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Acute myocardial infarction (AMI) represents the leading cause of cardiovascular death worldwide, and it is thus pivotal to develop effective approaches for the timely detection of AMI markers, especially possessing the characteristics of antibody-free, signal amplification, and manipulation convenience. We herein construct a MoS 2 nanosheet-powered CRISPR/Cas12a sensing strategy for sensitive determination of miR-499, a superior AMI biomarker to protein markers. The presence of miR-499 at a trace level is able to induce a significantly enhanced fluorescence signal in a DNA-based molecular engineering platform, which consists of CRISPR/Cas12a enzymatic reactions and MoS 2 nanosheet-controllable signal reporting components. The MoS 2 nanosheets were characterized by using atomic force microscopy (AFM) and transmission electron microscope (TEM). The detection feasibility was verified by using polyacrylamide gel electrophoresis (PAGE) analysis and fluorescence measurements. The detection limit is determined as 381.78 pM with the linear range from 0.1 ⅹ 10 -9 to 13.33 ⅹ 10 -9  M in a fast manner (about 30 min). Furthermore, miRNA detection in real human serum is also conducted with desirable recovery rates (89.5 %-97.6 %), which may find potential application for the clinic diagnosis. We describe herein the first example of MoS 2 nanosheet-based signal amplified fluorescence sensor for effective detection of AMI-related miRNA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aims: We have shown that human cardiac muscle patches (hCMPs) containing three different types of cardiac cells-cardiomyocytes (CMs), smooth muscle cells (SMCs), and endothelial cells (ECs), all of which were differentiated from human pluripotent stem cells (hPSCs)-significantly improved cardiac function, infarct size, and hypertrophy in a pig model of myocardial infarction (MI). However, hPSC-derived CMs (hPSC-CMs) are phenotypically immature, which may lead to arrhythmogenic concerns; thus, since hPSC-derived cardiac fibroblasts (hPSC-CFs) appear to enhance the maturity of hPSC-CMs, we compared hCMPs containing hPSC-CMs, -SMCs, -ECs, and -CFs (4TCC-hCMPs) with a second hCMP construct that lacked hPSC-CFs but was otherwise identical [hCMP containing hPSC-CMs, -AECs, and -SMCs (3TCC-hCMPs)]., Methods and Results: hCMPs were generated in a fibrin scaffold. MI was induced in severe combined immunodeficiency (SCID) mice through permanent coronary artery (left anterior descending) ligation, followed by treatment with cardiac muscle patches. Animal groups included: MI heart treated with 3TCC-hCMP; with 4TCC-hCMP; MI heart treated with no patch (MI group) and sham group. Cardiac function was evaluated using echocardiography, and cell engraftment rate and infarct size were evaluated histologically at 4 weeks after patch transplantation. The results from experiments in cultured hCMPs demonstrate that the inclusion of cardiac fibroblast in 4TCC-hCMPs had (i) better organized sarcomeres; (ii) abundant structural, metabolic, and ion-channel markers of CM maturation; and (iii) greater conduction velocities (31 ± 3.23 cm/s, P < 0.005) and action-potential durations (APD50 = 365 ms ± 2.649, P < 0.0001; APD = 408 ms ± 2.757, P < 0.0001) than those (velocity and APD time) in 3TCC-hCMPs. Furthermore, 4TCC-hCMPs transplantation resulted in better cardiac function [ejection fraction (EF) = 49.18% ± 0.86, P < 0.05], reduced infarct size (22.72% ± 0.98, P < 0.05), and better engraftment (15.99% ± 1.56, P < 0.05) when compared with 3TCC-hCMPs (EF = 41.55 ± 0.92%, infarct size = 39.23 ± 4.28%, and engraftment = 8.56 ± 1.79%, respectively)., Conclusion: Collectively, these observations suggest that the inclusion of hPSC-CFs during hCMP manufacture promotes hPSC-CM maturation and increases the potency of implanted hCMPs for improving cardiac recovery in mice model of MI., Competing Interests: Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Ubiquitin carboxyl-terminal esterase L1 (UCHL1) has been thought to be a neuron specific protein and shown to play critical roles in Parkinson's Disease and stroke via de-ubiquiting and stabilizing key pathological proteins, such as α-synuclein. In the present study, we found that UCHL1 was significantly increased in both mouse and human cardiomyocytes following myocardial infarction (MI). When LDN-57444, a pharmacological inhibitor of UCHL1, was used to treat mice subjected to MI surgery, we found that administration of LDN-57444 compromised cardiac function when compared with vehicle treated hearts, suggesting a potential protective role of UCHL1 in response to MI. When UCHL1 was knockout by CRISPR/Cas 9 gene editing technique in human induced pluripotent stem cells (hiPSCs), we found that cardiomyocytes derived from UCHL1-/- hiPSCs were more susceptible to hypoxia/re-oxygenation induced injury as compared to wild type cardiomyocytes. To study the potential targets of UCHL1, a BioID based proximity labeling approach followed by mass spectrum analysis was performed. The result suggested that UCHL1 could bind to and stabilize HIF-1α following MI. Indeed, expression of HIF-1α was lower in UCHL1-/- cells as determined by Western blotting and HIF-1α target genes were also suppressed in UCHL1-/- cells as quantified by real time RT-PCR. Recombinant UCHL1 (rUCHL1) protein was purified by E. Coli fermentation and intraperitoneally (I.P.) delivered to mice. We found that administration of rUCHL1 could significantly preserve cardiac function following MI as compared to control group. Finally, adeno associated virus mediated cardiac specific UCHL1 delivery (AAV9-cTNT-m-UCHL1) was performed in neonatal mice. UCHL1 overexpressing hearts were more resistant to MI injury as compare to the hearts infected with control virus. In summary, our data revealed a novel protective role of UCHL1 on MI via stabilizing HIF-1α and promoting HIF-1α signaling., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The Nutritional Risk Screening 2002 (NRS-2002) scale is a rapid and effective screening instrument that assesses nutritional risk among hospitalized patients., Objective: The present study aimed to explore the prognostic value of the NRS-2002 scale in acute myocardial infarction (AMI) considering its uncertain role in this particular condition., Methods: Patients with AMI included in the Retrospective Multicenter Study for Early Evaluation of Acute Chest Pain were investigated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to analyze the association between NRS-2002 and mortality in patients with AMI. The primary and secondary endpoints were all-cause and cardiac mortality during the follow-up period., Results: A total of 2307 patients were enrolled, among whom 246 (10.7%) died within a median follow-up duration of 10.67 (8.04-14.33) months. Kaplan-Meier analysis revealed that patients with an NRS-2002 score of 3 or higher had poorer cumulative survival than those with an NRS-2002 score lower than 3 (P < .001). In the multivariate Cox regression analysis, patients with an NRS-2002 score of 3 or higher had more than double the risk for all-cause mortality (hazard ratio, 2.25; 95% confidence interval, 1.50-3.40; P < .001) and twice the risk for cardiac-related mortality (hazard ratio, 2.01; 95% confidence interval, 1.29-3.13; P = .002) than did patients with lower scores., Conclusions: Our results showed that the NRS-2002 screening instrument was an independent prognostic predictor for both all-cause and cardiac mortality in patients with AMI. Nutritional risk assessment based on the NRS-2002 scale may provide useful prognostic information of early nutritional risk stratification in patients with AMI., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and is implicated in cardiac pathology, the contribution of its cytoplasmic domain to post-infarct myocardial injury and adverse left ventricular (LV) remodeling remains unknown. Methods: Myocardial infarction was induced in wild-type mice or mice lacking the TF cytoplasmic domain (TF∆CT) by occlusion of the left anterior descending coronary artery. Heart function was monitored with echocardiography. Heart tissue was collected at different time-points for histological, molecular and flow cytometry analysis. Results: Compared with wild-type mice, TF∆CT had a higher survival rate during a 28-day follow-up after myocardial infarction. Among surviving mice, TF∆CT mice had better cardiac function and less LV remodeling than wild-type mice. The overall improvement of post-infarct cardiac performance in TF∆CT mice, as revealed by speckle-tracking strain analysis, was attributed to reduced myocardial deformation in the peri-infarct region. Histological analysis demonstrated that TF∆CT hearts had in the infarct area greater proliferation of myofibroblasts and better scar formation. Compared with wild-type hearts, infarcted TF∆CT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1) - Rac1 axis. In particular, infarcted TF∆CT hearts displayed markedly lower ratios of inflammatory M1 macrophages and reparative M2 macrophages (M1/M2). In vitro experiment with primary macrophages demonstrated that deletion of the TF cytoplasmic domain inhibited macrophage polarization toward the M1 phenotype. Furthermore, infarcted TF∆CT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Finally, the overall cardioprotective effects observed in TF∆CT mice could be abolished by subcutaneously infusing a cocktail of PAR1-activating peptide and PAR2-inhibiting peptide via osmotic minipumps. Conclusions: Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Rationale: Previous studies have shown that human embryonic stem cell-derived cardiomyocytes improved myocardial recovery when administered to infarcted pig and non-human primate hearts. However, the engraftment of intramyocardially delivered cells is poor and the effectiveness of clinically relevant doses of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in large animal models of myocardial injury remains unknown. Here, we determined whether thymosin β4 (Tb4) could improve the engraftment and reparative potency of transplanted hiPSC-CMs in a porcine model of myocardial infarction (MI). Methods: Tb4 was delivered from injected gelatin microspheres, which extended the duration of Tb4 administration for up to two weeks in vitro . After MI induction, pigs were randomly distributed into 4 treatment groups: the MI Group was injected with basal medium; the Tb4 Group received gelatin microspheres carrying Tb4; the CM Group was treated with 1.2 × 10 8 hiPSC-CMs; and the Tb4+CM Group received both the Tb4 microspheres and hiPSC-CMs. Myocardial recovery was assessed by cardiac magnetic resonance imaging (MRI), arrhythmogenesis was monitored with implanted loop recorders, and tumorigenesis was evaluated via whole-body MRI. Results : In vitro , 600 ng/mL of Tb4 protected cultured hiPSC-CMs from hypoxic damage by upregulating AKT activity and BcL-XL and promoted hiPSC-CM and hiPSC-EC proliferation. In infarcted pig hearts, hiPSC-CM transplantation alone had a minimal effect on myocardial recovery, but co-treatment with Tb4 significantly enhanced hiPSC-CM engraftment, induced vasculogenesis and the proliferation of cardiomyocytes and endothelial cells, improved left ventricular systolic function, and reduced infarct size. hiPSC-CM implantation did not increase incidence of ventricular arrhythmia and did not induce tumorigenesis in the immunosuppressed pigs. Conclusions : Co-treatment with Tb4-microspheres and hiPSC-CMs was safe and enhanced the reparative potency of hiPSC-CMs for myocardial repair in a large-animal model of MI., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Aims: To examine whether transient over-expression of angiopoietin-1 (Ang-1) increases the potency of hiPSC-CMs for treatment of heart failure., Methods and Results: Atrial hiPSC-CMs (hiPSC-aCMs) were differentiated from hiPSCs and purified by lactic acid and were transfected with Ang-1 (Ang-1-hiPSC-aCMs) plasmid using lipoSTEM. Ang-1 gene transfection efficiency was characterized in vitro. Gene transfected CMs (1×106) were seeded into a fibrin/thrombin patch and implanted on the rat-infarcted left ventricular (LV) anterior wall after myocardial infarction (MI). Echo function was determined at 1- and 6 weeks post-MI. Immunohistochemistry study was performed at 6 weeks post-MI. Ang-1 (20 and 40 ng/mL) protected hiPSC-aCMs from hypoxia through up-regulating pERK1/2 and inhibiting Bax protein expressions. Ang-1-hiPSC-aCMs transiently secreted Ang-1 protein up to 14 days, with peak level on day-2 post-transfection (24.39 ± 13.02 ng/mL) in vitro. Animal study showed that transplantation of Ang-1-hiPSC-aCM seeded patch more effectively limited rat heart apoptosis at 1 day post-MI as compared with LipoSTEM-Ang-1 or hiPSC-aCMs transplantation. Ang-1-hiPSC-aCMs transplantation induced host (rat) and donor (human) CM mitosis and arteriole formation, improved cell engraftment rate, more effectively limited LV dilation (EDV = 460.7 ± 96.1 μL and ESV = 219.8 ± 72.9 μL) and improved LV global pump function (EF = 53.1 ± 9%) as compared with the MI (EDV = 570.9 ± 91.8 μL, P = 0.033; ESV = 331.6 ± 71.2 μL, P = 0.011; EF = 42.3 ± 4.1%, P = 0.02) or the LipoSTEM-Ang-1 injected (EDV = 491.4 ± 100.4 μL, P = 0.854; ESV = 280.9 ± 71.5 μL, P = 0.287; EF = 43.2 ± 4.6, P = 0.039) or hiPSC-CM transplanted (EDV = 547.9 ± 55.5 μL, P = 0.095; ESV = 300.2 ± 88.4 μL, P = 0.075; EF = 46 ± 10.9%, P = 0.166) animal groups at 6 weeks post-MI and treatment., Conclusion: Transient over-expression of Ang-1 enhanced hiPSC-aCM mitosis and engraftment and increased the reparability potency of hiPSC-aCMs for treatment of MI., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Background: A more accurate and simpler scoring systems for early risk stratification of acute myocardial infarction at admission can accelerate and improve decision-making., Aim: To develop and validate a simple physiological prognostic scoring system for early risk stratification in patients with acute myocardial infarction., Methods: Easily accessible physiological vital signs and demographic characteristics of patients with acute myocardial infarction at the time of presentation in the multicentre Retrospective Evaluation of Acute Chest Pain study were used to develop a multivariate logistic regression model predicting 12 and 24-month mortality. The study population consisted of 2619 patients from seven hospitals and was divided into a 70% sample for model derivation and a 30% sample for model validation. A nomogram was created to enable prospective risk stratification for clinical care., Results: The simple physiological prognostic scoring system consisted of age, heart rate, body mass index and Killip class. The area under the receiver operating characteristic curve of the simple physiological prognostic scoring system was superior to that of several risk scoring systems in clinical use. Net reclassification improvement, integrated discrimination improvement and decision curve analysis of the derivation set also revealed superior performance to the Global Registry of Acute Coronary Events score, and the Hosmer-Lemeshow test indicated good calibration for predicting mortality in patient with acute myocardial infarction in the validation set (P = 0.612)., Conclusion: This simple physiological prognostic scoring system may be a useful risk stratification tool for early assessment of patients with acute myocardial infarction., (Published by Oxford University Press on behalf of the European Society of Cardiology 2020.)

Aims: Recently, we demonstrated that the hearts of neonatal pigs (2-day old) have regenerative capacity, likely driven by cardiac myocyte division, but this potential is lost immediately after postnatal day 3. However, it is unknown if corticosteroid, a broad anti-inflammatory agent, will abrogate the regenerative capacity in the hearts of neonatal pigs. The aim of the current study is to evaluate the effect Dexamethasone (Dex), a broad anti-inflammatory agent, on heart regeneration, structure, and function of the neonatal pigs' post-myocardial infarction (MI)., Methods and Results: Dex (0.2 mg/kg/day) was injected intramuscularly into the neonatal pig (age: 2 days postnatal) during the first week post-MI. Myocardial scar and left ventricular function were determined by cardiac magnetic resonance (CMR) imaging. Bromodeoxyuridine (BrdU) pulse-chase labeling, histology, immunohistochemistry, and flow cytometry were performed to determine inflammatory cell infiltration, CM cytokinesis, and myocardial fibrosis. Dex injection during the first-week suppressed acute inflammation post-MI in the pig hearts. It inhibited BrdU incorporation to pig CMs and CM cytokinesis via inhibiting aurora-B protein expression which was associated with mature scar formation and thinned walls at the infarct site. CMR imaging showed Dex caused left ventricular aneurysm and poor ejection fraction., Conclusions: Dex inhibited CM cytokinesis and functional recovery and caused ventricular aneurysm in the hearts of 2-day old pigs post-MI., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Endothelial progenitor cells (EPCs) are bone-marrow derived cells that are critical in the maintenance of endothelial wall integrity and protection of ischemic myocardium through the formation of new blood vessels (vasculogenesis) or proliferation of pre-existing vasculature (angiogenesis). Diabetes mellitus (DM) and the metabolic syndrome are commonly associated with ischemic heart disease through its pathological effects on the endothelium and consequent endothelial dysfunction. Thymosin-β4 (Tβ4) which expressed in the embryonic heart is critical in epicardial and coronary artery formation. In this study, we explored the effects of Tβ4 treatment on diabetic EPCs in vitro and intramyocardial injection of Tβ4-treated and non-Tβ4 treated EPCs following acute myocardial infarction (MI) of diabetic rats in vivo. It was found that 10 ng/mL Tβ4 increased migration, tubule formation, and angiogenic factor secretion of diabetic EPCs in vitro. In vivo, although implantation of Tβ4 treated diabetic EPCs significantly increased capillary density and attracted more c-Kit positive progenitor cells into the infarcted hearts as compared with implantation of non-Tβ4 treated diabetic EPCs, the significantly improved left ventricular ejection fraction was only found in the rats which received non-Tβ4 treated EPCs. The data suggests that a low dose Tβ4 increases diabetic EPC migration, tubule formation, and angiogenic factor secretion. However, it did not improve the effects of EPCs on left ventricular pump function in diabetic rats with MI.

Background: The adult mammalian heart has limited ability to repair itself after injury. Zebrafish, newts, and neonatal mice can regenerate cardiac tissue, largely by cardiac myocyte (CM) proliferation. It is unknown whether hearts of young large mammals can regenerate., Methods: We examined the regenerative capacity of the pig heart in neonatal animals (ages 2, 3, or 14 days postnatal) after myocardial infarction or sham procedure. Myocardial scar and left ventricular function were determined by cardiac magnetic resonance imaging and echocardiography. Bromodeoxyuridine pulse-chase labeling, histology, immunohistochemistry, and Western blotting were performed to study cell proliferation, sarcomere dynamics, and cytokinesis and to quantify myocardial fibrosis. RNA-sequencing was also performed., Results: After myocardial infarction, there was early and sustained recovery of cardiac function and wall thickness in the absence of fibrosis in 2-day-old pigs. In contrast, older animals developed full-thickness myocardial scarring, thinned walls, and did not recover function. Genome-wide analyses of the infarct zone revealed a strong transcriptional signature of fibrosis in 14-day-old animals that was absent in 2-day-old pigs, which instead had enrichment for cytokinesis genes. In regenerating hearts of the younger animals, up to 10% of CMs in the border zone of the myocardial infarction showed evidence of DNA replication that was associated with markers of myocyte division and sarcomere disassembly., Conclusions: Hearts of large mammals have regenerative capacity, likely driven by cardiac myocyte division, but this potential is lost immediately after birth.

A major goal of tissue engineering is the creation of pre-vascularized tissues that have a high density of organized microvessels that can be rapidly perfused following implantation. This is especially critical for highly metabolic tissues like myocardium, where a thick myocardial engineered tissue would require rapid perfusion within the first several days to survive transplantation. In the present work, tissue patches containing human microvessels that were either randomly oriented or aligned were placed acutely on rat hearts post-infarction and for each case it was determined whether rapid inosculation could occur and perfusion of the patch could be maintained for 6 days in an infarct environment. Patches containing self-assembled microvessels were formed by co-entrapment of human blood outgrowth endothelial cells and human pericytes in fibrin gel. Cell-induced gel contraction was mechanically-constrained resulting in samples with high densities of microvessels that were either randomly oriented (with 420 ± 140 lumens/mm(2)) or uniaxially aligned (with 940 ± 240 lumens/mm(2)) at the time of implantation. These patches were sutured onto the epicardial surface of the hearts of athymic rats following permanent ligation of the left anterior descending artery. In both aligned and randomly oriented microvessel patches, inosculation occurred and perfusion of the transplanted human microvessels was maintained, proving the in vivo vascularization potential of these engineered tissues. No difference was found in the number of human microvessels that were perfused in the randomly oriented (111 ± 75 perfused lumens/mm(2)) and aligned (173 ± 97 perfused lumens/mm(2)) patches. Our results demonstrate that tissue patches containing a high density of either aligned or randomly oriented human pre-formed microvessels achieve rapid perfusion in the myocardial infarct environment - a necessary first-step toward the creation of a thick, perfusable heart patch., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unlabelled: A tissue-engineered cardiac patch provides a method to deliver cardiomyoctes to the injured myocardium with high cell retention and large, controlled infarct coverage, enhancing the ability of cells to limit remodeling after infarction. The patch environment can also yield increased survival. In the present study, we sought to assess the efficacy of a cardiac patch made from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to engraft and limit left ventricular (LV) remodeling acutely after infarction. Cardiac patches were created from hiPSC-CMs and human pericytes (PCs) entrapped in a fibrin gel and implanted acutely onto athymic rat hearts. hiPSC-CMs not only remained viable after in vivo culture, but also increased in number by as much as twofold, consistent with colocalization of human nuclear antigen, cardiac troponin T, and Ki-67 staining. CM+PC patches led to reduced infarct sizes compared with myocardial infarction-only controls at week 4, and CM+PC patch recipient hearts exhibited greater fractional shortening over all groups at both 1 and 4 weeks after transplantation. However, a decline occurred in fractional shortening for all groups over 4 weeks, and LV thinning was not mitigated. CM+PC patches became vascularized in vivo, and microvessels were more abundant in the host myocardium border zone, suggesting a paracrine mechanism for the improved cardiac function. PCs in a PC-only control patch did not survive 4 weeks in vivo. Our results indicate that cardiac patches containing hiPSC-CMs engraft onto acute infarcts, and the hiPSC-CMs survive, proliferate, and contribute to a reduction in infarct size and improvements in cardiac function., Significance: In the present study, a cardiac patch was created from human induced pluripotent stem cell-derived cardiomyocytes and human pericytes entrapped in a fibrin gel, and it was transplanted onto infarcted rat myocardium. It was found that a patch that contained both cardiomyocytes and pericytes survived transplantation and resulted in improved cardiac function and a reduced infarct size compared with controls., (©AlphaMed Press.)

Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cellular therapy for myocardial repair has been one of the most intensely investigated interventional strategies for acute myocardial infarction. Although the therapeutic potential of stem cells has been demonstrated in various studies, the underlying mechanisms for such improvements are poorly understood. In the present study, we investigated the long-term effects of stem cell therapy on both myocardial fiber organization and regional contractile function using a rat model of postinfarct remodeling. Human nonhematopoietic umbilical cord blood stem cells (nh-UCBSCs) were administered via tail vein to rats 2 days after infarct surgery. Animals were maintained without immunosuppressive therapy. In vivo and ex vivo MR imaging was performed on infarct hearts 10 months after cell transplantation. Compared to the age-matched rats exposed to the identical surgery, both global and regional cardiac functions of the nh-UCBSC-treated hearts, such as ejection fraction, ventricular strain, and torsion, were significantly improved. More importantly, the treated hearts exhibited preserved fiber orientation and water diffusivities that were similar to those in sham-operated control hearts. These data provide the first evidence that nh-UCBSC treatment may prevent/delay untoward structural remodeling in postinfarct hearts, which supports the improved LV function observed in vivo in the absence of immunosuppression, suggesting a beneficial paracrine effect occurred with the cellular therapy.

Human induced pluripotent stem cells (hiPSCs) hold promise for myocardial repair following injury, but preclinical studies in large animal models are required to determine optimal cell preparation and delivery strategies to maximize functional benefits and to evaluate safety. Here, we utilized a porcine model of acute myocardial infarction (MI) to investigate the functional impact of intramyocardial transplantation of hiPSC-derived cardiomyocytes, endothelial cells, and smooth muscle cells, in combination with a 3D fibrin patch loaded with insulin growth factor (IGF)-encapsulated microspheres. hiPSC-derived cardiomyocytes integrated into host myocardium and generated organized sarcomeric structures, and endothelial and smooth muscle cells contributed to host vasculature. Trilineage cell transplantation significantly improved left ventricular function, myocardial metabolism, and arteriole density, while reducing infarct size, ventricular wall stress, and apoptosis without inducing ventricular arrhythmias. These findings in a large animal MI model highlight the potential of utilizing hiPSC-derived cells for cardiac repair., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cell therapies have emerged as a promising treatment for the prevention of heart failure after myocardial infarction (MI). This study evaluated the capacity of an aligned, fibrin-based, stretch-conditioned cardiac patch consisting of either the native population or a cardiomyocyte (CM)-depleted population (i.e., CM+ or CM- patches) of neonatal rat heart cells to ameliorate left ventricular (LV) remodeling in the acute-phase postinfarction in syngeneic, immunocompetent rats. Patches were exposed to 7 days of static culture and 7 days of cyclic stretching prior to implantation. Within 1 week of implantation, both patches became vascularized, and non-CMs began migrating from CM+ patches. By week 4, patches had been remodeled into collagenous tissue, and live, elongated, donor CMs were found within grafted CM+ patches. Significant improvement in cardiac contractile function was seen with the administration of the CM+ patch (ejection fraction increased from 35.1% ± 4.0% for MI only to 58.8% ± 7.3% with a CM+ patch, p<0.05) associated with a 77% reduction in infarct size (61.3% ± 7.9% for MI only, 13.9% ± 10.8% for CM+ patch, p<0.05), and the elimination of LV free-wall thinning. Decreased infarct size and reduced wall thinning also occurred with the administration of the CM- patch (infarct size 36.9% ± 10.2%, LV wall thickness: 1058.2 ± 135.4 μm for CM- patch, 661.3 ± 37.4 μm for MI only, p<0.05), but without improvements in cardiac function. Approximately 36.5% of the transplanted CMs survived at 4 weeks; however, they remained separated and electrically uncoupled from the host myocardium by a layer of CM-free tissue, which suggests that the benefits of CM+ patch transplantation resulted from paracrine mechanisms originating from CMs. Collectively, these observations suggest that the transplantation of CM-containing engineered heart tissue patches can lead to dramatic improvements in cardiac function and remodeling after acute MI.

Background: Thymosin β4 (Tβ4) has been shown to enhance the survival of cultured cardiomyocytes. Here, we investigated whether the cytoprotective effects of Tβ4 can increase the effectiveness of transplanted swine mesenchymal stem cells (sMSCs) for cardiac repair in a rat model of myocardial infarction (MI)., Methods and Results: Under hypoxic conditions, cellular damage (lactate dehydrogenase leakage), apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelingc cells), and caspase-8 activity were significantly lower, whereas B-cell lymphoma-extra large protein expression was significantly higher, in sMSCs cultured with Tβ4 (1 μg/mL) than in sMSCs cultured without Tβ4, and Tβ4 also increased sMSC proliferation. For in vivo experiments, animals were treated with basal medium (MI: n=6), a fibrin patch (Patch: n=6), a patch containing sMSCs (sMSC: n=9), or a patch containing sMSCs and Tβ4 (sMSC/Tβ4: n=11); Tβ4 was encapsulated in gelatin microspheres to extend Tβ4 delivery. Four weeks after treatment, echocardiographic assessments of left-ventricular ejection fraction and fractional shortening were significantly better (P<0.05) in sMSC/Tβ4 animals (left-ventricular ejection fraction=51.7 ± 1.1%; fractional shortening=26.7 ± 0.7%) than in animals from MI (39 ± 3%; 19.5 ± 1.7%) and Patch (43 ± 1.4%; 21.6 ± 0.9%) groups. Histological assessment of infarct wall thickness was significantly higher (P<0.05) in sMSC/Tβ4 animals (50%, [45%, 80%]) than in animals from MI (25%, [20%, 25%]) group. Measurements in sMSC (left-ventricular ejection fraction=45 ± 2.6%; fractional shortening=22.9 ± 1.6%; TH = 43% [25%, 45%]), Patch, and MI animals were similar. Tβ4 administration also significantly increased vascular growth, the retention/survival of the transplanted sMSCs, and the recruitment of endogenous c-Kit(+) progenitor cells to the infarcted region., Conclusions: Extended-release Tβ4 administration improves the retention, survival, and regenerative potency of transplanted sMSCs after myocardial injury.

Background: The use of cells derived from human induced pluripotent stem cells as cellular therapy for myocardial injury has yet to be examined in a large-animal model., Methods and Results: Immunosuppressed Yorkshire pigs were assigned to 1 of 3 groups: A myocardial infarction group (MI group; distal left anterior descending coronary artery ligation and reperfusion; n=13); a cell-treatment group (MI with 4×10(6) vascular cells derived from human induced pluripotent stem cells administered via a fibrin patch; n=14); and a normal group (n=15). At 4 weeks, left ventricular structural and functional abnormalities were less pronounced in hearts in the cell-treated group than in MI hearts (P<0.05), and these improvements were accompanied by declines in scar size (10.4±1.6% versus 8.3±1.1%, MI versus cell-treatment group, P<0.05). The cell-treated group displayed a significant increase in vascular density and blood flow (0.83±0.11 and 1.05±0.13 mL·min(-1)·g(-1), MI versus cell-treatment group, P<0.05) in the periscar border zone (BZ), which was accompanied by improvements in systolic thickening fractions (infarct zone, -10±7% versus 5±5%; BZ, 7±4% versus 23±6%; P<0.05). Transplantation of vascular cells derived from human induced pluripotent stem cells stimulated c-kit(+) cell recruitment to BZ and the rate of bromodeoxyuridine incorporation in both c-kit(+) cells and cardiomyocytes (P<0.05). Using a magnetic resonance spectroscopic saturation transfer technique, we found that the rate of ATP hydrolysis in BZ of MI hearts was severely reduced, and the severity of this reduction was linearly related to the severity of the elevations of wall stresses (r=0.82, P<0.05). This decline in BZ ATP utilization was markedly attenuated in the cell-treatment group., Conclusions: Transplantation of vascular cells derived from human induced pluripotent stem cells mobilized endogenous progenitor cells into the BZ, attenuated regional wall stress, stimulated neovascularization, and improved BZ perfusion, which in turn resulted in marked increases in BZ contractile function and ATP turnover rate.

Rationale: The mechanism by which endogenous progenitor cells contribute to functional and beneficial effects in stem cell therapy remains unknown., Objective: Utilizing a novel (31)P magnetic resonance spectroscopy-2-dimensional chemical shift imaging method, this study examined the heterogeneity and bioenergetic consequences of postinfarction left ventricular (LV) remodeling and the mechanisms of endogenous progenitor cell contribution to the cellular therapy., Methods and Results: Human embryonic stem cell-derived vascular cells (hESC-VCs) that stably express green fluorescent protein and firefly luciferase (GFP(+)/Luc(+)) were used for the transplantation. hESC-VCs may release various cytokines to promote angiogenesis, prosurvival, and antiapoptotic effects. Both in vitro and in vivo experiments demonstrated that hESC-VCs effectively inhibit myocyte apoptosis. In the mouse model, a fibrin patch-based cell delivery resulted in a significantly better cell engraftment rate that was accompanied by a better ejection fraction. In the swine model of ischemia-reperfusion, the patch-enhanced delivery of hESC-VCs resulted in alleviation of abnormalities including border zone myocardial perfusion, contractile dysfunction, and LV wall stress. These results were also accompanied by a pronounced recruitment of endogenous c-kit(+) cells to the injury site. These improvements were directly associated with a remarkable improvement in myocardial energetics, as measured by a novel in vivo (31)P magnetic resonance spectroscopy-2-dimensional chemical shift imaging technology., Conclusions: The findings of this study demonstrate that a severely abnormal heterogeneity of myocardial bioenergetics in hearts with postinfarction LV remodeling can be alleviated by the hESC-VCs therapy. These findings suggest an important therapeutic target of peri-scar border zone and a promising therapeutic potential for using hESC-VCs together with the fibrin patch-based delivery system.

Objective: We sought to investigate immune cell kinetics in relation to skeletal myoblast survival and heart function improvement after nonautologous skeletal myoblast transplantation in a rat model of myocardial infarction., Methods: One week after myocardial infarction, 208 Wistar rats were grouped into group 1 (n = 24, receiving 150 muL of medium only), group 2 (n = 24, receiving 150 muL of medium and cyclosporine [INN: ciclosporin]), group 3 (n = 40, human skeletal myoblast transplantation), group 4 (n = 40, human skeletal myoblast transplantation with cyclosporine treatment), group 5 (n = 40, rat skeletal myoblast transplantation), and group 6 (n = 40, rat skeletal myoblast transplantation with cyclosporine treatment). The hearts were harvested at 10 minutes and 1, 4, 7, and 28 days after cell transplantation. Skeletal myoblast survival was confirmed by means of immunohistochemical studies and quantified by using real-time polymerase chain reaction. Host immune responses were assessed by immunostaining for macrophages and CD4+ and CD8+ lymphocytes. Heart function was evaluated by means of echocardiographic analysis., Results: The majority of macrophages and lymphocytes infiltrated in the acute phase (from day 1 to day 7) and then subsided by day 28. The donor skeletal myoblasts survived and differentiated well in all skeletal myoblast transplantation groups. Allogeneic skeletal myoblasts showed a superior survival rate than xenogeneic skeletal myoblasts (P < .01). Cyclosporine inhibited the infiltration of the immunocytes, enhanced skeletal myoblast survival, and improved heart performance compared with that seen in the groups not receiving cyclosporine treatment (P < .05)., Conclusions: Allomyoblasts survive better than do xenomyoblasts after transplantation into infarcted myocardium. After inhibition of immunocyte infiltration by means of immunosuppressive treatment, skeletal myoblast survival is enhanced, with improved heart performance. These findings suggest the feasibility of nonautologous myoblast transplantation with immunosuppressive treatment.

Treatment of patients with heart failure secondary to myocardial infarction remains a therapeutic challenge. Extensive myocyte death in the heart and post-ischemic remodeling accentuate progressive expansion of the scar area and compromise left ventricular contractile function. The scarcity of resident stem cells in the heart and limited proliferative capacity of adult cardiomyocytes warrant novel strategies of outside intervention to supplement the inept intrinsic repair mechanism. Heart cell therapy using patient's own skeletal muscle derived myoblasts (SkMs) provides a relatively simple and inexpensive therapeutic option. Phase-I and II clinical trials supported by plethora of pre-clinical studies have shown the safety and effectiveness of SkMs engraftment in the treatment of infarcted heart. However, before widespread application of this approach in the clinical settings, there remain some fundamental issues including extensive donor cell death during acute phase after SkMs engraftment, failure of SkMs to adopt cardiac phenotype and transient ventricular arrhythmias subsequent to SkMs transplantation which require serious considerations. This review will provide profound analysis of merits and limitations of SkMs as the choice cells for heart cell therapy and will summarize the potential of genetic and pharmacological manipulation SkMs to enhance their therapeutic efficacy for myocardial repair. Present article also includes recent patent review coverage on this topic.

Background: We investigated the feasibility and efficacy of polyethylenimine (PEI) based human vascular endothelial growth factor-165 (hVEGF165) gene transfer into human skeletal myoblasts (HSM) for cell based delivery to the infarcted myocardium., Methods and Results: Based on optimized transfection procedure using enhanced green fluorescent protein (pEGFP), HSM were transfected with plasmid-hVEGF165 (phVEGF165) carried by PEI (PEI-phVEGF165) nanoparticles. The transfected HSM were characterized for transfection and expression of hVEGF165 in vitro and transplanted into rat heart model of acute myocardial infarction (AMI): group-1=DMEM injection, group-2= HSM transplantation, group-3= PEI-phVEGF165-transfected HSM (PEI-phVEGF165 myoblast) transplantation. A total of 48 rats received cyclosporine injection from 3 days before and until 4 weeks after cell transplantation. Echocardiography was performed to assess the heart function. Animals were sacrificed for molecular and histological studies on the heart tissue at 4 weeks after treatment. Based on optimized transfection conditions, transfected HSM expressed hVEGF165 for 18 days with >90% cell viability in vitro. Apoptotic index was reduced in group-2 and group-3 as compared with group-1. Blood vessel density (x400) by immunostaining for PECAM-1 in group-3 was significantly higher (P=0.043 for both) as compared with group-1 and group-2 at 4 weeks. Regional blood flow (ml/min/g) in the left ventricular anterior wall was higher in group-3 (P=0.043 for both) as compared with group-1 and group-2. Improved ejection fraction was achieved in group-3 (58.44+/-4.92%) as compared with group-1 (P=0.004)., Conclusion: PEI nanoparticle mediated hVEGF165 gene transfer into HSM is feasible and safe. It may serve as a novel and efficient alternative for angiomyogenesis in cardiac repair.

Unlabelled: We compare the effectiveness of direct adenoviral angiopoietin-1 (Ad-Ang-1) injection with transplantation of skeletal myoblasts (SkMs) over-expressing angiopoietin-1 (Ang-1) for angiogenic response and improvement of heart function in an experimental porcine model of myocardial infarction (MI)., Methods: Ad-Ang-1 was used for intramyocardial injection or transduction of SkMs. Three weeks after coronary artery ligation in 32 female pigs, animals were grouped to receive multiple intramyocardial injections of DMEM without cells (group-1; n=7), or containing 3 x 10(8)Lac-z labelled SkMs transduced with Ad-Null vector carrying no gene (group-2; n=7), or 1 x 10(10) PFU Ad-Ang-1 (group-3; n=9), or 3 x 10(8)Lac-z labelled SkMs transduced with Ad-Ang-1 (group-4; n=9). The animals were immunosuppressed for 6-weeks. After euthanasia, their heart tissue was processed for histological studies., Results: Extensive survival of Lac-z positive SkMs was observed in and around the infarct 6 and 12-weeks after transplantation. Fluorescent immunostaining for vWF-VIII at 6-weeks revealed increased blood vessel density (x100) in group-4 (p<0.05) as compared with other groups. Regional blood flow (ml/g/min) in the peri-infarct area was improved in group-4 (2.7; p<0.05) as compared with group-1 (1.2+/-0.1), group-2 (1.1+/-0.4) and group-3 (1.7+/-0.1) at 6-weeks. Similarly, ejection fraction was significantly higher in group-4 (49.2+/-5.9%, p=0.03) as compared with group-1 (36.8+/-3%) at 6 weeks., Conclusion: SkMs mediated Ang-1 delivery is associated with improved angiogenic response, regional myocardial perfusion and heart function as compared with direct Ad-Ang-1 administration.

Objective: To achieve angiogenic interaction between VEGF(165) and angiopoietin-1 (Ang-1) using a novel adenoviral bicistronic vector (Ad-Bic) encoding the two factors and delivered ex vivo using sex-mismatched human skeletal myoblasts., Methods and Results: A myocardial infarction model was developed in 29 female pigs; randomised into four groups: DMEM (group-1, n=6); Adenovirus null (Ad-null) vector-myoblast (group-2, n=5); Ad-Ang-1 myoblast (group 3, n=7) and Ad-Bic-myoblast (group-4, n=11). Three weeks later, 5 ml DMEM without myoblasts or containing 3 x 10(8) myoblasts carrying lac-z gene and transduced with Ad-null, Ad-Ang-1 or Ad-Bic were injected intra-myocardially in and around the infarct. 2D-echocardiography and fluorescent microsphere studies 6- and 12-weeks post-treatment revealed significantly improved cardiac performance and regional blood flow in groups 3 and 4. Histological studies and Y-chromosome analysis revealed extensive survival of lac-z positive myoblasts staining positive for human proteins in the pig heart. ELISA, immunostaining and RT-PCR revealed that Ad-Bic transduced myoblasts concomitantly but transiently expressed hVEGF(165) and Ang-1 both in vitro and in vivo. Double fluorescent immunostaining of the tissue sections for vWFactor-III and smooth muscle actin showed significantly higher vascular density of mature blood vessels per low power microscopic field in groups 3 and 4 at 6- and 12-weeks., Conclusion: Our combined approach led to enhanced angiogenesis with a greater percentage of functionally mature blood vessels in a porcine heart.

This study investigated the potential of human skeletal myoblast carrying human VEGF(165) for angiomyogenesis for cardiac repair. A porcine heart model of chronic infarction was created in 18 female swine by coronary artery ligation. The animals were randomized into: group 1, DMEM injected ( n=6), group 2, myoblast transplanted ( n=5) and group 3, VEGF(165) myoblast transplanted ( n=7). Three weeks later 5 ml DMEM containing 3x10(8) myoblast carrying exogenous genes were injected into 20 sites in left ventricle intramyocardially in groups 2 and 3. Group 1 animals were injected 5 ml DMEM without cells. Animals were kept on 5 mg/kg cyclosporine per day for 6 weeks. Regional blood flow was measured using fluorescent microspheres. The heart was explanted between 6-12 weeks after transplantation for histological studies. Histological examination showed survival of lac-z expressing myoblasts in host tissue. Capillary density at low power field (x100) was 57.13+/-4.20 in group 3 which was significantly higher than the other groups. Regional blood flow was significantly improved 6 and 12 weeks after transplantation, which was 2.41+/-0.11 and 3.39+/-0.11 ml(-1) min(-1) g(-1), respectively, in group 3. Left ventricular ejection fraction increased from 31.25+/-4.09% to 43.0+/-2.68% at 6 weeks in group 3. Human myoblasts are potential transgene carriers for the myocardium, in addition to strengthening the weakened myocardium through myogenesis.

Abstract: A regulated promoter system to control gene expression is desirable for safe and efficacious over-expression of therapeutic transgene. Combined with skeletal myoblast (SkMs), we report the efficacy of hypoxia-regulated VEGF gene delivery for myocardial repair during acute myocardial infarction (AMI). A hypoxia-regulated VEGF plasmid (pHRE-VEGF) was developed. After optimization, ∼30% SkMs were transfected using polyethyleneimine (PEI) nanoparticles. The peak VEGF expression was higher in pHRE-VEGF transfected SkMs (VEGFSkMs) under hypoxia (151.34 ± 8.59 ng/ml) than that with normoxia (16.92 ± 2.74 ng/ml). The efficacy of hypoxia-regulated gene expression system was assessed in a rabbit model of AMI. The animals were grouped to receive basal M199 without cells (group-1) or containing non-transfected SkMs (group-2) or VEGFSkMs (group-3). In group-4, VEGFSkMs were injected into normal heart to serve as normoxia control. Improved SkM survival was observed in group-3 and -4 (p < 0.05 vs group-2) at day-3 and 7 after transplantation. Blood vessel density was 20.1 ± 1.3 in group-3 which was significantly higher than any other groups (p < 0.05) at 2 weeks after treatment. Improved blood flow (ml/min/g) in the left ventricle (LV) anterior wall was observed in group-3 (1.28 ± 0.09, p < 0.05) as compared with group-1 (0.76 ± 0.05) and group-2 (0.96 ± 0.06), and similar to group-4 (1.26 ± 0.05). LV ejection fraction was best preserved in group-3 (58.4 ± 1.75%) which was insignificantly different from group-4 (61.1 ± 1.8%), and group-2 (52.8 ± 1.4%), but significantly improved compared with group-1 (44.7 ± 2.2%, p < 0.05). The study demonstrates that nanoparticle based delivery of hypoxia-regulated VEGF transgene combined with SkMs during AMI effectively preserves LV regional blood flow and contractile function of the heart. [Copyright &y& Elsevier]

Abstract: Objective: To achieve angiogenic interaction between VEGF165 and angiopoietin-1 (Ang-1) using a novel adenoviral bicistronic vector (Ad-Bic) encoding the two factors and delivered ex vivo using sex-mismatched human skeletal myoblasts. Methods and results: A myocardial infarction model was developed in 29 female pigs; randomised into four groups: DMEM (group-1, n =6); Adenovirus null (Ad-null) vector-myoblast (group-2, n =5); Ad-Ang-1 myoblast (group 3, n =7) and Ad-Bic-myoblast (group-4, n =11). Three weeks later, 5 ml DMEM without myoblasts or containing 3×108 myoblasts carrying lac-z gene and transduced with Ad-null, Ad-Ang-1 or Ad-Bic were injected intra-myocardially in and around the infarct. 2D-echocardiography and fluorescent microsphere studies 6- and 12-weeks post-treatment revealed significantly improved cardiac performance and regional blood flow in groups 3 and 4. Histological studies and Y-chromosome analysis revealed extensive survival of lac-z positive myoblasts staining positive for human proteins in the pig heart. ELISA, immunostaining and RT-PCR revealed that Ad-Bic transduced myoblasts concomitantly but transiently expressed hVEGF165 and Ang-1 both in vitro and in vivo. Double fluorescent immunostaining of the tissue sections for vWFactor-III and smooth muscle actin showed significantly higher vascular density of mature blood vessels per low power microscopic field in groups 3 and 4 at 6- and 12-weeks. Conclusion: Our combined approach led to enhanced angiogenesis with a greater percentage of functionally mature blood vessels in a porcine heart. [Copyright &y& Elsevier]