Galpha(i2) but not Galpha(i3) is required for muscarinic inhibition of contractility and calcium currents in adult cardiomyocytes

Parasympathetic stimulation of the heart acts through M(2)-muscarinic acetylcholine receptors to regulate ion channel activity and subsequent inotropic status. Although muscarinic signal transduction is mediated via pertussis toxin-sensitive G proteins Galpha(i/o), the specific signal transduction requirements of Galpha(i2) and Galpha(i3) in mediating muscarinic regulated L-type calcium currents (I(Ca, L)), intracellular calcium, and cell contractility remain to be determined. Adult ventricular myocytes were isolated from Galpha(i2)-null mice, Galpha(i3)-null mice, and their wild-type littermates. Cell shortening, intracellular calcium levels, and I(Ca, L) were all measured in response to isoproterenol, a beta-adrenergic receptor agonist, and carbachol, a cholinergic receptor agonist. With isoproterenol stimulation, myocytes from all groups demonstrated a marked increase in calcium currents, correlating with augmented intracellular calcium transient amplitude and cell shortening. Carbachol significantly attenuated the isoproterenol response in wild-type and Galpha(i3)-null cells but had no effect in Galpha(i2)-null cells. This study demonstrates that Galpha(i2), but not Galpha(i3), is required for muscarinic inhibition of the beta-adrenergic response in adult murine ventricular myocytes.     

Galpha(i2), Galpha(i3)and Galpha(o) are all required for normal muscarinic inhibition of the cardiac calcium channels in nodal/atrial-like cultured cardiocytes.

The cardiac L-type calcium current (I(Ca,L)) is an important regulator of myocardial contractility. It is activated by sympathetic stimulation and inhibited by parasympathetic activity via muscarinic acetylcholine receptors. Muscarinic inhibition of I(Ca,L) occurs via activation of pertussis toxin (PTX)-sensitive heterotrimeric G-proteins. Although recent studies have shown that expression of G(oalpha) is important for this effect in adult mouse ventricular cells, two other PTX-sensitive G-proteins (G(i2) and G(i3)) are also expressed in cardiocytes and are activated. Their role in the regulation of I(Ca,L) has not been examined. In addition, it is not known whether nodal/atrial cardiac cells use the same G-proteins. We show that gene inactivation of each of the three PTX-sensitive Galpha-proteins (alpha(i2), alpha(i3), and alpha(o)) affects muscarinic inhibition of cardiac I(Ca,L) in embryonic stem (ES) cell-derived cardiocytes. Inactivation of either alpha(i2) or alpha(i3) markedly slows the time course of muscarinic inhibition of I(Ca,L), and in cells where both alpha(i2) and alpha(i3) are inactivated the effects are not additive. We also establish an essential role for alpha(o)in this atrial/nodal-like cardiocyte system and show that alpha(o)acts proximal to NO generation. NO generation plays a critical role in I(Ca,L) regulation since the nitric oxide synthase (NOS) antagonist, l -NMMA, blocked the inhibition of I(Ca,L) in WT and in alpha(i2)/alpha(i3)-null cells. In WT cells, the NO generating agent SIN-1 inhibited I(Ca,L) and the addition of carbachol resulted in faster inhibition, suggesting that pathways in addition to NO are also activated. This study shows that alpha(i2) and alpha(i3) play a critical role in the normal inhibition of cardiocyte I(Ca,L). Thus, all muscarinic receptor activated G-proteins (G(i2), G(i3) and G(o)) are necessary for normal inhibition and act through both NO and non-NO signaling pathways.     

Role of sterol regulatory element binding proteins in the regulation of Galpha(i2) expression in cultured atrial cells

We have previously demonstrated that growth of embryonic chick atrial cells in medium supplemented with lipoprotein-depleted serum (LPDS) resulted in a coordinate increase in the expression of genes involved in the parasympathetic response of the heart (the M2 muscarinic receptor; the alpha-subunit of the heterotrimeric G protein, Galpha(i2); and the inward rectifying K+ channel protein, GIRK1) and a marked increase in the negative chronotropic response of atrial cells to muscarinic stimulation. In the present study, we demonstrate that regulation of Galpha(i2) promoter activity by LPDS is mediated by the binding of a sterol regulatory element binding protein (SREBP) to a sterol regulatory element (SRE) in the Galpha(i2) promoter. Deletion and point mutation of this putative SRE interfered with the regulation of the Galpha(i2) promoter by SREBP and LPDS. Furthermore gel shift assays demonstrated that point mutations in the putative Galpha(i2) SRE markedly inhibited the binding of purified SREBP to oligonucleotides containing the Galpha(i2) SRE sequence. The expression of a dominant-negative SREBP mutant interfered with LPDS stimulation of Galpha(i2) promoter activity. Finally, we demonstrate that SREBP-1 is markedly more potent than SREBP-2 for the stimulation of Galpha(i2) promoter activity, suggesting that SREBP1 may play a role in the regulation of Galpha(i2) expression. These are the first data to demonstrate SREBP regulation of a protein not involved in lipid homeostasis and suggest a new relationship between lipid metabolism and the parasympathetic response of the heart.     

Potentiation of beta-adrenergic inotropic response by pyruvate in failing human myocardium.

BACKGROUND: Pyruvate has been shown to increase contractile function in isolated myocardium and to improve hemodynamics in patients with congestive heart failure. We tested the hypothesis that pyruvate potentiates the inotropic response to beta-adrenergic stimulation and to elevated extracellular calcium, since this may be of potential therapeutic value in the clinical setting of acute heart failure in order to circumvent deleterious effects on energy demand as can occur during catecholamine therapy. METHODS AND RESULTS: We investigated isometrically contracting isolated multicellular muscle preparations from terminal failing human hearts at 37 degrees C, pH 7.4, and a stimulation frequency of 1 Hz. At an extracellular calcium concentration of 1.25 mM, pyruvate (10 mM) alone increased developed force (F(dev)) from 9.0+/-2.3 to 21.1+/-4.3 mN/mm(2) (n=9, P<0.001) and isoproterenol (1 microM) alone increased F(dev) from 9.5+/-2.0 to 31.3+/-5.4 mN/mm(2) (P<0.001), whereas the combination of pyruvate and isoproterenol increased F(dev) over-proportionally from 9.0+/-2.3 to 47.4+/-6.4 mN/mm(2) (P<0.01). In a separate series we assessed the combination of pyruvate and calcium. Although F(dev) did not increase from 12 to 16 mM [Ca(2+)](o), 10 mM pyruvate further increased F(dev) from 25.8+/-5.0 to 30.6+/-4.7 mN/mm(2) (P<0.01). Rapid cooling contractures revealed that altered myofilament responsiveness and/or sarcoplasmic reticulum (SR) calcium load must underlie the positive inotropic effect of pyruvate. CONCLUSION: A combination of pyruvate and beta-adrenergic stimulation may be of therapeutic value in acute heart failure by reducing the concentrations of potential deleterious catecholamines that are currently necessary to maintain adequate tissue perfusion.     

Reduction of [Ca(2+)](i) restores uncoupled beta-adrenergic signaling in isolated perfused transgenic mouse hearts

The effects of alterations in calcium in the perfusion media were studied on ss-adrenergic coupling in isolated hearts from 3 different transgenic mice: cardiac-specific overexpressed alpha(1) subunit of L-type calcium channel, overexpressed Galpha(q), and phospholamban knockout. Isolated hearts from all 3 models, when studied at [Ca(2+)] of 2 mmol/L in the perfusate, showed the usual blunted or no response to ss-adrenergic stimulation. Lowering [Ca(2+)] to 0.75 to 1.5 mmol/L unloaded the hearts of calcium and restored to nearly normal the responsiveness to ss-agonist stimulation.     

Assessment of the beta-adrenergic receptor pathway in the intact failing human heart: progressive receptor down-regulation and subsensitivity to agonist response

We developed methods for identifying beta-adrenergic receptors in human right ventricular endomyocardial biopsy tissue with the radioligand (-)[125I]iodocyanopindolol (ICYP). Specific ICYP binding in a crude, high-yield membrane preparation derived from endomyocardial biopsy tissue was high (specificity greater than 90%), of high affinity (KD around 20 pM), saturable and stereospecific for the (-) vs the (+) isomer of isoproterenol. Subjects with mild-moderate and severe biventricular dysfunction had respective decreases in beta-adrenergic receptor density of 38.2% and 57.7% when normalization methods were averaged, with no significant differences in ICYP dissociation constant. A subgroup of subjects was subdivided by left ventricular ejection fraction (LVEF) into those with mild cardiac dysfunction (LVEF less than 0.50 greater than 0.40) and severe heart failure (LVEF less than 0.20) and given graded sequential infusions of dobutamine and calcium gluconate. Those with severe cardiac dysfunction had marked impairment of the dobutamine dP/dt and stroke work index response, whereas these responses to calcium did not differ in the two groups. These data indicate that in the intact human heart endomyocardial biopsy may be used for direct analysis of beta-adrenergic receptors, heart failure-associated myocardial beta-adrenergic down-regulation begins with mild-moderate ventricular dysfunction, reduction in myocardial beta-receptor density is related to degree of heart failure, and beta-receptor down-regulation is associated with pharmacologically specific impairment of the beta-agonist-mediated contractile response.     

Intracellular distribution of adrenoceptors in the failing human myocardium

Although it is increasingly recognized that the density of cardiac membrane-bound beta adrenoceptors declines in heart failure, the mechanisms involved are unclear. Furthermore, it is not known whether cardiac alpha-1 adrenoceptors are similarly affected. Inasmuch as agonist-induced desensitization results in translocation of adrenoceptors from the plasma membrane to an intracellular vesicular fraction, we determined the intracellular distribution of cardiac adrenoceptors in two groups: group 1 (n = 9) consisted of papillary muscles from patients with mild-to-moderate heart failure undergoing valve replacement, and group 2 (n = 8) consisted of severely failing hearts removed during orthotopic cardiac transplantation. The density of cardiac beta adrenoceptors was lower in membranes from group 2 (17.8 +/- 3.3 fmol/mg protein vs 27.8 +/- 3.7 fmol/mg in group 1; (p less than 0.01), and the percentage of beta receptors recovered in the vesicular fraction was higher in group 2 (47.1 +/- 3.3% vs 36.8 +/- 5.0% in group 1; p less than 0.01). In group 1 but not group 2 there was a significant inverse correlation (r = -0.87; p less than 0.001) between the density of membrane-bound beta receptors and the percentage of beta receptors recovered in the vesicular fraction. Alpha-1 adrenoceptors were lower in both membrane and vesicular fraction of group 2 compared to group 1; in group 2 but not group 1 there was a significant negative correlation between the density of membrane-bound alpha-1 adrenoceptors and the percentage of alpha-1 receptors in the vesicular fraction (r = -0.8; p less than 0.01). These results suggest that the regulation of alpha-1 and beta adrenoceptors differs in the failing myocardium. Furthermore, agonist-induced desensitization may play a predominant role only in mild-to-moderate heart failure.     

Delineation of the distribution of beta-adrenergic receptor subtypes in canine myocardium

beta 2-Receptors constitute only 10-30% of the total beta-adrenergic receptors in mammalian ventricular myocardium, but their precise tissue location cannot be determined easily by measuring physiological variables. To delineate the distribution of beta-receptor subtypes in myocytic and vascular components of the heart, we incubated transmural sections of canine left ventricle with [125Iodo]cyanopindolol and selected concentrations of the beta 1-selective antagonist betaxolol or the beta 2-selective antagonist ICI 118,551. Detailed competition binding data were best accounted for by a two-site model in which approximately 75% of total sites were beta 1- and 25% were beta 2-receptors. The relative proportions of beta-receptor subtypes in myocytic and vascular components were assessed autoradiographically by analyzing the density of binding sites in transmural sections incubated with radioligand and subtype-selective displacers. Betaxolol (10(-7) M) reduced the density of radioligand binding sites by 44% in regions composed primarily of ventricular myocytes but by less than 5% in small coronary arterioles. ICI 118,551 (10(-7) M) reduced radioligand binding-site density by 18% in myocytic regions and by 55% in small arterioles. In myocytic regions, these data indicated a subtype composition of approximately 85% beta 1- and 15% beta 2-sites. In contrast, arterioles contained almost exclusively the beta 2-subtype. The diameters of coronary vessels in which beta 2-receptors were found to be selectively increased fell within a narrow range (mean +/- SD, 35 +/- 11 microns; range, 16-55 microns). Small mural arteries and venules did not contain a significantly higher proportion of beta 2-receptors than adjacent myocytic regions.     

Autoimmune reactivity against the 20S-proteasome includes immunosubunits LMP2 (beta1i), MECL1 (beta2i) and LMP7 (beta5i)

Objectives. Autoantibodies against the 20S-proteasome displaya broad diversity with a remarkably low frequency of individualcross-reactivity against the different subunits of the proteasome.Although their pathogenic and diagnostic significance remainsobscure, an involvement in the clearance of circulating proteasomesas well as an interaction with the activity of the proteolyticcomplex was assumed in previous studies. Methods. To investigate the anti-proteasome response in moredetail and to disclose reactivities against former neglectedsubunits, two-dimensional electrophoresis followed by immunoblottingwas used. As a novel antigen source, the immunosubunits LMP2(β1i) and LMP7 (β5i) were expressed as recombinantproteins and employed in ELISA. Results. The subunits Iota (1) and Zeta (5) of the outer ringsas well as the catalytic subunit Delta (β1) and all threeimmunosubunits [MECL-1 (β2i), LMP2 (β1i) and LMP7(β5i)] of the inner rings of the proteasome were identifiedas autoantigens for the first time. Using a panel of anti-proteasomeantibody-positive sera of patients with SLE, autoimmune myositis(PM/DM) and primary Sjögren's syndrome (pSS), an autoimmuneresponse was documented against LMP2 (β1i) and LMP7 (β5i)in all three patient groups in ELISA. Conclusions. The frequent autoimmune response against LMP2 (β1i)and LMP7 (β5i) might indicate a role of inflammatory processesin the primary induction of the anti-proteasomal immune reaction,while the diversity of the humoral response against the proteasomesystem supports the assumption of a specific antigen-drivenprocess leading to these extended autoimmune reactivities. KEY WORDS: Proteasome, Immunosubunits, Autoantibodies, Autoimmunity Submitted 18 September 2007; revised version accepted 16 January 2008.     

Estrogen reduces serotonin-1A receptor-mediated oxytocin release and Galpha(i/o/z) proteins in the hypothalamus of ovariectomized rats

The present study examined the effect of estradiol on hypothalamic serotonin-1A (5-HT(1A)) receptor signaling in female rats. We first examined the time-course effects of a single injection of the 5-HT(1A) receptor agonist (+/-)8-OH-DPAT (5, 15 or 30 min prior to decapitation), and dose response of (+)8-OH-DPAT (50, 100, 200 or 500 microg/kg, s.c.) on plasma hormones in ovariectomized rats that received a daily injection of beta-estradiol 3-benzoate (10 microg/day, s.c.) or vehicle (sesame oil) for 2 days. In vehicle- and estrogen-treated rats, the peak response of hormones occurred at 15 min after injection and the time-course of oxytocin and adrenocorticotropic hormone (ACTH) responses to an injection of 8-OH-DPAT were comparable. However, only the oxytocin response was reduced by estrogen treatment. A second experiment compared the ACTH and oxytocin responses with doses of 50 or 200 microg/kg, s.c. of (+)8-OH-DPAT vs. (+/-)8-OH-DPAT in ovariectomized rats that were treated with oil or beta-estradiol 3-benzoate (10 microg/day, s.c.) for 2 days. (+)8-OH-DPAT and (+/-)8-OH-DPAT produced a similar magnitude of increase in plasma levels of ACTH and oxytocin. Treatment with beta-estradiol 3-benzoate produced a significant and comparable reduction in the oxytocin response to the highest dose (200 microg/kg, s.c.) of both (+)8-OH-DPAT and (+/-)8-OH-DPAT but did not alter the ACTH response to either (+)8-OH-DPAT or (+/-)8-OH-DPAT. In the dose-response experiment, a dose of 50 microg/kg of (+)8-OH-DPAT produced a maximal increase in plasma levels of ACTH, while the maximal oxytocin response was achieved with a dose of 200 microg/kg, s.c. Treatment with beta-estradiol 3-benzoate reduced the maximal oxytocin response to (+)8-OH-DPAT (by 29%) but did not alter the ACTH response to any doses of (+)8-OH-DPAT. To examine potential mechanisms mediating the effects of estrogen on 5-HT(1A) receptor signaling, we measured the levels of Galpha(i), Galpha(o) and Galpha(z) proteins, which couple 5-HT(1A) receptors to their effector enzymes, in two subregions of the hypothalamus. The levels of Galpha(z) protein were reduced in the mediobasal hypothalamus (containing the ventromedial and arcuate nuclei), which mainly expresses estrogen receptor-alpha, but not in the paraventricular hypothalamus, which mainly expresses estrogen receptor-beta. Estradiol reduced the levels of Galpha(i2) and Galpha(i3 )proteins in both hypothalamic regions but did not affect Galpha(i1) levels in either area. Combined, the data suggest that racemic and stereoselective 8-OH-DPAT have similar neuroendocrine effects and that both estrogen receptor-alpha and estrogen receptor-beta mediate the reduction in levels of Galpha(i2,3) proteins.     

Intracellular and extracellular targets of molecular imaging in the myocardium

Utilization of molecular imaging has significantly advanced the field of cardiovascular medicine. In addition to the targets currently in use, novel targets are being developed, including those involved in the processes of myocardial metabolism, myocardial injury, cardiac neurotransmission, and interstitial dysregulation. Further development of these imaging targets may lead to improved characterization of disease processes and guide provision of individualized therapies.     

Transgenic overexpression of insulin-like growth factor I prevents streptozotocin-induced cardiac contractile dysfunction and beta-adrenergic response in ventricular myocytes

Diabetic cardiomyopathy is characterized by cardiac dysfunction and altered level/function of insulin-like growth factor I (IGF-I). Both endogenous and exogenous IGF-I have been shown to effectively alleviate diabetes-induced cardiac dysfunction and oxidative stress. This study was designed to examine the effect of cardiac overexpression of IGF-I on streptozotocin (STZ)-induced cardiac contractile dysfunction in mouse myocytes. Both IGF-I heterozygous transgenic mice and their wild-type FVB littermates were made diabetic with a single injection of STZ (200 mg/kg, i.p.) and maintained for 2 weeks. The following mechanical indices were evaluated in ventricular myocytes: peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening (+/- dL/dt). Intracellular Ca2+ was evaluated as resting and peak intracellular Ca2+ levels, Ca2+-induced Ca2+ release and intracellular Ca2+ decay rate (tau). STZ led to hyperglycemia in FVB and IGF-I mice. STZ treatment prolonged TPS and TR90, reduced Ca2+-induced Ca2+ release, increased resting intracellular Ca2+ levels and slowed tau associated with normal PS and +/- dL/dt. All of which, except the elevated resting intracellular Ca2+, were prevented by the IGF-I transgene. In addition, myocytes from STZ-treated FVB mice displayed an attenuated contractile response to the beta-adrenergic agonist isoproterenol, which was restored by the IGF-I transgene. Contractile response to the alpha-adrenergic agonist phenylephrine and angiotensin II was not affected by either STZ treatment or IGF-I. These results validate the beneficial role of IGF-I in diabetic cardiomyopathy, possibly due to an improved beta-adrenergic response.     

Localization of a peripheral membrane protein: Gbetagamma targets Galpha(Z)

To explore the relative roles of protein-binding partners vs. lipid modifications in controlling membrane targeting of a typical peripheral membrane protein, Galpha(z), we directed its binding partner, betagamma, to mislocalize on mitochondria. Mislocalized betagamma directed wild-type Galpha(z) and a palmitate-lacking Galpha(z) mutant to mitochondria but did not alter localization of a Galpha(z) mutant lacking both myristate and palmitate. Thus, in this paradigm, a protein-protein interaction controls targeting of a peripheral membrane protein to the proper compartment, whereas lipid modifications stabilize interactions of proteins with membranes and with other proteins.     

Intracellular Ca2+ and delay of ischemia-induced electrical uncoupling in preconditioned rabbit ventricular myocardium

OBJECTIVE: Short periods of ischemia and reperfusion alter myocardial Ca2+ handling and temporarily induce a mild increase of [Ca2+]i. We hypothesized that these alterations are involved in the cardioprotective mechanism of ischemic preconditioning, possibly via a Ca(2+)-dependent activation of protein kinase C (PKC). METHODS AND RESULTS: In arterially perfused rabbit papillary muscles, we determined Ca2+ transients (indo 1) and indicators of the onset of irreversible ischemic damage, including [Ca2+]i rise, electrical uncoupling and contracture. We tested three protocols of ischemic preconditioning (1-3). In addition, the effects of infusion of staurosporine, a blocker of PKC (4), or glibenclamide, a blocker of K+ATP channels (5) were analyzed. Furthermore, pretreatment with phorbol 12-myrisate 13-acetate (PMA), an activator of PKC (6), or cyclopiazonic acid (CPA), an inhibitor of the SR Ca2+ pump (7) was tested. During periods of reperfusion in the preconditioning protocols, the duration of the Ca2+ transient and the diastolic Ca2+ level temporarily increased. Only if sustained ischemia was induced during these changes of the transients, cardioprotection was present. Similar alterations of the Ca2+ transient concurring with cardioprotection were induced by pretreatment with PMA as well as CPA. Staurosporine and glibenclamide antagonized the reperfusion-induced changes of the Ca2+ transients as well as cardioprotection. If reperfusion was extended until the Ca2+ transient had normalized, cardioprotection was also absent. Under all conditions tested, the diastolic Ca2+ elevation or the Ca2+ transient prolongation prior to sustained ischemia correlated with the postponement of ischemic injury. CONCLUSIONS: A pre-ischemic mild increase of [Ca2-]i presents a common effector of preconditioning. Our data suggest that activation of PKC or opening of K+ATP channels may initiate the pathway leading to an alteration of Ca2+ metabolism and a protected status of the myocardium.