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Lamin A/C (LMNA) is an important component of nuclear lamina. Mutations cause arrhythmia, heart failure, and sudden cardiac death. While LMNA-associated cardiomyopathy typically has an aggressive course that responds poorly to conventional heart failure therapies, there is variability in severity and age of penetrance between and even within specific mutations, which is poorly understood at the cellular level. Further, this heterogeneity has not previously been captured to mimic the heterozygous state, nor have the hundreds of clinical LMNA mutations been represented. Herein, we have overexpressed cardiopathic LMNA variants in HEK cells and utilized state-of-the-art quantitative proteomics to compare the global proteomic profiles of (1) aggregating Q353 K alone, (2) Q353 K coexpressed with WT, (3) aggregating N195 K coexpressed with WT, and (4) nonaggregating E317 K coexpressed with WT to help capture some of the heterogeneity between mutations. We analyzed each data set to obtain the differentially expressed proteins (DEPs) and applied gene ontology (GO) and KEGG pathway analyses. We found a range of 162 to 324 DEPs from over 6000 total protein IDs with differences in GO terms, KEGG pathways, and DEPs important in cardiac function, further highlighting the complexity of cardiac laminopathies. Pathways disrupted by LMNA mutations were validated with redox, autophagy, and apoptosis functional assays in both HEK 293 cells and in induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) for LMNA N195 K. These proteomic profiles expand our repertoire for mutation-specific downstream cellular effects that may become useful as druggable targets for personalized medicine approach for cardiac laminopathies.

Hypoxic-ischemic injury has been linked with increased risk for developing Alzheimer’s disease (AD). The underlying mechanism of this association is poorly understood. Here, we report distinct roles for hypoxia-inducible factor-1α (Hif-1α) in the regulation of BACE1 and γ-secretase activity, two proteases involved in the production of amyloid-beta (Aβ). We have demonstrated that Hif-1α upregulates both BACE1 and γ-secretase activity for Aβ production in brain hypoxia-induced either by cerebral hypoperfusion or breathing 10% O2. Hif-1α binds to γ-secretase, which elevates the amount of active γ-secretase complex without affecting the level of individual subunits in hypoxic-ischemic mouse brains. Additionally, the expression of full length Hif-1α increases BACE1 and γ-secretase activity in primary neuronal culture, whereas a transcriptionally incompetent Hif-1α variant only activates γ-secretase. These findings indicate that Hif-1α transcriptionally upregulates BACE1 and nontranscriptionally activates γ-secretase for Aβ production in hypoxic-ischemic conditions. Consequently, Hif-1α-mediated Aβ production may be an adaptive response to hypoxic-ischemic injury, subsequently leading to increased risk for AD. Preventing the interaction of Hif-1α with γ-secretase may therefore be a promising therapeutic strategy for AD treatment.

Membrane proteins represent a large family of proteins that perform vital physiological roles and represent key drug targets. Despite their importance, bioanalytical methods aiming to comprehensively characterize the post-translational modification (PTM) of membrane proteins remain challenging compared to other classes of proteins in part because of their inherent low expression and hydrophobicity. The inward rectifier potassium channel (Kir) 2.1, an integral membrane protein, is critical for the maintenance of the resting membrane potential and phase-3 repolarization of the cardiac action potential in the heart. The importance of this channel to cardiac physiology is highlighted by the recognition of several sudden arrhythmic death syndromes, Andersen–Tawil and short QT syndromes, which are associated with loss or gain of function mutations in Kir2.1, often triggered by changes in the β-adrenergic tone. Therefore, understanding the PTMs of this channel (particularly β-adrenergic tone-driven phosphorylation) is important for arrhythmia prevention. Here, we developed a proteomic method, integrating both top-down (intact protein) and bottom-up (after enzymatic digestion) proteomic analyses, to characterize the PTMs of recombinant wild-type and mutant Kir2.1, successfully mapping five novel sites of phosphorylation and confirming a sixth site. Our study provides a framework for future work to assess the role of PTMs in regulating Kir2.1 functions.

DNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care.

The GTPase OPA1 and the AAA-protease OMA1 serve well-established roles in mitochondrial stress responses and mitochondria-initiated cell death. In addition to its role in mitochondrial membrane fusion, cristae structure, and bioenergetic function, OPA1 controls apoptosis by sequestering cytochrome c (cyt c) in mitochondrial cristae. Cleavage of functional long OPA1 (L-OPA1) isoforms by OMA1 inactivates mitochondrial fusion and primes apoptosis. OPA1 cleavage is regulated by the prohibitin (PHB) complex, a heteromeric, ring-shaped mitochondrial inner membrane scaffolding complex composed of PHB1 and PHB2. In neurons, PHB plays a protective role against various stresses, and PHB deletion destabilizes OPA1 causing neurodegeneration. While deletion of OMA1 prevents OPA1 destabilization and attenuates neurodegeneration in PHB2 KO mice, how PHB levels regulate OMA1 is still unknown. Here, we investigate the effects of modulating neuronal PHB levels on OMA1 stability and OPA1 cleavage. We demonstrate that PHB promotes OMA1 turnover, effectively decreasing the pool of OMA1. Further, we show that OMA1 binds to cardiolipin (CL), a major mitochondrial phospholipid. CL binding promotes OMA1 turnover, as we show that deleting the CL-binding domain of OMA1 decreases its turnover rate. Since PHB is known to stabilize CL, these data suggest that PHB modulates OMA1 through CL. Furthermore, we show that PHB decreases cyt c release induced by tBID and attenuates caspase 9 activation in response to hypoxic stress in neurons. Taken together, our results suggest that PHB-mediated CL stabilization regulates stress responses and cell death through OMA1 turnover and cyt c release.

The nine-banded armadillo (Dasypus novemcinctus) is unique among mammals because females produce litters of genetically identical quadruplets via monozygotic polyembryony. This unusual form of reproduction could have profound impacts on the spatial genetic structure of populations of armadillos, but at present it is unclear whether littermates remain together as adults, or if sex-biased dispersal occurs. The goal of our study was to determine whether fine-scale spatial genetic structure (FSGS) within a population of armadillos diminishes with age (i.e., from juvenile to adult due to the dispersal of littermates away from one another), and if the degree of FSGS for a given age class differs between the sexes. We obtained genotype data at seven microsatellite DNA loci for 421 individuals in a wild population of armadillos inhabiting the Yazoo National Wildlife Refuge in western Mississippi. Correlogram analyses based on measures of spatial genetic autocorrelation showed weak but significant FSGS that was driven by positive spatial genetic autocorrelation among both male and female juveniles and adult males, but not adult females. Positive spatial genetic autocorrelation among adult males could be due to either female-biased dispersal or high variance in male reproductive success. Further work is required to discriminate between these two possibilities.

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The molecular mechanisms underlying congenital long QT syndrome (LQTS) are now beginning to be understood. New insights into the etiology and therapeutic strategies are emerging from heterologous expression studies of LQTS-linked mutant proteins, as well as inducible pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from LQTS patients. This review focuses on the major molecular mechanism that underlies LQTS type 2 (LQT2). LQT2 is caused by loss of function (LOF) mutations in KCNH2(also known as the human Ether-à-go-go-Related Geneor hERG). Most LQT2-linked mutations are missense mutations and functional studies suggest that ~90% of them disrupt the intracellular transport (trafficking) of KCNH2-encoded Kv11.1 proteins to the cell membrane. Trafficking deficient LQT2 mutations disrupt Kv11.1 protein folding and misfolded Kv11.1 proteins are retained in the endoplasmic reticulum (ER) until they are degraded in the ER associated degradation pathway (ERAD). This review focuses on the quality control mechanisms in the ER that contribute to the folding and ERAD of Kv11.1 proteins; the mechanism for ER export of Kv11.1 proteins in the secretory pathway; different subclasses of trafficking deficient LQT2 mutations; and strategies being developed to mitigate or correct trafficking deficient LQT2-related phenotypes.

The molecular mechanisms underlying congenital long QT syndrome (LQTS) are now beginning to be understood. New insights into the etiology and therapeutic strategies are emerging from heterologous expression studies of LQTS-linked mutant proteins, as well as inducible pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from LQTS patients. This review focuses on the major molecular mechanism that underlies LQTS type 2 (LQT2). LQT2 is caused by loss of function (LOF) mutations in KCNH2(also known as the human Ether-à-go-go-Related Geneor hERG). Most LQT2-linked mutations are missense mutations and functional studies suggest that ~90% of them disrupt the intracellular transport (trafficking) of KCNH2-encoded Kv11.1 proteins to the cell membrane. Trafficking deficient LQT2 mutations disrupt Kv11.1 protein folding and misfolded Kv11.1 proteins are retained in the endoplasmic reticulum (ER) until they are degraded in the ER associated degradation pathway (ERAD). This review focuses on the quality control mechanisms in the ER that contribute to the folding and ERAD of Kv11.1 proteins; the mechanism for ER export of Kv11.1 proteins in the secretory pathway; different subclasses of trafficking deficient LQT2 mutations; and strategies being developed to mitigate or correct trafficking deficient LQT2-related phenotypes.

Inherited arrhythmia syndromes comprise an increasingly complex group of diseases involving mutations in multiple genes encoding ion channels, ion channel accessory subunits and channel interacting proteins, and various regulatory elements. These mutations serve to disrupt normal electrophysiology in the heart, leading to increased arrhythmogenic risk and death. These diseases have added impact as they often affect young people, sometimes without warning. Although originally thought to alter ion channel function, it is now increasingly recognized that mutations may alter ion channel protein and messenger RNA processing, to reduce the number of channels reaching the surface membrane. For many of these mutations, it is also known that several interventions may restore protein processing of mutant channels to increase their surface membrane expression toward normal. In this article, we reviewed inherited arrhythmia syndromes, focusing on long QT syndrome type 2, and discuss the complex biology of ion channel trafficking and pharmacological rescue of disease-causing mutant channels. Pharmacological rescue of misprocessed mutant channel proteins, or their transcripts providing appropriate small molecule drugs can be developed, has the potential for novel clinical therapies in some patients with inherited arrhythmia syndromes.

Mitochondrial cardiomyopathies are fatal diseases, with no effective treatment. Alterations of heart mitochondrial function activate the mitochondrial integrated stress response (ISRmt), a transcriptional program affecting cell metabolism, mitochondrial biogenesis, and proteostasis. In humans, mutations in CHCHD10, a mitochondrial protein with unknown function, were recently associated with dominant multi-system mitochondrial diseases, whose pathogenic mechanisms remain to be elucidated. Here, in CHCHD10 knockin mutant mice, we identify an extensive cardiac metabolic rewiring triggered by proteotoxic ISRmt. The stress response arises early on, before the onset of bioenergetic impairments, triggering a switch from oxidative to glycolytic metabolism, enhancement of transsulfuration and one carbon (1C) metabolism, and widespread metabolic imbalance. In parallel, increased NADPH oxidases elicit antioxidant responses, leading to heme depletion. As the disease progresses, the adaptive metabolic stress response fails, resulting in fatal cardiomyopathy. Our findings suggest that early interventions to counteract metabolic imbalance could ameliorate mitochondrial cardiomyopathy associated with proteotoxic ISRmt.

Women Hunting I JUST WANTED TO SAY I REALLY appreciate the article on the Women Hunt program in the September/October Issue. Kaycee Smith Smoke-Colored Turkey I READ YOUR ARTICLE ONLINE about turkey color phases. Albino Hog NOTE: In the TF&G newsletter we posted an article looking for photos of white or albino feral hogs. [Extracted from the article]

Mutations in the KCNH2 or human ether-a-go-go–related gene-encoded K+ channel reduce functional KCNH2 current (IKCNH2) to cause long QT syndrome (LQT2) by multiple mechanisms, including defects in intracellular transport (trafficking). Trafficking-deficient, or class 2, LQT2 mutations reduce the Golgi processing and surface membrane expression of KCNH2 channel proteins. Drugs that associate with pore-S6 intracellular drug binding domain of KCNH2 channel proteins to cause high-affinity block of IKCNH2 also can increase the processing of class 2 LQT2 channel proteins through the secretory pathway. We used a strategy of intragenic suppression to test the hypothesis that amino acid substitutions in the putative drug binding domain at residue Y652 could compensate for protein folding abnormalities caused by class 2 LQT2 mutations. We found that the Y652C substitution, and to lesser extent the Y652S substitution, resulted in intragenic suppression of the class 2 LQT2 G601S phenotype; these substitutions increased Golgi processing of G601S channel proteins. The Y652C substitution also caused intragenic suppression of the class 2 LQT2 V612L and F640V phenotypes but not the LQT2 N470D or F805C phenotypes. These are the first findings to demonstrate that a single amino acid substitution in the putative KCNH2 drug binding domain can cause intragenic suppression of several LQT2 mutations.

Background:Idiopathic ventricular fibrillation (IVF) is a clinically challenging disease entity as phenotypic characteristics are lacking. We studied induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) from a patient with stress-induced IVF to define the cellular arrhythmic behavior and create a path to test therapeutics.Methods:iPS-CMs were generated from control (WT) and stress-induced IVF (si-IVF) patient-specific iPSC lines. Cells were characterized by single-cell optical mapping using calcium sensitive dye (Fluo-4) and electrical pacing (0.5Hz), with β-adrenergic stimulation (1x10-6mM isoproterenol (ISO)).Results:si-IVF cells had longer CaT rise time (ms) at baseline (BSL) (p<0.05) (86 +/- 5.45, n=54) and after ISO (83.88 +/- 5.12, n=41) than WT cells (BSL: 64.5 +/- 3.72, n=40) (ISO: 72 +/- 9.92, n=10). si-IVF cells had shorter CaT duration (ms) at BSL (1,069 +/- 36.00, n=54) than WT cells at BSL (1,280 +/- 37.59, n=40), with no significant difference after ISO (IVF: 1,073 +/- 44.51, n= 41 and WT: 1,230 +/- 32.79, n=10). Following ISO, 11.5% of si-IVF cells showed arrhythmic behavior with rapid continuous spontaneous Ca2+release (Fig. 1), compared to 0.0% of WT cells.Conclusion:We demonstrate feasibility of differentiating iPS-CMs from patients while investigating arrhythmogenicity of si-IVF cells compared to controls using optical mapping. Insights from CaT duration, rise time, and arrhythmogenic behavior helps establish the arrhythmogenic behavior, and begins to provide mechanistic insights into si-IVF. This methodology can be applied to test therapeutics for IVF patients with β-adrenergic triggers. Figure 1:Patient #1-specific si-IVF iPS-CM. A)Representative optical mapping recordings of iPS-CMs baseline paced at 1Hz (each trace is a separate cell) B)Application of ISO caused arrhythmic behavior and failure to capture paced beats in black, red, blue, and yellow traces but retention of paced response in green and grey traces.

Background: A trend towards decreased peripheral corneal flattening with increasing myopia has recently been demonstrated. The present study was conducted to determine whether corneal asphericity also varies significantly with hyperopic refractive error.Methods: Thirty‐five eyes with spherical equivalent refractive error ranging from ‐0.37 D to +6.00 D were examined. A conicoid equation was fitted to videokeratoscopic (Topographic Modeling System) data and corneal asphericity and apical radius of curvature values were calculated for each subject. Axial length measurements were made using a hand‐held biometric ruler. Keratometry was also performed on each eye.Results: The relationship between corneal asphericity (Q) and spherical equivalent refractive error was not statistically significant (p = 0.7419). In addition, no association could be demonstrated between Q and corneal radius of curvature or between Q and axial length. Corneal radius of curvature was positively correlated with axial length (r = 0.367, p = 0.0298). Axial length was found to decrease as hyperopic refractive error increased (r = 0.753, p = 0.0001).

Background: A trend towards decreased peripheral corneal flattening with increasing myopia has recently been demonstrated. The present study was conducted to determine whether corneal asphericity also varies significantly with hyperopic refractive error.

Introduction:Lamin A/C (LMNA) are intermediate filament proteins that form the nuclear lamina and have several functions including regulating transcription, organizing chromatin and providing structure to the nucleus. More than 200 missense LMNA variants have been linked to over a dozen diseases including cardiomyopathy. We previously reported that nuclear Lamin A aggregation is a major factor underlying skeletal and cardiac muscle laminopathies using an HEK overexpression model. Validating these results in more disease-relevant skeletal and cardiac muscle cells is important towards understanding their tissue specific effects. We performed a comparative Lamin A aggregation analysis using HEK, C2C12 mouse myoblast and iPSC-derived cardiomyocyte overexpression models.Results:Using a GFP tagged Lamin A construct, we systematically characterized nuclear aggregation for 172 disease-linked LMNA variants in each of its four coiled-coil domains (CCD 1A, 1B, 1C and 1D) and Ig-like domain (IgD) using HEK and C2C12 cells (n?2). CCD variants linked to cardiac muscle disease aggregated less in C2C12 cells (18/47(38%)) compared to HEK cells (27/48 (56%), while skeletal muscle disease variants aggregated similarly in C2C12 cells (50/66 (76%)) compared to HEK cells (53/68 (78%). In contrast, only 7/49 (14%) of IgD variants linked to cardiac and skeletal muscle disease aggregated compared to 35/48 (73%) in HEK cells. Less prevalent variants linked to premature aging syndromes and lipodystrophy showed little to no aggregation ranging from 0-26%. Interestingly, several skeletal disease LMNA variants that did not aggregate (e.g. R298C and N459S) are present in genotyping control cohorts (Exome Aggregation Consortium) supporting misclassification. Finally, we observed nuclear aggregation for a subset of eight representative LMNA variants using an iPSC-CM overexpression model.Conclusions:This study 1) validates LMNA aggregation as an important determinant in skeletal and cardiac laminopathies and highlights the variability between expression models, 2) demonstrates an iPSC-CM experimental platform for characterizing laminopathic mutations, and 3) underscores the importance of functional assays to assess the pathogenicity for variants.