Reduction in Kv Current Enhances the Temporal Dispersion of the Action Potential in Diabetic Myocytes: Insights From a Novel Repolarization Algorithm

BACKGROUND: Diabetes is associated with prolongation of the QT interval of the electrocardiogram and enhanced dispersion of ventricular repolarization, factors that, together with atherosclerosis and myocardial ischemia, may promote the occurrence of electrical disorders. Thus, we tested the possibility that alterations in transmembrane ionic currents reduce the repolarization reserve of myocytes, leading to action potential (AP) prolongation and enhanced beat-to-beat variability of repolarization. METHODS AND RESULTS: Diabetes was induced in mice with streptozotocin (STZ), and effects of hyperglycemia on electrical properties of whole heart and myocytes were studied with respect to an untreated control group (Ctrl) using electrocardiographic recordings in vivo, ex vivo perfused hearts, and single-cell patch-clamp analysis. Additionally, a newly developed algorithm was introduced to obtain detailed information of the impact of high glucose on AP profile. Compared to Ctrl, hyperglycemia in STZ-treated animals was coupled with prolongation of the QT interval, enhanced temporal dispersion of electrical recovery, and susceptibility to ventricular arrhythmias, defects observed, in part, in the Akita mutant mouse model of type I diabetes. AP was prolonged and beat-to-beat variability of repolarization was enhanced in diabetic myocytes, with respect to Ctrl cells. Density of Kv K(+) and L-type Ca(2+) currents were decreased in STZ myocytes, in comparison to cells from normoglycemic mice. Pharmacological reduction of Kv currents in Ctrl cells lengthened AP duration and increased temporal dispersion of repolarization, reiterating features identified in diabetic myocytes. CONCLUSIONS: Reductions in the repolarizing K(+) currents may contribute to electrical disturbances of the diabetic heart

Phenotypically heterogeneous podoplanin-expressing cell populations are associated with the lymphatic vessel growth and fibrogenic responses in the acutely and chronically infarcted myocardium

Cardiac lymphatic vasculature undergoes substantial expansion in response to myocardial infarction (MI). However, there is limited information on the cellular mechanisms mediating post-MI lymphangiogenesis and accompanying fibrosis in the infarcted adult heart. Using a mouse model of permanent coronary artery ligation, we examined spatiotemporal changes in the expression of lymphendothelial and mesenchymal markers in the acutely and chronically infarcted myocardium. We found that at the time of wound granulation, a three-fold increase in the frequency of podoplanin-labeled cells occurred in the infarcted hearts compared to non-operated and sham-operated counterparts. Podoplanin immunoreactivity detected LYVE-1-positive lymphatic vessels, as well as masses of LYVE-1-negative cells dispersed between myocytes, predominantly in the vicinity of the infarcted region. Podoplanin-carrying populations displayed a mesenchymal progenitor marker PDGFRα, and intermittently expressed Prox-1, a master regulator of the lymphatic endothelial fate. At the stages of scar formation and maturation, concomitantly with the enlargement of lymphatic network in the injured myocardium, the podoplanin-rich LYVE-1-negative multicellular assemblies were apparent in the fibrotic area, aligned with extracellular matrix deposits, or located in immediate proximity to activated blood vessels with high VEGFR-2 content. Of note, these podoplanin-containing cells acquired the expression of PDGFRβ or a hematoendothelial epitope CD34. Although Prox-1 labeling was abundant in the area affected by MI, the podoplanin-presenting cells were not consistently Prox-1-positive. The concordance of podoplanin with VEGFR-3 similarly varied. Thus, our data reveal previously unknown phenotypic and structural heterogeneity within the podoplanin-positive cell compartment in the infarcted heart, and suggest an alternate ability of podoplanin-presenting cardiac cells to generate lymphatic endothelium and pro-fibrotic cells, contributing to scar development

CD45 regulates retention, motility, and numbers of hematopoietic progenitors, and affects osteoclast remodeling of metaphyseal trabecules

The CD45 phosphatase is uniquely expressed by all leukocytes, but its role in regulating hematopoietic progenitors is poorly understood. We show that enhanced CD45 expression on bone marrow (BM) leukocytes correlates with increased cell motility in response to stress signals. Moreover, immature CD45 knockout (KO) cells showed defective motility, including reduced homing (both steady state and in response to stromal-derived factor 1) and reduced granulocyte colony-stimulating factor mobilization. These defects were associated with increased cell adhesion mediated by reduced matrix metalloproteinase 9 secretion and imbalanced Src kinase activity. Poor mobilization of CD45KO progenitors by the receptor activator of nuclear factor κB ligand, and impaired modulation of the endosteal components osteopontin and stem cell factor, suggested defective osteoclast function. Indeed, CD45KO osteoclasts exhibited impaired bone remodeling and abnormal morphology, which we attributed to defective cell fusion and Src function. This led to irregular distribution of metaphyseal bone trabecules, a region enriched with stem cell niches. Consequently, CD45KO mice had less primitive cells in the BM and increased numbers of these cells in the spleen, yet with reduced homing and repopulation potential. Uncoupling environmental and intrinsic defects in chimeric mice, we demonstrated that CD45 regulates progenitor movement and retention by influencing both the hematopoietic and nonhematopoietic compartments

Cardiac stem cell niches

The critical role that stem cell niches have in cardiac homeostasis and myocardial repair following injury is the focus of this review. Cardiac niches represent specialized microdomains where the quiescent and activated state of resident stem cells is regulated. Alterations in niche function with aging and cardiac diseases result in abnormal sites of cardiomyogenesis and inadequate myocyte formation. The relevance of Notch1 signaling, gap-junction formation, HIF-1α and metabolic state in the regulation of stem cell growth and differentiation within the cardiac niches are discussed

LpMab-12 Established by CasMab Technology Specifically Detects Sialylated O-Glycan on Thr52 of Platelet Aggregation-Stimulating Domain of Human Podoplanin

Podoplanin (PDPN), also known as Aggrus, possesses three tandem repeat of platelet aggregation-stimulating (PLAG) domains in its N-terminus. Among the PLAG domains, sialylated O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2) and the platelet-aggregating activity of human PDPN (hPDPN). Although various anti-hPDPN monoclonal antibodies (mAbs) have been generated, no specific mAb has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins. Herein, we report the development of a novel anti-glycopeptide mAb (GpMab), LpMab-12. LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells were clearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49–Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38–54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54), which carries α2–6 sialylated N-acetyl-D-galactosamine (GalNAc) on Thr52. LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on Thr52, a site-specific post-translational modification critical for the hPDPN association with CLEC-2

LpMab-12 Established by CasMab Technology Specifically Detects Sialylated O-Glycan on Thr52 of Platelet Aggregation-Stimulating Domain of Human Podoplanin

Podoplanin (PDPN), also known as Aggrus, possesses three tandem repeat of platelet aggregation-stimulating (PLAG) domains in its N-terminus. Among the PLAG domains, sialylated O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2) and the platelet-aggregating activity of human PDPN (hPDPN). Although various anti-hPDPN monoclonal antibodies (mAbs) have been generated, no specific mAb has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins. Herein, we report the development of a novel anti-glycopeptide mAb (GpMab), LpMab-12. LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells were clearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49–Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38–54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54), which carries α2–6 sialylated N-acetyl-D-galactosamine (GalNAc) on Thr52. LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on Thr52, a site-specific post-translational modification critical for the hPDPN association with CLEC-2

Low expression of monocyte-macrophage markers in the populations of podoplanin-positive cells in the infarcted myocardium.

<p>(<b>A</b>,<b>B</b>) Thin cardiac sections were indirectly immunolabeled with MOMA-2 (<b>A</b>) or F4/80 (<b>B</b>) antibodies and counterstained with hematoxylin and eosin. Time after MI is indicated. Spleen sections were included as positive controls. Areas in rectangles with the corresponding numbers are shown at a higher magnification in the adjacent insets. Note the presence of cells immunoreactive for MOMA-2 or F4/80 (arrows) in the inflamed epicardium (2 days, insets 2), myocardial interstitium (2 weeks, insets 2) and spleen, but not in the BZ of necrotic myocardium (2 days, insets 1) or the maturing scar (2 weeks, insets 1). (<b>C</b>) Flow-cytometry analysis of the F4/80 or CD11b expression in the podoplanin-positive cohorts populating the hearts of non-infarcted (NO), sham-operated (SHAM) and infarcted (MI) mice at 2 days after surgery. Graphs displaying individual values and the respective means (left), as well as representative scatterplots (right) are shown. Data represent frequency of double-positive cells within podoplanin-labeled populations (calculated as % cells in gate 2 out of the sum of cells in gate 1 and gate 2).</p