Genes use a simple language—written in the molecules of DNA—to build thousands of proteins in a dizzying variety of sizes and shapes. With only four different nucleotide building blocks, DNA codes for the 20 different amino acids (each with their own structures and properties) that provide the foundation for the enormous diversity of protein form and function. This diversity makes the systematic study of all the proteins of a given organism (called proteomics), a challenging enterprise.Interactions between proteins underlie nearly every fundamental process within the cell. They can form higher-order multiprotein complexes like those involved in transcription and replication, help transport proteins to their proper location in the cell, and participate in signaling pathways. Because of their importance, disruption of these interactions can have disastrous consequences. For example, the loss of the ability of a normal cellular protein called Src to bind to certain other proteins can be associated with cancer progression. The determinants of these interactions are poorly understood, but in many cases these interactions are mediated by small pieces of the proteins, which are called peptides. Peptides serve as the starting point for the novel strategy reported in this issue.Gianni Cesareni and colleagues have added to the repertoire of proteomic analysis by devising a global strategy to investigate protein–protein interactions on an organismal level using yeast as a model organism. The authors select a protein of interest from yeast, which can be thought of as the “bait” for which they wish to identify protein-binding partners. They start by looking at a number of different previously identified peptides that bind the bait protein. Commonalities between the sequences of these peptides form the “consensus” binding sequence, a base framework of protein sequence from which many possible variations can be derived. Since the protein sequences of all proteins (the proteome) in yeast can be deduced from the sequenced genome, the authors can scan the proteome for proteins that contain the consensus, or a closely related, sequence. These proteins could potentially bind the bait protein. Peptide sequences from these identified proteins are synthesized chemically and arrayed on a membrane, which is bathed in a solution containing the bait protein. After washing off the excess bait protein, they can figure out where it remains on the membrane and therefore tell which peptides the bait protein has bound. The proteins corresponding to these peptides are candidate binding proteins that are validated by further experimentation.The protein–protein interactions identified by this approach can be used to extend the network of known interactions in the proteome. This will enable researchers to draw functional linkages between proteins, whether they are involved in a basic biological process or in human disease. By examining whole families of proteins, it may also aid in elucidating the underlying determinants of binding specificity, which would provide clues to the biomechanisms underlying cell processes. These insights could lead to methods for manipulating these interactions, perhaps even in cases of human disease, as in the case of Src and cancer. This approach can readily be applied to the proteomes of more complex organisms like humans and adds to the growing number of experimental strategies available to researchers in proteomics.


Nucleo-cytoplasmic transport of RNA is one of many cellular pathways whose illumination has progressed hand in hand with understanding of retroviral mechanisms. A recent paper in  Cell  reports the involvement of an RNA helicase in the pathway by which HIV exports partially spliced and unspliced RNA out of the nucleus. This suggests the ubiquity of RNA helicases in RNA export from the nucleus, and has novel mechanistic implications.Cell

HIV must export to the cytoplasm not only full length genomic and structural gene-encoding RNA species, but also partially and fully spliced RNAs which code for a variety of overlapping structural and accessory genes. Whereas the fully spliced HIV RNA species readily exit the nucleus and undergo translation, the partially spliced and unspliced species require the interaction of the HIV-1 Rev protein with the cis-acting, viral RRE (Rev responsive element) for expression. Simpler retroviruses, such as the type-D Mason-Pfizer monkey virus (MPMV) solve a similar problem by encoding a cis-acting, constitutively active CTE (constitutive transporter element) in the 3'UTR of the viral genome, which uses a distinct pathway [ 1 , 2 ]. Of less clear significance is the existence of CRM-1 dependent nuclear export signals in gag proteins of HIV and RSV. It is theoretical possibility that shuttling gag proteins might contribute to nucleocytoplasmic export of genomic RNA, but it is equally possible that the only need for nuclear export of gag is to overcome the nuclear targeting signals late in viral replication [ 3 , 4 ]. Over the last seven years or so, tremendous progress has been made in characterizing nucleocytoplasmic transport pathways. The Rev/RRE pathway involves the Ran-CRM-1, shuttling system: In the nucleus, Ran-GEF converts Ran-GDP to Ran-GTP. Ran-GTP bound CRM-1 binds the NES (nuclear export signal) domain of Rev, which is in turn bound to the RRE, and enables CRM-1 to transport the resulting RNA/protein complex as cargo into the cytoplasm, presumably through CRM-1 interactions with nucleoporins. In the cytoplasm, Ran-GAP converts Ran-GTP to Ran-GDP, releasing the REV/RNA cargo. The asymmetric distribution of Ran-GEF and Ran-GAP between nucleus and cytoplasm ensures a constant RAN-GTP/GDP gradient to facilitate CRM-1 recycling and continued Rev/RRE export. CTE-mediated RNA export is based on binding to the Tap-NXT complex, and export through an unrelated transport pathway, which, ironically, is responsible for the bulk of mRNA transport [ 2 ]. Enter the laboratories of Kuan-Teh Jeang and Larry Kleiman, who, in a comprehensive study in the October 29th issue of  Cell , report the involvement of the RNA helicase, DDX3, in the Rev/RRE pathway [ 5 ]. Interestingly, DDX3 attracted their attention in preliminary screens indicating it was upregulated by the HIV-1 Tat protein, which is directly involved in transcriptional, not post-transcriptional regulation. Studying the effects of DDX3 overexpression, expression of dominant negative DDX3 mutants, or antisense knockdown of DDX3, variously on expression of Rev/RRE dependent reporter genes, Rev/RRE dependent subgenomic HIV constructs, or full length HIV genomes, Yedavalli et al. determined that in all cases, DDX3 had effects consistent with its playing a significant role in Rev/RRE dependent expression. These effects included expression of proteins, and nucleocytoplasmic partitioning of Rev/RRE dependent and Rev/RRE independent RNAs. In parallel studies, they determined that DDX3 had no effect on expression from CTE dependent constructs. A number of findings not only provided evidence consistent with a role for DDX3 in the Rev export pathway, but also provided mechanistic insights. DDX3, though constitutively cytoplasmic, shuttles between nucleus and cytoplasm in a manner dependent on CRM-1, as evidenced by inhibition by leptomycin B (LMB), which inhibits CRM-1 export, but not Tap export. DDX3 also localizes to nuclear outer membranes in a speckle formation and co-immunoprecipitates with nucleoporins, in addition to having a distribution in the cytoplasm. In cell lysates, DDX3 co-immunoprecipitates with either Rev protein or CRM-1. Although the studies on cell lysates fail to distinguish between pairwise binding and ternary complex formation, ternary complex formation is a distinct possibility. DDX3 directly binds CRM-1  in vitro  (in a manner independent of its NES). Although no data are presented concerning direct  in vitro  binding of DDX3 to Rev, for such an interaction to exist and to be significant, DDX3 would have to bind one of the three identified functional regions in Rev. These include the NES, the coincident NLS/RBD (nuclear localization signal/RNA binding domain) and the Rev multimerization domain, which flanks the RBD [ 6 ]. No regions in Rev besides these have been identified by mutagenesis or by any other techniques to be critical for function. Such an interaction would seem unlikely (though not impossible) in a complex of oligomerized Rev bound to RNA and CRM-1. Conceptually it might be easier to accommodate all the binding interactions in some kind of "pass-along" mechanism. This could allow some of the binding interactions to take place sequentially, rather than simultaneously. In the tradition of good research, the results of Yedavalli et al. [ 5 ] pose as many questions as they answer. Though the binding interactions so far discovered, and DDX3's ability to shuttle between nucleus and cytoplasm could be consistent with a quarternary complex of Rev, RNA, CRM-1 and DDX3, the existence of such a complex remains to be proven (and the authors avoid implying its existence). It is possible there is some sort of "pass along" mechanism in which some of the binding interactions are sequential. It is tempting to suggest that DDX3 plays a role in relieving torsional tension to facilitate RNA passage through a confining nuclear pore, or that it helps remove certain proteins from RNA before, as, or after it reaches the cytoplasmic side of the nuclear membrane. Two other helicases have been implicated in RNA nuclear export pathways. In yeast, Dbp5p helicase, binds nucleoporins, localizes to nuclear pore complexes on the cytoplasmic side of the nuclear membrane, and is involved in mRNA export [ 7 - 9 ]. RNA helicase A (RHA) has been implicated in CTE-mediated transport [ 10 - 13 ]. A general requirement for RNA helicases in RNA transport is an attractive concept. Conceivably they could even be a significant molecular motor contributing to RNA transport: some RNA helicases are highly processive [see [ 14 ] and references therein] and may work by translocating along ssRNA, displacing bound RNA and protein (the "snowplow" model). At least one highly processive RNA helicase has been show to displace tightly bound protein from dsRNA [ 15 ]. An energy-dependent, highly processive RNA helicase tethered to the cytoplasmic side of the nucleopore could thus pull a long RNA molecule through the pore (see Figure  1 ). Theoretically this could solve the problem of how to get the rest of the RNA through the pore once the region initially targeted by CRM-1 (the RRE in the case of HIV) is transported and released. Other possibilities could, of course, include other molecular motors such as kinesin, in association with protein/RNA complexes [ 16 ]. Figure 1 Schematic diagram outlining Rev mediated RNA export from nucleus to cytoplasm.  a , Viral RNA develops secondary structure before binding critical protein components of the export pathway.  b , Rev protein binds the RRE, forming a complex of REV, viral RNA, CRM-1 and DDX3, which begins to unwind secondary structure.  c , The export complex enters the nucleopore, where both CRM-1 and DDX3 interact with nucleoporins.  d , Even after CRM-1 and Rev are released from the export complex, DDX3 may still pull RNA through the complex by virtue of its processivity. On a highly speculative note, the intimate involvement of a DEAD box helicase in the Rev pathway, and the observation that DDX3 has a cytoplasmic localization, in addition to its nuclear membrane localization, recalls some old data from two independent laboratories, indicating that in some systems Rev may not act on export, but on post export events, to promote Rev/RRE dependent expression [ 17 , 18 ]. The eIF4A family has been called the "godfather" of DEAD box helicases [ 19 ]. EIF4A3 can be targeted to the exon junction complex of spliced mRNA, and it has been speculated that it may have a splicing-dependent influence on mRNA translation [ 20 ]. In cell types where Rev does not act through export, could it act by specifically targeting to the ribosome a related DEAD box RNA helicase, translation initiation factor? Time will tell. Finally there is the possibility that specific helicases will be valid therapeutic targets for antiretroviral chemotherapy. Although targeting a cellular gene involved in a viral pathway risks inhibiting a necessary cellular function, it avoids the overwhelming problem posed by rapid viral mutation to resistance.

HIV must export to the cytoplasm not only full length genomic and structural gene-encoding RNA species, but also partially and fully spliced RNAs which code for a variety of overlapping structural and accessory genes. Whereas the fully spliced HIV RNA species readily exit the nucleus and undergo translation, the partially spliced and unspliced species require the interaction of the HIV-1 Rev protein with the cis-acting, viral RRE (Rev responsive element) for expression. Simpler retroviruses, such as the type-D Mason-Pfizer monkey virus (MPMV) solve a similar problem by encoding a cis-acting, constitutively active CTE (constitutive transporter element) in the 3'UTR of the viral genome, which uses a distinct pathway [ 1 , 2 ]. Of less clear significance is the existence of CRM-1 dependent nuclear export signals in gag proteins of HIV and RSV. It is theoretical possibility that shuttling gag proteins might contribute to nucleocytoplasmic export of genomic RNA, but it is equally possible that the only need for nuclear export of gag is to overcome the nuclear targeting signals late in viral replication [ 3 , 4 ]. Over the last seven years or so, tremendous progress has been made in characterizing nucleocytoplasmic transport pathways.The Rev/RRE pathway involves the Ran-CRM-1, shuttling system: In the nucleus, Ran-GEF converts Ran-GDP to Ran-GTP. Ran-GTP bound CRM-1 binds the NES (nuclear export signal) domain of Rev, which is in turn bound to the RRE, and enables CRM-1 to transport the resulting RNA/protein complex as cargo into the cytoplasm, presumably through CRM-1 interactions with nucleoporins. In the cytoplasm, Ran-GAP converts Ran-GTP to Ran-GDP, releasing the REV/RNA cargo. The asymmetric distribution of Ran-GEF and Ran-GAP between nucleus and cytoplasm ensures a constant RAN-GTP/GDP gradient to facilitate CRM-1 recycling and continued Rev/RRE export. CTE-mediated RNA export is based on binding to the Tap-NXT complex, and export through an unrelated transport pathway, which, ironically, is responsible for the bulk of mRNA transport [ 2 ].Enter the laboratories of Kuan-Teh Jeang and Larry Kleiman, who, in a comprehensive study in the October 29th issue of  Cell , report the involvement of the RNA helicase, DDX3, in the Rev/RRE pathway [ 5 ]. Interestingly, DDX3 attracted their attention in preliminary screens indicating it was upregulated by the HIV-1 Tat protein, which is directly involved in transcriptional, not post-transcriptional regulation. Studying the effects of DDX3 overexpression, expression of dominant negative DDX3 mutants, or antisense knockdown of DDX3, variously on expression of Rev/RRE dependent reporter genes, Rev/RRE dependent subgenomic HIV constructs, or full length HIV genomes, Yedavalli et al. determined that in all cases, DDX3 had effects consistent with its playing a significant role in Rev/RRE dependent expression. These effects included expression of proteins, and nucleocytoplasmic partitioning of Rev/RRE dependent and Rev/RRE independent RNAs. In parallel studies, they determined that DDX3 had no effect on expression from CTE dependent constructs.CellA number of findings not only provided evidence consistent with a role for DDX3 in the Rev export pathway, but also provided mechanistic insights. DDX3, though constitutively cytoplasmic, shuttles between nucleus and cytoplasm in a manner dependent on CRM-1, as evidenced by inhibition by leptomycin B (LMB), which inhibits CRM-1 export, but not Tap export. DDX3 also localizes to nuclear outer membranes in a speckle formation and co-immunoprecipitates with nucleoporins, in addition to having a distribution in the cytoplasm. In cell lysates, DDX3 co-immunoprecipitates with either Rev protein or CRM-1. Although the studies on cell lysates fail to distinguish between pairwise binding and ternary complex formation, ternary complex formation is a distinct possibility. DDX3 directly binds CRM-1  in vitro  (in a manner independent of its NES). Although no data are presented concerning direct  in vitro  binding of DDX3 to Rev, for such an interaction to exist and to be significant, DDX3 would have to bind one of the three identified functional regions in Rev. These include the NES, the coincident NLS/RBD (nuclear localization signal/RNA binding domain) and the Rev multimerization domain, which flanks the RBD [ 6 ]. No regions in Rev besides these have been identified by mutagenesis or by any other techniques to be critical for function. Such an interaction would seem unlikely (though not impossible) in a complex of oligomerized Rev bound to RNA and CRM-1. Conceptually it might be easier to accommodate all the binding interactions in some kind of "pass-along" mechanism. This could allow some of the binding interactions to take place sequentially, rather than simultaneously.in vitroin vitroIn the tradition of good research, the results of Yedavalli et al. [ 5 ] pose as many questions as they answer. Though the binding interactions so far discovered, and DDX3's ability to shuttle between nucleus and cytoplasm could be consistent with a quarternary complex of Rev, RNA, CRM-1 and DDX3, the existence of such a complex remains to be proven (and the authors avoid implying its existence). It is possible there is some sort of "pass along" mechanism in which some of the binding interactions are sequential. It is tempting to suggest that DDX3 plays a role in relieving torsional tension to facilitate RNA passage through a confining nuclear pore, or that it helps remove certain proteins from RNA before, as, or after it reaches the cytoplasmic side of the nuclear membrane. Two other helicases have been implicated in RNA nuclear export pathways. In yeast, Dbp5p helicase, binds nucleoporins, localizes to nuclear pore complexes on the cytoplasmic side of the nuclear membrane, and is involved in mRNA export [ 7 - 9 ]. RNA helicase A (RHA) has been implicated in CTE-mediated transport [ 10 - 13 ]. A general requirement for RNA helicases in RNA transport is an attractive concept. Conceivably they could even be a significant molecular motor contributing to RNA transport: some RNA helicases are highly processive [see [ 14 ] and references therein] and may work by translocating along ssRNA, displacing bound RNA and protein (the "snowplow" model). At least one highly processive RNA helicase has been show to displace tightly bound protein from dsRNA [ 15 ]. An energy-dependent, highly processive RNA helicase tethered to the cytoplasmic side of the nucleopore could thus pull a long RNA molecule through the pore (see Figure  1 ). Theoretically this could solve the problem of how to get the rest of the RNA through the pore once the region initially targeted by CRM-1 (the RRE in the case of HIV) is transported and released. Other possibilities could, of course, include other molecular motors such as kinesin, in association with protein/RNA complexes [ 16 ].On a highly speculative note, the intimate involvement of a DEAD box helicase in the Rev pathway, and the observation that DDX3 has a cytoplasmic localization, in addition to its nuclear membrane localization, recalls some old data from two independent laboratories, indicating that in some systems Rev may not act on export, but on post export events, to promote Rev/RRE dependent expression [ 17 , 18 ]. The eIF4A family has been called the "godfather" of DEAD box helicases [ 19 ]. EIF4A3 can be targeted to the exon junction complex of spliced mRNA, and it has been speculated that it may have a splicing-dependent influence on mRNA translation [ 20 ]. In cell types where Rev does not act through export, could it act by specifically targeting to the ribosome a related DEAD box RNA helicase, translation initiation factor? Time will tell.Finally there is the possibility that specific helicases will be valid therapeutic targets for antiretroviral chemotherapy. Although targeting a cellular gene involved in a viral pathway risks inhibiting a necessary cellular function, it avoids the overwhelming problem posed by rapid viral mutation to resistance.Abbreviations RRE: Rev Responsive Element MPMV: Mason-Pfizer monkey virus CTE: Constitutive Transport Element NES: Nuclear Export Signal LMB: Leptomycin B NLS: Nuclear Localization Signal RBD: RNA Binding Domain
Abbreviations
RRE: Rev Responsive ElementMPMV: Mason-Pfizer monkey virusCTE: Constitutive Transport ElementNES: Nuclear Export SignalLMB: Leptomycin BNLS: Nuclear Localization SignalRBD: RNA Binding DomainCompeting Interests None. The opinions expressed are those of the author and do not necessarily express the opinion of the FDA.
Competing Interests
None. The opinions expressed are those of the author and do not necessarily express the opinion of the FDA.


Anti-ischemic therapy, in particular beta-blockers, is the most commonly employed drug for the control of myocardial ischemia in patients with stable coronary artery disease. Its widespread use also in patients with suspected coronary artery disease has important practical, clinical diagnostic and prognostic implications because diagnostic tests are heavily influenced by its effects. In the present review, the pathophysiological mechanisms of ischemia protection by antianginal therapy are described. Not all stressors are created equal in front of the different classes of antianginal drugs and on their turn the different classes of drugs exert different levels of protection on inducible ischemia. Several clinical implications can be drawn: From the diagnostic viewpoint antianginal therapy decreases test sensitivity, offsetting the real ischemic burden for a too high percentage of false negative tests. From the prognostic viewpoint test positivity in medical therapy identifies a group of subjects at higher risk of experiencing cardiac death and positivity on medical therapy can be considered a parameter of ischemia severity. Nonetheless in patients with known coronary artery disease the ability of antianginal therapy to modify the ischemic threshold at stress testing represent a powerful means to assess therapy efficacy. From a practical viewpoint, the use of antianginal therapy at time of testing has advantages and disadvantages which are largely dependent on the purpose a test is performed: if the purpose of testing is to diagnose ischemia, it should be performed in the absence of antianginal medications. If the purpose of testing is to assess the protective effects of antianginal therapy, the test should be performed on medications.

Background Anti-ischemic therapy, in particular beta-blockers, is the most commonly employed drug for the control of myocardial ischemia in patients with stable coronary artery disease. Its widespread use also in patients with suspected coronary artery disease has important practical, clinical diagnostic and prognostic implications because diagnostic tests are heavily influenced by its effects. The diagnostic and prognostic impact of anti-ischemic therapy on stress testing is largely ignored but not negligible. The issue raises several questions: How to evaluate patients at time of testing for myocardial ischemia? How to interpret a stress test performed on anti-ischemic therapy? Are the stressors employed for the detection of myocardial ischemia created equal in relation to the different classes of drugs used in clinical practice? Is stress testing able to assess the efficacy of medical therapy in patients with known coronary artery disease? Has the protection of anti-ischemic therapy on inducible myocardial ischemia any impact on long-term survival?
Background
Anti-ischemic therapy, in particular beta-blockers, is the most commonly employed drug for the control of myocardial ischemia in patients with stable coronary artery disease. Its widespread use also in patients with suspected coronary artery disease has important practical, clinical diagnostic and prognostic implications because diagnostic tests are heavily influenced by its effects. The diagnostic and prognostic impact of anti-ischemic therapy on stress testing is largely ignored but not negligible. The issue raises several questions: How to evaluate patients at time of testing for myocardial ischemia? How to interpret a stress test performed on anti-ischemic therapy? Are the stressors employed for the detection of myocardial ischemia created equal in relation to the different classes of drugs used in clinical practice? Is stress testing able to assess the efficacy of medical therapy in patients with known coronary artery disease? Has the protection of anti-ischemic therapy on inducible myocardial ischemia any impact on long-term survival?Pathophysiologic implications of anti-ischemic therapy during stress testing The answer to all these issues relies on the mechanism through which myocardial ischemia is induced by the different stressors (exercise or pharmacologic such as dipyridamole and dobutamine) employed during stress testing. Test exploring organic coronary artery stenosis can induce ischemia by two basic mechanisms: 1. an increase in oxygen demand, exceeding the fixed supply and 2. flow maldistribution due to inappropriate coronary arteriolar triggered by a metabolic/pharmacologic stimulus [ 1 ]. The mechanism of increased demand can be easily fitted into the familiar concept framework of ischemia as a supply-demand mismatch, deriving from an increase in oxygen requirements in the presence of a fixed reduction in coronary flow reserve. The different stresses can determine increases in demand through different mechanisms (Fig.  1 ). In resting conditions, myocardial oxygen consumption is dependent mainly upon heart rate, inotropic state, and the left ventricular wall stress (which is proportional to the systolic blood pressure) [ 2 ]. Following dipyridamole or adenosine administration, a slight increase in myocardial function, a modest decrease in blood pressure, and mild tachycardia can be observed, overall determining only a trivial increase in myocardial oxygen demand [ 3 ]. During exercise, the increase in heart rate, blood pressure, and inotropic state accounts for the overall increase in myocardial oxygen consumption [ 4 ]. Pacing and dobutamine also increase – to a lesser degree – myocardial oxygen demand [ 5 ]. During pacing, the increase is mainly due to the increased heart rate. Dobutamine markedly increases contractility and heart rate. Further augment in myocardial oxygen consumption for heart rate increase occurs with the co-administration of atropine with dobutamine [ 6 ]. and dipyridamole [ 7 ]. (Fig.  2 ). Figure 1 Major determinants of myocardial oxygen consumption in resting conditions (left) and during stress commonly employed with echocardiography. Figure 2 Conceptual allocation of tests employed in combination with echocardiography to detect coronary artery disease stenosis inducing ischemia via steal effect (left) or increased myocardial oxygen demand (right), or both mechanisms. In the presence of coronary atherosclerosis, appropriate arteriolar dilation can paradoxically exert detrimental effects on regional myocardial perfusion, causing overperfusion of myocardial layers or regions already well perfused in resting conditions at the expense of regions or layers with a precarious flow balance in resting conditions [ 8 ]. Anti-ischemic therapy can interfere with all the above mentioned mechanisms of ischemia induction, although in a very different fashion. The mechanism of action of anti-ischemic drugs is easily fitted within the familiar framework of supply-demand mismatch. In particular, beta-blockers are credited with reducing exercise-induced ischemia by decreasing myocardial oxygen demand and possibly by increasing supply through a reduction in extravascular forces [ 9 ]. Experimental studies demonstrated that beta-blockers reduce dipyridamole-induced ischemia by an alteration of regional myocardial blood flow [ 10 ]. However, this straightforward explanation seems inadequate in justifying the protective effects of beta-blockers on dipyridamole-induced ischemia. Experimental data show that beta-blockers do not affect the dipyiridamole-induced increase in flow [ 12 ]. On the other hand, the increase in myocardial oxygen consumption does not play any significant role in the induction of dipyridamole-induced ischemia, which is due to an absolute reduction in subendocardial flow (tightly linked to regional wall thickening) mostly for "vertical" and "horizontal" steal phenomena. However, experimental studies on the model of the exercising dog have shown that beta-blockers protect myocardium from stress-induced myocardial blood flow-function relation: for a given transmural flow, there is a rise of subendocardial and a fall of subepicardial flow, with an improved regional performance [ 10 ]. This same mechanism has also been documented with some calcium antagonists, such as diltiazem and may explain, in part, the beneficial effects of this class of drugs on dipyridamole-induced ischemia. Calcium antagonists can effectively prevent ischemia provoked by dipyridamole also through other mechanisms, which they share with nitrates, and they tend to increase the coronary flow supply during stress. In this case, the prevention of steal phenomena may be due to the increase in collateral flow (which has been shown with nitrates and, to a much lesser extent, with some calcium antagonists) [ 12 ]. and to the dilation of epicardial coronary lumen size. The pronounced increase in collateral flow can prevent horizontal steal phenomena due to dipyridamole, wheras even a small increase of the coronary diameter can dramatically reduce the blood pressure drop across the stenosis, therefore preventing vertical steal phenomena. Beta-blockers exert a direct and competitive action on beta-1 receptors, as they are employed as specific antagonists of dobutamine-induced ischemia. Dobutamine, through its beta-1 agonist action determines the increase in oxygen consumption, but it induces flow maldistribution through beta-2 arteriolar receptors.
Pathophysiologic implications of anti-ischemic therapy during stress testing
The answer to all these issues relies on the mechanism through which myocardial ischemia is induced by the different stressors (exercise or pharmacologic such as dipyridamole and dobutamine) employed during stress testing. Test exploring organic coronary artery stenosis can induce ischemia by two basic mechanisms: 1. an increase in oxygen demand, exceeding the fixed supply and 2. flow maldistribution due to inappropriate coronary arteriolar triggered by a metabolic/pharmacologic stimulus [ 1 ]. The mechanism of increased demand can be easily fitted into the familiar concept framework of ischemia as a supply-demand mismatch, deriving from an increase in oxygen requirements in the presence of a fixed reduction in coronary flow reserve. The different stresses can determine increases in demand through different mechanisms (Fig.  1 ). In resting conditions, myocardial oxygen consumption is dependent mainly upon heart rate, inotropic state, and the left ventricular wall stress (which is proportional to the systolic blood pressure) [ 2 ]. Following dipyridamole or adenosine administration, a slight increase in myocardial function, a modest decrease in blood pressure, and mild tachycardia can be observed, overall determining only a trivial increase in myocardial oxygen demand [ 3 ]. During exercise, the increase in heart rate, blood pressure, and inotropic state accounts for the overall increase in myocardial oxygen consumption [ 4 ]. Pacing and dobutamine also increase – to a lesser degree – myocardial oxygen demand [ 5 ]. During pacing, the increase is mainly due to the increased heart rate. Dobutamine markedly increases contractility and heart rate. Further augment in myocardial oxygen consumption for heart rate increase occurs with the co-administration of atropine with dobutamine [ 6 ]. and dipyridamole [ 7 ]. (Fig.  2 ).In the presence of coronary atherosclerosis, appropriate arteriolar dilation can paradoxically exert detrimental effects on regional myocardial perfusion, causing overperfusion of myocardial layers or regions already well perfused in resting conditions at the expense of regions or layers with a precarious flow balance in resting conditions [ 8 ]. Anti-ischemic therapy can interfere with all the above mentioned mechanisms of ischemia induction, although in a very different fashion. The mechanism of action of anti-ischemic drugs is easily fitted within the familiar framework of supply-demand mismatch. In particular, beta-blockers are credited with reducing exercise-induced ischemia by decreasing myocardial oxygen demand and possibly by increasing supply through a reduction in extravascular forces [ 9 ]. Experimental studies demonstrated that beta-blockers reduce dipyridamole-induced ischemia by an alteration of regional myocardial blood flow [ 10 ]. However, this straightforward explanation seems inadequate in justifying the protective effects of beta-blockers on dipyridamole-induced ischemia. Experimental data show that beta-blockers do not affect the dipyiridamole-induced increase in flow [ 12 ]. On the other hand, the increase in myocardial oxygen consumption does not play any significant role in the induction of dipyridamole-induced ischemia, which is due to an absolute reduction in subendocardial flow (tightly linked to regional wall thickening) mostly for "vertical" and "horizontal" steal phenomena.However, experimental studies on the model of the exercising dog have shown that beta-blockers protect myocardium from stress-induced myocardial blood flow-function relation: for a given transmural flow, there is a rise of subendocardial and a fall of subepicardial flow, with an improved regional performance [ 10 ]. This same mechanism has also been documented with some calcium antagonists, such as diltiazem and may explain, in part, the beneficial effects of this class of drugs on dipyridamole-induced ischemia. Calcium antagonists can effectively prevent ischemia provoked by dipyridamole also through other mechanisms, which they share with nitrates, and they tend to increase the coronary flow supply during stress. In this case, the prevention of steal phenomena may be due to the increase in collateral flow (which has been shown with nitrates and, to a much lesser extent, with some calcium antagonists) [ 12 ]. and to the dilation of epicardial coronary lumen size. The pronounced increase in collateral flow can prevent horizontal steal phenomena due to dipyridamole, wheras even a small increase of the coronary diameter can dramatically reduce the blood pressure drop across the stenosis, therefore preventing vertical steal phenomena. Beta-blockers exert a direct and competitive action on beta-1 receptors, as they are employed as specific antagonists of dobutamine-induced ischemia. Dobutamine, through its beta-1 agonist action determines the increase in oxygen consumption, but it induces flow maldistribution through beta-2 arteriolar receptors.Diagnostic implications of the use of anti-ischemic therapy during stress testing On the basis of these premises and taking into consideration that the markers of inducible myocardial ischemia (electrocardiogram, perfusion, wall motion) are very different therefore expressing a different sensitivity to the action of anti-ischemic therapy at time of testing, it is clear that medical therapy affects test results (see table  1 . In fact, the AHA/ACC Guidelines on Chronic stable angina state that [ 13 ]. "whenever possible, it is recommended that beta-blockers (and other anti-ischemic drugs) be withheld for four to five half-lives (usually about 48 h) before exercise imaging studies for the diagnosis and initial risk stratification of patients with suspected CAD". Ideally, these drugs should be withdrawn gradually to avoid a withdrawal phenomenon that may precipitate events. When beta-blockers cannot be stopped, stress testing may detect myocardial ischemia less reliably, but it usually will still be positive in patients at the highest risk. The same recommendations apply to imaging stress testing. Nonetheless, in patients who exercise to a submaximal level because of the effect of drugs, perfusion or echocardiographic imaging still affords higher sensitivity than the exercise ECG alone [ 14 ]. On the basis of these recommendations patients undergoing a stress testing for diagnostic purposes should be evaluated off therapy not to offset test results. Exercise imaging stress (nuclear perfusion or ultrasound) testing have a lower sensitivity when performed on anti-ischemic therapy [ 14 - 19 ]. due to the limited increase in heart rate and blood pressure which determine oxygen consumption. Antianginal therapy lowers the sensitivity of exercise echocardiography as it does with vasodilator stress testing [ 19 , 20 ]. Antianginal therapy with beta-blockers, calcium-antagonists or nitrates in various combinations prevent dipyridamole-induced ischemia by delaying the appearance of the transient dyssynergy [ 21 ]. this variation on dipyridamole time parallels variations in exercise time at exercise stress testing [ 21 ]. (Fig.  3 ). Dipyridamole stress sensitivity was 91% off therapy and fell to 65% under therapy in various combinations (beta-blockers and/or calcium antagonists and/or nitrates). The same reduction of dipyridamole test sensitivity is obtained with monotherapy with beta-blockers at time of testing (100% off therapy vs. 38% on therapy) [ 22 , 23 ]. (Fig.  4 ). Interestingly, the positive effects of beta-blockers on dipyridamole stress are largely independent of the effect on heart rate, possibly involving a direct anti-steal effect [ 21 - 23 ]. Angiotensin-converting enzyme inhibitors have no effect on dipyridamole stress echocardiography results [ 24 ]. The sensitivity of dobutamine is heavily affected by concomitant beta-blocker therapy. Beta-blockers effect a rightward shift in the dose-response curve to dobutamine and sharply lower test sensitivity, unless atropine is used [ 25 ]. (Fig.  5 ). Calcium antagonists and/or nitrates only mildly reduce dobutamine stress sensitivity (100% off therapy vs.88% on therapy, p=ns) (Fig.  6 ). Non-beta-blocker antianginal therapy reduces the severity of dobutamine-induced ischemia by reducing the value of peak wall motion score index and time of ischemia appearance. However, these changes are not correlated to variations in exercise tolerance [ 26 ]. Figure 3 Correlation between the therapy-induced variations in dipyridamole and exercise time in the 38 patients with positivity of both tests off treatment (Modified from 21). Figure 4 Dipyridamole test sensitivity on and off beta-blocking therapy (Modified from 23). Test sensitivity is significantly reduced in patients studied on beta-blocking therapy. Figure 5 Heart rate during dobutamine-atropine stress testing on and off beta-blockers (Modified from 25). Figure 6 Dobutamine-atropine stress echocardiography test sensitivity in patients on and off non-beta blocking therapy (Modified from 26). Dipyridamole stress nuclear imaging techniques do not seem to be influenced by anti-ischemic therapy [ 27 ]. However, it has been recently demonstrated that acute administration of beta-blockers in patients with known coronary artery disease reduces dipyridamole SPECT sensitivity from 69% with placebo to 52% (p = 0.039) with 10 or 20 mg of metoprolol in a per-vessel analysis, but not overall sensitivity [ 28 ]. The reason for this difference between stress echocardiography and nuclear imaging is likely to be related to the different markers of ischemia, i.e. wall motion abnormalities vs. perfusion, in the face of the same pathophysiologic mechanism of ischemia: the reduction of coronary reserve. In fact, in the presence of a coronary stenosis, during stress, flow remains elevated in the subepicardial layer but falls in the subepicardium. This selective stress-induced hypoperfusion is important for stress echocardiography, since the regional systolic thickening is linearly and closely related to subendocardial perfusion and only loosely related to subepicardial perfusion [ 29 , 30 ]. (Fig.  7 ). Figure 7 Schematic illustration of the principle underlying the impact of antianginal therapy on different markers of ischemia: regional function and perfusion imaging. At rest, perfusion is homogeneously distributed between endocardial and epicardial layers. In the presence of a significant coronary stenosis, vasodilation induced by pharmacologic stress, provokes a subendocardial underperfusion with a relative epicardial overperfusion which is translated into an impairment of function (echocardiographic dyssynergy) and perfusion (reversible defect at scintigraphy). Medical therapy at time of testing re-equilibrates the imbalance between subendocardial and subepicardial layers, but it affects only function. Figure 8 Kaplan-Meier survival curves (considering total mortality as an endpoint) in patients stratified according to presence (DET +) or absence (DET -) of myocardial ischemia at pharmacological stress echocardiography on and off antianginal medical therapy. The best survival is observed in patients with no inducible ischemia off therapy; the worst survival in patients with inducible ischemia on therapy (Positive DET vs. Negative DET off antianginal medical therapy, p < 0.000; Positive DET vs. Negative DET on antianginal medical therapy, p < 0.074) (Modified from 31).
Diagnostic implications of the use of anti-ischemic therapy during stress testing
On the basis of these premises and taking into consideration that the markers of inducible myocardial ischemia (electrocardiogram, perfusion, wall motion) are very different therefore expressing a different sensitivity to the action of anti-ischemic therapy at time of testing, it is clear that medical therapy affects test results (see table  1 . In fact, the AHA/ACC Guidelines on Chronic stable angina state that [ 13 ]. "whenever possible, it is recommended that beta-blockers (and other anti-ischemic drugs) be withheld for four to five half-lives (usually about 48 h) before exercise imaging studies for the diagnosis and initial risk stratification of patients with suspected CAD". Ideally, these drugs should be withdrawn gradually to avoid a withdrawal phenomenon that may precipitate events. When beta-blockers cannot be stopped, stress testing may detect myocardial ischemia less reliably, but it usually will still be positive in patients at the highest risk. The same recommendations apply to imaging stress testing. Nonetheless, in patients who exercise to a submaximal level because of the effect of drugs, perfusion or echocardiographic imaging still affords higher sensitivity than the exercise ECG alone [ 14 ]. On the basis of these recommendations patients undergoing a stress testing for diagnostic purposes should be evaluated off therapy not to offset test results. Exercise imaging stress (nuclear perfusion or ultrasound) testing have a lower sensitivity when performed on anti-ischemic therapy [ 14 - 19 ]. due to the limited increase in heart rate and blood pressure which determine oxygen consumption. Antianginal therapy lowers the sensitivity of exercise echocardiography as it does with vasodilator stress testing [ 19 , 20 ]. Antianginal therapy with beta-blockers, calcium-antagonists or nitrates in various combinations prevent dipyridamole-induced ischemia by delaying the appearance of the transient dyssynergy [ 21 ]. this variation on dipyridamole time parallels variations in exercise time at exercise stress testing [ 21 ]. (Fig.  3 ). Dipyridamole stress sensitivity was 91% off therapy and fell to 65% under therapy in various combinations (beta-blockers and/or calcium antagonists and/or nitrates). The same reduction of dipyridamole test sensitivity is obtained with monotherapy with beta-blockers at time of testing (100% off therapy vs. 38% on therapy) [ 22 , 23 ]. (Fig.  4 ). Interestingly, the positive effects of beta-blockers on dipyridamole stress are largely independent of the effect on heart rate, possibly involving a direct anti-steal effect [ 21 - 23 ]. Angiotensin-converting enzyme inhibitors have no effect on dipyridamole stress echocardiography results [ 24 ]. The sensitivity of dobutamine is heavily affected by concomitant beta-blocker therapy. Beta-blockers effect a rightward shift in the dose-response curve to dobutamine and sharply lower test sensitivity, unless atropine is used [ 25 ]. (Fig.  5 ). Calcium antagonists and/or nitrates only mildly reduce dobutamine stress sensitivity (100% off therapy vs.88% on therapy, p=ns) (Fig.  6 ). Non-beta-blocker antianginal therapy reduces the severity of dobutamine-induced ischemia by reducing the value of peak wall motion score index and time of ischemia appearance. However, these changes are not correlated to variations in exercise tolerance [ 26 ].Dipyridamole stress nuclear imaging techniques do not seem to be influenced by anti-ischemic therapy [ 27 ]. However, it has been recently demonstrated that acute administration of beta-blockers in patients with known coronary artery disease reduces dipyridamole SPECT sensitivity from 69% with placebo to 52% (p = 0.039) with 10 or 20 mg of metoprolol in a per-vessel analysis, but not overall sensitivity [ 28 ]. The reason for this difference between stress echocardiography and nuclear imaging is likely to be related to the different markers of ischemia, i.e. wall motion abnormalities vs. perfusion, in the face of the same pathophysiologic mechanism of ischemia: the reduction of coronary reserve. In fact, in the presence of a coronary stenosis, during stress, flow remains elevated in the subepicardial layer but falls in the subepicardium. This selective stress-induced hypoperfusion is important for stress echocardiography, since the regional systolic thickening is linearly and closely related to subendocardial perfusion and only loosely related to subepicardial perfusion [ 29 , 30 ]. (Fig.  7 ).Prognostic implications of anti-ischemic therapy during stress testing The protective effect of anti-ischemic therapy on inducible myocardial ischemia might exert a powerful impact on prognosis. From the EPIC-EDIC Data bank, it has been analyzed the prognostic impact of antianginal therapy at time of testing in 7333 patients with suspected or known coronary artery disease undergoing pharmacologic stress echocardiography with either dipyridamole or dobutamine. The results show that a positive test on medical therapy is an additional marker of ischemia severity at stress testing whereas a negative test on medical therapy is less prognostically benign, being a false negative result [ 31 ]. (Fig.  8 ). No prognostic difference was found among the various forms of anti-ischemic drugs at time of testing, but the presence per se of antinaginal therapy at time of testing is an independent predictor of death. It is worth noting that in the study only a very low percentage of patients was taking beta-blockers: if on one side this aspect represent a clear lack of adherence to recommendations [ 13 ]., on the other it is an observed pattern of prescription in our data base, which simply reflected the clinical practice and the lack of a universally accepted policy of testing regarding concomitant therapy [ 32 , 33 ]. Marwick et al. [ 34 ]. have demonstrated a protective effect on mortality of beta-blocker therapy in patients with a negative exercise echocardiography whereas specificity and negative predictive value is increased for the prediction of cardiac events (cardiac death, myocardial infarction and unstable angina) during exercise scintigraphy in patients evaluated off medical therapy at time of testing [ 35 ]. The clinical implications of these results are far-reaching. Inducible myocardial ischemia during pharmacological stress testing on medical therapy identifies the subset of patients at highest risk of death. On these patients an aggressive approach has to be undertaken in order to change the natural history of coronary artery disease. On the far opposite end the incidence of death in patients with a negative pharmacologic test off therapy is so low that no intervention could lower the spontaneous rate of death any further. At intermediate risk are those patients with a negative test on medical therapy or with a positive test off medical therapy. Different clinical scenarios can be foreseen on the basis of the present results: 1) A negative test on medical therapy might represent a false negative result, therefore it is advisable to repeat the test off therapy in order to assess the real ischemic burden through the conventional stress echocardiographic parameters [ 36 , 37 ]. – i.e. number of ischemic segments, severity of induced dysfunction, (both expressed by peak wall motion score index), pharmacologic load and time of onset of ischemia. This is in line with the recommendations of the American Heart Association in patients with stable angina [ 13 ].; 2) In the case of a positive test off medical therapy, the effect of therapy can be assessed with the advantage of using an objective, primary ischemic end point such as changes in wall motion during stress.
Prognostic implications of anti-ischemic therapy during stress testing
The protective effect of anti-ischemic therapy on inducible myocardial ischemia might exert a powerful impact on prognosis. From the EPIC-EDIC Data bank, it has been analyzed the prognostic impact of antianginal therapy at time of testing in 7333 patients with suspected or known coronary artery disease undergoing pharmacologic stress echocardiography with either dipyridamole or dobutamine. The results show that a positive test on medical therapy is an additional marker of ischemia severity at stress testing whereas a negative test on medical therapy is less prognostically benign, being a false negative result [ 31 ]. (Fig.  8 ). No prognostic difference was found among the various forms of anti-ischemic drugs at time of testing, but the presence per se of antinaginal therapy at time of testing is an independent predictor of death. It is worth noting that in the study only a very low percentage of patients was taking beta-blockers: if on one side this aspect represent a clear lack of adherence to recommendations [ 13 ]., on the other it is an observed pattern of prescription in our data base, which simply reflected the clinical practice and the lack of a universally accepted policy of testing regarding concomitant therapy [ 32 , 33 ]. Marwick et al. [ 34 ]. have demonstrated a protective effect on mortality of beta-blocker therapy in patients with a negative exercise echocardiography whereas specificity and negative predictive value is increased for the prediction of cardiac events (cardiac death, myocardial infarction and unstable angina) during exercise scintigraphy in patients evaluated off medical therapy at time of testing [ 35 ].The clinical implications of these results are far-reaching. Inducible myocardial ischemia during pharmacological stress testing on medical therapy identifies the subset of patients at highest risk of death. On these patients an aggressive approach has to be undertaken in order to change the natural history of coronary artery disease. On the far opposite end the incidence of death in patients with a negative pharmacologic test off therapy is so low that no intervention could lower the spontaneous rate of death any further. At intermediate risk are those patients with a negative test on medical therapy or with a positive test off medical therapy. Different clinical scenarios can be foreseen on the basis of the present results: 1) A negative test on medical therapy might represent a false negative result, therefore it is advisable to repeat the test off therapy in order to assess the real ischemic burden through the conventional stress echocardiographic parameters [ 36 , 37 ]. – i.e. number of ischemic segments, severity of induced dysfunction, (both expressed by peak wall motion score index), pharmacologic load and time of onset of ischemia. This is in line with the recommendations of the American Heart Association in patients with stable angina [ 13 ].; 2) In the case of a positive test off medical therapy, the effect of therapy can be assessed with the advantage of using an objective, primary ischemic end point such as changes in wall motion during stress.Conclusions Patients may be undergoing various forms of antianginal therapy at the time of testing, both an advantage and a disadvantage for stress echocardiography testing. The disadvantage is that antianginal therapy reduces sensitivity, since stress-induced wall motion abnormalities are caused by the development of obligatory myocardial ischemia. The advantage is that the effect of therapy can be assessed using an objective, primary ischemic end-point such as changes in stress-induced wall motion abnormalities. The presence of ischemia can be titrated on the basis of the ischemic-free stress time and the extent and severity of the induced dyssynergy. The various forms of stress are differently affected by various forms of therapy. In patients with known or suspected coronary artery disease, ongoing anti-ischemic therapy at the time of testing heavily modulates the prognostic value of pharmacological stress echo. In presence of concomitant anti-ischemic therapy, a positive test is more prognostically malignant and a negative test less prognostically benign. However, the decision to remove a patient from beta-blocker therapy for stress testing should be made on an individual basis and should be done carefully to avoid a potential hemodynamic rebound effect, which can lead to accelerated angina or hypertension [ 38 ]. No major side effects were recognized when medical therapy was withdrawn in large series of consecutive patients undergoing pharmacologic stress echocardiography [ 39 ]. In practical terms, when a test is performed for diagnostic purposes it should be done off medical therapy in order to avoid the influence of medical therapy (in case of hypertensive patients it is possible to prescribe ACE-inhibitors or Angiotensin II receptor blockers that do not exert any protective effect on myocardial ischemia). In patients with known coronary artery disease the decision to suspend medical therapy should be taken on an individual basis in view also of the fact that pharmacologic stress echocardiography is a versatile tool that can assess medical therapy efficacy in the long term prognosis [ 31 ]. Table 1 Effects of oral therapy on stress testing sensitivity STRESS Exercise Dipyridamole Dobutamine Beta-blockers ↓ ↓ ↓↓ Calcium channel blockers ↓ ↓ ↓↔ Nitrates ↓ ↓ ↓↔ ACE-inhibitors ↔ ↔ ↔ Aminophylline ↓↔ ↓↓ ↔ ACE, angiotensin-converting enzyme; ↓, decreased sensitivity; ↓↓ markedly decreased sensitivity; ↔, no effect on sensitivity; ↓↔, mild decrease in sensitivity.
Conclusions
Patients may be undergoing various forms of antianginal therapy at the time of testing, both an advantage and a disadvantage for stress echocardiography testing. The disadvantage is that antianginal therapy reduces sensitivity, since stress-induced wall motion abnormalities are caused by the development of obligatory myocardial ischemia. The advantage is that the effect of therapy can be assessed using an objective, primary ischemic end-point such as changes in stress-induced wall motion abnormalities. The presence of ischemia can be titrated on the basis of the ischemic-free stress time and the extent and severity of the induced dyssynergy. The various forms of stress are differently affected by various forms of therapy. In patients with known or suspected coronary artery disease, ongoing anti-ischemic therapy at the time of testing heavily modulates the prognostic value of pharmacological stress echo. In presence of concomitant anti-ischemic therapy, a positive test is more prognostically malignant and a negative test less prognostically benign. However, the decision to remove a patient from beta-blocker therapy for stress testing should be made on an individual basis and should be done carefully to avoid a potential hemodynamic rebound effect, which can lead to accelerated angina or hypertension [ 38 ]. No major side effects were recognized when medical therapy was withdrawn in large series of consecutive patients undergoing pharmacologic stress echocardiography [ 39 ]. In practical terms, when a test is performed for diagnostic purposes it should be done off medical therapy in order to avoid the influence of medical therapy (in case of hypertensive patients it is possible to prescribe ACE-inhibitors or Angiotensin II receptor blockers that do not exert any protective effect on myocardial ischemia). In patients with known coronary artery disease the decision to suspend medical therapy should be taken on an individual basis in view also of the fact that pharmacologic stress echocardiography is a versatile tool that can assess medical therapy efficacy in the long term prognosis [ 31 ].Effects of oral therapy on stress testing sensitivitySTRESSExerciseDipyridamoleDobutamineACE, angiotensin-converting enzyme; ↓, decreased sensitivity; ↓↓ markedly decreased sensitivity; ↔, no effect on sensitivity; ↓↔, mild decrease in sensitivity.