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Proc Natl Acad Sci U S A. Aug 8, 2006; 103(32): 11999–12004.
Published online Aug 1, 2006. doi:  10.1073/pnas.0603098103
PMCID: PMC1567687
Developmental Biology

The ATP-sensitive potassium (KATP) channel-encoded dSUR gene is required for Drosophila heart function and is regulated by tinman

Abstract

The homeobox transcription factor Tinman plays an important role in the initiation of heart development. Later functions of Tinman, including the target genes involved in cardiac physiology, are less well studied. We focused on the dSUR gene, which encodes an ATP-binding cassette transmembrane protein that is expressed in the heart. Mammalian SUR genes are associated with KATP (ATP-sensitive potassium) channels, which are involved in metabolic homeostasis. We provide experimental evidence that Tinman directly regulates dSUR expression in the developing heart. We identified a cis-regulatory element in the first intron of dSUR, which contains Tinman consensus binding sites and is sufficient for faithful dSUR expression in the fly’s myocardium. Site-directed mutagenesis of this element shows that these Tinman sites are critical to dSUR expression, and further genetic manipulations suggest that the GATA transcription factor Pannier is synergistically involved in cardiac-restricted dSUR expression in vivo. Physiological analysis of dSUR knock-down flies supports the idea that dSUR plays a protective role against hypoxic stress and pacing-induced heart failure. Because dSUR expression dramatically decreases with age, it is likely to be a factor involved in the cardiac aging phenotype of Drosophila. dSUR provides a model for addressing how embryonic regulators of myocardial cell commitment can contribute to the establishment and maintenance of cardiac performance.

Keywords: aging, hypoxia, sulfonylurea receptor

The heart is the first organ to be formed during embryogenesis and is necessary to circulate blood systemically and support the progression of organogenesis. In adulthood, heart performance is directly associated with quality of life, and its insufficiency is a major cause of mortality. Therefore, it is important to elucidate the molecular basis of both development and functional maturation of the heart to improve clinical treatment. Although some of the early regulators of heart development have been studied in detail, it is not fully understood how these regulators coordinate cardiac function and how each component contributes to the various aspects of cardiac performance. In this study, we use Drosophila as a model to address these issues because formation of the contractile heart tube during embryogenesis and the genetic cascade that specifies the Drosophila heart are highly conserved in species with a heart (13). Because embryonic heart specification is conserved, it is possible that molecular controls of later heart function are also conserved and relevant for vertebrate heart function and diseases (4).

Of particular interest to cardiac studies is Tinman (Tin), an NK homeodomain-containing transcriptional factor that is the earliest cardiac lineage cell marker and is necessary for the early specification of the primordial heart (5, 6). Tin is initially expressed throughout the mesoderm, and, later, its expression domain is restricted to the cardiac mesoderm by Decapentaplegic (Dpp, a bone morphogenetic protein homolog) and Wingless (Wg, a fly Wnt) signaling from the overlying ectoderm (79). Flies mutant for tin exhibit a lack of all heart precursor cells, suggesting that Tin is a key gene for determining cardiac cell lineage (5, 6). Homologs of Tin have been found in all vertebrates examined, and considerable evidence suggests that Tin’s role in cardiac induction is also functionally conserved (3). Tin has previously been shown to bind to the specific DNA sequence TNAAGTG (10). Until now, only seven genes have been suggested to be direct downstream targets of Tin: cardiac Tin itself, zinc-finger GATA transcription factor Pannier (Pnr), MADS box transcription factor Dmef2, structural protein β3-tublin, helix–loop–helix transcription factor dHAND, and the membrane proteins Tincar and Toll in cardiac primordial cells (1016). Another potential Tin target is dSUR, which has a heart-specific segmental expression pattern that is identical to the late cardiac-restricted expression of Tin (17) and is speculated to be an effector molecule for cardiac performance.

dSUR has been identified as a homolog of the mammalian SUR2 protein, which is a subunit of the ATP-sensitive potassium (KATP) channel complex. The forced expression of dSUR protein in Xenopus oocytes shows an inwardly rectifying potassium current, and this current is abolished by the sulfonylurea glybenclamide, a KATP channel blocker, indicating that dSUR might be a functional ortholog of the mammalian SUR2 protein (17). KATP channels are thought to sense the intracellular metabolic state [i.e., they are regulated by the kinetics of the intracellular ATP and ADP ratio, which in turn is affected by various important metabolic stresses (18)]. Therefore, one possible function of the KATP channel is to connect the metabolic state and the level of membrane excitation for various biological responses, such as tolerance against hypoxia in cardiac muscle and brain and also hormone secretion in pancreatic islet cells.

The mammalian KATP channel complex is a heterooctamer that consists of two subunits: a sulfonylurea receptor (SURx) and an inward rectifier (Kir6.x). A number of recent studies have elucidated that mammalian cardiac contractile cells posses two distinguishable KATP channel activities on a basis of pharmacological responses. These channels are sarcolemmal KATP (sarcKATP) and mitochondrial KATP, both of which are thought to be involved in ischemic preconditioning (IPC), a powerful biological system that makes heart muscle cells tolerant to hypoxia/ischemia (1921). Kir6.2 is a component of the mammalian cardiac sarcKATP channel. In mice lacking Kir6.2, the protective effect of IPC on cardiac hypoxia was not observed. Thus, sarcKATP may be an essential effector of hypoxic protection in mammals (22).

In this study, we demonstrate that dSUR expression is directly regulated by cardiac Tin and that cardiac Pnr is likely involved in transactivation by means of protein–protein interaction. Consistent with the similarities to mammalian SUR2, dSUR plays a protective role against both hypoxic stress and electrical pacing-induced heart failure. Moreover, reduction of this potassium channel function may contribute to the deterioration of cardiac performance with age in Drosophila.

Results

dSUR Expression in the Developing Heart Is Regulated by Cardiac Tinman.

In our search for direct targets of cardiac Tin, we focused on a single copy gene, dSUR, an ATP-binding cassette gene product that is expressed in the embryonic dorsal vessel during embryogenesis (17). The expression of dSUR was examined in detail by whole-mount in situ hybridization. Its expression is initiated at late stage 12 in the cardiac primordial cells and then continues throughout embryogenesis (Fig. 1A and C). tin expression is restricted to the cardiac mesoderm just after stage 11 by means of the ectodermally secreted signaling molecules Wg and Dpp and then becomes restricted to four of six cardiac cells (7, 9, 23, 24) (Fig. 1 B and D). From stage 13 on, dSUR expression is exclusively expressed in the Tin-positive myocardial cells (Fig. 1E). One-week-old adult flies were dissected into different body parts to examine dSUR expression by quantitative RT-PCR (Fig. 1I). We detected dSUR expression in the isolated hearts, abdomen (which contains the heart), and head. The head contains the corpora cardiaca (CC) cells, which produce adipokinetic hormone and have been previously shown to express dSUR (25).

Fig. 1.
The sulfonylurea receptor gene dSUR is a likely cardiac target for Tinman. (AH) Whole-mount in situ hybridization of late stage-11 (A and B), stage-14 (CF), and stage-16 embryos (G and H). Anterior is to the left throughout. (A and ...

The embryonic pattern of dSUR expression suggests that it may be a Tin target. We examined this possibility by expressing tin in all mesoderm by using the upstream activating sequence (UAS)-Gal4 overexpression system (26) and the twist:24B-Gal4 driver (26). As a result, an expansion of dSUR expression to all six myocardial cells per hemisegment (Fig. 1F), but not to the entire mesoderm, was observed. Alternatively, dSUR expression was not observed in mesodermal cells of tin mutant stage-16 embryos (data not shown), suggesting that dSUR is a potential direct target gene of Tin but requires the context of the cardiac-specific mesoderm.

Previous work suggested that a combination of patterned expression of the inductive signals encoded by dpp and wg determines the mesodermal positioning of the cardiac primordia, which includes maintenance of tin expression in the heart-forming cells (9). To test this hypothesis, we examined whether mesodermal overexpression of dpp also caused ectopic expression of dSUR. Indeed, panmesodermal dpp induced ectopic expression of dSUR in the ventral region of lateral mesoderm (Fig. 1 G and H) in a pattern similar to dpp-induced tin (9).

A Distinct Enhancer Is Sufficient for Cardiac-Restricted dSUR Expression.

Because cardiac dSUR expression depends on Tin, we scanned 40 kb of the dSUR locus for Tin consensus binding sites (TNAAGTG; circles in Fig. 2A Upper). Three large genomic fragments were chosen based on the high density of potential Tin-binding sites (En1, 4,095 bp; En2, 2,151 bp; and En3, 2,291 bp) (Fig. 2A). The enhancer activity of these En fragments was then examined in transgenic flies. Two fragments located upstream of the ATG start (En1 and En2) do not show any reporter activity in the embryonic heart (data not shown). In contrast, En3 exhibits a pattern of reporter gene expression identical to the endogenous dSUR pattern (Fig. 2B). This En3 fragment is downstream of the ATG start and contains six Tin sites. To determine whether these Tin sites are required for cardiac expression, we mutated them (Fig. 2A). Of the mutated Tin sites, only a mutated T3 site reduced the enhancer’s transcriptional activity (Fig. 2 AD). Mutations in both T2 and T3 (241 bp apart) abolished reporter gene expression in the cardiac progenitor cells (Fig. 2E), suggesting that Tin is capable of directly activating dSUR expression in the appropriate myocardial cells. We then tested shorter fragments (S, 890 bp; SS, 359 bp; and SSS, 297 bp; Fig. 2 A, F, and H) containing both T2- and T3-binding sites for enhancer activity. These three fragments mimicked the cardiac dSUR expression and showed a similar expression level as seen with En3. Within the context of the short SSS fragment, the T3-binding site is absolutely essential for reporter gene activation (Fig. 2I). We also scanned the En3 fragment for Mad/Media (Dpp pathway)-binding sites (GCCGCGACG) (11). We did not find Mad/Media sites with appreciable conservation within this enhancer, which is consistent with Dpp signaling only indirectly regulating dSUR expression, possibly by means of tin. However, we cannot exclude a direct regulation by Dpp by means of degenerate or not well conserved sites.

Fig. 2.
A Tinman-responsive element of dSUR directs cardiac expression. (A) Genomic organization of the dSUR locus and transgenic constructs for testing the enhancer activity are shown. The consensus Tin (TNAAGTG) and Pnr (WGATAR) sites are marked by circles ...

We then performed an EMSA to test whether Tin can directly bind to the T3 site. A DNA template (28 bp) composed of dSUR genomic sequence containing the T3-binding site produced a specific Tin-binding complex (Fig. 2J, arrow). Thus, Tin can directly associate with the T3 element in dSUR, which is consistent with the possibility that dSUR expression is directly controlled by Tin.

Pannier and Tinman Act Synergistically in Activating dSUR Expression.

The Tin expression pattern varies by developmental stage, and, likewise, its downstream target genes may also change during development. In vertebrates, GATA-4 provides the binding efficiency to Nkx2.5 in cardiomyocytes; therefore, these two transcription factors can act cooperatively to activate cardiac genes (2730). Similarly, the Drosophila counterparts Pnr and Tin physically interact and synergistically control cardiac gene expression of genes such as Dmef2 (12). To further characterize the role of Pnr in dSUR activation, we expressed Pnr panmesodermally and compared the expression of dSUR to that of dHand, which marks all cardiac lineages (31). Panmesodermally expressed Pnr activates both ectopic dHand and dSUR expression but only to a moderate extent (Fig. 3FH). In contrast, a dominant-negative Pnr (Pnr-EnR) did not induce, and instead reduced, dSUR and dHand expression (Fig. 3 IK). Moreover, both dSUR and dHand were strongly activated when tin and pnr were coexpressed (Fig. 3 L and M), suggesting that, like dHand, dSUR activation depends on genetic synergy between Tin and Pnr (14, 32).

Fig. 3.
Tinman and Pannier synergistically induce dSUR expression. (AP) Panmesodermal expression in the progeny of crosses between twist-Gal4;24B-Gal4 driver and UAS-cDNA-containing transgenic flies. Stage-13 in situ stained embryos for dSUR (Left), ...

Next, we examined whether a reduction of Pnr activity could be compensated for by overexpression of tin. We coexpressed tin and pnr-EnR throughout the mesoderm and found that the reduced dSUR and dHand expression, which was due to Pnr-EnR, was not rescued by forced panmesodermal tin expression (Fig. 3 NP). This finding suggests that dSUR activation requires not only Tin but also Pnr activity.

Furthermore, we searched the Pnr consensus binding site (WGATAR) within the En3 fragment to explain synergistic activation by Tin and Pnr. There are two well conserved Pnr sites in the SS fragment (Fig. 2A, squares). However, when we examined the enhancer activity when both of these Pnr consensus sites were mutated [SS(P2P3)], we found that this enhancer was equivalent to the WT SS fragment (Fig. 2 F and G). This finding implies that Pnr could bind to Tin directly or to other nonconsensus Pnr-binding sites, such as TGATA (which exists in the SSS fragment), to activate dSUR expression in the embryonic heart.

To address the possibility that Tin and Pnr may be acting in a complex in regulating cardiac dSUR transcription, an in vitro reporter assay with a luciferase plasmid was used, in which expression was driven by six concurrent T3 sites (Fig. 3Q; see also Fig. 2J). Cotransfection of the T3 reporter plasmid with Tin but not the Pnr expression vector into Drosophila Schneider cells resulted in a 3-fold activation of luciferase activity compared with the reporter construct alone. In contrast, when Tin and Pnr were cotransfected, the luciferase activity was elevated 9-fold compared with the reporter construct alone (or with a mutant T3-binding site), suggesting that Pnr acts as a cofactor with Tin to synergistically activate dSUR transcription.

dSUR Protects Cardiac Function from Stress-Induced Heart Failure.

dSUR and tin expression patterns are restricted to the contractile cardiac myocytes after specification of the heart field and continue to be expressed in the adult heart (Fig. 1 I and J). Thus, we postulate that dSUR function contributes to cardiac performance. To test this possibility, we generated transgenic flies that permitted expression of RNAi (33) specific for dSUR. Unfortunately, classical dSUR loss-of-function mutants are not available, and the locus apparently is in the vicinity of a dominant female sterile locus without suitable available deficiencies (G. Reuter, personal communication). Ubiquitous dSUR-RNAi expression in lines 1 and 2 was driven by tubulin-Gal4 and resulted in a 50–60% reduction of endogenous dSUR mRNA levels as assayed by real time RT-PCR (LightCycler system; Roche, Indianapolis, IN) (data not shown). Consistent with prior reports (25), expression of dSUR-RNAi in the CC cells of the ring gland led to reduced dSUR mRNA and increased glucose levels (data not shown). Ubiquitous or panmesodermal expression of these dSUR-RNAi constructs resulted in viable flies.

Our ability to knock down dSUR function provided an opportunity to assess heart physiology. We examined the performance of dSUR-RNAi hearts under stress by using an external electrical pacing assay in adult flies (34, 35). We expressed dSUR-RNAi with the heart-specific driver GMH5 (36) and determined the percentage of flies with heart failure. Remarkably, flies with cardiac expression of four lines of dSUR-RNAi exhibit an increased rate of pacing-induced heart failure compared with controls (Fig. 4A). Thus, dSUR function in the adult fly may be required to prevent cardiac failure under conditions of tachycardic stress.

Fig. 4.
Cardiac dSUR protects heart performance against electrical pacing. (A) Heart failure rate after 30-s high-frequency (6 Hz) external pacing (36) in 1-week-old progeny of the cross between the heart-specific GMH5 driver and UAS-dSUR-RNAi lines. Controls ...

dSUR Expression Is Reduced in the Heart of Drosophila with Age.

A progressive elevation of pacing-induced heart failure rate is also observed during the natural age-related decline in fly cardiac function (36). We compared the level of dSUR expression in the heart of young (1-week-old) and aged (5-week-old) flies by using real-time RT-PCR with RNA from isolated hearts (Fig. 1J). As reference points, we included tin, GFP driven by the cardiac-specific GMH5, and SH3β, all of which exhibit only a moderate decline in expression (to 65–80% compared with young flies). In contrast, dSUR expression declines to 15% by 5 weeks of age. Thus, the increase in heart failure with age correlates with a decrease in dSUR expression.

To further study the contribution of dSUR to cardiac aging, we measured the effect of tolbutamide, which is a member of the sulfonylurea drug family and has previously been shown to inhibit the dSUR-mediated potassium channel activity in the secretory CC cells (25). Young flies fed with tolbutamide exhibit a higher incidence of heart failure compared with the nontreated cohort (Fig. 4B), supporting the previous interpretation that the increase in heart failure rate is due to a reduction in KATP channel activity, either directly in the heart or by an indirect mechanism (25, 36). Cardiac-specific knock-down of dSUR has the same effect as short treatment with tolbutamide, suggesting that the sulfonylurea drug-induced increase in heart failure is likely due to a direct effect on the heart (Fig. 4 A and B). To test this idea further, we exposed adult flies to pinacidil, a KATP channel activator. We asked whether pinacidil could reduce the heart failure in older flies that is accompanied by reduced dSUR expression. Measures of heart failure showed a significant reduction in older flies (3–4 weeks) exposed to pinacidil compared with the nontreated group (Fig. 4C). These results suggest that increased dSUR activity may improve cardiac performance and further support the idea that dSUR loss of function contributes to declining cardiac performance in aging.

dSUR Protects Cardiac Function During and After Anoxia.

To test the current view that the main function of the mammalian KATP channels in the cardiovascular system is to protect the heart from hypoxia-induced damage, we examined the heart rate of WT and dSUR knock-down flies after exposure to hypoxia (Fig. 5). Because Drosophila is remarkably tolerant of hypoxia, we subjected the flies to stringent hypoxic stress by anoxia exposure for 2 hours, which stops the heartbeat, and monitored the recovery time of the heart rate. We found that with both panmesodermal and cardiac-specific dSUR knock-down, the rate of recovery was significantly slower than in WT flies (Fig. 5), suggesting that dSUR functions to protect the heart from damage caused by anoxia. Consistent with this interpretation is the observation that the reduction in heart rate during short-term anoxia is accentuated with mesodermal dSUR knock-down compared with WT (Fig. 7, which is published as supporting information on the PNAS web site). Taken together, these data suggest that dSUR plays a protective role against hypoxic stress in the fly’s cardiac muscles, which is reminiscent of the roles of mammalian KATP channels (22). These findings also demonstrate that the Drosophila heart can now be exploited to screen for modifiers of cardiac ischemia relevant to the human heart.

Fig. 5.
dSUR plays a protective role against hypoxia. RNAi-mediated knock-down mutants were derived from crossing twist-Gal4;24B-Gal4 or GMH5 driver flies with the UAS-dSUR-RNAi lines, and progeny were exposed to anoxia. (A) Experimental protocol: Young pupae ...

Discussion

In this study, we have identified a putative cis-regulatory element for dSUR expression in the heart and shown that dSUR expression is synergistically controlled by Tin and Pnr, which are both key regulators of the cardiac lineage. In addition to this early cardiac specification process, Tin and Pnr seem to also have later functions in which they regulate structural genes [e.g., β3-tubulin (13)] and, as we show in this study, genes that have a role in maintaining the physiological properties for normal heart function. For example, in mammals, Nkx2–5 is required for controlling Connexin43 expression, which forms gap junctions in the conduction system and thus influences cardiac performance (37). dSUR regulation by Tin is thus an avenue by which Tin regulates cardiac physiology and allows us to examine the link between early cardiac specification and maintenance of cardiac performance.

We have found a 295-bp cis element with two essential Tin sites that mimics the endogenous dSUR expression in the developing heart but not in the adipokinetic hormone-releasing CC cells of the ring gland. Pnr augments ectopic Tin-induced dSUR expression, as it does with Dmef-2 and dHand (12, 14, 32), but the Pnr consensus sites in the cardiac enhancer do not seem to be required. Nevertheless, Tin and Pnr act synergistically in activating dSUR both in vitro and in vivo, and forced expression of tin is unable to restore dSUR expression in flies expressing dominant-negative pnr-EnR. Thus, both Tin and Pnr are essential for dSUR activation in vivo, and our results suggest that Pnr is involved in DNA-bound Tin complexes. This interpretation is consistent with our EMSA and cotransfection experiments as well as previous reports that show that Tin binds to Pnr (12). However, we cannot rule out the possibility that Pnr is exerting its effects by means of “nonconsensus” DNA-binding sites.

It was recently shown that in CC cells of Drosophila, dSUR controls glucose homeostasis by increasing secretion of adipokinetic hormone (AKH) in response to low glucose concentration in the hemolymph (25). Evidence indicates that AKH release likely is increased by the SUR inhibitor sulfonylurea and is decreased by ectopic expression of constitutively active (and thus ATP-independent) ion channel Kir2.1, suggesting striking parallels between endocrine cells in Drosophila and mammals in controlling blood glucose. Therefore, we also examined the role of dSUR in cardiac physiology and heart homeostasis in adults. Our findings suggest that there are striking functional similarities between Drosophila and mammalian SUR in heart function. In the mammalian heart, there are two types of KATP channels, sarcKATP and mitochondrial KATP, which are candidate regulators of acute hypoxia and IPC. Impairing sarcKATP channel activity by genetic manipulation of mouse Kir6.2 results in compromised recovery of contractile function after hypoxia (22). Our data are consistent, with dSUR in Drosophila providing a similar protective mechanism against hypoxia. Moreover, a recent study in goldfish KATP channel function (38) revealed that the involvement of KATP in IPC is widely conserved, including in highly hypoxia-tolerant species.

To further address dSUR function, we performed external electrical pacing of the heart in dSUR knock-down mutants. Rapid electrical pacing per se is a nonhypoxic stimulus that may induce an IPC effect in mammals by activating KATP channels (3941). Indeed, Kir6.2 mutant hearts exhibit diminished electrical tolerance against catecholamine-induced ventricular arrhythmia because of a failure to achieve action potential shortening and by causing early after-depolarization (42). Thus, the elevated heart failure rate in dSUR knock-down hearts may be due to KATP channel insufficiency. Interestingly, IPC is no longer observed in older human patients (43), and in female guinea pigs, SUR2A expression is reduced in old ventricular tissue compared with young ventricular tissue (44). Moreover, human SUR2 mutations found in two independent families were recently shown to cause dilated cardiomyopathy, with an onset around middle age (45). These mutations result in the structural abnormalities of the KATP channel and impair the ATP-dependent channel gating. Patients carrying these mutations showed ventricular tachycardia with normal coronary angiography, suggesting that human cardiomyocyte KATP channels play a role in maintaining membrane electrical stability and that the reduction of the KATP channel activity causes electrical disturbance, especially in older hearts. Here, we observed that pacing-induced heart failure steeply increases in aging flies (36), which can be reversed by exposure to the KATP channel activator pinacidil. These observations, together with the drastically reduced dSUR expression in old flies, suggest that dSUR serves as an indicator of cardiac aging. Given the experimental advantages of Drosophila, such as a small genome size and short life span, dSUR and cardiac aging provide a unique model not only for assessing the control of physiological heart functions, such as the response to hypoxia, but also for the analysis of age-related human diseases.

Materials and Methods

Drosophila Strains and Constructs.

Detailed experimental procedures and a description of the Drosophila strains are available in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. Briefly, overexpression of the transgene was performed by using the UAS-Gal4 system (26). yw was used as a reference strain in this study. Whole-mount in situ hybridization was performed as described in ref. 32. Primer sequences for cloning dSUR enhancer fragments, generation of UAS-dSUR-RNAi transgenic flies, and quantitative RT-PCR (Roche) are described in Supporting Materials and Methods. Site-directed mutagenesis was performed by using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA).

Physiological Assays.

The external electrical pacing assay was performed according to the method described in ref. 36. For the hypoxia assay, the rate of the rhythmical heartbeat was measured at early pupal stages on a temperature-controlled stage (25°C). The pupae were placed in a small temperature-controlled chamber with one end open and supplied with a constant moisturized stream of 20%/80% oxygen/nitrogen gas (“normoxia”) or nitrogen gas only (0% oxygen, “anoxia”) at 25°C, and the heart rate was determined by visual inspection 10, 15, and 30 min after the 2-h exposure to anoxia.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Dr. P. Thomas (University of Michigan, Ann Arbor, MI) for dSUR cDNA; Dr. Z. Han (University of Texas, Dallas, TX) for the Tinman and Pannier expression vectors; Drs. H. Nakaya and P. Ruiz-Lozano for critical reading of the manuscript; and G. Hogg for technical assistance. This work was supported by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (to R.B.).

Abbreviations

KATP
ATP-sensitive potassium
sarcKATP
sarcolemmal KATP
IPC
ischemic preconditioning
CC
corpora cardiaca
UAS
upstream activating sequence.

Footnotes

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

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