Robert D. Harvey

Muscarinic regulation of cardiac ion channels

The parasympathetic component of the autonomic nervous system plays an important role in the physiological regulation of cardiac function by exerting significant influence over the initiation as well as propagation of electrical impulses, in addition to being able to regulate contractile force. These effects are mediated in whole or in part through changes in ion channel activity that occur in response to activation of M2 muscarinic cholinergic receptors following release of the neurotransmitter acetylcholine. The coupling of M2 receptor activation to most changes in cardiac ion channel function can be explained by one of two general paradigms. The first involves direct G protein‐dependent regulation of ion channel activity. The second involves indirect regulation of ion channel activity through modulation of cAMP‐dependent responses. This review focuses on recent advances in our understanding of the mechanisms by which M2 muscarinic receptor activation both inhibits and facilitates cAMP‐dependent ion channel responses in the heart.

Cytoplasmic cAMP concentrations in intact cardiac myocytes

In cardiac myocytes there is evidence that activation of some receptors can regulate protein kinase A (PKA)-dependent responses by stimulating cAMP production that is limited to discrete intracellular domains. We previously developed a computational model of compartmentalized cAMP signaling to investigate the feasibility of this idea. The model was able to reproduce experimental results demonstrating that both beta(1)-adrenergic and M(2) muscarinic receptor-mediated cAMP changes occur in microdomains associated with PKA signaling. However, the model also suggested that the cAMP concentration throughout most of the cell could be significantly higher than that found in PKA-signaling domains. In the present study we tested this counterintuitive hypothesis using a freely diffusible fluorescence resonance energy transfer-based biosensor constructed from the type 2 exchange protein activated by cAMP (Epac2-camps). It was determined that in adult ventricular myocytes the basal cAMP concentration detected by the probe is approximately 1.2 muM, which is high enough to maximally activate PKA. Furthermore, the probe detected responses produced by both beta(1) and M(2) receptor activation. Modeling suggests that responses detected by Epac2-camps mainly reflect what is happening in a bulk cytosolic compartment with little contribution from microdomains where PKA signaling occurs. These results support the conclusion that even though beta(1) and M(2) receptor activation can produce global changes in cAMP, compartmentation plays an important role by maintaining microdomains where cAMP levels are significantly below that found throughout most of the cell. This allows receptor stimulation to regulate cAMP activity over concentration ranges appropriate for modulating both higher (e.g., PKA) and lower affinity (e.g., Epac) effectors.

Caveolae compartmentalise β2-adrenoceptor signals by curtailing cAMP production and maintaining phosphatase activity in the sarcoplasmic reticulum of the adult ventricular myocyte

Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of β1-, but not β2-, adrenoceptors (ARs). Compartmentation of β2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae (specialised sarcolemmal invaginations rich in cholesterol and caveolin-3) contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of β2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca2+ current, but instead to a discrete PKA-mediated phosphorylation of phospholamban at Ser16. Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during β2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed β2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of β2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit β2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled “Local Signaling in Myocytes”.

cAMP microdomains and L‐type Ca2+ channel regulation in guinea‐pig ventricular myocytes

Many different receptors can stimulate cAMP synthesis in the heart, but not all elicit the same functional responses. For example, it has been recognized for some time that prostaglandins such as PGE1 increase cAMP production and activate PKA, but they do not elicit responses like those produced by β‐adrenergic receptor (βAR) agonists such as isoproterenol (isoprenaline), even though both stimulate the same signalling pathway. In the present study, we confirm that isoproterenol, but not PGE1, is able to produce cAMP‐dependent stimulation of the L‐type Ca2+ current in guinea pig ventricular myocytes. This is despite finding evidence that these cells express EP4 prostaglandin receptors, which are known to activate Gs‐dependent signalling pathways. Using fluorescence resonance energy transfer‐based biosensors that are either freely diffusible or bound to A kinase anchoring proteins, we demonstrate that the difference is due to the ability of isoproterenol to stimulate cAMP production in cytosolic and caveolar compartments of intact cardiac myocytes, while PGE1 only stimulates cAMP production in the cytosolic compartment. Unlike other receptor‐mediated responses, compartmentation of PGE1 responses was not due to concurrent activation of a Gi‐dependent signalling pathway or phosphodiesterase activity. Instead, compartmentation of the PGE1 response in cardiac myocytes appears to be due to transient stimulation of cAMP in a microdomain that can communicate directly with the bulk cytosolic compartment but not the caveolar compartment associated with βAR regulation of L‐type Ca2+ channel function.

Genistein Increases the Sensitivity of Cardiac Ion Channels to β-Adrenergic Receptor Stimulation

The whole-cell patch-clamp technique was used to monitor the effects of genistein, a tyrosine kinase inhibitor, on membrane currents recorded from isolated guinea pig ventricular myocytes. Under control conditions, genistein (50 micromol/L) did not activate the latent cAMP-regulated Cl- current (ICl). However, in the presence of a subthreshold concentration (1 nmol/L) of the beta-adrenergic agonist isoproterenol (Iso), genistein caused a near-maximal activation of this current. In the absence of genistein, Iso activated ICl with an EC50 of 5 nmol/L. In the presence of genistein, Iso activated ICl with an EC50 of 0.3 nmol/L. This facilitatory effect was not observed in the presence of daidzein (50 micromol/L), an analogue of genistein that only weakly inhibits tyrosine kinase activity. Furthermore, peroxovanadate, a potent inhibitor of phosphotyrosine phosphatase activity, inhibited ICl activated by Iso alone, and it blocked the stimulatory effect of genistein in the presence of Iso. To determine whether the stimulatory effect of genistein was specific for ICl, we also studied its action on the cAMP-regulated delayed rectifier K+ current (IK) and L-type Ca2+ current (ICa-L) present in these cells. Basal IK and ICa-L were partially (approximately 30% to 40%) inhibited by genistein. However, this inhibitory effect was mimicked by daidzein, suggesting that inhibition of tyrosine kinase activity is not involved. In addition to the nonspecific inhibitory effect, genistein also caused a significant increase in the beta-adrenergic sensitivity of the unblocked cationic currents. In the absence of genistein, 1 nmol/L Iso had no effect on either IK or ICa-L. However, in the presence of genistein, 1 nmol/L Iso significantly increased the magnitude of both currents. These results suggest that tyrosine kinase activity may play an important role in regulating beta-adrenergic responsiveness of the heart.

Effects of cholesterol depletion on compartmentalized cAMP responses in adult cardiac myocytes.

β1-Adrenergic receptors (β1ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. β1ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, β1AR activation enhanced the L-type Ca2+ current, intracellular Ca2+ transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-β-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by β1ARs without having any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. β1AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected β1AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between β1AR and EPR responses. They also suggest that β1AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.

Muscarinic inhibitory and stimulatory regulation of the L‐type Ca2+ current is not altered in cardiac ventricular myocytes from mice lacking endothelial nitric oxide synthase

1 Using conventional and perforated patch‐clamp techniques, the inhibitory and stimulatory effects of acetylcholine (ACh) on β‐adrenergic regulation of the L‐type Ca2+ current (ICa) were studied in ventricular myocytes from wild‐type mice (WT) and from mice lacking endothelial nitric oxide synthase (eNOS or NOS3; NOS3‐KO mice). 2 To validate the direct comparison of ACh effects on β‐adrenergic responses, the sensitivity of ICa to the β‐adrenergic agonist isoprenaline (Iso) was studied in both WT and NOS3‐KO mouse myocytes. ICa sensitivity to Iso was not found to be significantly different in WT and NOS3‐KO myocytes: Iso increased ICa with an EC50 of 4.9 and 3.7 nm in WT and NOS3‐KO myocytes, respectively. 3 ACh‐induced inhibition of ICa did not significantly differ in ventricular myocytes from WT and NOS3‐KO mice. ACh (10 μm) inhibited the stimulatory effect of 3 nm Iso by 39 and 35 % in WT and NOS3‐KO myocytes, respectively. 4 Exposure to and subsequent washout of ACh in the continuous presence of submaximally stimulating concentrations of Iso (1–3 nm) resulted in a transient rebound stimulation of ICa in both WT and NOS3‐KO mouse myocytes. The magnitude of the stimulatory effect of ACh did not significantly differ in WT and NOS3‐KO mice. 5 These results indicate that nitric oxide (NO) generated by NOS3 does not significantly affect the β‐adrenergic responsiveness of ICa. The results also confirm previous work indicating that NO generated by NOS3 is not obligatory for muscarinic inhibition of the β‐adrenergically regulated ICa in ventricular myocytes. Finally these results demonstrate for the first time that NO generated by NOS3 is not involved in muscarinic rebound stimulation of ICa in ventricular myocytes.

CaV1.2 signaling complexes in the heart.

L-type Ca(2+) channels (LTCCs) are essential for generation of the electrical and mechanical properties of cardiac muscle. Furthermore, regulation of LTCC activity plays a central role in mediating the effects of sympathetic stimulation on the heart. The primary mechanism responsible for this regulation involves β-adrenergic receptor (βAR) stimulation of cAMP production and subsequent activation of protein kinase A (PKA). Although it is well established that PKA-dependent phosphorylation regulates LTCC function, there is still much we do not understand. However, it has recently become clear that the interaction of the various signaling proteins involved is not left to completely stochastic events due to random diffusion. The primary LTCC expressed in cardiac muscle, CaV1.2, forms a supramolecular signaling complex that includes the β2AR, G proteins, adenylyl cyclases, phosphodiesterases, PKA, and protein phosphatases. In some cases, the protein interactions with CaV1.2 appear to be direct, in other cases they involve scaffolding proteins such as A kinase anchoring proteins and caveolin-3. Functional evidence also suggests that the targeting of these signaling proteins to specific membrane domains plays a critical role in maintaining the fidelity of receptor mediated LTCC regulation. This information helps explain the phenomenon of compartmentation, whereby different receptors, all linked to the production of a common diffusible second messenger, can vary in their ability to regulate LTCC activity. The purpose of this review is to examine our current understanding of the signaling complexes involved in cardiac LTCC regulation.

Muscarinic Receptor Agonists and Antagonists: Effects on Cardiovascular Function

Muscarinic receptor activation plays an essential role in parasympathetic regulation of cardiovascular function. The primary effect of parasympathetic stimulation is to decrease cardiac output by inhibiting heart rate. However, pharmacologically, muscarinic agonists are actually capable of producing both inhibitory and stimulatory effects on the heart as well as vasculature. This reflects the fact that muscarinic receptors are expressed throughout the cardiovascular system, even though they are not always involved in mediating parasympathetic responses. In the heart, in addition to regulating heart rate by altering the electrical activity of the sinoatrial node, activation of M₂ receptors can affect conduction of electrical impulses through the atrioventricular node. These same receptors can also regulate the electrical and mechanical activity of the atria and ventricles. In the vasculature, activation of M₃ and M₅ receptors in epithelial cells can cause vasorelaxation, while activation of M₁ or M₃ receptors in vascular smooth muscle cells can cause vasoconstriction in the absence of endothelium. This review focuses on our current understanding of the signaling mechanisms involved in mediating these responses.

PKC regulation of cardiac CFTR Cl− channel function in guinea pig ventricular myocytes

The role of protein kinase C (PKC) in regulating the protein kinase A (PKA)-activated Cl- current conducted by the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (cCFTR) was studied in guinea pig ventricular myocytes using the whole cell patch-clamp technique. Although stimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu) alone did not activate this Cl- current, even when intracellular dialysis was limited with the perforated patch-clamp technique, activation of PKC did elicit a significant response in the presence of PKA-dependent activation of the current by the beta-adrenergic receptor agonist isoproterenol. PDBu increased the magnitude of the Cl- conductance activated by a supramaximally stimulating concentration of isoproterenol by 21 +/- 3.3% (n = 9) when added after isoproterenol and by 36 +/- 16% (n = 14) when introduced before isoproterenol. 4alpha-Phorbol 12, 13-didecanoate, a phorbol ester that does not activate PKC, did not mimic these effects. Preexposure to chelerythrine or bisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl- current by 79 +/- 7.7% (n = 11) and 52 +/- 10% (n = 8), respectively. Our results suggest that although acute activation of endogenous PKC alone does not significantly regulate cCFTR Cl- channel activity in native myocytes, it does potentiate PKA-dependent responses, perhaps most dramatically demonstrated by basal PKC activity, which may play a pivotal role in modulating the function of these channels.The role of protein kinase C (PKC) in regulating the protein kinase A (PKA)-activated Cl- current conducted by the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (cCFTR) was studied in guinea pig ventricular myocytes using the whole cell patch-clamp technique. Although stimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu) alone did not activate this Cl- current, even when intracellular dialysis was limited with the perforated patch-clamp technique, activation of PKC did elicit a significant response in the presence of PKA-dependent activation of the current by the β-adrenergic receptor agonist isoproterenol. PDBu increased the magnitude of the Cl- conductance activated by a supramaximally stimulating concentration of isoproterenol by 21 ± 3.3% ( n = 9) when added after isoproterenol and by 36 ± 16% ( n= 14) when introduced before isoproterenol. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate PKC, did not mimic these effects. Preexposure to chelerythrine or bisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl- current by 79 ± 7.7% ( n = 11) and 52 ± 10% ( n = 8), respectively. Our results suggest that although acute activation of endogenous PKC alone does not significantly regulate cCFTR Cl- channel activity in native myocytes, it does potentiate PKA-dependent responses, perhaps most dramatically demonstrated by basal PKC activity, which may play a pivotal role in modulating the function of these channels.