Faculty Bibliography

Introduction: The main function of connexins is to form gap junctions; yet, recent studies show that Cx43 is not only a gap junction protein. In fact, Cx43 is a part of a protein interacting network (the connexome), likely to regulate other functions in a gap junction-independent manner. Recently, it was reported that loss of the last five amino acids of Cx43 (Cx43D378stop) leads to lethal ventricular arrhythmias in mice. Localization of Cx43 at the membrane and electrical coupling between cells was normal. Interestingly, there was a significant loss of sodium current amplitude. These observations linked two fundamental steps in action potential propagation, excitability and electrical coupling, through a common molecular mechanism. Here, we explore the hypothesis that the microtubular network at the cell end is part of the common link. Methods: N/A Results: Functional assays: Macropatch, and super-resolution scanning patch clamp in ventricular myocytes isolated from Cx43D378stop and Cre-negative (control) mice revealed a reduction in the amplitude of sodium current exclusively at the intercalated disc (ID), without a change in channel unitary conductance. Super-resolution fluorescence microscopy: direct stochastic optical reconstruction microscopy (20 nm resolution) showed Nav1.5 clusters in close proximity (or overlapping) with N-cadherin plaques. The distance between NaV1.5 clusters and the cell end increased from 57.2+12nm, n=365 in control to 111.7+11nm, n=446 in Cx43D378stop myocytes (p<0.001), indicating that mutation Cx43D378stop reduced NaV1.5 surface expression. This coincided with separation of the microtubule plus-end protein EB1 from N-cadherin-rich cell ends, from 23.7+31.9nm, n=665 in control, to 123.5+13.5nm, n=502 in Cx43D378stop cells (p<0.05). Conclusions: Functional surface expression of NaV1.5 at the ID depends on preservation of the Cx43 C-end. Cx43 is part of a molecular complex that anchors the microtubule plus-end to the cell end, thus allowing proper delivery of its ca!

This review summarizes data in support of the notion that the cardiac intercalated disc is the host of a protein interacting network, called "the connexome", where molecules classically defined as belonging to one particular structure (e.g., desmosomes, gap junctions, sodium channel complex) actually interact with others, and together, control excitability, electrical coupling and intercellular adhesion in the heart. The concept of the connexome is then translated into the understanding of the mechanisms leading to two inherited arrhythmia diseases: arrhythmogenic cardiomyopathy, and Brugada syndrome. The cross-over points in these two diseases are addressed to then suggest that, though separate identifiable clinical entities, they represent "bookends" of a spectrum of manifestations that vary depending on the effect that a particular mutation has on the connexome as a whole.

Telomere length homeostasis is essential for genomic stability and unlimited self-renewal of embryonic stem cells (ESCs). We show that telomere-associated protein Rif1 is required to maintain telomere length homeostasis by negatively regulating Zscan4 expression, a critical factor for telomere elongation by recombination. Depletion of Rif1 results in terminal hyperrecombination, telomere length heterogeneity, and chromosomal fusions. Reduction of Zscan4 by shRNA significantly rescues telomere recombination defects of Rif1-depleted ESCs and associated embryonic lethality. Further, Rif1 negatively modulates Zscan4 expression by maintaining H3K9me3 levels at subtelomeric regions. Mechanistically, Rif1 interacts and stabilizes H3K9 methylation complex. Thus, Rif1 regulates telomere length homeostasis of ESCs by mediating heterochromatic silencing.

We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr(166)), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr(166) nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO2-Tyr(166)-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNACUA pair for functional studies. Studies with mAb 4G11.2 showed that NO2-Tyr(166)-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for approximately 8% of total apoA-I within the artery wall but was nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO2-Tyr(166)-apoA-I existed as a lipid-poor lipoprotein with <3% recovered within the HDL-like fraction (d = 1.063-1.21). NO2-Tyr(166)-apoA-I in plasma showed a similar distribution. Recovery of NO2-Tyr(166)-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 +/- 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr(166) is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall.

Mitochondrial respiratory chain disorders are characterized by loss of electron transport chain (ETC) activity. Although the causes of many such diseases are known, there is a lack of effective therapies. To identify genes that confer resistance to severe ETC dysfunction when inactivated, we performed a genome-wide genetic screen in haploid human cells with the mitochondrial complex III inhibitor antimycin. This screen revealed that loss of ATPIF1 strongly protects against antimycin-induced ETC dysfunction and cell death by allowing for the maintenance of mitochondrial membrane potential. ATPIF1 loss protects against other forms of ETC dysfunction and is even essential for the viability of human rho degrees cells lacking mitochondrial DNA, a system commonly used for studying ETC dysfunction. Importantly, inhibition of ATPIF1 ameliorates complex III blockade in primary hepatocytes, a cell type afflicted in severe mitochondrial disease. Altogether, these results suggest that inhibition of ATPIF1 can ameliorate severe ETC dysfunction in mitochondrial pathology.

Accumulation of lipofuscin bisretinoids (LBs) in the retinal pigment epithelium (RPE) is the alleged cause of retinal degeneration in genetic blinding diseases (e.g., Stargardt) and a possible etiological agent for age-related macular degeneration. Currently, there are no approved treatments for these diseases; hence, agents that efficiently remove LBs from RPE would be valuable therapeutic candidates. Here, we show that beta cyclodextrins (beta-CDs) bind LBs and protect them against oxidation. Computer modeling and biochemical data are consistent with the encapsulation of the retinoid arms of LBs within the hydrophobic cavity of beta-CD. Importantly, beta-CD treatment reduced by 73% and 48% the LB content of RPE cell cultures and of eyecups obtained from Abca4-Rdh8 double knock-out (DKO) mice, respectively. Furthermore, intravitreal administration of beta-CDs reduced significantly the content of bisretinoids in the RPE of DKO animals. Thus, our results demonstrate the effectiveness of beta-CDs to complex and remove LB deposits from RPE cells and provide crucial data to develop novel prophylactic approaches for retinal disorders elicited by LBs.