Young Wook Chun

Screening for Acute IKr Block Is Insufficient to Detect Torsades de Pointes Liability Role of Late Sodium Current

Background— New drugs are routinely screened for IKr blocking properties thought to predict QT prolonging and arrhythmogenic liability. However, recent data suggest that chronic (hours) drug exposure to phosphoinositide 3-kinase inhibitors used in cancer can prolong QT by inhibiting potassium currents and increasing late sodium current (INa-L) in cardiomyocytes. We tested the extent to which IKr blockers with known QT liability generate arrhythmias through this pathway. Methods and Results— Acute exposure to dofetilide, an IKr blocker without other recognized electropharmacologic actions, produced no change in ion currents or action potentials in adult mouse cardiomyocytes, which lack IKr. By contrast, 2 to 48 hours of exposure to the drug generated arrhythmogenic afterdepolarizations and ≥15-fold increases in INa-L. Including phosphatidylinositol 3,4,5-trisphosphate, a downstream effector for the phosphoinositide 3-kinase pathway, in the pipette inhibited these effects. INa-L was also increased, and inhibitable by phosphatidylinositol 3,4,5-trisphosphate, with hours of dofetilide exposure in human-induced pluripotent stem cell–derived cardiomyocytes and in Chinese hamster ovary cells transfected with SCN5A, encoding sodium current. Cardiomyocytes from dofetilide-treated mice similarly demonstrated increased INa-L and afterdepolarizations. Other agents with variable IKr-blocking potencies and arrhythmia liability produced a range of effects on INa-L, from marked increases (E-4031, D-sotalol, thioridazine, and erythromycin) to little or no effect (haloperidol, moxifloxacin, and verapamil). Conclusions— Some but not all drugs designated as arrhythmogenic IKr blockers can generate arrhythmias by augmenting INa-L through the phosphoinositide 3-kinase pathway. These data identify a potential mechanism for individual susceptibility to proarrhythmia and highlight the need for a new paradigm to screen drugs for QT prolonging and arrhythmogenic liability.

EMT-inducing biomaterials for heart valve engineering: taking cues from developmental biology

Although artificial prostheses for diseased heart valves have been around for several decades, viable heart valve replacements have yet to be developed due to their complicated nature. The majority of research in heart valve replacement technology seeks to improve decellularization techniques for porcine valves or bovine pericardium as an effort to improve current clinically used valves. The drawback of clinically used valves is that they are nonviable and thus do not grow or remodel once implanted inside patients. This is particularly detrimental for pediatric patients, who will likely need several reoperations over the course of their lifetimes to implant larger valves as the patient grows. Due to this limitation, additional biomaterials, both synthetic and natural in origin, are also being investigated as novel scaffolds for tissue-engineered heart valves, specifically for the pediatric population. Here, we provide a brief overview of valves in clinical use as well as of the materials being investigated as novel tissue-engineered heart valve scaffolds. Additionally, we focus on natural-based biomaterials for promoting cell behavior that is indicative of the developmental biology process that occurs in the formation of heart valves in utero, such as epithelial-to-mesenchymal transition or transformation. By engineering materials that promote native developmental biology cues and signaling, while also providing mechanical integrity once implanted, a viable tissue-engineered heart valve may one day be realized. A viable tissue-engineered heart valve, capable of growing and remodeling actively inside a patient, could reduce risks and complications associated with current valve replacement options and improve overall quality of life in the thousands of patients who received such valves each year, particularly for children.

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

RATIONALE The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. OBJECTIVE To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. METHODS AND RESULTS Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa). CONCLUSIONS The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.Rationale: The lack of measurable single-cell contractility of human-induced pluripotent stem cell–derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. Objective: To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Methods and Results: Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current ( I Na). Conclusions: The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing. # Novelty and Significance {#article-title-25}

In situ forming gelatin-based tissue adhesives and their phenolic content-driven properties

The present study describes enzymatically cross-linked gelatin-based hydrogels as in situ forming tissue adhesives. A series of gelatin derivatives with different phenolic contents were synthesized by conjugating hydroxyphenyl propionic acid and tyramine to gelatin backbones. Two gelatin derivatives, gelatin-hydroxyphenyl propionic acid (GH) and gelatin-hydroxyphenyl propionic acid-tyramine (GHT) with maximum obtainable phenolic contents (146.6 μmol g-1 GH and 395.7 μmol g-1 GHT), were used to prepare gelatin-based hydrogels via horseradish peroxidase (HRP)-mediated reactions in the presence of hydrogen peroxide (H2O2). By changing the HRP and H2O2 concentrations, the gelation time, mechanical strength, and degradation rate of the hydrogels were fairly well controlled, indicating a tunable rate and degree of cross-linking. In addition, we found that an increase in phenolic content led to increased mechanical strength of the hydrogels. Lap-shear test results clearly showed that the GH and GHT hydrogels exhibited 2-3 times greater tissue adhesiveness compared to fibrin glues. On the basis of these results, we conclude that in situ forming gelatin-based hydrogels, which are both injectable and sprayable, can be used as an alternative to conventional tissue adhesives.

A temperature-sensitive, self-adhesive hydrogel to deliver iPSC-derived cardiomyocytes for heart repair

Induced pluripotent stem cells (iPSCs) from patients’ somatic tissues provide a viable source to create autologous cardiomyocytes (CMs) for potential cardiac-related cell therapies. However, a gap between the generation of iPSC-derived cardiomyocytes (iPSC-CMs) and the successful intra-cardiac engraftment of the cells to restore heart function remains to be bridged. Clinical data reporting engraftment of cells within human heart tissue has not been without its challenges, with significant cell loss from the site of delivery due to the physical stress of the cardiac cycle and the hostile inflammatory response within the infarct zone.[1–3] Hydrogels have been proven to support the survival of multiple cell types and have served as a platform for cell transplantation.[4, 5] Yet the use of tissue-adhesive, temperature-sensitive hydrogels to deliver iPSC-derived cardiomyocytes to infarcted heart remains to be explored. Therefore, we developed a polymer hydrogel to encapsulate, deliver, and integrate iPSC-CMs into infarcted myocardium to restore heart function. A temperature-sensitive biodegradable copolymer (polyethylene glycol-co-poly-e-caprolactone (PEG-PCL)) was synthesized [6] and conjugated with a collagen-binding peptide (SYIRIADTNIT). [7] The polymer was soluble in aqueous solutions at room temperature and underwent solution-to-gel transition at 37°C (Fig. 1 a,b).[6] As the peptide-modified polymer had a strong affinity to collagen I in vitro (Fig. 1c), it was expected that it would significantly increase the binding of the hydrogel to an infarcted heart, thus immobilizing iPSC-CMs within the damaged myocardium. Functional, beating cardiomyocytes were derived from patient fibroblast-derived iPSCs using a “Matrigel sandwich” method.[8] Cardiac differentiation was confirmed by fluorescence-activated cell sorting (FACS) and by immunostaining with known cardiac markers cardiac troponin T (cTNT), ryanodine receptor 2 (RYR) and α-actinin (Fig. 1f). The iPSC-CMs encapsulated in the polymer hydrogel at 37°C for two weeks maintained their viability (Fig. 1g) and cardiac phenotype, as evidenced by strong expression of troponin T and Nkx2.5 (Fig. 1h); thus, PEG-PCL does not appear to have obvious toxic effects on iPSC-CMs. Fig. 1 PEG-PCL copolymer was (a) dissolved in PBS at RT and (b) gelled at 37°C within 5 min (Sol-gel transition). Collagen-binding study was performed by adding peptide-modified polymer to collagen-coated surface at 37°C, and unbound polymer ... Engraftment of iPSC-CMs into infarcted myocardium using the peptide-modified hydrogel and its impact on infarcted heart function and structure were assessed with a rat myocardial infarction (MI) model. All animal surgery and animal care were approved by the Institutional Animal Care and Use Committee (IACUC) at Vanderbilt University (protocol: M/12/074). The left anterior descending (LAD) coronary artery of nude rats was ligated to induce MI. At 30 minutes post-MI, iPSC-CMs alone, or modified copolymer solution with or without iPSC-CMs (2–4 million/rat) were injected around the infarct border zone. A negative control group received the LAD ligation and phosphate-buffered saline (PBS) injection without cells or copolymer. At two weeks post-injection, heart dimensions and functional output were assessed by echocardiography. All groups had ventricular dilation and reduced fractional shortening (FS) (Fig. 2). However, rats treated with iPSC-CM-encapsulated copolymer demonstrated significantly less decline in FS (Δ= −6.37±0.49%) compared to other groups (Δ= −12.77±2.04%, −11.44±2.04% and −12.65±1.53% for PBS control, iPSC-CM only, and polymer only groups, respectively (Fig. 2a). p=0.016 vs. PBS, p=0.021 vs. iPSC-CM only, and p=0.005 vs. polymer only). Overall, the iPSC-CM plus polymer group demonstrated 50.1%, 28.2% and 49.6% improvement in LV systolic function over PBS, iPSC-CM only and polymer only groups (Fig. 2b). Moreover, rats treated with iPSC-CM plus polymer demonstrated a trend toward less LV enlargement (Δ= 15.07±3.24%) compared to other groups (Δ=24.44±3.99%, 25.02±2.03% and 23.17±4.51% for PBS control, iPSC-CM only, and polymer only groups, respectively; Fig. 2c) although this reached statistical significance only in comparison to the iPSC-CM group (p=0.032). Overall, iPSC-CM plus polymer group demonstrated 38.3%, 39.8% and 35.0% less LV enlargement over PBS, iPSC-CM only and polymer only groups, suggesting that iPSC-CM encapsulated in polymer curtailed adverse ventricular remodeling better than other treatment modalities. Fig. 2 The effects of hydrogel-encapsulated iPSC-CMs on left ventricle function and remodeling. (a–c) Echocardiography was performed before the ligation of rat LAD coronary arteries (baseline) and again 2 weeks post-delivery (n=5 rats per group). (a, ... Histological examination of the hearts was performed, and the LV anterior free wall thickness was measured using ImageJ and averaged from 3 randomly selected regions in each rat heart. Result demonstrated that, in addition to LV chamber enlargement, the LAD ligation resulted in dramatic thinning and significant fibrosis of the LV anterior free wall at two weeks in control groups (Fig. 2e). In contrast, heart injected with polymer-encapsulated iPSC-CMs had smaller LV chamber, thicker LV free wall and less fibrosis (Fig. 2e). Overall, hearts in the iPSC-CM plus polymer group were significantly thicker than all other groups; the average LV anterior wall thickness of cell plus polymer group was 2.46±0.06mm, compared with 0.48±0.07, 0.35±0.05 and 0.39±0.02mm in PBS, cell only and polymer only groups, respectively (Fig. 2d, p<0.001). In summary, implantation of iPSC-CMs encapsulated in polymer hydrogel was much more effective at limiting adverse LV remodeling and preserving cardiac function after MI than other treatment modalities. Importantly, as implantation of iPSC-CMs or polymer alone did not elicit as favorable outcomes as the iPSC-CMs plus polymer group, we attribute the latter group’s synergistic effects to enhanced survival of transplanted iPSC-CMs in vivo. Consistent with this notion, staining for human nuclei confirmed the presence of iPSC-CMs, delivered with the polymer hydrogel, in the peri-infarct region of the host rat myocardium at 2 weeks (Fig. 2f); moreover, the implanted cells maintained their cardiac phenotype, as demonstrated by positive staining of cardiac α-actinin (Fig. 2f). By contrast, no human nuclei were detected in hearts of control groups at 2 weeks. In conclusion, we describe a temperature-sensitive, collagen-binding hydrogel based system to deliver human iPSC-derived cardiomyocytes to improve cardiac structure and function in infarcted rat heart. Moreover, our studies indicate that the beneficial effects of encapsulating iPSC-CMs in hydrogel are mediated through enhanced survival of transplanted iPSC-CMs in vivo. While future studies are needed to demonstrate long-term functional engraftment of transplanted cells, our study illustrates a promising biomaterial-based approach to overcome a commonly recognized obstacle to the potentially revolutionary cell-based approaches to repair failing hearts: survival of donor cells in the infarcted heart.

THERAPEUTIC APPLICATION OF NANOTECHNOLOGY IN CARDIOVASCULAR AND PULMONARY REGENERATION

Recently, a wide range of nanotechnologies has been approached for material modification by realizing the fact that the extracellular matrix (ECM) consists of nanoscale components and exhibits nanoscale architectures. Moreover, cell-cell and cell- ECM interactions actively occur on the nanoscale and ultimately play large roles in determining cell fate in tissue engineering. Nanomaterials have provided the potential to preferentially control the behavior and differentiation of cells. The present paper reviews the need for nanotechnology in regenerative medicine and the role of nanotechnology in repairing, restoring, and regenerating damaged body parts, such as blood vessels, lungs, and the heart.

Gradient release of cardiac morphogens by photo-responsive polymer micelles for gradient-mediated variation of embryoid body differentiation

Retinoic acid (RA) is a well-known morphogen in human development. However, how an RA gradient distribution influences cardiac development remains obscure due to the lack of appropriate experimental apparatus. To address this issue, a polymeric micelle system with covalently attached RA was engineered to deliver gradient quantities of RA upon photo-irradiation. A photo-degradable polymeric nanoparticle (NP) composed of an amphiphilic methoxy(polyethylene glycol)-b-poly(ε-caprolactone)-co-poly(azido-ε-caprolactone-g-ortho nitrobenzyl retinoic ester) copolymer was synthesized, and hanging RA was covalently attached through a photo-sensitive o-nitrobenzyl (ONB) linker. The ONB linker was efficiently cleaved when exposed to a light (365 nm)-gradient, and the consequent gradient release of RA from the micelles was demonstrated. The efficacy of the photo-gradient-mediated RA release was validated across different concentrations of polymer micelles over varied irradiation periods. It was confirmed that polymer micelles demonstrated minimal cytotoxicity when exposed to mouse embryoid bodies (EBs). Finally, when the photo-gradient release of polymer micelles was applied, GFP-cardiac troponin T reporter mouse EBs demonstrated a concurrent gradient-like pattern of cardiac differentiation, verifying the utility of our novel photo-gradient approach to study morphogen gradients not only for cardiac development but also for other potential biological microenvironments subject to morphogen presentation with highly defined spatial and temporal geometries.

Polymeric stent materials dysregulate macrophage and endothelial cell functions: Implications for coronary artery stent

BACKGROUND Biodegradable polymers have been applied as bulk or coating materials for coronary artery stents. The degradation of polymers, however, could induce endothelial dysfunction and aggravate neointimal formation. Here we use polymeric microparticles to simulate and demonstrate the effects of degraded stent materials on phagocytic activity, cell death and dysfunction of macrophages and endothelial cells. METHODS Microparticles made of low molecular weight polyesters were incubated with human macrophages and coronary artery endothelial cells (ECs). Microparticle-induced phagocytosis, cytotoxicity, apoptosis, cytokine release and surface marker expression were determined by immunostaining or ELISA. Elastase expression was analyzed by ELISA and the elastase-mediated polymer degradation was assessed by mass spectrometry. RESULTS We demonstrated that poly(D,L-lactic acid) (PLLA) and polycaprolactone (PCL) microparticles induced cytotoxicity in macrophages and ECs, partially through cell apoptosis. The particle treatment alleviated EC phagocytosis, as opposed to macrophages, but enhanced the expression of vascular cell adhesion molecule (VCAM)-1 along with decreased nitric oxide production, indicating that ECs were activated and lost their capacity to maintain homeostasis. The activation of both cell types induced the release of elastase or elastase-like protease, which further accelerated polymer degradation. CONCLUSIONS This study revealed that low molecule weight PLLA and PCL microparticles increased cytotoxicity and dysregulated endothelial cell function, which in turn enhanced elastase release and polymer degradation. These indicate that polymer or polymer-coated stents impose a risk of endothelial dysfunction after deployment which can potentially lead to delayed endothelialization, neointimal hyperplasia and late thrombosis.

Investigating pediatric disorders with induced pluripotent stem cells

The study of disease pathophysiology has long relied on model systems, including animal models and cultured cells. In 2006, Shinya Yamanaka achieved a breakthrough by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). This revolutionary discovery provided new opportunities for disease modeling and therapeutic intervention. With established protocols, investigators can generate iPSC lines from patient blood, urine, and tissue samples. These iPSCs retain ability to differentiate into every human cell type. Advances in differentiation and organogenesis move cellular in vitro modeling to a multicellular model capable of recapitulating physiology and disease. Here, we discuss limitations of traditional animal and tissue culture models, as well as the application of iPSC models. We highlight various techniques, including reprogramming strategies, directed differentiation, tissue engineering, organoid developments, and genome editing. We extensively summarize current established iPSC disease models that utilize these techniques. Confluence of these technologies will advance our understanding of pediatric diseases and help usher in new personalized therapies for patients.

Genome Editing and Induced Pluripotent Stem Cell Technologies for Personalized Study of Cardiovascular Diseases

Purpose of ReviewThe goal of this review is to highlight the potential of induced pluripotent stem cell (iPSC)-based modeling as a tool for studying human cardiovascular diseases. We present some of the current cardiovascular disease models utilizing genome editing and patient-derived iPSCs.Recent FindingsThe incorporation of genome-editing and iPSC technologies provides an innovative research platform, providing novel insight into human cardiovascular disease at molecular, cellular, and functional level. In addition, genome editing in diseased iPSC lines holds potential for personalized regenerative therapies.SummaryThe study of human cardiovascular disease has been revolutionized by cellular reprogramming and genome editing discoveries. These exceptional technologies provide an opportunity to generate human cell cardiovascular disease models and enable therapeutic strategy development in a dish. We anticipate these technologies to improve our understanding of cardiovascular disease pathophysiology leading to optimal treatment for heart diseases in the future.