Partial purification and characterization of microtubular protein from Trypanosoma brucei

The tubulin proteins of the parasitic hemoflagellate Trypanosoma brucei brucei were purified and characterized. Cytoskeletal microtubules of trypanosomes do not disrupt under conditions used to solubilize brain tubulins. Trypanosomal tubulins, solubilized by extensive sonication, were partially purified from the crude cell extracts by taxol-mediated polymerization. Taxolinduced microtubules were identified by electron microscopy and analyzed biochemically. They consist predominantly of two proteins of about 52,000 and 56,000 Da. Their mobilities on sodium dodecyl sulfate gels differ slightly from those of bovine brain tubulins. Immunological cross-reactivity with antibodies raised against bovine brain tubulins confirmed the nature of the trypanosomal proteins. Peptide mapping of bovine and trypanosomal alpha- and beta-tubulins was performed by enzymatic digestion with staphylococcal protease V8 and chemical cleavage with N-chlorosuccinimide. In both cases, the peptide patterns generated from the trypanosomal alpha- and beta-tubulins were closely related to each other. This suggests that the trypanosomal alpha- and beta-tubulins may have remained more conserved during evolution than the tubulins from higher eukaryotes. The trypanosomal alpha-tubulin is post-translationally modified in vivo by the reversible addition of a tyrosine residue at its COOH terminus. As in higher eukaryotes, this reaction is completely specific for the alpha-polypeptide chain. Our observation represents the first documentation of the occurrence of COOH-terminal tyrosinolation of alpha-tubulin in an eukaryotic microorganism.     

Cellular localization of integrin isoforms in phenylephrine-induced hypertrophic cardiac myocytes

Cardiac hypertrophy is characterized by remodeling of the extracellular matrix (ECM). Integrins are cell-surface molecules that link the ECM to the cellular cytoskeleton where they play roles as signaling molecules and transducers of mechanical force. To clarify the possible roles of integrins in cardiac myocyte hypertrophy, we investigated the cellular localization and expression of ECM proteins and integrins in both normal cardiac myocytes and phenylephrine-induced hypertrophic myocytes. Addition of phenylephrine (PE) to cultured neonatal cardiac myocytes induced sarcomeric organization, increase in cell size, and synthesis of the hypertrophic marker, atrial natriuretic factor (ANF). In particular, fibronectin and collagen underwent dramatic localization changes during PE-induced cardiac hypertrophy. Significant changes were noted in the cellular localization of the respective collagen and fibronectin receptors, integrin alpha1 and alpha5, from diffuse to a sarcomeric banding pattern. Expression levels of integrins were also increased during hypertrophy. Treatment with okadaic acid (OA), an inhibitor of protein phosphatase 2A (PP2A), resulted in inhibition of hypertrophic response. These results suggest that dephosphorylation of integrin beta1 may be important in the induction of cardiac hypertrophy.     

Adapter molecule DOC-2 is differentially expressed in pressure and volume overload hypertrophy and inhibits collagen synthesis in cardiac fibroblasts.

DOC-2 (differentially expressed in ovarian carcinoma) is involved in Ras-, beta-integrin-, PKC-, and transforming growth factor-beta-mediated cell signaling. These pathways are implicated in the accumulation of extracellular matrix proteins during progression of hypertrophy to heart failure; however, the role of DOC-2 in cardiac pathophysiology has never been examined. This study was undertaken to 1) analyze DOC-2 expression in primary cultures of cardiac fibroblasts and cardiac myocytes and in the heart following different types of hemodynamic overloads and 2) examine its role in growth factor-mediated ERK activation and collagen production. Pressure overload and volume overload were induced for 10 wk in Sprague-Dawley rats by aortic constriction and by aortocaval shunt, respectively. ANG II (0.3 mg.kg(-1).day(-1)) was infused for 2 wk. Results showed that, compared with myocytes, DOC-2 was found abundantly expressed in cardiac fibroblasts. Treatment of cardiac fibroblasts with ANG II and TPA resulted in increased expression of DOC-2. Overexpression of DOC-2 in cardiac fibroblasts led to inhibition of hypertrophy agonist-stimulated ERK activation and collagen expression. An inverse correlation between collagen and DOC-2 was observed in in vivo models of cardiac hypertrophy; in pressure overload and after ANG II infusion, increased collagen mRNA correlated with reduced DOC-2 levels, whereas in volume overload increased DOC-2 levels were accompanied by unchanged collagen mRNA. These data for the first time describe expression of DOC-2 in the heart and demonstrate its modulation by growth-promoting agents in cultured cardiac fibroblasts and in in vivo models of heart hypertrophy. Results suggest a role of DOC-2 in cardiac remodeling involving collagen expression during chronic hemodynamic overload.     

Emodin and emodin-rich rhubarb inhibits histone deacetylase (HDAC) activity and cardiac myocyte hypertrophy

Pathological cardiac hypertrophy is a classical hallmark of heart failure. At the molecular level, inhibition of histone deacetylase (HDAC) enzymes attenuate pathological cardiac hypertrophy in vitro and in vivo. Emodin is an anthraquinone that has been implicated in cardiac protection. However, it is not known if the cardio-protective actions for emodin are mediated through HDAC-dependent regulation of gene expression. Therefore, we hypothesized that emodin would attenuate pathological cardiac hypertrophy via inhibition of HDACs, and that these actions would be reflected in an emodin-rich food like rhubarb. In this study, we demonstrate that emodin and Turkish rhubarb containing emodin inhibit HDAC activity in vitro, with fast-on, slow-off kinetics. Moreover, we show that emodin increased histone acetylation in cardiomyocytes concomitant to global changes in gene expression; gene expression changes were similar to the well-established pan-HDAC inhibitor trichostatin A (TSA). We additionally present evidence that emodin inhibited phenylephrine (PE) and phorbol myristate acetate (PMA)-induced hypertrophy in neonatal rat ventricular myocytes (NRVMs). Lastly, we demonstrate that the cardioprotective actions of emodin are translated to an angiotensin II (Ang) mouse model of cardiac hypertrophy and fibrosis and are linked to HDAC inhibition. These data suggest that emodin blocked pathological cardiac hypertrophy, in part, by inhibiting HDAC-dependent gene expression changes.     

Mechanotransduction in Stretch-Induced Hypertrophy of Cardiac Myocytes

Abstract

Mechanical loading of cardiac muscles causes rapid activation of a number of immediate-early (IE) genes and hypertrophy. However, little is known as to how muscle cells sense mechanical load and regulate gene expression. We examined roles of several putative mechanotransducers in stretch-induced hypertrophy of cardiac myocytes grown on a deformable silicone sheet. Using the patch-clamp technique, we found a single class of stretch-activated cation channels which was completely and reversibly blocked by gadolinium. The inhibition of this channel by gadolinium did not affect either stretch-induced expression of the IE genes or hypertrophy. Neither disruption of microtubules with colchicine nor that of actin microfilaments by cytochalasin D prevented the stretch-induced IE gene expression. Arresting contractile activity by tetrodotoxin did not affect the stretch-induced IE gene expression or hypertrophy. These results suggest that stretch-activated cation channels, microtubules, microfilaments, and contractile activity are not the mechanotransducers. Preliminary results suggest that cell stretch may cause a release of a growth factor(s), which in turn initiates a cascade of hypertrophic response of cardiac myocytes.     

Effects of all-trans retinoic acid on angiotensin II-induced myocyte hypertrophy.

We used cultured neonatal rat cardiac myocytes to test the hypothesis that all-trans retinoic acid (atRA) may act to modulate ANG II actions in inducing myocyte hypertrophy. Our observations were as follows. 1) atRA (10(-7) to approximately 10(-5) M ) inhibited ANG II-induced hyperplasia of fibroblasts in a dose-dependent manner. 2) Treatment of atRA attenuated the ANG II-induced increase in total cell protein content. 3) Treated with ANG II (10(-7) M) for 5 days, the cultured neonatal rat cardiac myocytes demonstrated an apparent accumulation of sarcomeric fiber proteins and Golgi's complex, as well as reorganization of the sarcomeric unit within individual myocytes. atRA (10(-6) M) treatment reduced the accumulation of contractile proteins and Golgi's complex without affecting the ANG II-induced reorganization of the sarcomeric unit. 4) atRA attenuated the ANG II-induced increase in intracellular Ca2+. Our results show that atRA inhibits some effects of ANG II on neonatal rat cardiac myocytes and suggest that atRA may be a therapeutic candidate for the prevention and therapy of cardiac hypertrophy and remodeling.     

Activation of SIRT1, a class III histone deacetylase, contributes to fructose feeding-mediated induction of the alpha-myosin heavy chain expression

Fructose feeding has been shown to induce the cardiac alpha-myosin heavy chain (MHC) expression and protect the heart from ischemia- and reperfusion-mediated cell injury. This study was designed to investigate the mechanism involved in the effect of this sugar on MHC gene expression and cardiac protection. Adult mice were fed with a 6-propyl-2-thiouracil (PTU) diet or PTU combined with a fructose-rich diet. PTU treatment made animals hypothyroid and that resulted in total replacement of cardiac alpha-MHC with the beta-MHC isoform. Addition of fructose in the PTU diet led to reexpression of the alpha-MHC isoform to a significant level. Similar induction of alpha-MHC expression was also seen when PTU diet was combined with resveratrol, an agonist of sirtuin (SIRT) 1 deacetylase. Analysis of heart lysate of these animals indicated that fructose feeding augmented the NAD-to-NADH ratio and the cardiac SIRT1 levels, thus suggesting a role of SIRT1 in fructose-mediated activation of alpha-MHC isoform. To analyze a direct effect of SIRT1 on MHC isoform expression, we generated transgenic mice expressing SIRT1 in the heart. Treatment of these transgenic mice with PTU diet did not lead to disappearance of alpha-MHC, as it did in the nontransgenic animals. SIRT1 overexpression also activated the alpha-MHC gene promoter in transient transfection assays, thus confirming a role of SIRT1 in the induction of alpha-MHC expression. Fructose feeding also attenuated the MHC isoform shift and blocked the cardiac hypertrophy response associated with pressure overload, which was again associated with the induction of cardiac SIRT1 levels. These results demonstrate that fructose feeding protects the heart by induction of the SIRT1 deacetylase and highlight its role in the induction of alpha-MHC gene expression.     

S100A6 is a negative regulator of the induction of cardiac genes by trophic stimuli in cultured rat myocytes

S100A6 (calcyclin), a member of the S100 family of EF-hand Ca2+ binding proteins, has been implicated in the regulation of cell growth and proliferation. We have previously shown that S100B, another member of the S100 family, is induced postinfarction and limits the hypertrophic response of surviving cardiac myocytes. We presently report that S100A6 expression is also increased in the periinfarct zone of rat heart postinfarction and in cultured neonatal rat myocytes by treatment with several trophic agents, including platelet-derived growth factor (PDGF), the alpha1-adrenergic agonist phenylephrine (PE), and angiotensin II (AII). Cotransfection of S100A6 in cultured neonatal rat cardiac myocytes inhibits induction of the cardiac fetal gene promoters skeletal alpha-actin (skACT) and beta-myosin heavy chain (beta-MHC) by PDGF, PE, AII, and the prostaglandin F2alpha (PGF2alpha), induction of the S100B promoter by PE, and induction of the alpha-MHC promoter by triiodothyronine (T3). By contrast, S100B cotransfection selectively inhibited only PE induction of skACT and beta-MHC promoters. Fluorescence microscopy demonstrated overlapping intracellular distribution of S100B and S100A6 in transfected myocytes and in postinfarct myocardium but heterodimerization of the two proteins could not be detected by co-immunoprecipitation. We conclude that S100A6 may function as a global negative modulator of differentiated cardiac gene expression comparable to its putative role in cell cycle progression of dividing cells.     

Post-translational modifications of tubulin and microtubule stability in adult rat ventricular myocytes and immortalized HL-1 cardiomyocytes

Little is known about the subcellular distribution and the dynamics of tubulins in adult cardiac myocytes although both are modified during cardiac hypertrophy and heart failure. Using confocal microscopy, we examined post-translational modifications of tubulin in fully differentiated ventricular myocytes isolated from adult rat hearts, as well as in immortalized and dividing HL-1 cardiomyocytes. Detyrosinated Glu-alpha-tubulin was the most abundant post-translationally modified tubulin found in ventricular myocytes, while acetylated- and delta2-alpha-tubulins were found in lower amounts or absent. In contrast, dividing HL-1 cardiomyocytes exhibited high levels of tyrosinated or acetylated alpha-tubulins. A mild nocodazole treatment (0.1 microM, 1 h) disrupted microtubules in HL-1 myocytes, but not in adult ventricular myocytes. A stronger treatment (10 microM, 2 h) was required to disassemble tubulins in adult myocytes. Glu-alpha-tubulin containing microtubules were more resistant to nocodazole treatment in HL-1 cardiomyocytes than in ventricular myocytes. Endogenous activation of the cAMP pathway with the forskolin analog L858051 (20 microM) or the beta-adrenergic agonist isoprenaline (10 microM) disrupted the most labile microtubules in HL-1 cardiomyocytes. In contrast, isoprenaline (10 microM), cholera toxin (200 ng/ml, a G(S)-protein activator), L858051 (20 microM) or forskolin (10 microM) had no effect on the microtubule network in ventricular myocytes. In addition, intracellular Ca2+ accumulation induced either by thapsigargin (2 microM) or caffeine (10 mM) did not modify microtubule stability in ventricular myocytes. Our data demonstrate the unique stability of the microtubule network in adult cardiac myocytes. We speculate that microtubule stability is required to support cellular integrity during cardiac contraction.     

Angiotensin-(1-7) inhibits growth of cardiac myocytes through activation of the mas receptor

Peptide hormones such as ANG II and endothelin contribute to cardiac remodeling after myocardial infarction by stimulating myocyte hypertrophy and myofibroblast proliferation. In contrast, angiotensin-(1-7) [ANG-(1-7)] infusion after myocardial infarction reduced myocyte size and attenuated ventricular dysfunction and remodeling. We measured the effect of ANG-(1-7) on protein and DNA synthesis in cultured neonatal rat myocytes to assess the role of the heptapeptide in cell growth. ANG-(1-7) significantly attenuated either fetal bovine serum- or endothelin-1-stimulated [(3)H]leucine incorporation into myocytes with no effect on [(3)H]thymidine incorporation. [d-Ala(7)]-ANG-(1-7), the selective ANG type 1-7 (AT(1-7)) receptor antagonist, blocked the ANG-(1-7)-mediated reduction in protein synthesis in cardiac myocytes, whereas the AT(1) and AT(2) angiotensin peptide receptors were ineffective. Serum-stimulated ERK1/ERK2 mitogen-activated protein kinase activity was significantly decreased by ANG-(1-7) in myocytes, a response that was also blocked by [d-Ala(7)]-ANG-(1-7). Both rat heart and cardiac myocytes express the mRNA for the mas receptor, and a 59-kDa immunoreactive protein was identified in both extracts of rat heart and cultured myocytes by Western blot hybridization with the use of an antibody to mas, an ANG-(1-7) receptor. Transfection of cultured myocytes with an antisense oligonucleotide to the mas receptor blocked the ANG-(1-7)-mediated inhibition of serum-stimulated MAPK activation, whereas a sense oligonucleotide was ineffective. These results suggest that ANG-(1-7) reduces the growth of cardiomyocytes through activation of the mas receptor. Because ANG-(1-7) is elevated after treatment with angiotensin-converting enzyme inhibitors or AT(1) receptor blockers, ANG-(1-7) may contribute to their beneficial effects on cardiac dysfunction and ventricular remodeling after myocardial infarction.     

Heterogeneity and structure of brain tubulins from cold-adapted Antarctic fishes. Comparison to brain tubulins from a temperate fish and a mammal

Tubulins purified from brain tissue of Antarctic fishes assemble in vitro to form microtubules at the low temperatures experienced by these extreme psychrophiles (Williams, R. C., Jr., Correia, J. J., and DeVries, A. L. (1985) Biochemistry 24, 2790-2798). We have initiated studies to determine the structural requirements for assembly of Antarctic fish tubulins at low temperatures. As a first step we have compared the heterogeneity, structures, amino acid compositions, and net charge of brain tubulins purified from three Antarctic fishes (Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus), from the temperate channel catfish (Ictalurus punctatus), and from a mammal (the cow). Each preparation contained the alpha- and beta-tubulins and was free of microtubule-associated proteins. When examined by isoelectric focusing and by two-dimensional electrophoresis, brain tubulins from the Antarctic fishes were found to be highly heterogeneous; each was resolved into approximately 20 isoelectric variants. The distributions of the isotubulins from the cold-adapted fishes were similar but differed significantly from those of tubulins from catfish and cow. The average isoelectric points of the alpha- and beta-tubulins from the Antarctic fishes were more basic than the isoelectric points of the corresponding tubulins from bovine brain. Peptide mapping confirmed that tubulins from the Antarctic fishes and the mammal differed in structure. The amino acid compositions of fish and mammalian tubulins were similar, but Antarctic fish tubulins apparently contained fewer Glx residues than did catfish or bovine tubulins. Finally, native tubulins from an Antarctic fish and the cow differed slightly in net negative charge. Thus, brain tubulins from the cold-adapted fishes differ structurally from the tubulins of a temperate fish and of a mammal.     

Construction of phylogenetic tree of plant tubulins basing on the homology of their protein sequences

We have built phylogenetic tree of alpha-, beta- and gamma-tubulins of plant kingdom, alpha- and beta-tubulins of pig, and all epsilon- and delta-tubulins with known primary sequences and analyzed the levels of homology of tubulin sequences for repersentatives of different groups of organisms. It has been established low heterogeneity of alpha-, gamma-tubulin families and more than two fold higher heterogeneity of beta-tubulins, based on the sequence speciality of alga tubulins. We have showed that sequences of animal tubulins do not fit any cluster formed by on the plant tubulins. Presence in primary tubulin sequences of three major families of insignificant specific differences which are specific for such phylogenetic items of plant kingdom as alga and angiosperms has been also demostrated. The cladogramm shows clear clasterization of investigated sequences according to their belonging to the tubulin families.     

Altered microtubule dynamics by expression of modified alpha-tubulin protein causes right-handed helical growth in transgenic Arabidopsis plants

The proper organization of cortical microtubule arrays is essential for anisotropic growth in plants but how distinct array patterns are formed is not understood. Here, we report a relationship between microtubule dynamics and array organization using transgenic plants expressing modified tubulins. When green fluorescent protein (GFP) or a hemaglutinin epitope tag was fused to the N-terminus of tubulins and expressed in Arabidopsis plants, these tubulins were incorporated into microtubules along with endogenous tubulins. Plants expressing the modified beta-tubulins were phenotypically normal and possessed transversely oriented cortical arrays in the epidermal cells of the root elongation zone; however, the expression of modified alpha-tubulins caused right-handed helical growth, increased trichome branching, and a shallow left-handed (S-form) helical array organization. In cells expressing the modified alpha-tubulins, microtubule dynamicity was suppressed and polymerization was promoted, and GFP-EB1 (End Binding 1) labeled larger regions of the microtubule end more frequently, when compared with control cells. We propose that the N-terminal appendage introduced into alpha-tubulin inhibits GTP hydrolysis, thus producing polymerization-prone microtubules with an extended GTP cap. Consistent with this interpretation, plants expressing an alpha-tubulin mutated in the GTPase-activating domain exhibited similar microtubule properties, with regard to dynamics and the localization of GFP-EB1, and showed right-handed helical growth.     

The secondary endosymbiont of the cryptomonad Guillardia theta contains alpha-, beta-, and gamma-tubulin genes.

Cryptomonads have acquired photosynthesis through secondary endosymbiosis: they have engulfed and retained a photosynthetic eukaryote. The remnants of this autotrophic symbiont are severely reduced, but a small volume of cytoplasm surrounding the plastid persists, along with a residual nucleus (the nucleomorph) that encodes only a few hundred genes. We characterized tubulin genes from the cryptomonad Guillardia theta. Despite the apparent absence of microtubules in the endosymbiont, we recovered genes encoding alpha-, beta-, and gamma-tubulins from the nucleomorph genome of G. theta. The presence of tubulin genes in the nucleomorph indicates that some component of the cytoskeleton is still present in the cryptomonad symbiont despite the fact that very little cytoplasm remains, no mitosis is known in the nucleomorph, and microtubules have never been observed anywhere in the symbiont. Phylogenetic analyses with nucleomorph alpha- and beta-tubulins support the origin of the cryptomonad nucleomorph from a red alga. We also characterized alpha and beta-tubulins from the host nucleus of G. theta and compared these with tubulins we isolated from two flagellates, Goniomonas truncata and Cyanophora paradoxa, previously proposed to be related to the cryptomonad host. Phylogenetic analyses support a relationship between the cryptomonad host and Goniomonas but do not support any relationship between cryptomonads and Cyanophora.     

Inhibition of phenylephrine-induced cardiac hypertrophy by docosahexaenoic acid

Many of the cardiovascular benefits of fish oil result from the antiarrhythmic actions of the n-3 polyunsaturated lipids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The beneficial effects of DHA/EPA in patients with coronary artery disease and myocardial infarction may also result from modulation of the myocardial hypertrophic response. Hypertrophy was assessed in neonatal cardiomyocytes exposed to phenylephrine (PE) by measuring cell surface area, total protein synthesis ((14)C leucine incorporation), and the organization of sarcomeric alpha-actinin and by monitoring expression of atrial natriuretic factor (ANF). We report that PE induced a twofold increase in cell surface area and protein synthesis in cardiomyocytes. The hypertrophied cardiomyocytes also exhibited increased expression of ANF in perinuclear regions and organization of sarcomeric alpha-actinin into classical z-bands. Treatment of cardiomyocytes with 5 microM DHA effectively prevented PE-induced hypertrophy as shown by inhibition of surface area expansion and protein synthesis, inhibition of ANF expression, and prevention of alpha-actinin organization into z-bands. DHA treatment prevented PE-induced activation of Ras and Raf-1 kinase. The upstream inhibition of Ras --> Raf-1 effectively prevented translocation and nuclear localization of phosphorylated extracellularly regulated kinase 1 and 2 (Erk1/2). These effects consequently led to inhibition of nuclear translocation, and hence, activation of the downstream signaling enzyme p90 ribosomal S6 kinase (p90(rsk)). These results indicate that PE-induced cardiac hypertrophy can be minimized by DHA. Our results suggest that inhibition of Ras --> Raf-1 --> Erk1/2 --> p90(rsk) --> hypertrophy is one possible pathway by which DHA can inhibit cardiac hypertrophy. In vivo studies are needed to confirm these in vitro effects of DHA.