Myocyte Autophagy in Heart Disease: Friend or Foe?

In the setting of hemodynamic stress, such as occurs in hypertension or following myocardial infarction, the heart undergoes a compensatory hypertrophic growth response. Left unchecked, this hypertrophic response triggers myocyte death, ventricular dilation, diminished contractile performance, and a clinical syndrome of heart failure. For some years, autophagy has been implicated in heart failure. More recently, mechanistic studies have emerged which provide new insights into the molecular underpinnings of hemodynamic stress-induced cardiomyocyte autophagy. Further, these studies have begun to provide clues as to whether cardiomyocyte autophagy is adaptive, mitigating disease pathogenesis, or maladaptive, contributing to disease progression. Here, we discuss recent studies that both answer questions and pose new ones.

Addendum to:

Cardiac Autophagy is a Maladaptive Response to Hemodynamic Stress

H. Zhu, P. Tannous, J.L. Johnstone, Y. Kong, J.M. Shelton, J.A. Richardson, V. Le, B. Levine, B.A. Rothermel and J.A. Hill

J Clin Invest 2007;117:1782-93     

Major oligomeric structural proteins of the HeLa nucleus

The three predominant polypeptides of the insoluble proteinaceous fraction from the HeLa cell nucleus polymerize in vitro upon oxidation of intrinsic sulfhydryl groups. The ease and specificity of this reaction indicate that these polypeptides exist as ordered oligomers in vivo. The comparable insoluble fraction from the rat liver nucleus also contains three predominant polypeptides of the same molecular weights, 65,000 71,000, and 75,000. The insoluble protein of the avian erythrocyte nuclear envelope consists principally of the 71,000- and the 75,000-dalton polypeptides. Indeed, in the avian erythrocyte nucleus these are the predominant polypeptides of the entire nucleus (Shelton, K., Cobbs, C., Povlishock, J. and Burkat, R., 1976, Arch. Biochem. Biophys.174, 177). Further, these avian polypeptides each form homogeneous covalently linked oligomers upon sulfhydryl oxidation (Cochran, D., Cobbs, C. and Shelton, K., 1977, J. Cell Biol.75, 151a). The insolubility, oligomeric disposition, and relative prominence of these polypeptides in a wide variety of cells indicate a fundamental structural role in the nucleus. Morphological features which may reflect this structural or skeletal role could be the nuclear envelope, the fibrous lamina, or perhaps an intrachromatinic matrix. The metabolism of the oligomeric polypeptides has been investigated in HeLa cells. Turnover of the HeLa insoluble nuclear protein is similar to that of the histones which are known to be stable proteins. The insoluble protein, including the oligomeric polypeptides, is synthesized in G1, S, and G2 phases of the cell cycle. This metabolic behavior indicates that the oligomeric polypeptides are reutilized in successive cell cycles and that synthesis accompanies nuclear and cellular expansion rather than deoxyribonucleohistone synthesis. This suggests that neither degradation nor selective synthesis of oligomeric polypeptides at a particular phase of the cell cycle are responsible for the breakdown and reformation of the interphase cell morphological features that occur during mitosis.     

Multiple forms and cellular localization of Drosophila DNA topoisomerase II

Purified type II topoisomerase from Drosophila melanogaster embryos was reported earlier to contain a major polypeptide of 166,000 daltons and several smaller peptides between 132,000 and 145,000 daltons (Shelton, E. R., Osheroff, N. and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9530-9535). Using purified topoisomerase II we have raised antibodies against the 132,000-166,000-dalton cluster of polypeptides. In this paper we demonstrate that at least three of these polypeptides are also present in embryos immediately upon lysis. Using antigen-affinity purified antibody from the cluster of purified topoisomerase II antigens, we have also discovered several smaller polypeptides in the molecular size range of 30,000-40,000 daltons in embryo extracts. These observations suggest the presence of multiple forms of DNA topoisomerases in the cell. In addition, we demonstrate that purified Drosophila topoisomerase II antibody recognizes yeast topoisomerase II antigens expressed by lambda gt 11-yeast topoisomerase II recombinants (Goto, T. and Wang, J. C. (1984) Cell 36, 1073-1080) establishing a structural homology between yeast and Drosophila enzymes. Antibody preparations were also used to localize the distribution of topoisomerase II in polytene nuclei. In contrast with the distribution of topoisomerase I which is located primarily at puffs, the Drosophila topoisomerase II is distributed generally along the chromosomes paralleling the distribution of DNA itself.     

Immunological evidence that band 3 is the major glucose transporter of the human erythrocyte membrane

We have previously reported that human erythrocyte band 3 contains 90-95% of the reconstitutable glucose transport activity of the erythrocyte membrane (Shelton, R.L. and Langdon, R.G. (1983) Biochim. Biophys. Acta 733, 25-33). We have now found that monoclonal and polyclonal antibodies to epitopes on band 3 specifically removed band 3 and more than 90% of the reconstitutable glucose transport activity from unfractionated octylglucoside extracts of erythrocyte membranes; nonimmune serum removed neither. Western blots of whole membrane extracts revealed that the polyclonal antibody to band 4.5 used to isolate cDNA clones presumed to code for the transporter (Mueckler, M., Caruso, C., Baldwin, C.A., Pancio, M., Blench, J., Morris, H.B., Allard, W.J., Lienhard, G.E. and Lodish, H.F. (1985) Science 229, 941-945) reacts strongly with six discrete bands in the 4.5 region. A monoclonal antibody to band 3 also reacts with a Mr 55,000 component of band 4.5. We conclude that band 3 contains the major glucose transporter of human erythrocytes, and that the transport activity in band 4.5 might be attributable to a band 3 fragment. Band 3 is probably a multifunctional transport protein responsible for transport of glucose, anions, and water.     

Cover Picture: J. Biophotonics 7/2014

Handheld OCT scanner in use in a primary care physician's office. The handheld scanner is equipped with an LCD touch screen, providing the physician with a traditional surface image as well as a corresponding depth‐resolved OCT image. The device‐mounted display enables the physician to maintain attention on the patient, rather than look at a traditional monitor display. (Picture: R. L. Shelton et al., pp. 525–533 in this issue)     

Binding of ferric heme by the recombinant globin from the cyanobacterium Synechocystis sp. PCC 6803

The product of the cyanobacterium Synechocystis sp. PCC 6803 gene slr2097 is a 123 amino acid polypeptide chain belonging to the truncated hemoglobin family. Recombinant, ferric heme-reconstituted Synechocystis sp. PCC 6803 hemoglobin is a low-spin complex whose endogenous hexacoordination gives rise to optical and NMR characteristics reminiscent of cytochrome b(5) [Scott, N. L., and Lecomte, J. T. J. (2000) Protein Sci. 9, 587-597]. In this work, the sequential assignments using (15)N-(13)C-labeled protein, (1)H nuclear Overhauser effects, and longitudinal relaxation data identified His70 as the proximal histidine and His46 as the sixth ligand to the iron ion. It was also found that one of two possible heme orientations within the protein matrix is highly preferred (>90%) and that this orientation is the same as in vertebrate myoglobins. The rate constant for the 180 degrees rotation of the heme within a protein cage to produce the favored isomer was 0.5 h(-1) at 25 degrees C, approximately 35 times faster than in sperm whale myoglobin. Variable temperature studies revealed an activation energy of 132 +/- 4 kJ mol(-1), similar to the value in metaquomyoglobin at the same pH. The rate constant for heme loss from the major isomer was estimated to be 0.01 h(-1) by optical spectroscopy, close to the value for myoglobin and decades slower than in the related Nostoc commune cyanoglobin. The slow heme loss was attributed in part to the additional coordination bond to His46, whereas the relatively fast rate of heme reorientation suggested that this bond was weaker than the proximal His70-Fe bond. The standard reduction potential of the hexacoordinated protein was measured with and without poly-L-lysine as a mediator and found to be approximately -150 mV vs SHE, indicating a stabilization of the ferric state compared to most hemoglobins and b(5) cytochromes.     

Structural interpretation of the amino acid sequence of a second domain from the Artemia covalent polymer globin

Artemia has a complex extracellular hemoglobin of Mr 260,000 comprising two globin chains (Mr 130,000) each of which is a polymer of eight covalently linked domains of Mr 16,000. The primary structure of this polymeric globin was studied to understand how globin folded domains are ordered within a globin chain and, in turn, how the latter associate into a functional hemoglobin molecule. Here we report the amino acid sequence of a second domain, E7 (Mr 16,081, excluding the heme), and interpretations of sequence data by computer-assisted alignment and modeling. This clearly shows that, as with domain E1 (Moens, L., Van Hauwaert, M.-L., De Smet, K., Geelen, D., Verpooten, G., Van Beeumen, J., Wodak, S., Alard, P., & Trotman, C. (1988) J. Biol. Chem. 263, 4679-4685), domain E7 is compatible with a globin folded structure of the beta-type chain. Several specific differences of domains E7 and E1 from the classic globins are identified. They possibly can be interpreted in terms of specific requirements for a double octameric functional molecule.     

Ligand binding in a hierarchy of globin complexes. The hexagonal bilayer hemoglobin of Lumbricus terrestris and its heme-containing subunits

The kinetics of CO and NO recombination with the giant approximately 3600-kDa hexagonal bilayer hemoglobin of Lumbricus terrestris and its subunits, the approximately 200-kDa dodecamer of globin chains (3 x chains (I + II + III + IV] (see preceding paper (Vinogradov S.N., Sharma, P.K., Qabor, A.N., Wall, J.S., Westrick, J.A., Simmons, J.H., and Gill, S.J. (1991) J. Biol. Chem. 266, 13091-13096], the 50-kDa disulfide-bonded trimer (chains II-IV), the monomer (chain I), and the approximately 30-kDa linker (chains VA, VB, and VI), were measured following photolysis over time scales ranging from picosecond to millisecond. CO recombination at 436 nm subsequent to excitation (9 ns) at 532 nm showed three phases covering a 100-fold range for the Hb, dodecamer, trimer, and linker protein. The proportion of the fast phase was 0.1-0.2 for the trimer, dodecamer, and Hb. The relative rates and amplitudes of the phases were not affected by changes in CO concentration or excitation intensity. The monomer showed a single phase with a rate of 2 x 10(6) M-1 s-1. The second-order reaction with NO showed two rates. The faster rate was 90 x 10(6) M-1 s-1 and accounted for approximately 0.7 of the reaction for all species except the monomer, where it accounted for the full reaction. The slower rate was 15 x 10(6) M-1 s-1 for all species except the monomer.     

Block duplications of a zeta-zeta-alpha-theta gene set in the rabbit alpha-like globin gene cluster

In order to understand the coordinate regulation between the alpha-like and beta-like globins during the developmental switches in hemoglobin synthesis, we have studied the rabbit alpha-like globin gene family. A cluster of six linked genes arranged 5'-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3' has been isolated as a set of overlapping clones from a library of rabbit genomic DNA. Blot-hybridization analysis of genomic DNA not only confirms this linkage arrangement but also reveals the presence of additional zeta and theta genes. We propose that this gene cluster was generated by a block duplication of a set of alpha-like genes; the proposed duplication unit is zeta-zeta-alpha-theta. Further duplications of a zeta-zeta-theta set are also proposed to have occurred. As expected for a duplicated locus, the rabbit alpha-like gene cluster contains long blocks of internal homology. The Z homology block is about 7.2 kilobase pairs long and contains the zeta genes; the T homology block is about 4.7 kilobase pairs long and contains a theta gene. Surprisingly, both Z and T homology blocks are flanked by a common junction sequence (J) which contains a region very similar to the 3'-untranslated sequence of an alpha-globin gene. Analysis of the J sequences suggests a recombination mechanism by which the alpha gene could have been deleted from the second set of genes in the cluster (zeta 2-zeta 3-theta 2). The relationships among the genes in characterized alpha-like gene clusters in mammals are summarized. The rabbit gene cluster differs from those of other mammals principally in the loss of a gene orthologous to the human psi alpha 1 and in the block duplication of the zeta-zeta-alpha-theta gene set.     

A membrane-bound vertebrate globin

The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2)-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O(2) binding with a variable affinity (P(50)～1.3-12.5 torr), depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein.     

Sensitive detection of human globin chains by microbore high-performance liquid chromatography and its forensic application

This paper describes a simple and sensitive high-performance liquid chromatographic (HPLC) method for the detection of human globin chains in blood and bloodstains. The method involves direct injection of the filtered samples of dilute hemolysates or bloodstain extracts onto a microbore C₄ reversed-phase column (2.1 mm I.D.) with UV detection at 220 nm. Microbore HPLC offers a significant improvement in sensitivity with little loss of the resolution of globin chains and only small variations in the determination of γ chain composition. The detection limit of hemoglobin (Hb) was 0.1 μg, which is equivalent to about 1 nl of fresh whole blood. Umbilical cord blood could be differentiated from adult blood in stains that were up to twenty weeks old, by the presence of γ globin chains. The present method will be useful for detection of abnormal Hbs and for the determination of γ chain composition in clinical laboratories, as well as in the practice of forensic science for the analysis of minute amounts of blood and bloodstains.     

A fusion gene in man: DNA sequence analysis of the abnormal globin gene of Hemoglobin Miyada

An abnormal globin gene from a patient heterozygous for Hemoglobin Miyada was cloned and sequenced. The results indicated that the 5′ flanking region and the 5′ side of the gene were identical to those of a β-globin gene and that the 3′ side was identical to that of a γ-globin gene. The part of the gene identical to a β-globin gene shifted to the part identical to the δ-globin gene somewhere in a homologous sequence region between the third nucleotide of the 17th codon and the second nucleotide of the 22nd codon of these two genes. Thus, results of analysis of the nucleotide sequence support the idea that the abnormal globin gene of Hemoglobin Miyada was generated as a fusion gene by unequal crossing over between a β- and a δ-globin gene.     

Replies to my critics

This paper contains replies to the reviews of my book by Steven Downes, Massimo Pigliucci and Deborah Shelton & Rick Michod.     

A large deletion encompassing the entire alpha-like globin gene cluster in a family of northern European extraction.

We describe a new deletional form of alpha thalassemia segregating in three generations of a family of northern European origin. A full-term female girl had hypochromic, microcytic anemia since early infancy associated with delayed language development, slow growth and weight gain. Hematologic studies suggested the presence of alpha thalassemia. Gene-blotting studies showed no abnormal alpha-like globin gene fragments; however, studies of inheritance of informative polymorphic restriction fragments using zeta, alpha and 3'-alpha-hypervariable region (3'-HVR) probes showed evidence for an extensive deletion encompassing the entire alpha-like globin gene cluster. The 3' breakpoint of this deletion maps beyond the 3'-HVR, a region implicated as a hot spot for the generation of other large deletional events within the alpha-like cluster. The 5' breakpoint maps at least 10 kilobases (kb) 5' to the zeta-globin gene. The minimum size estimate for this deletion is greater than 47 kilobases.     

Kinetic analysis of a cytoplasmic casein kinase II from Artemia sp.

• 1.1. A cytoplasmic casein kinase II (CKII) has been purified more than 10,000-fold from Artemia sp.
• 2.2. The reaction mechanism of the cytoplasmic CKII was determined to be random bi bi, using ATP and casein as substrates, which is in agreement with the results obtained for a DrosophilaCKII [Glover, Shelton and Brutlag (1983) J. biol. Chem.258, 3258–3265]. K_m values for ATP and casein are 8 μM and 0.2 mg/ml respectively. The binding of either substrate lowers the enzyme-affinity for the other by a factor α = 1.65.
• 3.3. In vitro, the enzyme is inhibited by poly(A)²-mRNA and 2,3-diphosphoglyceric acid (2,3-DPG). The inhibition by 2,3-DPG is due to competition with the protein substrate.
• 4.4. The possible in vivo effects of these inhibitors in CKII-mediated translational regulation is discussed.

     

Organization of non-vertebrate globin genes.

The organization of non-vertebrate globin genes exhibits substantially more variability than the three-exon, two-intron structure of the vertebrate globin genes. (1) The structures of genes of the single-domain globin chains of the annelid Lumbricus and the mollusc Anadara, and the globin gene coding for the two-domain chains of the clam Barbatia, are similar to the vertebrate plan. (2) Genes for single-domain chains exist in bacteria and protozoa. Although the globin gene is highly expressed in the bacterium Vitreoscilla, the putative globin gene hmp in E. coli, which codes for a chimeric protein whose N-terminal moiety of 139 residues contains 67 residues identical to the Vitreoscilla globin, may be either unexpressed or expressed at very low levels, despite the presence of normal regulatory sequences. The DNA sequence of the globin gene of the protozoan Paramecium, determined recently by Yamauchi and collaborators, appears to consist of two exons separated by a short intron. (3) Among the lower eukaryotes, the yeasts Saccharomyces and Candida have chimeric proteins consisting of N-terminal globin and C-terminal flavoprotein moieties of about the same size. The structure of the gene for the chimeric protein of Saccharomyces exhibits no introns. According to Riggs, the presence of chimeric proteins in E. coli and other prokaryotes, such as Alcaligenes and Rhizobium, as well as in yeasts, suggests a previously unrecognized evolutionary pathway for hemoglobin, namely that of a multipurpose heme-binding domain attached to a variety of unrelated proteins with diverse functions. (4) The published globin gene sequences of the insect larva Chironomus have an intron-less structure and are present as clusters of multiple copies; the expression of the globin genes is tissue and developmental stage-specific. Furthermore, the expression of many of these genes has not yet been demonstrated despite the presence of apparently normal regulatory sequences in the two flanking regions. Unexpectedly, Bergtrom and collaborators have recently shown that at least three Ctt globin II beta genes contain putative introns. (5) Pohajdak and collaborators have found a seven-exon and six-intron structure for the globin gene of the nematode Pseudoterranova which codes for a two-domain globin chain. Although the second and fourth introns of the N-terminal domain correspond to the two introns found in vertebrate globin genes, the position of the third intron is close to that of the central intron in plant hemoglobins.(ABSTRACT TRUNCATED AT 400 WORDS)