Adenylyl cyclase 5/6 underlie PIP3 dependent regulation

A wide variety of signaling substances such as hormones, neurotransmitters, odorants and chemokines control intracellular signaling by regulating the production of the second messenger cAMP. By activating Epac, PKA and cyclic nucleotide-gated ion channels, the production of cAMP alters a wide range of biological processes including cell division and metabolism. A number of GPCRs controls intracellular cAMP levels via stimulatory or inhibitory G proteins via adenylyl cyclases. The function of the broadly expressed AC5 and AC6 is enhanced by stimulatory (Gαs) or attenuated by inhibitory (Gαi) G proteins. Mechanistically both inhibition and stimulation is mediated via a direct protein-protein interaction. In addition to this direct regulation, several previous studies reported a cAMP rebound stimulation after withdrawal of Gi stimulation in cardiac myocytes for which the mechanism is debated (Hartzell, 1988; Wang & Lipsius, 1995). A similar cAMP rebound response was observed previously in our lab after termination of α2A-AR adrenergic receptor activation in HEK293T cells (Markus et al., 2013). The present study was aimed at investigating mechanisms underlying Gi-induced cAMP rebound effects. Many genetically encoded biosensors have been developed based on fluorescence resonance energy transfer (FRET) to visualize the spatiotemporal dynamics of various intracellular signals including second messengers. FRET-based cAMP biosensor (Epac1-camps) as well as heterologous overexpression system was used to investigate the mechanisms underlying Gi-mediated cAMP rebound stimulation in cardiac myocytes and also in heterologous expression system. When studying the mechanism of the long-known phenomenon of cAMP rebound stimulation after withdrawal of Gi stimulation in cardiac myocytes, we observed a PTX-sensitive/Gi-mediated/ adenylyl cyclase (type 5/6)/ cAMP-dependent pathway for this cAMP rebound stimulation. In addition, we observed that inhibition of Gβγ by gallein led to an attenuation of the AC5- mediated cAMP rebound response, although, overexpression of AC4 did not produce additional cAMP stimulation. This implies that different Gβγ-mediated signaling pathways may exist. Interestingly, we observed that PI3K inhibitor attenuates AC5/6-dependent cAMP rebound effects. This indicated that Gi-mediated cAMP rebound response was mediated via the PI3K-dependent pathway. Indeed, overexpression of PIP3-specific phosphatase PTEN confirmed that PIP3 itself either directly or indirectly mediated Gi-dependent cAMP rebound responses. Additionally, inhibition of PIP2-specific phosphatase SHIP and downstream events of PIP3-dependent regulation of Akt further confirm the influence of PIP3 on cAMP rebound levels. Indeed, surpassing Gi-mediated PI3K activation through PDGF-receptor stimulation strengthens this pathway. In addition, we confirmed that inhibition of PI3K also prevented cAMP rebound response after withdrawal of ACh in atrial myocytes. We suppose that the novel PIP3 dependent regulation of AC5/6 might represent a missing mechanism that explains physiological phenomena such as post vagal tachycardia

Illuminating cell signaling with genetically encoded FRET biosensors in adult mouse cardiomyocytes.

FRET-based biosensor experiments in adult cardiomyocytes are a powerful way of dissecting the spatiotemporal dynamics of the complicated signaling networks that regulate cardiac health and disease. However, although much information has been gleaned from FRET studies on cardiomyocytes from larger species, experiments on adult cardiomyocytes from mice have been difficult at best. Thus the large variety of genetic mouse models cannot be easily used for this type of study. Here we develop cell culture conditions for adult mouse cardiomyocytes that permit robust expression of adenoviral FRET biosensors and reproducible FRET experimentation. We find that addition of 6.25 µM blebbistatin or 20 µM (S)-nitro-blebbistatin to a minimal essential medium containing 10 mM HEPES and 0.2% BSA maintains morphology of cardiomyocytes from physiological, pathological, and transgenic mouse models for up to 50 h after adenoviral infection. This provides a 10-15-h time window to perform reproducible FRET readings using a variety of CFP/YFP sensors between 30 and 50 h postinfection. The culture is applicable to cardiomyocytes isolated from transgenic mouse models as well as models with cardiac diseases. Therefore, this study helps scientists to disentangle complicated signaling networks important in health and disease of cardiomyocytes

Intracellular β\u3csub\u3e1\u3c/sub\u3e-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility

Rationale: β1ARs (β1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β1AR in cardiac contractility remains to be elucidated. Objective: Test localization and function of intracellular β1AR on cardiac contractility. Methods and Results: Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of β1ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant βAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant βAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility. Conclusions: Functional β1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular β1ARs requires catecholamine transport via OCT3

Adenylyl cyclase 5/6 underlie PIP3 dependent regulation

A wide variety of signaling substances such as hormones, neurotransmitters, odorants and chemokines control intracellular signaling by regulating the production of the second messenger cAMP. By activating Epac, PKA and cyclic nucleotide-gated ion channels, the production of cAMP alters a wide range of biological processes including cell division and metabolism. A number of GPCRs controls intracellular cAMP levels via stimulatory or inhibitory G proteins via adenylyl cyclases. The function of the broadly expressed AC5 and AC6 is enhanced by stimulatory (Gαs) or attenuated by inhibitory (Gαi) G proteins. Mechanistically both inhibition and stimulation is mediated via a direct protein-protein interaction. In addition to this direct regulation, several previous studies reported a cAMP rebound stimulation after withdrawal of Gi stimulation in cardiac myocytes for which the mechanism is debated (Hartzell, 1988; Wang & Lipsius, 1995). A similar cAMP rebound response was observed previously in our lab after termination of α2A-AR adrenergic receptor activation in HEK293T cells (Markus et al., 2013). The present study was aimed at investigating mechanisms underlying Gi-induced cAMP rebound effects. Many genetically encoded biosensors have been developed based on fluorescence resonance energy transfer (FRET) to visualize the spatiotemporal dynamics of various intracellular signals including second messengers. FRET-based cAMP biosensor (Epac1-camps) as well as heterologous overexpression system was used to investigate the mechanisms underlying Gi-mediated cAMP rebound stimulation in cardiac myocytes and also in heterologous expression system. When studying the mechanism of the long-known phenomenon of cAMP rebound stimulation after withdrawal of Gi stimulation in cardiac myocytes, we observed a PTX-sensitive/Gi-mediated/ adenylyl cyclase (type 5/6)/ cAMP-dependent pathway for this cAMP rebound stimulation. In addition, we observed that inhibition of Gβγ by gallein led to an attenuation of the AC5- mediated cAMP rebound response, although, overexpression of AC4 did not produce additional cAMP stimulation. This implies that different Gβγ-mediated signaling pathways may exist. Interestingly, we observed that PI3K inhibitor attenuates AC5/6-dependent cAMP rebound effects. This indicated that Gi-mediated cAMP rebound response was mediated via the PI3K-dependent pathway. Indeed, overexpression of PIP3-specific phosphatase PTEN confirmed that PIP3 itself either directly or indirectly mediated Gi-dependent cAMP rebound responses. Additionally, inhibition of PIP2-specific phosphatase SHIP and downstream events of PIP3-dependent regulation of Akt further confirm the influence of PIP3 on cAMP rebound levels. Indeed, surpassing Gi-mediated PI3K activation through PDGF-receptor stimulation strengthens this pathway. In addition, we confirmed that inhibition of PI3K also prevented cAMP rebound response after withdrawal of ACh in atrial myocytes. We suppose that the novel PIP3 dependent regulation of AC5/6 might represent a missing mechanism that explains physiological phenomena such as post vagal tachycardia

Intracellular β1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility

Rationaleβ1ARs (β1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β1AR in cardiac contractility remains to be elucidated.ObjectiveTest localization and function of intracellular β1AR on cardiac contractility.Methods and resultsMembrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of β1ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant βAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant βAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility.ConclusionsFunctional β1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular β1ARs requires catecholamine transport via OCT3

GRK5 Controls SAP97-Dependent Cardiotoxic β1 Adrenergic Receptor-CaMKII Signaling in Heart Failureβ

Rationale: Cardiotoxic β1 adrenergic receptor (β1AR)-CaMKII signaling is a major and critical feature associated with development of heart failure. Synapse-associated protein 97 (SAP97) is a multi-functional scaffold protein that binds directly to the C-terminus of β1AR and organizes a receptor signalosome. Objective: We aim to elucidate the dynamics of β1AR-SAP97 signalosome and its potential role in chronic cardiotoxic β1AR-CaMKII signaling that contributes to development of heart failure. Methods and Results: The integrity of cardiac β1AR-SAP97 complex was examined in heart failure. Cardiac specific deletion of SAP97 was developed to examine β1AR signaling in ageing mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β1AR-SAP97 signaling complex is reduced in heart failure. Cardiac specific deletion of SAP97 yields an ageing-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA-dependent association of β1AR with arrestin2 and CaMKII and turns on an Epac-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 is necessary to promote agonist-induced dissociation of SAP97 from β1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β1AR-SAP97 complex and increases in CaMKII activity in hearts. Conclusions: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac β1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy

Adenylyl cyclase isoform 1 contributes to sinoatrial node automaticity via functional microdomains

Sinoatrial node (SAN) cells are the heart's primary pacemaker. Their activity is tightly regulated by β-adrenergic receptor (β-AR) signaling. Adenylyl cyclase (AC) is a key enzyme in the β-AR pathway that catalyzes the production of cAMP. There are current gaps in our knowledge regarding the dominant AC isoforms and the specific roles of Ca2+-activated ACs in the SAN. The current study tests the hypothesis that distinct AC isoforms are preferentially expressed in the SAN and compartmentalize within microdomains to orchestrate heart rate regulation during β-AR signaling. In contrast to atrial and ventricular myocytes, SAN cells express a diverse repertoire of ACs, with ACI as the predominant Ca2+-activated isoform. Although ACI-KO (ACI-/-) mice exhibit normal cardiac systolic or diastolic function, they experience SAN dysfunction. Similarly, SAN-specific CRISPR/Cas9-mediated gene silencing of ACI results in sinus node dysfunction. Mechanistically, hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channels form functional microdomains almost exclusively with ACI, while ryanodine receptor and L-type Ca2+ channels likely compartmentalize with ACI and other AC isoforms. In contrast, there were no significant differences in T-type Ca2+ and Na+ currents at baseline or after β-AR stimulation between WT and ACI-/- SAN cells. Due to its central characteristic feature as a Ca2+-activated isoform, ACI plays a unique role in sustaining the rise of local cAMP and heart rates during β-AR stimulation. The findings provide insights into the critical roles of the Ca2+-activated isoform of AC in sustaining SAN automaticity that is distinct from contractile cardiomyocytes