Rapid beta-adrenergic modulation of cardiac calcium channel currents by a fast G protein pathway.

beta-Adrenergic agonists activate the G protein, Gs, which stimulates cardiac calcium currents by both cytoplasmic, indirect and membrane-delimited, direct pathways. To test whether beta-adrenergic agonists might use both pathways in the heart, isoproterenol was rapidly applied to cardiac myocytes, resulting in a biphasic increase in cardiac calcium channel currents that had time constants of 150 milliseconds and 36 seconds. beta-Adrenergic antagonists of a G protein inhibitor blocked both the fast and slow responses, whereas the adenylyl cyclase activator forskolin produced only the slow response. The presence of a fast pathway in the heart can explain what the slow pathway cannot account for: the ability of cardiac sympathetic nerves to change heart rate within a single beat.

Direct activation of mammalian atrial muscarinic potassium channels by GTP regulatory protein Gk.

The mammalian heart rate is regulated by the vagus nerve, which acts via muscarinic acetylcholine receptors to cause hyperpolarization of atrial pacemaker cells. The hyperpolarization is produced by the opening of potassium channels and involves an intermediary guanosine triphosphate-binding regulatory (G) protein. Potassium channels in isolated, inside-out patches of membranes from atrial cells now are shown to be activated by a purified pertussis toxin-sensitive G protein of subunit composition alpha beta gamma, with an alpha subunit of 40,000 daltons. Thus, mammalian atrial muscarinic potassium channels are activated directly by a G protein, not indirectly through a cascade of intermediary events. The G protein regulating these channels is identified as a potent Gk; it is active at 0.2 to 1 pM. Thus, proteins other than enzymes can be under control of receptor coupling G proteins.

A monoclonal antibody to the alpha subunit of Gk blocks muscarinic activation of atrial K+ channels.

The activated heterotrimeric guanine nucleotide binding (G) protein Gk, at subpicomolar concentrations, mimics muscarinic stimulation of a specific atrial potassium current. Reconstitution studies have implicated the alpha and beta gamma subunits as mediators, but subunit coupling by the endogenous G protein has not been analyzed. To study this process, a monoclonal antibody (4A) that binds to alpha k but not to beta gamma was applied to the solution bathing an inside-out patch of atrial membrane; the antibody blocked carbachol-activated currents irreversibly. The state of the endogenous Gk determined its susceptibility to block by the antibody. When agonist was absent or when activation by muscarinic stimulation was interrupted by withdrawal of guanosine triphosphate (GTP) in the presence or absence of guanosine diphosphate (GDP), the effects of the antibody did not persist. Thus, monoclonal antibody 4A blocked muscarinic activation of potassium channels by binding to the activated G protein in its holomeric form or by binding to the dissociated alpha subunit.

The alpha subunit of the GTP binding protein Gk opens atrial potassium channels.

Guanine nucleotide binding (G) proteins (subunit composition alpha beta gamma) dissociate on activation with guanosine triphosphate (GTP) analogs and magnesium to give alpha-guanine nucleotide complexes and free beta gamma subunits. Whether the opening of potassium channels by the recently described Gk in isolated membrane patches from mammalian atrial myocytes was mediated by the alpha k subunit or beta gamma dimer was tested. The alpha k subunit was found to be active, while the beta gamma dimer was inactive in stimulating potassium channel activity. Thus, Gk resembles Gs, the stimulatory regulatory component of adenylyl cyclase, and transducin, the regulatory component of the visual system, in that it regulates its effector function--the activity of the ligand-gated potassium channel--through its guanine nucleotide binding subunit.

Atrotoxin: a specific agonist for calcium currents in heart.

A specific label for voltage-dependent calcium channels is essential for the isolation and purification of the membrane protein that constitutes the calcium channel and for a better understanding of its function. A fraction of Crotalus atrox that increases voltage-dependent calcium currents in single, dispersed guinea pig ventricular cells was isolated. In the doses used, neither sodium nor potassium currents were changed. The fraction was active in the absence of detectable phospholipase or protease activity, and the active component, designated atrotoxin, produced its effect rapidly and reversibly. The effect was produced by extracellular but not intracellular application of the agent. The increase in Ca2+ current was blocked by the Ca2+ channel blockers cobalt and nitrendipine. The active fraction completely blocked specific [3H]nitrendipine binding to guinea pig ventricular membrane preparations. The inhibition of nitrendipine binding by atrotoxin was apparently via an allosteric mechanism. Thus atrotoxin was shown to bind to the Ca2+ channel and to act as a specific Ca2+ channel agonist.

Heart rate regulation by G proteins acting on the cardiac pacemaker channel.

Heart rate is determined by pacemaker currents, of which the most important is the hyperpolarization-activated current I(f). Heart rate and I(f) are increased by beta-adrenergic agonists and decreased by muscarinic agonists released from cardiac sympathetic and vagal nerves, respectively. The hypothesis that the receptors for each agonist are directly coupled to I(f) channels by G proteins was tested. Under substrate-free conditions, preactivated G protein Gs stimulated and preactivated G protein G(o) inhibited I(f) channels of sinoatrial node pacemaker cells. These effects were mimicked by the corresponding preactivated alpha subunits of the G proteins. Unexpectedly, the two G proteins acted simultaneously, with G(o) being the more potent. This result may explain in molecular terms the classical observation in cardiac physiology, that vagal inhibition of heart rate is much greater on a background of sympathetic stimulation.

A G protein directly regulates mammalian cardiac calcium channels.

A possible direct effect of guanine nucleotide binding (G) proteins on calcium channels was examined in membrane patches excised from guinea pig cardiac myocytes and bovine cardiac sarcolemmal vesicles incorporated into planar lipid bilayers. The guanosine triphosphate analog, GTP gamma S, prolonged the survival of excised calcium channels independently of the presence of adenosine 3',5'-monophosphate (cAMP), adenosine triphosphate, cAMP-activated protein kinase, and the protein kinase C activator tetradecanoyl phorbol acetate. A specific G protein, activated Gs, or its alpha subunit, purified from the plasma membranes of human erythrocytes, prolonged the survival of excised channels and stimulated the activity of incorporated channels. Thus, in addition to regulating calcium channels indirectly through activation of cytoplasmic kinases, G proteins can regulate calcium channels directly. Since they also directly regulate a subset of potassium channels, G proteins are now known to directly gate two classes of membrane ion channels.

GAP domains responsible for ras p21-dependent inhibition of muscarinic atrial K+ channel currents.

The interaction between the low molecular weight G protein ras p21 and a guanosine triphosphatase activating protein (GAP) uncouples a heterotrimeric G protein (Gk) from muscarinic receptors. Through the use of isolated atrial cell membranes and genetically engineered GAP deletion mutants, the src homology regions (SH2-SH3) at the amino terminus of GAP have been identified as the domains responsible for this effect. Deletion of the domain required to stimulate the guanosine triphosphatase activity of ras p21 relieves the requirement for ras p21 in this system. A model is presented that suggests that ras p21 induces a conformational change in GAP, which allows the SH2-SH3 regions of GAP to function.

Splice variants of the alpha subunit of the G protein Gs activate both adenylyl cyclase and calcium channels.

Signal transducing guanine nucleotide binding (G) proteins are heterotrimers with different alpha subunits that confer specificity for interactions with receptors and effectors. Eight to ten such G proteins couple a large number of receptors for hormones and neurotransmitters to at least eight different effectors. Although one G protein can interact with several receptors, a given G protein was thought to interact with but one effector. The recent finding that voltage-gated calcium channels are stimulated by purified Gs, which stimulates adenylyl cyclase, challenged this concept. However, purified Gs may have four distinct alpha-subunit polypeptides, produced by alternative splicing of messenger RNA. By using recombinant DNA techniques, three of the splice variants were synthesized in Escherichia coli and each variant was shown to stimulate both adenylyl cyclase and calcium channels. Thus, a single G protein alpha subunit may regulate more than one effector function.

Molecular localization of regions in the L-type calcium channel critical for dihydropyridine action.

Sensitivity to dihydropyridines (DHPs) is a distinct characteristic that differentiates L-type Ca2+ channels from T-, N-, and P-type Ca2+ channels. To identify regions necessary for the functional effects of DHPs, chimeric Ca2+ channels were constructed in which portions of motif III or motif IV of a DHP-insensitive brain Ca2+ channel, BI-2, were introduced into the DHP-sensitive cardiac L-type Ca2+ channel. The resultant chimeric Ca2+ channels were expressed in Xenopus oocytes, and the effects of a DHP agonist and antagonist were studied. The results show that the linker region between S5 and S6 in motif IV of the L-type Ca2+ channel is a major site for DHP action. The DHP agonist and antagonist molecules interact with distinct sites on the alpha 1 subunit of the L-type Ca2+ channel. The data further show that the SS2-S6 region of motif III is not involved in DHP action but may be an important structural component of inactivation.

cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits.

The cardiac L-type Ca2+ channel is a textbook example of an ion channel regulated by protein phosphorylation; however, the molecular events that underlie its regulation remain unknown. Here, we report that in transiently transfected HEK293 cells expressing L-type channels, elevations in cAMP resulted in phosphorylation of the alpha1C and beta2a channel subunits and increases in channel activity. Channel phosphorylation and regulation were facilitated by submembrane targeting of protein kinase A (PKA), through association with an A-kinase anchoring protein called AKAP79. In transfected cells expressing a mutant AKAP79 that is unable to bind PKA, phosphorylation of the alpha1C subunit and regulation of channel activity were not observed. Furthermore, we have demonstrated that the association of an AKAP with PKA was required for beta-adrenergic receptor-mediated regulation of L-type channels in native cardiac myocytes, illustrating that the events observed in the heterologous expression system reflect those occurring in the native system. Mutation of Ser1928 to alanine in the C-terminus of the alpha1C subunit resulted in a complete loss of cAMP-mediated phosphorylation and a loss of channel regulation. Thus, the PKA-mediated regulation of L-type Ca2+ channels is critically dependent on a functional AKAP and phosphorylation of the alpha1C subunit at Ser1928.

Differential regulation of inotropy and lusitropy in overexpressed Gsalpha myocytes through cAMP and Ca2+ channel pathways.

We investigated the mechanisms responsible for altered contractile and relaxation function in overexpressed Gsalpha myocytes. Although baseline contractile function (percent contraction) in Gsalpha mice was similar to that of wild-type (WT) mice, left ventricular myocyte contraction, fura-2 Ca2+transients, and Ca2+ channel currents (ICa) were greater in Gsalpha mice in response to 10(-8) M isoproterenol (ISO) compared with WT mice. The late phase of relaxation of the isolated myocytes and fura-2 Ca2+ transients was accelerated at baseline in Gsalpha but did not increase further with ISO. In vivo measurements using echocardiography also demonstrated enhanced relaxation at baseline in Gsalpha mice. Forskolin and CaCl2 increased contraction similarly in WT and Gsalpha mice. Rp-cAMP, an inhibitor of protein kinase, blocked the increases in contractile response and Ca2+ currents to ISO in WT and to forskolin in both WT and Gsalpha. It also blocked the accelerated relaxation in Gsalpha at baseline but not the contractile response to ISO in Gsalpha myocytes. Baseline measurements of cAMP and phospholambation phosphorylation were enhanced in Gsalpha compared with WT. These data indicate that overexpression of Gsalpha accelerates relaxation at end diastolic but does not affect baseline systolic function in isolated myocytes. However, the enhanced responses to sympathetic stimulation partly reflect increased Ca2+ channel activity; i.e the cellular mechanisms mediating these effects appear to involve a cAMP-independent as well as a cAMP-dependent pathway.

Sarcoplasmic reticulum Ca(2+) atpase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca(2+) handling capacity.

Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

Cardiotrophic effects of protein kinase C epsilon: analysis by in vivo modulation of PKCepsilon translocation.

Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (epsilonV1) and translocation activator (psiepsilonRACK [receptors for activated C kinase]) specifically targeting PKCepsilon. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCepsilon signaling. As expected for a PKCepsilon RACK binding peptide, confocal microscopy showed that epsilonV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCepsilon by epsilonV1 at lower expression levels upregulated alpha-skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, epsilonV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCepsilon translocation with psiepsilonRACK resulted in selectively increased beta myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCepsilon signaling in normal postnatal maturational myocardial development and suggest the potential for PKCepsilon activators to stimulate "physiological" cardiomyocyte growth.

Increased myocardial Rab GTPase expression: a consequence and cause of cardiomyopathy.

The Ras-like Rab GTPases regulate vesicle transport in endocytosis and exocytosis. We found that cardiac Rabs1, 4, and 6 are upregulated in a dilated cardiomyopathy model overexpressing beta(2)-adrenergic receptors. To determine if increased Rab GTPase expression can contribute to cardiomyopathy, we transgenically overexpressed in mouse hearts prototypical Rab1a, the small G protein that regulates vesicle transport from endoplasmic reticulum to and through Golgi. In multiple independent mouse lines, Rab1a overexpression caused cardiac hypertrophy that progressed in a time- and transgene dose-dependent manner to heart failure. Isolated cardiac myocytes were hypertrophied and exhibited contractile depression with impaired calcium reuptake. Ultrastructural analysis revealed enlarged Golgi stacks and increased transitional vesicles in ventricular myocytes, with increased secretory atrial natriuretic peptide granules and degenerative myelin figures in atrial myocytes; immunogold studies localized Rab1a to these abnormal vesicular structures. A survey of hypertrophy signaling molecules revealed increased protein kinase C (PKC) alpha and delta, and confocal microscopy showed abnormal subcellular distribution of PKCalpha in Rab1a transgenics. These results indicate that increased expression of Rab1 GTPase in myocardium distorts subcellular localization of proteins and is sufficient to cause cardiac hypertrophy and failure.

A novel mechanism for myocardial stunning involving impaired Ca(2+) handling.

The mechanism of myocardial stunning has been studied extensively in rodents and is thought to involve a decrease in Ca(2+) responsiveness of the myofilaments, degradation of Troponin I (TnI), and no change in Ca(2+) handling. We studied the mechanism of stunning in isolated myocytes from chronically instrumented pigs. Myocytes were isolated from the ischemic (stunned) and nonischemic (normal) regions after 90-minute coronary stenosis followed by 60-minute reperfusion. Baseline myocyte contraction was reduced, P<0.01, in stunned myocytes (6.3+/-0.4%) compared with normal myocytes (8.8+/-0.4%). The time for 70% relaxation was prolonged, P<0.01, in stunned myocytes (131+/-8 ms) compared with normal myocytes (105+/-5 ms). The impaired contractile function was associated with decreased Ca(2+) transients (stunned, 0.33+/-0.04 versus normal, 0.49+/-0.05, P<0.01). Action potential measurements in stunned myocytes demonstrated a decrease in plateau potential without a change in resting membrane potential. These changes were associated with decreased L-type Ca(2+)-current density (stunned, -4.8+/-0.4 versus normal, -6.6+/-0.4 pA/pF, P<0.01). There were no differences in TnI, sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a), and phospholamban protein quantities. However, the fraction of phosphorylated phospholamban monomer was reduced in stunned myocardium. In rats, stunned myocytes demonstrated reduced systolic contraction but actually accelerated relaxation and no change in Ca(2+) transients. Thus, mechanisms of stunning in the pig are radically different from the widely held concepts derived from studies in rodents and involve impaired Ca(2+) handling and dephosphorylation of phospholamban, but not TnI degradation.

Early and delayed consequences of beta(2)-adrenergic receptor overexpression in mouse hearts: critical role for expression level.

BACKGROUND: Transgenic cardiac beta(2)-adrenergic receptor (AR) overexpression has resulted in enhanced signaling and cardiac function in mice, whereas relatively low levels of transgenically expressed G(alphas) or beta(1)AR have resulted in phenotypes of ventricular failure. Potential relationships between the levels of betaAR overexpression and biochemical, molecular, and physiological consequences have not been reported. METHODS AND RESULTS: We generated transgenic mice expressing beta(2)AR at 3690, 7120, 9670, and 23 300 fmol/mg in the heart, representing 60, 100, 150, and 350 times background betaAR expression. All lines showed enhanced basal adenylyl cyclase activation but a decrease in forskolin- and NaF-stimulated adenylyl cyclase activities. Mice of the highest-expressing line developed a rapidly progressive fibrotic dilated cardiomyopathy and died of heart failure at 25+/-1 weeks of age. The 60-fold line exhibited enhanced basal cardiac function without increased mortality when followed for 1 year, whereas 100-fold overexpressors developed a fibrotic cardiomyopathy and heart failure, with death occurring at 41+/-1 weeks of age. Adenylyl cyclase activation did not correlate with early or delayed decompensation. Propranolol administration reduced baseline +dP/dt(max) to nontransgenic levels in all beta(2)AR transgenics except the 350-fold overexpressors, indicating that spontaneous activation of beta(2)AR was present at this level of expression. CONCLUSIONS: These data demonstrate that the heart tolerates enhanced contractile function via 60-fold beta(2)AR overexpression without detriment for a period of >/=1 year and that higher levels of expression result in either aggressive or delayed cardiomyopathy. The consequences for enhanced betaAR function in the heart appear to be highly dependent on which signaling elements are increased and to what extent.

The nature and origin of spontaneous noise in G protein-gated ion channels.

Arrival of agonist is generally thought to initiate the signal transduction process in G protein-receptor coupled systems. However, the muscarinic atrial K+ (K+[ACh]) channel opens spontaneously in the absence of applied agonist, giving a noisy appearance to the current records. We investigated the nature and origin of the noise by measuring single channel currents in cell-attached or excised, inside-out membrane patches. Guanosine triphosphate (GTP) produced identical single channel currents in a concentration- and Mg(2+)-dependent manner in the presence or absence of carbachol, but the requirements for GTP were greater in the absence of agonist. Hence the agonist-independent currents appeared to be produced by an endogenous G protein, Gk. This prediction was confirmed when an affinity-purified, sequence-specific Gi-3 alpha antibody or pertussis toxin (PTX) blocked the agonist-independent currents. Candidate endogenous agonists were ruled out by the lack of effect of their corresponding antagonists. Thus agonist-independent currents had the same nature as agonist-dependent K+[ACh] currents and seemed to originate in the same way. We have developed a hypothesis in which agonist-free, empty receptors prime Gk with GTP and Gk activates atrial K+ [ACh] channels producing basal currents or noise. Agonist-independent activation by G proteins of effectors including ion channels appears to be a common occurrence.

The properties of batrachotoxin-modified cardiac Na channels, including state-dependent block by tetrodotoxin.

Batrachotoxin (BTX) modification and tetrodotoxin (TTX) block of BTX-modified Na channels were studied in single cardiac cells of neonatal rats using the whole-cell patch-clamp recording technique. The properties of BTX-modified Na channels in heart are qualitatively similar to those in nerve. However, quantitative differences do exist between the modified channels of these two tissues. In the heart, the shift of the conductance-voltage curve for the modified channel was less pronounced, the maximal activation rate constant, (tau m)max, of modified channels was considerably slower, and the slow inactivation of the BTX-modified cardiac Na channels was only partially abolished. TTX blocked BTX-modified mammalian cardiac Na channels and the block decreased over the potential range of -80 to -40 mV. The apparent dissociation constant of TTX changed from 0.23 microM at -50 mV to 0.69 microM at 0 mV. No further reduction of block was observed at potentials greater than -40 mV. This is the potential range over which gating from closed to open states occurred. These results were explained by assuming that TTX has a higher affinity for closed BTX-modified channels than for open modified channels. Hence, the TTX-binding rate constants are considered to be state dependent rather than voltage dependent. This differs from the voltage dependence of TTX block reported for BTX-modified Na channels from membrane vesicles incorporated into lipid bilayers and from amphibian node of Ranvier.

Endothelium-dependent vasodilating effect of substance P during flow-reducing coronary stenosis in the dog.

The effects of substance P, a putative central and peripheral neurotransmitter, on coronary vasculature and its mechanisms were studied in 31 anesthetized open chest dogs. Without coronary stenosis, intracoronary infusion of substance P (0.001 to 1 pmol/kg per min) for 40 s increased coronary blood flow up to 173 +/- 10.7% in dose-dependent fashion. Application of coronary stenosis created by an inflated intraluminal microballoon that preserved active vasomotion of the stenosed segment produced a pressure gradient of 34 +/- 2 mm Hg, a decrease in rest coronary blood flow of 21 +/- 1.6% and significant depression of the rate of rise in left ventricular pressure (dP/dt). During coronary stenosis, substance P increased coronary blood flow up to 150 +/- 9.4%, lowered mean distal coronary pressure and decreased stenosis resistance in dose-dependent fashion. After endothelial denudation of the proximal part of the coronary artery, the substance P-induced increments in coronary blood flow during coronary stenosis were abolished. In vitro measurements of isometric tension from both intact and denuded portions of coronary arteries confirmed a marked inhibition of substance P-induced relaxation in the denuded segments. These results show the obligatory role of the endothelium in substance P-induced coronary artery dilation. Furthermore, intracoronary infusion of substance P (1 pmol/kg per min) from the site distal to coronary stenosis that precluded the responsiveness of the large coronary artery decreased coronary blood flow by 24 +/- 4%, lowered mean distal coronary pressure by 15 +/- 1.9 mm Hg and intensified stenosis resistance by 77 +/- 7.2%. Thus, substance P exerts a direct potent dilating effect on both large and small coronary arteries. However, because of its strict endothelium-dependency, this peptide may play a detrimental role in the regulation of coronary blood flow when an atherosclerotic stenotic lesion with endothelial damage or dysfunction is present in the proximal part of the coronary artery.