Phosphoinositide 3-kinase γ Regulates Cardiac Contractility by Locally Controlling Cyclic Adenosine Monophosphate Levels

doi:10.1016/j.tcm.2006.04.006

Trends in Cardiovascular Medicine

Volume 16, Issue 7, October 2006, Pages 250-256

https://doi.org/10.1016/j.tcm.2006.04.006 Get rights and content

Class I phosphoinositide 3-kinases (PI3Ks) are enzymes with both protein and lipid kinase activities that regulate important cellular functions in many tissues. In the heart, subclass IA PI3Ks (mainly PI3Kα) regulate cell growth, apoptosis, cell division and cell size, whereas PI3Kγ, the only member of subclass IB, has been shown to regulate cardiac contractility. We have shown that the loss of PI3Kγ (PI3Kγ^−/− mice) enhances cardiac excitation–contraction coupling by modulating cyclic adenosine monophosphate (cAMP) levels in subcellular domains containing the sarcoplasmic reticulum. Specifically, PI3Kγ^−/− mice show enhanced sarcoplasmic reticulum Ca²⁺ cycling in association with increased cAMP. Surprisingly, L-type Ca²⁺ current, a prototypic target of cAMP-dependent protein kinase A phosphorylation, is largely unchanged in PI3Kγ^−/− mice. In this article, we discuss the consequences and implications of cAMP compartmentation in cardiomyocytes. We also review the different roles of PI3Kγ in the heart, particularly as they relate to cardiac contractility, intracellular cAMP levels, and the regulation of β-adrenergic receptor signaling in physiologic and pathologic states.

Section snippets

cAMP Compartmentation in the Heart

In addition to the β-ARs, there are several other hormones and compounds that bind to G-protein-coupled receptors and increase cAMP; however, not all of these hormones produce the inotropic and lusitropic responses seen with β-AR stimulation in the heart. For example, both glucagon-like peptide-1 and prostaglandin E₁ increase cAMP to a level comparable with that elicited by the β-AR agonist isoproterenol (ISO), but neither of these hormones elicit the contractile responses observed with ISO (

PI3Kγ and cAMP Compartmentation in Ventricular Cardiomyocytes

Two separate mouse strains with targeted deletion of the PI3Kγ gene (PI3Kγ^−/−) have been created (Hirsch et al. 2000, Sasaki et al. 2000). These transgenic mice are viable and fertile, with normal heart rates and normal mean arterial pressures (Crackower et al. 2002). PI3Kγ^−/− mice show significantly enhanced cardiac function and increased cardiomyocyte contractility in association with elevated basal intracellular cAMP levels (Crackower et al. 2002, Nienaber et al. 2003, Patrucco et al. 2004,

Implications and Possible Mechanism(s) of PI3Kγ Regulation of cAMP

Intracellular cAMP levels are highly regulated in heart and other tissues by the β-AR signaling pathways (Bers 2002). Previous studies have indicated that PI3K activity is enhanced by β₁-ARs (Leblais et al. 2004) or β₂-ARs (Chesley et al. 2000, Zhu et al. 2001, Jo et al. 2002) (or both), as well as by AC activation (Leblais et al. 2004). Although it appears that PI3Kγ is not able to directly hydrolyze cAMP (Patrucco et al. 2004), genetic ablation of PI3Kγ clearly increases cAMP levels in

Perspectives and Significance

A prominent feature of diseased myocardium is reduced Ca²⁺ transient amplitude resulting from decreased SR Ca²⁺ uptake without changes in I_Ca,L density (Gwathmey et al. 1987, Beuckelmann et al. 1992, Gomez et al. 1997, Benitah et al. 2002, Bers et al. 2003). These observations suggest that alterations in the regulation of cAMP by PI3Kγ may contribute to the functional changes observed in heart disease. Consistent with this idea, PI3Kγ activity and expression is increased in cardiac disease (

Acknowledgments

This study was supported by funding from the Canadian Institutes of Health Research (CIHR) to PHB, who is a Career Investigator with the Heart and Stroke Foundation (HSF) of Ontario. B-GK holds a postdoctoral fellowship from the HSF of Canada and the TACTICS-CIHR program at the University of Toronto. RAR holds postdoctoral fellowships from the HSF of Canada and the Alberta Heritage Foundation for Medical Research.

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Recent reports, however, suggest that PI3Kβ as well might be GPCR-controlled [36]. Most of the interest in PI3Kγ and PI3Kβ concerns their role in inflammation as they are involved in chemokine receptor-induced recruitment of inflammatory cells [36], and in the cardiovascular system, being implicated in the regulation of cardiac contractility [37,38]. In this regard, it has been proposed that cardiac PI3Kγ negatively regulates cAMP levels, as a part of a macromolecular complex including β2AR, Gi, PKA, and PDEs [38].
The acylated peptide ghrelin (AG) and its endogenous non-acylated isoform (UAG) protect cardiomyocytes, pancreatic β-cells, and preadipocytes from apoptosis, and induce preadipocytes differentiation into adipocytes. These events are mediated by AG and UAG binding to a still unidentified receptor, which determines the activation of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) ERK1/2. AG and UAG also possess antilipolytic activity in vitro, but the underlying mechanism remains unknown. Thus, the objective of the current study was to characterize the molecular events involved in AG/UAG receptor signaling cascade. We treated rat primary visceral adipocytes with isoproterenol (ISO) and forskolin (FSK) to stimulate lipolysis, simultaneously incubating them with or without AG or UAG. Both peptides blocked ISO- and FSK-induced lipolysis. By direct measurement of cAMP intracellular content, we demonstrated that AG/UAG effect was associated to a reduction of ISO-induced cAMP accumulation. Moreover, the cAMP analog 8Br-cAMP abolished AG/UAG effect. As AG and UAG were ineffective against lipolysis induced by db-cAMP, another poorly hydrolyzable cAMP analog, phosphodiesterase (PDE) involvement was hypothesized. Indeed, cilostamide, a specific PDE3B inhibitor, blocked AG/UAG effect on ISO-induced lipolysis. Furthermore, the PI3K inhibitor wortmannin and AKT inhibitor 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4piperidinyl)-2H-benzimidazol-2-one trifluoroacetate also blocked AG/UAG action, suggesting a role in PDE3B activation. In particular, PI3K isoenzyme gamma (PI3Kγ) selective inhibition through the compound AS605240 prevented AG/UAG effect on ISO-stimulated lipolysis, hampering AKT phosphorylation on Ser⁴⁷³. Taken together, these data demonstrate for the first time that AG/UAG attenuation of ISO-induced lipolysis involves PI3Kγ/AKT and PDE3B.
Role of prostanoid IP and EP receptors in mediating vasorelaxant responses to PGI<inf>2</inf> analogues in rat tail artery: Evidence for G<inf>i/o</inf> modulation via EP<inf>3</inf> receptors
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Secondly, Gi/o could activate PI3 kinase via its βγ subunit (Zhang et al., 1995) which in turn could activate L-type Ca2+ channels (Viard et al., 1999) leading to an increase in intracellular Ca2+. PI3 kinase can also activate phosphodiesterases 3 and 4 (PDE3 and PDE4) leading to enhanced cAMP degradation (Kerfant et al., 2006; Alfranca et al., 2006) which could further limit relaxation. In line with this and similar to PTX, the PI3 kinase inhibitor LY-294002 enhanced the relaxation to these analogues, though to a lesser extent than PTX.
Prostanoid IP receptors coupled to Gs are thought to be the primary target for prostacyclin (PGI₂) analogues. However, these agents also activate prostanoid EP_1–4 receptor subtypes to varying degrees, which are positively (EP_2/4) or negatively (EP₃) coupled to adenylate cyclase through Gs or Gi, respectively. We investigated the role of these receptors in modulating relaxation to PGI₂ analogues cicaprost, iloprost and treprostinil in pre-contracted segments of rat tail artery. Prostanoid IP (RO1138452), EP₄ (GW627368X), EP₃ (L-798106), EP_1–3 (AH6809), and EP₁ (SC-51322) receptor antagonists were used to determine each receptor contribution. The role of G_i/o was investigated using pertussis toxin (PTX), while dependence on cAMP was determined using adenylate cyclase (2′5′dideoxyadenosine, DDA) and protein kinase A (2′-O-monobutyryladenosine- 3′,5′-cyclic monophosphorothioate, Rp- isomer, Rp-2′-O-MB-cAMPS) inhibitors, and by measurement of tissue cAMP. All analogues caused relaxation which was significantly (P < 0.01) inhibited by RO1138452; with maximum response to cicaprost, iloprost and treprostinil reduced by 51%, 66% and 37%, respectively. GW627368X had no effect when used alone, but in combination with RO1138452, caused a rightward shift of the curves for cicaprost and iloprost but not treprostinil. PTX treatment potentiated relaxation to all 3 analogues (P < 0.01), as did L798106 and AH6809 but not SC-51322. Basal cAMP levels were higher in PTX-treated tissues and DDA- and Rp-2'-O-MB-cAMPs--sensitive responses increased to analogue concentrations < 0.1 μM. In conclusion, prostanoid EP₃ receptors via G_i/o negatively modulate prostanoid IP receptor-mediated relaxation to cicaprost, iloprost and treprostinil. However, other pathways contribute to analogue-induced vasorelaxation, the nature of which remains unclear for treprostinil.
Activation of the Renin-Angiotensin System in Heart Failure
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PI-(3)Kɛ regulates basal levels of cAMP and phospholamban and could be an important modulator of ECC and contractility [80]. Nevertheless, PI-(3)Kɛ-lacking mice cardiomyocytes showed no alterations in ICaL [81,82]. Overexpression of human AC8 in mice enhanced cAMP levels, Ca2+ transients and contractility but left ICaL unaltered due to compartmentation of cAMP hydrolysis by phosphodiesterase (PDE) [83].
L-type Ca²⁺ channels are mediators of Ca²⁺ influx and the regulatory events accompanying it and are pivotal in the function and dysfunction of ventricular cardiac myocytes. L-type Ca²⁺ channels are located in sarcolemma, including the T-tubules facing the sarcoplasmic reticulum junction, and are activated by membrane depolarization, but intracellular Ca²⁺-dependent inactivation limits Ca²⁺ influx during action potential. I_CaL is important in heart function because it triggers excitation–contraction coupling, modulates action potential shape and is involved in cardiac arrhythmia. L-type Ca²⁺ channels are multi-subunit complexes that interact with several molecules involved in their regulations, notably by β-adrenergic signaling. The present review highlights some of the recent findings on L-type Ca²⁺ channel function, regulation, and alteration in acquired pathologies such as cardiac hypertrophy, heart failure and diabetic cardiomyopathy, as well as in inherited arrhythmic cardiac diseases such as Timothy and Brugada syndromes.
Molecular basis for zinc transporter 1 action as an endogenous inhibitor of L-type calcium channels
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The L-type calcium channel (LTCC) has a variety of physiological roles that are critical for the proper function of many cell types and organs. Recently, a member of the zinc-regulating family of proteins, ZnT-1, was recognized as an endogenous inhibitor of the LTCC, but its mechanism of action has not been elucidated. In the present study, using two-electrode voltage clamp recordings in Xenopus oocytes, we demonstrate that ZnT-1-mediated inhibition of the LTCC critically depends on the presence of the LTCC regulatory β-subunit. Moreover, the ZnT-1-induced inhibition of the LTCC current is also abolished by excess levels of the β-subunit. An interaction between ZnT-1 and the β-subunit, as demonstrated by co-immunoprecipitation and by fluorescence resonance energy transfer, is consistent with this result. Using surface biotinylation and total internal reflection fluorescence microscopy in HEK293 cells, we show a ZnT-1-dependent decrease in the surface expression of the pore-forming α₁-subunit of the LTCC. Similarly, a decrease in the surface expression of the α₁-subunit is observed following up-regulation of the expression of endogenous ZnT-1 in rapidly paced cultured cardiomyocytes. We conclude that ZnT-1-mediated inhibition of the LTCC is mediated through a functional interaction of ZnT-1 with the LTCC β-subunit and that it involves a decrease in the trafficking of the LTCC α₁-subunit to the surface membrane.

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¹: Both authors contributed equally to this work.

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Review articlePhosphoinositide 3-kinase γ Regulates Cardiac Contractility by Locally Controlling Cyclic Adenosine Monophosphate Levels

Section snippets

cAMP Compartmentation in the Heart

PI3Kγ and cAMP Compartmentation in Ventricular Cardiomyocytes

Implications and Possible Mechanism(s) of PI3Kγ Regulation of cAMP

Perspectives and Significance

Acknowledgments

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beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from Gs to Gi

Proc Natl Acad Sci U S A

Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo

Nat Cell Biol

Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms

Physiol Rev

Voltage-gated Ca2+ currents in the human pathophysiologic heart: a review

Basic Res Cardiol

Cardiac excitation–contraction coupling

Nature

When is cAMP not cAMP? Effects of compartmentalization

Circ Res

Sarcoplasmic reticulum Ca2+ and heart failure: roles of diastolic leak and Ca2+ transport

Circ Res

Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure

Circulation

Beta-adrenergic signal transduction in the failing and hypertrophied myocardium

J Mol Med

Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis

Nat Med

The beta(2)-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through G(i)-dependent coupling to phosphatidylinositol 3'-kinase

Circ Res

Nitric oxide signalling by selective beta(2)-adrenoceptor stimulation prevents ACh-induced inhibition of beta(2)-stimulated Ca(2+) current in cat atrial myocytes

J Physiol

Cyclic AMP compartmentation due to increased cAMP-phosphodiesterase activity in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type 8 (AC8)

FASEB J

Defective excitation–contraction coupling in experimental cardiac hypertrophy and heart failure

Science

Review article
Phosphoinositide 3-kinase γ Regulates Cardiac Contractility by Locally Controlling Cyclic Adenosine Monophosphate Levels

beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from G_s to G_i

Voltage-gated Ca²⁺ currents in the human pathophysiologic heart: a review

Sarcoplasmic reticulum Ca²⁺ and heart failure: roles of diastolic leak and Ca²⁺ transport