Ischemic Heart Research Articles

Electrocardiogram signal patterns detection of myocardial ischemia rhythm using an artificial neural network based on MATLAB/Simulink

This study aims to design a computer program to detect myocardial ischemic heart defects through electrocardiogram (ECG) signal patterns and their accuracy. Myocardial ischemia is a heart disorder caused by the narrowing of the blood vessels in the walls of the heart. The method used is a backpropagation-based artificial neural network (ANN) based on MATLAB/Simulink. The input data is trained to recognize the target pattern of the ECG signal based on the potential and time in the ST segment. The optimal weight of the results of the ANN backpropagation algorithm is used in the process of testing the ECG signal pattern to obtain the ANN output. The ANN output was analyzed for potential depression or elevation to identify normal heart or myocardial ischemia. The results of the training show that from several architectures that have been tested, the optimal ANN architecture is 1 hidden layer with 11 hidden units. These results are obtained from the epoch parameter and the mean square error (MSE) value as well as the accuracy of each architecture. The backpropagation ANN learning process requires 8 epochs to achieve the performance goal with MSE 4.03 × 10-9. The system can recognize target patterns with a training accuracy of 99.82%. The test results of the ANN program identification system can detect myocardial ischemia and normal heart abnormalities with an accuracy of 86.7%. Some data were not detected because the ANN output did not meet the criteria for cardiac ischemia or normal myocardium on the ECG signal. Based on the accuracy of the ANN program identification system, the detection of myocardial ischemia rhythm ECG signal patterns using ANN can be said to work well.

Untangling the influence of depression on clinical risks: role of leukocytes and somatic symptoms

Abstract Background Depression has been associated with increased hospitalization and mortality risk, especially for cardiovascular causes. We previously found a composite circulating inflammation score (INFLA-score) to explain part of this link, although the role of its component and of depressive symptoms domains in this relationship is unexplored. Methods In an Italian population cohort (N = 13,191; age≥35 years; 51.7 % women; 4,856 hospitalizations and 471 deaths, median follow-up 7.28/8.24 years), we estimated the proportion of association explained by C-reactive protein levels (CRP), platelet count, granulocyte-to-lymphocyte ratio (GLR) and white blood cell counts (WBC), in multivariable Cox regressions modelling first hospitalization/mortality for all and cardiovascular (CVD), ischemic heart (IHD) and cerebrovascular disease (CeVD) causes vs depression severity based on an alternative validated version of PHQ-9. We also estimated the proportion of association explained by INFLA-score in the associations of polychoric factors tagging somatic and cognitive depressive symptoms with clinical risks. Results In models adjusted for age, sex and education, significant proportions of the positive influence of depression on clinical risks were explained by CRP (4.8% on IHD hospitalizations), GLR (11% on all-cause mortality) and WBC (24% on IHD/CeVD hospitalizations). Stable associations of somatic but not of cognitive depressive symptoms were observed with increased hospitalization risk (+16% for all causes, +14% for CVD causes), with INFLA-score again explaining small but significant proportions of these associations (2.5% for all causes, 8.6% for IHD causes). Conclusions These findings suggest a prominent explanatory role of leukocytes in the link between depression and clinical (especially CVD) risks, and highlight the importance of inflammation in the influence of somatic depressive symptoms. Therefore, acting on these factors may reduce clinical risks associated with depression. Key messages

Alternation of heart microRNA-mRNA network by high-intensity interval training and proanthocyanidin in myocardial ischemia rats: Artificial intelligence and validation experimental.

Heart ischemia is an irreversible condition that occurs via decreased blood flow in vessels by genetic factors, molecular regulators, and environmental conditions. The microRNAs binding to 3´UTR of target genes can influence gene expression and play pivotal roles in several mechanisms identified as a potential biomarker to the pathogenesis. We have screened a pool of microRNAs and mRNAs according to their potential correlation to myocardial ischemia based on artificial intelligence. We constructed the hub genes and mRNA-microRNA networks by R programing language and in silico analysis. Moreover, we calculated the binding affinity of the 3D structure of proanthocyanidin on VEGFα and GATA4 to ameliorate heart tissue after ischemia. Then we treated rats with 300 mg/kg proanthocyanidins and exercised in different intensity and duration times (low, moderate, and high-intensity interval training) for 14 weeks. In the second step, after 14 weeks, isoproterenol hydrochloride was injected into the rats, and myocardial ischemia was induced. We indicated that VEGFα, GATA4, and GJA1 axis associated with miR-27a-3p, miR-499-5p, miR-206-3p, miR-208a-3p are regulatable after 14 weeks of exercise training and proanthocyanidin extract consumption and could prevent myocardial injuries in ischemia. Moreover, we revealed different intensity and duration times, and proanthocyanidin modulated the microRNA-mRNA interaction in rats with myocardial ischemia. Proanthocyanidin consumption as a bioactive compound may significantly ameliorate myocardial dysfunction and offset pathological hallmarks of myocardial ischemia. Moreover, exercise has protective effects on myocardial tissue by reprograming genes and genetic regulator factors. PRACTICAL APPLICATIONS: Complimentary medicine identified Proanthocyanidin and exercise are recognized as effective methods to prevent and improve Myocardial ischemia. According to medical biology servers, we explored the VEGFα, GATA4, and GJA1 axis associated with miR-27a-3p, miR-499-5p, miR-206-3p, miR-208a-3p as a vital pathomechanism of myocardial ischemia. Furthermore, proanthocyanidin extract is the effective compound that could has protective effects on myocardial tissue by reprograming genes and genetic regulator factors. Furthermore, proanthocyanidin and swimming training might recover myocardial dysfunction and regulate the hub genes and mRNA-microRNA networks.

Angiogenic stem cell delivery platform to augment post-infarction neovasculature and reverse ventricular remodeling

Many cell-based therapies are challenged by the poor localization of introduced cells and the use of biomaterial scaffolds with questionable biocompatibility or bio-functionality. Endothelial progenitor cells (EPCs), a popular cell type used in cell-based therapies due to their robust angiogenic potential, are limited in their therapeutic capacity to develop into mature vasculature. Here, we demonstrate a joint delivery of human-derived endothelial progenitor cells (EPC) and smooth muscle cells (SMC) as a scaffold-free, bi-level cell sheet platform to improve ventricular remodeling and function in an athymic rat model of myocardial infarction. The transplanted bi-level cell sheet on the ischemic heart provides a biomimetic microenvironment and improved cell–cell communication, enhancing cell engraftment and angiogenesis, thereby improving ventricular remodeling. Notably, the increased density of vessel-like structures and upregulation of biological adhesion and vasculature developmental genes, such as Cxcl12 and Notch3, particularly in the ischemic border zone myocardium, were observed following cell sheet transplantation. We provide compelling evidence that this SMC-EPC bi-level cell sheet construct can be a promising therapy to repair ischemic cardiomyopathy.

Role of leukocytes, gender, and symptom domains in the influence of depression on hospitalization and mortality risk: Findings from the Moli-sani study.

BackgroundMajor depressive disorder is a mental illness associated with chronic conditions like cardiovascular disease (CVD). Circulating inflammation has been proposed as a potential mechanism underlying this link, although the role of specific biomarkers, gender, and symptom domains is not well elucidated.MethodsWe performed multivariable Cox regressions of first hospitalization/all-cause mortality and CVD, ischemic heart (IHD), and cerebrovascular disease (CeVD) causes vs. depression severity in an Italian population cohort (N = 13,191; age ≥ 35 years; 49.3% men; 4,856 hospitalizations and 471 deaths, median follow-up 7.28 and 8.24 years, respectively). In models adjusted for age, sex, and socioeconomic status, we estimated the proportion of association explained by C-reactive protein (CRP), platelet count, granulocyte-to-lymphocyte ratio (GLR), and white blood cell count (WBC). Gender-by-depression interaction and gender-stratified analyses were performed. Associations of polychoric factors tagging somatic and cognitive symptoms with incident clinical risks were also tested, as well as the proportion explained by a composite index of circulating inflammation (INFLA score).ResultsSignificant proportions of the influence of depression on clinical risks were explained by CRP (4.8% on IHD hospitalizations), GLR (11% on all-cause mortality), and WBC (24% on IHD/CeVD hospitalizations). Gender-by-depression interaction was significantly associated only with all-cause mortality (p = 0.03), with moderate depression showing a + 60% increased risk in women, but not in men. Stable associations of somatic, but not of cognitive, symptoms with increased hospitalization risk were observed (+ 16% for all causes, + 14% for CVD causes), with INFLA score explaining small but significant proportions of these associations (2.5% for all causes, 8.6% for IHD causes).ConclusionsThese findings highlight the importance of cellular components of inflammation, gender, and somatic depressive symptoms in the link between depression and clinical (especially CVD) risks, pointing to the existence of additional pathways through which depression may play a detrimental effect on the cardiovascular system.

Role of CD40(L)-TRAF signaling in inflammation and resolution-a double-edged sword.

Cardiovascular diseases (CVD) and cardiovascular risk factors are the leading cause of death in the world today. According to the Global Burden of Disease Study, hypertension together with ischemic heart and cerebrovascular diseases is responsible for approximately 40% of all deaths worldwide. The major pathomechanism underlying almost all CVD is atherosclerosis, an inflammatory disorder of the vascular system. Recent large-scale clinical trials demonstrated that inflammation itself is an independent cardiovascular risk factor. Specific anti-inflammatory therapy could decrease cardiovascular mortality in patients with atherosclerosis (increased markers of inflammation). Inflammation, however, can also be beneficial by conferring so-called resolution, a process that contributes to clearing damaged tissue from cell debris upon cell death and thereby represents an essential step for recovery from, e.g., ischemia/reperfusion damage. Based on these considerations, the present review highlights features of the detrimental inflammatory reactions as well as of the beneficial process of immune cell-triggered resolution. In this context, we discuss the polarization of macrophages to either M1 or M2 phenotype and critically assess the role of the CD40L-CD40-TRAF signaling cascade in atherosclerosis and its potential link to resolution. As CD40L can bind to different cellular receptors, it can initiate a broad range of inflammatory processes that may be detrimental or beneficial. Likewise, the signaling of CD40L downstream of CD40 is mainly determined by activation of TRAF1-6 pathways that again can be detrimental or beneficial. Accordingly, CD40(L)-based therapies may be Janus-faced and require sophisticated fine-tuning in order to promote cardioprotection.

Surgery on a Primary Cranial Vault Lymphoma

Abstract AIMS Unique cranial involvement in Lymphomas is extraordinary and when it occurs, it is usually accompanied by systemic involvement. The role of the surgery in these cases is still unclear. METHOD Case report of a 72-year-old female with a primary cranial vault lymphoma with primary surgical management at time of recurrence. RESULTS 72-year-old female, with a background of treated breast cancer and ischemic heart, presented with a right parieto-occipital lytic bone lesion with a soft tissue mass with a diagnosis of Non-Hodgkin’s Lymphoma Type B, compatible with follicular lymphoma, with no other symptoms Systemic involvement was ruled out in PET CT bone marrow biopsy. First-line chemotherapy was precluded due to the cardiotoxicity, and she was started on Rituximab-Bendamustine and radiotherapy. PET-CT one year later shows local progression with lytic destruction. The MRI showed an imprint on the brain and a lesion in intimate contact with the superior longitudinal sinus. As an apparently localized lesion and the patient could not receive the optimal chemotherapy treatment, a craniectomy was proposed. Fifteen months after surgery the patient is free of disease on PET CT. CONCLUSION Primary cranial lymphoma is an unusual entity which precludes clear algorithms. The primary treatment is the chemotherapy, nonetheless, If the patient cannot receive the optimal treatment due to his background or in the presence of recurrent disease with limited treatment options, surgery should be considered if technically plausible and adequate to patient’s performance status.

The Klotho protein supports redox balance and metabolic functions of cardiomyocytes during ischemia/reperfusion injury.

BackgroundAcute heart ischemia followed by reperfusion leads to overproduction of reactive oxygen/nitrogen species (ROS/RNS), disrupted expression of nitric oxide synthase (NOS) and unbalanced glucose metabolism. Klotho is a membrane-bound or soluble protein that exerts protective activity in many organs. While Klotho is produced mainly in the kidneys and brain, it has been recently proven that Klotho is expressed in the cardiomyocytes as well. This study aimed to show the influence of the Klotho protein on oxidative/nitrosative stress and metabolic function of the cardiomyocytes subjected to ischemia/reperfusion (I/R) injury.MethodsHuman cardiac myocytes underwent in vitro chemical I/R (with sodium cyanide and 2-deoxyglucose), in the presence or absence of the recombinant human Klotho protein. The present study included an investigation of cell injury markers, level of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), level of oxidative/nitrosative stress and metabolic processes of the cardiomyocytes.ResultsAdministration of Klotho protein resulted in mitigation of injury, decreased level of NOX2 and NOX4, reduced generation of ROS/RNS and hydrogen peroxide (H2O2), decreased expression of inducible NOS and limited production of nitrates/nitrites in cells under I/R. Glucose uptake and lactate production in the cardiomyocytes subjected to I/R were normalized after Klotho supplementation.ConclusionsThe Klotho protein participates in the regulation of redox balance and supports metabolic homeostasis of the cardiomyocytes and hence, contributes to protection against I/R injury.

The presence of sodium glucose co-transporter 2 in mesangial cells and pericytes and its roles in mesangial lesions and in capillaries under diabetic and ischemic conditions

SGLT2 is expressed in mesangial cells and pericytes, and is upregulated byhigh glucose and ischemia. Upregulated SGLT2 in both cells might directly worsen ischemia in kidney interstitial legion, heart and brain. The overexpression of SGLT2 in these cells could induce various organ failures via damages or loss of capillaries and dysfunctions of mesangial cells, which are attenuated by SGLT2 inhibitors.

Synthesis of the Antagonist of the GalR2 Galanin Receptor and Studies of Its Biological Activity in Ischemia and Reperfusion of the Rat Heart In Vivo

Synthesis of the Antagonist of the GalR2 Galanin Receptor and Studies of Its Biological Activity in Ischemia and Reperfusion of the Rat Heart In Vivo

FSTL1-knockdown improves neural oscillation via decreasing neuronal-inflammation regulating apoptosis in Aβ1–42 induced AD model mice

Follistatin like protein 1 (FSTL1) is a famous growth regulatory protein. FSTL1 has been noticed in many diseases, including heart and lung ischemia, cerebral ischemia, glioma, schizophrenia, and Autism. The role of FSTL1 has been declared in the genetics and development of the central nervous system. Therefore, we designed this study to investigate the function and the role of FSTL1 in Alzheimer's disease. Firstly, we noticed upregulated expression level of FSTL1 among four to six-month-old 5XFAD AD mice. Accordingly, we hypothesized that FSTL1-Knockdown improved AD model mice's cognitive function and recover from Alzheimer's disease. Thus, AD model mice were made by single intracerebroventricular injections of Aβ1–42 peptides in FSTL1+/− and CON mice. Next, our results concluded that FSTL1-knockdown effectively improved cognitive functions. FSTL1-knockdown enhanced the pattern of neural oscillations, and synaptic plasticity in Aβ1–42 treated FSTL1-Knockdown mice compared to Aβ1–42 induced AD model mice. Next, FSTL1-Knockdown inhibited the activation of microglia and binding of TLR-4 with microglia. Further, inactivated microglia stopped the formation of MyD88. Thus, our data revealed that FSTL1-Knockdown is slowing down the caspase/BAX/Bcl-2/TLR-4 regulating apoptosis pathway, and the expression of inflammatory cytokines in the hippocampus of Aβ1–42 inserted FSTL1-Knockdown mice. Overall, all these data illuminate the clinical significance role of down-regulated FSTL1. FSTL1-Knockdown reduced the amyloid-beta by affecting microglia, neural-inflammation and apoptosis in AD-like model mice. Finally, down regulation of FSTL1 improved synaptic plasticity, neural oscillations, and cognitive behaviours in the Aβ1–42 induced AD model mice.

Integrated Bioinformatics and Validation of lncRNA-Mediated ceRNA Network in Myocardial Ischemia/Reperfusion Injury.

Background Myocardial ischemia/reperfusion (MI/R) injury is a common pathology in ischemia heart disease. Long noncoding RNAs (lncRNAs) are significant regulators related to many ischemia/reperfusion conditions. This study is aimed at exploring the molecule mechanism of lncRNA-mediated competing endogenous RNA (ceRNA) network in MI/R. Methods The dataset profiles of MI/R and normal tissues (GSE130217 and GSE124176) were obtained from the GEO database. Integrated bioinformatics were performed to screen out differentially expressed genes (DEGs). Thereafter, an lncRNA-mediated ceRNA network was constructed by the starBase database. The GO annotations and KEGG pathway analysis were conducted to study action mechanism and related pathways of DEGs in MI/R. A model of hypoxia/reoxygenation- (H/R-) treated HL-1 cell was performed to verify the expression of lncRNAs through qRT-PCR. Results 2406 differentially expressed- (DE-) mRNAs, 70 DE-lncRNAs, and 156 DE-miRNAs were acquired. These DEGs were conducted to construct an lncRNA-mediated ceRNA network, and a subnetwork including lncRNA Xist/miRNA-133c/mRNA (Slc30a9) was screen out. The functional enrichment analyses revealed that the lncRNAs involved in the ceRNA network might functions in oxidative stress and calcium signaling pathway. The lncRNA Xist expression is reduced under H/R conditions, followed by the increased level of miRNA-133c, thus downregulating the expression of Slc30a9. Conclusion In sum, the identified ceRNA network which included the lncRNA Xist/miR-133c/Slc30a9 axis might contribute a better understanding to the pathogenesis and development of MI/R injury and offer a novel targeted therapy way.

Thymosin β4 and prothymosin α promote cardiac regeneration post-ischaemic injury in mice.

The adult mammalian heart is a post-mitotic organ. Even in response to necrotic injuries, where regeneration would be essential to reinstate cardiac structure and function, only a minor percentage of cardiomyocytes undergo cytokinesis. The gene programme that promotes cell division within this population of cardiomyocytes is not fully understood. In this study, we aimed to determine the gene expression profile of proliferating adult cardiomyocytes in the mammalian heart after myocardial ischaemia, to identify factors to can promote cardiac regeneration. Here, we demonstrate increased 5-ethynyl-2'deoxyuridine incorporation in cardiomyocytes 3 days post-myocardial infarction in mice. By applying multi-colour lineage tracing, we show that this is paralleled by clonal expansion of cardiomyocytes in the borderzone of the infarcted tissue. Bioinformatic analysis of single-cell RNA sequencing data from cardiomyocytes at 3 days post ischaemic injury revealed a distinct transcriptional profile in cardiomyocytes expressing cell cycle markers. Combinatorial overexpression of the enriched genes within this population in neonatal rat cardiomyocytes and mice at postnatal day 12 (P12) unveiled key genes that promoted increased cardiomyocyte proliferation. Therapeutic delivery of these gene cocktails into the myocardial wall after ischaemic injury demonstrated that a combination of thymosin beta 4 (TMSB4) and prothymosin alpha (PTMA) provide a permissive environment for cardiomyocyte proliferation and thereby attenuated cardiac dysfunction. This study reveals the transcriptional profile of proliferating cardiomyocytes in the ischaemic heart and shows that overexpression of the two identified factors, TMSB4 and PTMA, can promote cardiac regeneration. This work indicates that in addition to activating cardiomyocyte proliferation, a supportive environment is a key for regeneration to occur.

Inhibition of intraflagellar transport protein-88 promotes epithelial-to-mesenchymal transition and reduces cardiac remodeling post-myocardial infarction

The epicardium is a potential source of cardiac progenitors to support reparative angiogenesis after myocardial infarction (MI) through epithelial-to-mesenchymal transition (EMT). Primary cilia are recognized as hubs of cellular signaling, and their presence can alter downstream pathways to modulate EMT. The present study aimed to examine the effects of inhibiting intraflagellar transport protein-88 (Ift88), a protein vital to ciliary assembly, on epicardial EMT and cardiac remodeling post-MI. Epicardium derived cells (EPDCs) were cultured from E13.5 heart explants and treated with adenoviral vector encoding short-hairpin RNA against the mouse Ift88 (Ad-shIft88) to disassemble the primary cilium. Effects of Ad-shIft88 on epicardial EMT and cardiac remodeling were examined in mice post-MI. Our results show that Ad-shIft88 enhanced EMT of cultured EPDCs. In adult mice, intra-myocardial administration of Ad-shIft88 increased the number of Wilms tumor 1 (Wt1) positive cells in the epicardium and myocardium, promoted expression of genes associated with epicardial EMT, and enhanced capillary and arteriolar densities post-MI. Additionally, intra-myocardial Ad-shIft88 treatment attenuated cardiac hypertrophy and improved myocardial function three weeks post-MI. In conclusion, knockdown of Ift88 improves epicardial EMT, neovascularization and cardiac remodeling in the ischemic heart. Our study highlights the primary cilium as a potential therapeutic target post-MI.

Quantitative aspects of nitric oxide production in the heart.

Nitric oxide (NO) has essential roles in heart physiology, including the regulation of myocardial contractility and coronary blood flow, and in heart pathophysiology, particularly in the ischemic heart. NO is produced by both NO synthase (NOS)-dependent and NOS-independent pathways in the heart. This review summarizes quantitative aspects of NO production in the heart; the contribution of cardiomyocytes, endothelial cells (ECs), red blood cells (RBCs), and neurons are also discussed. Based on the available data, under normal conditions, the human heart produces about 50-70 µmol NO per day, primarily attributed to eNOS activity; ECs produce the highest amount of NO compared to other cell types in the heart. On the other hand, during ischemic conditions, NOS-independent NO production participates a significant role in the heart NO production that can exceed NOS-dependent NO generation. These data are relevant as most cardiovascular disorders are associated with NO dysfunction, and increasing NO bioavailability and signaling is a potential therapeutic approach for cardiovascular diseases.

Dynamic regulation of HIF-1 signaling in the rhesus monkey heart after ischemic injury

BackgroundHypoxia inducible factor-1 (HIF-1) plays a key role in modulating post-infarct healing after myocardial ischemic injury through transcriptional regulation of hundreds of genes involved in diverse cardiac remodeling processes. However, the dynamic changes in HIF-1 target gene expression in the ischemic heart after myocardial infarction (MI) have not been well characterized.MethodsWe employed a rhesus monkey model of MI induced by left anterior descending artery ligation and examined the expression pattern of HIF-1 target genes in the ischemic heart at 1, 7, and 28 days after injury by bulk RNA-sequencing analysis.ResultsMyocardial transcriptomic analysis demonstrated a temporal-specific regulation of genes associated with the inflammatory response, cell proliferation, fibrosis and mitochondrial metabolism during the pathological progression of MI. HIF-1 target genes involved in processes related to glycolysis, angiogenesis, and extracellular matrix (ECM) remodeling also exhibited distinct expression patterns during MI progression. Copper concentrations were gradually decreased in the heart after ischemic injury, which was positively correlated with the expression of HIF-1-mediated angiogenic and glycolytic genes but negatively correlated with the expression of HIF-1-mediated ECM remodeling genes. Moreover, genes related to intracellular copper trafficking and storage were suppressed along with the loss of myocardial copper in the ischemic heart.ConclusionsThis study demonstrated a dynamic, functional-specific regulation of HIF-1 target gene expression during the progression of MI. The fine-tuning of HIF-1 signaling in the ischemic heart may be relate to the alteration in myocardial copper homeostasis. These findings provide transcriptomic insights into the distinct roles of HIF-1 signaling in the heart after ischemic injury, which will help determine the beneficial cutoff point for HIF-1 targeted therapy in ischemic heart diseases.

Multi-species meta-analysis identifies transcriptional signatures associated with cardiac endothelial responses in the ischaemic heart.

Myocardial infarction remains the leading cause of heart failure. The adult human heart lacks the capacity to undergo endogenous regeneration. New blood vessel growth is integral to regenerative medicine necessitating a comprehensive understanding of the pathways that regulate vascular regeneration. We sought to define the transcriptomic dynamics of coronary endothelial cells following ischaemic injuries in the developing and adult mouse and human heart and to identify new mechanistic insights and targets for cardiovascular regeneration. We carried out a comprehensive meta-analysis of integrated single cell RNA-sequencing data of coronary vascular endothelial cells from the developing and adult mouse and human heart spanning healthy and acute and chronic ischaemic cardiac disease. We identified species-conserved gene regulatory pathways aligned to endogenous neovascularisation. We annotated injury-associated temporal shifts of the endothelial transcriptome and validated four genes: VEGF-C, KLF4, EGR1 and ZFP36. Moreover, we showed that ZFP36 regulates human coronary endothelial cell proliferation and defined that VEGF-C administration in vivo enhances clonal expansion of the cardiac vasculature post-myocardial infarction. Finally, we constructed a coronary endothelial cell meta-atlas, CrescENDO, to empower future in-depth research to target pathways associated with coronary neovascularisation. We present a high-resolution single cell meta-atlas of healthy and injured coronary endothelial cells in the mouse and human heart, revealing a suite of novel targets with great potential to promote vascular regeneration, and providing a rich resource for therapeutic development. Myocardial infarction is the leading cause of heart failure, a condition with high morbidity and mortality. Vascular regeneration is vital to tissue survival and functional restoration while a comprehensive understanding of the underpinning mechanisms is lacking. We show that the meta-analysis of single cell RNA-sequencing data excels at deciphering multi-faceted vascular responses conserved between mouse and human following ischaemic injury. We envision that these data will inform future strategies and accelerate therapeutic development for promoting vascular regeneration in the ischaemic heart.

Housekeeping Proteins Exhibit a High Level of Expression Variability Within Control Group and Between Ischemic Human Heart Biopsies.

Background Human cardiac biopsies are widely used in clinical and fundamental research to decipher molecular events that characterize cardiac physiological and pathophysiological states. One of the main approaches relies on the analysis of semiquantitative immunoblots that reveals alterations in protein expression levels occurring in diseased hearts. To maintain semiquantitative results, expression level of target proteins must be standardized. The expression of HKP (housekeeping proteins) is commonly used to this purpose. Methods and Results We evaluated the stability of HKP expression (actin, β-tubulin, GAPDH, vinculin, and calsequestrin) and total protein staining within control (coefficient of variation) and comparatively with ischemic human heart biopsies (P value). All HKP exhibited a high level of intragroup (ie, actin, β-tubulin, and GAPDH) and/or intergroup variability (ie, GAPDH, vinculin, and calsequestrin). Among all, we found total protein staining to exhibit the highest degree of stability within and between groups, which makes this reference the best to study protein expression level in human biopsies from ischemic hearts and age-matched controls. In addition, we illustrated that using an inappropriate reference protein marker misleads interpretation on SERCA2 (sarco/endoplasmic reticulum Ca2+ ATPase)and cMyBPC (cardiac myosin binding protein-C)expression level after myocardial infarction. Conclusions These reemphasize the need to standardize the level of protein expression with total protein staining in comparative immunoblot studies on human samples from control and diseased hearts.

Fibroblast GSK-3α Promotes Fibrosis via RAF-MEK-ERK Pathway in the Injured Heart.

Heart failure is the leading cause of mortality, morbidity, and health care expenditures worldwide. Numerous studies have implicated GSK-3 (glycogen synthase kinase-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms seem to play overlapping, unique and even opposing functions in the heart. Previously, we have shown that of the 2 isoforms of GSK-3, cardiac fibroblast GSK-3β acts as a negative regulator of myocardial fibrosis in the ischemic heart. However, the role of cardiac fibroblast-GSK-3α in the pathogenesis of cardiac diseases is completely unknown. To define the role of cardiac fibroblast-GSK-3α in myocardial fibrosis and heart failure, GSK-3α was deleted from fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or Postn-promoter-driven Cre recombinase. Control and GSK-3α KO mice were subjected to cardiac injury and heart parameters were evaluated. The fibroblast kinome mapping was carried out to delineate molecular mechanism followed by in vivo and in vitro analysis. Fibroblast-specific GSK-3α deletion restricted fibrotic remodeling and preserved function of the injured heart. We observed reductions in cell migration, collagen gel contraction, α-SMA protein levels, and expression of ECM genes in TGFβ1-treated KO fibroblasts, indicating that GSK-3α is required for myofibroblast transformation. Surprisingly, GSK-3α deletion did not affect SMAD3 activation, suggesting the profibrotic role of GSK-3α is SMAD3 independent. The molecular studies confirmed decreased ERK signaling in GSK-3α-KO CFs. Conversely, adenovirus-mediated expression of a constitutively active form of GSK-3α (Ad-GSK-3αS21A) in fibroblasts increased ERK activation and expression of fibrogenic proteins. Importantly, this effect was abolished by ERK inhibition. GSK-3α-mediated MEK-ERK activation is a critical profibrotic signaling circuit in the injured heart, which operates independently of the canonical TGF-β1-SMAD3 pathway. Therefore, strategies to inhibit the GSK-3α-MEK-ERK signaling circuit could prevent adverse fibrosis in diseased hearts.

415.4: Ischemia Minimization Improves Cardiac Function in an Ex Vivo Xeno Working Heart Model

Introduction: Previous studies showed that ischemia minimization (IM), accomplished by perfusing the heart xenograft during storage, prevents ‘initial cardiac xenograft dysfunction’ (ICXD) and enables prolonged survival following orthotopic cardiac xenotransplantation. Here we report initial observations in a model designed to evaluate effects on heart performance associated with IM and genetic modifications targeted to address xeno-injury mechanisms in a working ex vivo model of ICXD. Method: Hearts from genetically modified and wildtype (WT) pigs were procured after flushing with cold preservation solution (UW, 4oC) and stored for 3 hours either in cold saline (0.9%, 4oC: cold storage (CS)) or were perfused with oxygenated STEEN Solution with RBCs (IM). IM perfusion at 40 mmHg was initiated at room temperature for 20 minutes to facilitate homogeneous graft perfusion before cooling to 4oC for the remainder of the storage period. The genetically modified hearts either had combined knockouts of three specific xenogeneic carbohydrate genes (GTKO, CMAHKO, b4GALNT2KO: TKO) with variable expression of human transgenes (hTG) including complement and thrombo-regulatory proteins (n=29); or GTKO with additional expression of hCD55 (GTKO.hCD55; n=3). Heart function and laboratory parameters were assessed at specific timepoints on a working heart rig while perfused with freshly collected heparinized whole human blood. Troponin I was used as a marker for myocardial injury. Results: In total, 41 hearts were perfused ex vivo, 23 after CS (TKO + hTG n=14, GTKO.hCD55 n=2, WT n=7), and 18 after IM (TKO + hTG n=15, GTKO.hCD55 n=1, WT n=2). Cardiac output (mL/min) was significantly improved for the IM group at all loading conditions relative to CS, (see figure 1). There was a strong trend toward reduced troponin I release, particularly within the first 60 minutes of perfusion (see figure 2). Conclusion: IM is protective with respect to cardiac function following pig heart ischemia and subsequent reperfusion with human blood in an ex vivo model of heart xenotransplantation. The hypothesis that variation in physiologic and biochemical outcomes within groups is influenced by variable expression of complement and coagulation pathway regulatory genes is being investigated.