Scaffolding proteins in cardiac myocytes.

Post-translational modification, such as protein phosphorylation, plays a critical role to reversibly amplify and modulate signaling pathways. Since kinases and phosphatases have broad substrate recognition motifs, compartmentalization and localization of signaling complexes are required to achieve specific signals. Scaffolds are proteins that associate with two or more binding partners and function to enhance the efficiency and/or specificity of cellular signaling pathways. The identification of scaffolding proteins that control the tempo and/or spatial organization of signaling pathways in cells has benefited enormously from recent technological advances that allow for the detection of protein-protein interactions, including in vivo in intact cells. This review will focus on scaffolding proteins that nucleate multi-protein complexes (and could represent novel entry points into signaling pathways that might be amenable to therapeutic manipulation) in cardiomyocytes.

Sustained augmentation of cardiac alpha1A-adrenergic drive results in pathological remodeling with contractile dysfunction, progressive fibrosis and reactivation of matricellular protein genes.

We previously reported that transgenic (TG) mice with cardiac-restricted alpha(1A)-adrenergic receptor (alpha(1A)-AR)-overexpression showed enhanced contractility, but no hypertrophy. Since chronic inotropic enhancement may be deleterious, we investigated if long-term, cardiac function and longevity are compromised. alpha(1A)-TG mice, but not their non-TG littermates (NTLs), showed progressive loss of left ventricular (LV) hypercontractility (dP/dt(max): 14,567+/-603 to 11,610+/-915 mmHg/s, P<0.05, A1A1 line: 170-fold overexpression; and 13,625+/-826 to 8322+/-682 mmHg/s, respectively, P<0.05, A1A4 line: 112-fold overexpression, at 2 and 6 months, respectively). Both TG lines developed LV fibrosis, but not LV dilatation or hypertrophy, despite activation of hypertrophic signaling pathways. Microarray and real time RT-PCR analyses revealed activation of matricellular protein genes, including those for thrombospondin 1, connective tissue growth factor and tenascin C, but not transforming growth factor beta1. Life-span was markedly shortened (mean age at death: 155 days, A1A1 line; 224 days, A1A4 line compared with NTLs: >300 days). Telemetric electrocardiography revealed that death in the alpha(1A)-AR TG mice was due to cardiac standstill preceded by a progressive diminution in QRS amplitude, but not by arrhythmias. The QRS changes and sudden death could be mimicked by alpha(1)-AR activation, and reversed preterminally by alpha(1)-AR blockade, suggesting a relationship to stress- or activity-associated catecholamine release. Thus, long-term augmentation of cardiac alpha(1A)-adrenergic drive leads to premature death and progressive LV fibrosis with reactivation of matricellular protein genes. To our knowledge this is the first evidence in vivo for a role of the alpha(1A)-AR in ventricular fibrosis and in pathological cardiac remodeling.

Abnormal calcium and protein kinase C-epsilon signaling in hypertrophied atrial tumor myocytes (AT-1 cells).

Cardiac hypertrophy leads to contractile dysfunction and altered hormone responsiveness through incompletely understood mechanisms. Atrial tumor (AT-1) myocytes (AT-1 cells) are a cardiomyocyte lineage that proliferates but hypertrophies when proliferation is prevented with mitomycin C. Because both states maintain a highly differentiated phenotype, AT-1 cells were used to explore the signaling pathways that accompany and/or contribute to hypertrophic cardiomyocyte growth. Mitomycin C-induced AT-1 cell enlargement is associated with a pronounced increase in the amplitude and the duration of both electrically stimulated calcium transients and endothelin receptor-dependent calcium responses. Studies with caffeine indicate that the intracellular pool of releasable calcium is similar in control and hypertrophied AT-1 cells. This agrees with the results of Northern analyses that show similar steady-state levels of transcripts encoding the sarcoplasmic reticulum Ca-ATPase (and higher levels of transcripts encoding the Na+/Ca2+ exchanger) in hypertrophied AT-1 cells, relative to proliferating control cultures. However, immunoblot analyses reveal a marked increase in the expression of protein kinase C (PKC)-epsilon (a critical intermediate in the signaling pathway for endothelin receptor-dependent modulation of intracellular calcium) during AT-1 cell hypertrophy; the abundance of other PKC isoforms is not changed. Collectively, these results identify reciprocal regulation between calcium/PKC signaling and hypertrophic growth. The evidence that AT-1 cell hypertrophy leads to abnormalities in calcium regulation and specific changes in PKC-epsilon expression that alter endothelin receptor responsiveness supports the notion that pathophysiological changes in PKC-epsilon abundance lead to functionally important changes in hormonal modulation of cardiomyocyte function.

Compartmentation of G protein-coupled signaling pathways in cardiac myocytes.

There is a large body of functional data that supports the existence of subcellular compartmentation of the components of cyclic AMP action in the heart. Data from isolated perfused hearts and from purified ventricular myocytes imply a fixed and hormone-specific spatial relationship amongst components of cyclic AMP synthesis, response, and degradation. Available data demonstrate that within a cardiac myocyte, not all cyclic AMP gains access to all cyclic AMP-dependent protein kinase (PKA), that not all PKA interacts with all possible cellular substrates of PKA, and that only a subset of the myocyte's phosphodiesterases (PDEs) may degrade cyclic AMP after a given synthetic stimulus. Molecular mechanisms contributing to compartmentation are being discovered: localization of receptors, G proteins, and adenylyl cyclases in caveolar versus noncaveolar regions of the sarcolemma; localization of PKA by A-kinase anchoring proteins; localization of PKA substrates, PDE isoforms, and phosphoprotein phosphatases in discrete subcellular regions; and differential regulation of multiple isoforms of adenylyl cyclase, phosphoprotein phosphatase, and PDE in distinct subcellular compartments.

beta(1) and beta(2)-adrenergic receptor subtype effects in German shepherd dogs with inherited lethal ventricular arrhythmias.

OBJECTIVE: Delayed afterdepolarization-induced triggered activity originating in ventricular myocardium is a mechanism for some age-dependent, inherited ventricular tachycardias in a colony of German shepherd dogs. METHODS: We used standard microelectrode techniques to study beta-adrenergic receptor subtype modulation of the triggered activity in anteroseptal left ventricular myocardium from eleven of these dogs and seven unafflicted, age-matched German shepherd controls. RESULTS: During sustained stimulation at cycle lengths of 300-4000 ms, 10(-9)-10(-7) M isoproterenol concentration-dependently shortened action potential duration (APD) to 90% repolarization more in myocardium from afflicted than from unafflicted dogs. This shortening was prevented by a beta(1)-blocker CGP20712A (10(-7) M) while a beta(2)-blocker ICI118551 (10(-7) M) did not modify the effect of isoproterenol in either group. The beta(2)-agonist zinterol 10(-8)-10(-6) M had no effect on APD. Stimulation at a cycle length of 250 ms in the presence of 10(-7) M isoproterenol induced more triggered AP in myocardium from afflicted than unafflicted dogs. beta(1)-Blockade completely eliminated, while beta(2)-blockade facilitated, and the beta(2)-agonist zinterol did not induce triggered activity in the two groups. CONCLUSION: Isoproterenol effects on APD and triggered activity in the myocardium of dogs with inherited arrhythmias are due primarily to an abnormality of beta(1)-adrenoceptor mediated signaling that is subject to beta(2)-adrenergic modulation.

Differential targeting of beta -adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway.

Differential modes for beta(1)- and beta(2)-adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdomains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in cardiomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in beta(2)-ARs, Galpha(i), protein kinase A RIIalpha subunits, caveolin-3, and flotillins (caveolin functional homologues); beta(1)-ARs, m(2)-muscarinic cholinergic receptors, Galpha(s), and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RIalpha subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface beta(2)-ARs localize to caveolae in cardiomyocytes and cardiac fibroblasts (with markedly different beta(2)-AR expression levels), indicating that the fidelity of beta(2)-AR targeting to caveolae is maintained over a physiologic range of beta(2)-AR expression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of beta(2)-ARs (but not beta(1)-ARs) in caveolae. Other studies show co-immunoprecipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. However, caveolin is not required for adenylyl cyclase targeting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Nevertheless, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accumulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domain-specific differences in the stoichiometry of individual elements in the beta-AR signaling cascade represent important modifiers of cAMP-dependent signaling in the heart.

The alpha(1)-adrenoceptor subtype- and protein kinase C isoform-dependence of Norepinephrine's actions in cardiomyocytes.

Catecholamines modulate cardiac function at least in part through alpha(1)-adrenergic receptors linked to the activation of protein kinase C (PKC). This study examines the molecular forms of the alpha(1)-receptor and PKC that mediate norepinephrine's actions in cardiomyocytes; distinct approaches (activation-dependent down-regulation of PKC isoforms) and novel reagents (A61603, an alpha(1A/c)-receptor agonist) are used to resolve this issue which has been the focus of dispute in previous studies. Norepinephrine (NE) induces a rise in diacylglycerol levels which is sustained for 24 h and is associated with the translocation (at 5 min) and down-regulation (at 24 h) of PKC delta and PKC xi (but not PKC alpha). The selective targeting of the alpha(1)-adrenergic receptor to activate novel PKC isoforms is remarkable, given an 8-fold greater abundance of PKC alpha relative to PKC xi in this preparation. NE activates the extracellular signal-regulated protein kinase (ERK) subfamily of mitogen-activated protein kinases through a PKC delta/PKC xi -dependent pathway. WB-4101 (alpha(1A/c)- and alpha(1D)-receptor antagonist) and 5-methylurapidil (alpha(1A/c)-receptor antagonist) inhibit norepinephrine-dependent accumulation of inositol phosphate and diacylglycerol, down-regulation of PKC delta and PKC xi, and activation of ERK. Each of these responses is stimulated by A61603, but not attenuated by high concentrations of chloroethylclonidine (which irreversibly inactivates the alpha(1B)-, and to a lesser extent, the alpha(1D)-receptor) or BMY 7378 (selective alpha(1D)-receptor antagonist). A61603 also activates p38-MAPK and induces hypertrophy. These studies establish that NE's actions in cardiomyocytes can be attributed to the alpha(1A/c)-adrenergic receptor subtype and nPKC isoforms, thereby identifying specific targets for the development of pharmaceuticals to influence cardiac contractile function and/or growth responses.

Coupling function of endogenous alpha(1)- and beta-adrenergic receptors in mouse cardiomyocytes.

Genetically altered mouse models constitute unique systems to delineate the role of adrenergic receptor (AR) signaling mechanisms as modulators of cardiomyocyte function. The interpretation of results from these models depends on knowledge of the signaling properties of endogenous ARs in mouse cardiomyocytes. In the present study, we identify for the first time several defects in AR signaling in cardiomyocytes cultured from mouse ventricles. beta(1)-ARs induce robust increases in cAMP accumulation and the amplitude of the calcium and cell motion transients in mouse cardiomyocytes. Selective beta(2)-AR stimulation increases the amplitude of calcium and motion transients, with only a trivial rise in cAMP accumulation in comparison. beta(2)-AR responses are not influenced by pertussis toxin in cultured mouse cardiomyocytes. alpha(1)-ARs fail to activate phospholipase C, the extracellular signal-regulated protein kinase, p38-MAPK, or stimulate hypertrophy in mouse cardiomyocytes. Control experiments establish that this is not due to a lesion in distal elements in the signaling machinery, because these responses are induced by protease-activated receptor-1 agonists and phospholipase C is activated by Pasteurella multocida toxin (a G(q) alpha-subunit agonist). Surprisingly, norepinephrine activates p38-MAPK via beta-ARs in mouse cardiomyocytes, but beta-AR activation of p38-MAPK alone is not sufficient to induce cardiomyocyte hypertrophy. Collectively, these results identify a generalized defect in alpha(1)-AR signaling and a defect in beta(2)-AR linkage to cAMP (although not to an inotropic response) in cultured mouse cardiomyocytes. These naturally occurring vagaries in AR signaling in mouse cardiomyocytes provide informative insights into the requirements for hypertrophic signaling and impact on the value of mouse cardiomyocytes as a reconstitution system to investigate AR signaling in the heart.

Signaling properties and functions of two distinct cardiomyocyte protease-activated receptors.

Previous studies have established that cardiomyocytes express protease-activated receptor (PAR)-1, a high-affinity receptor for thrombin, which is also activated by the tethered-ligand domain sequence (SFLLRN) and which promotes inositol trisphosphate accumulation, stimulates extracellular signal-regulated protein kinase, and modulates contractile function. A single previous report identified PAR-1 as a hypertrophic stimulus, but there have been no subsequent investigations of the mechanism. This study reveals the coexpression of PAR-1 and PAR-2 (a second PAR, which is activated by trypsin/tryptase but not thrombin) by Northern blot analysis and compares their signaling properties in neonatal rat ventricular cardiomyocytes. SFLLRN and SLIGRL (an agonist peptide for PAR-2) promote inositol trisphosphate accumulation, stimulate mitogen-activated protein kinases (extracellular signal-regulated protein kinase and p38-mitogen-activated protein kinase), elevate calcium concentration, and increase spontaneous automaticity. SFLLRN (but not SLIGRL) also activates c-Jun NH(2)-terminal kinase and AKT. In keeping with their linkage to pathways that have been associated with growth and/or survival, SFLLRN and SLIGRL both induce hypertrophy. However, PAR agonists promote cell elongation, a morphology that is distinct from the uniform increase in cell dimension induced by alpha(1)-adrenergic receptor activation. These studies provide novel evidence that cardiomyocytes coexpress 2 functional PARs, which link to a common set of signals that culminate in changes in contractile function and hypertrophic growth. PAR actions may assume clinical importance in the border zone surrounding an infarction, where local proteolysis of PARs by serine proteases generated during inflammatory or thrombogenic pathways would elevate calcium concentration (setting the stage for arrhythmias), promote hypertrophic growth, and/or influence cardiomyocyte survival.

Changes in adenylyl cyclase isoforms as a mechanism for thyroid hormone modulation of cardiac beta-adrenergic receptor responsiveness.

Although thyroid hormones are known to modulate cardiac beta-adrenergic receptor expression, the physiologic implications of these changes in the cardiac manifestations of altered thyroid hormone metabolism have been disputed. This study examined whether thyroid hormone modulates signaling via the cyclic adenosine monophosphate (cAMP) pathway by regulating cardiac adenylyl cyclase (AC) isoform expression. Northern blot analyses and AC enzyme assays were performed on preparations from hypothyroid, euthyroid, and hyperthyroid rat ventricles. Steady-state levels of cardiac AC mRNA types V and VI in hypothyroid ventricles were 173% +/- 8% and 149% +/- 12%, respectively, of the values in euthyroid ventricles (P < .01). This increase in AC mRNA isoforms was accompanied by a 1.5-fold increase (P < .05) in the activation of catalytic AC by forskolin and Mn. In contrast, the relative abundance of transcripts for types V and VI AC was similar in hyperthyroid and euthyroid ventricles, but catalytic AC activation by forskolin and Mn was significantly reduced by 35% in membranes obtained from hyperthyroid ventricles. AC activation through beta-adrenergic receptor stimulation by isoproterenol was not altered by thyroid hormone status. Thus, the effect of thyroid hormone to repress AC catalytic activity would be anticipated to offset the increase in beta-adrenergic receptor expression in hyperthyroidism. These studies identify cardiac AC enzymes as important targets for thyroid hormone-dependent regulation of signaling via the cAMP pathway, and support the finding that cardiac adrenergic responsiveness is unaltered in thyroid disease states.

Abnormal cardiac repolarization and impulse initiation in German shepherd dogs with inherited ventricular arrhythmias and sudden death.

OBJECTIVE: We tested the hypothesis that delayed afterdepolarization (DAD)-associated rhythms in German shepherd dogs with reduced anteroseptal left ventricular (LV) sympathetic innervation derive from abnormal beta-adrenergic receptor effector coupling. METHODS AND RESULTS: In anteroseptal LV midmyocardium of afflicted dogs, beta-receptor density was greater than that in normal dogs (P < .05), with affinity being equal in both groups. Basal and maximum isoproterenol (ISO) stimulated adenylyl cyclase activity of anteroseptal LV of afflicted dogs was greater than that in normal dogs (P < .05). Isolated anteroseptal M cell preparations of afflicted dogs studied with microelectrodes showed abnormal lengthening, rather than shortening of action potential duration in response to ISO, as well as a 61% incidence of 10(-7) mol/l ISO-induced triggered activity as compared to 12% in normals (P < .05). In contrast, there was no difference between afflicted and control dogs in triggered activity, beta-receptors or adenylyl cyclase activity in a normally innervated region of the ventricles. CONCLUSION: In this model there is an increase in beta-receptor density and beta-adrenergic stimulation of adenylyl cyclase and of triggered activity in anteroseptal myocardium but not in a normally innervated region of the heart. Hence, abnormal beta-adrenergic signal transduction appears associated with the neural abnormality identified in dogs with inherited VT.

Activated protein kinase C isoforms target to cardiomyocyte caveolae : stimulation of local protein phosphorylation.

Protein kinase C (PKC) isoforms constitute an important component of the signal transduction pathway used by cardiomyocytes to respond to a variety of extracellular stimuli. Translocation to distinct intracellular sites represents an essential step in the activation of PKC isoforms, presumably as a prerequisite for stable access to substrate. Caveolae are specialized subdomains of the plasma membrane that are reported to concentrate key signaling proteins and may represent a locus for PKC action, given that PKC activators have been reported to dramatically alter caveolae morphology. Accordingly, this study examines whether PKC isoforms initiate signaling in cardiomyocyte caveolae. Phorbol ester-sensitive PKC isoforms were detected at very low levels in caveolae fractions prepared from unstimulated cardiomyocytes; phorbol 12-myristate 13-acetate (PMA) (but not 4alpha-PMA, which does not activate PKC) recruited calcium-sensitive PKCalpha and novel PKCdelta and PKCepsilon to this compartment. The subcellular localization of the phorbol ester-insensitive PKClambda isoform was not influenced by PMA. Endothelin also induced the selective translocation of PKCalpha and PKCepsilon (but not PKCdelta or PKClambda) to caveolae. Multiple components of the extracellular signal-regulated protein kinase (ERK) cascade, including A-Raf, c-Raf-1, mitogen-activated protein kinase kinase, and ERK, were detected in caveolae under resting conditions. Although levels of these proteins were not altered by PMA, translocation of phorbol ester-sensitive PKC isoforms to caveolae was associated with the activation of a local ERK cascade as well as the phosphorylation of a approximately 36-kDa substrate protein in this fraction. Finally, a minor fraction of a protein that has been designated as a receptor for activated protein kinase C resides in caveolae and (along with caveolin-3) could represent a mechanism to target PKC isoforms to cardiomyocyte caveolae. These studies identify cardiomyocyte caveolae as a meeting place for activated PKC isoforms and their downstream target substrates.

Hypoxia-associated induction of early growth response-1 gene expression.

The paradigm for the response to hypoxia is erythropoietin gene expression; activation of hypoxia-inducible factor-1 (HIF-1) results in erythropoietin production. Previously, we found that oxygen deprivation induced tissue factor, especially in mononuclear phagocytes, by an early growth response (Egr-1)-dependent pathway without involvement of HIF-1 (Yan, S.-F., Zou, Y.-S., Gao, Y., Zhai, C., Mackman, N., Lee, S., Milbrandt, J., Pinsky, D., Kisiel, W., and Stern, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8298-8303). Now, we show that cultured monocytes subjected to hypoxia (pO2 approximately 12 torr) displayed increased Egr-1 expression because of de novo biosynthesis, with a approximately 10-fold increased rate of transcription. Transfection of monocytes with Egr-1 promoter-luciferase constructs localized elements responsible for hypoxia-enhanced expression to -424/-65, a region including EBS (ets binding site)-SRE (serum response element)-EBS and SRE-EBS-SRE sites. Further studies with each of these regions ligated to the basal thymidine kinase promoter and luciferase demonstrated that EBS sites in the element spanning -424/-375 were critical for hypoxia-enhanceable gene expression. These data suggested that an activated ets factor, such as Elk-1, in complex with serum response factor, was the likely proximal trigger of Egr-1 transcription. Indeed, hypoxia induced activation of Elk-1, and suppression of Elk-1 blocked up-regulation of Egr-1 transcription. The signaling cascade preceding Elk-1 activation in response to oxygen deprivation was traced to activation of protein kinase C-betaII, Raf, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase and mitogen-activated protein kinases. Comparable hypoxia-mediated Egr-1 induction and activation were observed in cultured hepatoma-derived cells deficient in HIF-1beta and wild-type hepatoma cells, indicating that the HIF-1 and Egr-1 pathways are initiated independently in response to oxygen deprivation. We propose that activation of Egr-1 in response to hypoxia induces a different facet of the adaptive response than HIF-1, one component of which causes expression of tissue factor, resulting in fibrin deposition.

Characterization of the alpha1-adrenergic chronotropic response in neuropeptide Y-treated cardiomyocytes.

The cardiac alpha1-adrenergic chronotropic response changes from stimulatory to inhibitory post-natally. The mature inhibitory response is mediated by the alpha1B-adrenoceptor and a pertussis toxin sensitive G protein. In vivo and in vitro studies identify sympathetic innervation as critical for the maturation of this inhibitory response. Additional experiments in a culture model indicate the effect of innervation is dependent on neurally released neuropeptide Y. The present study establishes that the individual signaling elements in the neuropeptide Y induced alpha1-adrenergic cascade are the same as those appearing during normal in vivo development. In addition, the data demonstrate that the effect of neuropeptide Y does not result from activation of the putative cardiac Y3 neuropeptide Y receptor subtype, since it is reproduced by the peptide fragment neuropeptide Y-(13-36) but not by [Leu31, Pro34]neuropeptide Y.

The thrombin receptor elevates intracellular calcium in adult rat ventricular myocytes.

While there is evidence that thrombin receptor activation leads to contractile dysfunction and induces arrhythmias in ischemic/reperfused cardiac tissue, thrombin is variably reported to modulate intracellular calcium in cardiomyocytes. The present study demonstrates that thrombin receptor activation leads to a rise in intracellular calcium in adult ventricular myocytes and serves to reconcile previous discrepant findings. The thrombin receptor-derived agonist peptide (SFLLRN, a portion of the tethered ligand created by thrombin's proteolytic actions) increases cytosolic calcium and twitch amplitude in cardiomyocytes isolated from adult ventricles. The truncated control peptide FLLRN has no effect, establishing that the response to SFLLRN results from a specific agonist peptide-receptor interaction. However, the response to SFLLRN occurs only at high agonist peptide concentrations and thrombin itself is inactive. This result is not compatible with an action of SFLLRN at a distinct protease-activated receptor (PAR-2; which is activated by SFLLRN, but not by thrombin), since SLIGRL (a ligand which is selective for PAR-2, but not the thrombin receptor) has no effect. Rather, the enzyme-based cell isolation procedure may partially cleave the thrombin receptor and influence cell responses, since concentrations of SFLLRN which are sub-threshold in enzymatically disaggregated myocytes significantly increase the force of isometric contraction of intact rat papillary muscles. These studies provide the first evidence that thrombin receptor activation leads to a change in intracellular calcium and a positive inotropic response in adult ventricular myocardium.