Differential assay reactivity of immunglobulin A anti-ß2 glycoprotein I antibodies: implications for the clinical interpretation of antiphospholipid antibody testing.

OBJECTIVE: The routine measurement of IgA anticardiolipin (aCL) and IgA anti-ß2 glycoprotein I (anti-ß2 GPI) antibodies remain controversial despite several studies demonstrating an association with thromboembolic disease in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). This controversy may be a contributing factor for the current under use of IgA antiphospholipid antibodies. We aimed to investigate the nature of discrepant IgA anti-ß2 GPI reactivity to help define the diagnostic value of IgA antiphospholipid antibodies. MATERIAL AND METHODS: Four sera selected from SLE/APS patients and positive for antiphospholipid antibodies but having discrepant IgA anti-ß2 GPI reactivity on two commercial assays were studied. IgA antibodies were affinity purified to investigate anti-ß2 GPI reactivity. Column wash through and eluent fractions were tested on both IgA anti-ß2 GPI assays. Results were normalized to total protein. Assay conjugates and standards from the discrepant assays were interchanged. RESULTS: The diseased samples were strongly positive in one assay [144-388 IgA antiphospholipid (APL) units] and negative or weakly positive in another assay (9.9-53 APL units). IgA eluents from IgA anti-ß2 GPI positive samples reacted 10 times stronger on the reactive assay. When normalized to protein content, the eluents showed no cross-reactivity for IgG or IgM anti-ß2 GPI antibodies, confirming IgA isotype specificity. Conjugate interchange confirmed that both assays bound IgA anti-ß2 GPI antibodies, but the anti-IgA conjugate from the reactive assay was 4 times stronger, suggesting that its ability to detect IgA anti-ß2 GPI antibodies was partially dependent on the anti-IgA conjugate and calibration. CONCLUSION: These results confirm not only the presence of IgA anti-ß2 GPI antibodies in the selected patient samples but also highlight an IgA conjugate issue for the unreactive assay, causing an underestimation of IgA anti-ß2 GPI. This finding may assist in the ongoing standardization efforts of APS antibody testing. In addition, conclusions from published clinical studies may need to be revised as some assays may understate IgA significance.

Identification of potent and selective amidobipyridyl inhibitors of protein kinase D.

The synthesis and biological evaluation of potent and selective PKD inhibitors are described herein. The compounds described in the present study selectively inhibit PKD among other putative HDAC kinases. The PKD inhibitors of the present study blunt phosphorylation and subsequent nuclear export of HDAC4/5 in response to diverse agonists. These compounds further establish the central role of PKD as an HDAC4/5 kinase and enhance the current understanding of cardiac myocyte signal transduction. The in vivo efficacy of a representative example compound on heart morphology is reported herein.

A novel kinase inhibitor establishes a predominant role for protein kinase D as a cardiac class IIa histone deacetylase kinase.

Class IIa histone deacetylases (HDACs) repress genes involved in pathological cardiac hypertrophy. The anti-hypertrophic action of class IIa HDACs is overcome by signals that promote their phosphorylation-dependent nuclear export. Several kinases have been shown to phosphorylate class IIa HDACs, including calcium/calmodulin-dependent protein kinase (CaMK), protein kinase D (PKD) and G protein-coupled receptor kinase (GRK). However, the identity of the kinase(s) responsible for phosphorylating class IIa HDACs during cardiac hypertrophy has remained controversial. We describe a novel and selective small molecule inhibitor of PKD, bipyridyl PKD inhibitor (BPKDi). BPKDi blocks signal-dependent phosphorylation and nuclear export of class IIa HDACs in cardiomyocytes and concomitantly suppresses hypertrophy of these cells. These studies define PKD as a principal cardiac class IIa HDAC kinase.

Suppression of HDAC nuclear export and cardiomyocyte hypertrophy by novel irreversible inhibitors of CRM1.

Histone deacetylase 5 (HDAC5) represses expression of nuclear genes that promote cardiac hypertrophy. Agonism of a variety of G protein coupled receptors (GPCRs) triggers phosphorylation-dependent nuclear export of HDAC5 via the CRM1 nuclear export receptor, resulting in derepression of pro-hypertrophic genes. A cell-based high-throughput screen of a commercial compound collection was employed to identify compounds with the ability to preserve the nuclear fraction of GFP-HDAC5 in primary cardiomyocytes exposed to GPCR agonists. A hit compound potently inhibited agonist-induced GFP-HDAC5 nuclear export in cultured neonatal rat ventricular myocytes (NRVMs). A small set of related compounds was designed and synthesized to evaluate structure-activity relationship (SAR). The results demonstrated that inhibition of HDAC5 nuclear export was a result of compounds irreversibly reacting with a key cysteine residue in CRM1 that is required for its function. CRM1 inhibition by the compounds also resulted in potent suppression of cardiomyocyte hypertrophy. These studies define a novel class of anti-hypertrophic compounds that function through irreversible inhibition of CRM1-dependent nuclear export.

Canonical transient receptor potential channels promote cardiomyocyte hypertrophy through activation of calcineurin signaling.

The calcium/calmodulin-dependent phosphatase calcineurin plays a central role in the control of cardiomyocyte hypertrophy in response to pathological stimuli. Although calcineurin is present at high levels in normal heart, its activity appears to be unaffected by calcium during the course of a cardiac cycle. The mechanism(s) whereby calcineurin is selectively activated by calcium under pathological conditions has remained unclear. Here, we demonstrate that diverse signals for cardiac hypertrophy stimulate expression of canonical transient receptor potential (TRPC) channels. TRPC consists of a family of seven membrane-spanning nonselective cation channels that have been implicated in the nonvoltage-gated influx of calcium in response to G protein-coupled receptor signaling, receptor tyrosine kinase signaling, and depletion of internal calcium stores. TRPC3 expression is up-regulated in multiple rodent models of pathological cardiac hypertrophy, whereas TRPC5 expression is induced in failing human heart. We demonstrate that TRPC promotes cardiomyocyte hypertrophy through activation of calcineurin and its downstream effector, the nuclear factor of activated T cells transcription factor. These results define a novel role for TRPC channels in the control of cardiac growth, and suggest that a TRPC-derived pool of calcium contributes to selective activation of calcineurin in diseased heart.

The CRM1 nuclear export receptor controls pathological cardiac gene expression.

Diverse pathological insults trigger a cardiac remodeling process during which myocytes undergo hypertrophy, with consequent decline in cardiac function and eventual heart failure. Multiple transcriptional regulators of pathological cardiac hypertrophy are controlled at the level of subcellular distribution. For example, prohypertrophic transcription factors belonging to the nuclear factor of activated T cells (NFAT) and GATA families are subject to CRM1-dependent nuclear export but are rapidly relocalized to the nucleus in response to cues for hypertrophic growth. Here, we demonstrate that the antihypertrophic chromatin-modifying enzyme histone deacetylase 5 (HDAC5) is shuttled out of the cardiomyocyte nucleus via a CRM1-mediated pathway in response to diverse signals for hypertrophy. CRM1 antagonists block the agonist-mediated nuclear export of HDAC 5 and repress pathological gene expression and associated hypertrophy of cultured cardiomyocytes. Conversely, CRM1 activity is dispensable for nonpathological cardiac gene activation mediated by thyroid hormone and insulin-like growth factor 1, agonists that fail to trigger the nuclear export of HDAC5. These results suggest a selective role for CRM1 in derepression of pathological cardiac genes via its neutralizing effects on antihypertrophic factors such as HDAC5. Pharmacological approaches targeting CRM1-dependent nuclear export in heart muscle may have salutary effects on cardiac function by suppressing maladaptive changes in gene expression evoked by stress signals.