Proof-of-concept study demonstrating the pathogenicity of affinity-purified IgG antibodies directed to domain I of ß2-glycoprotein I in a mouse model of anti-phospholipid antibody-induced thrombosis.

OBJECTIVE: IgG aPL against domain I of ß2-glycoprotein I (ß2GPI) [anti-DI (aDI)] is associated with the pathogenesis of APS, an autoimmune disease defined by thrombosis and pregnancy morbidity. To date, however, no study has demonstrated direct pathogenicity of IgG aDI in vivo. In this proof-of-concept study, we designed a novel system to affinity purify polyclonal aDI aPL in order to assess its prothrombotic ability in a well-characterized mouse microcirculation model for APS. METHODS: Two polyclonal IgG fractions were isolated from serum of a patient with APS, both with high aPL activity but differing in aDI activity (aDI-rich and aDI-poor). These IgG fractions were tested for their pathogenic ability in an in vivo mouse model of thrombosis. Male CD1 mice were injected intraperitoneally with either aDI-rich or aDI-poor IgG; as a control, IgG isolated from healthy serum was used. A pinch injury was applied to the right femoral vein and thrombus dynamics and tissue factor activity in isolated tissue were evaluated. RESULTS: Both aDI-rich and aDI-poor IgG retained aCL and anti-ß2GPI activity, while only aDI-rich IgG displayed high aDI activity, as defined by our in-house cut-offs for positivity in each assay. aDI-rich IgG induced significantly larger thrombi in vivo compared with aDI-poor IgG (P < 0.0001). Similarly, aDI-rich IgG significantly enhanced the procoagulant activity of carotid artery endothelium and peritoneal macrophages isolated from experimental animals (P < 0.01). CONCLUSION: These data directly demonstrate that the ability to cause thrombosis in vivo is concentrated in the aDI fraction of aPL.

Post-Myocardial Infarction T-tubules Form Enlarged Branched Structures With Dysregulation of Junctophilin-2 and Bridging Integrator 1 (BIN-1).

BACKGROUND: Heart failure is a common secondary complication following a myocardial infarction (MI), characterized by impaired cardiac contraction and t-tubule (t-t) loss. However, post-MI nano-scale morphological changes to the remaining t-ts are poorly understood. METHOD AND RESULTS: We utilized a porcine model of MI, using a nonlethal microembolization method to generate controlled microinfarcts. Using serial block face scanning electron microscopy, we report that post-MI, after mild left-ventricular dysfunction has developed, t-ts are not only lost in the peri-infarct region, but also the remnant t-ts form enlarged, highly branched disordered structures, containing a dense intricate inner membrane. Biochemical and proteomics analyses showed that the calcium release channel, ryanodine receptor 2 (RyR2), abundance is unchanged, but junctophilin-2 (JP2), important for maintaining t-t trajectory, is depressed (-0.5×) in keeping with the t-ts being disorganized. However, immunolabeling shows that populations of RyR2 and JP2 remain associated with the remodeled t-ts. The bridging integrator 1 protein (BIN-1), a regulator of tubulogensis, is upregulated (+5.4×), consistent with an overdeveloped internal membrane system, a feature not present in control t-ts. Importantly, we have determined that t-ts, in the remote region, are narrowed and also contain dense membrane folds (BIN-1 is up-regulated +3.4×), whereas the t-ts have a radial organization comparable to control JP2 is upregulated +1.7×. CONCLUSIONS: This study reveals previously unidentified remodeling of the t-t nano-architecture in the post-MI heart that extends to the remote region. Our findings highlight that targeting JP2 may be beneficial for preserving the orientation of the t-ts, attenuating the development of hypocontractility post-MI.

Targeting caveolin-3 for the treatment of diabetic cardiomyopathy.

Diabetes is a global health problem with more than 550 million people predicted to be diabetic by 2030. A major complication of diabetes is cardiovascular disease, which accounts for over two-thirds of mortality and morbidity in diabetic patients. This increased risk has led to the definition of a diabetic cardiomyopathy phenotype characterised by early left ventricular dysfunction with normal ejection fraction. Here we review the aetiology of diabetic cardiomyopathy and explore the involvement of the protein caveolin-3 (Cav3). Cav3 forms part of a complex mechanism regulating insulin signalling and glucose uptake, processes that are impaired in diabetes. Further, Cav3 is key for stabilisation and trafficking of cardiac ion channels to the plasma membrane and so contributes to the cardiac action potential shape and duration. In addition, Cav3 has direct and indirect interactions with proteins involved in excitation-contraction coupling and so has the potential to influence cardiac contractility. Significantly, both impaired contractility and rhythm disturbances are hallmarks of diabetic cardiomyopathy. We review here how changes to Cav3 expression levels and altered relationships with interacting partners may be contributory factors to several of the pathological features identified in diabetic cardiomyopathy. Finally, the review concludes by considering ways in which levels of Cav3 may be manipulated in order to develop novel therapeutic approaches for treating diabetic cardiomyopathy.