Richard T. Mathias

Intermediate Filaments Regulate Tissue Size and Stiffness in the Murine Lens

PURPOSEnTo define the contributions of the beaded filament (BF), a lens-specific intermediate filament (IF), to lens morphology and biomechanics.nnnMETHODSnWild-type and congenic CP49 knockout (KO) mice were compared by using electrophysiological, biomechanical, and morphometric approaches, to determine changes that occurred because of the absence of this cytoskeletal structure.nnnRESULTSnElectrophysiological assessment established that the fiber cells lacking the lens-specific IFs were indistinguishable from wild-type fiber cells. The CP49 KO mice exhibited lower stiffness, and an unexpected higher resilience than the wild-type lenses. The absence of these filaments resulted in lenses that were smaller, and exhibited a higher ratio of lens:lens nucleus size. Finally, lens shape differed as well, with the CP49 KO showing a higher ratio of axial:equatorial diameter.nnnCONCLUSIONSnPrevious work has shown that BFs are necessary in maintaining fiber cell and lens structural phenotypes with age, and that absence of these filaments results in a loss of lens clarity. This work demonstrates that several tissue-level properties that are critical to lens function are also dependent, at least in part, on the presence of these lens-specific IFs.

Fluid Circulation Determined In The Isolated Bovine Lens

PURPOSEnIn 1997, a theoretical model was developed that predicted the existence of an internal, Na(+)-driven fluid circulation from the poles to the equator of the lens. In the present work, we demonstrate with a novel system that fluid movement can be measured across the polar and equatorial surface areas of isolated cow lenses. We have also determined the effects of ouabain and reduced bath [Na(+)].nnnMETHODSnLenses were isolated in a chamber with three compartments separated by two thin O-rings. Each compartment, anterior (A), equatorial (E), and posterior (P), was connected to a vertical capillary graduated in 0.25 μL. Capillary levels were read every 15 minutes. The protocols consisted of 2 hours in either open circuit or short circuit. The effects of ouabain and low-Na(+) solutions were determined under open circuit.nnnRESULTSnIn 21 experiments, the E capillary increased at a mean rate of 0.060 μL/min while the A and P levels decreased at rates of 0.044 and 0.037 μL/min, respectively, closely accounting for the increase in E. The first-hour flows under short circuit were approximately 40% larger than those in open-circuit conditions. The first-hour flows were always larger than those during the second hour. Preincubation of lenses with either ouabain or low-[Na(+)] solutions resulted in reduced rates of fluid transport. When KCl was used to replace NaCl, a transitory stimulation of fluid transport occurred.nnnCONCLUSIONSnThese experiments support that a fluid circulation consistent with the 1997 model is physiologically active.

Connexin 46 (cx46) gap junctions provide a pathway for the delivery of glutathione to the lens nucleus.

Background: Delivery of the anti-oxidant, glutathione, to the lens nucleus is vital for its transparency. Results: Gap junction channels, which couple central fiber cells to outer lens cells, are permeable to glutathione. Conclusion: Glutathione diffuses from cortical cells to the nucleus via gap junctions. Significance: The age-dependent decline of glutathione levels, an initiating factor in cataract formation, might be due to alterations in coupling. Maintenance of adequate levels of glutathione (GSH) in the lens nucleus is critical for protection of lens proteins from the effects of oxidative stress and for lens transparency. How GSH is transported to the nucleus is unknown. We show that GSH diffuses to the nucleus from the outer cortex, where a high concentration of the anti-oxidant is established by synthesis or uptake, via the network of gap junctions. Using electrophysiological measurements, we found that channels formed by Cx46 and Cx50, the two connexin isoforms expressed in the lens, were moderately cation-selective (PNa/PCl ∼5 for Cx46 and ∼3 for Cx50). Single channel permeation of the larger GSH anion was low but detectable (PNa/PGSH ∼12 for Cx46 and ∼8 for Cx50), whereas permeation of divalent anion glutathione disulfide (GSSG) was undetectable. Measurement of GSH levels in the lenses from connexin knock-out (KO) mice indicated Cx46, and not Cx50, is necessary for transport of GSH to the core. Levels of GSH in the nucleus were markedly reduced in Cx46 KO, whereas they were unaffected by Cx50 KO. We also show that GSH delivery to the nucleus is not dependent on the lens microcirculation, which is believed to be responsible for extracellular transport of other nutrients to membrane transporters in the core. These results indicate that glutathione diffuses from cortical fiber cells to the nucleus via gap junction channels formed by Cx46. We present a model of GSH diffusion from outer cells to inner fiber cells through gap junctions.

Altered ubiquitin causes perturbed calcium homeostasis, hyperactivation of calpain, dysregulated differentiation, and cataract

Significance Eye lens opacification or cataract is the most prevalent age-related disease, blinding 18 million people. Cataractogenesis involves accumulation and precipitation of damaged proteins from the normally clear lens. The ubiquitin proteolytic system is the main cytoplasmic degradative pathway that is charged with selectively removing damaged proteins. Ubiquitin has seven lysines. Although lysine 6 is involved in less than 3% of ubiquitin conjugates and we find few changes in lens proteins when lysine 6 is unavailable, we observed that mutating ubiquitin lysine 6 alters cell coupling, resulting in Ca2+ elevation, hyperactivation of calpain, and associated cleavage of substrates, culminating in developmental defects and a cataractous lens. The data show previously unidentified connections between ubiquitin proteasome systems (UPSs) and calpain-based degradative systems and illuminate roles for ubiquitin lysine 6 in development. Although the ocular lens shares many features with other tissues, it is unique in that it retains its cells throughout life, making it ideal for studies of differentiation/development. Precipitation of proteins results in lens opacification, or cataract, the major blinding disease. Lysines on ubiquitin (Ub) determine fates of Ub-protein substrates. Information regarding ubiquitin proteasome systems (UPSs), specifically of K6 in ubiquitin, is undeveloped. We expressed in the lens a mutant Ub containing a K6W substitution (K6W-Ub). Protein profiles of lenses that express wild-type ubiquitin (WT-Ub) or K6W-Ub differ by only ∼2%. Despite these quantitatively minor differences, in K6W-Ub lenses and multiple model systems we observed a fourfold Ca2+ elevation and hyperactivation of calpain in the core of the lens, as well as calpain-associated fragmentation of critical lens proteins including Filensin, Fodrin, Vimentin, β-Crystallin, Caprin family member 2, and tudor domain containing 7. Truncations can be cataractogenic. Additionally, we observed accumulation of gap junction Connexin43, and diminished Connexin46 levels in vivo and in vitro. These findings suggest that mutation of Ub K6 alters UPS function, perturbs gap junction function, resulting in Ca2+ elevation, hyperactivation of calpain, and associated cleavage of substrates, culminating in developmental defects and a cataractous lens. The data show previously unidentified connections between UPS and calpain-based degradative systems and advance our understanding of roles for Ub K6 in eye development. They also inform about new approaches to delay cataract and other protein precipitation diseases.

Mefloquine effects on the lens suggest cooperative gating of gap junction channels

Mefloquine (MFQ) selectively blocks exogenously expressed gap junction channels composed of C×50 but not C×46. The purpose of the current study was to evaluate MFQ effects on wild-type (WT) mouse lenses that express both C×50 and C×46 in their outer shell of differentiating fibers (DFs). Lenses in which C×46 was knocked into both C×50 alleles (KI) were used as controls; MFQ had no effect on coupling in these lenses. When WT lenses were exposed to MFQ, the DF coupling conductance decreased significantly, suggesting that C×50 contributes about 57% of the coupling conductance in DF and C×46 contributes 43%. Remarkably, in the presence of MFQ, the 43% of the channels that remained open did not gate closed in response to a reduction in pH, whereas in the absence of MFQ, the same pH change caused all the DF channels to gate closed. Since MFQ is a selective blocker of C×50 channels, it appears that C×46 channels lack pH-mediated gating in the absence of functional C×50 channels but are pH-sensitive in the presence of C×50 channels. These results suggest the two types of channels interact and gate cooperatively.

Chapter 3 The Role of Gap Junction Channels in the Ciliary Body Secretory Epithelium

Publisher Summary The secretory epithelium of the ciliary body is responsible for generating the aqueous humor (AH). The epithelium comprises two cell layers: the pigmented epithelium (PE) and non pigmented epithelium (NPE), whose apical surfaces appose one another. At the apical–apical interface, the two layers are in communication through gap junctions formed from connexins Cx40 and Cx43. Cells of the PE are not in direct gap junctional communication with each other; however, the cells of the NPE are coupled by gap junctions made from Cx26 and Cx32. This chapter discusses the properties of the ciliary body and gap junction channels, with special emphasis on channels made from the connexins present in the ciliary epithelium. An important question arises concerning the gap junction channels at the apical–apical interface: that is, whether these channels conduct the water that is ultimately secreted? Model calculations are presented in the chapter that suggest the channels could do so, but only if they provide a rather high degree of ion coupling. Finally, experimental animal models that might help test this hypothesis are reviewed.

Connexins in Lens Development and Disease

Gap junctions are responsible for the coupling of cells that enables intercellular exchange of ions, small metabolites, and nutrients; lens homeostasis depends on these intercellular connections. Aberrant expression of the genes encoding lens connexins (Cx43, Cx46, and Cx50), and function of the connexins themselves, have been linked to cataractogenesis and ocular growth defects. The use of in vivo and in vitro experimental models has provided significant advances in understanding the function of gap junctions in the lens. Current data suggest that Cx46 is required for maintenance of lens clarity via its effects on Ca2+ homeostasis, and that Cx50 is required for proper lens growth. The substitution of either of these connexins for the other only partially ameliorates the pathological effects. In spite of these advances, questions about the mechanisms by which connexin-related pathologies occur in the mammalian lens remain largely unanswered.

Integration of Stem Cells into the Cardiac Syncytium: Formation of Gap Junctions

The heart is a functional syncytium. This means that each myocyte is electrically connected to other myocytes in its vicinity. Functional electrical coupling requires gap junctions. Gap junctions are composed of connexins. There are over 20 connexins in the human genome. If either electrical or mechanical regeneration of cardiac function is to be achieved via cell therapy (exclusive of paracrine effects), then the delivery cells must couple to the existing myocytes via gap junctions. In this chapter we review the basic physiology of gap junctions and what is known about their expression in cardiac myocytes and in stem cells. Given that multiple connexins are expressed in myocytes, we consider the types of gap junction channels that can be formed and their prevalence in a calculation of independent assortment of equally expressed connexins. Finally, myocytes are not the only cells present in the heart; there is a substantial presence of fibroblasts and endothelial cells. Fibroblasts do not express the same assortment of connexins as myocytes and do not in general form gap junctions with them. When stem cells are considered for cardiac regeneration, their expression of cardiac connexins is usually confirmed. However, it is just as important to confirm the absence of fibroblast connexins which could potentially create a sink for the local circuit currents that generate the cardiac impulse. Given the excitement generated by induced pluripotent stem cells which are derived from fibroblasts, it will be particularly important to demonstrate that the new myocytes generated from these cells express only cardiac-myocyte-specific connexins.

A Transmural Gradient in Angiotensin II Concentration in the Canine Ventricle

Transmural differences in the heart have long been known and their importance in normal heart function emphasized. Angiotensin II is a potent effector that is regularly targeted in heart failure treatments. We used the whole-cell patch clamp to investigate the effects of angiotensin II on NaK-ATPase activity in the canine left ventricle and we show that transmural gradients in NaK-ATPase activity and contractile force are established by a corresponding gradient in angiotensin II concentration.