Rationale Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase (MPO), a heme-enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. Objective To reveal the role of MPO for the development of ventricular arrhythmias. Methods and Results In different murine models of myocardial ischemia MPO deficiency profoundly decreased vulnerability for ventricular tachycardia (VT) upon programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry following isoproterenol injection. Experiments employing CD11b/CD18-integrin-deficient (CD11b-/-) mice and intravenous MPO infusion revealed that neutrophil infiltration is a prerequisite for myocardial MPO accumulation. Ventricles from MPO-deficient (Mpo-/-) mice showed less pronounced slowing and decreased heterogeneity of electrical conduction in the periinfarct zone than WT mice. Expression of the redox sensitive gap-junctional protein connexin43 (Cx43) was reduced in the periinfarct area of WT compared to Mpo-/- mice. In isolated WT cardiomyocytes, Cx43 protein content decreased upon MPO/H2O2-incubation. Mapping of induced pluripotent stem-cell-derived cardiomyocyte (iPSCM) networks and in vivo investigations linked Cx43 breakdown to MPO-dependent activation of matrix-metalloproteinase 7. Moreover, Mpo-/- mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, MPO was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases (MAPK) resulting in upregulated collagen generation. In support of our experimental findings, baseline MPO plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction above 35% undergoing elective diagnostic cardiac evaluation. Conclusions MPO emerges as a crucial mediator of post-ischemic myocardial remodeling, and may evolve as a novel pharmacological target for secondary disease prevention following myocardial ischemia.
Gαi2 deficiency combined with cardiac β1-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, β1-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking Gαi2. We propose an enhanced effect of increased β1-adrenergic drive by the lack of protection via Gαi2. Gαi3 up-regulation was not sufficient to compensate for Gαi2 deficiency, suggesting an isoform-specific or a concentration-dependent mechanism.
The aim of this study was to investigate whether continuous electrical stimulation affects electrophysiological properties and cell morphology of fetal cardiomyocytes (FCMs) in culture. Fetal cardiomyocytes at day 14.5 post coitum were harvested from murine hearts and electrically stimulated for 6 days in culture using a custom-made stimulation chamber. Subsequently, action potentials of FCM were recorded with glass microelectrodes. Immunostainings of α-Actinin, connexin 43, and vinculin were performed. Expression of ion channel subunits Kcnd2, Slc8a1, Cacna1, Kcnh2, and Kcnb1 was analyzed by quantitative reverse-transcriptase polymerase chain reaction. Action potential duration to 50% and 90% repolarization (APD50 and APD90) of electrically stimulated FCMs were significantly decreased when compared to nonstimulated control FCM. Alignment of cells was significantly higher in stimulated FCM when compared to control FCM. The expression of connexin 43 was significantly increased in stimulated FCM when compared to control FCM. The ratio between cell length and cell width of the stimulated FCM was significantly higher than in control FCM. Kcnh2 and Kcnd2 were upregulated in stimulated FCM when compared to control FCM. Expression of Slc8a1, Cacna1c, and Kcnb1 was not different in stimulated and control FCMs. The decrease in APD50 observed after electrical stimulation of FCM in vitro corresponds to the electrophysiological maturation of FCM in vivo. Expression levels of ion channels suggest that some important but not all aspects of the complex process of electrophysiological maturation are promoted by electrical stimulation. Parallel alignment, increased connexin 43 expression, and elongation of FCM are signs of a morphological maturation induced by electrical stimulation.
Transplanted iPSCM can persist in the long term and integrate electrically into host tissue, supporting their potential for cell replacement therapy. Quality of electrical integration improves between 6-12 days and 6-8 months after transplantation, and there are signs of an electrophysiological maturation. However, even after 6-8 months, AP properties of transplanted iPSCM differ from those of recipient cardiomyocytes.
Glomerular biology is dependent on tightly controlled signal transduction networks that control phosphorylation of signaling proteins such as cytoskeletal regulators or slit diaphragm proteins of kidney podocytes. Cross-species comparison of phosphorylation events is a powerful mean to functionally prioritize and identify physiologically meaningful phosphorylation sites. Here, we present the result of phosphoproteomic analyses of cow and rat glomeruli to allow cross-species comparisons. We discovered several phosphorylation sites with potentially high biological relevance, e.g. tyrosine phosphorylation of the cytoskeletal regulator synaptopodin and the slit diaphragm protein neph-1 (Kirrel). Moreover, cross-species comparisons revealed conserved phosphorylation of the slit diaphragm protein nephrin on an acidic cluster at the intracellular terminus and conserved podocin phosphorylation on the very carboxyl terminus of the protein. We studied a highly conserved podocin phosphorylation site in greater detail and show that phosphorylation regulates affinity of the interaction with nephrin and CD2AP. Taken together, these results suggest that species comparisons of phosphoproteomic data may reveal regulatory principles in glomerular biology. All MS data have been deposited in the ProteomeXchange with identifier PXD001005 (http://proteomecentral.proteomexchange.org/dataset/PXD001005).
Cardiac cell replacement therapy is a promising therapy to improve cardiac function in heart failure. Persistence, structural and functional maturation, and integration of transplanted cardiomyocytes into recipients' hearts are crucial for a safe and efficient replacement of lost cells. We studied histology, electrophysiology, and quantity of intramyocardially transplanted rat neonatal cardiomyocytes (NCMs) and performed a detailed functional study with repeated invasive (pressure-volume catheter) and noninvasive (echocardiography) analyses of infarcted female rat hearts including pharmacological stress before and 3 weeks after intramyocardial injection of 5 × 106 (low NCM) or 25 × 106 (high NCM) syngeneic male NCMs or medium as placebo (Ctrl). Quantitative real-time polymerase chain reaction (PCR) for Y-chromosome confirmed a fivefold higher persisting male cell number in high NCM versus low NCM after 3 weeks. Sharp electrode measurements within viable slices of recipient hearts demonstrated that transplanted NCMs integrate into host myocardium and mature to an almost adult phenotype, which might be facilitated through gap junctions between host myocardium and transplanted NCMs as indicated by connexin43 in histology. Ejection fraction of recipient hearts was severely impaired after ligation of left anterior descending (LAD; pressure-volume catheter: 39.2 ± 3.6%, echocardiography: 39.9 ± 1.4%). Repeated analyses revealed a significant further decline within 3 weeks in Ctrl and a dose-dependent stabilization in cell-treated groups. Consistently, stabilized cardiac function/morphology in cell-treated groups was seen in stroke volume, cardiac output, ventricle length, and wall thickness. Our findings confirm that cardiac cell replacement is a promising therapy for ischemic heart disease since immature cardiomyocytes persist, integrate, and mature after intramyocardial transplantation, and they dose-dependently stabilize cardiac function after myocardial infarction.
Myocardial infarction (MI) is a leading cause of morbidity and mortality worldwide. Improved survival has led to an increasing incidence of ischemic cardiomyopathy, making it a major reason for hospitalization in the western world. The inflammatory response in the ischemic myocardium determines the extent of structural remodeling and functional deterioration, with neutrophils (PMN) being a key modulator of the propagation and resolution of inflammation. The heme enzyme myeloperoxidase (MPO) is abundantly expressed in PMN and is an important mediator of their inflammatory capacities. Here, we examine the effects of PMN reduction, MPO deficiency and MPO inhibition in two murine models of MI. Reduction in PMN count resulted in less scar formation and improved cardiac function. Similar results were obtained in genetically MPO deficient mice, suggesting that MPO is a critical factor in PMN-mediated cardiac remodeling. To test our findings in a therapeutic approach, we orally administered the MPO inhibitor AZM198 in the context of MI and could demonstrate improved cardiac function and reduced structural remodeling. Therefore, MPO appears to be a favorable pharmacological target for the prevention of long-term morbidity after MI.
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