Modeling acute myocardial infarction and cardiac fibrosis using human induced pluripotent stem cell-derived multi-cellular heart organoids.

Heart disease involves irreversible myocardial injury that leads to high morbidity and mortality rates. Numerous cell-based cardiac in vitro models have been proposed as complementary approaches to non-clinical animal research. However, most of these approaches struggle to accurately replicate adult human heart conditions, such as myocardial infarction and ventricular remodeling pathology. The intricate interplay between various cell types within the adult heart, including cardiomyocytes, fibroblasts, and endothelial cells, contributes to the complexity of most heart diseases. Consequently, the mechanisms behind heart disease induction cannot be attributed to a single-cell type. Thus, the use of multi-cellular models becomes essential for creating clinically relevant in vitro cell models. This study focuses on generating self-organizing heart organoids (HOs) using human-induced pluripotent stem cells (hiPSCs). These organoids consist of cardiomyocytes, fibroblasts, and endothelial cells, mimicking the cellular composition of the human heart. The multi-cellular composition of HOs was confirmed through various techniques, including immunohistochemistry, flow cytometry, q-PCR, and single-cell RNA sequencing. Subsequently, HOs were subjected to hypoxia-induced ischemia and ischemia-reperfusion (IR) injuries within controlled culture conditions. The resulting phenotypes resembled those of acute myocardial infarction (AMI), characterized by cardiac cell death, biomarker secretion, functional deficits, alterations in calcium ion handling, and changes in beating properties. Additionally, the HOs subjected to IR efficiently exhibited cardiac fibrosis, displaying collagen deposition, disrupted calcium ion handling, and electrophysiological anomalies that emulate heart disease. These findings hold significant implications for the advancement of in vivo-like 3D heart and disease modeling. These disease models present a promising alternative to animal experimentation for studying cardiac diseases, and they also serve as a platform for drug screening to identify potential therapeutic targets.

Proposal for considerations during human iPSC-derived cardiac organoid generation for cardiotoxicity drug testing.

Human iPSC-derived cardiac organoids (hiPSC-COs) for cardiotoxicity drug testing via the variety of cell lines and unestablished protocols may lead to differences in response results due to a lack of criteria for generation period and size. To ensure reliable drug testing, it is important for researchers to set optimal generation period and size of COs according to the cell line and protocol applied in their studies. Hence, we sought to propose a process to establish minimum criteria for the generation duration and size of hiPSC-COs for cardiotoxic drug testing. We generated hiPSC-COs of different sizes based on our protocol and continuously monitored organoids until they indicated a minimal beating rate change as a control that could lead to more accurate beating rate changes on drug testing. Calcium transients and physiological tests to assess the functionality of hiPSC-COs on selected generation period, which showed regular cardiac beating, and immunostaining assays to compare characteristics were performed. We explained the generation period and size that exhibited and maintained regular beating rate changes on hiPSC-COs, and lead to reliable response results to cardiotoxicity drugs. We anticipate that this study will offer valuable insights into considering the appropriate generation period and size of hiPSC-COs ensuring reliable outcomes in cardiotoxicity drug testing.

Efficacy Evaluation of SDF-1α-Based Polypeptides in an Acute Myocardial Infarction Model Using Structure-Based Drug Design.

Stromal cell-derived factor-1 alpha (SDF-1α, CXCL12) mediates the migration of circulating cells to desired sites for tissue development, homeostasis, and regeneration and can be used to promote cardiac regeneration by recruiting stem cells. However, the use of SDF-1α in the injured heart necessitates not only higher binding affinity to its receptor, CXCR4+, but also better robustness against enzymatic degradation than other SDF-1 isoforms. Here, we conduct a screening of SDF-1α analog peptides that were designed by structure-based drug design (SBDD), a type of computer-aided drug design (CADD). We have developed in vitro and in vivo methods that enable us to estimate the effect of peptides on the migration of human mesenchymal stem cells (hMSCs) and cardiac regeneration in acute myocardial infarction (AMI)-induced animals, respectively. We demonstrate that one type of SDF-1α analog peptide, SDP-4, among the four analog peptides preselected by SBDD, is more potent than native SDF-1α for cardiac regeneration in myocardial infarction. It is interesting to note that the migratory effects of SDP-4 determined by a wound healing assay, a Transwell assay, and a 2D migration assay are comparable to those of SDF-1α. These results suggest that in vivo, as well as in vitro, screening of peptides developed by SBDD is a quintessential process to the development of a novel therapeutic compound for cardiac regeneration. Our finding also has an implication that the SDP-4 peptide is an excellent candidate for use in the regeneration of an AMI heart.

Cyclic Stretching Induces Maturation of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes through Nuclear-Mechanotransduction.

BACKGROUND: During cardiogenesis, cardiac cells receive various stimuli, such as biomechanical and chemical cues, from the surrounding microenvironment, and these signals induce the maturation of heart cells. Mechanical force, especially tensile force in the heart, is one of the key stimuli that induce cardiomyocyte (CM) maturation through mechanotransduction, a process through which physical cues are transformed into biological responses. However, the effects and mechanisms of tensile force on cell maturation are poorly studied. METHODS: In this study, we developed a cyclic stretch system that mimics the mechanical environment of the heart by loading tensile force to human-induced pluripotent stem cell (hiPSC)-derived CMs. hiPSC-CMs cultured with the cyclic stretch system analyzed morphological change, immunofluorescent staining, expression of maturation markers in mRNA, and beating properties compared to static cultures. RESULTS: hiPSC-CMs cultured with the cyclic stretch system showed increased cell alignment, sarcomere length and expression of maturation markers in mRNA, such as TNNI3, MYL2 and TTN, compared to static cultures. Especially, the expression of genes related to nuclear mechanotransduction, such as Yap1, Lamin A/C, plectin, and desmin, was increased in the cyclically stretched hiPSC-CMs. Furthermore, the volume of the nucleus was increased by as much as 120% in the cyclic stretch group. CONCLUSION: These results revealed that nuclear mechanotransduction induced by tensile force is involved in CM maturation. Together, these findings provide novel evidence suggesting that nuclear mechanotransduction induced by tensile force is involved in the regulation of cardiac maturation.

Development of Magnetic Torque Stimulation (MTS) Utilizing Rotating Uniform Magnetic Field for Mechanical Activation of Cardiac Cells.

Regulation of cell signaling through physical stimulation is an emerging topic in biomedicine. BACKGROUND: While recent advances in biophysical technologies show capabilities for spatiotemporal stimulation, interfacing those tools with biological systems for intact signal transfer and noncontact stimulation remains challenging. Here, we describe the use of a magnetic torque stimulation (MTS) system combined with engineered magnetic particles to apply forces on the surface of individual cells. MTS utilizes an externally rotating magnetic field to induce a spin on magnetic particles and generate torsional force to stimulate mechanotransduction pathways in two types of human heart cells-cardiomyocytes and cardiac fibroblasts. METHODS: The MTS system operates in a noncontact mode with two magnets separated (60 mm) from each other and generates a torque of up to 15 pN µm across the entire area of a 35-mm cell culture dish. The MTS system can mechanically stimulate both types of human heart cells, inducing maturation and hypertrophy. RESULTS: Our findings show that application of the MTS system under hypoxic conditions induces not only nuclear localization of mechanoresponsive YAP proteins in human heart cells but also overexpression of hypertrophy markers, including ß-myosin heavy chain (ßMHC), cardiotrophin-1 (CT-1), microRNA-21 (miR-21), and transforming growth factor beta-1 (TGFß-1). CONCLUSIONS: These results have important implications for the applicability of the MTS system to diverse in vitro studies that require remote and noninvasive mechanical regulation.

Early red blood cell abnormalities as a clinical variable in sepsis diagnosis.

BACKGROUND: Sepsis is a medical emergency during which early detection is closely associated with mortality. In sepsis, red blood cell (RBC) abnormalities have been reported. However, it is not known how early RBC abnormalities are expressed compared with various clinical manifestations used in sepsis-related organ failure assessment (SOFA). OBJECTIVE: Therefore, using a lipopolysaccharide (LPS)-induced sepsis model we investigated the clinical significance of RBC abnormalities as an early indicator in the detection of septic injury compared with clinical variables. METHODS: Sprague-Dawley rats received LPS (20 mg/kg) intraperitoneally. Aggregation indices (AIs) and aggregation half-time (T1/2), and elongation indices (EI max) were measured. Clinical data-related SOFA and lactate were measured at 2 h, 4 h, 8 h and 12 h after LPS injection. RESULTS: AIs increased at 4 h, and T1/2 decreased at 2 h after LPS injection. Platelet counts decreased at 4 h, and lactate increased at 2 h after LPS injection. AIs showed strong correlations with T1/2 and platelets, EI max increased at 2 h after LPS injection, while EI max had a positive correlation with lactate. CONCLUSIONS: RBC aggregation appears to be an early indicator of clinical deterioration in sepsis and may represent a diagnostic indicator in sepsis.

Comparison of Angiogenic Activities of Three Neuropeptides, Substance P, Secretoneurin, and Neuropeptide Y Using Myocardial Infarction.

BACKGROUND: The interplay between neurogenesis and angiogenesis is crucial during the development mediated by neuro-angiogenic morphogens. In particular, the angiogenic activity of neuropeptides and their role in tissue regeneration have long been investigated for better understanding of their biological mechanisms and further applications. However, there have been few studies for direct comparison of angiogenic activities of neuropeptides for in vitro and in vivo models. In this study, we report that direct comparison of the angiogenic activities of neuropeptide Y, secretoneurin, and substance P (SP) immobilized on hydrogels in in vitro and in vivo experiments. METHODS: A hyaluronic acid-based hydrogel is prepared by utilizing acrylated hyaluronic acid and thiolated peptides as a crosslinker and angiogenic factors, respectively. Angiogenic activities of three neuropeptides are evaluated not only by in vitro angiogenic and gene expression assays, but also by an in vivo chronic myocardial infarction model. RESULTS: The comparison of in vitro angiogenic activities of three peptides demonstrates that the SP-immobilized hydrogel shows a higher degree of cell network formation and angiogenic-specific genes than those of the other peptides and the control case. In addition, a three-dimensional angiogenic assay illustrates that more sprouting is observable in the SP group. Evaluation of regenerative activity in the chronic myocardial infarction model reveals that all three peptide-immobilized hydrogels induce increased cardiac function as well as structural regeneration. Among all the cases, the SP group provided the highest regenerative activity both in vitro and in vivo. CONCLUSION: In our comparison study, the SP-immobilized hydrogel shows the highest angiogenic activity and tissue regeneration among the test groups. This result suggests that nerve regeneration factors help angiogenesis in damaged tissues, which also highlights the importance of the neuro-angiogenic peptides as an element of tissue regeneration.

Combination of three angiogenic growth factors has synergistic effects on sprouting of endothelial cell/mesenchymal stem cell-based spheroids in a 3D matrix.

Combinations of angiogenic growth factors have been shown to have synergistic effects on angiogenesis and natural wound healing in various animal models. Each growth factor has unique roles during angiogenesis; vascular endothelial growth factor (VEGF) plays a key role during the initial step of angiogenesis, whereas PDGF functions in the maturation of blood vessels. We used a combination of three angiogenic growth factors to increase angiogenesis in vitro and in vivo. We chose VEGF as a basic factor and added platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) to induce angiogenesis in three in vitro and in vivo models: 3D angiogenesis assay, 3D co-culture, and matrigel plug implantation assay. Cell proliferation was significantly higher in co-cultured cells treated with PDGF + VEGF + FGF than in the control, single, or dual combination groups. mRNA expression of α-smooth muscle actin (α-SMA), von Willebrand factor (vWF), and CD105 was higher in the triple group (PDGF + VEGF + FGF) than in control, single, or dual combination groups. In the PDGF + VEGF + FGF group, the length and number of branches of spheroids was also significantly higher than in the control, single, or dual combination groups. Furthermore, in a nude mouse model, α-SMA expression was significantly higher in the PDGF + VEGF + FGF group than in other groups. In conclusion, the addition of PDGF and FGF to VEGF showed synergistic effects on angiogenesis in vitro and in vivo. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1535-1543, 2016.

Differential regeneration of myocardial infarction depending on the progression of disease and the composition of biomimetic hydrogel.

Hydrogel has been used for regenerating myocardial infraction (MI) as a delivery vehicle for cells and growth factors. This study showed that injectable hyaluronic acid (HA)-based hydrogels alone would effectively regenerate the damaged infarcted heart tissue. We found that there are two major factors of regeneration in MI. One is molecular weight of HA and another is the progression of MI; sub-acute and chronic. Rat MI model was prepared by ligating the left anterior descending coronary artery (LAD). Four weeks after injection of hydrogel, functional analysis of the heart and histological analysis was assessed. When different molecular weight HA-based hydrogels with 50 kDa, 130 kDa, and 170 kDa were applied to the infarcted area in the sub-acute model, 50 kDa HA-based hydrogel showed the most significant regeneration of myocardium as well as functional recovery among samples. For the disease progression, 50 kDa HA-based hydrogels were injected to sub-acute and chronic MI models. The regeneration activity was significantly decreased in the chronic models reflecting that injection timing of the therapeutic agents is also major determinants in the regeneration process. These results suggest that injection time and composition of hydrogel are two major points treating MI.

Regeneration of chronic myocardial infarction by injectable hydrogels containing stem cell homing factor SDF-1 and angiogenic peptide Ac-SDKP.

Regeneration of chronic myocardial infarction (CMI) is one of the challenging issues due to its limited regeneration activity compared to acute or sub-acute stage. In this study, we examined whether combination of stem cell homing factor (SDF-1) and angiogenic peptides (Ac-SDKP) injected with biomimetic hydrogels promote regeneration of cardiac function in a CMI model. We evaluated the regeneration of chronically infarcted myocardium using injectable biomimetic hydrogels containing two therapeutic factors; stromal-derived factor-1 (SDF-1) and Ac-SDKP for stem cell homing and angiogenesis, respectively. Injection of the two therapeutic factors into the infarct region of the left ventricle showed that the biomimetic hydrogels containing two therapeutic factor exhibited significantly improved left ventricle function, increased angiogenesis, decreased infarct size and greatest wall thickness within the infarct region at 4 weeks post-treatment. From these results, it is clear that hydrogels containing two therapeutic factors showed synergistic effects on regeneration in the chronic heart failure model. In conclusion, these results suggest that combination of stem cell homing factor with angiogenic peptides recruit stem cells to the microenvironments, increase the expression of angiogenic genes, enhance the matured vessel formation and improve the cardiac function in chronic MI.

Role of inflammation in the pathogenesis of cardiorenal syndrome in a rat myocardial infarction model.

BACKGROUND: Cardiorenal syndrome is now frequently recognized, and the combined dysfunction of heart and kidney increases morbidity and mortality. This study aimed to investigate possible mechanisms that underlie renal damage following heart dysfunction using a rat myocardial infarction model, focusing on the inflammatory pathway. METHODS: Rats were randomized into four groups: normal, volume depletion, sham operation and myocardial infarction (MI). MI was induced by the ligation of the left coronary artery and a volume depletion model was produced by low-salt diet and furosemide injection. Biochemical, histological and flow cytometric analyses were performed at 3 days and 4 and 8 weeks after MI. RESULTS: On Day 3 following MI, the development of subclinical acute kidney injury was identified through significantly increased serum and urine neutrophil gelatinase-associated lipocalin level. We detected the increase of activated monocytes (CC chemokine receptor 2(+) ED-1(+)) in peripheral blood, along with the infiltration of ED-1(+) macrophages and the increment of nuclear p65 in the kidney of MI rats, suggesting the contribution of nuclear factor-kappa B-mediated inflammation in the development of Type 1 cardiorenal syndrome (CRS). The inflammatory cytokines, interleukin-6 and tumour necrosis factor-α (TNF-α) mRNA expression, as well as microvascular endothelial permeability and tubular cell apoptosis, significantly increased in the kidneys of MI rats. At 4 and 8 weeks after MI, tubular cell apoptosis, ED-1(+) macrophage infiltration and interstitial fibrosis increased in MI rats, and these chronic changes were significantly mitigated by systemic monocyte/macrophage depletion using liposome clodronate. CONCLUSION: This study identifies the possible important role of inflammatory response as a mediator of heart-kidney crosstalk in CRS.