Anterior STEMI associated with decreased strain in remote cardiac myocardium

To assess (1) global longitudinal strain (GLS) by feature tracking cardiac magnetic resonance (CMR) in the sub-acute and chronic phases after ST-elevation infarction (STEMI) and compare to GLS in healthy controls, and (2) the evolution of GLS and regional longitudinal strain (RLS) over time, and their relationship to infarct location and size. Seventy-seven patients from the CHILL-MI-trial (NCT01379261) who underwent CMR 2–6 days and 6 months after STEMI and 27 healthy controls were included for comparison. Steady state free precession (SSFP) long-axis cine images were obtained for GLS and RLS, and late gadolinium enhancement (LGE) images were obtained for infarct size quantifications. GLS was impaired in the sub-acute (− 11.8 ± 3.0%) and chronic phases (− 14.3 ± 2.9%) compared to normal GLS in controls (− 18.4 ± 2.4%; p < 0.001 for both). GLS improved from sub-acute to chronic phase (p < 0.001). GLS was to some extent determined by infarct size (sub-acute: r2 = 0.2; chronic: r2 = 0.2, p < 0.001). RLS was impaired in all 6 wall-regions in LAD infarctions in both the sub-acute and chronic phase, while LCx and RCA infarctions had preserved RLS in remote myocardium at both time points. Global longitudinal strain is impaired sub-acutely after STEMI and improvement is seen in the chronic phase, although not reaching normal levels. Global longitudinal strain is only moderately determined by infarct size. Regional longitudinal strain is most impaired in the infarcted region, and LAD infarctions have effects on the whole heart. This could explain why LAD infarcts are more serious than the other culprit vessel infarctions and more often cause heart failure.

PLURIPOTENT STEM CELL-DERIVED CARDIOVASCULAR PROGENITORS DIFFFERENTIATED ON LAMININ 221 REGENERATE AND IMPROVE FUNCTION OF INFARCTED SWINE HEARTS

BackgroundIschemic heart disease is a huge global burden where patients often have irreversibly damaged heart muscle. State-of-the-art technology using stem cell-derived products for cellular therapy could potentially replace damaged heart muscle for regenerative cardiology.Methods and ResultsPluripotent human embryonic stem cells (hESCs) were differentiated on a laminin LN521+221 matrix to cardiovascular progenitors (CVPs). Global transcriptome analyses at multiple time points by single-cell RNA-sequencing demonstrated high reproducibility (R2 > 0.95) between two hESCs lines. We identified several CVP signature genes as quality batch control parameters which are highly specific to our CVPs as compared to canonical cardiac progenitor genes. A total of 200 million CVPs were injected into the infarcted region caused by permanent ligation of the coronary arteries of 10 immunosuppressed pigs and maintained for 4- and 12-weeks post transplantation. The transplanted cells engrafted and proliferated in the infarcted area as indicated by IVIS imaging, histology staining and spatial transcriptomic analysis. Spatial transcriptomic analysis at 1 week following transplantation showed that the infarcted region expressed human genes in the same area as immunohistology sections. Heart function was analyzed by magnetic resonance imaging (MRI) and computerized tomography (CT). Functional studies revealed overall improvement in left ventricular ejection fraction by 21.35 ± 3.3 %, which was accompanied by significant improvements in ventricular wall thickness and wall motion, as well as a reduction in infarction size after CVP transplantation as compared to medium control pigs (P < 0.05). Immunohistology analysis revealed maturation of the CVPs to cardiomyocytes (CMs) where the human grafts aligned with host tissue forming end-to-end connections typical for heart muscle. Electrophysiology analyses revealed electric continuity between injected and host tissue CMs. Episodes of ventricular tachyarrhythmia (VT) over a period of 25 days developed in four pigs, one pig had persistent VT, while the rest remained in normal sinus rhythm. All ten pigs survived the experiment without any VT-related death.ConclusionsWe report a highly reproducible, chemically defined and fully humanized differentiation method of hESCs for the generation of potent CVPs. This method may pave the way for lasting stem cell therapy of myocardial infarction (MI) in humans.Clinical PerspectiveWhat is New?We present a highly reproducible, chemically defined and fully humanized laminin-based differentiation method for generation of large amounts of cardiovascular progenitors (CVP); 20 million cells in a 10 cm2 culture dish which were used for a preclinical study in pigs.Transplantation of the CVPs into the myocardial infarcted pig hearts yields maturation of the progenitor cells to cardiomyocytes (CMs) and improved cardiac function (21.35 ± 3.3 % LVEF improvement) using only 200 million CVPs.Temporary episodes of ventricular arrhythmia (50%) were observed after CVP transplantation. No fatal ventricular arrhythmia occurred.What are the clinical implications?Our laminin-based approach generated potent CVPs in vivo and largely restored function of the damaged heart.Cardiovascular progenitors may provide a new and safe therapeutic strategy for myocardial infarction.The results may have a significant impact on regenerative cardiology.

Single cell genomics for cellular phenotyping: applications in brain disorder

Neurovascular unit (NVU) is an elaborated multicellular brain-vessel-blood interface supporting a controlled blood-brain communication through the selective-permeable blood-brain barrier (BBB) and an adequate neurovascular and neurometabolic coupling. Impairment of NVU during aging and neurologic disorders is accompanied by microvascular dysfunction, BBB opening, neurovascular uncoupling, and neuroinflammation, with deleterious effects on brain microenvironment and neuronal signaling. After stroke, neurons are usually lost in the infarct core and astrocytes become reactive and proliferative, dysregulating the balance between neuronal and non-neuronal cells of the NVU in the lesioned area. In this review, we present major cytological responses of the NVU to cerebral ischemia with an emphasis on the aged brain. Early responses of the neurovascular unit to chronic hypoxia include neutrophils infiltration, brain edema, blood vessel disintegration, astrocytes and endothelial cells proliferation as well as conversion of resident microglia to phagocytic microglia. Later responses include the confinement of the peri-infarcted region by a scar tissue composed mainly of reactive astrocytes and endothelial cells, and angiogenesis. However, the newly formed capillary network is disorganized and the blood vessels are leaky making a successful regeneration of the damaged area, unlikely.

Abstract 369: Infarct Macrophage Secretome Factors Regulate Neutrophil Degranulation

Following myocardial infarction (MI), the left ventricle undergoes wound healing that begins withan intense inflammatory response to recruit leukocytes to the infarcted region. Infiltratedneutrophils degranulate, releasing a series of proteases including matrix metalloproteinase(MMP)-9 into the extracellular space. Macrophages also infiltrate into the infarct region, and wehypothesized that the macrophage may secrete factors that regulate neutrophil degranulation.Stimulating bone marrow derived neutrophils from C57BL/6J mice (3-6 month old male) withphorbol myristate acetate (PMA) induced degranulation, as evidenced by release of MMP-9.Co-stimulation with MI day 1 macrophage secretome (10% by volume) reduced PMA-induceddegranulation of MMP-9 by 8-fold (p<0.0014). Transcriptomic analysis of day 1 MI macrophagesrevealed galectin-3, vimentin, fibronectin, and murinoglobulin-1 were highly expressed.Examination of the day 1 MI macrophage secretome also showed high expression of vimentinand fibronectin. To further unmask signaling connections, neutrophils were co-stimulated withPMA and day 1 MI macrophage secretome, along with blocking antibodies for the 4 proteinsindividually. Galectin-3 inhibition promoted the macrophage secretome effect, whilemurinoglobulin-1 inhibition reversed the macrophage secretome effect. Neutrophil stimulationwith recombinant galectin-3 showed no additive effect with PMA on MMP-9 release, suggestingthat galectin-3 works through the protein kinase C signaling pathway to induce neutrophildegranulation. Neither Vimentin inhibition nor stimulation had an effect on neutrophildegranulation Overall, our results uncovered a role for galectin-3 and fibronectin in inducingneutrophil degranulation while murinoglobulin-1 may be a potent inhibitor of degranulation. Theday 1 MI macrophage therefore secretes both factors that promote as well as factors thattemper neutrophil degranulation. The balance of these factors determines the net effect of themacrophage secretome on neutrophil degranulation.

Computational Representations of Myocardial Infarct Scars and Implications for Arrhythmogenesis

Image-based computational modeling is becoming an increasingly used clinical tool to provide insight into the mechanisms of reentrant arrhythmias. In the context of ischemic heart disease, faithful representation of the electrophysiological properties of the infarct region within models is essential, due to the scars known for arrhythmic properties. Here, we review the different computational representations of the infarcted region, summarizing the experimental measurements upon which they are based. We then focus on the two most common representations of the scar core (complete insulator or electrically passive tissue) and perform simulations of electrical propagation around idealized infarct geometries. Our simulations highlight significant differences in action potential duration and focal effective refractory period (ERP) around the scar, driven by differences in electrotonic loading, depending on the choice of scar representation. Finally, a novel mechanism for arrhythmia induction, following a focal ectopic beat, is demonstrated, which relies on localized gradients in ERP directly caused by the electrotonic sink effects of the neighboring passive scar.

Abstract 14071: In-vivo Assessment of the Peri-infarcted Region by a Novel Micro-electrode Array System in a Large Animal Model of Myocardial Infarction

Introduction: Ventricular arrhythmias are life threatening complications in ischemic cardiomyopathy associated with significant mortality. Inhomogeneity in conduction and dispersion of refractoriness are substrates for reentry tachycardias. Micro-electrode array (MEA) systems are currently used to study extracellular field potentials of myocytes in vitro. Hypothesis: Aim of this study was to validate and apply for the first time a novel epicardial MEA 128-channel electrode in a large animal model of myocardial infarction (MI). Methods: We induced MI by percutaneous coil occlusion of the proximal LAD in swine (body weight: 20±1.5 kg, n=6). Epicardial mapping in-vivo was performed by a lateral mini thoracotomy (length 5 cm) with placement of a flexible 128-channel-MEA (32x32 mm, 100 μm electrodes with 2.7 mm distance) on (a) healthy, (b) infarcted and (c) peri-infarcted areas of the left ventricle. Animals were stimulated with predefined pacing protocols. Results: We assed global as well as regional function after MI confirming its efficacy and impact. Application of the MEA - electrode was safe and feasible, showing reproducible results in all animals. Analyzing different ventricular regions in 2D- reconstruction maps we found the inhomogeneity of conduction velocity to be significantly increased creating a characteristic pattern in the peri-infarcted region ( Figure A ). At each electrode the local ECG was registered to calculate differences in activation time. In comparison to recordings prior to the MI the peri-infarct tissue exhibited significant aberrations in spontaneous impulse propagation ( Figure B ) as well as in pacing protocol measurements. Conclusions: We applied and demonstrated the feasibility of in-vivo epicardial MEA - mapping in a swine large animal model of MI. We believe this tool holds great potential for evaluating conduction velocity and impulse propagation for testing regenerative and anti-arrhythmic therapeutic strategies.

Abstract 018: Rat Ckit+ Cardiac Progenitor Cells Release Pro-survival Micrornas In Exosomes Following Hypoxic Stimulation.

Introduction: Cardiovascular disease is the leading cause of morbidity and mortality among developed nations, and acute myocardial infarction is the major subgroup. The need exists for cardiac therapeutic systems that mitigate tissue damage and induce regeneration within and around the infarcted region. Cardiac progenitor cells (CPCs) and other stem cell types have been attractive candidates for therapies. However, results suggest that regenerative or protective effects may occur through paracrine mechanisms. One such way may be through cell-cell transfer of microRNA (miR) via cell-secreted exosomes, which contain protein, mRNA, and miR. Cell-based therapies face substantial limitations, and therapies avoid these limitations and mimic paracrine efforts—such as delivery of harvested exosomes—have yet to be developed due in part to lack of characterization. Hypothesis: We hypothesized that in response to hypoxic conditions, CPCs secrete a pro-regenerative miRnome within exosomes. Methods: We used an Affymetrix MicroRNA GeneChip microarray to identify the miR populations that were present in CPC-conditioned media after hypoxic and normoxic treatments. Exosomes were isolated via ultracentrifugation (100,000xG) and validated by anti-CD9 immunohistochemistry and dynamic light scattering. We used RT-qPCR to quantify levels of miR upregulation in secreted exosomes. Results: We found seven miRs upregulated (1.3- to 7.9-fold) due to hypoxia stimulation. Within that miRnome, two miRs ([[Unable to Display Character: &#8208;]]20a, [[Unable to Display Character: &#8208;]]210) are known to exert cardioprotection in infarct models, providing evidence of a potential beneficial paracrine effect from CPCs. Four of the remaining miRs (-15b, -17, -103, -199a) have been shown to regulate angiogenesis, proliferation, apoptosis, and fibrosis in various cell and tissue types. One miR ([[Unable to Display Character: &#8208;]]292) has been largely unexplored, but predicted mRNA targets include CTGF, which is involved in cell adhesion and fibrosis. Conclusions: The strong evidence of anti-apoptotic and anti-fibrotic potential of this miRnome indicates that the cocktail may serve as a powerful modulator of cardiac remodeling. Future work will investigate applying miR-containing exosomes from hypoxia-stimulated CPCs as a therapy to rescue the infarcted heart.

Effect of simvastatin on collagen I deposition in non-infarcted myocardium: role of NF-κB and osteopontin

The novel biological effect of statins in alleviating myocardium fibrosis following infarction has been increasingly recognized, yet the underlying mechanisms are not fully understood. The purpose of this study was to characterize the effect of simvastatin on myocardial fibrosis and collagen I deposition in the non-infarcted region after myocardial infarction (MI) and to identify the role of NF-κB and osteopontin in simvastatin-mediated inhibition of post-MI collagen over-expression. A rat model of MI was generated by ligating the left anterior descending coronary artery. The rats surviving the MI operation were randomly divided into the following 3 groups: myocardial infarction (MI, vehicle), simvastatin (Sim, 30 mg·kg–1·day–1), and pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB, 100 mg·kg–1·day–1). Four weeks after MI, cardiac function, mRNAs, and protein expression in non-infarcted myocardium were analyzed. Myocardial fibrosis and collagen I over-expression were observed following MI, accompanied by an increase of NF-κB and osteopontin. Simvastatin improved post-MI left ventricular dysfunction and ameliorated post-MI associated changes to several cardiac parameters, including the left ventricular end diastolic pressure (LVEDP), the maximal rate of pressure development (+dP/dtmax), and the maximal rate of pressure decline (–dP/dtmax). Concurrently, simvastatin significantly suppressed the over-expression of NF-κB, osteopontin, and collagen I in the non-infarcted region following MI. Inhibition of NF-κB by PDTC also reduced osteopontin over-expression and excessive collagen I production and improved the above functional myocardial parameters. These results show that post-MI myocardial fibrosis and collagen I over-expression in the non-infarcted region is associated with activation of NF-κB and osteopontin up-regulation. The anti-fibrotic effect of simvastatin following MI is associated with the attenuation of the expression of osteopontin and NF-κB. The inhibition of NF-κB activation could be the process upstream of osteopontin suppression in the simvastatin-mediated effect.