Digitalis toxicity: ECG vignette

Abstract“Digitalis toxicity, often candidly indexed as poisoning, has plagued the medical profession for over 200 years. The situation qualifies as a professional disgrace on the basis of three items: the situation persists, physicians are often slow to recognize it and, over the decades, writers have been harsh in their denunciation of fellow physicians when toxicity has occurred….” These are the opening remarks of an essay published in 1983 on the 2nd centenary of William Withering's ‘magic potion from foxglove's extract for dropsy.’ Even today, after many decades, these words appear relevant! We present and discuss an interesting ECG of digitalis toxicity

Surfactant protein D induces immune quiescence and apoptosis of mitogen-activated peripheral blood mononuclear cells

Surfactant Protein D (SP-D) is an integral molecule of the innate immunity secreted by the epithelial cells lining the mucosal surfaces. Its C-type lectin domain offers pattern recognition functions while it binds to putative receptors on immune cells to modify cellular functions. Activated PBMCs and increased serum levels of SP-D are observed under a range of pathophysiological conditions including infections. Thus, we speculated if SP-D can modulate systemic immune response via direct interaction with activated PBMCs. Here, we have examined interaction of a recombinant fragment of human SP-D (rhSP-D) on PHA-activated PBMCs. We observed a significant downregulation of TLR2, TLR4, CD11c and CD69 upon rhSP-D treatment. rhSP-D inhibited production of Th1 (TNF-α and IFN-γ) and Th17 (IL-17) cytokines along with IL-6. Interestingly, levels of IL-2, Th2 (IL-4) and regulatory (IL-10 and TGF-β) cytokines were unaltered. Differential expression of co-stimulatory CD28 and co-inhibitory CTLA4 expression along with their ligands CD80 and CD86 revealed selective up-regulation of CTLA4 at both mRNA and protein level. In addition, rhSP-D induced apoptosis only in the activated but not in non-activated PBMCs. Blockade of CTLA4 inhibited rhSP-D mediated apoptosis, confirming an involvement of CTLA4 in induction of apoptosis. We conclude that SP-D restores immune homeostasis: it regulates expression of immunomodulatory receptors and cytokines, which is followed by apoptosis induction of immune-activated cells. These findings appear to suggest a general role for SPD in immune surveillance against activated immune cells

CIP4 is required for the hypertrophic growth of neonatal cardiac myocytes

BACKGROUND: CIP4 is a scaffold protein that regulates membrane deformation and tubulation, organization of the actin cytoskeleton, endocytosis of growth factor receptors, and vesicle trafficking. Although expressed in the heart, CIP4 has not been studied with regards to its potential function in cardiac myocytes. RESULTS: We now show using RNA interference that CIP4 expression in neonatal rat ventricular myocytes is required for the induction of non-mitotic, hypertrophic growth by the α-adrenergic agonist phenylephrine, the IL-6 cytokine leukemia inhibitor factor, and fetal bovine serum, as assayed using morphometry, immunocytochemistry for the hypertrophic marker atrial natriuretic factor and [(3)H]leucine incorporation for de novo protein synthesis. This requirement was consistent with the induction of CIP4 expression by hypertrophic stimulation. The inhibition of myocyte hypertrophy by CIP4 small interfering oligonucleotides (siRNA) was rescued by expression of a recombinant CIP4 protein, but not by a mutant lacking the N-terminal FCH domain responsible for CIP4 intracellular localization. CONCLUSIONS: These results imply that CIP4 plays a significant role in the intracellular hypertrophic signal transduction network that controls the growth of cardiac myocytes in heart disease

SP-D impedes transfer of HIV-1 from multi-layered vaginal epithelium with a distinct gene signature

Surfactant Protein (SP) D is a member of the collectin family of soluble pattern recognition receptors. We have previously shown that a recombinant fragment of SP-D (rhSP-D) inhibits gp120-CD4 interaction and HIV-1 entry in target cells. To potentiate its prophylactic use as a vaginal microbicide, we determined ex vivo efficacy using organotypic human vaginal-ectocervical epithelia (VEC-100) that closely resemble the native tissues of origin. VEC-100, stratified human vaginal-ectocervical tissues grown on membrane inserts were treated with rhSP-D followed by a challenge with HIV-1 to assess the transfer of HIV-1 through the VEC-100 tissues to PBMCs in the basal submucosal compartment. Treated VEC tissues were subjected to mRNA Illumina microarray analysis. Levels of transcripts encoding for immune mediators, adhesion and tight junction proteins were also evaluated. Effect of rhSP-D on viability, NFκB activation, cytokine secretion and bacterial colonization of cervical vaginal epithelial cells was determined. rhSP-D significantly inhibited HIV-1 transfer from the multi-layered epithelial tissues to the basal PBMCs as compared to HIV-1 alone. Global gene expression profile of HIV-1 challenged VEC-100 tissues revealed differential regulation of genes and pathways majorly involved in inflammation, cell survival and transcription factors. Levels of Guanylate-binding proteins (GBPs) and interferon-inducible proteins were significantly upregulated suggesting an interferon host defense response. rhSP-D showed an inhibition in the levels of GBPs and rescued the cell adhesion molecules such as Claudin 2, 3, 4, 5 and Occludin, known to be down regulated by HIV-1 in primary vaginal cells. Importantly, rhSP-D conditioned VEC tissue supernatants did not enhance susceptibility of target cells to HIV-1. rhSP-D treated vaginal epithelial cells did not show any significant alteration in viability, NFκB activation and levels of immune mediators like IL-1RA, Elafin, SLPI, TGFβ, GRO-α, MIP-3α and RANTES. Bacterial colonization and direct toxicity assays revealed that rhSP-D did not adversely affect growth of vaginal commensals. Blockade of viral movement within the vaginal epithelium, inhibition of detrimental early gene signature and safety profile of rhSP-D suggests that topical formulation comprising rhSP-D may significantly curb the sexual transmission of HIV-1

The scaffold protein muscle A-kinase anchoring protein β orchestrates cardiac myocyte hypertrophic signaling required for the development of heart failure

Cardiac myocyte hypertrophy is regulated by an extensive intracellular signal transduction network. In vitro evidence suggests that the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) serves as a nodal organizer of hypertrophic signaling. However, the relevance of mAKAPβ signalosomes to pathological remodeling and heart failure in vivo remains unknown. Using conditional, cardiac myocyte-specific gene deletion, we now demonstrate that mAKAPβ expression in mice is important for the cardiac hypertrophy induced by pressure overload and catecholamine toxicity. mAKAPβ targeting prevented the development of heart failure associated with long-term transverse aortic constriction, conferring a survival benefit. In contrast to 29% of control mice (n=24), only 6% of mAKAPβ knockout mice (n=31) died in the 16 weeks of pressure overload (P=0.02). Accordingly, mAKAPβ knockout inhibited myocardial apoptosis and the development of interstitial fibrosis, left atrial hypertrophy, and pulmonary edema. This improvement in cardiac status correlated with the attenuated activation of signaling pathways coordinated by the mAKAPβ scaffold, including the decreased phosphorylation of protein kinase D1 and histone deacetylase 4 that we reveal to participate in a new mAKAP signaling module. Furthermore, mAKAPβ knockout inhibited pathological gene expression directed by myocyte-enhancer factor-2 and nuclear factor of activated T-cell transcription factors that associate with the scaffold. mAKAPβ orchestrates signaling that regulates pathological cardiac remodeling in mice. Targeting of the underlying physical architecture of signaling networks, including mAKAPβ signalosome formation, may constitute an effective therapeutic strategy for the prevention and treatment of pathological remodeling and heart failure

The Scaffold Protein Muscle A-Kinase Anchoring Protein β Orchestrates Cardiac Myocyte Hypertrophic Signaling Required for the Development of Heart Failure

BACKGROUND: Cardiac myocyte hypertrophy is regulated by an extensive intracellular signal transduction network. In vitro evidence suggests that the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) serves as a nodal organizer of hypertrophic signaling. However, the relevance of mAKAPβ signalosomes to pathological remodeling and heart failure in vivo remains unknown. METHODS AND RESULTS: Using conditional, cardiac myocyte–specific gene deletion, we now demonstrate that mAKAPβ expression in mice is important for the cardiac hypertrophy induced by pressure overload and catecholamine toxicity. mAKAPβ targeting prevented the development of heart failure associated with long-term transverse aortic constriction, conferring a survival benefit. In contrast to 29% of control mice (n=24), only 6% of mAKAPβ knockout mice (n=31) died in the 16 weeks of pressure overload (P=0.02). Accordingly, mAKAPβ knockout inhibited myocardial apoptosis and the development of interstitial fibrosis, left atrial hypertrophy, and pulmonary edema. This improvement in cardiac status correlated with the attenuated activation of signaling pathways coordinated by the mAKAPβ scaffold, including the decreased phosphorylation of protein kinase D1 and histone deacetylase 4 that we reveal to participate in a new mAKAP signaling module. Furthermore, mAKAPβ knockout inhibited pathological gene expression directed by myocyte-enhancer factor-2 and nuclear factor of activated T-cell transcription factors that associate with the scaffold. CONCLUSIONS: mAKAPβ orchestrates signaling that regulates pathological cardiac remodeling in mice. Targeting of the underlying physical architecture of signaling networks, including mAKAPβ signalosome formation, may constitute an effective therapeutic strategy for the prevention and treatment of pathological remodeling and heart failure

p90 ribosomal S6 kinase 3 contributes to cardiac insufficiency in α-tropomyosin Glu180Gly transgenic mice

Myocardial interstitial fibrosis is an important contributor to the development of heart failure. Type 3 p90 ribosomal S6 kinase (RSK3) was recently shown to be required for concentric myocyte hypertrophy under in vivo pathological conditions. However, the role of RSK family members in myocardial fibrosis remains uninvestigated. Transgenic expression of α-tropomyosin containing a Glu180Gly mutation (TM180) in mice of a mixed C57BL/6:FVB/N background induces a cardiomyopathy characterized by a small left ventricle, interstitial fibrosis, and diminished systolic and diastolic function. Using this mouse model, we now show that RSK3 is required for the induction of interstitial fibrosis in vivo. TM180 transgenic mice were crossed to RSK3 constitutive knockout (RSK3(−/−)) mice. Although RSK3 knockout did not affect myocyte growth, the decreased cardiac function and mild pulmonary edema associated with the TM180 transgene were attenuated by RSK3 knockout. The improved cardiac function was consistent with reduced interstitial fibrosis in the TM180;RSK3(−/−) mice as shown by histology and gene expression analysis, including the decreased expression of collagens. The specific inhibition of RSK3 should be considered as a potential novel therapeutic strategy for improving cardiac function and the prevention of sudden cardiac death in diseases in which interstitial fibrosis contributes to the development of heart failure

Calcineurin Aβ–Specific Anchoring Confers Isoform-Specific Compartmentation and Function in Pathological Cardiac Myocyte Hypertrophy

Background: The Ca 2+ /calmodulin-dependent phosphatase calcineurin is a key regulator of cardiac myocyte hypertrophy in disease. An unexplained paradox is how the β isoform of the calcineurin catalytic A-subunit (CaNAβ) is required for induction of pathological myocyte hypertrophy, despite calcineurin Aα expression in the same cells. It is unclear how the pleiotropic second messenger Ca 2+ drives excitation–contraction coupling while not stimulating hypertrophy by calcineurin in the normal heart. Elucidation of the mechanisms conferring this selectivity in calcineurin signaling should reveal new strategies for targeting the phosphatase in disease. Methods: Primary adult rat ventricular myocytes were studied for morphology and intracellular signaling. New Förster resonance energy transfer reporters were used to assay Ca 2+ and calcineurin activity in living cells. Conditional gene deletion and adeno-associated virus–mediated gene delivery in the mouse were used to study calcineurin signaling after transverse aortic constriction in vivo. Results: CIP4 (Cdc42-interacting protein 4)/TRIP10 (thyroid hormone receptor interactor 10) was identified as a new polyproline domain-dependent scaffold for CaNAβ2 by yeast 2-hybrid screen. Cardiac myocyte–specific CIP4 gene deletion in mice attenuated pressure overload–induced pathological cardiac remodeling and heart failure. Blockade of CaNAβ polyproline-dependent anchoring using a competing peptide inhibited concentric hypertrophy in cultured myocytes; disruption of anchoring in vivo using an adeno-associated virus gene therapy vector inhibited cardiac hypertrophy and improved systolic function after pressure overload. Live cell Förster resonance energy transfer biosensor imaging of cultured myocytes revealed that Ca 2+ levels and calcineurin activity associated with the CIP4 compartment were increased by neurohormonal stimulation, but minimally by pacing. Conversely, Ca 2+ levels and calcineurin activity detected by nonlocalized Förster resonance energy transfer sensors were induced by pacing and minimally by neurohormonal stimulation, providing functional evidence for differential intracellular compartmentation of Ca 2+ and calcineurin signal transduction. Conclusions: These results support a structural model for Ca 2+ and CaNAβ compartmentation in cells based on an isoform-specific mechanism for calcineurin protein–protein interaction and localization. This mechanism provides an explanation for the specific role of CaNAβ in hypertrophy and its selective activation under conditions of pathologic stress. Disruption of CaNAβ polyproline-dependent anchoring constitutes a rational strategy for therapeutic targeting of CaNAβ-specific signaling responsible for pathological cardiac remodeling in cardiovascular disease deserving of further preclinical investigation

Regulation of Neuronal Survival and Axon Growth by a Perinuclear cAMP Compartment

cAMP signaling is known to be critical in neuronal survival and axon growth. Increasingly the subcellular compartmentation of cAMP signaling has been appreciated, but outside of dendritic synaptic regulation, few cAMP compartments have been defined in terms of molecular composition or function in neurons. Specificity in cAMP signaling is conferred in large part by A-kinase anchoring proteins (AKAPs) that localize protein kinase A and other signaling enzymes to discrete intracellular compartments. We now reveal that cAMP signaling within a perinuclear neuronal compartment organized by the large multivalent scaffold protein mAKAPα promotes neuronal survival and axon growth. mAKAPα signalosome function is explored using new molecular tools designed to specifically alter local cAMP levels as studied by live-cell FRET imaging. In addition, enhancement of mAKAPα-associated cAMP signaling by isoform-specific displacement of bound phosphodiesterase is demonstrated to increase retinal ganglion cell survival in mice of both sexes following optic nerve crush injury. These findings define a novel neuronal compartment that confers cAMP regulation of neuroprotection and axon growth and that may be therapeutically targeted in disease. cAMP is a second messenger responsible for the regulation of diverse cellular processes including neuronal neurite extension and survival following injury. Signal transduction by cAMP is highly compartmentalized in large part because of the formation of discrete, localized multimolecular signaling complexes by A-kinase anchoring proteins. Although the concept of cAMP compartmentation is well established, the function and identity of these compartments remain poorly understood in neurons. In this study, we provide evidence for a neuronal perinuclear cAMP compartment organized by the scaffold protein mAKAPα that is necessary and sufficient for the induction of neurite outgrowth and for the survival of retinal ganglion cells following optic nerve injury