doi: 10.1161/01.RES.0000091365.24799.DF
© 2003 American Heart Association, Inc.
Editorials |
"Cardiac Memory"
A Struggle Against Forgetting
From the Bioelectricity Laboratory (E.J.F., K.R., G.K.), Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass; Cardiovascular Engineering Inc (G.F.M.), Holliston, Mass.
Correspondence to Gideon Koren, MD, Bioelectricity Laboratory, Brigham and Women’s Hospital, Cardiovascular Division, 75 Francis St, Boston, MA 02115. E-mail koren{at}calvin.bwh.harvard.edu
Key Words: cAMP response element binding protein • inducible cAMP early repressor • calcium channels • potassium channels • cardiac memory
One of the most remarkable characteristics of living organisms is their ability to alter their behavior by learning. Human beings excel at processing new information, retaining it in their memory, and using it to develop new ideas. Studies in the mollusk Aplysia showed that learning involves the strengthening and increased effectiveness of preexisting synapses. Over time, it has become clear that synaptic plasticity is the fundamental mechanism for learning and memory. Short-term memory, lasting minutes to hours, involves to a large extent a serotonin-mediated release of cAMP, which activates protein kinase A (PKA), leading to the covalent modifications of S-type K+ channels and delayed-rectifier K+ channels. These modifications, which cause spike broadening, an increase in Ca2+ influx, and enhanced transmitter release, do not require protein synthesis. Long-term memory, lasting days to weeks, differs from short-term memory and requires the synthesis of new proteins. Studies in Aplysia, the fruit fly Drosophila, and later in rodents have revealed that this process involves activation of the cAMP response element binding protein (CREB)-mediated transcriptional cascade. Inactivation of CREB or inhibition of its phosphorylation interferes with learning and memory (see reviews1,2).
The CREB family of transcription factors includes CREB, CREM (cAMP response element modulator), and ATF-1 (activating transcription factor 1). Each member of the family contains a basic DNA-binding domain at the C-terminus and a leucine zipper domain that mediates homodimerization or heterodimerization of these factors (bZIP). All three factors bind to a cAMP response cis-regulatory element (CRE) that consists of the sequence TGACGTCA. The core sequence CGTCA is conserved in almost all CREs and is essential for their function. CREB
contains several additional domains including a kinase-inducible domain (KID) that separates the two glutamine-rich transcription activator domains, Q1 and Q2/CAD. Q2/CAD interacts with components of the basal transcriptional machinery and is necessary and sufficient for basal CRE-mediated gene expression. Phosphorylation of Ser133 within the KID facilitates the binding of CREB-binding protein (CBP) and p300, both of which serve as coactivators of the transcriptional machinery. Through its endogenous histone acetyl transferase activity, CBP acetylates histones, which leads to the unraveling of the chromatin and facilitates transcription. CREB has two main isoforms that function as transcription activators (CREB and CREB
). Alternative splicing of the CREM transcript determines whether CREM isoforms function as transcriptional activators (CREM
and CREM) or repressors (S-CREM or ICER). Thus, isoforms that lack the activation domains will bind to the CRE and repress transcription. This model of CREB/CREM activation is simple but far from conclusive, since not all Ser133 phosphorylation events lead to transcriptional activation. Of note, in all these models, the steady-state levels of CREB polypeptides in the cells remain unchanged (see reviews2–4). By contrast, the CREM isoform ICER (inducible cAMP early repressor) is a potent inhibitor of CREB- and CREM-mediated transcription and is rapidly induced by cAMP. On induction, ICER polypeptides are rapidly degraded by the ubiquitin-proteasome pathway.5
In addition to cAMP/PKA-inducible activation of CREB, Greenberg and colleagues have shown that CREB can function as a Ca2+-inducible transcription factor. Membrane depolarization in neurons leads to calcium entry through L-type Ca2+ channels (ICa,L). These calcium cations can bind to calmodulin (CaM) and activate CaMKI, CaMKII, and CaMKIV, each of which can phosphorylate Ser133. Calcium can also activate the Ras/ERK pathway with similar consequences. Neuronal growth factors can activate the CREB pathway through receptor tyrosine kinases, which after ligand binding and dimerization can also activate the Ras/ERK pathway. In addition, the phosphatidylinositol 3-kinase/Akt pathway can activate CREB-mediated transcription after stimulation with IGF-1. Finally, the stress-activated kinase, SAPK2/p38MAPK can also activate the CREB-mediated pathway through several downstream kinases (see reviews2–4).
There are more than 500 genes that contain a cis-CRE regulatory element in their promoter region. The list of channels that contain CRE includes Kv1.5, Kv3.1, Cav1.2, aquaporin 2, and CFTR.2,6 Na+/K+-ATPase also contains CRE. Of note, some of the genes that contain CRE are critical to cell survival. Indeed, in neuronal cells, CREB phosphorylation correlates with neuronal survival. In PC12 cells, hypoxia induces the expression of CREB-dependent Bcl-2 and protects the cells from apoptosis. Similarly, dentate gyrus neurons are protected by CREB-mediated induction of Bcl-2.7,8 In the heart, the overexpression of a dominant-negative CREB mutant where Ser133 was mutated to alanine resulted in transgenic mice with dilated cardiomyopathy with extensive myocyte loss and fibrosis.9 Recently, Tomita and colleagues showed that induction of ICER in the heart resulted in suppression of Bcl-2 and was associated with cardiac myocyte apoptosis.10 In both cases, the overexpression of the CREB mutant or ICER will result in loss of CREB-related transcriptional responses that likely involve many genes. Clearly, the properly balanced activation of the CREB family of transcription factors plays an important role in myocyte and neuronal cell survival, while its suppression promotes apoptosis.
Does the heart have memory? Levine and colleagues observed T-wave changes after extra systoles.11 Subsequently, Chatterjee et al reported persistent changes in T-wave morphology after pacing.12 Several groups later reported similar changes in posttachycardia syndrome, intermittent left bundle brunch block (LBBB), and after extra systoles.12–14 Rosenbaum and colleagues expanded these concepts and termed the changes in ST-T waves following pacing "cardiac memory" (CM) since the sinus beats followed the same vector as the paced beats.14 Rosen and colleagues demonstrated that CM could be further classified as either short-term (STCM) or long-term (LTCM) (see review15). These investigators showed that LTCM is associated with a reduction in epicardial action potential notch that correlates with a decrease in Ito and Kv4.3 transcript. In addition, there are marked changes in Ito kinetics of activation and recovery from inactivation. CM is also associated with changes in activation and recovery from inactivation of ICa,L, as well as changes in the distribution of Cx43. The changes in Ito and ICa,L lead to elevation and prolongation of the action potential plateau and are therefore associated with modulation of the transmural repolarization gradient. Both ACE inhibition and AT-1 receptor blockade suppress STCM but not LTCM. By contrast, ICa,L blockade inhibits both STCM and LTCM (see review15).
In this issue of Circulation Research, Patberg and colleagues16 studied STCM and LTCM in AV sequentially paced (AVP) or sham-operated dogs. STCM was assessed by ECG, VCG, and subepicardial biopsies. LTCM was studied in dogs paced for 3 weeks and in sham controls. The investigators observed a 50% reduction in nuclear CREB without changes in the level of phosphorylated CREB after 2 hours of AVP. Pretreatment with saralasin or nifedipine abolished STCM and the reduction in CREB. Of note, there was a suggestion that CREB downregulation was localized to the area nearest the pacing site. Three weeks of pacing led to a similar reduction in CRE binding activity in nuclear extracts obtained from AVP ventricles but not from controls. Previous reports indicated that STCM and LTCM are not associated with pathological changes. If pacing stimulates the CREB-mediated transcription, then it may have a protective effect against apoptosis. However, if pacing downregulates the CREB-mediated transcription, it may induce myocyte apoptosis at the site of pacing. Thus, the careful study of CREB-mediated transcription at the site of pacing may have important implications. The 50% reduction in nuclear CREB after 2 hours of pacing is an intriguing observation. However, the case for spatial dispersion of effect is minimal since the paced regions do not appear to differ significantly from each other, only from prepacing control. Of note, with the exception of ICER, no CREB-mediated gene activation or suppression involves changes in steady-state levels of CREB. Thus, any change in CREB needs to be interpreted in the context of the expression of all CREB and CREM isoforms as well as ATF-1. Each of these factors could compensate for the moderate decrease in CREB. ICER expression is upregulated by cAMP, but not by a depolarization-induced increase in the intracellular concentration of calcium.5 Furthermore, the 50% reduction in CREB may not be sufficient to modify CREB-mediated transcription, since the loss of function of one allele of CREB in mice does not modify the phenotype.17 In any case, even the complete inactivation of transcription factors may take many hours to be reflected in the levels of their respective gene products and of the downstream genes that they regulate, and therefore the relationship of this change to STCM after 2 hours of AVP needs to be supported by additional experiments documenting changes in downstream CREB-regulated gene products. Furthermore, the Kv4.3 CRE lacks the complete core consensus sequence required for optimal CRE function (CGTCT instead of CGTCA). Thus, the relatively minor gel retardation effect likely reflects nonspecific background or low-affinity binding of CREB to the Kv4.3 CRE, limiting the interpretation of these data. Additional studies are needed to determine whether the CREB family of transcription factors regulates the expression of Kv4.3.
Ventricular pacing may activate CREB-mediated transcriptional response by stretch that can stimulate receptor tyrosine kinases. Moreover, calcium entry during pacing may stimulate CREB phosphorylation by the activation of CaMK. Thus, it is puzzling that the authors did not detect an increase in phosphorylated CREB 5 minutes after the initiation of pacing, although the constant level of phosphorylated CREB in the setting of reduced nuclear CREB suggests increased relative phosphorylation of the existing CREB pool. The blocking effect of nifedipine and saralasin may represent direct effects on myocytes, as suggested by the authors, or may be secondary to either the marked reduction in afterload that was demonstrated or a change in preload, which was not evaluated.
In summary, the work of Patberg et al16 demonstrates STCM induction in AVP dogs with a pacing-related reduction in nuclear CREB polypeptides around the pacing electrode. Dogs with LTCM manifested reduced binding to CRE at the same location. As observed in neurons, STCM is more likely related to posttranslational modifications of calcium and potassium channels rather than to CREB-mediated transcriptional response. It is prudent to recognize that the long-term downregulation of CREB can trigger upregulation of CREM or ATF-1 polypeptides. Thus, the study of this dynamically regulated system and its effect on LTCM necessitates the careful assessment of the regional heterogeneity in the expression of all members of the CREB family of transcription factors. Importantly, pacing may regulate other transcriptional cofactors that could mediate the downregulation of Kv4.3 transcript. Undoubtedly, further research is necessary to extend these initial observations and ascertain their biological significance.
Acknowledgments
Dr Koren is a recipient of National Heart, Lung, and Blood Institute Grants HL-46005 and HL-62328. We thank Janine Chalk and Amit Koren for their help with the manuscript.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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