post-MI Tissue Research Articles

3D bioprinted aged human post-infarct myocardium tissue model.

Fibrotic tissue formed after myocardial infarction (MI) can be as detrimental as MI itself. However, current in vitro cardiac fibrosis models fail to recapitulate the complexities of post-MI tissue. Moreover, although MI and subsequent fibrosis is most prominent in the aged population, the field suffers from inadequate aged tissue models. Herein, an aged human post-MI tissue model, representing the native microenvironment weeks after initial infarction, is engineered using three-dimensional bioprinting via creation of individual bioinks to specifically mimic three distinct regions: remote, border, and scar. The aged post-MI tissue model is engineered through combination of gelatin methacryloyl, methacrylated hyaluronic acid, aged type I collagen, and photoinitiator at variable concentrations with different cell types, including aged human induced pluripotent stem cell-derived cardiomyocytes, endothelial cells, cardiac fibroblasts, and cardiac myofibroblasts, by introducing a methodology which utilizes three printheads of the bioprinter to model aged myocardium. Then, using cell-specific proteins, the cell types that comprised each region are confirmed using immunofluorescence. Next, the beating characteristics are analyzed. Finally, the engineered aged post-MI tissue model is used as a benchtop platform to assess the therapeutic effects of stem cell-derived extracellular vesicles on the scar region. As a result, high viability (>74%) was observedin each region of the printed model. Constructs demonstrated functional behavior, exhibiting a beating velocity of 6.7 μm/s and a frequency of 0.3 Hz. Finally, the effectiveness of hiPSC-EV and MSC-EV treatment was assessed. While hiPSC-EV treatment showed no significant changes, MSC-EV treatment notably increased cardiomyocyte beating velocity, frequency, and confluency, suggesting a regenerative potential. In conclusion, we envision that our approach of modeling post-MI aged myocardium utilizing three printheads of the bioprinter may be utilized for various applications in aged cardiac microenvironment modeling and testing novel therapeutics.

Mechanisms of Regenerative Potential Activation in Cardiac Mesenchymal Cells.

Recovery of the contractile function of the heart and the regeneration of the myocardium after ischemic injury are contemporary issues in regenerative medicine and cell biology. This study aimed to analyze early transcriptional events in cardiac tissue after infarction and to explore the cell population that can be isolated from myocardial tissue. We induced myocardial infarction in Wistar rats by permanent ligation of the left coronary artery and showed a change in the expression pattern of Notch-associated genes and Bmp2/Runx2 in post-MI tissues using RNA sequencing and RT-PCR. We obtained primary cardiac mesenchymal cell (CMC) cultures from postinfarction myocardium by enzymatic dissociation of tissues, which retained part of the activation stimulus and had a pronounced proliferative potential, assessed using a “xCELLigence” real-time system. Hypoxia in vitro also causes healthy CMCs to overexpress Notch-associated genes and Bmp2/Runx2. Exogenous activation of the Notch signaling pathway by lentiviral transduction of healthy CMCs resulted in a dose-dependent activation of the Runx2 transcription factor but did not affect the activity of the Bmp2 factor. Thus, the results of this study showed that acute hypoxic stress could cause short-term activation of the embryonic signaling pathways Notch and Bmp in CMCs, and this interaction is closely related to the processes of early myocardial remodeling after a heart attack. The ability to correctly modulate and control the corresponding signals in the heart can help increase the regenerative capacity of the myocardium before the formation of fibrotic conditions.

Cells of the Immune System in Cardiac Remodeling: Main Players in Resolution of Inflammation and Repair After Myocardial Infarction.

The burden of heart failure (HF), developing after myocardial infarction MI, still represents a major issue in clinical practice. Failure of appropriate resolution of inflammation during post-myocardial injury is associated with unsuccessful left ventricular remodeling and underlies HF pathogenesis. Cells of the immune system have been shown to mediate both protective and damaging effects in heart remodeling. This ambiguity of the role of the immune system and inconsistent results of the recent clinical trials question the benefits of anti-inflammatory therapies during acute MI. The present review will summarize knowledge of the roles that different cells of the immune system play in the process of post-infarct cardiac healing. Data on the phenotype, active molecules and functions of the immune cells, based on the results of both experimental and clinical studies, will be provided. For some cellular subsets, such as macrophages, neutrophils, dendritic cells and lymphocytes, an anti-inflammatory activity has been attributed to the specific subpopulations. Activity of other cells, such as eosinophils, mast cells, natural killer (NK) cells and NKT cells has been shown to be highly dependent of the signals created by micro-environment. Also, new approaches for classification of cellular phenotypes based on the single-cell RNA sequencing allow better understanding of the phenotype of the cells involved in resolution of inflammation. Possible perspectives of immune-mediated therapy for AMI patients are discussed in the conclusion. We also outline unresolved questions that need to be solved in order to implement the current knowledge on the role of the immune cells in post-MI tissue repair into practice.

Abstract 828: The Functional Role of Myofibroblast-Expressed Smooth Muscle Alpha Actin in Post-Myocardial Infarction Tissue Healing

After myocardial infarction (MI), dead cardiomyocytes are permanently replaced by scar tissue due to the lack of cardiomyocyte regeneration. In response to MI, cardiac fibroblasts (CFs) differentiate into myofibroblasts which possess highly organized smooth muscle α-actin (αSMA) stress fibers and secrete a large amount of extracellular matrix (ECM) proteins, essential for the post-MI infarct scar formation. It is well known that the accumulated ECM in the infarct scar stabilize the scar and prevents cardiac rupture. However, the functional role of αSMA stress fiber itself in CF-derived myofibroblasts is still largely unknown besides the wide use of it as a marker of myofibroblast differentiation. Our recent work demonstrated that during myofibroblast differentiation of CFs post-MI, the expression of αSMA reaches a peak a few days earlier than genes encoding ECM proteins. Myofibroblasts then lose αSMA expression and differentiate to the newly identified matrifibrocytes as the infarct scar becomes increasingly stable. These facts suggest a more important role of αSMA stress fibers than ECM proteins in the tissue healing process in early phase post-MI, possibly through providing a support lattice which in turn strengthens the infarcted myocardium. To test our hypothesis, we generated a mouse line with Acta2 (the gene encoding αSMA) flanked by LoxP sites ( Acta2 fl/fl ) to enable cre-mediated Acta2 knockout. Here we show that the loss of Acta2 in CFs leads to enhanced proliferation and reduced contractility upon TGFβ-induced myofibroblast differentiation in vitro . By using a mouse line with tamoxifen-inducible CF-specific Acta2 knockout and CF lineage tracing ( Tcf21 MerCreMer ;Acta2 fl/fl ;R26 GFP ) and a WT control mouse line ( Tcf21 MerCreMer ;R26 GFP ), we identified that CF-specific Acta2 knockout results in significantly exacerbated post-MI cardiac function, increased cardiac rupture incidence, and enhanced fibrosis. More lineage tracing experiments and transcriptome analyses are being conducted to better decipher the impact of Acta2 knockout on CF physiology and post-MI tissue healing.

Toward Regeneration of the Heart: Bioengineering Strategies for Immunomodulation.

Myocardial Infarction (MI) is the most common cardiovascular disease. An average-sized MI causes the loss of up to 1 billion cardiomyocytes and the adult heart lacks the capacity to replace them. Although post-MI treatment has dramatically improved survival rates over the last few decades, more than 20% of patients affected by MI will subsequently develop heart failure (HF), an incurable condition where the contracting myocardium is transformed into an akinetic, fibrotic scar, unable to meet the body's need for blood supply. Excessive inflammation and persistent immune auto-reactivity have been suggested to contribute to post-MI tissue damage and exacerbate HF development. Two newly emerging fields of biomedical research, immunomodulatory therapies and cardiac bioengineering, provide potential options to target the causative mechanisms underlying HF development. Combining these two fields to develop biomaterials for delivery of immunomodulatory bioactive molecules holds great promise for HF therapy. Specifically, minimally invasive delivery of injectable hydrogels, loaded with bioactive factors with angiogenic, proliferative, anti-apoptotic and immunomodulatory functions, is a promising route for influencing the cascade of immune events post-MI, preventing adverse left ventricular remodeling, and offering protection from early inflammation to fibrosis. Here we provide an updated overview on the main injectable hydrogel systems and bioactive factors that have been tested in animal models with promising results and discuss the challenges to be addressed for accelerating the development of these novel therapeutic strategies.

Abstract 17169: Computationally-Engineered Analog of Stromal Cell-Derived Factor 1α Preserves the Mechanical Properties of Infarcted Myocardium Under Planar Biaxial Tension

Introduction: Adverse remodeling of the left ventricle (LV) after myocardial infarction (MI) results in abnormal tissue biomechanics and impaired cardiac function, ultimately leading to heart failure. We hypothesized that intramyocardial delivery of engineered stromal cell-derived factor 1α analog (ESA), our previously-developed supra-efficient pro-angiogenic chemokine, preserves biaxial LV mechanical properties after MI. Methods: Male Wistar rats (n=46) underwent sham surgery (n=15) or permanent left anterior descending coronary artery ligation (n=31). Rats sustaining MI were randomized for intramyocardial injections of either saline (100 μL, n=15) or ESA (6 μg/kg, n=16), delivered at standardized peri-infarct sites. After 4 weeks, echocardiography was performed, and the hearts were explanted. Biaxial tensile testing of the anterior LV wall was performed using a strain-controlled biaxial load frame (Fig. 1A), producing up to physiologic circumferential and longitudinal strains (ε=20%, each). The modulus was determined along each axis, and maximum shear stress was calculated as a composite metric of the tissue’s response to physiologic strains. Data are expressed as mean±SEM. Results: At 4 weeks post-MI, ESA-treated hearts had smaller end-diastolic LV internal dimension (6.89±0.27 cm vs 7.69±0.22 cm, p=0.03) and improved ejection fraction (63.7±2.9% vs 49.4±4.4%, p=0.01) compared to saline-injected controls. Hearts treated with ESA exhibited lower moduli than saline controls in both the circumferential (269.4±33.2 kPa vs 431.5±55.6 kPa, p=0.02) and longitudinal axes (182.4±25.0 kPa vs 332.3±51.2 kPa, p=0.02, Fig. 1B). The maximum shear stress for ESA-treated hearts was significantly reduced compared to that for saline controls (5.8±0.8 kPa vs 9.0±1.2 kPa, p=0.03) and was similar to that for sham controls (Fig. 1C). Conclusion: Intramyocardial ESA injection mitigates post-MI tissue stiffening and preserves biaxial LV mechanical properties.

Impact of proteasome inhibitor MG-132 on expression of NF-κB, IL-1β and histological remodeling after myocardial infarction.

The aim of the present study was to investigate the impact of carbobenzoxy-Leu-Leu-leucinal (MG-132) on myocardial remodeling in rats with myocardial infarction (MI) and investigate the possible underlying mechanisms. The rat model of MI was established, followed by administration of MG-132 (MG group), pyrrolidine dithiocarbamic acid (PDTC group) or normal saline (MI group) for 28 days. The expression of nuclear factor-κB (NF-κB) p65, interleukin 1β (IL-1β) and matrix metalloproteinase 2 (MMP-2), as well as the total volume of collagen and the ratio of type I/III collagen were then detected. Total collagen, including type I and III collagen, and the ratio of type I/III collagen were significantly increased in MI rats compared with those in the sham group (P<0.01), while it was significantly decreased in the PDTC and MG groups compared with that in the MI group (P<0.01). A similar trend was identified for the expression of NF-κB, IL-1β and MMP-2, which was significantly increased in the MI group compared with that in the sham group (P<0.01), while it was significantly decreased in the MG and PDTC groups compared with that in the MI group (P<0.01). In conclusion, MG-132 was demonstrated to improve post-MI tissue remodeling, and the mechanism may be associated with the inhibition of NF-κB activation and the downregulation of inflammatory cytokines, such as IL-1β.

Alterations in the Eicosanoid Profile and Mitochondrial Injury in Human Ventricular Tissue Following Myocardial Infarction

Study objectiveMyocardial infarction (MI) accounts for a significant proportion of death and disability in the ageing population. n‐6 PUFAs linoleic acid and arachidonic acid have come under investigation for their role in mediating a cardiac response following ischemic injury. CYP450 metabolism of these fatty acids results in formation of numerous metabolites, called eicosanoids, which can be further metabolised by the enzyme soluble epoxide hydrolase (sEH). These lipid mediators exhibit a wide range of cellular effects including alterations to mitochondria structure and function which may impact cardiac performance. This study investigated alterations to CYP‐derived eicosanoids and mitochondria in heart tissue obtained from individuals who experienced a previous MI.MethodsSamples were obtained from male and female individuals included in the Human Explanted Heart Program and correlated to non‐failing control hearts (NFC) collected from unused transplant donors through the Human Organ Procurement and Exchange Program at the University of Alberta. Ventricular tissues were harvested from patients who had previously experienced a LAD infarct (≥2yrs), as determined by echocardiography, ECG and coronary angiograms. Protein expression was determined by immunoblotting techniques. Mitochondrial enzymatic activities were assessed by spectrophotometric methods while mitochondrial ultrastructure and cristae density were assessed by electron microscopy. A metabolic profile in ventricular tissue was obtained by LCMS.ResultsMarked differences in both LA and AA metabolic profiles were revealed in post‐MI tissues. There were significant increases in cardiotoxic metabolites correlating with decreased cardiac function and injury. Interestingly, there was a significant increase in both sEH activity and expression in post‐MI tissues compared to NFC. Expression of mitochondrial proteins remained unchanged, however enzymatic function declined in post‐MI tissues compared to NFC correlating with marked disruption in mitochondrial ultrastructure.ConclusionsThese data provide the first evidence demonstrating a marked shift in eicosanoid metabolism in post‐MI hearts that correlates with mitochondrial structural disruption and overall decreased mitochondrial function.Support or Funding InformationSupport for this project was provided by the Canadian Institutes of Health Research.KL Jamieson is supported by funding from Alberta Innovates‐Health Solutions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential effects of Akt1 signaling on short- versus long-term consequences of myocardial infarction and reperfusion injury

A specific role for Akt1 in events following myocardial infarction (MI) and ischemia/reperfusion (I/R) injury is not known. We aimed to determine whether Akt1 deletion in in vivo mouse models of MI and after ischemia I/R injury would alter myocyte survival, cardiac function, and fibrosis. Akt1+/+ and Akt1−/− mice were subjected to MI and I/R, followed by assessment of downstream signaling events and functional consequences. Although no difference in infarct size following short-term MI was observed between Akt1+/+ and Akt1−/− mice, I/R caused substantially more cardiomyocyte apoptosis and tissue damage in Akt1−/− mice compared with Akt1+/+. Importantly, these effects were reversed upon pretreatment with GSK-3 inhibitor SB415286. Counterintuitively, Akt1−/− hearts exhibited improved cardiac function following long-term MI compared with Akt1+/+ and were associated with reduced fibrosis in the left ventricle (LV). Our results demonstrate that Akt1-mediated inhibition of GSK-3 activity is critical for cardioprotection following I/R. However, in the long term, Akt1 contributes to fibrosis in post-MI hearts and might exacerbate cardiac dysfunction showing dichotomous role for Akt1 in cardiac remodeling after MI. Our data suggest that better understanding of the Akt1/GSK-3 pathway may provide insights for better therapeutic strategies in post-MI tissues.

Targeted delivery of vascular endothelial growth factor improves stem cell therapy in a rat myocardial infarction model

Rebuilding of infarcted myocardium by mesenchymal stem cells (MSCs) has not been successful because of poor cell survival due in part to insufficient blood supply after myocardial infarction (MI). We hypothesize that targeted delivery of vascular endothelial growth factor (VEGF) to MI can help regenerate vasculature in support of MSC therapy in a rat model of MI. VEGF-encapsulated immunoliposomes targeting overexpressed P-selectin in MI tissue were infused by tail vein immediately after MI. One week later, MSCs were injected intramyocardially. The cardiac function loss was moderated slightly by targeted delivery of VEGF or MSC treatment. Targeted VEGF+MSC combination treatment showed highest attenuation in cardiac function loss. The combination treatment also increased blood vessel density (80%) and decreased collagen content in post-MI tissue (33%). Engraftment of MSCs in the combination treatment group was significantly increased and the engrafted cells contributed to the restoration of blood vessels. From the Clinical EditorVEGF immunoliposomes targeting myocardial infarction tissue resulted in significantly higher attenuation of cardiac function loss when used in combination with mesenchymal stem cells. MSCs were previously found to have poor ability to restore cardiac tissue, likely as a result of poor blood supply in the affected areas. This new method counterbalances that weakness by the known effects of VEGF, as demonstrated in a rat model.

Mesenchymal Stem Cells Overexpressing CXCR4 Attenuate Remodeling of Postmyocardial Infarction by Releasing Matrix Metalloproteinase-9

Myocardial infarction (MI) results in loss of myofibers in the ischemic zone of the heart, followed by scar formation. These factors increase barriers to mobilization of mesenchymal stem cells (MSC), thereby impeding their effectiveness in cardiac repair. This study examined MSC overexpressing CXCR4 (MSC(CX4)) to determine penetration into infarcted myocardium by releasing collagen degrading enzyme, matrix metalloproteinase-9 (MMP-9). In vitro, mouse MSC were utilized, including MSC using adenoviral transduction, to express CXCR4/green fluorescent protein (GFP) (MSC(CX4)), Null/GFP (MSC(Null)), MSC treated with siRNA targeting CXCR4 (MSC(siR)), MSC treated with control siRNA(MSC(Con-siR)), MSC(CX4) treated with siRNA targeting MMP-9 (MSC(CX4-siRMP9)) and MMP-14 (MSC(CX4-siRMP14)), MSC derived from MMP-9 knockout mouse with adenoviral transduction for GFP (MSC(MP9-)), or MSC(MP9-) plus overexpressing CXCR4 (MSC(MP9-CX4)). The ability to cross the basement membrane was evaluated in all MSC using a trans-collagen gel invasion assay. The CXCR4 and MMP expression were analyzed by Western blot. In vivo, MSC with various treatments were infused into mice via tail vein injections 7 days after MI. Echocardiography was performed before harvesting hearts for analysis at 4 weeks after MSC injection. Both in vitro and in vivo studies demonstrated upregulation of MMP-9 induced by MSC(CX4), promoting increased GFP(+) cell migration into the infarcted area in comparison to control group. This enhanced response was associated with reduced left ventricular (LV) fibrosis, increased LV free wall thickness, angiogenesis, and improved LV function. Under hypoxic conditions, MMP-9 is upregulated in MSC(CX4), thus facilitating cross of the basement membrane, resulting in an improved remodeling of post-MI tissue.

A new in vivo/in vitro model for assessing the capacity of human derived oral mucosa stem cells to colonize the infarcted myocardium

Ischemic heart failure (IHF) is a leading cause for mortality and morbidity worldwide. Stem cell (SC) therapy is a promising strategy for IHF treatment. A major prerequisite for successful SC therapy is homogenous colonization of the infarcted myocardium. This process implies SC migration from the transplantation site. The purposes of the present study were to develop a new model system capable of quantifying SC migration within the infarcted myocardium and to assess the effect of post myocardial infarction (MI) remodeling on the migration of a newly identified neural crest SC population derived from the oral mucosa (hOMSC). MI was induced in rats. Myocardial scar tissue was collected at 0,1,3,7,14, and 28 days post-MI. DiI-labeledhOMSC were injected in the center of cylindrical scar tissue specimens harvested from each post-MI time point and transferred to organ culture. Changes in host and hOMSC cell densities were quantified in the center and periphery of specimens after 0, 3, 7, 14, and 28 days of culture by morphometric fluorescence microscopy. The results indicate a decrease in hOMSC density in the central zone and an increase in density in the peripheral zones indicating hOMSC migration from the central transplantation site to the rest of the infarcted myocardium. The average hOMSC density increased overtime due to cell proliferation. The level of hOMSC migration and proliferation was significantly affected by post-MI remodeling phase being higher in post-MI tissue that still comprised cardiomyofibers than in granulation and fibrotic tissues. Preliminary hOMSC transplantation in nude rat supports these findings. The present study shows that the new in vivo/in vitro model system is sufficiently sensitive to detect and quantify changes in the migratory capacity of stem cells within the infarcted myocardium and suggest that cell therapy applied in the early stages (up to 3 days) of post-MI remodeling facilitates the process of tissue colonization.

Targeting VEGF‐encapsulated immunoliposomes to MI heart improves vascularity and cardiac function

Recent attempts at rebuilding the myocardium using stem cells have yielded disappointing results. The lack of a supporting vasculature may, in part, explain these disappointing findings. However, concerns over possible side effects have hampered attempts at revascularizing the infarcted myocardium using systemic delivery of proangiogenic compounds. In this study, we develop the technology to enhance the morphology and function of postinfarct neovasculature. Previously, we have shown that the up-regulated expression of endothelial cell adhesion molecules in the myocardial infarction (MI) region provides a potential avenue for selectively targeting drugs to infarcted tissue. After treatment with anti-P-selectin-conjugated liposomes containing vascular endothelial growth factor (VEGF), changes in cardiac function and vasculature post-MI were quantified in a rat MI model. Targeted delivery of VEGF to post-MI tissue resulted in significant increase in fractional shortening and improved systolic function. These functional improvements were accompanied by a 21% increase in the number of anatomical vessels and a 74% increase in the number of perfused vessels in the MI region of treated animals. No significant improvements in cardiac function were observed in untreated, systemic VEGF-treated, nontargeted liposome-treated, or blank immunoliposome-treated animals. Targeted delivery of low doses of proangiogenic compounds to post-MI tissue results in significant improvements in cardiac function and vascular structure.

Residual post MI tissue viability contributes to determining exercise-induced ventricular arrhythmias

Residual post MI tissue viability contributes to determining exercise-induced ventricular arrhythmias