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Synaptopodin 2-like protein (SYNPO2L) is localized in the sarcomere of cardiomyocytes and is involved in heart morphogenesis. However, the molecular function of SYNPO2L in the heart is not fully understood. We investigated the interaction of SYNPO2L with sarcomeric α-actinin and actin filaments in cultured mouse cardiomyocytes. Immunofluorescence studies showed that SYNPO2L colocalized with α-actinin and actin filaments at the Z-discs of the sarcomere. Recombinant SYNPO2La or SYNPO2Lb caused a bundling of the actin filaments in the absence of α-actinin and enhanced the α-actinin-dependent formation of actin bundles. In addition, high-speed atomic force microscopy revealed that SYNPO2La directly bound to α-actinin via its globular ends. The interaction between α-actinin and SYNPO2La fixed the movements of the two proteins on the actin filaments. These results strongly suggest that SYNPO2L cooperates with α-actinin during actin bundle formation to facilitate sarcomere formation and maintenance.

Introduction: In ventricular myocytes, spontaneous release of calcium (Ca2+) from the sarcoplasmic reticulum via ryanodine receptors (“Ca2+ sparks”) is acutely increased by stretch, due to a stretch-induced increase of reactive oxygen species (ROS). In acute regional ischemia there is stretch of ischemic tissue, along with an increase in Ca2+ spark rate and ROS production, each of which has been implicated in arrhythmogenesis. Yet, whether there is an impact of ischemia on the stretch-induced increase in Ca2+ sparks and ROS has not been investigated. We hypothesized that ischemia would enhance the increase of Ca2+ sparks and ROS that occurs with stretch.Methods: Isolated ventricular myocytes from mice (male, C57BL/6J) were loaded with fluorescent dye to detect Ca2+ sparks (4.6 μM Fluo-4, 10 min) or ROS (1 μM DCF, 20 min), exposed to normal Tyrode (NT) or simulated ischemia (SI) solution (hyperkalemia [15 mM potassium], acidosis [6.5 pH], and metabolic inhibition [1 mM sodium cyanide, 20 mM 2-deoxyglucose]), and subjected to sustained stretch by the carbon fiber technique (~10% increase in sarcomere length, 15 s). Ca2+ spark rate and rate of ROS production were measured by confocal microscopy.Results: Baseline Ca2+ spark rate was greater in SI (2.54 ± 0.11 sparks·s−1·100 μm−2; n = 103 cells, N = 10 mice) than NT (0.29 ± 0.05 sparks·s−1·100 μm−2; n = 33 cells, N = 9 mice; p < 0.0001). Stretch resulted in an acute increase in Ca2+ spark rate in both SI (3.03 ± 0.13 sparks·s−1·100 μm−2; p < 0.0001) and NT (0.49 ± 0.07 sparks·s−1·100 μm−2; p < 0.0001), with the increase in SI being greater than NT (+0.49 ± 0.04 vs. +0.20 ± 0.04 sparks·s−1·100 μm−2; p < 0.0001). Baseline rate of ROS production was also greater in SI (1.01 ± 0.01 normalized slope; n = 11, N = 8 mice) than NT (0.98 ± 0.01 normalized slope; n = 12, N = 4 mice; p < 0.05), but there was an acute increase with stretch only in SI (+12.5 ± 2.6%; p < 0.001).Conclusion: Ischemia enhances the stretch-induced increase of Ca2+ sparks in ventricular myocytes, with an associated enhancement of stretch-induced ROS production. This effect may be important for premature excitation and/or in the development of an arrhythmogenic substrate in acute regional ischemia.

β2-Glycoprotein I (β2-GPI) is a major antigen for antiphospholipid antibodies (Abs) present in patients with the antiphospholipid syndrome (APS). We previously reported that β2-GPI specifically binds to oxidized low density lipoprotein (oxLDL), but not to native low density lipoprotein (LDL). In the present study, a ligand specific for β2-GPI, oxLig-1, was purified from the extracted lipids of oxLDL. The structure of oxLig-1 was shown to be identical to that of synthesized 7-ketocholesteryl-9-carboxynonanoate by mass spectroscopy and nuclear magnetic resonance analyses. Both purified and synthesized oxLig-1 were recognized by β2-GPI and subsequently by anti-β2-GPI auto-Abs, either in enzyme-linked immunosorbent assay (ELISA) or in ligand blot analysis. Binding of liposomes containing oxLig-1 (oxLig-1-liposomes) to mouse macrophages, J774A.1 cells, was relatively low, as compared with that of phosphatidylserine (PS)-liposomes. In contrast, binding of oxLig-1-liposomes was enhanced more than 10-fold in the presence of both β2-GPI and an anti-β2-GPI auto-Ab (WB-CAL-1), derived from (NZW x BXSB) F1 mouse, an animal APS model. Anti-β2-GPI auto-Abs derived from APS patients with episodes of arterial thrombosis were detected in ELISA, using a solid phase oxLig-1 complexed with β2-GPI.We suggest that autoimmune atherogenesis linked to β2-GPI interaction with oxLDL and Abs may be present in APS.

β2-Glycoprotein I (β2-GPI) is a major antigen for anti-cardiolipin antibodies and their epitopes are cryptic. Conformation of each domain of β2-GPI was optimized from its crystal structure by energy minimization and by molecular dynamics simulation. Three electrostatic interactions, i.e. D193–K246, D222–K317 and E228–K308, were observed between domains IV and V in the optimized structure that was constructed based on the consensus sequences obtained by the phage-displayed random peptide library. Antigenic structures determined by the epitope mapping mainly consisted of hydrophobic amino acids located on two discontinuous sequences in domain IV. These amino acid clusters, as an epitope, were covered by domain V and were of a hidden nature. A similar but incomplete counterpart to the epitopic clusters was found in domain I but was not in domains II or III. Binding of anti-β2-GPI auto-antibodies to solid-phase β2-GPI was significantly reduced either by L replacement for W235, a common amino acid component for the epitopes, or by V replacement for all of D193, D222 and E228. Structural analysis indicated a hypothesis that these electrostatic interactions between domains IV and V retained exposure to W235 and that epitope spreading occurred in the region surrounding W235. Thus, epitopic structures recognized by anti-β2-GPI auto-antibodies are cryptic and inter-domain electrostatic interactions are involved in their in exposure. [ABSTRACT FROM AUTHOR]

To clarify the consequences of the valine/leucine polymorphism at position 247 of the β2‐glycoprotein I (β2GPI) gene in patients with antiphospholipid syndrome (APS), by investigating the correlation between genotypes and the presence of anti‐β2GPI antibody. The reactivity of anti‐β2GPI antibodies was characterized using recombinant Val247 and Leu247 β2GPI.Sixty‐five Japanese patients with APS and/or systemic lupus erythematosus who were positive for antiphospholipid antibodies and 61 controls were analyzed for the presence of the Val/Leu247 polymorphism of β2GPI. Polymorphism assignment was determined by polymerase chain reaction followed by restriction enzyme digestion. Recombinant Val247 and Leu247 β2GPI were established to compare the reactivity of anti‐β2GPI antibodies to β2GPI between these variants. The variants were prepared on polyoxygenated plates or cardiolipin‐coated plates, and the reactivity of a series of anti‐β2GPI antibodies (immunized anti‐human β2GPI monoclonal antibodies [Cof‐19–21] and autoimmune anti‐β2GPI monoclonal antibodies [EY1C8, EY2C9, and TM1G2]) and IgGs purified from patient sera was investigated.A positive correlation between the Val247 allele and the presence of anti‐β2GPI antibodies was observed in the patient group. Human monoclonal/polyclonal anti‐β2GPI autoantibodies showed higher binding to recombinant Val247 β2GPI than to Leu247 β2GPI, although no difference in the reactivity of the immunized anti‐β2GPI between these variants was observed. Conformational optimization showed that the replacement of Leu247 by Val247 led to a significant alteration in the tertiary structure of domain V and/or the domain IV–V interaction.The Val247 β2GPI allele was associated with both a high frequency of anti‐β2GPI antibodies and stronger reactivity with anti‐β2GPI antibodies compared with the Leu247 β2GPI allele, suggesting that the Val247 β2GPI allele may be one of the genetic risk factors for development of APS. [ABSTRACT FROM AUTHOR]