Larry F. Lemanski

Morphology of developing heart in cardiac lethal mutant Mexican axolotls, Ambystoma mexicanum

Abstract In Ambystoma mexicanum, recessive mutant gene c results in an absence of embryonic heart function because of altered influences from surrounding tissues (Humphrey, 1972). The present light and electron microscope study compares heart development in normal and mutant embryos from Harrison stage 34 or 6 days (at which normal heart beat initiates) through stage 41 or 25 days (at which mutant embryos die). The hearts display increasing differences as development progresses, and by stage 41 mutant abnormalities are striking. The normal myocardium shows organized sarcomeres at stage 34 and numerous intercalated discs subsequently appear. By stage 41, the normal myocardium is composed of highly differentiated muscle cells and shows extensive trabeculation. The mutant myocardium throughout development remains only one cell layer thick with no indication of developing trabeculae. Mutant cells at stage 34 have a few 140 A and 60 A filaments along with what appear to be Z bodies. A partial organization of myofibrillar components is occasionally noted at stages 38–41; however, distinct sarcomeres are not apparent and intercalated discs are rarely seen. In general the mutant cells appear less differentiated than usual and in many respects are reminiscent of pre-heart-beat normal cells. Although most mutant cells show images characteristic of pathological conditions (e.g., pleomorphic mitochondria, membranous whorls, and numerous autophagic vacuoles), selective myocardial cell death, a phenomenon associated with normal trabeculation, is not evident. It is clear that gene c, in homozygous condition, results in an altered pattern of heart cell differentiation. The mutation, by way of abnormal inductive processes, appears to affect the synthesis and organization of heart contractile proteins.

Gating of mammalian cardiac gap junction channels by transjunctional voltage

Numerous two-cell voltage-clamp studies have concluded that the electrical conductance of mammalian cardiac gap junctions is not modulated by the transjunctional voltage (Vj) profile, although gap junction channels between low conductance pairs of neonatal rat ventricular myocytes are reported to exhibit Vj-dependent behavior. In this study, the dependence of macroscopic gap junctional conductance (gj) on transjunctional voltage was quantitatively examined in paired 3-d neonatal hamster ventricular myocytes using the double whole-cell patch-clamp technique. Immunolocalization with a site-specific antiserum directed against amino acids 252-271 of rat connexin43, a 43-kD gap junction protein as predicted from its cDNA sequence, specifically stained zones of contact between cultured myocytes. Instantaneous current-voltage (Ij-Vj) relationships of neonatal hamster myocyte pairs were linear over the entire voltage range examined (0 less than or equal to Vj less than or equal to +/- 100 mV). However, the steady-state Ij-Vj relationship was nonlinear for Vj greater than +/- 50 mV. Both inactivation and recovery processes followed single exponential time courses (tau inactivation = 100-1,000 ms, tau recovery approximately equal to 300 ms). However, Ij recovered rapidly upon polarity reversal. The normalized steady-state junctional conductance-voltage relationship (Gss-Vj) was a bell-shaped curve that could be adequately described by a two-state Boltzmann equation with a minimum Gj of 0.32-0.34, a half-inactivation voltage of -69 and +61 mV and an effective valence of 2.4-2.8. Recordings of gap junction channel currents (ij) yielded linear ij-Vj relationships with slope conductances of approximately 20-30 and 45-50 pS. A kinetic model, based on the Boltzmann relationship and the polarity reversal data, suggests that the opening (alpha) and closing (beta) rate constants have nearly identical voltage sensitivities with a Vo of +/- 62 mV. The data presented in this study are not consistent with the contingent gating scheme (for two identical gates in series) proposed for other more Vj-dependent gap junctions and alternatively suggest that each gate responds to the applied Vj independently of the state (open or closed) of the other gate.

Fine structure of the heart in the Japanese Medaka, Oryzias latipes

Heart morphology in the small freshwater teleost, Japanese Medaka, (oryzias latipes) was studied by light and electron microscopy. Particular attention was directed to the three heart layers, epicardium, myocardium, and endocardium in the atrial and ventricular portions of the organ. The trabeculae originate and insert into the myocardial wall forming many complex anastomoses and interdigitations. The entire ventricle is trabeculated giving the chamber a “spongy” appearance; the atrium is less extensively trabeculated. The epicardial layer forms an outer covering over the organ and is composed of simple squamous epithelial cells. The myocardial cells of the trabeculae have small diameters but extend for considerable distances. The myofibrils usually are located peripherally, while the nucleus, mitochondria, and other cellular organelles are located centrally. The myofibrils exhibit a typical vertebrate structure with distinct A-bands, I-bands, and Z-lines; H-zones are discernible but appear less prominent than in higher vertebrates. Intercalated disks have fasciae adherentes and maculae adherentes (desmosomes) as component parts. Although a distinct T-system is absent, prominent subsarcolemmal cisternae are located in the I-band areas and there is a moderately well-developed sarcoplasmic reticulum. The endocardium is composed of a continuous layer of cells that appear to be metabolically very active. Filling the cytoplasm of these cells are large numbers of rough endoplasmic reticulum cisternae and associated mitochondria, vesicles containing amorphous material, and large round bodies with consistencies varying from dense to amorphous to filamentous.

Ectopic expression of tropomyosin promotes myofibrillogenesis in mutant axolotl hearts

Expression of tropomyosin protein, an essential component of the thin filament, has been found to be drastically reduced in cardiac mutant hearts of the Mexican axolotl (Ambystoma mexicanum) with no formation of sarcomeric myofibrils. Therefore, this naturally occurring cardiac mutation is an appropriate model to examine the effects of delivering tropomyosin protein or tropomyosin cDNA into the deficient tissue. In this study, we describe the replacement of tropomyosin by using a cationic liposome transfection technique applied to whole hearts in vitro. When mouse α‐tropomyosin cDNA under the control of a cardiac‐specific α‐myosin heavy chain promoter was transfected into the mutant hearts, tropomyosin expression was enhanced resulting in the formation of well‐organized sarcomeric myofibrils. Transfection of a β‐tropomyosin construct under control of the same promoter did not result in enhanced organization of the myofibrils. Transfection of a β‐galactosidase reporter gene did not result in the formation of organized myofibrils or increased tropomyosin expression. These results demonstrate the importance of α‐tropomyosin to the phenotype of this mutation and to normal myofibril formation. Moreover, we have shown that a crucial contractile protein can be ectopically expressed in cardiac muscle that is deficient in this protein, with the resulting formation of organized sarcomeres. Dev. Dyn. 1998;213:412–420.

Protein kinase C-mediated desmin phosphorylation is related to myofibril disarray in cardiomyopathic hamster heart.

The cardiomyopathic (CM) Syrian golden hamster (strain UM-X7.1) exhibits a hereditary cardiomyopathy, which causes premature death resulting from congestive heart failure. The CM animals show extensive cardiac myofibril disarray and myocardial calcium overload. The present study has been undertaken to examine the role of desmin phosphorylation in myofibril disarray observed in CM hearts. The data from skinned myofibril protein phosphorylation assays have shown that desmin can be phosphorylated by protein kinase C (PKC). There is no significant difference in the content of desmin between CM and control hamster hearts. However, the desmin from CM hearts has a higher phosphorylation level than that of the normal hearts. Furthermore, we have examined the distribution of desmin and myofibril organization with immunofluorescent microscopy and immunogold electron microscopy in cultured cardiac myocytes after treatment with the PKC-activating phorbol ester, 12-O-tetradecanylphorbol-13-acetate (TPA). When the cultured normal hamster cardiac cells are treated with TPA, desmin filaments are disassembled and the myofibrils become disarrayed. The myofibril disarray closely mimics that observed in untreated CM cultures. These results suggest that disassembly of desmin filaments, which could be caused by PKC-mediated phosphorylation, may be a factor in myofibril disarray in cardiomyopathic cells and that the intermediate filament protein, desmin, plays an Important role in maintaining myofibril alignment in cardiac cells.

Immunoelectron microscopic localization of alpha-actinin on Lowicryl-embedded thin-sectioned tissues.

A procedure has been developed for the immunoelectron microscopic localization of intracellular antigens on thin-sectioned tissues. The tissues were fixed in a periodate-lysine-paraformaldehyde solution or a formaldehyde-glutaraldehyde combination and embedded in the acrylate-methacrylate mixture, Lowicryl K4M (Polaron), which was polymerized under ultraviolet irradiation at -35 degrees C. Thin sections were mounted on gold grids, immunostained using an indirect method with ferritin-labeled antibodies, and, optionally, counterstained with osmium tetroxide and/or lead citrate and uranyl acetate. The procedure provided good morphologic preservation of the cell architecture in adult and embryonic heart, and skeletal and smooth muscle tissue, as well as nonmuscle cells. At the same time it retained the antigenicities of several contractile proteins, including myosin, tropomyosin, actin, and alpha-actinin. The method has advantages over en bloc staining techniques in that the problem of antibody penetration into the cells is eliminated and careful controls can be performed on adjacent sections. This technique will be useful for localizing, at the ultrastructural level, contractile and other selected proteins in a variety of muscle and non-muscle cells. Details of the new protocol and a description of the results of using antibody against the contractile protein, alpha-actinin, are given.

Differential expression of a novel isoform of α-tropomyosin in cardiac and skeletal muscle of the Mexican axolotl (Ambystoma mexicanum)

Alternative mRNA splicing is a fundamental process in eukaryotes that contributes to tissue-specific and developmentally regulated patterns of tropomyosin (TM) gene expression. Northern blot analyses suggest the presence of multiple transcripts of tropomyosin in skeletal and cardiac muscle of adult Mexican axolotls. We have cloned and sequenced two tropomyosin cDNAs designated ATmC-1 and ATmC-2 from axolotl heart tissue and one TM cDNA from skeletal muscle, designated ATmS-1. Nucleotide sequence analyses suggest that ATmC-1 and ATmC-2 are the products of the same alpha-TM gene produced via alternate splicing, whereas ATmC-1 and ATmS-1 are the identical isoforms generated from the alpha-gene. RT-PCR analysis using isoform-specific primer pairs and detector oligonucleotides suggests that ATmC-2 is expressed predominantly in adult axolotl hearts. ATmC-2 is a novel isoform, which unlike ATmC-1 and other known striated muscle isoforms expresses exon 2a instead of exon 2b.

Low-Density Lipoprotein Stimulated Peroxide Production and Endocytosis in Cultured Human Endothelial Cells: Mechanisms of Action

The effects of arachidonic acid metabolism and NADPH oxidase inhibitor on the hydrogen peroxide (H2O2) generation and endocytotic activity of cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein (LDL) levels have been investigated. EC were incubated with 240 mg/dl LDL cholesterol and cellular H2O2 production and endocytotic activity measured in the presence and absence of the arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A, and NADPH oxidase inhibitor, apocynin. All inhibitors, with the exception of indomethacin, markedly reduced high LDL-induced increases in EC H2O2 generation and endocytotic activity. EC exposed to exogenously applied arachidonic acid had cellular functional changes similar to those induced by high LDL concentrations. EC incubated with 1-25 uM arachidonic acid had increased H2O2 production and heightened endocytotic activity. Likewise, EC pre-loaded with [3H]arachidonic acid when exposed to increasing LDL levels (90-330 mg/dl cholesterol) had a dose-dependent rise in cytosolic [3H]arachidonic acid. The phospholipase A2 inhibitors, 4-bromophenacyl bromide and 7,7-dimethyleicosadienoic acid, markedly inhibited H2O2 production in EC exposed to 240 mg/dl LDL cholesterol. These findings suggest that arachidonic acid contributes mechanistically to high LDL-perturbed EC H2O2 generation and heightened endocytosis. Such cellular functional changes add to our understanding of endothelial perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

Characterization of a TM‐4 type tropomyosin that is essential for myofibrillogenesis and contractile activity in embryonic hearts of the Mexican axolotl

A striated muscle isoform of a Tropomyosin (TM‐4) gene was characterized and found to be necessary for contractile function in embryonic heart. The full‐length clone of this isoform was isolated from the Mexican axolotl (Ambystoma mexicanum) and named Axolotl Tropomyosin Cardiac‐3 (ATmC‐3). The gene encoded a cardiac‐specific tropomyosin protein with 284 amino acid residues that demonstrated high homology to the Xenopus cardiac TM‐4 type tropomyosin. Northern blot analysis indicates a transcript of ∼1.25 kb in size. RT‐PCR and in situ hybridization demonstrated that this isoform is predominantly in cardiac tissue. Our laboratory uses an animal model that carries a cardiac lethal mutation (gene c), this mutation results in a greatly diminished level of tropomyosin protein in the ventricle. Transfection of ATmC‐3 DNA into mutant hearts increased tropomyosin levels and promoted myofibrillogenesis. ATmC‐3 expression was blocked in normal hearts by transfection of exon‐specific anti‐sense oligonucleotide (AS‐ODN). RT‐PCR confirmed lower transcript expression of ATmC‐3 and in vitro analysis confirmed the specificity of the ATmC‐3 exon 2 anti‐sense oligonucleotide. These AS‐ODN treated hearts also had a disruption of myofibril organization and disruption of synchronous contractions. These results demonstrated that a striated muscle isoform of the TM‐4 gene was expressed embryonically and was necessary for normal structure and function of the ventricle. J. Cell. Biochem. 85: 747–761, 2002.

Methionine sulfoxide reductase A (MsrA) protects cultured mouse embryonic stem cells from H2O2‐mediated oxidative stress

Methionine sulfoxide reductase A (MsrA), a member of the Msr gene family, can reduce methionine sulfoxide residues in proteins formed by oxidation of methionine by reactive oxygen species (ROS). Msr is an important protein repair system which can also function to scavenge ROS. Our studies have confirmed the expression of MsrA in mouse embryonic stem cells (ESCs) in culture conditions. A cytosol‐located and mitochondria‐enriched expression pattern has been observed in these cells. To confirm the protective function of MsrA in ESCs against oxidative stress, a siRNA approach has been used to knockdown MsrA expression in ES cells which showed less resistance than control cells to hydrogen peroxide treatment. Overexpression of MsrA gene products in ES cells showed improved survivability of these cells to hydrogen peroxide treatment. Our results indicate that MsrA plays an important role in cellular defenses against oxidative stress in ESCs. Msr genes may provide a new target in stem cells to increase their survivability during the therapeutic applications. J. Cell. Biochem. 111: 94–103, 2010.