Search

Summary: We investigated the role of the N-terminus (residues 1–43) of the myosin essential light chain (N-ELC) in regulating cardiac function in hypertrophic (HCM-A57G) and restrictive (RCM-E143K) cardiomyopathy mice. Both models were cross-genotyped with N-ELC-truncated Δ43 mice, and the offspring were studied using echocardiography and muscle contractile mechanics. In A57G×Δ43 mice, Δ43 expression improved heart function and reduced hypertrophy and fibrosis. No improvements were seen in E143K×Δ43 compared to RCM-E143K mice. HCM-mutant pathology involved an impaired N-ELC tension sensor, disrupted N-ELC-actin interactions, an altered force-pCa relationship, and a destabilized myosin’s super-relaxed state. Removal of the malfunctioning N-ELC sensor led to functional rescue in HCM-truncated mutant hearts. However, the RCM mutation could not be rescued by N-ELC deletion, likely due to its proximity to the myosin motor domain, affecting lever-arm rigidity and myosin function. This study provides insights into the role of N-ELC in the development and potential rescue of ELC-mutant cardiomyopathy.

Abstract The agreement between fluorescein breakup time (FBUT) estimates and noninvasive breakup time (NIBUT) estimates automatically acquired using two videokeratoscopes was assessed. Small-cone (E300, Medmont) and large-bowl (K5M, OCULUS) Placido-ring–based videokeratoscopes were utilized for automated NIBUT estimation and fluorescein strip method was used for FBUT estimation. 33 subjects completed the study. Clear instructions were given regarding the blink before measurements. Bland–Altman analysis was used to test the agreement between tear film breakup time estimates and repeated measure analysis of variance was used to test the differences between measurement types. In comparison to FBUT, E300 NIBUT estimate was shorter (− 0.6 ± 2.6 s), whereas K5M NIBUT estimate was longer (3.3 ± 2.4 s). Limits of agreement for FBUT vs. E300 was 29.8 s, for FBUT vs. K5M 26.4 s, and 31.4 s for E300 vs. K5M. There were significant differences between tear film breakup times (F = 3.59, df = 2, P = 0.032). E300 and K5M NIBUT measurements have poor agreement with FBUT, even when blinking is precisely clarified. Agreement was better for shorted break up times than that for longer ones but in general NIBUT measurements are not interchangeable.

The short form of the leptin receptor (LeptRa) plays a key role in the transport of leptin to the central nervous system (CNS). Here, MTS-leptin and recombinant ovine (ro) leptin-mediated expression of LeptRa and VEGFA and VEGFR2 concentration in selected hypothalamic nuclei, choroid plexus (ChP), and anterior pituitary (AP) were analyzed considering the photoperiod and acute-fasting (experiment 1), and nutritional status (experiment 2) of ewes. In experiment 1, 60 sheep were fed normally or fasted for 72 h and received one injection of saline, MTS-leptin, or roleptin 1 h prior to euthanasia. LeptRa mRNA transcript levels and VEGF system protein concentrations were detected in the ARC, ChP predominantly in the SD, and AP for the LD without detection of LeptRa in the POA and VMH/DMH. In experiment 2, an altered diet for 5 months created lean or fat sheep. Twenty sheep were divided into four groups: the lean and fat groups were given saline, while the lean-R and fat-R groups received resistin 1 h prior to euthanasia. Changes in adiposity influenced the lowering effect of resistin on the expression of LeptRa and VEGF system protein concentrations. Overall, both photoperiodic and nutritional signals influence the effects of MTS-leptin/roleptin and resistin-mediated leptin transport to the CNS via LeptRa. Resistin seems to be another adipokine involved in the adaptive/pathological phenomenon of leptin resistance in sheep.

Abstract This study evaluates the effectiveness of objective techniques in assessing dry eye disease (DED) treatment compared with the subjective assessment commonly used in clinical practice. Thirty subjects were recruited for two visits separated by 28(± 3) days of treatment with artificial tears. A buttery of common subjective assessment methods were accompanied by a set of objective techniques including measurement of noninvasive tear film break-up time (NIBUT), lipid layer thickness (LLT), and quantitative evaluation of tear film surface quality and dynamics (TFD). Additionally, meibography was performed. Two commercially available videokeratoscopes and a prototype of a lateral shearing interferometer were used for the measurements. Both subjective and objective techniques showed a positive effect of artificial tears in DED treatment. Statistically significant improvements were observed in subjective symptoms (from P

ABSTRACT Many eukaryotic membrane-dependent functions are often spatially and temporally regulated by membrane microdomains (FMMs), also known as lipid rafts. These domains are enriched in polyisoprenoid lipids and scaffolding proteins belonging to the stomatin, prohibitin, flotillin, and HflK/C (SPFH) protein superfamily that was also identified in Gram-positive bacteria. In contrast, little is still known about FMMs in Gram-negative bacteria. In Escherichia coli K-12, 4 SPFH proteins, YqiK, QmcA, HflK, and HflC, were shown to localize in discrete polar or lateral inner membrane locations, raising the possibility that E. coli SPFH proteins could contribute to the assembly of inner membrane FMMs and the regulation of cellular processes. Here, we studied the determinant of the localization of QmcA and HflC and showed that FMM-associated cardiolipin lipid biosynthesis is required for their native localization pattern. Using Biolog phenotypic arrays, we showed that a mutant lacking all SPFH genes displayed increased sensitivity to aminoglycosides and oxidative stress that is due to the absence of HflKC. Our study therefore provides further insights into the contribution of SPFH proteins to stress tolerance in E. coli. IMPORTANCE Eukaryotic cells often segregate physiological processes in cholesterol-rich functional membrane microdomains. These domains are also called lipid rafts and contain proteins of the stomatin, prohibitin, flotillin, and HflK/C (SPFH) superfamily, which are also present in prokaryotes but have been mostly studied in Gram-positive bacteria. Here, we showed that the cell localization of the SPFH proteins QmcA and HflKC in the Gram-negative bacterium E. coli is altered in the absence of cardiolipin lipid synthesis. This suggests that cardiolipins contribute to E. coli membrane microdomain assembly. Using a broad phenotypic analysis, we also showed that HflKC contribute to E. coli tolerance to aminoglycosides and oxidative stress. Our study, therefore, provides new insights into the cellular processes associated with SPFH proteins in E. coli.

Dilated cardiomyopathy (DCM) is a devastating heart disease that affects about 1 million people in the United States, but the underlying mechanisms remain poorly understood. In this study, we aimed to determine the biomechanical and structural causes of DCM in transgenic mice carrying a novel mutation in the MYL2 gene, encoding the cardiac myosin regulatory light chain. Transgenic D94A (aspartic acid-to-alanine) mice were created and investigated by echocardiography and invasive hemodynamic and molecular structural and functional assessments. Consistent with the DCM phenotype, a significant reduction of the ejection fraction (EF) was observed in ∼5- and ∼12-mo-old male and female D94A lines compared with respective WT controls. Younger male D94A mice showed a more pronounced left ventricular (LV) chamber dilation compared with female counterparts, but both sexes of D94A lines developed DCM by 12 mo of age. The hypocontractile activity of D94A myosin motors resulted in the rightward shift of the force-pCa dependence and decreased actin-activated myosin ATPase activity. Consistent with a decreased Ca2+ sensitivity of contractile force, a small-angle X-ray diffraction study, performed in D94A fibers at submaximal Ca2+ concentrations, revealed repositioning of the D94A cross-bridge mass toward the thick-filament backbone supporting the hypocontractile state of D94A myosin motors. Our data suggest that structural perturbations at the level of sarcomeres result in aberrant cardiomyocyte cytoarchitecture and lead to LV chamber dilation and decreased EF, manifesting in systolic dysfunction of D94A hearts. The D94A-induced development of DCM in mice closely follows the clinical phenotype and suggests that MYL2 may serve as a new therapeutic target for dilated cardiomyopathy.

Casirivimab and imdevimab (REGN-COV-2) are investigational monoclonal antibodies approved in November 2020 by the Food and Drug Administration for emergency use in mild and moderate COVID-19. These two noncompeting human IgG1 monoclonal antibodies can target the receptor-binding domain of the spike protein of SARSCoV-2, prevent its entry into human cells, and reduce viral load. The antibodies can be administered intravenously for mild-to-moderate COVID-19 patients who do not require hospitalization and supplemental oxygen. The purpose of the study is to review the latest available data on COVID-19 treatment using casirivimab and imdevimab. According to recent preclinical studies, the antibody cocktail presents optimal antiviral strength and has the potential to minimize the chances of the virus escaping. It was shown in animal studies that the cocktail reduces the pathological consequences caused by viruses, decreases the number of viruses in the respiratory system, and reduces lung titers and pneumonia symptoms. Casirivimab and imdevimab as a cocktail also prevents the rapid appearance of treatment-resistant mutants. In the clinical trial, REGN-COV-2 decreased viral load, particularly in patients with a non-initiated immune response (serum antibody-negative) and with high viral load at baseline. The adverse effects were comparable in the combined REGN-COV2 dose groups (2.4 g and 8.0 g), as well as in the placebo group. The cocktail caused few and mainly low-grade toxic effects. Casirivimab and imdevimab seem to be effective and safe antiviral therapy for nonhospitalized patients with COVID-19. Further observations and research are extremely necessary to assess the efficacy, security and indications in a wider group of patients.

In this study, we investigated the rescue potential of two phosphomimetic mutants of the myosin regulatory light chain (RLC, MYL2 gene), S15D, and T160D RLCs. S15D-RLC mimics phosphorylation of the established serine-15 site of the human cardiac RLC. T160D-RLC mimics the phosphorylation of threonine-160, identified by computational analysis as a high-score phosphorylation site of myosin RLC. Cardiac myosin and left ventricular papillary muscle (LVPM) fibers were isolated from a previously generated model of hypertrophic cardiomyopathy (HCM), Tg-R58Q, and Tg-wild-type (WT) mice. Muscle specimens were first depleted of endogenous RLC and then reconstituted with recombinant human cardiac S15D and T160D phosphomimetic RLCs. Preparations reconstituted with recombinant human cardiac WT-RLC and R58Q-RLC served as controls. Mouse myosins were then tested for the actin-activated myosin ATPase activity and LVPM fibers for the steady-state force development and Ca2+-sensitivity of force. The data showed that S15D-RLC significantly increased myosin ATPase activity compared with T160D-RLC or WT-RLC reconstituted preparations. The two S15D and T160D phosphomimetic RLCs were able to rescue Vmax of Tg-R58Q myosin reconstituted with recombinant R58Q-RLC, but the effect of S15D-RLC was more pronounced than T160D-RLC. Low tension observed for R58Q-RLC reconstituted LVPM from Tg-R58Q mice was equally rescued by both phosphomimetic RLCs. In the HCM Tg-R58Q myocardium, the S15D-RLC caused a shift from the super-relaxed (SRX) state to the disordered relaxed (DRX) state, and the number of heads readily available to interact with actin and produce force was increased. At the same time, T160D-RLC stabilized the SRX state at a level similar to R58Q-RLC reconstituted fibers. We report here on the functional superiority of the established S15 phospho-site of the human cardiac RLC vs. C-terminus T160-RLC, with S15D-RLC showing therapeutic potential in mitigating a non-canonical HCM behavior underlined by hypocontractile behavior of Tg-R58Q myocardium.

This study focuses on mimicking constitutive phosphorylation in the N-terminus of the myosin regulatory light chain (S15D-RLC) as a rescue strategy for mutation-induced cardiac dysfunction in transgenic (Tg) models of restrictive (RCM) and dilated (DCM) cardiomyopathy caused by mutations in essential (ELC, MYL3 gene) or regulatory (RLC, MYL2 gene) light chains of myosin. Phosphomimetic S15D-RLC was reconstituted in left ventricular papillary muscle (LVPM) fibers from two mouse models of cardiomyopathy, RCM-E143K ELC and DCM-D94A RLC, along with their corresponding Tg-ELC and Tg-RLC wild-type (WT) mice. The beneficial effects of S15D-RLC in rescuing cardiac function were manifested by the S15D-RLC-induced destabilization of the super-relaxed (SRX) state that was observed in both models of cardiomyopathy. S15D-RLC promoted a shift from the SRX state to the disordered relaxed (DRX) state, increasing the number of heads readily available to interact with actin and produce force. Additionally, S15D-RLC reconstituted with fibers demonstrated significantly higher maximal isometric force per cross-section of muscle compared with reconstitution with WT-RLC protein. The effects of the phosphomimetic S15D-RLC were compared with those observed for Omecamtiv Mecarbil (OM), a myosin activator shown to bind to the catalytic site of cardiac myosin and increase myocardial contractility. A similar SRX↔DRX equilibrium shift was observed in OM-treated fibers as in S15D-RLC-reconstituted preparations. Additionally, treatment with OM resulted in significantly higher maximal pCa 4 force per cross-section of muscle fibers in both cardiomyopathy models. Our results suggest that both treatments with S15D-RLC and OM may improve the function of myosin motors and cardiac muscle contraction in RCM-ELC and DCM-RLC mice.

AimsThe E143K (Glu → Lys) mutation in the myosin essential light chain has been associated with restrictive cardiomyopathy (RCM) in humans, but the mechanisms that underlie the development of defective cardiac function are unknown. Using transgenic E143K-RCM mice, we sought to determine the molecular and cellular triggers of E143K-induced heart remodelling.Methods and resultsThe E143K-induced abnormalities in cardiac function and morphology observed by echocardiography and invasive haemodynamics were paralleled by augmented active and passive tension measured in skinned papillary muscle fibres compared with wild-type (WT)-generated force. In vitro, E143K-myosin had increased duty ratio and binding affinity to actin compared with WT-myosin, increased actin-activated ATPase activity and slower rates of ATP-dependent dissociation of the acto-myosin complex, indicating an E143K-induced myosin hypercontractility. E143K was also observed to reduce the level of myosin regulatory light chain phosphorylation while that of troponin-I remained unchanged. Small-angle X-ray diffraction data showed a decrease in the filament lattice spacing (d1,0) with no changes in the equatorial reflections intensity ratios (I1,1/I1,0) in E143K vs. WT skinned papillary muscles. The hearts of mutant-mice demonstrated ultrastructural defects and fibrosis that progressively worsened in senescent animals and these changes were hypothesized to contribute to diastolic disturbance and to mild systolic dysfunction. Gene expression profiles of E143K-hearts supported the histopathology results and showed an upregulation of stress-response and collagen genes. Finally, proteomic analysis evidenced RCM-dependent metabolic adaptations and higher energy demands in E143K vs. WT hearts.ConclusionsAs a result of the E143K-induced myosin hypercontractility, the hearts of RCM mice model exhibited cardiac dysfunction, stiff ventricles and physiological, morphologic, and metabolic remodelling consistent with the development of RCM. Future efforts should be directed toward normalization of myosin motor function and the use of myosin-specific therapeutics to avert the hypercontractile state of E143K-myosin and prevent pathological cardiac remodelling.