The β-Adrenergic receptor (β-AR) pathways play a role in regulating cardiac function. Stimulation of these receptor pathways by the endogenous catecholamine, norepinephrine, results in a dramatic increase in contractile function. In individuals with heart failure, the activities of the adrenergic and renin-angiotensin systems are significantly elevated, resulting in chronic over-stimulation of the β-AR pathways which, if left unchecked, can severely damage cardiac tissues. As a protective mechanism, the β-AR pathways attempt to limit excess stimulation to the heart by undergoing the process of desensitization. There are several well-characterized components of β-AR desensitization. First and foremost, the β1-AR subtype, which is the dominant β-AR subtype (80%) in nonfailing heart undergoes subtype selective down-regulation. In contrast, the β2-AR subtype is unaffected. Additionally, both b1- and β2-AR subtypes become uncoupled from their signal transduction pathways due to phosphorylation by multiple protein kinases, PK-A, PK-C, and βARK(s).Other factors that we and others have found to be involved in uncoupling of the β-AR are an increase in the inhibitory G-protein, Gai, and a decrease in stimulatory G-protein, Gas, activity.

 

Since the β-AR genes were cloned by Dr. Robert Lefkowitz's laboratory in the late 1980s, the molecular mechanisms responsible for receptor down-regulation have undergone extensive investigation. In cell model systems and, subsequently, in human heart, it is now appreciated that the amount of β-AR protein correlated closely with the amount of β-AR mRNA. Thus, in the failing human heart, both β1-AR and its cognate mRNA are down-regulated to a similar degree.

 

The steady-state abundance of any mRNA is regulated by the rates of both its synthesis and degradation. Synthesis, or the process of transcription, is known to be important for many gene products including β-ARs. Far less well understood, but of considerable importance to highly regulated genes such as proto-oncogenes and cytokines, is regulation of mRNA stability. Recently, my laboratory has demonstrated, using cell model systems, that the human β1-AR mRNA undergoes agonist-mediated destabilization to a degree that could at least, theoretically, account for observed down-regulation of the β1-AR mRNA in the failing human heart (1,2). The mRNAs of several other G-protein coupled receptors have also been shown to be regulated similarly. The mRNA of a number of other G-protien coupled receptors are also regulated by medonism.

 

Like many proto-oncogenes and cytokines, the human β1-AR 3' untranslated region (3'UTR) has A+U-rich elements or AREs which are cis-acting nucleotide sequences known to be associated with rapid destabilization of mRNAs. A number of cytosolic and/or nuclear proteins interact with AREs with high-affinity. One such protein is AUF1 for which there is a strong correlation between its affinity for an mRNA and its intrinsic stability. AUF1 has also recently been demonstrated to be a component of a multi-protein complex associated with rapid destabilization of certain cytokine mRNAs, the association being dependent on phosphorylation by p38 MAP kinase.

 

We have recently established that AUF1 mRNA and protein expression are up-regulated in failing human heart and in cell lines treated with β-AR agonist (3). Further, we have demonstrated that AUF1 binds with high affinity to the A+U-rich regions of the human β1-AR and hamster β2-AR 3'UTRs, both mRNAs that undergo β-agonist-mediated destabilization, and with lower affinity to the human β2-AR 3'UTR, an mRNA that is not down-regulated in the failing human heart.

 

Making the picture less clear, it appears that the 3'UTR of the β1-AR mRNA may be "necessary but not sufficient" for agonist-mediated destabilization, inferring that other regions of the mRNA including the coding region or 5'UTR might also be necessary for this process. This trait is again similar to certain proto-oncogenes and cytokines where elements involved in "regulated" stability may not simply contained within the 3'UTR of the message (1).

 

Much remains to be understood regarding how mRNA binding proteins are regulated and how, in turn, they regulate the stability of their target mRNAs. We are asking questions regarding the effects of þ-agonist stimulation on the intracellular distribution of target mRNAs and trans-acting factors, as well as questions about the role translation plays in affecting mRNA stability (4-8).  In the near future, we hope to have answers to some of these questions using both in vitro biochemical and molecular biological techniques, as well as a genetic approach using transgenic mouse technology.

 

A second major aim of our laboratory is to investigate the role of β1-AR signaling in transgenic mouse models. The Lefkowitz laboratory was the first to produce a transgenic mouse over-expressing a G-protein-coupled receptor in ventricular myocardium, the human β2-AR. This mouse is significantly hyperdynamic and appears to have modest changes in histopathology. Since the β1-AR is predominant β-AR subtype in the human heart, and given the recognized differences in β1- and β2-AR signaling, we have produced a transgenic mouse over-expressing the human β1-AR in a cardiac-specific context. Our preliminary data indicate that the β1-AR mouse has significant histopathology, early on, with a progression to dilated cardiomyopathy and decreased contractile function in older mice. Thus, preliminarily, the phenotype of β1-AR overexpression appears to be more severe than that of β2-AR overexpression (9).

 

We hope to use this model system to understand more completely the biochemical changes that occur in the failing heart, as well as to evaluate new therapeutic modalities.