in the level of passive strain is detected by a reduction in the bending of the cardiac tissue strips. In some embodiments, the candidate therapeutic agent reduces the passive strain of the cardiac tissue. In some embodiments, the candidate therapeutic agent reduces the tissue stiffness of the cardiac tissue. In some embodiments, the candidate therapeutic agent reduces the cardiomyocyte hypertrophy in the cardiac tissue. In some embodiments, the candidate therapeutic agent reduces the cardiac fibrosis in the cardiac tissue. In some embodiments, the candidate therapeutic agent increases the contractile kinetics of the cardiac tissue. In some embodiments, the candidate therapeutic agent increases the relaxation kinetics of the cardiac tissue. In some embodiments, the candidate therapeutic agent is a small molecule, a nucleic acid, or a cell.

[0013] Aspects related to the present disclosure relate to a method for identifying a therapeutic agent for treating heart failure with preserved ejection fraction, the method comprising: (a) contacting a cardiac organoid with an effective amount of transforming growth factor-β1 and an effective amount of endothelin-1 (ET-1) to induce heart failure with preserved ejection fraction in cardiac tissue; (b) exposing the cardiac organoid exhibiting heart failure with preserved ejection fraction to a candidate therapeutic agent, and (c) if, compared to the absence of the candidate therapeutic agent, in the presence of the candidate therapeutic agent, (i) the passive strain of the cardiac organoid, (ii) the cardiac tissue stiffness in the cardiac organoid, (iii) the cardiomyocyte hypertrophy in the cardiac organoid, or (iv) the level of fibrosis in the cardiac organoid is reduced, or (v) the contractile kinetics of the cardiac organoid or (vi) the relaxation kinetics of the cardiac organoid is increased, or any combination thereof, then identifying the candidate therapeutic agent as a therapeutic agent for heart failure with preserved ejection fraction. In some embodiments, the candidate therapeutic agent reduces the passive strain of the cardiac organoid. In some embodiments, the candidate therapeutic agent reduces the stiffness of the cardiac organoid. In some embodiments, the candidate therapeutic agent reduces cardiomyocyte hypertrophy in the cardiac organoid. In some embodiments, the candidate therapeutic agent reduces cardiac fibrosis in the cardiac organoid. In some embodiments, the candidate therapeutic agent increases the contractile kinetics of the cardiac organoid. In some embodiments, the candidate therapeutic agent increases the relaxation kinetics of the cardiac organoid. In some embodiments, the candidate therapeutic agent is a small molecule, nucleic acid, or cell.

[0014] Another aspect of the present disclosure is a method for assessing the toxicity of a compound to cardiomyocytes, the method comprising: (a) contacting cardiac tissue with an effective amount of transforming growth factor-β1 and an effective amount of endothelin-1 (ET-1) to induce heart failure with preserved ejection fraction in the cardiac tissue; (b) exposing the cardiac tissue exhibiting heart failure with preserved ejection fraction to the compound, and (c) if, compared to the absence of the compound, in the presence of the compound, (i) the passive strain of the cardiac tissue, (ii) the cardiac tissue stiffness, (iii) the cardiomyocyte hypertrophy, or (iv) the level of cardiac fibrosis is reduced, or (v) the contractile kinetics or (vi) the relaxation kinetics is increased, or any combination thereof, then determining or confirming the toxicity of the compound to the cardiac tissue. In some embodiments, the toxicity of the compound to the cardiac tissue is determined. In some embodiments, the cardiac tissue is a cardiac tissue strip. In some embodiments, the cardiac tissue is a cardiac organoid.

[0015] Another aspect of the present disclosure is a method for determining the efficacy of a compound in alleviating at least one symptom of heart failure with preserved ejection fraction, the method comprising: (a) contacting cardiac tissue with an effective amount of transforming growth factor-β1 and an effective amount of endothelin-1 (ET-1) to induce heart failure with preserved ejection fraction in the cardiac tissue; (b) exposing the cardiac tissue exhibiting heart failure with preserved ejection fraction to the compound, and (c) determining or confirming the efficacy of the compound in alleviating at least one symptom of heart failure with preserved ejection fraction in the cardiac tissue if, compared to the absence of the compound, in the presence of the compound, (i) the passive strain of the cardiac tissue, (ii) the cardiac tissue stiffness, (iii) the cardiomyocyte hypertrophy, or (iv) the level of cardiac fibrosis is reduced, or (v) the level of systolic kinetics or (vi) the level of relaxation kinetics is increased, or any combination thereof. In some embodiments, the efficacy of the compound in alleviating at least one symptom of heart failure with preserved ejection fraction is determined. In some embodiments, the cardiac tissue is a cardiac tissue strip. In some embodiments, the cardiac tissue is a cardiac organoid.

[0016] Other features and advantages of the present disclosure will become apparent from the following detailed description, including the drawings. However, it should be understood that when indicating embodiments, the provision of the detailed description and specific examples is for illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1.Functional outcomes of the combined action of ET-1 / TGF-β1 on the contractility of hvCTS. (A) Representative images of time-matched hvCTS under control conditions and treated with the combination endothelin-1 / transforming growth factor-β1 (ET-1 / TGF-β1). Note that the pillars of the treated group are curved, indicating higher passive strain of the corresponding mounted hvCTS. (B) Representative normalized force traces recorded from hvCTS of the same groups as in (A). The group treated with ET-1 / TGF-β1 (dashed line) shows slower contraction and relaxation kinetics compared to the control group (solid line). (C) List plot of the contractile force. Control group (solid circles); group treated with ET-1 / TGF-β1 (dashed line). (D) Contraction time and relaxation time of the same groups as in (A)-(B) recorded by post-trace measurement. n = 4-20. Control group (solid circles); group treated with ET-1 / TGF-β1 (open circles). **p < 0.01 and ***p < 0.001. (E) Plot of the change in stiffness, generated force, maximum dF / dt, and maximum -dF / dt against % strain recorded using a length-controlled equidistant system for control and ET-1 / TGF-β1-treated hvCTS stiffness. n = 7-12.

[0018] Figure 2 .ET-1 / TGF-β1 confers HFpEF phenotypic characteristics to hvCOC. (A) Representative images of time-matched hvCOC under control conditions and treated with the combination of ET-1 / TGF-β1. Note that the treated group has a more compact appearance, consistent with a stiffer hvCOC. (B) After hydrostatic loading (100 μl), the change in diastolic pressure is plotted against the change in diastolic area. The slope of the plotted graph represents stiffness. Control (solid line); HFpEF (dashed line). (C) hvCOC treated with ET-1 / TGF-β1 (open circles) is significantly stiffer than the control group (solid circles). (D) Representative pressure-volume loops of control (solid line) and ET-1 / TGF-β1-treated (dashed line) hvCOC. (E) Relationship of pressure change (left) to time and volume change (right) to time for control (solid line) and ET-1 / TGF-β1-treated (dashed line) hvCOC. (F) List plot of the generated pressure, stroke volume, stroke work, and ejection fraction of control (solid circles) and ET-1 / TGF-β1-treated (open circles) hvCOC, indicating that although stiffness increases, there is no significant change in contractile function. In summary, the treated hvCOC shows a phenotype consistent with HFpEF characteristics. (B), (C), and (F) n = 8-13. Student t-test. *p < 0.05. Higher concentrations do impair the contractility of hvCOC.

[0019] Figure 3 . Transcriptomic and bioinformatics analysis of normal and HFpEF patients. hvCTS and hvCOC heart models reveal unique downregulation of SERCA2a in HFpEF. (A) Venn diagram showing 29% shared differentially expressed genes (DEGs) between HFpEF patients and the HFpEF-hvCTS heart model disclosed herein, and 33% shared DEGs between HFpEF patients and the HFpEF-hvCOC heart model disclosed herein. Comparative functional analysis using Ingenuity Pathway Analysis showed that, based on hierarchical clustering of enriched canonical pathways, biological functions, and cardiotoxicity functions, the heart organoids of the HFpEF-hvCOC model were more similar to human HFpEF patients than the heart tissue strips of the HFpEF-hvCTS model. (B) The calcium signaling pathway was similarly enriched in human HFpEF patients and in both engineered HFpEF-hvCTS and HFpEF-hvCOC tissue models and native human heart tissue from HFpEF patients relative to their respective controls. Notably, SERCA2a was the most highly expressed gene in the gene ontology or GO term "regulation of calcium transmembrane transport activity coupled to ATPase" in normal patients and control engineered hvCTS and models. Interestingly, SERCA2a (ATP2A2) was also most significantly downregulated in HFpEF patients and in both engineered HFpEF-hvCTS and HFpEF-hvCOC heart models compared to their respective controls. These transcriptomic and bioinformatics results are consistent with the HFpEF phenotype observed in HFpEF patients and in engineered HFpEF-hvCTS and HFpEF-hvCOC models, indicating that SERCA2A is a target candidate for the amelioration, rescue, or reversal of HFpEF disease or its symptoms in humans.

[0020] Figure 4 . AAV transduction titer-dependence and time-dependence. (A) Representative fluorescence and bright-field images of hvCMs 14 days after AAV-GFP transduction with different viral genomes (vg) / cardiomyocytes. (B) Effect of time and viral titer on the percentage of GFP-expressing cells after AAV-GFP transduction. (C) Effect of SERCA gene expression 14 days after AAV-GFP or AAV1.SERCA2A transduction. (C) n = 1 - 3. One-way ANOVA. *p < 0.05.

[0021] Figure 5. AAV1-SERCA2a rescues the HFpEF phenotype in hvCTS. (A) Representative images of HFpEF-hvCTS with AAV-GFP or AAV1.SERCA2A. (B) Representative force tracings of HFpEF-hvCTS with AAV-GFP (solid line) or AAV1.SERCA2A (dashed line). (C) Contractile force. HFpEF-hvCTS with AAV-GFP (solid circles); HFpEF-hvCTS with AAV1.SERCA2A (open circles). (D) Contraction time and relaxation time of HFpEF-GFP and HFpEF-SERCA hvCTS obtained by post-trace measurement. HFpEF-hvCTS with AAV-GFP (solid circles); HFpEF-hvCTS with AAV1.SERCA2A (open circles). (C)-(D) n = 5-8. Student's t-test.

[0022] Figure 6 . Effects of ET-1 on hvCTS. (A) Representative images of hvCTS treated with 0-100 nM ET-1. (B) Representative post-trace normalized force tracings of hvCTS. 0 nM ET-1 (black line); 30 nM ET-1 (gray line); 100 nM ET-1 (dashed line). (C) Contractile force. 0 nM ET-1 (black circles); 30 nM ET-1 (gray circles); 100 nM ET-1 (open circles). (D) Contraction time and relaxation time of control and ET-1 hvCTS obtained by post-trace measurement. 0 nM ET-1 (black circles); 30 nM ET-1 (gray circles); 100 nM ET-1 (open circles). (E) Effects of % strain on hvCTS stiffness, force generated, maximum dF / dt, and maximum -dF / dt, as measured using an isometric system. (C)-(D) n = 4. Student's t-test. (E) n = 2. Multiple t-test. Control (solid circles); ET-1 (open circles). *p < 0.05 and **p < 0.01.

[0023] Figure 7. Effects of TGF-β1 on hvCTS. (A) Representative images of hvCTS treated with 0 - 30 ng / ml TGF-β1. (B) Representative normalized force traces after tracing of hvCTS without TGF-β1 (control; solid line) and 1 ng / ml TGF-β1 (dashed line). (C) Contractile force. 0 ng / ml TGF-β1 (solid circles); 1 ng / ml TGF-β1 (open circles); 3 ng / ml TGF-β1 (upright open triangles); 10 ng / ml TGF-β1 (inverted open triangles); 30 ng / ml TGF-β1 (open squares). (D) Contraction time and relaxation time of hvCTS at different TGF-β1 concentrations obtained by post-tracing measurement. 0 ng / ml TGF-β1 (solid circles); 1 ng / ml TGF-β1 (open circles); 3 ng / ml TGF-β1 (upright open triangles); 10 ng / ml TGF-β1 (inverted open triangles); 30 ng / ml TGF-β1 (open squares). (E) Effects of % strain on hvCTS stiffness, force generated, maximum dF / dt, and maximum -dF / dt in the control and 1 ng / ml TGF-β1 groups, as measured using an isometric system. Control (solid circles); TGF-β1 (open circles). (C) - (D) n = 2 - 16. Student's t-test. (E) n = 12 - 16. Multiple t-tests. *p < 0.05, **p < 0.01, and ***p < 0.001. Detailed implementation

[0024] In mammalian hearts, HFpEF is associated with impaired cardiac relaxation [26, 27]. Cardiomyocytes isolated from HFpEF patients and experimental models are characterized by prolonged relaxation, reduced contraction velocity, decreased β-adrenergic response, and increased myocardial stiffness [26, 27]. Many cellular and molecular mechanisms may contribute to disease-related defects. It is believed that abnormal cardiac relaxation is attributed to defects in the sarcoplasmic reticulum (SR) Ca 2+ -ATPase pump activity, which is mainly responsible for controlling the rate at which Ca 2+ is taken up into the SR during relaxation [28 - 31]. In experimental models of HFpEF, the reduced SR Ca 2+ uptake that causes prolonged contraction during relaxation is associated with SR Ca 2+-ATPase pump content and activity. In addition, SERCA2a protein levels were found to be significantly reduced in the aging human myocardium characteristic of HFpEF. This reduction in SERCA2a levels was associated with impaired myocardial function at baseline and was further exacerbated by higher heart rates and hypoxic conditions [32-34]. Studies of human left ventricular hypertrophy have shown a relationship between elevated calcium load and diastolic tension, which is amplified at faster heart rates. Calcium that dissociates slowly from the troponin-actin-myosin complex after contraction may contribute to a "rigor-like" state that increases diastolic stiffness. Additionally, slow reuptake is directly related to the relaxation phase of stress. In experimental models of HFpEF, SERCA2a has been found to be reduced and intracellular calcium elevated. Thus, the consequences of slowed calcium reuptake affect not only myocardial relaxation but also contribute to the development of diastolic stiffness, a key feature in the pathophysiology of HFpEF.

[0025] In most patients with HFpEF, abnormal lusitropy (myocardial relaxation), measured by impaired tissue relaxation, is evident at rest and during exercise, but the diagnostic sensitivity is reduced when measured only at rest. During exercise, when both preload and heart rate are elevated, reduced distensibility is compounded by impaired lusitropy, resulting in a rapid increase in left ventricular (LV) pressure in the incompletely relaxed ventricle. Many cellular and molecular mechanisms may contribute to disease-related defects. Abnormalities in cardiac relaxation are attributed to defects in sarcoplasmic reticulum (SR) Ca 2+ -ATPase pump activity, which is primarily responsible for controlling the rate at which Ca 2+ is taken up into the SR during relaxation [28-31]. In experimental models of HFpEF, reduced SR Ca 2+ uptake during relaxation causes prolonged contraction and is associated with SR Ca 2+-ATPase pump content and activity. In addition, SERCA2a protein levels were found to be significantly reduced in the aging human myocardium characteristic of HFpEF. This reduction in SERCA2a levels was associated with impaired myocardial function at baseline and was further exacerbated by higher heart rates and hypoxic conditions [32-34]. Another approach to restoring intracellular calcium homeostasis is to enhance SERCA calcium uptake. This can be achieved by modulating SERCA activity or increasing SERCA pump expression. It has been shown that in an HFpEF animal model characterized by abnormal lusitropy and reduced SERCA2a, delivery of SERCA2a by gene transfer restored relaxation parameters such as -dP / dt, the left ventricular time constant τ of isovolumic (i.e., constant volume) relaxation, and the passive stiffness of the left ventricle to adult levels

[31] . These results confirm that viral delivery of SERCA2a in the HFpEF heart can improve relaxation parameters

[31] . In other studies, Otsuka-Long-Evans Tokushima fatty rats were used as a model of HFpEF, which represents a model for spontaneous non-insulin-dependent type II diabetes (DM), characterized by diastolic dysfunction and associated with reduced SERCA2a expression. In multiple studies using short-term expression of SERCA2a, oxygen consumption and relaxation parameters were restored to normal levels in these HFpEF models [28-30]. The experiments disclosed herein established HFpEF-hvCTS and HF-EF-hvCOC models of heart failure with preserved ejection fraction in diseased human hearts, and the data further relate to a method of treating HFpEF that involves increasing or restoring intracellular calcium homeostasis in cardiomyocytes by increasing or enhancing SERCA calcium uptake to improve or reverse HFpEF, for example, in a human.

[0026] There are currently no human clinical trials targeting relaxation in HFpEF. To date, only two clinical trials, both in chronic systolic heart failure (HFrEF), have tested the effect of improving SERCA2a function in HF. The HORIZONS-HF study investigated the effect of istaoxime (an intravenous agent that increases SERCA2a activity) in hospitalized patients with acute decompensated heart failure. Pulmonary capillary wedge pressure (PCWP) decreased by 3-5 mmHg in patients receiving a 6-hour infusion of istaoxime, while right atrial pressure or cardiac index remained unchanged

[35] . The CUPID-1 study tested the effect of SERCA2a gene therapy in patients with HFrEF. Subjects randomly receiving the highest dose of a single intracoronary infusion of AAV1-SERCA2a had less deterioration in 6-minute walk time, peak VO2, and NT-proBNP levels after 6 months [36-38]. In a larger study testing only this high dose (1013 In a follow-up study of 15 patients receiving 1×10¹² genome particles of AAV1.SERCA2a per patient, the safety of this method was confirmed [39-40]. However, SERCA2a transduction had no effect on the primary outcomes of HF hospitalization or decompensated HF. In patients with available cardiac tissue, the median AAV1-SERCA2a DNA level measured was 43 copies / μg DNA (range 10-192 copies / μg DNA), which was below the dose of 2,000-50,000 copies / μg DNA that was effective in preclinical models. This very low transduction efficiency may explain the neutral results

[41] . Although the results from the study were disappointing, the strategy of the CUPID-2 trial was to increase SERCA2a activity as a way to improve the systolic dysfunction unique to HFrEF. The effect of SERCA2a activity on cardiac relaxation was not evaluated. Increasing SERCA2a activity or expression is expected to be an attractive therapeutic target in HFpEF. Indeed, using the human heart tissues and chambers disclosed herein has enabled us to titrate an optimized titer for transducing the human heart, which is much higher than the optimized titer that works in animals. Shortening LVτ would likely allow for tolerance of higher heart rates by modulating the rapid rise in ventricular chamber pressure, thus allowing the ventricles to fill more completely with blood and improving exercise capacity and peak VO2. Therefore, transduction with higher doses of AAV1.SERCA2A is also expected to produce a more effective and robust dose response. Additionally, as increased SERCA2a activity restores myocardial calcium homeostasis, over time, increasing or restoring SERCA protein expression is expected to lead to reversal of other maladaptive pathways. Combining the above with the observation that higher viral titers disclosed herein are beneficial in transducing hCM, it is expected that higher doses of AAV1.SERCA2a than those used in CUPID2 will provide effective treatment for patients with HFpEF, with precise characterization of myocardial relaxation and filling pressures during exercise before and after treatment.

[0027] In summary, the disclosed HFpEF-hvCTS and HFpEF-hvCOC are of human origin in the disease context (HFpEF) and are suitable for complex phenotypic measurements, as revealed in the following examples. Thus, the HFpEF-hvCTS and hvHFpEF-hvCOC cardiac models are able to recapitulate the characteristic phenotypes seen in patients. Using the SERCA2a transgene as an example, it was also demonstrated how the disclosed human HFpEF models can be used to identify novel druggable targets, followed by therapeutic screening for cardiac improvement. As needed, the general HFpEF model can be further customized for the discovery of additional novel targets and screening of therapeutic agents. Overall, the preclinical human HFpEF models disclosed herein can be used to study disease mechanisms and facilitate the discovery of novel druggable targets and screening of therapeutic agents to identify the most promising agents by accurately predicting the clinical efficacy and safety of new drugs prior to human trials.

[0028] Example

[0029] Example 1

[0030] Materials and Methods

[0031] Induction of HFpEF in human ventricular cardiac tissue strips (hvCTS)

[0032] Engineer three-dimensional (3D) multicellular human ventricular cardiac tissue strips (hvCTS) containing cardiac tissue in a manner consistent with that previously described ([6,10]). Briefly, cardiac clusters from day 15 of hPSC cardiac differentiation are dissociated into single cells and allowed to recover in an incubator for 3 days prior to hvCTS construction. Each hvCTS consists of 1.3×10 6 cardiac cells differentiated from hPSCs in 100 μl of an ice-cold solution of 2 mg / ml collagen I (0.80 - 0.95 mg / ml Matrigel, 0.6X PBS, 20 mM NaOH, 0.8X minimum essential medium (Sigma-Aldrich), 1.6 mM HEPES, and 0.1X hvCTS maintenance medium (see composition below)) and 1.3×10 5Composed of human foreskin fibroblasts. Then, 100 μl of the final cell-collagen mixture was added to each polydimethylsiloxane (PDMS) bioreactor, which consisted of force-sensing cantilever columns at each end of a rectangular well, and returned to the incubator to form hvCTS attached between the two end columns. The hvCTS was maintained in DMEM medium supplemented with 10% newborn calf serum (Gibco), with half of the medium changed daily for 5 days, and then switched to RPMI + B27 containing TGFβ1 (1 ng / mL) and ET-1 (100 nM) for an additional 5.5 days to induce the HFpEF phenotype, thus generating hvCTS with the HFpEF phenotype ready for testing.

[0033] Measurement results of stress and strain in hvCTS

[0034] At 37 °C, the force generated by hvCTS was measured using an isometric muscle bath system in phenol red-free DMEM medium with HEPES buffer. Using the isometric muscle bath system, the force and other contractile parameters were obtained at 0 - 50% of the natural length of the tissue (L0) at 5% intervals, under spontaneous (unstimulated) and electrically stimulated conditions (field stimulation at 1.0 Hz, 1.5 Hz, 2.0 Hz, 2.5 Hz, and 3.0 Hz). Stiffness was calculated as stress / strain.

[0035] HFpEF induction in human heart organoid ventricles (hvCOC)

[0036] The 3D multicellular human ventricular cardiac organoid chamber (hvCOC) myocardial tissue was engineered using a super-compliant indwelling elastomeric balloon as previously described ([8],

[42] ). For ease of explanation, hvCOC is used herein to refer to the human ventricular cardiac organoid chamber and the human ventricular cardiac organoids contained within such a chamber, as will be apparent from the context. Briefly, each hvCOC consisted of 1.0×10 7 cardiac cells differentiated from hPSCs and 1.0×10 6Composed of human foreskin fibroblasts. The cell-collagen mixture was added to the space between the agarose mold and the balloon, ensuring that the porous polyethylene ring was placed just below the O-ring (5.8 mm) 3 mm above the bottom of the balloon to enhance tissue attachment and was immersed in the cell suspension. The bioreactor was incubated for 1 hour for gel curing before filling it with NCS medium (8 mL) up to the top of the bioreactor. The medium was changed every 24 hours when the hvCOC was compacted. Five days after removing the hvCOC from the agarose gel, the medium was changed every other day, switched to RPMI + B27 (1 ng / mL) containing TGFβ1 for 4 days and then switched to RPMI + B27 (1 ng / mL) containing TGFβ1 + ET-1 (100 nM) for an additional 1.5 days to induce the HFpEF phenotype.

[0037] Measurement results of stiffness and function in hvCOC

[0038] A high-sensitivity pressure catheter was advanced into the lumen of the hvCOC chamber for pressure measurement. A digital camera was mounted outside the bioreactor and allowed for direct tissue monitoring to determine the chamber area. Chamber pressure and digital video were acquired simultaneously under spontaneous (unstimulated) and electrically paced conditions (field stimulation at 1.0 Hz, 1.5 Hz, 2.0 Hz, 2.5 Hz, and 3.0 Hz) at 0 μL, 25 μL, 50 μL, and 100 μL loads. Stiffness was calculated by plotting the slope of the change in diastolic pressure against the change in diastolic area of the chamber.

[0039] Bulk RNA sequencing of HFpEF hvCOC and hvCTS

[0040] Differentially expressed genes (DEGs) from RNAseq analysis of the HFpEF-hvCTS and HFpEF-hvCOC models (TGFB1 + ET1 vs. control) were compared with human heart failure transcriptomic data

[11] for gene-level analysis and comparative functional enrichment analysis.

[0041] AAV1.SERCA2A infection in hvCTS

[0042] On day 15 after differentiation, at 1 x 10 2 viral genomes (vg) / cardiomyocyte to 1 x 10 5Individual vg / cardiomyocytes were transduced with AAV1.SERCA2A (encoding region expressing SERCA2a) into dissociated human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to deliver SERCA2a mRNA into hvCTS. The hPSC-CMs were then prepared into hvCTS and induced to HFpEF with HFpEF induction medium containing TGFB1 + ET1 as described above and maintained in this medium until ready for testing on day 11 after preparation. In some experiments, SERCA2a mRNA was delivered into hvCTS by directly transducing hvCTS with AAV1.SERCA2A without HFpEF induction and maintained for 8 days until ready for testing.

[0043] Example 2

[0044] hvCTS exhibiting the HFpEF phenotype

[0045] Given the biological roles of ET-1 and TGF-β1 in HFpEF, the combination of hypertrophic stimuli and profibrotic signals was tested by examining the functional outcome of their combined effect on the contractility of hvCTS to reproduce the HFpEF phenotype, an issue relevant to the establishment of a human HFpEF cardiac model. Figure 1 A shows representative images of time-matched hvCTS under control conditions and after combined ET-1 / TGF-β1 treatment (5.5 days). Note that the columns of the treatment group are curved, indicating higher passive strain in the corresponding mounted hvCTS. Consistent with this, the ET-1 / TGF-β1-treated group showed slower contraction and relaxation kinetics compared to the control, as evaluated by post-trace measurements ( Figure 1 B - D; n = 4 - 20, **p < 0.01 and ***p < 0.001). For further mechanistic insights, length-controlled hvCTS recordings were obtained using an isometric system. Figure 1 E shows the relationship between stiffness, force generated, maximum dF / dt, and maximum -dF / dt as a function of strain % for control and ET-1 / TGF-β1-treated hvCTS (n = 7 - 12). Collectively, these data show that ET-1 / TGF-β1-treated, but not control or single-treated ( Figure 7 and 8) hvCTS uniquely showed significantly increased stiffness, as well as slowed relaxation and contraction kinetics, all of which are characteristics observed in HFpEF patients.

[0046] Example 3

[0047] Then, the ability of ET-1 / TGF-β1 administration to confer HFpEF phenotypic properties on hvCOCs (i.e., hv cardiac organoids contained within hvCOCs) was evaluated. Figure 2 A shows representative images of time-matched hvCOCs under control conditions and after combined ET-1 / TGF-β1 treatment. Figure 2 B shows that, compared to time-matched control hvCOCs, ventricular stiffness, as derived from the plot of change in diastolic pressure against change in diastolic area after hydrostatic loading (100 μL), was significantly higher and the appearance more compact in ET-1 / TGF-β1-treated hvCOCs. Figure 2 Representative pressure–volume loops of control (black) and ET-1 / TGF-β1-treated (red) hvCOCs are given in D. Despite the increased stiffness, no changes in developed pressure, stroke volume, stroke work, or ejection fraction were observed between control (black) and ET-1 / TGF-β1-treated (red) hvCOCs, indicating that systolic function was not significantly altered. Thus, ET-1 / TGF-β1-treated hvCOCs showed a phenotype consistent with HFpEF characteristics ( Figure 2 E–F; n = 8–13, *p < 0.05).

[0048] Example 4

[0049] To gain molecular insights into HFpEF tissue, transcriptomic and bioinformatics analyses were performed to systematically compare normal and HFpEF patients, as well as control and HFpEF-induced hvCTS and hvCOC ( Figure 3 ). The data were organized into a Venn diagram that showed a 29% overlap in differentially expressed genes (DEGs) between HFpEF patients and the HFpEF-hvCTS model ( Figure 3 A). This overlap between HFpEF patients and the HFpEF-hvCOC model was 33%. Comparative functional analysis using the Ingenuity Pathway Analysis further revealed that, based on hierarchical clustering of enriched canonical pathways, biological functions, and cardio-toxicity functions, the HFpEF-hvCOC model was more similar to human HFpEF patients compared to the HFpEF-hvCTS model ( Figure 3B). Interestingly, the calcium signaling pathway was similarly enriched in both human HFpEF patients and engineered HFpEF-hvCTS and HFpEF-hvCOC models relative to their respective controls. Notably, the ATP2A2 gene (encoding SERCA2a) was the most highly expressed gene in the GO term "regulation of ATPase-coupled calcium transmembrane transport activity" in normal patients as well as control engineered hvCTS and hvCOC. Interestingly, ATP2A2 (SERCA2a) was also the most significantly downregulated in HFpEF patients as well as engineered HFpEF-hvCTS and HFpEF-hvCOC models compared to their respective controls. These transcriptomic and bioinformatics results are consistent with the phenotypes observed in HFpEF patients, suggesting that SERCA2a is a target candidate for rescuing or reversing disease traits.

[0050] To test this hypothesis, the titer-dependence and time-dependence of recombinant adeno-associated virus type 1 (AAV1) transduction of hCM were first investigated. Figure 4 Panel A shows that after AAV-GFP transduction, the percentage of green fluorescent protein-positive (GFP-positive) hvCM gradually increased over time until reaching a plateau ([ Figure 4 Panels A and 4B). The time required to reach the plateau and the plateau level were dependent on the virus titer (viral genomes or vg / cell). Similarly, the effect on SERCA2a gene expression 14 days after AAV1-SERCA2a transduction was time-dependent and titer-dependent (n = 1 - 3; one-way ANOVA, *p < 0.05). Based on these data, a titer of 1 x 10 5 viral genomes / cell and day 14 were selected to study the effect of AAV1-SERCA2a-mediated overexpression in HFpEF-hvCTS. Figure 5 Panel A shows representative images of time-matched hvCOC under HFpEF conditions and after combined AAV1.SERCA2a treatment. Figure 5 Panels C - D show that AAV1-SERCA2a transduction rescues the disease phenotype in HFpEF-hvCTS by restoring slowed contraction kinetics. Isometric measurements further revealed the effect of strain % on the stiffness, force generated, maximum dF / dt, and maximum -dF / dt of AAV1.SERCA2A-transduced HFpEF-hvCTS (n = 5 - 8, p < 0.05).

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[0094] For the purposes of describing and disclosing methods, such as those described in such publications, that may be used in connection with the information described herein, all patents and other publications identified are hereby expressly incorporated by reference in their entirety.

Claims

1. A method for identifying a therapeutic agent for treating heart failure with preserved ejection fraction, the method comprising: (a) contacting cardiac tissue with an effective amount of transforming growth factor-β1 and an effective amount of endothelin-1 (ET-1) to induce heart failure with preserved ejection fraction in the cardiac tissue; (b) exposing the cardiac tissue exhibiting heart failure with preserved ejection fraction to a candidate therapeutic agent, and (c) identifying the candidate therapeutic agent as a therapeutic for heart failure with preserved ejection fraction if, in the presence of the candidate therapeutic agent, the level of (i) passive strain of the cardiac tissue, (ii) cardiac tissue stiffness, (iii) cardiomyocyte hypertrophy, or (iv) cardiac fibrosis is reduced, or the level of (v) contraction kinetics or (vi) relaxation kinetics is increased, or any combination thereof, is increased. The method of claim 1 , wherein the cardiac tissue is formed into cardiac tissue strips.

3. The method of claim 2, wherein the cardiac tissue strip is formed between two posts, and the reduced level of passive strain is detected by a reduction in bending of the cardiac tissue strip. The method of claim 1 , wherein the candidate therapeutic agent reduces passive strain on the cardiac tissue.

5. The method of claim 1, wherein the candidate therapeutic agent reduces tissue stiffness of the cardiac tissue. The method of claim 1 , wherein the candidate therapeutic agent reduces cardiomyocyte hypertrophy in the cardiac tissue.

7. The method of claim 1, wherein the candidate therapeutic agent reduces cardiac fibrosis in the cardiac tissue.

8. The method of claim 1, wherein the candidate therapeutic agent increases the contractile kinetics of the cardiac tissue.

9. The method of claim 1, wherein the candidate therapeutic agent increases the relaxation kinetics of the cardiac tissue.

10. The method of claim 1, wherein the candidate therapeutic agent is a small molecule, a nucleic acid, or a cell.

11. A method for identifying a therapeutic agent for treating heart failure with preserved ejection fraction, the method comprising: (a) contacting the cardiac organoid with an effective amount of transforming growth factor-β1 and an effective amount of endothelin-1 (ET-1) to induce heart failure with preserved ejection fraction in cardiac tissue; (b) exposing the cardiac organoid exhibiting heart failure with preserved ejection fraction to a candidate therapeutic agent, and (c) identifying the candidate therapeutic agent as a therapeutic for heart failure with preserved ejection fraction if, in the presence of the candidate therapeutic agent, the level of (i) passive strain in the cardiac organoid, (ii) cardiac tissue stiffness in the cardiac organoid, (iii) cardiomyocyte hypertrophy in the cardiac organoid, or (iv) fibrosis in the cardiac organoid is reduced, or the level of (v) contraction kinetics in the cardiac organoid or (vi) relaxation kinetics in the cardiac organoid is increased, or any combination thereof.

12. The method according to claim 11, wherein the candidate therapeutic agent reduces the passive strain of the cardiac organoid.

13. The method according to claim 11, wherein the candidate therapeutic agent reduces the stiffness of the cardiac organoid.

14. The method according to claim 11, wherein the candidate therapeutic agent reduces cardiomyocyte hypertrophy in the cardiac organoid.

15. The method according to claim 11, wherein the candidate therapeutic agent reduces cardiac fibrosis in the cardiac organoid.

16. The method according to claim 11, wherein the candidate therapeutic agent increases the contractile kinetics of the cardiac organoid.

17. The method according to claim 11, wherein the candidate therapeutic agent increases the relaxation kinetics of the cardiac organoid.

18. The method according to claim 11, wherein the candidate therapeutic agent is a small molecule, nucleic acid, or cell.

19. A method for evaluating the toxicity of a compound to cardiomyocytes, the method comprising: (a) contacting cardiac tissue with an effective amount of transforming growth factor-β1 and an effective amount of endothelin-1 (ET-1) to induce heart failure with preserved ejection fraction in the cardiac tissue; (b) exposing the cardiac tissue exhibiting heart failure with preserved ejection fraction to the compound, and (c) determining or confirming the toxicity of the compound to the cardiac tissue if, compared to the absence of the compound, in the presence of the compound, (i) the passive strain of the cardiac tissue, (ii) the cardiac tissue stiffness, (iii) the level of cardiomyocyte hypertrophy, or (iv) the level of cardiac fibrosis is reduced, or (v) the level of contractile kinetics or (vi) the level of relaxation kinetics is increased, or any combination thereof.

20. The method according to claim 19, wherein the toxicity of the compound to the cardiac tissue is determined.

21. The method according to claim 19, wherein the cardiac tissue is a cardiac tissue strip.

22. The method according to claim 19, wherein the cardiac tissue is a cardiac organoid.

23. A method for determining the efficacy of a compound in alleviating at least one symptom of heart failure with preserved ejection fraction, the method comprising: (a) contacting cardiac tissue with an effective amount of transforming growth factor-β1 and an effective amount of endothelin-1 (ET-1) to induce heart failure with preserved ejection fraction in the cardiac tissue; (b) exposing the cardiac tissue exhibiting heart failure with preserved ejection fraction to the compound, and (c) determining or confirming the efficacy of the compound in alleviating at least one symptom of heart failure with preserved ejection fraction in the cardiac tissue if, compared to the absence of the compound, in the presence of the compound, (i) the passive strain of the cardiac tissue, (ii) the cardiac tissue stiffness, (iii) the level of cardiomyocyte hypertrophy, or (iv) the level of cardiac fibrosis is reduced, or (v) the level of contractile kinetics or (vi) the level of relaxation kinetics is increased, or any combination thereof.

24. The method according to claim 23, wherein the efficacy of the compound in alleviating at least one symptom of heart failure with preserved ejection fraction is determined.

25. The method according to claim 23, wherein the cardiac tissue is a strip of cardiac tissue.

26. The method according to claim 23, wherein the cardiac tissue is a cardiac organoid.

Images