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Emerging Roles for Myoglobin in the Heart
Emerging Roles for Myoglobin in the Heart Daniel J. Garry,* Shane B. Kanatous, and Pradeep P.A. Mammen
Myoglobin (Mb) is an intensely studied hemoprotein that is restricted mainly to the heart and oxidative myofibers in skeletal muscle. Previous physiologic and pharmacologic studies have supported a role for Mb in facilitated oxygen transport or as an oxygen reservoir in striated muscle. Transgenic and gene disruption technologies have been utilized to produce mice that lack Mb. Studies utilizing these transgenic mouse models support the notion that Mb may have multiple, diverse functions in the heart. Future studies using these emerging technologies will further enhance the understanding of the role of Mb and other hemoproteins in cardiovascular biology. (Trends Cardiovasc Med 2003;13:111–116) © 2003, Elsevier Science Inc.
Tissue hemoglobins are distributed widely among diverse organisms including plants, mollusks, and mammals. Two hundred years ago, investigators proposed that the red color of muscle tissue in vertebrates was due to an intrinsic pigment distinct from hemoglobin (reviewed in Wittenberg and Wittenberg 1989). Initially, this muscle pigment was referred to as myochrome, and was eventually named myoglobin (Mb) to reflect its functional, structural, and evolutionary relationship with hemoglobin (Kendrew et al. 1954, Wittenberg and Wittenberg 1989). The functional role for Mb has been ex-
Daniel J. Garry, Shane B. Kanatous, and Pradeep P.A. Mammen are at the Department of Internal Medicine, University of Texas, Southwestern Medical Center, Dallas, Texas, USA. Daniel J. Garry also is affiliated with the Department of Molecular Biology, University of Texas, Southwestern Medical Center, Dallas, Texas, USA. * Address correspondence to: Daniel J. Garry, MD, PhD, 5323 Harry Hines Blvd., NB11.118A, University of Texas, Southwestern Medical Center, Dallas, TX 75390-8573, USA. Tel.: (1) 214-648-1175; fax: (1) 214-648-1450; e-mail: [email protected]. © 2003, Elsevier Science Inc. All rights reserved. 1050-1738/03/$-see front matter
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amined extensively since its discovery, and recent studies using a gene disruption strategy to produce mice that lack Mb support a role for this hemoprotein as an important modulator of cardiac function. This review summarizes the results of numerous studies that examined the structural, molecular, and physiologic roles for Mb in the heart. • Structural Organization Mb was the first protein to be subjected to definitive structural analysis (Kendrew et al. 1958). This structural analysis was undertaken by John Kendrew and coworkers with the use of x-ray diffraction techniques (Kendrew et al. 1958 and 1960). Subsequently, a number of structural studies confirmed Kendrew’s initial findings and enhanced the understanding of the physical structure of Mb (Frauenfelder et al. 2001, Shoenborn et al. 1965, Tilton et al. 1984). Collectively, these studies established that the backbone of Mb consists of eight helices (designated A–H). These helices wrap around a central pocket containing a prosthetic protoheme group that is bracketed by histidine residues (His64, which is above the heme residue; and
His93, which is below the heme residue) (Figure 1). The prosthetic protoheme group is a stable compound of iron with protoporphyrin IX. Four out of the six coordination sites of the iron atom are involved with bonds to porphyrin (Kendrew 1963, Wittenberg and Wittenberg 1989). The linking of the fifth coordination site of the iron atom to the histidine moiety of Mb (referred to as the proximal histidine or His93, which is located below the heme group) stabilizes the heme group, and the final iron coordination (sixth) site is available for the binding of ligands (e.g., dioxygen, nitric oxide [NO], carbon monoxide, etc.) (Frauenfelder and McMahon 2001, Wittenberg and Wittenberg 1989). With the use of x-ray diffraction and xenon binding studies, four cavities were identified within the protein (Xe1, Xe2, Xe3, and Xe4) and were shown to be evolutionarily conserved (Frauenfelder et al. 2001, Shoenborn et al. 1965, Tilton et al. 1984) (Figure 1). The significance of these cavities is unclear, but studies (Chu et al. 2000, Frauenfelder et al. 2001, Ostermann et al. 2000, Scott et al. 2001) suggest that they may function either as escape routes for ligands or as cavities that are able to concentrate ligands and promote their binding to the heme residue. • Genomic Organization Mb and hemoglobin evolved from a common ancestral gene more than 500 million years ago. The genomic organization for the Mb gene has been defined in numerous species, including the grey seal, sperm whale, mouse, and human (Blanchetot et al. 1986, Weller et al. 1984, Wittenberg and Wittenberg 1989). Although considerable homology exists among various species, the genomic structure is relatively simple, because the Mb gene consists of three exons and two introns. In the mouse, exon 2 encodes the heme-binding domain and amino acids 31 through 105 of the total 154 residues that comprise this hemoprotein (Blanchetot et al. 1986). Previous studies (Bassel-Duby et al. 1993, Parsons et al. 1993, Yan et al. 2001) utilizing transgenic strategies have established that sequences contained within a 0.4-kb proximal 5 flanking region of either the human or mouse Mb genes are sufficient to confer expression in the heart or oxidative skeletal myofibers. Transgenic
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and mutational analyses (Grayson et al. 1995, Yan et al. 2001) have identified the importance of a CCAC box Sp1 binding element, an A/T-rich myocyte enhancer factor-2 (MEF2) binding motif, and a proximal E-box motif for the transcriptional regulation of this hemoprotein (Figure 2). Additional studies (Chin et al. 1998) suggest that a combinatorial mechanism involving nuclear factor of activated T cells (NFAT) and MEF2 proteins are important in the transcriptional activation of the Mb gene. Collectively, these studies support the conclusion that functional interactions between transcription factors such as Sp1, MEF2, NFAT proteins, and basic helix–loop– helix proteins modulate the transcriptional regulation of the Mb gene and confer expression in the myocardium and oxidative skeletal myofibers (Figure 2). • Gene Disruption/Knockout Studies In vertebrates, Mb is restricted mainly to the myocardium and oxidative skeletal myofibers (Figure 3). Mb is expressed early during cardiogenesis and is upregulated during the postnatal period and in the adult (Figure 3) (Garry et al. 1996). This distinctive temporal and spatial expression pattern coincides with increasing metabolic demands on the heart associated with growth and development. Because of the structural similarity (i.e., the heme-binding domain) between Mb and hemoglobin, numerous pharmacologic studies supported the concept that Mb facilitates oxygen flux from the erythrocyte to the mitochondria in the heart and is essential for survival (reviewed in Wittenberg and Wittenberg 1989). To further examine the role of Mb in the heart, we and others (Garry et al. 1998, Gödecke et al. 1999) independently utilized a gene-disruption strategy and deleted exon 2, which encodes the hemebinding domain from the Mb gene. Homozygous mutant mice lacked fulllength or truncated Mb protein in heart and oxidative skeletal muscle. We observed that many Mb-deficient embryos died in utero at midgestation with signs of cardiac failure. Fetal mice that lacked Mb and survived to gestational day 12.5 suffered no subsequent excess mortality, developed normally, and were capable of tolerating the physiologic challenges associated with pregnancy (Meeson et al.
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2001). In addition, viable Mb null mice had preserved cardiac performance under normal conditions. These results supported the conclusion that Mb was an important hemoprotein during cardiac development. Furthermore, the survival of a subpopulation of Mb-deficient mice suggested that mutant embryos survived by mounting complex adaptations that were sufficient to promote viability and cardiac function under normal conditions. In the absence of Mb, no apparent structural changes were observed to be associated with the sarcomeric organization or the mitochondrial content of the heart or skeletal muscle. Gödecke et al. (1999) identified a number of cellular adaptive mechanisms in the Mb-deficient heart, including increased capillary density, increased coronary flow, and elevated hematocrits that, in part, were capable of substituting for Mb to preserve myocardial performance and cardiac energetics. These results were supported further by the observations of increased capillary density in hearts and oxidative skeletal muscle of viable Mb null mice (Grange et al. 2001, Meeson et al. 2001). In addition to the cellular adaptive mechanisms, increased expression of the hypoxia-inducible gene program that includes hypoxia-inducible factor 1 (HIF1), endothelial per-ARNT-sim (PAS) domain protein (HIF-2), vascular endothelial growth factor, stress proteins (Hsp27), and NO metabolism are observed during the later stages of development and persist in the Mb null heart (Meeson et al. 2001). The induction of the hypoxiainducible gene program presumably is in response to intracellular hypoxia and in part promotes the cellular adaptations, including increased vasculogenesis (Grange et al. 2001, Meeson et al. 2001). Collectively, the induction of these molecular and cellular adaptive mechanisms suggests that Mb functions as a mediator of oxygen transport in the heart. Future studies using both pharmacologic and physiologic stimuli in conjunction with these knockout mouse models will enhance the understanding of Mb biology. • Functional Roles for Mb Since the early 1900s, tissue hemoglobins such as Mb were believed to function in the storage of dioxygen and in facilitated oxygen transport from the periph-
ery of the cell to the mitochondria (Wittenberg 1959). The physiologic role as a store for dioxygen was supported by the observation that mammals adapted for either breath-hold diving (e.g., Northern elephant seal or Sperm whale) or those living at high altitude had a 10-fold higher (0.5 mM in mammals at sea level vs. 4–5 mM in marine mammals such as the seal) Mb concentration in skeletal muscle (Table 1) (Hochachka 1999). Mb also has been shown to function in facilitated oxygen diffusion in the cardiomyocyte and typically remains 40% to 70% saturated with dioxygen (Mb O2 I MbO2) (Figure 4). This function as a mediator of O2 flux has been verified further in isolated cell and tissue preparations in combination with various pharmacologic inhibitors of Mb. Utilizing isolated cardiomyocyte preparations exposed to various oxygen conditions, Merx et al. (2001) observed decreased oxygen consumption of Mb/ cardiomyocytes at low oxygen tension compared with wild type controls, further establishing a role for Mb-facilitated oxygen diffusion. An additional role for Mb is the regulation of NO bioavailability within the cardiomyocyte (Figure 4). Based on the results of kinetic and spectral studies as well as the structural characterization of this hemoprotein, Frauenfelder et al. (2001) proposed that Mb functions as an allosteric enzyme, because the cavities are capable of concentrating ligands (i.e., NO, O2, CO, etc.) and affecting the rate of binding of these ligands to the heme residue (Figure 5). NO avidly binds to hemoproteins such as hemoglobin, Mb, cytochrome c oxidase, and soluble guanylate cyclase, and these proteins are essential in the modulation of NO bioactivity and responsiveness to the concentration of NO (oxymyoglobin NO → ferrin myoglobin nitrate) (Figure 4) (Eich et al. 1996). NO is an important inter- and intracellular physiologic messenger that is capable of evoking a number of cellular responses that are beneficial or potentially toxic. Previous studies (Brunori 2001, Flögel et al. 2001b) have established that NO is an important regulator of mitochondrial oxidative phosphorylation. NO competes with O2 to reversibly bind and inhibit cytochrome c oxidase (complex IV of the electron transport system), thereby inhibiting mitochondrial respiration (Poderoso TCM Vol. 13, No. 3, 2003
Figure 2. The 2.0-kb myoglobin (Mb) promoter confers expression within the cardiomyocyte and contains essential transcriptional regulatory motifs. Schematic of the 2.0-kb Mb promoter containing essential motifs and the putative transcription factors that bind the regulatory elements [i.e., CCAC box, E-boxes, A/T motif, and nuclear factor of activated T-cell NFAT response elements (NRE), and myocyte enhancer factor-2 (MEF2)].
Figure 1. Structure of myoglobin consists of eight helices wrapped around a heme residue (red). Xenon binding studies using x-ray diffraction have identified four xenon cavities (designated 1–4), which may serve to concentrate ligands or serve as escape routes for the ligands. The evolutionarily conserved histidine residues serve to stabilize the heme (red) group (H93) or serve as a gate (H64, yellow) regulating the entry and exit of ligands.
et al. 1998, Shiva et al. 2001). In a previous study (Grange et al. 2001), we observed that NO generation is altered in skeletal muscle of Mb null mice following an electrical stimulation. Moreover, we challenged the Mb/ mice with exposure to chronic hypoxia and observed that in the absence of Mb, perturbed NO homeostasis resulted in left ventricular systolic dysfunction (Mammen et al. 2001). These results supported the conclusion that Mb functions as a modulator of NO bioavailability in the heart (Figure 5) (Flögel et al. 2001b, Mammen et al. 2001).
Figure 3. Myoglobin (Mb) is temporally and spatially expressed in the heart and oxidative skeletal myofibers. Using in situ hybridization and 35S-labeled antisense Mb riboprobes, expression was evaluated in the embryonic and adult mouse. (A) Brightfield hematoxylin-stained section corresponds to the field shown in panel (B). (B) In situ hybridization of a transverse section of an E10.0 mouse embryo reveals Mb expression within the ventricular myocardium and Mb absence within the atrium. Mb is expressed at lower levels within the myotome of the somite. (C) Transverse section of an adult mouse heart probed with the Mb riboprobe reveals expression in the ventricle and atrium. (D) Transverse section of the adult mouse hindlimb reveals heterogeneous expression of Mb in oxidative myofibers of the adult hindlimb. Note the increased expression of Mb within the oxidative soleus muscle (arrow). a, atrium; f, fibula; m, myotome of somite; s, soleus muscle; v, ventricle.
Table 1. Mb concentrations among species Organism Northern elephant seal Sperm whale Bottlenose dolphin New Zealand white rabbit Mouse (129) Human Human
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mg/g wet weight 64 57 26 8 1.9 2.4 2.1
Tissue
Reference
Longissimus dorsi muscle Skeletal muscle Longissimus dorsi muscle Skeletal muscle Skeletal muscle Heart Psoas muscle
Noren et al. (2001) Dolar et al. (1999) Noren and Williams (2000) Noren and Williams (2000) Perkoff and Tyler (1958) Swaanenburg et al. (2001) Perkoff and Tyler (1958)
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Figure 4. Myoglobin (Mb) is a regulator of dioxygen and nitric oxide (NO) in the cardiomyocyte. Dioxygen (O2) binds to deoxymyoglobin (Mb) to yield oxymyoglobin (MbO2), which transports oxygen to sites of use. MbO2 reacts with or scavenges NO to form ferric Mb (metMb) and nitrate (NO3). MetMb-reductase reduces metMb to yield Mb. Finally, Mb may bind NO to yield nitrosylmyoglobin (MbNO), which may react with oxygen to produce metmyoglobin (Brunori 2001).
A recent study by McMahon et al. (2002) provided data supporting the hypothesis that NO may function as a hormone, because it can be alternately captured or released by hemoglobin in red blood cells for bioavailability remote from the site of NO synthesis. Similarly, Mb may function not only as a scavenger of NO, but may also shuttle NO to compartments within the cardiomyocyte for bioavailability (Figure 5). Mb also has been reported to have peroxidase activity (Figure 5) (George and Irvine 1955). Although the peroxidase activity of Mb is lower than that of established endogenous peroxidases, studies (Cadenas 1989, Khan et al. 1998)
suggest that Mb has the potential for catalytic oxidation of substrates in the presence of hydrogen peroxide. Kinetic studies of Mb with mutations at histidine 64, tyrosine 103, and cystine 110 have indicated that these sites are essential in providing Mb with peroxidase activity (Khan et al. 1998, Witting et al. 2000 and 2002). Furthermore, recent studies by Schrader’s laboratory (Flögel et al. 2001a, Flögel et al. 2001b) support a role for Mb as a scavenger of reactive oxygen species. These studies indicate that Mb may play a critical role in limiting the toxic effects of reactive oxygen species in the cardiomyocyte (Figure 5).
• Tissue Globins Although Mb was the first tissue hemoglobin identified in vertebrates, two novel globins—neuroglobin and cytoglobin— recently have been shown to be expressed in adult vertebrate tissues. Neuroglobin is relatively dissimilar to Mb (21% identity) and hemoglobin (25% identity) and does not appear to share a common ancestral gene with either of these globins (Figure 6) (Burmester et al. 2000). Neuroglobin is a monomeric 151 amino acid hemoprotein that reversibly binds oxygen (although less avidly than does Mb) and is expressed primarily in focal regions of the brain in relatively low
Figure 5. Possible roles for myoglobin (Mb) in the heart. Mb has been proposed to function in the transport and storage of dioxygen. Recent studies suggest that Mb may function as an inactivator or a transporter of nitric oxide (NO). Alternatively, Mb may function to scavenge reactive oxygen species or function as an allosteric enzyme by concentrating ligands in the xenon pockets (yellow spheres) to accelerate the binding of the ligands (i.e., O2, NO, CO) to the heme residue.
Figure 6. Sequence homology of mouse myoglobin (Mb), cytoglobin (Cygb), neuroglobin (Ngb), and hemoglobin A (Hba) hemoproteins. The amino acid sequences for Mb, Cygb, Ngb, and Hba for the mouse proteins are shown with shading of homologous sequences. Note that the amino acids His-93 and His-64 (arrowhead) are conserved and stabilize the heme group. The eight helices (A–H) are shown in schematic fashion (red) and are above the corresponding amino acid sequence that constitutes each helix.
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concentrations (approximately 0.01% of the total protein content or 1 M) (Burmester et al. 2000, Mammen et al. 2002). The focal expression of neuroglobin suggests that this hemoprotein may function as a modulator of NO (in a fashion similar to that of hemoglobin and Mb) and/or as an oxygen sensor, rather than as a mediator of oxygen transport or storage (Couture et al. 2001, Dewilde et al. 2001, Trent et al. 2001). Cytoglobin is the fourth tissue globin that has been identified and appears to be ubiquitously expressed in the zebrafish, mouse, and human (Burmester et al. 2002). This novel member of the globin family constitutes a 190 amino acid protein that appears to share an ancestral gene with Mb (Figure 6). Whereas the functional role for cytoglobin is unclear, a recent study (Kawada et al. 2001) has reported that this protein is localized to the stellate cells in the liver and may function as a novel endogenous peroxidase. Further biochemical and molecular studies using transgenic strategies will be helpful in the definition of the functional roles for both of these novel hemoproteins. • Future Directions Over the past 4 decades, multiple disciplines have contributed significantly to the understanding of the role of Mb in the heart using structural, biochemical, physiologic, and molecular techniques. The results of these studies support the hypothesis that Mb is a multifunctional hemoprotein that participates in diverse functional roles, including oxygenmediated transport and NO inactivation, and as a reservoir of dioxygen, an allosteric enzyme, and a scavenger of reactive oxygen species. Future studies will continue to utilize emerging technologies to define the molecular details of the biological role for Mb. These studies will examine broadly the role of Mb as an allosteric enzyme using structural and biochemical techniques, the transcriptional regulation of the Mb gene under hypoxic and normoxic conditions, and a role for Mb as a scavenger or transporter of NO that participates in NO homeostasis. Furthermore, the availability of sequenced species-specific genomic programs will allow investigators to continue to identify novel hemoproteins and determine their functional role in the heart or as a partner/substitute for Mb. Collectively, these TCM Vol. 13, No. 3, 2003
future studies will further enhance our understanding of the role of hemoproteins in cardiovascular biology. • Acknowledgments The authors thank Drs. R. Sanders Williams, Rhonda Bassel-Duby, and Annette Meeson for helpful discussions throughout the course of these studies. They also acknowledge funding support from the National Institutes of Health (HL63788) and the Donald W. Reynolds Foundation.
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Insights from Angiogenesis Trials Using Fibroblast Growth Factor for Advanced Arteriosclerotic Disease Rohit Khurana and Michael Simons*
The aim of therapeutic angiogenesis in cardiovascular disease states is to improve myocardial and peripheral extremity perfusion and function within ischemic regions that are not amenable to traditional modes of revascularization. Substantial “proof of concept,” efficacy, and safety data have emerged from numerous animal models and clinical trials that fibroblast growth factor (FGF), when administered by various delivery strategies, has a therapeutic angiogenic capacity. This initial excitement has been replaced by cautious optimism in the wake of results from larger, randomized, double-blinded placebo-controlled trials of both FGF gene and protein administration. A greater understanding of the profound placebo effect, careful patient selection, and improved endpoint assessment are factors that need to be addressed in this rapidly evolving era of molecular therapeutics. (Trends Cardiovasc Med 2003;13:116–122) © 2003, Elsevier Science Inc.
Rohit Khurana and Michael Simons are at the Section of Cardiology and Angiogenesis Research Center, Dartmouth Hitchcock Medical Center, Dartmouth Medical School, Lebanon, New Hampshire, USA. * Address correspondence to: Michael Simons, MD, Section of Cardiology, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756, USA. Tel.: (1) 603-650-3540; fax: (1) 603-650-5171; e-mail: [email protected]. © 2003, Elsevier Science Inc. All rights reserved. 1050-1738/03/$-see front matter
• FGF Biology Fibroblast growth factors (FGFs) are a family of 22 closely related proteins that share certain structural features and the ability to interact with specific receptors (Ornitz and Itoh 2001). FGFs elicit diverse biologic effects on numerous cell types (fibroblasts, endothelial cells, smooth muscle cells, and keratinocytes, among others). These effects encompass stimulation of growth, proliferation, migration, TCM Vol. 13, No. 3, 2003
Myoglobin (Mb) is an intensely studied hemoprotein that is restricted mainly to the heart and oxidative myofibers in skeletal muscle. Previous physiologic and pharmacologic studies have supported a role for Mb in facilitated oxygen transport or as an oxygen reservoir in striated muscle. Transgenic and gene disruption technologies have been utilized to produce mice that lack Mb. Studies utilizing these transgenic mouse models support the notion that Mb may have multiple, diverse functions in the heart. Future studies using these emerging technologies will further enhance the understanding of the role of Mb and other hemoproteins in cardiovascular biology. (Trends Cardiovasc Med 2003;13:111–116) © 2003, Elsevier Science Inc.
Tissue hemoglobins are distributed widely among diverse organisms including plants, mollusks, and mammals. Two hundred years ago, investigators proposed that the red color of muscle tissue in vertebrates was due to an intrinsic pigment distinct from hemoglobin (reviewed in Wittenberg and Wittenberg 1989). Initially, this muscle pigment was referred to as myochrome, and was eventually named myoglobin (Mb) to reflect its functional, structural, and evolutionary relationship with hemoglobin (Kendrew et al. 1954, Wittenberg and Wittenberg 1989). The functional role for Mb has been ex-
Daniel J. Garry, Shane B. Kanatous, and Pradeep P.A. Mammen are at the Department of Internal Medicine, University of Texas, Southwestern Medical Center, Dallas, Texas, USA. Daniel J. Garry also is affiliated with the Department of Molecular Biology, University of Texas, Southwestern Medical Center, Dallas, Texas, USA. * Address correspondence to: Daniel J. Garry, MD, PhD, 5323 Harry Hines Blvd., NB11.118A, University of Texas, Southwestern Medical Center, Dallas, TX 75390-8573, USA. Tel.: (1) 214-648-1175; fax: (1) 214-648-1450; e-mail: [email protected]. © 2003, Elsevier Science Inc. All rights reserved. 1050-1738/03/$-see front matter
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amined extensively since its discovery, and recent studies using a gene disruption strategy to produce mice that lack Mb support a role for this hemoprotein as an important modulator of cardiac function. This review summarizes the results of numerous studies that examined the structural, molecular, and physiologic roles for Mb in the heart. • Structural Organization Mb was the first protein to be subjected to definitive structural analysis (Kendrew et al. 1958). This structural analysis was undertaken by John Kendrew and coworkers with the use of x-ray diffraction techniques (Kendrew et al. 1958 and 1960). Subsequently, a number of structural studies confirmed Kendrew’s initial findings and enhanced the understanding of the physical structure of Mb (Frauenfelder et al. 2001, Shoenborn et al. 1965, Tilton et al. 1984). Collectively, these studies established that the backbone of Mb consists of eight helices (designated A–H). These helices wrap around a central pocket containing a prosthetic protoheme group that is bracketed by histidine residues (His64, which is above the heme residue; and
His93, which is below the heme residue) (Figure 1). The prosthetic protoheme group is a stable compound of iron with protoporphyrin IX. Four out of the six coordination sites of the iron atom are involved with bonds to porphyrin (Kendrew 1963, Wittenberg and Wittenberg 1989). The linking of the fifth coordination site of the iron atom to the histidine moiety of Mb (referred to as the proximal histidine or His93, which is located below the heme group) stabilizes the heme group, and the final iron coordination (sixth) site is available for the binding of ligands (e.g., dioxygen, nitric oxide [NO], carbon monoxide, etc.) (Frauenfelder and McMahon 2001, Wittenberg and Wittenberg 1989). With the use of x-ray diffraction and xenon binding studies, four cavities were identified within the protein (Xe1, Xe2, Xe3, and Xe4) and were shown to be evolutionarily conserved (Frauenfelder et al. 2001, Shoenborn et al. 1965, Tilton et al. 1984) (Figure 1). The significance of these cavities is unclear, but studies (Chu et al. 2000, Frauenfelder et al. 2001, Ostermann et al. 2000, Scott et al. 2001) suggest that they may function either as escape routes for ligands or as cavities that are able to concentrate ligands and promote their binding to the heme residue. • Genomic Organization Mb and hemoglobin evolved from a common ancestral gene more than 500 million years ago. The genomic organization for the Mb gene has been defined in numerous species, including the grey seal, sperm whale, mouse, and human (Blanchetot et al. 1986, Weller et al. 1984, Wittenberg and Wittenberg 1989). Although considerable homology exists among various species, the genomic structure is relatively simple, because the Mb gene consists of three exons and two introns. In the mouse, exon 2 encodes the heme-binding domain and amino acids 31 through 105 of the total 154 residues that comprise this hemoprotein (Blanchetot et al. 1986). Previous studies (Bassel-Duby et al. 1993, Parsons et al. 1993, Yan et al. 2001) utilizing transgenic strategies have established that sequences contained within a 0.4-kb proximal 5 flanking region of either the human or mouse Mb genes are sufficient to confer expression in the heart or oxidative skeletal myofibers. Transgenic
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and mutational analyses (Grayson et al. 1995, Yan et al. 2001) have identified the importance of a CCAC box Sp1 binding element, an A/T-rich myocyte enhancer factor-2 (MEF2) binding motif, and a proximal E-box motif for the transcriptional regulation of this hemoprotein (Figure 2). Additional studies (Chin et al. 1998) suggest that a combinatorial mechanism involving nuclear factor of activated T cells (NFAT) and MEF2 proteins are important in the transcriptional activation of the Mb gene. Collectively, these studies support the conclusion that functional interactions between transcription factors such as Sp1, MEF2, NFAT proteins, and basic helix–loop– helix proteins modulate the transcriptional regulation of the Mb gene and confer expression in the myocardium and oxidative skeletal myofibers (Figure 2). • Gene Disruption/Knockout Studies In vertebrates, Mb is restricted mainly to the myocardium and oxidative skeletal myofibers (Figure 3). Mb is expressed early during cardiogenesis and is upregulated during the postnatal period and in the adult (Figure 3) (Garry et al. 1996). This distinctive temporal and spatial expression pattern coincides with increasing metabolic demands on the heart associated with growth and development. Because of the structural similarity (i.e., the heme-binding domain) between Mb and hemoglobin, numerous pharmacologic studies supported the concept that Mb facilitates oxygen flux from the erythrocyte to the mitochondria in the heart and is essential for survival (reviewed in Wittenberg and Wittenberg 1989). To further examine the role of Mb in the heart, we and others (Garry et al. 1998, Gödecke et al. 1999) independently utilized a gene-disruption strategy and deleted exon 2, which encodes the hemebinding domain from the Mb gene. Homozygous mutant mice lacked fulllength or truncated Mb protein in heart and oxidative skeletal muscle. We observed that many Mb-deficient embryos died in utero at midgestation with signs of cardiac failure. Fetal mice that lacked Mb and survived to gestational day 12.5 suffered no subsequent excess mortality, developed normally, and were capable of tolerating the physiologic challenges associated with pregnancy (Meeson et al.
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2001). In addition, viable Mb null mice had preserved cardiac performance under normal conditions. These results supported the conclusion that Mb was an important hemoprotein during cardiac development. Furthermore, the survival of a subpopulation of Mb-deficient mice suggested that mutant embryos survived by mounting complex adaptations that were sufficient to promote viability and cardiac function under normal conditions. In the absence of Mb, no apparent structural changes were observed to be associated with the sarcomeric organization or the mitochondrial content of the heart or skeletal muscle. Gödecke et al. (1999) identified a number of cellular adaptive mechanisms in the Mb-deficient heart, including increased capillary density, increased coronary flow, and elevated hematocrits that, in part, were capable of substituting for Mb to preserve myocardial performance and cardiac energetics. These results were supported further by the observations of increased capillary density in hearts and oxidative skeletal muscle of viable Mb null mice (Grange et al. 2001, Meeson et al. 2001). In addition to the cellular adaptive mechanisms, increased expression of the hypoxia-inducible gene program that includes hypoxia-inducible factor 1 (HIF1), endothelial per-ARNT-sim (PAS) domain protein (HIF-2), vascular endothelial growth factor, stress proteins (Hsp27), and NO metabolism are observed during the later stages of development and persist in the Mb null heart (Meeson et al. 2001). The induction of the hypoxiainducible gene program presumably is in response to intracellular hypoxia and in part promotes the cellular adaptations, including increased vasculogenesis (Grange et al. 2001, Meeson et al. 2001). Collectively, the induction of these molecular and cellular adaptive mechanisms suggests that Mb functions as a mediator of oxygen transport in the heart. Future studies using both pharmacologic and physiologic stimuli in conjunction with these knockout mouse models will enhance the understanding of Mb biology. • Functional Roles for Mb Since the early 1900s, tissue hemoglobins such as Mb were believed to function in the storage of dioxygen and in facilitated oxygen transport from the periph-
ery of the cell to the mitochondria (Wittenberg 1959). The physiologic role as a store for dioxygen was supported by the observation that mammals adapted for either breath-hold diving (e.g., Northern elephant seal or Sperm whale) or those living at high altitude had a 10-fold higher (0.5 mM in mammals at sea level vs. 4–5 mM in marine mammals such as the seal) Mb concentration in skeletal muscle (Table 1) (Hochachka 1999). Mb also has been shown to function in facilitated oxygen diffusion in the cardiomyocyte and typically remains 40% to 70% saturated with dioxygen (Mb O2 I MbO2) (Figure 4). This function as a mediator of O2 flux has been verified further in isolated cell and tissue preparations in combination with various pharmacologic inhibitors of Mb. Utilizing isolated cardiomyocyte preparations exposed to various oxygen conditions, Merx et al. (2001) observed decreased oxygen consumption of Mb/ cardiomyocytes at low oxygen tension compared with wild type controls, further establishing a role for Mb-facilitated oxygen diffusion. An additional role for Mb is the regulation of NO bioavailability within the cardiomyocyte (Figure 4). Based on the results of kinetic and spectral studies as well as the structural characterization of this hemoprotein, Frauenfelder et al. (2001) proposed that Mb functions as an allosteric enzyme, because the cavities are capable of concentrating ligands (i.e., NO, O2, CO, etc.) and affecting the rate of binding of these ligands to the heme residue (Figure 5). NO avidly binds to hemoproteins such as hemoglobin, Mb, cytochrome c oxidase, and soluble guanylate cyclase, and these proteins are essential in the modulation of NO bioactivity and responsiveness to the concentration of NO (oxymyoglobin NO → ferrin myoglobin nitrate) (Figure 4) (Eich et al. 1996). NO is an important inter- and intracellular physiologic messenger that is capable of evoking a number of cellular responses that are beneficial or potentially toxic. Previous studies (Brunori 2001, Flögel et al. 2001b) have established that NO is an important regulator of mitochondrial oxidative phosphorylation. NO competes with O2 to reversibly bind and inhibit cytochrome c oxidase (complex IV of the electron transport system), thereby inhibiting mitochondrial respiration (Poderoso TCM Vol. 13, No. 3, 2003
Figure 2. The 2.0-kb myoglobin (Mb) promoter confers expression within the cardiomyocyte and contains essential transcriptional regulatory motifs. Schematic of the 2.0-kb Mb promoter containing essential motifs and the putative transcription factors that bind the regulatory elements [i.e., CCAC box, E-boxes, A/T motif, and nuclear factor of activated T-cell NFAT response elements (NRE), and myocyte enhancer factor-2 (MEF2)].
Figure 1. Structure of myoglobin consists of eight helices wrapped around a heme residue (red). Xenon binding studies using x-ray diffraction have identified four xenon cavities (designated 1–4), which may serve to concentrate ligands or serve as escape routes for the ligands. The evolutionarily conserved histidine residues serve to stabilize the heme (red) group (H93) or serve as a gate (H64, yellow) regulating the entry and exit of ligands.
et al. 1998, Shiva et al. 2001). In a previous study (Grange et al. 2001), we observed that NO generation is altered in skeletal muscle of Mb null mice following an electrical stimulation. Moreover, we challenged the Mb/ mice with exposure to chronic hypoxia and observed that in the absence of Mb, perturbed NO homeostasis resulted in left ventricular systolic dysfunction (Mammen et al. 2001). These results supported the conclusion that Mb functions as a modulator of NO bioavailability in the heart (Figure 5) (Flögel et al. 2001b, Mammen et al. 2001).
Figure 3. Myoglobin (Mb) is temporally and spatially expressed in the heart and oxidative skeletal myofibers. Using in situ hybridization and 35S-labeled antisense Mb riboprobes, expression was evaluated in the embryonic and adult mouse. (A) Brightfield hematoxylin-stained section corresponds to the field shown in panel (B). (B) In situ hybridization of a transverse section of an E10.0 mouse embryo reveals Mb expression within the ventricular myocardium and Mb absence within the atrium. Mb is expressed at lower levels within the myotome of the somite. (C) Transverse section of an adult mouse heart probed with the Mb riboprobe reveals expression in the ventricle and atrium. (D) Transverse section of the adult mouse hindlimb reveals heterogeneous expression of Mb in oxidative myofibers of the adult hindlimb. Note the increased expression of Mb within the oxidative soleus muscle (arrow). a, atrium; f, fibula; m, myotome of somite; s, soleus muscle; v, ventricle.
Table 1. Mb concentrations among species Organism Northern elephant seal Sperm whale Bottlenose dolphin New Zealand white rabbit Mouse (129) Human Human
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mg/g wet weight 64 57 26 8 1.9 2.4 2.1
Tissue
Reference
Longissimus dorsi muscle Skeletal muscle Longissimus dorsi muscle Skeletal muscle Skeletal muscle Heart Psoas muscle
Noren et al. (2001) Dolar et al. (1999) Noren and Williams (2000) Noren and Williams (2000) Perkoff and Tyler (1958) Swaanenburg et al. (2001) Perkoff and Tyler (1958)
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Figure 4. Myoglobin (Mb) is a regulator of dioxygen and nitric oxide (NO) in the cardiomyocyte. Dioxygen (O2) binds to deoxymyoglobin (Mb) to yield oxymyoglobin (MbO2), which transports oxygen to sites of use. MbO2 reacts with or scavenges NO to form ferric Mb (metMb) and nitrate (NO3). MetMb-reductase reduces metMb to yield Mb. Finally, Mb may bind NO to yield nitrosylmyoglobin (MbNO), which may react with oxygen to produce metmyoglobin (Brunori 2001).
A recent study by McMahon et al. (2002) provided data supporting the hypothesis that NO may function as a hormone, because it can be alternately captured or released by hemoglobin in red blood cells for bioavailability remote from the site of NO synthesis. Similarly, Mb may function not only as a scavenger of NO, but may also shuttle NO to compartments within the cardiomyocyte for bioavailability (Figure 5). Mb also has been reported to have peroxidase activity (Figure 5) (George and Irvine 1955). Although the peroxidase activity of Mb is lower than that of established endogenous peroxidases, studies (Cadenas 1989, Khan et al. 1998)
suggest that Mb has the potential for catalytic oxidation of substrates in the presence of hydrogen peroxide. Kinetic studies of Mb with mutations at histidine 64, tyrosine 103, and cystine 110 have indicated that these sites are essential in providing Mb with peroxidase activity (Khan et al. 1998, Witting et al. 2000 and 2002). Furthermore, recent studies by Schrader’s laboratory (Flögel et al. 2001a, Flögel et al. 2001b) support a role for Mb as a scavenger of reactive oxygen species. These studies indicate that Mb may play a critical role in limiting the toxic effects of reactive oxygen species in the cardiomyocyte (Figure 5).
• Tissue Globins Although Mb was the first tissue hemoglobin identified in vertebrates, two novel globins—neuroglobin and cytoglobin— recently have been shown to be expressed in adult vertebrate tissues. Neuroglobin is relatively dissimilar to Mb (21% identity) and hemoglobin (25% identity) and does not appear to share a common ancestral gene with either of these globins (Figure 6) (Burmester et al. 2000). Neuroglobin is a monomeric 151 amino acid hemoprotein that reversibly binds oxygen (although less avidly than does Mb) and is expressed primarily in focal regions of the brain in relatively low
Figure 5. Possible roles for myoglobin (Mb) in the heart. Mb has been proposed to function in the transport and storage of dioxygen. Recent studies suggest that Mb may function as an inactivator or a transporter of nitric oxide (NO). Alternatively, Mb may function to scavenge reactive oxygen species or function as an allosteric enzyme by concentrating ligands in the xenon pockets (yellow spheres) to accelerate the binding of the ligands (i.e., O2, NO, CO) to the heme residue.
Figure 6. Sequence homology of mouse myoglobin (Mb), cytoglobin (Cygb), neuroglobin (Ngb), and hemoglobin A (Hba) hemoproteins. The amino acid sequences for Mb, Cygb, Ngb, and Hba for the mouse proteins are shown with shading of homologous sequences. Note that the amino acids His-93 and His-64 (arrowhead) are conserved and stabilize the heme group. The eight helices (A–H) are shown in schematic fashion (red) and are above the corresponding amino acid sequence that constitutes each helix.
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concentrations (approximately 0.01% of the total protein content or 1 M) (Burmester et al. 2000, Mammen et al. 2002). The focal expression of neuroglobin suggests that this hemoprotein may function as a modulator of NO (in a fashion similar to that of hemoglobin and Mb) and/or as an oxygen sensor, rather than as a mediator of oxygen transport or storage (Couture et al. 2001, Dewilde et al. 2001, Trent et al. 2001). Cytoglobin is the fourth tissue globin that has been identified and appears to be ubiquitously expressed in the zebrafish, mouse, and human (Burmester et al. 2002). This novel member of the globin family constitutes a 190 amino acid protein that appears to share an ancestral gene with Mb (Figure 6). Whereas the functional role for cytoglobin is unclear, a recent study (Kawada et al. 2001) has reported that this protein is localized to the stellate cells in the liver and may function as a novel endogenous peroxidase. Further biochemical and molecular studies using transgenic strategies will be helpful in the definition of the functional roles for both of these novel hemoproteins. • Future Directions Over the past 4 decades, multiple disciplines have contributed significantly to the understanding of the role of Mb in the heart using structural, biochemical, physiologic, and molecular techniques. The results of these studies support the hypothesis that Mb is a multifunctional hemoprotein that participates in diverse functional roles, including oxygenmediated transport and NO inactivation, and as a reservoir of dioxygen, an allosteric enzyme, and a scavenger of reactive oxygen species. Future studies will continue to utilize emerging technologies to define the molecular details of the biological role for Mb. These studies will examine broadly the role of Mb as an allosteric enzyme using structural and biochemical techniques, the transcriptional regulation of the Mb gene under hypoxic and normoxic conditions, and a role for Mb as a scavenger or transporter of NO that participates in NO homeostasis. Furthermore, the availability of sequenced species-specific genomic programs will allow investigators to continue to identify novel hemoproteins and determine their functional role in the heart or as a partner/substitute for Mb. Collectively, these TCM Vol. 13, No. 3, 2003
future studies will further enhance our understanding of the role of hemoproteins in cardiovascular biology. • Acknowledgments The authors thank Drs. R. Sanders Williams, Rhonda Bassel-Duby, and Annette Meeson for helpful discussions throughout the course of these studies. They also acknowledge funding support from the National Institutes of Health (HL63788) and the Donald W. Reynolds Foundation.
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PII S1050-1738(02)00256-6
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Insights from Angiogenesis Trials Using Fibroblast Growth Factor for Advanced Arteriosclerotic Disease Rohit Khurana and Michael Simons*
The aim of therapeutic angiogenesis in cardiovascular disease states is to improve myocardial and peripheral extremity perfusion and function within ischemic regions that are not amenable to traditional modes of revascularization. Substantial “proof of concept,” efficacy, and safety data have emerged from numerous animal models and clinical trials that fibroblast growth factor (FGF), when administered by various delivery strategies, has a therapeutic angiogenic capacity. This initial excitement has been replaced by cautious optimism in the wake of results from larger, randomized, double-blinded placebo-controlled trials of both FGF gene and protein administration. A greater understanding of the profound placebo effect, careful patient selection, and improved endpoint assessment are factors that need to be addressed in this rapidly evolving era of molecular therapeutics. (Trends Cardiovasc Med 2003;13:116–122) © 2003, Elsevier Science Inc.
Rohit Khurana and Michael Simons are at the Section of Cardiology and Angiogenesis Research Center, Dartmouth Hitchcock Medical Center, Dartmouth Medical School, Lebanon, New Hampshire, USA. * Address correspondence to: Michael Simons, MD, Section of Cardiology, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756, USA. Tel.: (1) 603-650-3540; fax: (1) 603-650-5171; e-mail: [email protected]. © 2003, Elsevier Science Inc. All rights reserved. 1050-1738/03/$-see front matter
• FGF Biology Fibroblast growth factors (FGFs) are a family of 22 closely related proteins that share certain structural features and the ability to interact with specific receptors (Ornitz and Itoh 2001). FGFs elicit diverse biologic effects on numerous cell types (fibroblasts, endothelial cells, smooth muscle cells, and keratinocytes, among others). These effects encompass stimulation of growth, proliferation, migration, TCM Vol. 13, No. 3, 2003