Myocardial Extracellular Matrix Research Articles

In Situ Structural Characterization of Cardiomyocyte Microenvironment by Multimodal STED Microscopy

Within the myocardium, cardiomyocytes reside in a complex and dynamic extracellular matrix (ECM) consisting of a basement membrane (BM) and interstitial matrix. The interactions between cardiomyocytes and the myocardial ECM play a critical role in maintaining cardiac geometry and function throughout cardiac development and in adult hearts. Understanding how the structural changes of the myocardial ECM affect cardiomyocyte function requires knowledge of pericellular structures. These structures are of a size beyond the resolution of conventional optical microscopy. Here, we demonstrated multi-scale and multi-aspect characterization of the cardiomyocyte microenvironment in myocardial tissue sections using multimodal stimulated emission depletion (STED) microscopy. Second harmonic generation and autofluorescence facilitated multiplexed imaging, enabling the interpretation of protein distribution in 3D. STED imaging modality revealed BM structures of cardiomyocytes and myocardial capillaries at the subdiffractional level. Moreover, meaningful measurements retrieved from acquired images, such as sarcomere length and capillary density, enabled quantitative assessment of myocardial structures.

Hypertension-induced cardiac pro-arrhythmic disorders of connexin-43 and extracellular matrix are attenuated in hairless versus wild-type of spontaneously hypertensive rats

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Development Agency under the contract no. 21-0410 VEGA grants no. 2/0002/20, 2/0006/23 Background Mechanical dysfunction and occurrence of malignant ventricular fibrillation (VF) are hallmarks of hypertensive heart disease and major health issue. Spontaneously hypertensive rats (SHR) that imitates human primary hypertension are prone to develop VF, mainly due to myocardial remodelling associated with disorders of electrical coupling protein connexin-43 (Cx43) and extracellular matrix (ECM) resulting in electrical instability. However, it is not known whether postnatal acclimation of hairless strain of SHR to surrounding temperature may induce cardio-protection. Therefore, we aimed to explore Cx43 and ECM alterations as well as susceptibility to inducible VF in hairless versus wild type SHR. Design and Methods Six-month-old wild type SHR males and females and hairless SHR housed at standard 22°C were examined. Biometric parameters, including electro- and echocardiography were registered. Left ventricular tissue was used for immunolabeling of Cx43 to reveal its topology and for western blotting to determine protein levels of Cx43 and its function modulating protein kinases, PKCε, PKCδ and PKCα as well as for ECM markers, TGF-β, SMAD2/3, MMP-2, collagen-1 and its histological deposition. Susceptibility of the heart to electrically-inducible VF was tested using isolated perfused heart technique. Key results Comparing to wild type males, hairless SHR exhibited less pronounced prolongation of QT and QRS interval as well as an increase of ejection fraction. Myocardial Cx43 and its variant phosphorylated at serine-368, which preserve electrical coupling, were increased and pro-arrhythmic cardiomyocyte Cx43 topology was attenuated in males and females hairless vs wild type SHR. Moreover, the level of β-catenin, which ensures mechano-electrical coupling, was also increased in hairless SHR. On the other hand, collagen-1 and hydroxyproline were lower and the TGF-β1 and SMAD2/3 proteins were suppressed in males hairless vs wild type SHR. Myocardial ECM remodelling was less pronounced in females regardless rat strain. Hairless SHR males were less susceptible to inducible VF compared to wild type SHR, while females were less prone to develop VF regardless the strain. In conclusion, our findings imply that postnatal acclimation of hairless SHR is accompanied by attenuation of hypertension-induced adverse down-regulation of Cx43 and up-regulation of extracellular matrix proteins. It may contribute, at least in part, to the protection from malignant cardiac arrhythmias and mechanical dysfunction.

Evaluation of Different Decellularization Protocols for Obtaining and Characterizing Canine Cardiac Extracellular Matrix.

Cardiovascular diseases are considered the leading cause of mortality globally; even with low mortality in dogs, such diseases are described in the same way in companion animals and humans. This study aimed to devise an effective decellularization protocol for the canine myocardium through the association of physical, chemical, and enzymatic methods, assessing resultant alterations in the myocardial extracellular matrix to obtain a suitable scaffold. Two canine hearts were collected; the samples were sectioned into ±1 cm2 fragments, washed in distilled water and 1× PBS solution, and followed by treatment under four distinct decellularization protocols. Sodium Dodecyl Sulfate (SDS) 1% 7 days + Triton X-100 1% for 48 h (Protocol I); Sodium Dodecyl Sulfate (SDS) 1% 5 days + Triton X-100 1% for 48 h (Protocol II); Trypsin 0.05% for 1 h at 36 °C + freezing -80 °C overnight + Sodium Dodecyl Sulfate (SDS) 1% for 3 days, Triton-X-100 for 48 h hours (Protocol III); 0.05% trypsin for 1 h at 36 °C + freezing at -80 °C overnight + 1% Sodium Dodecyl Sulfate (SDS) for 2 days + 1% Triton-X-100 for 24 h (Protocol IV). After analysis, Protocols I and II showed the removal of cellular content and preservation of extracellular matrix (ECM) contents, unlike Protocols III and IV, which retracted the ECM and removed essential elements of the matrix. In theory, although Protocols I and II have similar results, Protocol II stands out for the preservation of the architecture and components of the extracellular matrix, along with reduced exposure time to reagents, making it the recommended protocol for the development of a canine myocardial scaffold.

Production of Cardiac Extracellular Matrix from Adult Human Fibroblasts for Culture Dish Coating.

The myocardium is composed of cardiomyocytes and an even greater number of fibroblasts, the latter being responsible for extracellular matrix production. From the early stages of heart development throughout the lifetime, in both normal and pathological conditions, the composition of the extracellular matrix changes and influences myocardium structure and function. The purpose of the method described here is to obtain the substrate for the culture of cardiac cells in vitro (termed cardiac ECM), mimicking the myocardial extracellular matrix in vivo. To this end, fibroblasts isolated from the adult human heart were cultured to confluence on gelatin-coated dishes to produce the myocardium-specific extracellular matrix. The subsequent removal of cardiac fibroblasts, while preserving the deposited cardiac ECM, produced the substrate for studying the influence of the myocardium-specific extracellular matrix on other cells. Importantly, the composition of the fibroblast-derived coating of the culture dish changes according to the in vivo activity of the fibroblasts isolated from the heart, allowing subsequent studies of cell-matrix interactions in different normal and pathological conditions.

BIOMARKERS OF CARDIOVASCULAR COMPLICATIONS ASSOCIATED WITH COVID-19 DISEASE

The coronavirus disease (COVID-19), which caused a global pandemic in the world, is now becoming an endemic, seasonal disease. Therefore, the search for new biomarkers to predict the course of the disease and its outcomes remains extremely important. It has been found that COVID-19 affects not only the respiratory system but also other organs and systems of the body. In particular, the target of the SARS-CoV-2 virus is the cardiovascular system. Because the severity of COVID-19 disease is highly variable, laboratory biomarkers can provide important prognostic information already at the emergency stage, especially in patients with atypical manifestations. It has been demonstrated that viral infections, and in particular COVID-19, can cause changes in the host's lipid profile. Changes in the processes of intracellular cholesterol biosynthesis and its transport in the bloodstream of patients with COVID-19 have been reported. Thus, in patients suffering from COVID-19, significantly reduced levels of total іукгь cholesterol (C), HDL, and LDL cholesterol, and an increased ratio of triglycerides/C have been recorded. Patients with COVID-19 are characterized by an increased content of residual (remnant) cholesterol, which is defined as cholesterol present in residual lipoproteins rich in triglycerides and, as shown by the results of genetic, observational, and clinical studies, is associated with the development of atherosclerosis and overall mortality. Several peptide cardiac biomarkers have become other predictors of cardiovascular prognosis in patients with COVID-19: highly sensitive cardiac troponin – successfully used in clinical practice as a marker of myocyte damage for diagnosis and prognostic assessment of acute coronary syndrome; N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a biomarker of myocardial stretch; peptide ST2 – a marker of remodeling of myocardial extracellular matrix, which prevents fibrosis, cardiac hypertrophy and apoptosis; copeptin is a biomarker of neurohumoral activation. The review provides a concise analysis of currently known cardiac biomarkers in COVID-19 and their prognostic significance.

Fabrication and In Vitro Characterization of Polycaprolactone/Graphene Oxide/Collagen Nanofibers for Myocardial Repair

AbstractThis study is focused on fabricating tissue‐engineered electrospun nanofibers that contain polycaprolactone (PCL), graphene oxide (GO), and collagen (COL) to get an alternative treatment for cardiac injuries. GO (1.5 wt%) is used to support the contraction‐elongation of cardiomyocytes by improving electrical stimulation. The COL (1, 3, and 5 wt%) is the main component of the myocardial extracellular matrix have led to their frequent use in cardiac tissue engineering (CTE). The scanning electron microscope (SEM) images show the homogeneous and bead‐free morphologies of the nanofibers. Adding a high amount (3% and 5%) of COL decreases the tensile strength value of 17% PCL/1.5% GO nanofiber. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) assay demonstrates that the COL addition increases cell viability compared to that in 17% PCL/1.5% GO nanofibers on the third day. The response of the nanofibers to alternating current (AC) signal is studied between the frequencies 40 and 105 Hz. The direct current (DC) conductivity values of the films are determined to be between 1.10−10 and 6.10−10 S m−1 at 25 °C. The AC conductivity values show frequency‐dependent behavior. Among the PCL/GO‐based electrospun nanofibers, 17% PCL/1.5% GO/5% COL nanofiber shows greater DC and AC conductivity than 17% PCL/1.5% GO nanofiber.

Abstract 17724: Myocardial Extracellular Matrix Proteins Involved in the Overloaded and Unloaded Heart After Myocardial Infarction

Introduction: The cardiac extracellular matrix (ECM) is a dynamic scaffold that transmits and responds to forces that act on the heart during the cardiac cycle. While acute left ventricle (LV) unloading by catheter-based LV assist device (pLVAD) is now a therapeutic option, its impact on ECM is unknown. Hypothesis: The composition of LV ECM proteins changes in response to wall stress. Methods: Ten Yorkshire pigs underwent the protocol in figure 1. Non-ischemic remote myocardium was processed for ECM specific proteomics. Volcano plots were constructed to determine proteins of interest and differential expression was considered as (-log10(p)>1.3 and |log2(fold-change)| >0.5). Gene ontology (GO) analysis was performed to identify associated molecular processes and biological functions. A group of age-matched pigs (n=6) that did not undergo MI induction served as reference. Immunofluorescence microscopy was performed to validate differential expression. Results: Of the 986 proteins analyzed, 39 were significantly differentially expressed between overloaded and unloaded hearts. GO analysis by molecular function revealed that differences tending towards RNA binding and lipid binding proteins. Analysis by biological process showed that cell differentiation, vesicle transport, and programmed cell death were the processes most favored. Staining for three of the identified differentially expressed proteins (figure 2) had results congruent with proteomic analysis. Conclusions: There were significant differences in the protein composition of the cardiac ECM between acutely unloaded and overloaded myocardium, suggesting that it actively responds to altered LV load.

Abstract 18671: Cardiac Magnetic Resonance Radiomics Characterization of Myocardial Extracellular Matrix in Dilated Cardiomyopathy

Introduction: Current CMR sequences cannot discriminate between different myocardial extracellular space (ECS), including collagen, non-collagen, and inflammation. We sought to investigate if CMR radiomics analysis can distinguish between non-collagen and inflammation from collagen in dilated cardiomyopathy (DCM). Methods: 132 DCM patients were scheduled for an invasive septal biopsy and underwent CMR at 3T incorporating native and post-contrast T1 mapping and late gadolinium enhancement (LGE). The radiomic features were computed from the mid-septal myocardium, near the biopsy region, on native T1, extracellular volume (ECV) map, and LGE images. Principal component analysis was used to reduce the number of calculated radiomic features to five. These features were then used in a regression model to discriminate between non-collagen and collagen ECS. Biopsy samples were used to quantify ECS, myocardial fibrosis, and inflammation. Results: Four histopathological phenotypes were identified as normal (G1, n=20), non-collagenous ECS expansion (G2, n=49), collagenous ECS expansion (G3, n=42), and severe fibrosis (G4, n=21). G2 was associated with the highest risk of myocardial inflammation (65%). While native T1 and ECV provided high diagnostic performance in differentiating G4 (AUC= 0.90 and 0.90), their performance in differentiating between G2 and G3 decreased (AUC= 0.59 and 0.55, respectively). Integration of ECV radiomics provided better discrimination and reclassification between G2 and G3 (AUC= 0.79, net reclassification index 0.83; 95% CI 0.45-1.22, p<0.001). A similar trend was observed with the addition of native T1 radiomics and LGE radiomics. Conclusions: The radiomic features extracted from native T1, ECV, and LGE provided incremental information that may improve our capability to discriminate non-collagenous ECS from collagen and could be useful for detecting subtle chronic inflammation in DCM patients.

Remodeling in early myocardial infarction: alteration of extracellular matrix; Collagen-1, Collagen-3, α-SMA, and α-Actinin in Porcine heart model

Link of Video Abstract: https://youtu.be/lThOW40upEc Background: Myocardial infarction (MI) leads to remodeling due to altered cardiac structure and function. The study of MI histopathology in humans or large animals mimicking the human heart is limited. Using the porcine heart model, our study investigated the composition of myocardial extracellular matrix (ECM) in collagen-1, collagen-3, α-smooth muscle actin (α-SMA), and α-actinin before and after myocardial infarction. Methods: This study used two groups of domestic pigs: the Infarct group (n=4) and the Sham group (n=4). MI was induced by permanent ligation of the proximal branch of the posterior left ventricular artery. Cardiac enzymes, electrocardiography, and echocardiography data were collected before and after ligation. Cardiac tissue was harvested from the infarcted area after 60 minutes of ligation and stained with hematoxylin-eosin and Movat's Pentachrome. Collagen-1, collagen-3, and α-SMA were identified with immunohistochemical labeling, and the labeled area was measured using ImageJ. Meanwhile, α-actinin was visualized using immunofluorescence. Results: Expression of collagen-1, collagen-3, and α-SMA in the infarct group were significantly decreased after 60 minutes of infarction compared with those in the sham group (p<0.01). The α-actinin was fragmented and diminished in the infarct group. Conclusion: Myocardial remodeling was detected 60 minutes after infarction with mild alteration in myocardial histoarchitecture and significant deterioration of ECM composition of collagen-1, collagen-3, α-SMA, and fragmented α-actinin fibers in the porcine heart model.

Management of Obesity-Related Inflammatory and Cardiovascular Diseases by Medicinal Plants: From Traditional Uses to Therapeutic Targets.

Inflammation is a crucial factor in the development and progression of cardiovascular diseases (CVD). Cardiac remodeling in the presence of persistent inflammation leads to myocardial fibrosis and extracellular matrix changes, which reduce cardiac function, induce arrhythmias, and finally, cause heart failure. The majority of current CVD treatment plans concentrate on reducing risk factors such as hyperlipidemia, type 2 diabetes, and hypertension. One such strategy could be inflammation reduction. Numerous in vitro, animal, and clinical studies indicate that obesity is associated with low-grade inflammation. Recent studies have demonstrated the potential of medicinal plants and phytochemicals to cure and prevent obesity and inflammation. In comparison to conventional therapies, the synergistic effects of several phytochemicals boost their bioavailability and impact numerous cellular and molecular targets. Focusing on appetite, pancreatic lipase activity, thermogenesis, lipid metabolism, lipolysis and adipogenesis, apoptosis in adipocytes, and adipocyte life cycle by medicinal plants and phytochemicals represent an important goal in the development of new anti-obesity drugs. We conducted an extensive review of the literature and electronic databases, including Google Scholar, PubMed, Science Direct, and MedlinePlus, for collecting data on the therapeutic effects of medicinal plants/phytochemicals in curing obesity and its related inflammation and CVD diseases, including cellular and molecular mechanisms, cytokines, signal transduction cascades, and clinical trials.

Natriuretic Peptide Levels and Stages of Left Ventricular Dysfunction in Heart Failure with Preserved Ejection Fraction.

In heart failure with preserved ejection fraction (HFpEF), natriuretic peptide (NP) levels are frequently lower. In several trials, the outcome differed between patients with low and high NP levels. This suggests that NP could be used to identify distinct stages of left ventricular (LV) remodeling and myocardial tissue composition. This study investigated cardiac remodeling/dysfunction and myocardial tissue characteristics assessed by echocardiography and cardiac magnetic resonance (CMR) in HFpEF patients in relation to NP levels. Clinical and echocardiographic data of 152 HFpEF patients were derived from outpatient visits. A total of 71 HFpEF patients underwent CMR-derived T1-mapping. Multivariable regression analyses were performed to examine the association of NT-proBNP categories (</> median) and NT-proBNP as continuous variable with echocardiography and CMR-derived T1-mapping. Mean age was 71 ± 9, 93% of patients were women and median NT-proBNP was 195 pg/mL, with 35% of patients below the diagnostic cut-off value (<125 pg/mL). Patients with high NT-proBNP had comparable LV systolic function and LV relaxation but significantly worse LV stiffness and left atrial function compared with patients with low NT-proBNP. Higher NT-proBNP was significantly associated with higher LV stiffness and extracellular volume fraction (ECV) (β = 1.82, 95% CI: 0.19;3.44, p = 0.029). Higher NT-proBNP levels identify HFpEF patients with worse LV stiffness because of more severe myocardial extracellular matrix remodeling, representing an advanced stage of HFpEF.

Involvement and possible role of transglutaminases 1 and 2 in mediating fibrotic signalling, collagen cross-linking and cell proliferation in neonatal rat ventricular fibroblasts.

Transglutaminase (TG) isoforms control diverse normal and pathophysiologic processes through their capacity to cross-link extracellular matrix (ECM) proteins. Their functional and signalling roles in cardiac fibrosis remain poorly understood, despite some evidence of TG2 involvement in abnormal ECM remodelling in heart diseases. In this study, we investigated the role of TG1 and TG2 in mediating fibrotic signalling, collagen cross-linking, and cell proliferation in healthy fibroblasts by siRNA-mediated knockdown. siRNA for TG1, TG2 or negative control was transfected into cultured neonatal rat ventricular fibroblasts and cardiomyocytes. mRNA expression of TGs and profibrotic, proliferation and apoptotic markers was assessed by qPCR. Cell proliferation and soluble and insoluble collagen were determined by ELISA and LC-MS/MS, respectively. TG1 and TG2 were both expressed in neonatal rat cardiomyocytes and fibroblasts before transfection. Other TGs were not detected before and after transfection. TG2 was predominantly expressed and more effectively silenced than TG1. Knocking down TG1 or TG2 significantly modified profibrotic markers mRNA expression in fibroblasts, decreasing connective tissue growth factor (CTGF) and increasing transforming growth factor-β1 compared to the negative siRNA control. Reduced expression of collagen 3A1 was found upon TG1 knockdown, while TG2 knockdown raised α-smooth muscle actin expression. TG2 knockdown further increased fibroblast proliferation and the expression of proliferation marker cyclin D1. Lower insoluble collagen content and collagen cross-linking were evidenced upon silencing TG1 or TG2. Transcript levels of collagen 1A1, fibronectin 1, matrix metalloproteinase-2, cyclin E2, and BCL-2-associated X protein/B-cell lymphoma 2 ratio were strongly correlated with TG1 mRNA expression, whereas TG2 expression correlated strongly with CTGF mRNA abundance. These findings support a functional and signalling role for TG1 and TG2 from fibroblasts in regulating key processes underlying myocardial ECM homeostasis and dysregulation, suggesting that these isoforms could be potential and promising targets for the development of cardiac fibrosis therapies.

Hypothalamic Oxytocin Neuron Activation Attenuates Intermittent Hypoxia-Induced Hypertension and Cardiac Dysfunction in an Animal Model of Sleep Apnea.

Obstructive sleep apnea is a prevalent and poorly treated cardiovascular disease that leads to hypertension and autonomic imbalance. Recent studies that restore cardiac parasympathetic tone using selective activation of hypothalamic oxytocin neurons have shown beneficial cardiovascular outcomes in animal models of cardiovascular disease. This study aimed to determine if chemogenetic activation of hypothalamic oxytocin neurons in animals with existing obstructive sleep apnea-induced hypertension would reverse or blunt the progression of autonomic and cardiovascular dysfunction. Two groups of rats were exposed to chronic intermittent hypoxia (CIH), a model of obstructive sleep apnea, for 4 weeks to induce hypertension. During an additional 4 weeks of exposure to CIH, 1 group was treated with selective activation of hypothalamic oxytocin neurons while the other group was untreated. Hypertensive animals exposed to CIH and treated with daily hypothalamic oxytocin neuron activation had lower blood pressure, faster heart rate recovery times after exercise, and improved indices of cardiac function compared with untreated hypertensive animals. Microarray analysis suggested that, compared with treated animals, untreated animals had gene expression profiles associated with cellular stress response activation, hypoxia-inducible factor stabilization, and myocardial extracellular matrix remodeling and fibrosis. In animals already presenting with CIH-induced hypertension, chronic activation of hypothalamic oxytocin neurons blunted the progression of hypertension and conferred cardioprotection after an additional 4 weeks of CIH exposure. These results have significant clinical translation for the treatment of cardiovascular disease in patients with obstructive sleep apnea.

Morphological and immunological markers of repeated heart remodeling after surgical reconstruction of the left ventricle in ischemic cardiomyopathy.

Effective treatment of patients with ischemic cardiomyopathy (ICM) is one of the most challenging issues in modern cardiac surgery. The aim of this study was to assess the state of cardiomyocytes and myocardial extracellular matrix, as well as to identify informative markers of an unfavorable prognosis for surgical treatment of ICM. We retrospectively reviewed patients who underwent surgical treatment of ICM between 2011 and 2018 at a single center. Patients were divided into groups depending on the presence of repeated left ventricle (LV) remodeling in one-year follow-up after surgical reconstruction of the LV in ICM patients. A total of 45 patients with ICM were reviewed. The mean age of the patients was 57.9 ± 7.8 years. According to the results of the study, the area of cardiomyocyte nuclei differed statistically significantly among the regions with varying degrees of impaired local contractility (p = 0.042). According to the results of the pairwise comparison in dyskinetic areas of the myocardium, the area of cardiomyocyte nuclei was higher than in normokinetic areas (p = 0.042). A moderate positive correlation was found between the LV ejection fraction measured in one-year follow-up period after surgery and the number of CD163-positive cells (p = 0.012). In the myocardium of patients with LV reverse remodeling in the long-term postoperative period, perivascular fibrosis occurs more frequently than in patients with progressive LV remodeling. The number of M2-anti-inflammatory macrophages prevails in the myocardium of the patients with reverse remodeling compared with patients with progressive remodeling.

Inhibition of long noncoding RNA Gm41724 alleviates pressure overload-induced cardiac fibrosis by regulating lamina-associated polypeptide 2α

Cardiac fibrosis is a pathological process underlying myocardial remodeling and is characterized by excessive deposition of the myocardial extracellular matrix. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of various biological processes. In this study, we investigated the role of a novel lncRNA, Gm41724, in cardiac fibrosis induced by pressure overload. High-throughput whole transcriptome sequencing analysis was performed to detect differentially expressed lncRNAs in cardiac fibroblasts (CFs) with or without TGF-β1 treatment. Differential expression analysis and gene set enrichment analysis identified Gm41724 as a potential molecule targeting fibrosis. Gm41724 positively regulated the activation of CFs induced by TGF-β1 and pressure overload. Knocking down Gm41724 could inhibit the differentiation of CFs into myofibroblasts and alleviate cardiac fibrosis induced by pressure overload. Mechanistically, comprehensive identification of RNA-binding proteins by mass spectrometry (CHIRP-MS) and RNA immunoprecipitation (RIP) assay combined with other methods of molecular biological revealed the important role of Gm41724 binding to lamina-associated polypeptide 2α (lap2α) for the activation of CFs. Further mechanistic studies indicated that the regulator of G protein signaling 4 (Rgs4), as the downstream effector of Gm41724/lap2α, regulated CFs activation. Our results implicated the involvement of Gm41724 in cardiac fibrosis induced by pressure overload and it is expected to be a promising target for anti-fibrotic therapy.

From Macro to Micro: Comparison of Imaging Techniques to Detect Vascular Network Formation in Left Ventricle Decellularized Extracellular Matrix Hydrogels.

Background: Angiogenesis is a crucial process in physiological maintenance and tissue regeneration. To understand the contribution of angiogenesis, it is essential to replicate this process in an environment that reproduces the biochemical and physical properties which are largely governed by the extracellular matrix (ECM). We investigated vascularization in cardiac left ventricular ECM hydrogels to mimic post-myocardial repair. We set out to assess and compare different destructive and non-destructive methods, optical as well as non-optical, to visualize angiogenesis and associated matrix remodeling in myocardial ECM hydrogels. Methods: A total of 100,000, 300,000, and 600,000 Human Pulmonary Microvascular Endothelial Cells (HPMEC) were seeded in left ventricular cardiac ECM hydrogel in 48-well plates. After 1, 7, and 14 days of culture, the HPMEC were imaged by inverted fluorescence microscopy and 3D confocal laser scanning microscopy (Zeiss Cell Discoverer 7). In addition, cell-seeded ECM hydrogels were scanned by optical coherence tomography (OCT). Fixed and paraffin-embedded gels were thin-sectioned and assessed for ECM components via H&E, picrosirius red histochemical staining, and immunostaining for collagen type I. ImageJ-based densitometry was used to quantify vascular-like networks and GraphPad was used for statistical analyses. Results: Qualitative analyses were realized through fluoromicrographs obtained by the confocal laser scanning microscope which allowed us to visualize the extensive vascular-like networks that readily appeared at all seeding densities. Quantification of networks was only possible using fluoromicrographs from inverted microscopy. These showed that, after three days, the number of master junctions was seeding density-dependent. The resolution of optical coherence tomography was too low to distinguish between signals caused by the ECM and cells or networks, yet it did show that gels, irrespective of cells, were heterogeneous. Interestingly, (immuno)histochemistry could clearly distinguish between the cast cardiac-derived matrix and newly deposited ECM in the hydrogels. The H&E staining corroborated the presence of vascular-like network structures, albeit that sectioning inevitably led to the loss of 3D structure. Conclusions: Except for OCT, all methods had complementary merit and generated qualitative and quantitative data that allowed us to understand vascular network formation in organ-derived ECM hydrogels.

Assessment of Myocardial Diastolic Dysfunction as a Result of Myocardial Infarction and Extracellular Matrix Regulation Disorders in the Context of Mesenchymal Stem Cell Therapy.

The decline in cardiac contractility due to damage or loss of cardiomyocytes is intensified by changes in the extracellular matrix leading to heart remodeling. An excessive matrix response in the ischemic cardiomyopathy may contribute to the elevated fibrotic compartment and diastolic dysfunction. Fibroproliferation is a defense response aimed at quickly closing the damaged area and maintaining tissue integrity. Balance in this process is of paramount importance, as the reduced post-infarction response causes scar thinning and more pronounced left ventricular remodeling, while excessive fibrosis leads to impairment of heart function. Under normal conditions, migration of progenitor cells to the lesion site occurs. These cells have the potential to differentiate into myocytes in vitro, but the changed micro-environment in the heart after infarction does not allow such differentiation. Stem cell transplantation affects the extracellular matrix remodeling and thus may facilitate the improvement of left ventricular function. Studies show that mesenchymal stem cell therapy after infarct reduces fibrosis. However, the authors did not specify whether they meant the reduction of scarring as a result of regeneration or changes in the matrix. Research is also necessary to rule out long-term negative effects of post-acute infarct stem cell therapy.

Combined role for YAP-TEAD and YAP-RUNX2 signalling in substrate-stiffness regulation of cardiac fibroblast proliferation

Cardiac fibrosis is associated with increased stiffness of the myocardial extracellular matrix (ECM) in part mediated by increased cardiac fibroblast proliferation However, our understanding of the mechanisms regulating cardiac fibroblast proliferation are incomplete. Here we characterise a novel mechanism involving a combined activation of Yes-associated protein (YAP) targets RUNX Family Transcription Factor 2 (RUNX2) and TEA Domain Transcription Factor (TEAD).We demonstrate that cardiac fibroblast proliferation is enhanced by interaction with a stiff ECM compared to a soft ECM. This is associated with activation of the transcriptional co-factor, YAP. We demonstrate that this stiffness induced activation of YAP enhances the transcriptional activity of both TEAD and RUNX2 transcription factors. Inhibition of either TEAD or RUNX2, using gene silencing, expression of dominant-negative mutants or pharmacological inhibition, reduces cardiac fibroblast proliferation. Using mutants of YAP, defective in TEAD or RUNX2 activation ability, we demonstrate a dual role of YAP-mediated activation of TEAD and RUNX2 for substrate stiffness induced cardiac fibroblast proliferation.Our data highlights a previously unrecognised role of YAP mediated RUNX2 activation for cardiac fibroblast proliferation in response to increased ECM stiffness.

Impact of Myocardial Fibrosis on Cardiovascular Structure, Function and Functional Status in Heart Failure with Preserved Ejection Fraction

Myocardial fibrosis, measured using cardiovascular magnetic resonance extracellular volume (ECV), is associated with adverse outcome in heart failure with preserved ejection fraction, but the mechanisms by which myocardial fibrosis exerts this deleterious effect are unclear. We performed mediation analyses of data from the Pirfenidone in Patients with Heart Failure and Preserved Left Ventricular Ejection Fraction (PIROUETTE) trial to determine whether myocardial fibrotic regression causes changes in cardiovascular function and functional status following antifibrotic therapy. Regression of myocardial fibrosis correlated with improvements in 6-min walk test and KCCQ clinical summary score. The only outcome variable that demonstrated a treatment effect was an increase in left ventricular ejection fraction (LVEF). The estimated average causal mediation effects of myocardial ECV, absolute myocardial extracellular matrix volume and absolute myocardial cellular volume on LVEF were 6.1%, 21.5% and 13.7%, respectively, none of which was significant and therefore not mediated by myocardial fibrosis. (PIROUETTE; NCT02932566).Graphical

Development of conductive polymeric nanofiber patches for cardiac tissue engineering application

AbstractElectrically conductive patches using biocompatible polymeric nanofibers have a beneficial effect on electroresponsive tissues such as the brain, heart, and nervous system. Recently, conductive nanofiber patches with electromechanical properties gained more attention as a promising and a well‐effective technology in tissue engineering due to their conductive and flexible nature. In the present study, proposed conductive nanofibrous patches were developed using the electrospinning technique to mimic native myocardial extracellular matrix (ECM) biological and mechanical properties. Different patches of polyurethane (PU) and polylactic acid (PLA) biopolymer were developed by blending and dual syringe electrospinning followed by coating with interfacial polymerization of polyaniline (PANI). The developed nanofibrous patches were evaluated in terms of morphology, physicochemical properties, mechanical flexibility, and in vitro biocompatibility using human EA. hy926 endothelial cells. Results indicated an appropriate surface wettability and mechanical stability of the developed patches compared to reported human myocardium tissue properties. In addition, the electrochemical impedance spectroscopy (EIS) test showed that patches coated with PANI as conductive films can enhance conductivity compared to the non‐conductive (i.e pure polymers) and resulted in better cell proliferation and attachment. Interestingly, the electrical stimulation (ES) during in vitro test can be electrically stimulated and promote cell proliferation as well as mimic the myocardium bioelectricity. Our findings showed that the developed conductive patches can provide a robust conductive system for electroresponsive tissue indicated by cell proliferation shown in FESEM images of attached cells.