Search

Positioned within the eye, the lens supports vision by transmitting and focusing light onto the retina. As an adaptive glassy material, the lens is constituted primarily by densely-packed, polydisperse crystallin proteins that organize to resist aggregation and crystallization at high volume fractions, yet the details of how crystallins coordinate with one another to template and maintain this transparent microstructure remain unclear. The role of individual crystallin subtypes (α, β, and γ) and paired subtype compositions, including how they experience and resist crowding-induced turbidity in solution, is explored using combinations of spectrophotometry, hard-sphere simulations, and surface pressure measurements. After assaying crystallin combinations, β-crystallins emerged as a principal component in all mixtures that enabled dense fluid-like packing and short-range order necessary for transparency. These findings helped inform the design of lens-like hydrogel systems, which are used to monitor and manipulate the loss of transparency under different crowding conditions. When taken together, the findings illustrate the design and characterization of adaptive materials made from lens proteins that can be used to better understand mechanisms regulating transparency., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Crystallins comprise the protein-rich tissue of the eye lens. Of the three most common vertebrate subtypes, β-crystallins exhibit the widest degree of polydispersity due to their complex multimerization properties in situ. While polydispersity enables precise packing densities across the concentration gradient of the lens for vision, it is unclear why there is such a high degree of structural complexity within the β-crystallin subtype and what the role of this feature is in the lens. To investigate this, we first characterized β-crystallin polydispersity and then established a method to dynamically disrupt it in a process that is dependent on isoform composition and the presence of divalent cationic salts (CaCl 2 or MgCl 2 ). We used size-exclusion chromatography together with dynamic light scattering and mass spectrometry to show how high concentrations of divalent cations dissociate β-crystallin oligomers, reduce polydispersity, and shift the overall protein surface charge-properties that can be reversed when salts are removed. While the direct, physiological relevance of these divalent cations in the lens is still under investigation, our results support that specific isoforms of β-crystallin modulate polydispersity through multiple chemical equilibria and that this native state is disrupted by cation binding. This dynamic process may be essential to facilitating the molecular packing and optical function of the lens.

Background: Collagenous tissues are composed of precisely oriented, tightly packed collagen fibril bundles to confer the maximal strength within the smallest volume. While this compact form benefits mobility, it consequentially restricts vascularity and cell density to a minimally viable level in some regions. These tissues reside in a homeostatic state with an unstable equilibrium, where perturbations to structure or molecular milieu cause descension into a long-term compromised state. Several studies have shown that glycosaminoglycans are key molecules required for healthy tissue maintenance. Our long-term goal is to determine if glycosaminoglycans serve a critical function of stabilizing soluble monomeric collagen in the interstitial fluid that bathes tissue for immediate availability in tissue development and repair in vivo. Materials and Methods: To test glycosaminoglycan and collagen interactions at the most fundamental level, we have explored the effect of the monosaccharides that populate the glycosaminoglycans of the extracellular matrix on collagen assembly kinetics, pre-established matrix stability, and collagen incorporation into a preassembled matrix. Results: Results showed that monosaccharides increased the threshold concentration required for spontaneous polymerization by at least three orders of magnitude. When the monosaccharides were introduced to a pre-existing collagen network, fibrillar dissociation was undetectable. Fluorescent-labeling studies illustrated that in the presence of the saccharide solution, soluble collagen maintains the functional capacity to integrate into a pre-existing network. Conclusion: This work demonstrates a feasible role for glycosaminoglycans in supporting tissue remodeling and highlights the potential importance of age-related deterioration of glycosaminoglycan biosynthesis in reference to the homeostasis of collagen-based tissues., Competing Interests: No competing financial interests exist., (Copyright 2020, Mary Ann Liebert, Inc., publishers.)

Light chain (AL) amyloidosis is a devastating, complex, and incurable protein misfolding disease. It is characterized by an abnormal proliferation of plasma cells (fully differentiated B cells) producing an excess of monoclonal immunoglobulin light chains that are secreted into circulation, where the light chains misfold, aggregate as amyloid fibrils in target organs, and cause organ dysfunction, organ failure, and death. In this article, we will review the factors that contribute to AL amyloidosis complexity, the findings by our laboratory from the last 16 years and the work from other laboratories on understanding the structural, kinetics, and thermodynamic contributions that drive immunoglobulin light chain-associated amyloidosis. We will discuss the role of cofactors and the mechanism of cellular damage. Last, we will review our recent findings on the high resolution structure of AL amyloid fibrils. AL amyloidosis is the best example of protein sequence diversity in misfolding diseases, as each patient has a unique combination of germline donor sequences and multiple amino acid mutations in the protein that forms the amyloid fibril.

Although enhanced cardiac matrix metalloproteinase (MMP)-2 synthesis has been associated with ventricular remodeling and failure, whether MMP-2 expression is a direct mediator of this process is unknown. We generated transgenic mice expressing active MMP-2 driven by the alpha-myosin heavy chain promoter. At 4 mo MMP-2 transgenic hearts demonstrated expression of the MMP-2 transgene, myocyte hypertrophy, breakdown of Z-band registration, lysis of myofilaments, disruption of sarcomere and mitochondrial architecture, and cardiac fibroblast proliferation. Hearts from 8-mo-old transgenic mice displayed extensive myocyte disorganization and dropout with replacement fibrosis and perivascular fibrosis. Older transgenic mice also exhibited a massive increase in cardiac MMP-2 expression, representing recruitment of endogenous MMP-2 synthesis, with associated expression of MMP-9 and membrane type 1 MMP. Increases in diastolic [control (C) 33 +/- 3 vs. MMP 51 +/- 12 microl; P = 0.003] and systolic (C 7 +/- 2 vs. MMP 28 +/- 14 microl; P = 0.003) left ventricular (LV) volumes and relatively preserved stroke volume (C 26 +/- 4 vs. MMP 23 +/- 3 microl; P = 0.16) resulted in markedly decreased LV ejection fraction (C 78 +/- 7% vs. MMP 48 +/- 16%; P = 0.0006). Markedly impaired systolic function in the MMP transgenic mice was demonstrated in the reduced preload-adjusted maximal power (C 240 +/- 84 vs. MMP 78 +/- 49 mW/microl(2); P = 0.0003) and decreased end-systolic pressure-volume relation (C 7.5 +/- 1.5 vs. MMP 4.7 +/- 2.0; P = 0.016). Expression of active MMP-2 is sufficient to induce severe ventricular remodeling and systolic dysfunction in the absence of superimposed injury.

Matrix metalloproteinase-2 (MMP-2) is a central component of the response to injury in the heart. During ischemia, MMP-2 influences ventricular performance and is a determinant of postinfarction remodeling. Elevation of MMP-2 during reperfusion after ischemia suggests that new protein is synthesized, but the molecular regulation of MMP-2 generation during ischemia-reperfusion (I/R) injury has not been studied. Using the MMP-2 promoter linked to a beta-galactosidase reporter in transgenic mice, we investigated the transcriptional regulation and cellular sources of MMP-2 in isolated, perfused mouse hearts subjected to acute global I/R injury. I/R injury induced a rapid activation of MMP-2 promoter activity with the appearance of beta-galactosidase antigen in cardiomyocytes, fibroblasts, and endothelial cells. Activation of intrinsic MMP-2 transcription and translation was confirmed by real-time PCR and quantitative Western blot analyses. MMP-2 transcription and translation were inhibited by perfusion with 1.0 mM hydroxyl radical scavenger N-(-2-mercaptopropionyl)-glycine. Nuclear extracts demonstrated increased abundance of two activator proteins-1 (AP-1) components JunB and FosB following I/R injury. Immunohistochemical staining localized JunB and FosB proteins to the nuclei of all three cardiac cell types following I/R injury, consistent with enhanced nuclear transport of these transcription factors. Chromatin immunoprecipitation (ChIP) of the AP-1 binding site in the intrinsic murine MMP-2 promoter yielded only JunB under control conditions, whereas ChIP following I/R injury recovered both JunB and FosB, consistent with a change in occupancy from JunB homodimers in controls to JunB/FosB heterodimers following I/R injury. We conclude that enhanced MMP-2 transcription and translation following I/R injury are mediated by induction, via oxidant stress, of discrete AP-1 transcription factor components.

Objective: Matrix metalloproteinase-2 (MMP-2) plays a major role in dysfunctional ventricular remodeling following myocardial injury induced by ischemia/reperfusion and heart failure. To directly assess the role of MMP-2 in the absence of superimposed injury, we generated cardiac-specific, constitutively active MMP-2 transgenic mice., Methods: Morphologic and functional studies were carried out using both intact and demembranated (skinned) right ventricular trabeculae dissected from hearts of 8-month-old MMP-2 transgenic mice and wild-type controls (WT)., Results: Electron micrographs showed that compared to WT, MMP-2 myocardium had no gross, ultrastructural changes (no myocyte dropout or gross fibrosis). However, MMP-2 myocardium contained fibroblasts with abundant rough endoplasmic reticulum, consistent with an activated synthetic phenotype, suggesting extracellular matrix remodeling in MMP-2 trabeculae. Consistent with remodeling, mechanical studies found increased stiffness of intact unstimulated trabeculae (increasing sarcomere lengths from 2 to 2.3 microm caused a greater rise of passive muscle force for MMP-2 trabeculae versus WT). With electrical stimulation, MMP-2 trabeculae generated substantially less active force at all sarcomere lengths. Moreover, inotropic responses to increases of bath [Ca2+], pacing frequency, and isoproterenol were all significantly reduced versus WT trabeculae. Skinned fiber assessment of myofilament function revealed that maximum Ca2+-activated force of skinned MMP-2 trabeculae was reduced to approximately 50% of WT, suggesting a myofilament contraction defect., Conclusion: Cardiac-specific, constitutively active MMP-2 expression leads to impaired contraction and diminished responses to inotropic stimulation. These findings indicate that MMP-2 can directly impair ventricular function in the absence of superimposed injury.

Enhanced synthesis of a specific matrix metalloproteinase, MMP-2, has been demonstrated in experimental models of ventricular failure and in cardiac extracts from patients with ischaemic cardiomyopathy. Cultured neonatal rat cardiac fibroblasts and myocytes were used to analyse the determinants of MMP-2 synthesis, including the effects of hypoxia. Culture of rat cardiac fibroblasts for 24 h in 1% oxygen enhanced MMP-2 synthesis by more than 5-fold and augmented the MMP-2 synthetic responses of these cells to endothelin-1, angiotensin II and interleukin 1beta. A series of MMP-2 promoter-luciferase constructs were used to map the specific enhancer element(s) that drive MMP-2 transcription in cardiac cells. Deletion studies mapped a region of potent transactivating function within the 91 bp region from -1433 to -1342 bp, the activity of which was increased by hypoxia. Oligonucleotides from this region were cloned in front of a heterologous simian-virus-40 (SV40) promoter and mapped the enhancer activity to a region between -1410 and -1362 bp that included a potential activating protein 1 (AP-1)-binding sequence, C(-1394)CTGACCTCC. Site-specific mutagenesis of the core TGAC sequence (indicated in bold) eliminated the transactivating activity within the -1410 to -1362 bp sequence. Electrophoretic mobility shift assays (EMSAs) using the -1410 to -1362 bp oligonucleotide and rat cardiac fibroblast nuclear extracts demonstrated specific nuclear-protein binding that was eliminated by cold competitor oligonucleotide, but not by the AP-1-mutated oligonucleotide. Antibody-supershift EMSAs of nuclear extracts from normoxic rat cardiac fibroblasts demonstrated Fra1 and JunB binding to the -1410 to -1362 bp oligonucleotide. Nuclear extracts isolated from hypoxic rat cardiac fibroblasts contained Fra1, JunB and also included FosB. Co-transfection of cardiac fibroblasts with Fra1-JunB and FosB-JunB expression plasmids led to significant increases in transcriptional activity. These studies demonstrate that a functional AP-1 site mediates MMP-2 transcription in cardiac cells through the binding of distinctive Fra1-JunB and FosB-JunB heterodimers. The synthesis of MMP-2 is widely considered, in contrast with many members of the MMP gene family, to be independent of the AP-1 transcriptional complex. This report is the first demonstration that defined members of the Fos and Jun transcription-factor families specifically regulate this gene under conditions relevant to critical pathophysiological processes.

Acute increases in blood pressure (BP) increase myocardial tumor necrosis factor (TNF)-alpha production, but it is not known whether chronic hypertensive stress elevates myocardial TNF-alpha production, possibly contributing to cardiac remodeling, decreased cardiac function, and faster progression to heart failure. BP, cardiac function, and size were evaluated in normotensive [Sprague-Dawley (SD)], spontaneously hypertensive (SHR), and spontaneously hypertensive heart failure-prone (SHHF) rats at 6, 12, 15, and 18 mo of age and in failing SHHF. Left ventricular tissues were evaluated for secretion of bioactive TNF-alpha and inhibition of TNF-alpha secretion by phosphodiesterase inhibitors. All ventricles secreted bioactive and immunoreactive TNF-alpha, but secretion decreased with age. SHR and SHHF rats secreted more TNF-alpha than SD rats at 6 mo of age, but only failing SHHF rats secreted significantly more TNF-alpha at 18 mo. Amrinone inhibited TNF-alpha secretion in all rats and was less potent but more efficacious than RO-201724 in all strains. TNF-alpha secretion correlated with BP and left ventricular mass in 6-mo-old rats, but this relationship disappeared with age. Results suggest that hypertension and/or cardiac remodeling is associated with elevated myocardial TNF-alpha, and, although hypertension, per se, did not maintain elevated cardiac TNF-alpha levels, SHHF rats increase TNF-alpha production during the end stages of failure.

Phosphodiesterase (PDE) inhibitors are used as therapeutic agents for management of congestive heart failure. PDE inhibitors are potent inotropic and vasodilator drugs, which have also been shown to inhibit tumor necrosis factor alpha (TNF-alpha) production. TNF-alpha is a pleiotropic cytokine that has the ability to produce cardiac depressant and other cardiovascular effects in many disease conditions. TNF-alpha levels are elevated in patients with chronic congestive heart failure, and it is possible that TNF-alpha may play a role in this condition. The effects of PDE inhibitors on TNF-alpha secretion from rat heart were evaluated in this study. Rat left ventricle was minced and incubated for 4 hr with various PDE inhibitors, and the amount of TNF-alpha secretion was evaluated by cytotoxicity assay. Ro-20, 1724, etazolate, amrinone, milrinone and pentoxifylline inhibited unstimulated TNF-alpha production, with IC50 values of 1.87, 2.07, 13.9, 153 and 201 microM, respectively. Lipopolysaccharide-induced TNF-alpha secretion from rat left ventricle was also evaluated in this study. Amrinone, milrinone and pentoxifylline inhibited lipopolysaccharide-induced TNF-alpha secretion, with IC50 values of 14.8, 81.6 and 748 microM, respectively, whereas Ro-2D, 1724 and etazolate had no effect on lipopolysaccharide-induced TNF-alpha secretion. These results demonstrated that TNF-alpha was secreted from rat left ventricle after 4 hr and different pharmacological manipulations were able to inhibit the secretion of TNF-alpha from left ventricle. These initial pharmacological results may provide an important tool for further investigation into the beneficial effects of PDE inhibitors in congestive heart failure or other conditions where TNF-alpha levels are elevated.