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Cardiovascular Research 2004 62(3):450-459; doi:10.1016/j.cardiores.2004.01.035
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Copyright © 2004, European Society of Cardiology

Altered cAMP-mediated signalling and its role in the pathogenesis of dilated cardiomyopathy

Matthew A Movsesian*

Cardiology Section, VA Salt Lake City Health Care System, Departments of Internal Medicine (Cardiology) and Pharmacology, University of Utah, 500 Foothill Boulevard, Salt Lake City, UT 84148, USA

* Tel.: +1-801-582-1565x4156; fax: +1-801-584-2532. Email address: matthew.movsesian{at}hsc.utah.edu

Received 29 October 2003; revised 29 January 2004; accepted 30 January 2004


   Abstract
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 Abstract
1. Introduction
 2. Evidence that reductions...
 3. Evidence that reductions...
 4. What can be...
 5. A way to...
 6. Evidence for the...
 7. Evidence that different...
 8. Evidence for the...
 9. Future directions
 References
 
Alterations in the level and function of proteins involved in cAMP-mediated signalling are important in the pathophysiology and treatment of dilated cardiomyopathy. What is unclear is the extent to which these alterations, which attenuate receptor-stimulated cAMP generation, contribute to the pathogenesis of dilated cardiomyopathy and the extent to which they constitute a beneficial compensatory response. Studies in animals involving overexpression and ablation of proteins or peptides involved in cAMP-mediated signalling have yielded disparate results that are difficult to reconcile with a simple hypothesis. Our ability to understand these differences is limited by the lack of information on how these different genetic manipulations affect the phosphorylation of individual substrates of protein kinase A (PK-A) through which cAMP signals are transduced. This is important in view of evidence that the phosphorylation of individual PK-A substrates can be regulated selectively in different intracellular compartments, and that the phosphorylation of some PK-A substrates is increased in dilated cardiomyopathy while the phosphorylation of others is reduced. Approaches that quantify changes in the phosphorylation of individual PK-A substrates in models of dilated cardiomyopathy will provide information that may allow a better understanding of the pathogenesis of the syndrome and a more rational approach to its treatment.

KEYWORDS Altered cAMP-mediated signalling; Dilated cardiomyopathy; Protein kinase A


   1. Introduction
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 Abstract
 1. Introduction
2. Evidence that reductions...
 3. Evidence that reductions...
 4. What can be...
 5. A way to...
 6. Evidence for the...
 7. Evidence that different...
 8. Evidence for the...
 9. Future directions
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‘cAMP-mediated signalling’ comprises a ubiquitous set of mechanisms whereby hormones regulate intracellular events (Fig. 1). cAMP, which is formed from ATP by adenylate cyclase, binds to and activates protein kinase A (‘PK-A’), which phosphorylates diverse proteins and thereby modulates their function. Binding of catecholamines to β-adrenergic receptors stimulates adenylate cyclase through interactions with the G protein G{alpha}s. Catecholamine-stimulated cAMP generation is inhibited by β-adrenergic receptor kinase, which phosphorylates β-adrenergic receptors and interferes with their interactions with G proteins, and by G{alpha}i, which inhibits this stimulation through mechanisms that are not yet fully elucidated. Further downstream, cAMP-mediated signalling is reversed by phosphodiesterases that hydrolyse cAMP to AMP and by phosphatases that dephosphorylate PK-A substrates [1,2].


Figure 1
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  		Fig. 1 cAMP-mediated signalling. Binding of agonists to β-adrenergic receptors leads to stimulation of adenylate cyclase via interactions with G{alpha}s. cAMP content is increased, resulting in increased binding and activation of PK-A and increased phosphorylation of PK-A substrates. G{alpha}i has an inhibitory role on this process. Phosphorylation of β-adrenergic receptors by β-adrenergic receptor kinase (‘β-ARK’) interferes with interactions with G proteins and stimulation of adenylate cyclase. Phosphodiesterases (‘PDE’) and phosphatases reverse these processes.

 
Alterations in cAMP-mediated signalling are part of the syndrome of dilated cardiomyopathy (the term ‘dilated cardiomyopathy’ is used in this review to refer to both ischaemic and idiopathic forms of the syndrome). In dilated cardiomyopathy, the density of myocardial β1-adrenergic receptors is reduced, as is the coupling of β1- and β2-adrenergic receptor occupancy to stimulation of adenylate cyclase activity [3,4]. Several mechanisms contributing to receptor-adenylate cyclase uncoupling have been identified, including increases in the activity of β-adrenergic receptor kinase [5] and of G{alpha}i [5–8]. The increase in G{alpha}i activity reflects a combination of increased levels of the protein and increased activation by membrane-associated nucleoside diphosphate kinase activity [6,9]. Some of these changes, including decreases in the expression of β-adrenergic receptors and increases in the expression of G{alpha}i, can be brought about in experimental situations by exposure to β-adrenergic receptor agonists [10–12]. An increase in the activity of PP1 phosphatases, which dephosphorylate some PK-A substrates, has also been described in failing human myocardium [13,14].

These alterations, which result in a decrease in intracellular cAMP content and in the phosphorylation of PK-A substrates in heart muscle, have often been viewed as constituting an ‘impairment’ of cAMP-mediated signalling that contributes to the pathophysiology of dilated cardiomyopathy (Fig. 2). From this perspective, therapy aimed at overcoming this impairment—i.e., raising intracellular cAMP content using β-adrenergic receptor agonists to stimulate cAMP formation or using PDE3 cyclic nucleotide phosphodiesterase inhibitors to reduce cAMP hydrolysis—would be justified. But while cAMP-raising agents are effective in increasing myocardial contractility in patients with dilated cardiomyopathy in the short term, their long-term use has adverse effects on mortality [15–27]. In contrast, long-term treatment with β-adrenergic receptor antagonists, which act to decrease cAMP generation, results in improved survival and, in some cases, restoration of normal chamber size and contractility [28–37]. This response is accompanied by a rise in the level of myocardial β-adrenergic receptors and a decrease in the level of G{alpha}i [38–40]. These observations from clinical trials suggest that changes in cAMP-mediated signalling in dilated cardiomyopathy, in the aggregate, constitute a beneficial compensatory response that to some degree protects the diseased myocardium from the adverse effects of catecholamines (Fig. 2).


Figure 2
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  		Fig. 2 Possible roles of altered cAMP-mediated signalling in the pathogenesis of dilated cardiomyopathy. Changes (‘{Delta}’) in proteins involved in cAMP-mediated signalling, induced by catecholamines, may contribute to the pathogenesis of dilated cardiomyopathy. Alternatively, they may constitute a compensatory response that protects the myocardium from the adverse effects of catecholamines.

 
It is interesting in this context to review observations made in animals in whom the expression or function of proteins involved in cAMP generation in cardiac myocytes has been altered. Some of these observations offer evidence that reductions in cAMP-mediated signalling are harmful and that increases in cAMP content are beneficial, while other observations offer evidence to the contrary.


   2. Evidence that reductions in cAMP generation may be pathogenetic
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3. Evidence that reductions...
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 9. Future directions
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Several lines of investigation suggest that changes in the level or activity of proteins involved in cAMP generation have a causative role in dilated cardiomyopathy (Table 1). One set of experiments involved the expression of a modified {kappa}-opioid receptor, coupled to G{alpha}i and activated specifically by a synthetic ligand, in mouse hearts [41]. Expression of this modified receptor therefore provides a mechanism for stimulating G{alpha}i-mediated signalling and thereby inhibiting G{alpha}s-stimulated cAMP formation. Stimulation of G{alpha}i-mediated signalling resulted in the development of dilated cardiomyopathy in these mice. This suggests that an increase in G{alpha}i activity—which occurs in the human form of dilated cardiomyopathy—has a contributory rather than a compensatory role in its pathophysiology. Of note, however, the effects of modified {kappa}-opioid receptor stimulation on cAMP generation were not quantified in this study.


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  		Table 1 Animal models with changes in proteins involved in cAMP generation and signalling

 
Other studies offer evidence that increases in cAMP-mediated signalling in genetic forms of dilated cardiomyopathy can ‘rescue’ the phenotype. One such study examined the role of β-adrenergic receptor phosphorylation by β-adrenergic receptor kinase in a transgenic mouse in which ablation of the muscle LIM protein gene (‘MLP’) results in the development of dilated cardiomyopathy. Expression of a β-adrenergic receptor kinase inhibitor peptide in MLP–/– mice blocked the development of dilated cardiomyopathy [42–44]. This suggests that the uncoupling of β-adrenergic receptors from adenylate cyclase in conjunction with an increase in β-adrenergic receptor kinase activity—another phenomenon which occurs in dilated cardiomyopathy in humans—may have a pathogenetic role in the syndrome. It should be noted, though, that β-adrenergic receptor kinase may function additionally by interfering with the activity of Gβ/G{gamma}, so that some of the beneficial effects of β-adrenergic kinase inhibitor peptide expression may be cAMP-independent [45].

Other investigators, studying the dilated cardiomyopathy in mice overexpressing G{alpha}q, found that concurrent overexpression of adenylate cyclase type VI increased cAMP generation, improved contractile function and increased survival [46,47]. The benefit observed with this increase in cAMP generation in dilated cardiomyopathy would imply that a decrease in cAMP generation is likely to contribute to its pathogenesis. In other experiments, meanwhile, overexpression of adenylate cyclase type VIII by itself had no adverse effects on cardiac function despite a fourfold increase in basal PK-A activity [48]. This suggests that a rise in cAMP content and in PK-A activity is not likely to be harmful in dilated cardiomyopathy.

The role of β2-adrenergic receptor overexpression in G{alpha}q-overexpressing mice has also been studied [48,49]. At low levels of overexpression of β2-adrenergic receptors, an increase in cardiac contractility was noted, without accompanying adverse effects on survival. From this it may be inferred that a decrease in receptor-mediated cAMP generation may contribute to the contractile impairment in dilated cardiomyopathy. Overexpression of β2-adrenergic receptors did not, however, rescue the phenotype in MLP–/– mice [43], and higher levels of β2-adrenergic receptor overexpression in G{alpha}q-overexpressing mice had adverse effects (see below).


   3. Evidence that reductions in cAMP generation may constitute a compensatory response
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 1. Introduction
 2. Evidence that reductions...
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4. What can be...
 5. A way to...
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 7. Evidence that different...
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 9. Future directions
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Several other lines of investigation have yielded results suggesting that reductions in the level or activity of proteins involved in cAMP generation have a protective role in dilated cardiomyopathy (Table 1). In one study, cardiac-specific overexpression of β1-adrenergic receptors resulted in an increase in contractility in the near term, but in the subsequent development of hypertrophy and failure [50]. This pattern, which is not unlike the clinical response to cAMP-raising agents in humans with dilated cardiomyopathy, suggests that long-term increases in cAMP content are maladaptive in this disease. Other investigators also noted that cardiac-specific overexpression of β1-adrenergic receptors results in an increase in chamber size and a decrease in contractile function over time [51].

The effects of overexpression of G{alpha}s, another manipulation expected to increase receptor-mediated cAMP generation, have also been studied [52]. Mice overexpressing G{alpha}s develop hypocontractility and chamber dilatation, suggesting that an increase in receptor-mediated cAMP generation can contribute to the development of dilated cardiomyopathy. From this it may be inferred that reduced signalling through these receptors in dilated cardiomyopathy, owing to their downregulation and uncoupling from adenylate cyclase, should be beneficial.

Studies with mice overexpressing β2-adrenergic receptors have yielded more complex results. As noted earlier, overexpression of β2-adrenergic receptors at relatively low levels in G{alpha}q-overexpressing mice yielded an improvement in cardiac function. But overexpression of β2-adrenergic receptors at higher levels did not yield this improvement, and instead hastened the progression of myocardial fibrosis and heart failure [49,53]. This suggests that augmentation of cAMP generation to a modest degree may be beneficial in dilated cardiomyopathy, but that augmentation at a greater level is maladaptive.

Another line of investigation involved overexpression of the catalytic subunit of PK-A [54]. This manipulation, which bypasses cAMP-generating mechanisms, results in the expression of a form of PK-A that is active even in the absence of cAMP. Mice overexpressing the catalytic subunit of PK-A develop dilated cardiomyopathy with a reduction in cardiac contractility. If an increase in protein phosphorylation by PK-A contributes to the development of dilated cardiomyopathy, a decrease in cAMP generation should have a protective effect.


   4. What can be deduced from these studies in transgenic animals?
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These studies in animal models yield rather complex results. In one study, a modification that should reduce cAMP generation led to the development of dilated cardiomyopathy, while in other studies, modifications that increase cAMP generation ‘rescued’ or improved function in models of dilated cardiomyopathy. But in other examples, modifications that should augment cAMP generation led to the development of dilated cardiomyopathy.

What conclusions regarding the role of altered cAMP generation in the pathogenesis of dilated cardiomyopathy and the mechanisms in these effects can be drawn from these diverse results? The answer is not clear. Consider the results of studies involving overexpression of β1-adrenergic receptors and G{alpha}s, which are associated with an increase in β-adrenergic receptor-stimulated cAMP generation and yield a dilated cardiomyopathy phenotype, and of studies involving overexpression of adenylate cyclase, which yields a receptor-independent augmentation of cAMP-mediated signalling and rescues a dilated cardiomyopathy phenotype [46,47,50–52]. One possible inference is that adverse outcomes reflect changes in receptor-mediated stimulation of adenylate cyclase rather than receptor-independent adenylate cyclase activity. But if this were the explanation, overexpression of β-adrenergic receptor kinase inhibitor peptide, which should increase β-adrenergic receptor-mediated stimulation of adenylate cyclase, should contribute to the development of a dilated cardiomyopathy phenotype. Instead, overexpression of β-adrenergic receptor kinase inhibitor peptide prevents the development of this phenotype in MLP–/– mice [42–44]. It is difficult to formulate a simple hypothesis consistent with these observations.

The degree to which cAMP generation is augmented might be a consideration. In the G{alpha}q-associated cardiomyopathy, low levels of β2-adrenergic receptor overexpression appear helpful, while higher levels of β2-adrenergic receptor overexpression were not [49,53]. That a profound augmentation of cAMP generation is harmful in pathologic myocardium while a more subtle augmentation is helpful might be consistent with the notion that the reduction in cAMP generation in dilated cardiomyopathy in humans represents a compensatory response that is exaggerated to the point of having harmful features. But it is not clear that there is any other evidence from these transgenic models to support this hypothesis.

Another issue involves the possibility that the effects of changes in cAMP-mediated signalling in these transgenic models may depend upon the state of the myocardium: Increases in cAMP generation might have a pathogenetic effect in animals whose hearts are otherwise ‘normal’ but have a beneficial effect in animals with dilated cardiomyopathy. While some of the observations cited might be reconciled in this manner, this reasoning cannot account for the observation that increasing G{alpha}i-mediated signalling results in the development of dilated cardiomyopathy. Moreover, it is difficult to reconcile this theory with the results of clinical trials indicating that increases in cAMP content in response to β-adrenergic receptor agonists and PDE3 inhibitors have adverse long-term effects in dilated cardiomyopathy while decreases in cAMP content in response to β-adrenergic receptor antagonists have beneficial long-term effects.

Finally, there are issues involving differences among the transgenic models. Overexpression of β2-adrenergic receptors has different effects in dilated cardiomyopathies associated with MLP ablation than in dilated cardiomyopathies associated with G{alpha}q overexpression. This raises the possibility that some of the apparent discrepancies among observations may relate to the models used. The fact is that we do not know the extent to which different animal models, some of which are primarily hypertrophic, represent the mechanisms involved in the pathophysiology of dilated cardiomyopathy in humans. We cannot, therefore, in the face of disparate results in different models, be certain of the applicability of specific findings to the human disease.


   5. A way to reconcile seemingly disparate results
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There is perhaps one way in which these seemingly disparate results can be reconciled with each other and with the results in clinical trials in humans: by postulating that the changes in cAMP-mediated signalling in dilated cardiomyopathy in humans are, as a group, compensatory, and that this represents the sum of beneficial and adverse results that are represented to differing degrees in transgenic animal models.

How can this be? It is important to consider that changes in cAMP generation exert their effects through PK-A, which phosphorylates a large number of proteins involved in the regulation of diverse responses of cardiac myocytes. To name only a few examples: phosphorylation of L-type Ca2+ channels in the plasma membrane and ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum increases the rate and amplitude of the rise of intracellular [Ca2+] during systole [55,56]; phosphorylation of phospholamban stimulates Ca2+ sequestration by SERCA2, the Ca2+-transporting ATPase of the sarcoplasmic reticulum [57,58]; phosphorylation of phosphorylase kinase and glycogen synthase stimulates glycogen hydrolysis [59,60]; phosphorylation of troponin reduces the activation of contractile proteins by Ca2+ [61]; and phosphorylation of cAMP-response element-binding protein (CREB) and cAMP-response element modulator (CREM) regulates gene transcription [62]. Given these diverse processes regulated by cAMP in cardiac myocytes, it is not clear that the changes in cAMP-mediated signalling in dilated cardiomyopathy can be viewed as a functional unit that either contributes to the pathophysiology of the syndrome or represents a beneficial compensatory response. Instead, it may be that the changes in cAMP-mediated signalling in dilated cardiomyopathy in humans include a combination of contributory and compensatory features, i.e., increases in the phosphorylation of some of the protein substrates of PK-A may contribute to the pathophysiology of dilated cardiomyopathy, while increases in the phosphorylation of other protein substrates may constitute a beneficial response to the pathophysiologic process that limits or retards its progression (Fig. 3).


Figure 3
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  		Fig. 3 Role of altered cAMP-mediated signalling in the pathogenesis of dilated cardiomyopathy: theory derived from animal experiments. Rather than be either compensatory or contributory, the changes in cAMP-mediated signalling in dilated cardiomyopathy may have some effects which contribute to the pathogenesis of the disease and other effects which protect the myocardium. These effects may be attributable to changes in the phosphorylation of different PK-A substrates.

 
If this theory is correct, it will be very important to know whether differences among results in genetically manipulated animal models correlate with differences in the phosphorylation of individual PK-A substrates. The identification of such differences would offer an evidence-based rationale for formulating a hypothesis with respect to which downstream signalling events and physiologic sequelae are likely to be beneficial in dilated cardiomyopathy and which are likely to be maladaptive.

But it is important first to consider whether this theory is tenable. This rests upon the answers to two questions: Are there PK-A substrates whose phosphorylation may be beneficial in dilated cardiomyopathy? And are there mechanisms for regulating the phosphorylation of individual PK-A substrates selectively?


   6. Evidence for the potential benefits of increasing the phosphorylation of individual PK-A substrates
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That there are PK-A substrates whose phosphorylation contributes to the pathogenesis of dilated cardiomyopathy is a logical necessity: if this were not so, long-term treatment with cAMP-raising agents would not have adverse effects. But several studies in animal models suggest that an increase in the phosphorylation of some PK-A substrates may be desirable. One study dealt with CREB, whose phosphorylation results in its binding to cAMP response elements and thereby regulating the transcription of genes containing these elements [62]. Transgenic mice expressing a nonphosphorylatable CREB in heart muscle developed dilated cardiomyopathy [63]. This suggests that a reduction of CREB phosphorylation by PK-A may be pathogenetic in dilated cardiomyopathy, and that increasing CREB phosphorylation by PK-A may be beneficial. The observation that CREB phosphorylation is anti-apoptotic in cardiac myocytes is further evidence of the potential desirability of increasing CREB phosphorylation in dilated cardiomyopathy [64]. In other studies, mice in whom the cAMP-responsive transcription factor CREM was inactivated showed impaired myocardial contraction and relaxation, at rest or with increasing rates of contraction [65,66]. In the mice with CREM inactivation, however, progression to dilated cardiomyopathy was not observed, so that the potential desirability of increased CREM phosphorylation in the long term is less clear.

Other investigators have examined the effect of phospholamban phosphorylation on dilated cardiomyopathy in MLP–/– mice. Unphosphorylated phospholamban binds to SERCA2 and inhibits its activity; phosphorylation by PK-A relieves this inhibition and stimulates Ca2+ uptake by the sarcoplasmic reticulum [57,58]. Ablation of phospholamban—which, like its phosphorylation, relieves the inhibition of SERCA2—prevented the development of dilated cardiomyopathy in MLP–/– mice [67]. Similarly, expression of a phospholamban mutant with a serine-to-aspartic acid mutation at the PK-A phosphorylation site—in effect, a constitutively ‘pseudophosphorylated’ phospholamban—attenuated the progressive impairment of left ventricular function in a BIO14.6 cardiomyopathic hamster model of dilated cardiomyopathy [68]. These observations suggest that increasing the phosphorylation of phospholamban by PK-A may be desirable in dilated cardiomyopathy.

Other experimental results, though, call this into question. In G{alpha}q-overexpressing mice, and in mice expressing myosin-binding protein C with a mutation found in a subset of humans with familial hypertrophic cardiomyopathy, phospholamban ablation improved contractility in isolated cardiac myocytes but did not avert the development of myocardial hypertrophy in animals [69].


   7. Evidence that different mechanisms for raising intracellular cAMP content affect the phosphorylation of different PK-A substrates
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cAMP content is regulated selectively in functionally distinct spatial compartments of cardiac myocytes. Depending on the molecular mechanism by which cAMP is raised, cAMP content can be increased differentially in some of these compartments. These increases are associated with changes in the phosphorylation of different PK-A substrates, and result in different physiologic responses. Prostaglandin E1 and β-adrenergic receptor agonists raise intracellular cAMP content in cardiac myocytes by binding to their G{alpha}s-coupled receptors and stimulating adenylate cyclase. But prostaglandin E1 does not bring about inotropic responses, while β-adrenergic receptor agonists do [70–72]. The fact that β-adrenergic receptor agonists increase cAMP content in both cytosolic and microsomal fractions of cardiac myocytes while PGE1 increases cAMP content only in cytosolic fractions suggests that inotropic responses require an increase in cAMP content in a functional compartment that is enriched in microsomal fractions. Glucagon-like peptide-1, which also raises cAMP content without eliciting inotropic responses, may similarly fail to raise cAMP content in compartments represented in microsomal fractions [73].

More subtle differences have been documented in responses to β1- and β2-adrenergic receptors agonists. Agonists of both receptor subtypes elicit inotropic responses, but β1-adrenergic receptor agonists cause increases in systolic [Ca2+] that correlate with changes in membrane-bound cAMP content and are accompanied by increases in phospholamban phosphorylation. β2-adrenergic receptor agonists, in contrast, cause increases in systolic [Ca2+] that do not correlate with changes in cAMP content and are not accompanied by increases in phospholamban phosphorylation [74,75]. To some extent, the localisation of β1- and β2-adrenergic receptors to distinct regions of the plasma membranes of cardiac myocytes may be involved [76]. In addition, cAMP-independent effects of β2-adrenergic receptor agonists that involve the coupling of these receptors to G proteins other than G{alpha}s may interfere with protein phosphorylation by PK-A in some compartments [77–79].

Another difference in signalling mediated through β1- and β2-adrenergic receptors may be particularly relevant to the pathogenesis of dilated cardiomyopathy. Several studies indicate that stimulation of β1-adrenergic receptors results in a pro-apoptotic signal in cardiac myocytes, while stimulation of β2-adrenergic receptors results in a net anti-apoptotic signal [80–82]. Interestingly, both the pro-apoptotic effects of β1-adrenergic receptor stimulation and the anti-apoptotic effects of β2-adrenergic receptor stimulation may be cAMP- and PK-A-independent [82,83].

Finally, there are important differences between the effects of β-adrenergic receptor agonists and PDE3 cyclic nucleotide phosphodiesterase inhibitors, which raise cAMP content by inhibiting its hydrolysis. At doses that result in similar increases in intracellular cAMP content, the β-adrenergic receptor agonist isoprenaline elicits an increase in phospholamban phosphorylation, while the PDE3 cyclic nucleotide phosphodiesterase inhibitor milrinone does not [84].


   8. Evidence for the relevance of these considerations to the pathophysiology of dilated cardiomyopathy in humans
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The compartmentation of cAMP metabolism is likely to be a factor in the pathophysiology of dilated cardiomyopathy in humans. When cAMP content in cytosolic and microsomal fractions of failing human myocardium is compared to cAMP content in cytosolic and microsomal fractions of normal human myocardium, the diminution of cAMP content in failing hearts is much more pronounced in microsomal fractions than in cytosolic fractions (Table 2) [85]. This implies that changes in cAMP-mediated signalling in dilated cardiomyopathy in humans affect cAMP metabolism in a compartment-selective manner.


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  		Table 2 Compartment-selective changes in cAMP-mediated signalling in dilated cardiomyopathy in humans

 
In other studies, the phosphorylation of selected PK-A substrates has been compared in tissue from normal and failing human hearts. One group of investigators reported an increase in the phosphorylation of ryanodine-sensitive Ca2+ channels at its PK-A site, while other investigators identified a decrease in the phosphorylation of phospholamban at its PK-A site (Table 2) [86,87]. These findings imply that the changes in cAMP-mediated signalling in dilated cardiomyopathy in humans somehow affect the phosphorylation of individual PK-A substrates differentially.


   9. Future directions
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The observations discussed above are evidence for the plausibility of the hypothesis that changes in cAMP-mediated signalling in dilated cardiomyopathy have a combination of beneficial and adverse effects owing to changes in cAMP content in different intracellular compartments and in the level of phosphorylation of different PK-A substrates. The conflicting results that occur with different manipulations of proteins involved in cAMP-mediated signalling discussed above may therefore reflect differences in effects on compartmental cAMP metabolism and in the phosphorylation of different PK-A substrates. But this is precisely the information we are lacking: out of all the studies involving transgenic animals cited above, only one, which identified increases in the phosphorylation of ryanodine-sensitive Ca2+ channels and of phospholamban when PK-A catalytic subunits are overexpressed, addressed this question at all [54]. And since an increase in catalytic subunit expression would mimic diffuse rather than localised increases in intracellular cAMP content, the information that can be inferred from this study with regard to compartmentation and selective phosphorylation is unclear.

Knowing how manipulations of proteins involved in cAMP generation affect the phosphorylation of individual PK-A should be helpful in several ways. At a basic level, it will provide important detail on how cAMP-mediated signalling is regulated in a compartment-selective and protein-selective manner. As to the pathophysiology of dilated cardiomyopathy, correlations between changes in the phosphorylation of individual substrates of PK-A and effects on cardiac phenotype may help identify which specific PK-A substrates are involved in the beneficial effects of raising intracellular cAMP content in dilated cardiomyopathy and which are involved in the adverse effects. This could lead to the development of new therapeutic strategies for manipulating cAMP-mediated signalling in patients with dilated cardiomyopathy.

One such strategy may be to target individual PK-A substrates. Gene transfer of antisense phospholamban to myocytes from failing human hearts—a form of phospholamban ablation—was found to improve contractile function in these myocytes [88]. Subsequently, however, a mutation in phospholamban that interferes with its expression—a naturally occurring functional phospholamban ablation—has been found to be associated with the development of a particularly severe dilated cardiomyopathy in humans [89]. This raises some question as to the desirability of de-inhibiting SERCA2 by decreasing phospholamban expression as a long-term strategy in dilated cardiomyopathy. Alternatively, these observations might be consistent with a notion alluded to earlier, i.e., that the effects of increasing cAMP-mediated signalling may be different in normal and pathologic myocardium.

On the other hand, these observations with phospholamban may reflect a problem inherent in trying to remedy what is probably a complex array of changes in the phosphorylation of diverse PK-A substrates by increasing the phosphorylation (or mimicking the effect of increasing the phosphorylation) of a single PK-A substrate. It may be more ‘holistic’, at the cellular level, to target several PK-A substrates concurrently. The combination of β-adrenergic receptor antagonism and PDE3 inhibition, which has shown some promise in preliminary studies, may owe its benefits to increases in a different subset of PK-A substrates than would occur with either PDE3 inhibitors alone or with β-adrenergic receptor antagonists [90–94]. The recent identification of three isoforms of PDE3 localised to different intracellular compartments raises the possibility of targeting these individual isoforms in order to achieve more compartment-selective increases in cAMP content and protein phosphorylation [95,96].

At this point, the information needed to devise and select among such strategies on a rational basis will require the development of methods for quantifying changes in the phosphorylation of an array of proteins under a range of conditions. The field of proteomics is developing at a pace that makes it likely this will not be far off. The more difficult task will be to elucidate the cause-and-effect relationships between changes in the phosphorylation of specific PK-A substrates and the pathogenetic processes in dilated cardiomyopathy. When one considers how many proteins may be phosphorylated by PK-A, the mathematical complexity involved in assigning specific roles to specific phosphorylation events becomes evident. Moreover, the ability to derive meaningful information from such experiments requires the availability of an animal model in which the molecular pathophysiology mimics that of humans with dilated cardiomyopathy. It is not yet obvious that such a model exists. Mice, for example, with their rapid heart rates, may be unsuitable models for the arrhythmic complications of dilated cardiomyopathy in humans. Ultimately, it will probably be necessary to try to integrate observations from a combination of models that represent different aspects of the pathophysiology of dilated cardiomyopathy.

These experimental challenges are daunting, but they are not insurmountable. And it is very likely that the information gained by addressing them will constitute major advances in our understanding of the pathophysiology of this highly complex syndrome and in our approaches to its treatment. From this perspective, the apparent disparities among observations in the transgenic models discussed may be seen not as a frustrating ending but as an auspicious opportunity for increasing our understanding of the complexities of the pathogenesis of dilated cardiomyopathy.


   Acknowledgements
 
This work was supported by Medical Research Funds from the United States Department of Veterans Affairs, the American Heart Association and the University of Utah Research Foundation.


   Notes
 
Time for primary review 21 days


   References
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 1. Introduction
 2. Evidence that reductions...
 3. Evidence that reductions...
 4. What can be...
 5. A way to...
 6. Evidence for the...
 7. Evidence that different...
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 9. Future directions
 References
 

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