Ann Thorac Surg 2003;76:S2246-S2253
© 2003 The Society of Thoracic Surgeons
a Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania, USA
* Address reprint requests to Dr Feldman, Department of Medicine, Jefferson Medical College, 1025 Walnut St, Room 822, Philadelphia, PA 19107-5083, USA
Presented at the symposium, "Gibbon & His Heart-Lung Machine: 50 Years & Beyond," Philadelphia, PA, May 2, 2003.
Heart failure is a disease of epidemic proportions effecting more than 5 million people in the United States. It accounts for more than 1 million hospitalizations, represents the most common discharge diagnosis of patients over the age of 65, and is one of only a handful of human diseases that are increasing in frequency. The increase in frequency of heart failure is due at least in part to the aging of the US population. It is expected that more than 450,000 new cases will be recognized in the coming year and nearly an equal number of deaths will occur that are directly attributable to heart failure. Inclusive of both inpatient and outpatient costs, the care for heart failure will cost the US economy nearly 30 billion dollars in 2003.
Over the past 2 decades seminal information regarding the pathophysiology of heart failure has come from evaluation of the results of large multicenter, randomized, placebo-controlled clinical trials. These studies have validated the use of both angiotensin-converting enzyme (ACE) inhibitors and beta adrenergic-receptor blockers (ß-blockers) in the therapy of heart failure, findings that have resulted in improvements in both morbidity and mortality. However two fundamental observations have come from heart failure clinical trials: only some patients respond to a given drug; and different patients respond differently to the same drug. This variability in pharmacologic response provides important relevance to heart failure patients, many of whom take as many as nine medications. Identifying which drugs benefit selected patients can obviate adverse drug effects; decrease the total number of medications consumed by individual patients; decrease pharmaceutical costs; improve compliances; and provide an opportunity to optimize the dosing of those drugs that will be most effective.
The fact that selective patient groups respond differently to a pharmacologic agent could be explained by either their phenotype or their genotype. In patients with heart failure, phenotype is relatively easy to assess and can include measures such as ejection fraction, functional capacity (MVO2, 6-minute walk time), heart failure symptoms (New York Heart Association classification, Global Assessment scoring), quality of life (Minnesota Living with Heart Failure, Kansas City Questionnaire), or ventricular morphology (left ventricular volume, left ventricular mass, or left ventricular diameter). In addition historical information can also be important in patient phenotyping including the number of hospitalizations. By contrast identifying a patient's genotype has been far more challenging. However recent breakthroughs in molecular technology now provide the opportunity to identify both mutations and single nucleotide polymorphisms with a high degree of accuracy. These new techniques have resulted in the advancement of pharmacogenomics and functional genomics and their application to the treatment of patients with heart failure.
Effect of phenotype on heart failure outcomes
Several recent clinical trials clearly illustrate selective response in patients with different phenotypes. For example in an early trial involving 477 patients, the oral inotropic agent vesnarinone effected a 61% reduction in the risk of a patient dying during a 6-month period . By contrast in a second study involving a total of 3,833 patients, an identical dose of vesnarinone was associated with an increased number of deaths . While these differences might be attributed to the size of the two studies there were important phenotypic differences between the two study populations. For example in the initial study patients were required to have serum digoxin levels less than 1.8 ug/L, serum creatinine less than 2.4 mg/dL, and the ability to perform upright-bicycle exercise. Nearly 20% of enrolled patients had NYHA class II symptoms. By contrast there were no exercise criteria for entry into the Vesnarinone Trial, digoxin levels were not assessed, and patients were required to have NYHA class III or IV heart failure symptoms. Similar differences in drug response can be identified in recent studies assessing the efficacy of anticytokine therapy with the tumor necrosis factor (TNF) soluble receptor etanercept (Enbrel). In early studies assessing efficacy of etanercept in patients who were able to exercise, the recombinant protein effected improvements in cardiac size and function . By contrast etanercept showed no benefit in patients who were unable to exercise (D. Mann, unpublished data). Perhaps exercise served as a surrogate marker of functional reserve and fibrosis in these patients.
Evaluation of genotype in patients with heart failure
Recent clinical trials have also suggested the possibility that genotypic differences rather than simply phenotypic differences might also result in different outcomes in various patient groups. For example the ACE inhibitor enalapril was more effective in white patients with heart failure than in black patients  whereas the combination of hydralazine and isosorbide appeared to be more effective in black patients . Furthermore the ß1 selective ß-blocker bucindolol appears less effective in black patients whereas the nonselective ß-blocker carvedilol appears to be equally effective in black and white patients [6, 7] Figures 17 .
In cardiovascular disease these tools have been used to identify the role of gene structure in predicting the risk of developing heart failure as well as for determining an individual patient's response to pharmacologic therapy. To date this research has focused largely on the presence of polymorphisms in genes that are important in the pathophysiology of heart failuremost notably genes encoding proteins important in neurohormonal and cytokine activation. In each case the overexpression of various neurohormones, cytokines, and endogenous peptides have been shown to play some role in the pathogenesis of heart failure. In addition these proteins and peptides have also served as therapeutic targets. Thus the presence of polymorphisms that effect either an increase or a decrease in neurohormone/cytokine production or responsiveness would be predicted to modify either outcome or therapeutic response in a heart failure population.
Polymorphisms in adrenergic receptors
In response to diminished cardiac output the failing heart is characterized by increased adrenergic drive and the release of norepinephrine. This increased adrenergic drive is maladaptive as it leads to adrenergic receptor pathway desensitization and cardiotoxic effects on the ventricular myocardium [8, 9]. Indeed in animal models, cardiac targeted overexpression of either the ß1 or the ß2 adrenergic receptor lead to the development of cardiac dilatation and failure in a dose-responsive manner . These findings led investigators to assess whether the presence of polymorphisms or mutations in the genes encoding the ß-adrenergic receptors might have import in determining either drug response or outcome in patients with heart failure or alternatively the risk of developing heart failure in patients without obvious cardiac disease.
Several relatively common polymorphisms have been identified in the ß1-AR gene [11, 12]. An A to G substitution at nucleotide position 145 results in either a Ser or Gly at amino acid position 49. A G to A substitution at nucleotide 1165 results in an Arg instead of a Gly at amino acid position 389although it should be noted that the Gly is actually the minor allele (the original report of the cloning of the receptor having cloned the less common allele). When studied in kidney cells, the Gly 49 receptor underwent a 25% loss of receptor density after exposure to isoproterenol for 18 hours; however the Ser 49 receptor did not undergo receptor downregulation . Thus the Gly49 receptor exhibits wild-type coupling with enhanced agonist-promoted downregulation.
When expressed in vitro, receptors having a Gly at amino acid position 389 were less able to effect an increase in adenlyl cyclase activity in the presence of agonist than were receptors having an Arg. Thus it was not surprising that patients homozygous for Arg 389 had a more robust exercise response when compared with patients homozygous for Gly 389 . However because of the limited size of these studies, they were unable to provide information as to how ß1/ß2AR genotypic combinations might influence exercise capacity. Furthermore these studies raised the possibility that while patients having decreased adrenergic coupling due to a Gly at position 389 had a less robust exercise response, they might have improved survival because of diminished adrenergic receptor-adenlyl cyclase coupling. Interestingly the Arg 389 genotype alone was not associated with an increased risk for the development of heart failure . However there was a 10-fold increased risk of the development of heart failure in black subjects who were homozygous for ß1Arg389 and a common coding polymorphism of the gene for the 2c-ARthe deletion of four amino acids (Del322-325).
Found at an allele frequency of 0.411 in black subjects but only at an allele frequency of 0.038 in white subjects, 2c Del322-326 effects a substantial loss of synaptic autoinhibitory feedback resulting in enhanced presynaptic release of norepinephrine [16, 17]. Taken alone it results in a 5.65 odds ratio for heart failure. By contrast black subjects have an allele frequency for the ß1Arg389 of 0.560 while white subjects have an allele frequency of 0.762. Thus individuals harboring homozygous alleles for both variants would have enhanced receptor activation secondary to higher extracellular levels of norepinephrine and heightened responsiveness due to more effective receptor-effector coupling. Taken together these two changes would likely magnify any cardiotoxic effects of adrenergic drive. Furthermore these studies suggest that the presence of this two-locus genotype may be effectively treated with tailored therapy that combines a ß-adrenergic receptor antagonist and an 2-adrenergic receptor agonist. Studies to test this hypothesis are ongoing.
At least four polymorphic loci have been identified in the coding region of the ß2AR gene and several have also been identified in the five upstream regions . The most common polymorphisms are an A to G substitution at nucleotide 46 resulting in an Arg or Gly being encoded at amino acid 16; a C or a G at nucleotide 79 resulting in a Gln or Glu at amino acid 27; and a C or a T at nucleotide 492 resulting in a Thr or Ile at amino acid position 164 in the fourth transmembrane spanning domain of the receptor (homozygous Ile being extremely rare). Because of their location in the amino-terminal end of the receptor, polymorphisms at amino acids 16 and 27 did not influence agonist or antagonist binding or coupling to G proteins . However polymorphisms in these two regions (namely amino acids 16 and 27) did alter downregulation in response to chronic agonist exposure. For example wild-type (Arg-16/Glyn 27) receptors downregulated 26%, Gly-16/Glyn27 receptors downregulated 41%, and the Arg16/Glu27 receptor failed to downregulate when exposed long-term to isoproterenol; Ile164 has decreased binding affinity for ßAR agonists resulting in decreased basal and agonist-stimulated adenylyl cyclase activities when compared with Thr ßAR .
Evaluation of the influence of selected polymorphisms in patients with heart failure was consistent with studies in vitro (Fig 1). For example patients with Ile164, Gly 16, and the combination of Gly16 and Gln27 had diminished functional capacity when compared with Thr164, Arg16, and Gly16/Gln27 respectively . Thus patients having a genotype in which both loci were homozygous for receptors resistant to downregulation (namely Arg16/Glu27) had higher functional capacity on exercise as measured by a higher oxygen consumption than did patients in whom both receptor polymorphisms favored susceptibility to downregulation (namely Gly16/Gln27) . By contrast when comparing patients with Ile64 or Thr164 the lower functional capacity in patients with Ile164 was likely secondary to decreased ability of adrenergic drive to effect increased adenylyl cyclase and subsequent improvements in myocardial contractility due to decreased coupling of the Ile64 receptor. The Gly-16 ß2AR polymorphism has also been associated with attenuated vasodilatory response to catecholamines in normal individuals and thus might influence peripheral blood flow and vascular resistance in patients with heart failure. Thus assessment of ßAR genotype might have important implications in both prognosis and treatment of patients with heart failure. However identification of the response to ß-blockade or long-term outcomes in heart failure patients with varying ßAR genotypes has not yet been thoroughly evaluated.
Polymorphisms in the angiotensin-converting enzyme gene
Like adrenergic drive, increased levels of angiotensin II have also been associated with a worse outcome and higher mortality in patients with heart failure . Angiotensin II is produced in the heart, upregulated by hypertrophic signals and mechanical load, and effects changes in both myocyte structure and the extracellular matrix . However whether it is the AT1 or AT2 receptor that initiates deleterious myocardial or myocyte events is unclear  despite recent studies using genetic over-expression and knock-out models. However inhibition of the angiotensin-converting enzyme has become a mainstay in the therapy of patients with heart failure as ACE inhibition improves survival in patients with NYHA class II to IV symptoms and decreases the combined endpoint of all cause mortality and hospitalizations in patients with assymptomatic left ventricular dysfunction [28, 29]. Like ß-blockade, ACE inhibition appears to benefit some but not all patients with heart failure. This finding is most notable in the differential effect of ACE inhibitors in black patients and white patients with heart failure described earlier. In addition both normal patients and those with heart failure have great variability in circulating levels of ACE.
The variability in serum levels of ACE is due at least in part to genetics . A common biallelic polymorphism is found in intron 16 of the ACE gene . The two alleles differ in the presence or absence of a 287-base pair insertion (I, insertion; D, deletion) (Fig 2). Thus individuals can present with one of three genotypes: DD, ID, and II. The D allele has been consistently associated with higher ACE activity or angiotensin II levels in a variety of populations including normal persons , hypertensive subjects , and patients with heart failure after a myocardial infarction . Those homozygous for the D allele have the highest levels of angiotensin II, those homozygous for the I allele have the lowest levels, and heterozygous individuals have intermediate levels. However despite the obvious pathophysiologic relevance and early observational studies the relevance of the ACE polymorphism to clinical risk remains somewhat controversial .
There is less controversy regarding the influence of ACE genotype in patients with heart muscle disease. For example patients with the D/D genotype have decreased exercise tolerance . In addition patients in the Captopril and Thrombolysis study (CATS) with two D alleles had significantly more left ventricular dilatation one year after a myocardial infarction . However both of these studies were relatively small.
Recently we investigated whether the ACE deletion allele was associated with an adverse outcome in 328 patients with heart failure who were either treated or not treated with ß-blockade . Transplant-free survival was compared by genotype for the whole cohort and separately in patients with (n = 120) or without (n = 208) ß-blocker therapy (Fig 3). Transplant-free survival was significantly worse in patients with the D allelea finding that was consistent with an earlier study . This genetic heterogeneity did not seem to be influenced by ACE inhibitor therapy. Also consistent with earlier studies, the ACE D allele did not appear to increase the risk of myocardial injury and the development of heart failure but rather appeared to act as a disease modifier, altering the rate of disease progression . Importantly in patients not treated with ß-blockers the adverse impact of the ACE D allele was dramatically increased. By contrast in patients receiving ß-blocker therapy the influence of ACE genotype on transplant-free survival was not evident. Furthermore ß-blocker therapy appeared to have its most marked effect in patients with the DD genotype. Thus these results suggested important pharmacogenetic interactions between the ACE D/I polymorphism and therapy with ß-blockers in the determination of heart failure survival (Fig 4). Furthermore they suggested that in patients with the DD genotype, treatment with optimal doses of ß-blockade might be started before optimization of therapy with an ACE inhibitorin contrast to common therapeutic algorithms.
Influence of a polymorphism in the endothelial nitric oxide synthase gene
Another neurohormone that is expressed at high levels in patients with heart failure is nitric oxide (NO) . Nitric oxide is synthesized from L-arginine by a family of three enzymes, the nitric oxide synthases (NOS) . Endothelial nitric oxide synthase (NOS3) and the neuronal isoform (NOS1) are constitutive enzymes that produce low levels of basal NO production. By contrast inducible nitric oxide synthase (NOS2) produces high levels of NO in the presence of physiologic stimuli most notably tumor necrosis factor (TNF). Nitric oxide plays an important role in the pathophysiology of heart failure through relaxation of smooth muscle cells in the peripheral vasculature (although effects appear to be blunted in heart failure patients secondary to a decrease in the L-arginine-NO metabolic pathway and impairment of NOS activity)  and inhibition of adrenergic-activated myocyte toxicity . Thus pharmacologic or genetic reductions in NOS activity would be predicted to have an adverse impact on heart failure progression.
In NOS3 a common polymorphism switch from G to T in nucleotide 894 results in the conversion of glutamate to aspartate at codon 298. The Asp298 variant has a shorter half-life in endothelial cell cultures as the result of increased enzymatic cleavage . Thus it is not surprising that patients with Asp298 have an increased vasoconstrictive response to vasoconstrictors consistent with decreased NOS activity. Consistent with this finding patients with heart failure who harbored the Asp298 polymorphism had a significantly poorer event-free survival when compared with those having the wild-type genotype . That these interactions are complex was shown by the fact that the impact of the Asp298 variant was seen predominantly in patients with nonischemic cardiomyopathy and not in those with heart failure secondary to ischemic heart disease. While studies assessing the relevance of polymorphisms in the NOS2 gene will also be of importance, these early association studies suggest that polymorphisms in the NOS genes might be important in prognostic predictions as well as in the evaluation of the success of potential NO donors in the therapy of heart failure (Fig 6).
Aldosterone synthase and heart failure
Like angiotensin II and NO, an increase in aldosterone is also associated with heart failure. Similar to other neurohormones a direct relationship has been identified between aldosterone and mortality in this group of patients [50, 51]. Aldosterone has a variety of effects on both the heart and the vasculature. For example aldosterone induces rapid increases in intracellular calcium and thus may potentiate the effects of other vasoconstrictors and might also exert important effects on nitric oxide pathways in the endothelium. Fibroblasts residing in the interstitial spaces of the adventitia also respond to aldosterone and this peptide may be an important determinant of vessel and heart muscle remodeling [52, 53]. That inhibition of aldosterone can provide salutary benefits in patients with heart failure was first observed by the Randomized Aldactone Evaluation Study (RALES) . In patients with NYHA class III/IV heart failure symptoms treated with standard therapy including an ACE inhibitor, a diuretic, and a ß-blocker, aldosterone effected a 30% reduction in mortality that was the result of a decrease in progressive heart failure-related deaths as well as sudden cardiac death.
Interestingly a single nucleotide polymorphism has been recognized in the promoter of the aldosterone synthase gene. Located at position -344, this T to C transition results in a significant increase in aldosterone levels in patients harboring either one or two copies of the effected allele. Recently we found that survival was not adversely effected by the presence of patients having one allele containing the polymorphism. However when linked with the DD ACE genotype, patients who were homozygous for the DD ACE allele and who had one copy of the aldosterone synthase -344 single nucleotide polymorphism had a markedly worse transplant free survival than those having the ACE DD genotype alone . However additional studies will be needed to fully understand the relevance of this polymorphism to disease prognosis as well as to therapy with aldosterone inhibitors.
Cardioprotection effects of adenosine
Heart failure in humans is also characterized by an increase in plasma concentrations of adenosine, possibly through norepinephrine and angiotensin II-induced activation of ecto-5'nucleotidease [55, 56]. Produced by both cardiomyocytes and endothelial cells, adenosine effects salutary benefits on the heart through a variety of mechanisms including attenuation of release of catecholamine, increase in coronary blood flow, inhibition of platelet and leukocyte activation, and inhibition of rennin release and TNF expression . In addition adenosine is a critically important mediator of the cardioprotection afforded by ischemic preconditioning .
The enzyme adenosine monophosphate (AMP) deaminase is the rate-limiting step for entry into the purine nucleotide cycle and catalyzes the conversion of AMP to inosine monophosphate (Fig 5). Alternatively AMP is converted to adenosine by nucleotidase . In the presence of decreased activity of AMP deaminase, the stoichiometry of the reaction shifts toward increased production of adenosine. Thus patients with AMP deaminase deficiency have increased adenosine production. A relatively common mutation is found in at least 1 allele of the AMP deaminase 1 gene. This nonsense mutation results in diminished (heterozygous) or absent activity of AMP deaminase resulting in increased expression of adenosine. Putatively the presence of the AMPD1 mutation would be expected to have a better outcome in the presence of heart muscle dysfunction because endogenous muscle adenosine levels would be higher. Indeed Loh and colleagues  found that patients having at least one allele harboring the AMPD1 mutation experienced significantly longer duration of heart failure symptoms before referral for transplantation evaluation and had a greater chance of surviving without a cardiac transplant when compared with patients homozygous for the wild-type allele (Fig 7).
Another tumor necrosis factor- peptide that is expressed by the heart is tumor necrosis factor- (TNF). Expressed by failing heart but not by nonfailing myocardium, investigators have identified a direct relationship between levels of circulating TNF and both functional symptoms and mortality in patients with heart failure [63, 64]. That TNF participates in the development of dilated cardiomyopathy is evidenced by the fact that cardiac-specific overexpression of TNF in transgenic mice elicits the development of heart failure that recapitulates that seen in humans. Mice develop cardiac hypertrophy, dilation, interstitial infiltrates, fibrosis, ventricular dysrhythmias, reexpression of fetal genes, and early death .
The genes encoding both TNF and TNFß are located in tandem on the short arm of chromosome 6. Two biallelic polymorphisms have been identified: a G to A transition at position -308 in the promoter region of the TNF gene (G = TNF1, A = TNF2) and a G to A transition at position +252 in the first intron of the TNFß gene (G = TNFß1, A = TNFß2) [66, 67]. Both TNF2 and TNFß2 are associated with high TNF production [68, 69]. Furthermore, rheumatoid arthritis and systemic lupus erythematosus are associated with increased frequency of the TNF2 allele and patients homozygous for the TNF2 allele have a higher risk for death due to cerebral malaria [70, 71]. However we were not able to identify a relationship between either TNF or TNFß polymorphisms and the presence of either heart failure or circulating levels of TNF in a relatively large cohort of patients with chronic heart failure . However these studies were limited by the fact that all patients had class III heart failure symptoms and we did not assess the relevance of the genotype to long-term prognosis in this patient population. Furthermore because the causes of TNF elevation are multifactorial, genotype alone might not predict circulating TNF levels.
In conclusion a variety of recent clinical studies suggest that the presence of selected polymorphisms and mutations can in different situations predict the occurrence of disease, a patient's response to pharmacologic therapy, or an individual's long-term prognosis. These studies are facilitated by the accomplishments of the human genome project as well as by technologic breakthroughs including silicon-based techniques and high-throughput genotyping that is rapid and cost effective. It is believed that more than 100,000 functional polymorphisms and mutations exist in the more than 30,000 human genes and an increasing array of studies suggest that the overall response of any given person is probably predicated on the combination of more than one genotype. Thus not only is technology required but also sophisticated computational analysis will eventually be necessary to utilize the information now being gleaned from focused association studies in the actual treatment of patients with heart failure and other cardiac diseases. Nonetheless we should expect that in the not too distant future clinicians will choose their therapeutic drugs and devices not just based on patient phenotype but also on genotype. Perhaps one good example might be the treatment of heart failure. Physicians now choose between starting an ACE inhibitor and a ß-blocker based on their own experience and intuition and up-titrate that agent before adding a second drug. One can easily imagine a time in the future when such decisions will be based on genotype, allowing each patient to receive that specific agent (and have that agent up-titrated to appropriate levels) that will best facilitate improvements in their heart muscle performance. Of even greater importance will be the opportunity to identify patients at risk of developing heart failure and begin treatment before the onset of symptoms.
The author thanks Michele Breccia for preparation of the manuscript. This work was supported by NHLBI Grant UO1HL6900902.
This article has been cited by other articles:
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[Abstract] [Full Text] [PDF]
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