Myocardial Infarction Repair Research Articles

S100A9 Links Inflammation and Repair in Myocardial Infarction.

The alarmin S100A9 has been identified as a potential therapeutic target in myocardial infarction. Short-term S100A9 blockade during the inflammatory phase post-myocardial infarction inhibits systemic and cardiac inflammation and improves cardiac function long term. To evaluate the impact of S100A9 blockade on postischemic cardiac repair. We assessed cardiac function, hematopoietic response, and myeloid phagocyte dynamics in WT (wild type) C57BL/6 mice with permanent coronary artery ligation, treated with the specific S100A9 blocker ABR-238901 for 7 or 21 days. In contrast to the beneficial effects of short-term therapy, extended S100A9 blockade led to progressive deterioration of cardiac function and left ventricle dilation. The treatment reduced the proliferation of Lin-Sca-1+c-Kit+ hematopoietic stem and progenitor cells in the bone marrow and the production of proreparatory CD150+CD48-CCR2+ hematopoietic stem cells. Monocyte trafficking from the spleen to the myocardium and subsequent phenotype switching to reparatory Ly6CloMerTKhi macrophages was also impaired, leading to inefficient efferocytosis, accumulation of apoptotic cardiomyocytes, and a larger myocardial scar. The transcription factor Nur77 (Nr4a1 [nuclear receptor subfamily 4 group A member 1]) mediates the transition from inflammatory Ly6Chi monocytes to reparatory Ly6Clo macrophages. S100A9 upregulated the levels and activity of Nur77 in monocytes and macrophages in vitro and in Ly6Chi/int monocytes in vivo, and S100A9 blockade antagonized these effects. Finally, the presence of reparatory macrophages in the myocardium was also impaired in S100A9-/- mice with permanent myocardial ischemia, leading to depressed cardiac function long term. We show that S100A9 plays an important role in both the inflammatory and the reparatory immune responses to myocardial infarction. Long-term S100A9 blockade negatively impacts cardiac recovery and counterbalances the beneficial effects of short-term therapy. These results define a therapeutic window targeting the inflammatory phase for optimal effects of S100A9 blockade as potential immunomodulatory treatment in acute myocardial infarction.

Exogenous IL‐4 Promotes Myocardial Infarction Repair by Turning off Pro‐Inflammation in Neutrophils while Stimulating Anti‐Inflammation in Macrophages to Induce Neutrophil Phagocytosis

Macrophages and neutrophils are primary leukocytes involved in the inflammatory response to myocardial infarction (MI). While IL‐4 is an in vitro stimulus for anti‐inflammation, the MI myocardium does not express IL‐4. We hypothesized that continuous exogenous IL‐4 infusion starting 24 h after MI would promote a polarization switch in inflammatory cells towards a reparative phenotype. C57BL/6J male mice (3–6 months of age) were subcutaneously infused with either saline (n=17) or IL‐4 (20 ng/g/day; n=17) and evaluated at MI day 3. Macrophages and neutrophils were isolated ex vivo from the infarct region and examined for pro‐inflammatory genes (Ccl3, Ccl5, Il1b, Il12a, and Tnfa) and anti‐inflammatory genes (Arg1, Ym1, Mrc1, Il10, and Tgfb, Il6). Exogenous IL‐4 decreased pro‐inflammatory Ccl3, IL‐12a, Tnfa, and Tgfb1 in neutrophils while increasing anti‐inflammatory Arg1 and Ym1 in macrophages (all p<0.05); none of the other markers were different. Tissue clearance by IL‐4 treated neutrophils was not different, while phagocytosis of neutrophils, but not myocytes, doubled in IL‐4 treated macrophages (p<0.05). Because macrophage cell physiology was influenced by IL‐4, whole transcriptome analysis of macrophages isolated from the infarct regions of IL‐4 (n=10) or saline (n=11) treated mice was performed by RNA‐sequencing. Of the 24,341 genes sequenced, 2,042 genes were differentially expressed with IL‐4 stimulation (all p<0.05). Pdgfc gene expression was ranked first by p value, increasing from 1.8±0.3 in saline treated macrophages to 5.4±0.2 FPKM units in macrophages stimulated with IL‐4 (p=1×10−9). Importantly, changes in macrophage physiology and transcriptome occurred before differences in cardiac physiology evaluated by echocardiography, which allowed the isolation of effects in cell phenotype not induced by global myocardial effects. Bone marrow derived monocytes stimulated with mouse recombinant PDGF‐CC protein (10 μg/ml) or PDGF‐CC blocking antibody (10 μg/ml) revealed no effect on Arg1 or Ym1 expression, indicating the in vivo effect of IL‐4 to stimulate macrophage gene expression was independent of PDGF‐CC. Altogether, our results indicate that exogenous IL‐4 promotes inflammation resolution by turning off pro‐inflammation in neutrophils while stimulating anti‐inflammation in macrophages to induce neutrophil phagocytosis.Support or Funding InformationNational Institutes of Health under Award Numbers HL075360, HL129823, and HL137319, Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development under Award Numbers 5I01BX000505

Bioprinting and in vitro characterization of alginate dialdehyde–gelatin hydrogel bio-ink

Cell-laden cardiac patches have recently been emerging to renew cellular sources for myocardial infarction (MI, commonly know as a heart attack) repair. However, the fabrication of cell-laden patches with porous structure remains challenging due to the limitations of currently available hydrogels and existing processing techniques. The present study utilized a bioprinting technique to fabricate hydrogel patches and characterize them in terms of printability, mechanical and biological properties. Cell-laden hydrogel (or bio-ink) was formulated from alginate dialdehyde (ADA) and gelatin (GEL) to improve the printability, degradability as well as bioactivity. Five groups of hydrogel compositions were designed to investigate the influence of the oxidation degree of ADA and hydrogels concentration on the properties of printed scaffolds. ADA–GEL hydrogels have generally shown favorable for living cells (EA.hy926 cells and hybrid human umbilical vein endothelial cell line). The hydrogel with an oxidation degree of 10% and a concentration ratio of 70/30 (or 10%ADA70–GEL30) demonstrated the best printability among the groups examined. Formulated hydrogels were also bioprinted with the living cells (EA.hy926), and the scaffolds printed were then subject to the cell culture for 7 days. Our results illustrate that the scaffolds bioprinted from 10%ADA70–GEL30 hydrogels had the best homogenous cell distribution and also the highest cell viability. Taken together, in the present study we synthesized a newly formulated bio-ink from ADA and GEL and for the fist time, used them to bioprint cardiac patches, which have the potential to be used in MI repair.

Circulating angiogenic cells in glioblastoma: toward defining crucial functional differences in CAC-induced neoplastic versus reactive neovascularization.

BackgroundIn order to identify suitable therapeutic targets for glioma anti-angiogenic therapy, the process of neovascularization mediated by circulating angiogenic cells (CACs) needs to be scrutinized.MethodsIn the present study, we compared the expression of neovascularization-related genes by 3 circulating CAC subsets (hematopoietic progenitor cells [HPCs], CD34+, and KDR+ cells; internal controls: peripheral blood mononuclear cells and circulating endothelial cells) of treatment-naïve patients with glioblastoma (GBM) to those of patients undergoing reactive neovascularization (myocardial infarction (MI). CACs from umbilical cord (representing developmental neovascularization) and healthy subjects served as controls. Fluorescent-activated cell sorting was used to isolate CACs, RT-PCR to determine the expression levels of a panel of 48 neovascularization-related genes, and Luminex assays to measure plasma levels of 21 CAC-related circulating molecules.ResultsWe found essential differences in gene expression between GBM and MI CACs. GBM CACs had a higher expression of proangiogenic factors (especially, KITL, CXCL12, and JAG1), growth factor and chemotactic receptors (IGF1R, TGFBR2, CXCR4, and CCR2), adhesion receptor monomers (ITGA5 and ITGA6), and matricellular factor POSTN. In addition, we found major differences in the levels of neovascularization-related plasma factors. A strong positive correlation between plasma MMP9 levels and expression of CXCR4 in the CAC subset of HPCs was found in GBM patients.ConclusionsOur findings indicate that CAC-mediated neovascularization in GBM is characterized by more efficient CAC homing to target tissue and a more potent proangiogenic response than in physiologic tissue repair in MI. Our findings can aid in selecting targets for therapeutic strategies acting against GBM-specific CACs.

Homozygous MESP1 knock-in reporter hESCs facilitated cardiovascular cell differentiation and myocardial infarction repair.

Different populations of cardiovascular progenitor cells have been shown to possess varying differentiation potentials. They have also been used to facilitate heart repair. However, sensitive reporter cell lines that mark the human cardiovascular progenitors are in short supply.Methods: MESP1 marks the earliest population of cardiovascular progenitor cells during embryo development. Here, we generated a homozygous MESP1 knock-in reporter hESC line where mTomato gene joined to the MESP1 coding region via a 2A peptide, in which both MESP1 alleles were preserved. We performed transcriptome and functional analysis of human MESP1+ cardiovascular progenitor cells and tested their therapeutic potential using a rat model of myocardial infarction.Results: MESP1-mTomato knock-in reporter faithfully recapitulated the endogenous level of MESP1. Transcriptome analysis revealed that MESP1+ cells highly expressed early cardiovascular genes and heart development genes. The activation of MESP1 relied on the strength of canonical Wnt signaling, peak MESP1-mTomato fluorescence correlated with the window of canonical Wnt inhibition during in vitro differentiation. We further showed that MESP1 bound to the promoter of the WNT5A gene and the up-regulation of WNT5A expression suppressed canonical Wnt/β-CATENIN signaling. Moreover, induced MESP1 expression could substitute the canonical Wnt inhibition step and promote robust cardiomyocyte formation. We used a configurable, chemically defined, tri-lineage differentiation system to obtain cardiomyocytes, endothelial cells, and smooth muscle cells from MESP1+ cells at high efficiency. Finally, we showed that the engraftment of MESP1+ cells repaired rat myocardial infarction model.Conclusions: MESP1-mTomato reporter cells offered a useful platform to study cardiovascular differentiation from human pluripotent stem cells and explore their therapeutic potential in regenerative medicine.

Mussel-inspired conductive Ti2C-cryogel promotes functional maturation of cardiomyocytes and enhances repair of myocardial infarction.

Rationale: Researches on conductive engineering cardiac patch (ECP) for myocardial infarction (MI) treatment have achieved some progress in the animal while the availability of traditional conductive materials in ECP is still limited because of their controversial cytotoxicity. Here we aim to introduce a novel hydrophilic biocompatible conductive material: MXene Ti2C and mussel-inspired dopamine into PEGDA-GelMA cryogel to construct a bio-functional ECP of which the property closes to natural heart for the repair of MI.Method: MXene Ti2C was etched from MAX Ti2AlC, then uniformly dispersed into the prepolymer composed with dopamine-N′, N′-methylene-bisacrylamide, methacrylate-gelatin, and poly (ethylene glycol) diacrylate by simple water bath sonication. The resilient conductive Ti2C-cryogel was fabricated by chemical cryogelation. The conductive ECP was evaluated in vitro and transplanted to the MI rat model for MI treatment.Results: In vitro, the 3D vessels-shape framework was observed in Ti2C-8-cryogel which was seeded with rats aortic endothelial cells. When the Ti2C-cryogels were cocultured with CMs, remarkably aligned sarcomere and the primitive intercalated disc between the mature CMs were formed on day 7. The as-prepared Ti2C-8-cryogel ECP also demonstrated rapid calcium transients and synchronous tissue-like beating. When transplanted into the infarcted heart of the MI rat model, the Ti2C-8-cryogel ECP could improve the cardiac function, reduce the infarct size, and inhibit the inflammatory response. Obvious vasculation especially newly formed arteriole was also found.Conclusion: A novel conductive Ti2C-embedded cardiac patch with suitable conductivity and the mechanical property was developed and could be served as an ideal candidate for MI repair.

Engineered bioactive nanoparticles incorporated biofunctionalized ECM/silk proteins based cardiac patches combined with MSCs for the repair of myocardial infarction: In vitro and in vivo evaluations

The development of cardiac patches by the combination of bioactive nano- and bio-materials with mesenchymal stem cells signifies an auspicious approach for the treatment of cardiac repair in myocardial infarction. In the present investigation, we study about the cardiac function of morphology improved gold nanoparticles combined with extracellular matrix/silk proteins for the cell proliferation and expansion of cardiomyocytes. The physico-chemical and morphological characteristics demonstrated that spherical and homogeneous Au particles are distributed on the matrix porous surface for providing favorable conductivity and biological influences in cardiac repair. The in vitro cell studies of prepared patches have established enhanced cell compatibility and retention of cardiomyocytes survival. The in vivo determinations imply that Au-ESF group decreases infarct size to 65% from 89% in control group. These developed cardiac patches can be highly suitable in the cardiac regeneration and offer new platform in cardiac tissue engineering.

An Injectable Conductive Three-Dimensional Elastic Network by Tangled Surgical-Suture Spring for Heart Repair.

Designing scaffolds with persistent elasticity and conductivity to mimic microenvironments becomes a feasible way to repair cardiac tissue. Injectable biomaterials for cardiac tissue engineering have demonstrated the ability to restore cardiac function by preventing ventricular dilation, enhancing angiogenesis, and improving conduction velocity. However, limitations are still among them, such as poor mechanical stability, low conductivity, and complicated procedure. Here, we developed thermal plastic poly(glycolic acid) surgical suture and mussel-inspired conductive particle's adhesion into a highly elastic, conductive spring-like coils. The polypyrrole (PPy)-coated biospring acted as an electrode and then was assembled into a solid-state supercapacitor. After being injected through a syringe needle (0.33 mm inner diameter), the tangled coils formed an elastically conductive three-dimensional (3-D) network to modulate cardiac function. We found that cardiomyocytes (CMs) grew along the spring coils' track with elongated morphologies and formed highly oriented sarcomeres. The biospring enhanced the CMs' maturation in synchronous contraction accompanied by high expressions of cardiac-specific proteins, α-actinin, and connexin 43 (cx43). After the elastic, conductive biosprings were injected into the myocardial infarction (MI) area, the left ventricular fractional shortening was improved by about 12.6% and the infarct size was decreased by about 34%. Interestingly, the spring can be utilized as a sensor to measure the CMs' contractile force, which was 1.57 × 10-3 ± 0.26 × 10-3 mN (∼4.1 × 106 cells). Accordingly, this study highlights an injectable biospring to form a tangled conductive 3-D network in vivo for MI repair.

P5391Systemic administration of high-mobility group box 1 can suppress adverse post-infarction ventricular remodeling in a rat infarction model by enhancing self-regeneration

Abstract Background High-mobility group box 1 protein (HMGB1) reportedly enhances CXCR4-positive bone marrow-derived mesenchymal stem cell (BM-MSC) recruitment to damaged tissue to promote tissue regeneration. Purpose Our aim of this study is to evaluate whether systemic administration of HMGB1 might promote tissue repair in a rat myocardial infarction (MI) model. Methods We prepared 26 MI model rats with high ligation of the left coronary artery. Two weeks later, HMGB1 (3 mg/kg/day) or phosphate-buffered saline (control: 3 mL/kg/day) was administered for 4 days via femoral vein. Cardiac performance was evaluated by ultrasonography, left ventricular (LV) remodeling via immunostaining. We then used immunostaining to examine MSC recruitment to damaged tissue in green fluorescent protein bone marrow transplantation (GFP-BMT) model rats, and also performed intravital imaging using two-photon microscopy to visualize BM-cells recruitment in real time. Results Compared with control rats, there was a significant improvement in the left ventricular ejection fraction of the HMGB1 group (HMGB1 vs. control: 48.6% ± 5.5% vs. 33.6% ± 5.4%; p<0.01) at 4 weeks after each administration. LV remodeling, characterized by interstitial fibrosis, cardiomyocyte hypertrophy, and a decrease of capillary density, was significantly attenuated in the HMGB1 group compared with control rats. On QT-PCR analysis, VEGF mRNA expression was significantly higher in the HMGB1 group than in the control (border zone; 1.6±0.6 vs. 1.1±0.2; p=0.02, septal zone; 1.1±0.1 vs. 0.9±0.1; p<0.01). In GFP-BMT rats, GFP+/PDGFR+ cells were significantly mobilized to the border zone in the HMGB1 group compared with the control (1331±197 vs. 615±45 /mm2; p<0.01), leading to formation of newly developed vasculature (Figure 1). In intravital imaging, more GFP+ cells were mobilized to the infarction area in the HMGB1 group than in the control, which was further enhanced at 12h later. Additionally, SDF-1 expression in the peri-infarction area increased significantly in MI rats compared with normal rats (MI vs. normal; 2.1±0.4 vs. 0.9±0.1; p<0.01), in where some cell-adhesions of vascular endothelial cells were destroyed. Conclusions Systemic administration of HMGB1 mobilized BM-MSCs to the damaged myocardium via the SDF-1/CXCR4 signaling complex. Those BM-MSCs might migrate to extracellular matrix in the border zone via the gap of each endothelial cell, leading to induction of angiogenesis and reduced fibrosis. Acknowledgement/Funding None

A collagen hydrogel loaded with HDAC7-derived peptide promotes the regeneration of infarcted myocardium with functional improvement in a rodent model

Myocardial infarction (MI) leads to the loss of cardiomyocytes, left ventricle (LV) dilation, and cardiac dysfunction, eventually developing into heart failure. Most of the strategies for MI therapy require biomaterials that can support tissue regeneration. In this study, we hypothesized that the extracellular matrix (ECM)-derived collagen I hydrogel loaded with histone deacetylase 7 (HDAC7)-derived-phosphorylated 7-amino-acid peptide (7Ap) could restrain LV remodeling and improve cardiac function after MI. An MI model was established by ligation of the left anterior descending coronary artery (LAD) of C57/B6 mice. The 7Ap-loaded collagen I hydrogel was intramyocardially injected to the infarcted region of the LV wall of the heart. After local delivery, the 7Ap-collagen increased neo-microvessel formation, enhanced stem cell antigen-1 positive (Sca-1+) stem cell recruitment and differentiation, decreased cellular apoptosis, and promoted cardiomyocyte cycle progression. Furthermore, the 7Ap-collagen restricted the fibrosis of the LV wall, reduced the infarct wall thinning, and improved cardiac performance significantly at 2 weeks post-MI. These results highlight the promising implication of 7Ap-collagen as a novel candidate for MI therapy. Statement of SignificanceThe mammalian myocardium has a limited regenerative capability following myocardial infarction (MI). MI leads to extensive loss of cardiomyocytes, thus culminating in adverse cardiac remodeling and congestive heart failure. In situ tissue regeneration through endogenous cell mobilization has great potential for tissue regeneration. A 7-amino-acid-peptide (7A) domain encoded by a short open-reading frame (sORF) of the HDAC7 gene. The phosphorylated from of 7A (7Ap) has been reported to promote in situ tissue repair via the mobilization and recruitment of endogenous stem cell antigen-1 positive (Sca-l+) stem cells. In this study, 7Ap was shown to improve H9C2 cell survival, in vitro. In vivo investigations in a mouse MI model demonstrated that intra-myocardial delivery of 7Ap-loaded collagen hydrogel promoted neovascularization, stimulated Sca-l+ stem cell recruitment and differentiation, reduced cardiomyocyte apoptosis and promoted cell cycle progression. As a result, treated infarcted hearts had increased wall thickness, had improved heart function and exhibited attenuation of adverse cardiac remodeling, observed for up to 2 weeks. Overall, these results highlighted the positive impact of implanting 7Ap-collagen as a novel constituent for MI repair.

Paracrine Mechanisms Involved in Mesenchymal Stem Cell Differentiation into Cardiomyocytes.

Cardiovascular Disease (CVD) is one of the world-wide healthcare problem that involves the heart or blood vessels. CVD includes myocardial infarction and coronary artery diseases (CAD). Dysfunctional myocardial cells are leading causes of low cardiac output or ventricular dysfunction after cardiac arrest and may contribute to the progression of CVD which could not generate new cardiomyocytes in human adult heart. The mesenchymal stem cells (MSCs) which are present in adult marrow can self-renew and have the capacity of differentiation into multiple types of cells including cardiomyocytes. Recent biochemical analyses greatly revealed that several regulators of MSCs, such as HGF, PDGF, Wnt, and Notch-1 signaling pathways have been shown to be involved in the proliferation and differentiation into cardiomyocytes. Preclinical studies are paving the way for further applications of MSCs in the repair of myocardial infarction. In this study, we discuss and summarize the paracrine mechanisms involved in MSCs differentiation into cardiomyocytes.

A 6-week multidisciplinary tailored Cardiac Rehabilitation Program is able to improve Left-Ventricular Ejection Fraction in patients with Chronic Heart Failure

Background Cardiac rehabilitation (CR) is recommended to patients with chronic heart failure (CHF) and reduced Left-Ventricular ejection fraction (EF) at a class 1 evidence level. Purpose The aim of this prospective study was to assess the effect of a rehabilitation program on EF in patients with CHF. Methods One hundred and five patients (mean age: 5713 years, men: 67%, diabetic patients: 42%) with CHF (EF Reasons of admission were: coronary heart disease artery bypass graft surgery (17%), angioplasty percutaneous coronary interventions and stent (24%), angor pectoris or myocardial infarction (8%), aortic or mitral valve replacement or repair (10%), dilated cardiomyopathy (34%), other (7%). Patients received beta-blocker (96%), conversion enzyme inhibitor or angiotensine II receptor antagonist (42%), sacubitril-valsartan (48%), furosemide (60%), antialdosterone therapy (55%), ivabradine (12%), other (11%). EF assessed by echocardiography using the biplane Simpson's method, peak oxygen consumption (VO2), maximal walking distance (MWD) during 6 min walk test and NT-proBNP were assessed before and after rehabilitation. Results After 6 weeks, there were significantly improvements in EF (35% ± 7.23 vs. 40% ± 8.3 - P Conclusion Our study shows that a 6-week multidisciplinary tailored Cardiac Rehabilitation Program improves significantly Left-Ventricular ejection fraction in patients with Chronic Heart Failure. This should be relevant to improve prognosis.

Prevalence of iron deficiency in patients with chronic heart failure admitted in a Cardiac Rehabilitation Center in Reunion Island

Iron deficiency is common in patients with heart failure and its diagnosis using serum ferritin and transferrin saturation is recommended. The aim of this preliminary prospective study was to evaluate the prevalence of iron deficiency (ID) in patients with chronic heart failure (CHF) with reduced Left-Ventricular ejection fraction (LVEF), admitted in cardiac rehabilitation (CR), in Reunion Island. Sixty-one HF patients with LVEF (< = 40%) (58 years ± 13, women 41%) and 253 non HF cardiac patients, served as a control group (62 years ± 12, women 34%) was included. Ferritin, transferrin saturation (TSAT) and hemoglobin were accessed on admission. Iron deficiency was defined in accordance with the following criteria: serum ferritin < 100 μg/L; or serum ferritin 100–299 μg/L plus TSAT < 20%. Anemia was defined as more than 13 grams (g) of hemoglobin per deciliter (dL) of blood for men and 12 g/dL for women. Reasons of admission for HF patients vs non HF cardiac patient were: coronary heart disease artery bypass graft surgery (23% vs. 33%), angioplasty percutaneous coronary interventions and stent (20% vs. 32%), angor pectoris or myocardial infarction (11% vs. 4%), aortic or mitral valve replacement or repair (12% vs. 21%), dilated cardiomyopathy (34% vs. 0%), other (0% vs. 10%). Iron deficiency was founded in 52% in HF patients (with anemia in 38% among them), and in 53% in non HF cardiac patients (with anemia in 24% among them). See Table 1 . Iron deficiency is very common in cardiac patient admitted in Rehabilitation in Reunion Island, without difference between HF patients and non HF patients.

Cardiac Fibroblast-Specific Activating Transcription Factor 3 Promotes Myocardial Repair after Myocardial Infarction.

Background:Myocardial ischemia injury is one of the leading causes of death and disability worldwide. Cardiac fibroblasts (CFs) have central roles in modulating cardiac function under pathophysiological conditions. Activating transcription factor 3 (ATF3) plays a self-protective role in counteracting CF dysfunction. However, the precise function of CF-specific ATF3 during myocardial infarction (MI) injury/repair remains incompletely understood. The aim of this study was to determine whether CF-specific ATF3 affected cardiac repair after MI.Methods:Fifteen male C57BL/6 wild-type mice were performed with MI operation to observe the expression of ATF3 at 0, 0.5, 1.0, 3.0, and 7.0 days postoperation. Model for MI was constructed in ATF3TGfl/flCol1a2-Cre+ (CF-specific ATF3 overexpression group, n = 5) and ATF3TGfl/flCol1a2-Cre− male mice (without CF-specific ATF3 overexpression group, n = 5). In addition, five mice of ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre− were subjected to sham MI operation. Heart function was detected by ultrasound and left ventricular remodeling was observed by Masson staining (myocardial fibrosis area was detected by blue collagen deposition area) at the 28th day after MI surgery in ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre− mice received sham or MI operation. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect cell proliferation/cell cycle-related gene expression in cardiac tissue. BrdU staining was used to detect fibroblast proliferation.Results:After establishment of an MI model, we found that ATF3 proteins were increased in the heart of mice after MI surgery and dominantly expressed in CFs. Genetic overexpression of ATF3 in CFs (ATF3TGfl/flCol1a2-Cre+ group) resulted in an improvement in the heart function as indicated by increased cardiac ejection fraction (41.0% vs. 30.5%, t = 8.610, P = 0.001) and increased fractional shortening (26.8% vs. 18.1%, t = 7.173, P = 0.002), which was accompanied by a decrease in cardiac scar area (23.1% vs. 11.0%, t = 8.610, P = 0.001). qRT-PCR analysis of CFs isolated from ATF3TGfl/flCol1a2-Cre+ and ATF3TGfl/flCol1a2-Cre− ischemic hearts revealed a distinct transcriptional profile in ATF3-overexpressing CFs, displaying pro-proliferation properties. BrdU-positive cells significantly increased in ATF3-overexpressing CFs than control CFs under angiotensin II stimuli (11.5% vs. 6.8%, t = 31.599, P = 0.001) or serum stimuli (31.6% vs. 20.1%, t = 31.599, P = 0.001). The 5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester assay showed that the cell numbers of the P2 and P3 generations were higher in the ATF3-overexpressing CFs at 24 h (P2: 91.6% vs. 71.8%, t = 8.465, P = 0.015) and 48 h (P3: 81.6% vs. 51.1%, t = 9.029, P = 0.012) after serum stimulation. Notably, ATF3 overexpression-induced CF proliferation was clearly increased in the heart after MI injury.Conclusions:We identify that CF-specific ATF3 might contribute to be MI repair through upregulating the expression of cell cycle/proliferation-related genes and enhancing cell proliferation.

Correction: Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model

[This corrects the article DOI: 10.1371/journal.pone.0175807.].

UV-Assisted 3D Bioprinting of Nanoreinforced Hybrid Cardiac Patch for Myocardial Tissue Engineering.

Biofabrication of cell supportive cardiac patches that can be directly implanted on myocardial infarct is a potential solution for myocardial infarction repair. Ideally, cardiac patches should be able to mimic myocardium extracellular matrix for rapid integration with the host tissue, raising the need to develop cardiac constructs with complex features. In particular, cardiac patches should be electrically conductive, mechanically robust and elastic, biologically active and prevascularized. In this study, we aim to biofabricate a nanoreinforced hybrid cardiac patch laden with human coronary artery endothelial cells (HCAECs) with improved electrical, mechanical, and biological behavior. A safe ultraviolet (UV) exposure time with insignificant effect on cell viability was identified for methacrylated collagen (MeCol) micropatterning. The effects of carboxyl functionalized carbon nanotubes (CNTs) on MeCol and alginate matrix morphology, mechanical properties, electrical behavior, and cellular response were investigated at different CNT mass ratios. A UV-integrated 3D-bioprinting technique was implemented to create hybrid hydrogel constructs consisting of CNT-incorporated alginate framework and cell-laden MeCol. The compressive modulus, impedance, and swelling degree of hybrid constructs were assessed over 20 days of incubation in culture medium at 37°C for different CNT mass ratios. The HCAEC viability, proliferation, and differentiation in the context of the bioprinted hybrid constructs were assessed over 10 days in vitro. The functionalized CNTs provided a highly interconnected nanofibrous meshwork that significantly improved viscoelastic behavior and electrical conductivity of photo-cross-linked MeCol. Alginate-coated CNTs provided a nanofilamentous network with fiber size of ∼25-500 nm, improving not only electrical and mechanical properties but also HCAEC attachment and elongation compared to pristine alginate. The CNT-reinforced 3D-printed hybrid constructs presented significantly higher stiffness and electrical conductivity particularly in the physiologically relevant frequency range (∼5 Hz). The CNT-reinforced hybrid implants maintained a significantly higher swelling degree over 20 days of culturing compared to CNT-free hybrid constructs. For a selected CNT mass ratio, HCAECs presented significant cellular proliferation, migration, and differentiation (lumen-like formation) over 10 days of incubation in vitro. Findings from this study deliver essential steps toward developing conductive, robust, and potentially prevascularized hybrid cardiac patches.

Self-assembling peptide GFS-4 nanofiber scaffolds for three-dimensional cell cultures and myocardial infarction repair

The purpose of this study is to investigate the effects of self-assembling peptide GFS-4 on three-dimen-sional myocardial cell culture and tissue repair of myocardial infarction. The circular dichroism (CD) spectrum was used to detect secondary structure of GFS-4, and atomic force microscope (AFM) was used to analyze the microstructure of self-assembly. The nanofiber scaffolds self-assembled by GFS-4 were used as the three-dimensional culture material to observe the growth effect of cardiomyocytes. The model of myocardial infarction was established and the effect of GFS-4 on myocardial infarction was studied. The results indicated that self-assembling peptide GFS-4 could form mainly β-sheet structure that can form dense nanofiber scaffolds after 24 hours' self-assembling. The myocardial cells had a favorable growth status in GFS-4 nanofiber scaffold hydrogel when cells treated in three-dimen-sional cell culture. The experiment of repairing myocardial infarction in vitro proved that peptide GFS-4 hydrogel scaffold could alleviate tissue necrosis in a myocardial infarction area. As a new nanofiber scaffold material, self-assembling peptide GFS-4 can be used for three-dimensional cell culture and tissue repairing in myocardial infarction area.

Potential of propagation-based synchrotron X-ray phase-contrast computed tomography for cardiac tissue engineering.

Hydrogel-based cardiac tissue engineering offers great promise for myocardial infarction repair. The ability to visualize engineered systems in vivo in animal models is desired to monitor the performance of cardiac constructs. However, due to the low density and weak X-ray attenuation of hydrogels, conventional radiography and micro-computed tomography are unable to visualize the hydrogel cardiac constructs upon their implantation, thus limiting their use in animal systems. This paper presents a study on the optimization of synchrotron X-ray propagation-based phase-contrast imaging computed tomography (PCI-CT) for three-dimensional (3D) visualization and assessment of the hydrogel cardiac patches. First, alginate hydrogel was 3D-printed into cardiac patches, with the pores filled by fibrin. The hydrogel patches were then surgically implanted on rat hearts. A week after surgery, the hearts including patches were excised and embedded in a soft-tissue-mimicking gel for imaging by using PCI-CT at an X-ray energy of 25 keV. During imaging, the sample-to-detector distances, CT-scan time and the region of interest (ROI) were varied and examined for their effects on both imaging quality and radiation dose. The results showed that phase-retrieved PCI-CT images provided edge-enhancement fringes at a sample-to-detector distance of 147 cm that enabled visualization of anatomical and microstructural features of the myocardium and the implanted patch in the tissue-mimicking gel. For visualization of these features, PCI-CT offered a significantly higher performance than the dual absorption-phase and clinical magnetic resonance (3 T) imaging techniques. Furthermore, by reducing the total CT-scan time and ROI, PCI-CT was examined for lowering the effective dose, meanwhile without much loss of imaging quality. In effect, the higher soft tissue contrast and low-dose potential of PCI-CT has been used along with an acceptable overall animal dose to achieve the high spatial resolution needed for cardiac implant visualization. As a result, PCI-CT at the identified imaging parameters offers great potential for 3D assessment of microstructural features of hydrogel cardiac patches.

Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model.

BackgroundSkeletal myoblast transplantation seems a promising approach for the repair of myocardial infarction (MI). However, the low engraftment efficacy and impaired angiogenic ability limit the clinical efficiency of the myoblasts. Gene engineering with angiogenic growth factors promotes angiogenesis and enhances engraftment of transplanted skeletal myoblasts, leading to improved infarction recovery in myocardial ischemia. The present study evaluated the therapeutic effects of hepatocyte growth factor (HGF) gene-engineered skeletal myoblasts on tissue regeneration and restoration of heart function in a rat MI model.Methods and resultsThe skeletal myoblasts were isolated, expanded, and transduced with adenovirus carrying the HGF gene (Ad-HGF). Male SD rats underwent ligation of the left anterior descending coronary artery. After 2 weeks, the surviving rats were randomized into four groups and treated with skeletal myoblasts by direct injection into the myocardium. The survival and engraftment of skeletal myoblasts were determined by real-time PCR and in situ hybridization. The cardiac function with hemodynamic index and left ventricular architecture were monitored; The adenovirus-mediated-HGF gene transfection increases the HGF expression and promotes the proliferation of skeletal myoblasts in vitro. Transplantation of HGF-engineered skeletal myoblasts results in reduced infarct size and collagen deposition, increased vessel density, and improved cardiac function in a rat MI model. HGF gene modification also increases the myocardial levels of HGF, VEGF, and Bcl-2 and enhances the survival and engraftment of skeletal myoblasts.ConclusionsHGF engineering improves the regenerative effect of skeletal myoblasts on MI by enhancing their survival and engraftment ability.

Synthesis of Injectable Alginate Hydrogels with Muscle-Derived Stem Cells for Potential Myocardial Infarction Repair

Myocardial infarction (MI), caused by the occlusion of the left ventricular coronary artery, leads to the loss of cardiomyocytes and, potentially, heart failure. Cardiomyocytes in adult mammals proliferate at an extremely low rate and thus, a major challenge in MI treatment is supplementing exogenous cells and keeping them viable in MI areas. To address this challenge, injecting hydrogels encapsulating cells into MI areas, to compensate for the loss of cardiomyocytes, shows promise. This study synthesized two types of alginate hydrogels, based on self-crosslinking (SCL) and calcium ion crosslinking (Ca2+) in varying formulations. The hydrogels encapsulated living muscle-derived stem cells (MDSCs) and their performance was evaluated in terms of optimizing cell viability during the injection process, as well as the live/dead rate after long-term cultivation. The morphology of the hydrogel-encapsulated cells was characterized by scanning electronic microscopy (SEM) and live/dead cells were examined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining) assay. The mechanical properties of the hydrogels were also determined via a rheometer, to identify their influence on cell viability during the injection process and with respect to long-term cultivation. The SCL hydrogel with a 0.8% alginate and 20% gelatin formulation resulted in the highest cell viability during the injection process, and the Ca2+ hydrogel composed of 1.1% alginate and 20% gelatin maintained the highest cell survival rate after two months in culture.