RGS3L allows for an M2 muscarinic receptor-mediated RhoA-dependent inotropy in cardiomyocytes.

The role and outcome of the muscarinic M2 acetylcholine receptor (M2R) signaling in healthy and diseased cardiomyocytes is still a matter of debate. Here, we report that the long isoform of the regulator of G protein signaling 3 (RGS3L) functions as a switch in the muscarinic signaling, most likely of the M2R, in primary cardiomyocytes. High levels of RGS3L, as found in heart failure, redirect the Gi-mediated Rac1 activation into a Gi-mediated RhoA/ROCK activation. Functionally, this switch resulted in a reduced production of reactive oxygen species (- 50%) in cardiomyocytes and an inotropic response (+ 18%) in transduced engineered heart tissues. Importantly, we could show that an adeno-associated virus 9-mediated overexpression of RGS3L in rats in vivo, increased the contractility of ventricular strips by maximally about twofold. Mechanistically, we demonstrate that this switch is mediated by a complex formation of RGS3L with the GTPase-activating protein p190RhoGAP, which balances the activity of RhoA and Rac1 by altering its substrate preference in cardiomyocytes. Enhancement of this complex formation could open new possibilities in the regulation of the contractility of the diseased heart.

Constitutive inhibitory G protein activity upon adenylyl cyclase-dependent cardiac contractility is limited to adenylyl cyclase type 6.

PURPOSE: We previously reported that inhibitory G protein (Gi) exerts intrinsic receptor-independent inhibitory activity upon adenylyl cyclase (AC) that regulates contractile force in rat ventricle. The two major subtypes of AC in the heart are AC5 and AC6. The aim of this study was to determine if this intrinsic Gi inhibition regulating contractile force is AC subtype selective. METHODS: Wild-type (WT), AC5 knockout (AC5KO) and AC6 knockout (AC6KO) mice were injected with pertussis toxin (PTX) to inactivate Gi or saline (control).Three days after injection, we evaluated the effect of simultaneous inhibition of phosphodiesterases (PDE) 3 and 4 with cilostamide and rolipram respectively upon in vivo and ex vivo left ventricular (LV) contractile function. Also, changes in the level of cAMP were measured in left ventricular homogenates and at the membrane surface in cardiomyocytes obtained from the same mouse strains expressing the cAMP sensor pmEPAC1 using fluorescence resonance energy transfer (FRET). RESULTS: Simultaneous PDE3 and PDE4 inhibition increased in vivo and ex vivo rate of LV contractility only in PTX-treated WT and AC5KO mice but not in saline-treated controls. Likewise, Simultaneous PDE3 and PDE4 inhibition elevated total cAMP levels in PTX-treated WT and AC5KO mice compared to saline-treated controls. In contrast, simultaneous PDE3 and PDE4 inhibition did not increase in vivo or ex vivo rate of LV contractility or cAMP levels in PTX-treated AC6KO mice compared to saline-treated controls. Using FRET analysis, an increase of cAMP level was detected at the membrane of cardiomyocytes after simultaneous PDE3 and PDE4 inhibition in WT and AC5KO but not AC6KO. These FRET data are consistent with the functional data indicating that AC6 activity and PTX inhibition of Gi is necessary for simultaneous inhibition of PDE3 and PDE4 to elicit an increase in contractility. CONCLUSIONS: Together, these data suggest that AC6 is tightly regulated by intrinsic receptor-independent Gi activity, thus providing a mechanism for maintaining low basal cAMP levels in the functional compartment that regulates contractility.

Knockout of adenylyl cyclase isoform 5 or 6 differentially modifies the ß1-adrenoceptor-mediated inotropic response.

Although only ß2-adrenergic receptors (ßAR) dually couple with stimulatory G protein (Gs) and inhibitory G protein (Gi), inactivation of Gi enhances both ß1AR and ß2AR responsiveness. We hypothesize that Gi restrains spontaneous adenylyl cyclase (AC) activity independent of receptor activation. Subcellular localization of the AC5/6 subtypes varies contributing to the compartmentation of ßAR signaling. The primary objectives were to determine: (1) if ß1AR-mediated inotropic responses were dependent upon either AC5 or AC6; (2) if intrinsic Gi inhibition is AC subtype selective and (3) the role of phosphodiesterases (PDE) 3/4 to regulate ß1AR responsiveness. ß1AR-mediated increases in contractile force and cAMP accumulation in cardiomyocytes were measured from wild type, AC5 and AC6 knockout (KO) mice, with or without pertussis toxin (PTX) pretreatment to inactivate Gi and/or after selective inhibition of PDEs 3/4. Noradrenaline potency at ß1ARs was increased in AC6 KO. PDE4 inhibition increased noradrenaline potency in wild type and AC5 KO, but not AC6 KO. PTX increased noradrenaline potency only in wild type but increased the maximal ß1AR response in all mouse strains. PDE3 inhibition increased noradrenaline potency only in AC5 KO that was treated prior with PTX. ß1AR-evoked cAMP accumulation was increased more by PDE4 inhibition than PDE3 inhibition in wild type and AC5 KO that was amplified by Gi inhibition. These data indicate that ß1AR-mediated inotropic responses are not dependent upon either AC5 or AC6 alone. Inactivation of Gi enhanced ß1AR-mediated inotropic responses despite not coupling to Gi, consistent with Gi exerting a tonic receptor independent inhibition upon AC5/6. PDE4 seems the primary regulator of ß1AR signaling through AC6 in wild type. AC6 KO results in a reorganization of ß1AR compartmentation characterized by signaling through AC5 regulated by Gi, PDE3 and PDE4 that maintains normal contractile function.

Hypothermia elongates the contraction-relaxation cycle in explanted human failing heart decreasing the time for ventricular filling during diastole.

Targeted temperature management is part of the standardized treatment for patients in cardiac arrest. Hypothermia decreases cerebral oxygen consumption and induces bradycardia; thus, increasing the heart rate may be considered to maintain cardiac output. We hypothesized that increasing heart rate during hypothermia would impair diastolic function. Human left ventricular trabeculae obtained from explanted hearts of patients with terminal heart failure were stimulated at 0.5 Hz, and contraction-relaxation cycles were recorded. Maximal developed force (Fmax), maximal rate of development of force [(dF/d t)max], time to peak force (TPF), time to 80% relaxation (TR80), and relaxation time (RT = TR80 - TPF) were measured at 37, 33, 31, and 29°C. At these temperatures, stimulation frequency was increased from 0.5 to 1.0 and to 1.5 Hz. At 1.5 Hz, concentration-response curves for the ß-adrenergic receptor (ß-AR) agonist isoproterenol were performed. Fmax, TPF, and RT increased when temperature was lowered, whereas (dF/d t)max decreased. At all temperatures, increasing stimulation frequency increased Fmax and (dF/d t)max, whereas TPF and RT decreased. At 31 and 29°C, resting tension increased at 1.5 Hz, which was ameliorated by ß-AR stimulation. At all temperatures, maximal ß-AR stimulation increased Fmax, (dF/d t)max, and maximal systolic force, whereas resting tension decreased progressively with lowering temperature. ß-AR stimulation reduced TPF and RT to the same extent at all temperatures, despite the more elongated contraction-relaxation cycle at lower temperatures. Diastolic dysfunction during hypothermia results from an elongation of the contraction-relaxation cycle, which decreases the time for ventricular filling. Hypothermic bradycardia protects the heart from diastolic dysfunction and increasing the heart rate during hypothermia should be avoided. NEW & NOTEWORTHY Decreasing temperature increases the duration of the contraction-relaxation cycle in the human ventricular myocardium, significantly reducing the time for ventricular filling during diastole. During hypothermia, increasing heart rate further reduces the time for ventricular filling and in some situations increases resting tension further impairing diastolic function. Modest ß-adrenergic receptor stimulation can ameliorate these potentially detrimental changes during diastole while improving contractile force generation during targeted temperature management.

Related GPCRs couple differently to Gs: preassociation between G protein and 5-HT7 serotonin receptor reveals movement of Gαs upon receptor activation.

How GPCRs and G proteins interact is important for their biologic functions and their functions as pharmacologic targets. It is still an open question whether receptors and G proteins are preassembled in a complex or interact only after receptor activation. We compared the propensity of the two Gs-coupled serotonin (5-HT) receptors 5-HT4 and 5-HT7 to associate with G protein prior to agonist activation. Combining receptor-immobilized fluorescence recovery after photobleaching and fluorescence resonance energy transfer methodologies, we observed that 5-HT7 receptors markedly reduced the diffusion of both Gα and Gßγ at the cell surface, which indicated 5-HT7 receptor preassociation with Gs. This is in sharp contrast to the 5-HT4 receptor for which the diffusion of Gαßγ was not modified, and agonist activation brought together the receptor and Gγ, which is consistent with interaction by collision coupling. Agonist activation of 5-HT7 dissociated Gγ from the receptor, whereas Gαs underwent a rapid conformational change with respect to both Gγ and the receptor, followed by a slower dissociation of Gγ from both Gαs and the receptor. Taken together, these data demonstrate a different propensity among receptors to preassociate with G protein in the absence of ligand and reveals a rapid conformational change in Gαs upon activation by the receptor.-Andressen, K. W., Ulsund, A. H., Krobert, K. A., Lohse, M. J., Bünemann, M., Levy, F. O. Related GPCRs couple differently to Gs: preassociation between G protein and 5-HT7 serotonin receptor reveals movement of Gαs upon receptor activation.

Low ß2-adrenergic receptor level may promote development of castration resistant prostate cancer and altered steroid metabolism.

The underlying mechanisms responsible for the development of castration-resistant prostate cancer (CRPC) in patients who have undergone androgen deprivation therapy are not fully understood. This is the first study to address whether ß2-adrenergic receptor (ADRB2)- mediated signaling may affect CRPC progression in vivo. By immunohistochemical analyses, we observed that low levels of ADRB2 is associated with a more rapid development of CRPC in a Norwegian patient cohort. To elucidate mechanisms by which ADRB2 may affect CRPC development, we stably transfected LNCaP cells with shRNAs to mimic low and high expression of ADRB2. Two UDP-glucuronosyltransferases, UGT2B15 and UGT2B17, involved in phase II metabolism of androgens, were strongly downregulated in two LNCaP shADRB2 cell lines. The low-ADRB2 LNCaP cell lines displayed lowered glucuronidation activities towards androgens than high-ADRB2 cells. Furthermore, increased levels of testosterone and enhanced androgen responsiveness were observed in LNCaP cells expressing low level of ADRB2. Interestingly, these cells grew faster than high-ADRB2 LNCaP cells, and sustained their low glucuronidation activity in castrated NOD/SCID mice. ADRB2 immunohistochemical staining intensity correlated with UGT2B15 staining intensity in independent TMA studies and with UGT2B17 in one TMA study. Similar to ADRB2, we show that low levels of UGT2B15 are associated with a more rapid CRPC progression. We propose a novel mechanism by which ADRB2 may affect the development of CRPC through downregulation of UGT2B15 and UGT2B17.

Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis.

The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders. We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain (TMD) and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use.

The inotropic effect of the active metabolite of levosimendan, OR-1896, is mediated through inhibition of PDE3 in rat ventricular myocardium.

AIMS: We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca2+ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors. METHODS: Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors. RESULTS: OR-1896 evoked a maximum PIR of 33 ± 10% above basal at 1 µM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89 ± 14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5 ± 5.3%) or milrinone (3.2 ± 4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of ß-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca2+. Levosimendan, OR-1896 and EMD all increased the sensitivity to ß-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α1-AR response. CONCLUSION: The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca2+ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.

Non-classical regulation of ß1- and ß 2-adrenoceptor-mediated inotropic responses in rat heart ventricle by the G protein Gi.

Studies suggest that increased activity of Gi contributes to the reduced ß-adrenoceptor-mediated inotropic response (ßAR-IR) in failing cardiomyocytes and that ß2AR-IR but not ß1AR-IR is blunted by dual coupling to Gs and Gi. We aimed to clarify the role of Gi upon the ß1AR-IR and ß2AR-IR in Sham and failing myocardium by directly measuring contractile force and cAMP accumulation. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation in cardiomyocytes from rats with post-infarction heart failure (HF) or sham operates (Sham). The ß2AR-IR in Sham and HF was small and was amplified by simultaneously inhibiting phosphodiesterases 3 and 4 (PDE3&4). In HF, the inotropic response and cAMP accumulation evoked by ß1AR- or ß2AR-stimulation were reduced. Inactivation of Gi with pertussis toxin (PTX) did not restore the ß1AR-IR or ß2AR-IR in HF to Sham levels but did enhance the maximal ß2AR-IR. PTX increased both ß1AR- and ß2AR-evoked cAMP accumulation more in Sham than that in HF, and HF levels approached those in untreated Sham. The potency of agonists at ß1 and at ß2ARs (only under PDE3&4 inhibition) was increased in HF and by PTX in both HF and Sham. Without PDE3&4 inhibition, PTX increased only the maximal ß2AR-IR, not potency. We conclude that Gi regulates both ß1AR- and ß2AR-IR independent of receptor coupling with Gi. Gi together with PDE3&4 tonically restrict the ß2AR-IR. Gi inhibition did not restore the ßAR-IR in HF despite increasing cAMP levels, suggesting that the mechanism of impairment resides downstream to cAMP signalling.

Gi proteins regulate adenylyl cyclase activity independent of receptor activation.

BACKGROUND AND PURPOSE: Despite the view that only ß2- as opposed to ß1-adrenoceptors (ßARs) couple to G(i), some data indicate that the ß1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. EXPERIMENTAL APPROACH: We used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate ß2ARs (ß1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. KEY RESULTS: PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. CONCLUSIONS AND IMPLICATIONS: Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

NSC23766, a widely used inhibitor of Rac1 activation, additionally acts as a competitive antagonist at muscarinic acetylcholine receptors.

Small molecules interfering with Rac1 activation are considered as potential drugs and are already studied in animal models. A widely used inhibitor without reported attenuation of RhoA activity is NSC23766 [(N(6)-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride]. We found that NSC23766 inhibits the M2 muscarinic acetylcholine receptor (M2 mAChR)-induced Rac1 activation in neonatal rat cardiac myocytes. Surprisingly, NSC27366 concomitantly suppressed the carbachol-induced RhoA activation and a M2 mAChR-induced inotropic response in isolated neonatal rat hearts requiring the activation of Rho-dependent kinases. We therefore aimed to identify the mechanisms by which NSC23766 interferes with the differentially mediated, M2 mAChR-induced responses. Interestingly, NSC23766 caused a rightward shift of the carbachol concentration response curve for the positive inotropic response without modifying carbachol efficacy. To analyze the specificity of NSC23766, we compared the carbachol and the similarly Gißγ-mediated, adenosine-induced activation of Gi protein-regulated potassium channel (GIRK) channels in human atrial myocytes. Application of NSC23766 blocked the carbachol-induced K(+) current but had no effect on the adenosine-induced GIRK current. Similarly, an adenosine A1 receptor-induced positive inotropic response in neonatal rat hearts was not attenuated by NSC23766. To investigate its specificity toward the different mAChR types, we studied the carbachol-induced elevation of intracellular Ca(2+) concentrations in human embryonic kidney 293 (HEK-293) cells expressing M1, M2, or M3 mAChRs. NSC23766 caused a concentration-dependent rightward shift of the carbachol concentration response curves at all mAChRs. Thus, NSC23766 is not only an inhibitor of Rac1 activation, but it is within the same concentration range a competitive antagonist at mAChRs. Molecular docking analysis at M2 and M3 mAChR crystal structures confirmed this interpretation.

Connective tissue growth factor/CCN2 attenuates ß-adrenergic receptor responsiveness and cardiotoxicity by induction of G protein-coupled receptor kinase-5 in cardiomyocytes.

Myocardial connective tissue growth factor (CTGF/CCN2) is induced in heart failure, a condition associated with diminution of ß-adrenergic receptor (ß-AR) responsiveness. Accordingly, we aimed to investigate whether CTGF could play a mechanistic role in regulation of ß-AR responsiveness. Concentration-response curves of isoproterenol-stimulated cAMP generation in cardiomyocytes from transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) or cardiomyocytes pretreated with recombinant human CTGF (rec-hCTGF) revealed marked reduction of both ß1-AR and ß2-AR responsiveness. Consistently, ventricular muscle strips from Tg-CTGF mice stimulated with isoproterenol displayed attenuation of maximal inotropic responses. However, no differences of maximal inotropic responses of myocardial fibers from Tg-CTGF mice and nontransgenic littermate control (NLC) mice were discerned when stimulated with supramaximal concentrations of dibutyryl-cAMP, indicating preserved downstream responsiveness to cAMP. Congruent with a mechanism of desensitization of ß-ARs, mRNA and protein levels of G protein-coupled receptor kinase 5 (GRK5) were found isoform-selective upregulated in both cardiomyocytes from Tg-CTGF mice and cardiomyocytes exposed to rec-hCTGF. Corroborating a mechanism of GRK5 in CTGF-mediated control of ß-AR sensitivity, Chinese hamster ovary cells pretreated with rec-hCTGF displayed increased agonist- and biased ligand-stimulated ß-arrestin binding to ß-ARs. Despite increased sensitivity of cardiomyocytes from GRK5-knockout (KO) mice to ß-adrenergic agonists, pretreatment of GRK5-KO cardiomyocytes with rec-hCTGF, as opposed to cardiomyocytes from wild-type mice, did not alter ß-AR responsiveness. Finally, Tg-CTGF mice subjected to chronic exposure (14 days) to isoproterenol revealed blunted myocardial hypertrophy and preserved cardiac function versus NLC mice. In conclusion, this study uncovers a novel mechanism controlling ß-AR responsiveness in cardiomyocytes involving CTGF-mediated regulation of GRK5.

PDE3, but not PDE4, reduces ß1 - and ß2-adrenoceptor-mediated inotropic and lusitropic effects in failing ventricle from metoprolol-treated patients.

BACKGROUND AND PURPOSE: PDE3 and/or PDE4 control ventricular effects of catecholamines in several species but their relative effects in failing human ventricle are unknown. We investigated whether the PDE3-selective inhibitor cilostamide (0.3-1 µM) or PDE4 inhibitor rolipram (1-10 µM) modified the positive inotropic and lusitropic effects of catecholamines in human failing myocardium. EXPERIMENTAL APPROACH: Right and left ventricular trabeculae from freshly explanted hearts of 5 non-ß-blocker-treated and 15 metoprolol-treated patients with terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline, mediated through ß1 adrenoceptors (ß2 adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through ß2 adrenoceptors (ß1 adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of PDE inhibitors. Catecholamine potencies were estimated from -logEC50s. KEY RESULTS: Cilostamide did not significantly potentiate the inotropic effects of the catecholamines in non-ß-blocker-treated patients. Cilostamide caused greater potentiation (P = 0.037) of the positive inotropic effects of (-)-adrenaline (0.78 ± 0.12 log units) than (-)-noradrenaline (0.47 ± 0.12 log units) in metoprolol-treated patients. Lusitropic effects of the catecholamines were also potentiated by cilostamide. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline on right and left ventricular trabeculae from metoprolol-treated patients. CONCLUSIONS AND IMPLICATIONS: Metoprolol induces a control by PDE3 of ventricular effects mediated through both ß1 and ß2 adrenoceptors, thereby further reducing sympathetic cardiostimulation in patients with terminal heart failure. Concurrent therapy with a PDE3 blocker and metoprolol could conceivably facilitate cardiostimulation evoked by adrenaline through ß2 adrenoceptors. PDE4 does not appear to reduce inotropic and lusitropic effects of catecholamines in failing human ventricle.

Prostaglandin E1 facilitates inotropic effects of 5-HT4 serotonin receptors and ß-adrenoceptors in failing human heart.

Prostaglandins have displayed both beneficial and detrimental effects in clinical studies in patients with severe heart failure. Prostaglandins are known to increase cardiac output, but the mechanism is not clarified. Here, we tested the hypothesis that prostaglandins can increase contractility in human heart by amplifying cAMP-dependent inotropic responses. Contractility was measured ex vivo in isolated left ventricular strips and phosphodiesterase (PDE) and adenylyl cyclase (AC) activity was measured in homogenates or membranes from failing human left ventricles. PGE(1) (1 µM) alone did not modify contractility, but given prior, amplified maximal serotonin (5-HT)-evoked (10 µM) contractile responses mediated by 5-HT(4) receptors several fold (24 ± 7 % with PGE(1) vs. 3 ± 2 % above basal with 5-HT alone). The 5-HT(4)-mediated inotropic response was amplified by the PDE3 inhibitor cilostamide and further amplified in combination with PGE(1) (26 ± 6 vs. 56 ± 12 % above basal). PGE(1) reduced the time to reach 90 % of both the maximal 5-HT- and isoproterenol-evoked inotropic response compared to 5-HT or isoproterenol alone. PGE(1) did not modify PDE activity in the homogenate, either alone or when given simultaneously with PDE3 and/or PDE4 inhibitors. Neither 5-HT- nor isoproterenol-stimulated AC activity was significantly amplified by PGE(1). Sensitivity of ventricular strips to Ca(2+) was not enhanced in the presence of PGE(1). Our results show that PGE(1) can enhance cAMP-mediated responses in failing human left ventricle, through a mechanism independent of PDE inhibition, amplification of AC activity or increasing sensitivity to calcium. This effect of PGE(1) possibly contributes to the increase of cardiac output, independent of decreased afterload, observed after prostaglandin administration in humans.

Cardiomyocyte-restricted inhibition of G protein-coupled receptor kinase-3 attenuates cardiac dysfunction after chronic pressure overload.

Transgenic mice with cardiac-specific expression of a peptide inhibitor of G protein-coupled receptor kinase (GRK)3 [transgenic COOH-terminal GRK3 (GRK3ct) mice] display myocardial hypercontractility without hypertrophy and enhanced α(1)-adrenergic receptor signaling. A role for GRK3 in the pathogenesis of heart failure (HF) has not been investigated, but inhibition of its isozyme, GRK2, has been beneficial in several HF models. Here, we tested whether inhibition of GRK3 modulated evolving cardiac hypertrophy and dysfunction after pressure overload. Weight-matched male GRK3ct transgenic and nontransgenic littermate control (NLC) mice subjected to chronic pressure overload by abdominal aortic banding (AB) were compared with sham-operated (SH) mice. At 6 wk after AB, a significant increase of cardiac mass consistent with induction of hypertrophy was found, but no differences between GRK3ct-AB and NLC-AB mice were discerned. Simultaneous left ventricular (LV) pressure-volume analysis of electrically paced, ex vivo perfused working hearts revealed substantially reduced systolic and diastolic function in NLC-AB mice (n = 7), which was completely preserved in GRK3ct-AB mice (n = 7). An additional cohort was subjected to in vivo cardiac catheterization and LV pressure-volume analysis at 12 wk after AB. NLC-AB mice (n = 11) displayed elevated end-diastolic pressure (8.5 ± 3.1 vs. 2.9 ± 1.2 mmHg, P < 0.05), reduced cardiac output (3,448 ± 323 vs. 4,488 ± 342 µl/min, P < 0.05), and reduced dP/dt(max) and dP/dt(min) (both P < 0.05) compared with GRK3ct-AB mice (n = 16), corroborating the preserved cardiac structure and function observed in GRK3ct-AB hearts assessed ex vivo. Increased cardiac mass and myocardial mRNA expression of ß-myosin heavy chain confirmed the similar induction of cardiac hypertrophy in both AB groups, but only NLC-AB hearts displayed significantly elevated mRNA levels of brain natriuretic peptide and myocardial collagen contents as well as reduced ß(1)-adrenergic receptor responsiveness to isoproterenol, indicating increased LV wall stress and the transition to HF. Inhibition of cardiac GRK3 in mice does not alter the hypertrophic response but attenuates cardiac dysfunction and HF after chronic pressure overload.

Prostanoid-mediated inotropic responses are attenuated in failing human and rat ventricular myocardium.

Prostanoid-modulatory approaches in heart failure patients have displayed effects which may seem to be mutually incompatible. Both treatment with prostanoids and inhibition of prostanoid synthesis have resulted in increased mortality in heart failure patients. Currently, it is unknown if prostanoids mediate contractile effects in failing human heart and if this can explain some of the clinical effects seen after prostanoid modulatory treatments. Therefore, the objectives of this study were to determine if prostanoids could elicit direct inotropic responses in human ventricle, and if so to determine if they are modified in failing ventricle. Contractile force was measured in left ventricular strips from non-failing or failing human and rat hearts. The ratio of phosphorylated to non-phosphorylated myosin light chain 2 (MLC-2) was measured by Western blotting in myocardial strips, and the levels of prostanoid FP receptor mRNA and protein were measured in rat by real-time RT-PCR and receptor binding assays. In non-failing human hearts, prostanoids evoked a positive inotropic effect and an increase of MLC-2 phosphorylation which was absent in failing human hearts. In failing rat heart, the prostanoid FP receptor-mediated inotropic response and prostanoid FP receptor-density was reduced by ~40-50% compared to non-failing rat heart. Prostanoids mediate a sustained positive inotropic response in non-failing heart, which appears to be down regulated in failing heart. The pathophysiological significance of changes in prostanoid-mediated inotropic support in the failing heart remains to be determined.

Synthesis and pharmacological properties of novel hydrophilic 5-HT4 receptor antagonists.

Serotonin (5-hydroxytryptamine, 5-HT) is an important signalling molecule in the human body. The 5-HT(4) serotonin receptor, coupled to the G protein G(s), plays important physiological and pathophysiological roles in the heart, urinary bladder, gastrointestinal tract and the adrenal gland. Both 5-HT(4) antagonists and agonists have been developed in the aim to treat diseases in these organs. 5-HT(4) agonists might have beneficial effects in the central nervous system (CNS) and therefore, 5-HT(4) antagonists might cause CNS side effects. In this study, we have developed new amphoteric 5-HT(4) antagonists. A series of cyclic indole amide derivatives possessing an oxazine ring and a piperidine alkane carboxylic acid side chain and the corresponding prodrug esters were synthesized and their binding to 5-HT(4) receptors and antagonist properties were evaluated. In addition, an indole ester without the oxazine ring and the corresponding indole amide derivatives were also tested. Octanol-water distribution (LogD(Oct7.4)) was tested for some of the synthesized ligands. The main structure-affinity characteristics of the 5-HT(4) compounds tested were that the prodrug esters show higher affinity than their corresponding free acids, indole esters show higher affinity than the corresponding amides and ligands containing the oxazine ring in the indole skeleton show higher affinity than indole derivatives not containing the ring. One representative prodrug ester and its corresponding free acid were tested for binding on a panel of receptors and showed preserved selectivity for the 5-HT(4) receptor. These new molecules may be useful to target peripheral 5-HT(4) receptors.

Prostanoid F receptors elicit an inotropic effect in rat left ventricle by enhancing myosin light chain phosphorylation.

AIMS: The aims of this study were to determine if the prostanoid F receptor (FPR)-mediated inotropic effect in rat ventricle is mediated by increased phosphorylation of myosin light chain-2 (MLC-2) and to elucidate the signalling pathway(s) activated by FPRs to regulate MLC-2 phosphorylation. METHODS AND RESULTS: Contractility was measured in left ventricular strips from adult male rats. Strips were also snap-frozen, and changes in the phosphorylation level of both MLC-2 and myosin phosphatase targeting subunit-2 (MYPT-2) were quantified. FPR stimulation with fluprostenol increased contractility by approximately 100% above basal and increased phosphorylation of both MLC-2 (by approximately 30%) and MYPT-2 (by approximately 50%). The FPR-mediated inotropic effect and MLC-2 phosphorylation were reduced by a similar magnitude in the presence of the myosin light chain kinase (MLCK) inhibitor ML-7 (approximately 60-70%) and an inhibitor of Ca(2+)/calmodulin, W-7 (approximately 35%). Inhibition of Rho-associated kinase by Y-27632 reduced the FPR-mediated inotropic effect and MLC-2 phosphorylation by approximately 40-45% and MYPT-2 phosphorylation by approximately 70%. ML-7 and Y-27632 together reduced contractility and MLC-2 phosphorylation by approximately 70-80%. The FPR-mediated inotropic effect was only modestly affected by high concentrations of the inositol tris-phosphate (IP(3)) receptor blocker 2-APB, but not by the protein kinase C (PKC) inhibitor bisindolylmaleimide. CONCLUSION: The FPR-evoked inotropic effect is mediated by increasing the phosphorylation of MLC-2 through regulation of both MLCK and myosin light chain phosphatase activities. The second messenger IP(3) and PKC are unlikely to be involved in the signalling cascade of the FPR-mediated positive inotropic effect. Therefore, FPR signalling mechanism(s) regulating MLC-2 phosphorylation likely extend beyond those classically established for G(q/11)-coupled receptors.

Effects of serotonin in failing cardiac ventricle: signalling mechanisms and potential therapeutic implications.

Previously, cardioexcitation by serotonin (5-hydroxytryptamine, 5-HT) was believed to be confined to atria in mammals including man, and mediated through 5-HT(4) receptors in pig and man, but 5-HT(2A) receptors in rat. Recent studies, reviewed here, demonstrate that functional 5-HT(4) receptors can be revealed in porcine and human ventricular myocardium during phosphodiesterase inhibition, and that 5-HT(4) receptor mRNA is increased in human heart failure. In rats, functional 5-HT(4) and 5-HT(2A) receptors appear in the cardiac ventricle during heart failure and mediate inotropic responses through different mechanisms. 5-HT(2A) receptor signalling resembles that from alpha(1)-adrenoceptors and causes inotropic effects through increased myosin light chain phosphorylation, resulting in Ca(2+) sensitisation. 5-HT(4) receptor signalling resembles that from beta-adrenoceptors and causes inotropic effects through a pathway involving cAMP and PKA-mediated phosphorylation of proteins involved in Ca(2+) handling, resulting in enhanced contractility through increased Ca(2+) availability. Cyclic AMP generated through 5-HT(4) receptor stimulation seems more efficiently coupled to increased contractility than cAMP generated through beta-adrenoceptor stimulation. Increasing contractility through cAMP is considered less energy efficient than Ca(2+) sensitisation and this may be one reason why beta-adrenoceptor antagonism is beneficial in heart failure patients. Treatment of heart failure rats with the 5-HT(4) antagonist SB207266 (piboserod) resulted in potentially beneficial effects, although small. Further studies are needed to clarify if such treatment will be useful for patients with heart failure.

Diastolic dysfunction in alveolar hypoxia: a role for interleukin-18-mediated increase in protein phosphatase 2A.

AIMS: Chronic obstructive pulmonary disease with alveolar hypoxia is associated with diastolic dysfunction in the right and left ventricle (LV). LV diastolic dysfunction is not caused by increased afterload, and we recently showed that reduced phosphorylation of phospholamban at serine (Ser) 16 may explain the reduced relaxation of the myocardium. Here, we study the mechanisms leading to the hypoxia-induced reduction in phosphorylation of phospholamban at Ser16. METHODS AND RESULTS: In C57Bl/6j mice exposed to 10% oxygen, signalling molecules were measured in cardiac tissue, sarcoplasmic reticulum (SR)-enriched membrane preparations, and serum. Cardiomyocytes isolated from neonatal mice were exposed to interleukin (IL)-18 for 24 h. The beta-adrenergic pathway in the myocardium was not altered by alveolar hypoxia, as assessed by measurements of beta-adrenergic receptor levels, adenylyl cyclase activity, and subunits of cyclic AMP-dependent protein kinase. However, alveolar hypoxia led to a significantly higher amount (124%) and activity (234%) of protein phosphatase (PP) 2A in SR-enriched membrane preparations from LV compared with control. Serum levels of an array of cytokines were assayed, and a pronounced increase in IL-18 was observed. In isolated cardiomyocytes, treatment with IL-18 increased the amount and activity of PP2A, and reduced phosphorylation of phospholamban at Ser16 to 54% of control. CONCLUSION: Our results indicate that the diastolic dysfunction observed in alveolar hypoxia might be caused by increased circulating IL-18, thereby inducing an increase in PP2A and a reduction in phosphorylation of phospholamban at Ser16.