Gut microbiota-derived trimethylamine N-oxide (TMAO) plays a role in the pathogenesis of cardiovascular disease, but its role in the pathogenesis of atrial fibrillation (AF) remains uncertain. TMAO levels were quantified in plasma from serial subjects undergoing elective cardiac catheterizations and shown to independently associate with prevalent AF following adjustment for risk factors. Human cAMP response element modulator isoform IbΔC-X transgenic mice (CREM-IbΔC-X) supplemented with a TMAO diet developed AF sooner. C57BL/6J mice on and off a TMAO diet had more inducible AF via a transesophageal pacing study compared with chow-fed controls. Dietary choline supplementation increased circulating TMAO levels and significantly accelerated AF onset in CREM-IbΔC-X mice. Iodomethylcholine (IMC) reduced circulating TMAO levels and choline-induced AF onset. Cecal metagenomic analyses showed that choline supplementation induced changes in microbial communities associated with AF, while many of these changes were attenuated by IMC. Choline supplementation promoted overall adverse atrial remodeling with left atrial dilation. Optical mapping studies showed that mice supplemented with choline exhibited reduced conduction velocity, shortened action potential duration at 80% repolarization, and decreased wavelength. TMAO inhibited muscarinic receptor 2, resulting in autonomic dysfunction that promotes AF. In summary, TMAO, independently associated with AF risk in subjects, enhanced AF in multiple mouse models via autonomic dysfunction and is a therapeutic target for preventing AF.

Autoimmune response to self-antigens results in autoantibodies (AAbs), wherein AAbs against extra cellular loop (ECL) 2 of β1-adrenergic receptor (β1AR) is known to underlie dilated cardiomyopathy (DCM). Contrarily, recent studies show that patients with β1AR AAbs belonging to the IgG3 subclass have beneficial outcomes. However, the signaling mechanisms that underlie the beneficial outcomes are not well understood. We have previously shown that IgG3(+) β1AR AAbs facilitates uniquely biased β1AR signaling in response to β-blocker compared to non-IgG3(+) β1AR AAbs. Since IgG3(+) β1AR AAbs are associated with favorable patient outcomes, it is critical to determine the mechanistic basis of the unique beneficial signaling pathway that may have therapeutic potential. HEK 293 cells stably expressing human β1AR (HEK-β1AR) was generated and treated with β1AR AAbs. Minimal changes in phosphorylation of β1AR was observed following IgG3(+) β1AR AAbs, while significant phosphorylation of β1ARs was observed with non-IgG3(+) β1AR AAbs. To dissect the diverse signaling responses mediated by IgG3(+) and non-IgG(+) β1AR AAbs, HEK-β1AR cells were treated with IgG3(+) or non-IgG3(+) β1AR AAbs. β1ARs were immunoprecipitated and subjected to mass-spectrometry analysis to identify unique co-immunoprecipitating proteins that could complex with receptor in presence of IgG3(+) or non-IgG3(+) β1AR AAbs. One of the proteins that was differentially recruited to the β1AR complex was Insulin Receptor Substrate 4 (IRS4). Traditionally, IRS4 is a signaling hub downstream of insulin/insulin growth factor receptors mediating glucose uptake. The differential interaction of IRS4 with β1AR in the presence of IgG3(+) vs. non-IgG3(+) β1AR AAbs reflects that IRS4 could be a key signaling regulator of uniquely biased signal transduction pathway in the presence of IgG3(+) vs. non-IgG3(+) β1AR AAbs. Mechanisms underlying this unique IRS4 mediated signaling modulation will be discussed.

β-adrenergic receptor (βAR) is a key regulator of cardiac function. Agonist activation of βAR leads to phosphorylation-mediated desensitization resulting in βAR trafficking to endosomes. Endosomal βARs undergo resensitization through protein phosphatase 2A (PP2A)-mediated dephosphorylation. We have shown that stimulation of βARs activates phosphoinositide 3-kinase γ (PI3Kγ) that phosphorylates endogenous inhibitor of PP2A (I2PP2A) promoting robust binding to PP2A resulting in PP2A inhibition. βAR dysfunction due to desensitization is considered as a classical hallmark of heart failure, while less is known about resensitization. Plasma membrane and endosomal fractions were isolated from non-failing and failing patient samples to determine whether βAR resensitization is altered in human heart failure. Endosomes from heart failure samples showed significant accumulation of phosphorylated β2ARs which was associated with reduced adenylyl cyclase and βAR-associated PP2A activity compared to non-failing showing that resensitization is inhibited in human heart failure. Since PP2A activity is inhibited by robust binding of I2PP2A to PP2A, we hypothesized that disrupting I2PP2A interaction with PP2A would unlock PP2A inhibition normalizing βAR resensitization. In silico modeling and biochemical studies mapped the PP2A interaction with I2PP2A to the C-terminal region of PP2A (PP2Act). Agonist isoproterenol (ISO) treatment of cells expressing PP2Act showed significant reduction in β2AR phosphorylation along with increased cAMP generation and normalized PP2A activity showing preserved βAR resensitization. While expression of PP2A mutant with deletion in the PP2Act (ΔPP2Act) did not preserve βAR resensitization. Challenging mice with cardiomyocyte-specific expression of PP2Act to ISO showed preserved cardiac function providing insights that targeting resensitization could potentially be a novel therapeutic strategy. Key words: Resensitization, I2PP2A, PP2A, Cardiac hypertrophy

Dysfunction of beta-adrenergic receptor (βAR) is a key hallmark of heart failure, wherein mechanisms of phosphorylation-mediated desensitization of βARs are well understood. However, less is known about dephosphorylation-mediated resensitization of βARs that is regulated by protein phosphatase 2A (PP2A) in heart failure. Our previous studies have shown that agonist stimulation of βAR activates phosphoinositide 3-kinase γ (PI3Kγ) which phosphorylates endogenous inhibitor of PP2A (I2PP2A) on serine residues 9 & 93. Phosphorylated I2PP2A (pI2PP2A) binds to PP2A inhibiting its activity thereby, impairing βAR resensitization. Although structural studies have shown I2PP2A to be a dimer, yet little is understood about the mechanistic basis of PP2A inhibition by I2PP2A. We therefore hypothesized that phosphorylation of I2PP2A promotes dimerization driving robust interaction with PP2A resulting in inhibition of PP2A and impairment in βAR resensitization. To test for dimerization, hemagglutinin (HA)-tagged I2PP2A and Myc-tagged I2PP2A were generated and co-expressed in HEK 293 cells. Immunoprecipitation of HA-I2PP2A led to significant co-immunoprecipitation of Myc-I2PP2A reflecting the ability of I2PP2A to dimerize. To determine whether phosphorylation is key for dimerization, phospho-mimetic mutants of I2PP2A (S9,93A (inactive) or S9,93D (active)) were generated. Immunoblotting of cellular lysates with expression of these phospho-mimetics showed that S9,93D exclusively formed dimers, while S9,93A I2PP2A only formed monomers suggesting that phosphorylation is key facilitator of dimerization. The role of I2PP2A phosphorylation in its dimerization and PP2A interaction with consequences on βAR resensitization will be presented. Determining the mechanistic basis of this understudied pathway is critical as human heart failure is associated with increased PI3Kγ activity, elevated expression of I2PP2A and reduced βAR function. Key words: Desensitization, Resensitization, I2PP2A, PP2A, Dimerization

Although phosphoinositide 3-kinase (PI3Kγ) null mice (PI3Kγ -/- ) show increased ventricular rate/contraction, it is unknown whether PI3Kγ regulates calcium recycling machinery underlying this phenotype. Primary adult cardiomyocytes from PI3Kγ -/- mice show altered sarcoendoplasmic reticulum (SR) calcium cycling following caffeine. Unexpectedly, PI3Kγ -/- cardiomyocytes showed significant reduction in phosphorylation of phospholamban (PLN) at Thr17, a key regulator of SR calcium re-uptake without changes in phosphorylation at Ser16. Furthermore, loss in PLN phosphorylation in PI3Kγ -/- cardiomyocytes was associated with augmented interaction with SR calcium ATPase (SERCA). Surprisingly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ inact ) in global PI3Kγ -/- mice (PI3Kγ inact /PI3Kγ -/- ) normalized caffeine-induced calcium re-uptake, PLN phosphorylation at Thr17 and decreased PLN-SERCA interaction. These data suggested kinase-independent function of PI3Kγ in regulation of SR calcium load and PLN phosphorylation. Since phosphorylation of Thr17 of PLN is carried out by Ca 2+ /Calmodulin dependent protein kinase (CamKII), we probed for the role of PI3Kγ in regulation of CamKII in PLN phosphorylation. Mechanistically, PI3Kγ exhibits scaffolding function in recruitment of CamKII to PLN at SR to mediate PLN phosphorylation. Furthermore, we showed that PI3Kγ directly interacts with CamKII. The study unravels a yet to be recognized kinase-independent role of PI3Kγ in regulating PLN with implications in cardiac function.

Background: The clinical data suggest an association of autoantibodies (AAb) targeting angiotensin II type1 receptor (AT1R) and outcomes in heart failure (HF) and preeclampsia. Notably, patient’s risk increases 2 to 4-fold for heart diseases and hypertension. Although AT1R blockers (ARBs) have emerged as a key component in the therapeutic arsenal against CVD, we believe that rather than completely blocking AT1R signaling, maintaining a balanced signaling is crucial for cardiovascular homeostasis. Therefore, we posit that reducing AT1R overactivation and blocking AAb-mediated activation could benefit cardiac health and attenuate the progression of CVD. In our previous study (PMID:36440576), we identified small molecules capable of inhibiting AAb-induced AT1R signaling. Leveraging these observations, we have developed a novel AT1R allosteric ligand that inhibits autoantibody binding to AT1R while still retaining normal AT1R signaling. Methods: Using computational and medicinal chemistry approaches, we designed a series of novel AT1R ligands and obtained compounds with >98% purity through chemical synthesis. Monoclonal antibodies mimicking AT1R-AAb have been generated. Serum samples from dilated cardiomyopathy (DCM) and preeclampsia (PE) patient cohorts were collected and presence of AT1R-AAb was determined using ELISA and epitope-peptide competition studies. Pharmacological characterization of compounds was performed using calcium mobilization, 125I -AngII binding, ex vivo vasoconstriction, and ELISA assays. Results: We identified an AT1R allosteric compound that reduced AngII-mediated calcium signaling with a 5-fold shift in EC 50 . The IC 50 was determined to be 18 µM. It reduces the AngII-mediated vasoconstriction while blocking both monoclonal and patient-derived autoantibody binding with an IC 50 of 5 µM. Conclusion: We have identified a novel AT1R allosteric ligand with negative allosteric modulator (NAM) properties, which has the potential to block autoantibodies and could serve as a new generation of ARBs. Uniquely, it retains beneficial AT1R signaling without competing with AngII, and its allosteric ligand property can be tailored to achieve optimal AT1R signaling.

Allosteric modulation is a central mechanism for metabolic regulation but has yet to be described for a gut microbiota-host interaction. Phenylacetylglutamine (PAGln), a gut microbiota-derived metabolite, has previously been clinically associated with and mechanistically linked to cardiovascular disease (CVD) and heart failure (HF). Here, using cells expressing β1- versus β2-adrenergic receptors (β1AR and β2AR), PAGln is shown to act as a negative allosteric modulator (NAM) of β2AR, but not β1AR. In functional studies, PAGln is further shown to promote NAM effects in both isolated male mouse cardiomyocytes and failing human heart left ventricle muscle (contracting trabeculae). Finally, using in silico docking studies coupled with site-directed mutagenesis and functional analyses, we identified sites on β2AR (residues E122 and V206) that when mutated still confer responsiveness to canonical β2AR agonists but no longer show PAGln-elicited NAM activity. The present studies reveal the gut microbiota-obligate metabolite PAGln as an endogenous NAM of a host GPCR. Allosteric modulation is crucial in metabolic regulation but unexplored in gut microbehost interactions. Here the authors show gut microbe-derived phenylacetylglutamine acts as a negative allosteric modulator of β2-adrenergic receptors, impacting heart function.

Introduction Autoimmune response to self-antigens results in autoantibodies (AAbs), wherein AAbs against extra cellular loop 2 (ECL2) of β1AR is known to underlie dilated cardiomyopathy (DCM). Contrarily, recent studies show that patients with β1AR AAbs belonging to the IgG3 subclass have beneficial outcomes. However, the signaling mechanisms that underlie the beneficial outcomes are not well understood. Hypothesis IgG3(+) β1AR AAbs facilitates uniquely biased β1AR signaling in response to β-blockers. Methods Since IgG3(+) β1AR AAbs are associated with favorable patient outcomes, in silico studies were conducted to evaluate the interaction between the IgG3(+) antibody and the known structure of β1AR. In silico epitope prediction suggested that the extracellular loop 1 (ECL1) could be a potential epitope, wherein binding by IgG3(+) AAbs at ECL1 could facilitate unique biased signaling in response to β-blockers. Peptides representing human β1AR ECL 1 was synthesized and antibodies targeting the human β1AR ECL 1 was generated by immunizing the mice. HEK 293 cells expressing human β1AR were pre-treated with serum containing antibodies against ECL 1 of β1AR followed by either β1AR selective agonist dobutamine (DOB) or β-blocker metoprolol stimulation. Downstream β1AR signaling was measured by cAMP generation. To test for specificity of ECL1 in the modulating β1AR responses, the cells were incubated with ECL 1 peptides along with antibodies against ECL1 of human β1AR as competitors and cAMP generation assessed following metoprolol. Results Treatment of human β1AR expressing HEK 293 cells with serum from mouse immunized with ECL1 of human β1AR showed no baseline changes in the cAMP generation. However, pretreatment of these cells with ECL1 antibodies followed by stimulation with DOB surprisingly showed marked reduction in cAMP generation in contrast to adjuvant controls which showed significant increase in cAMP. While, treatment of these ECL1 antibody pretreated cells with metoprolol unexpectedly resulted in cAMP generation which is blocked in the adjuvant controls. This shows that antibodies against the ECL1 of human β1AR binds to the β1AR and is able to allosterically modulate downstream signaling mediated by agonist (DOB) or β-blocker (metoprolol) as at baseline it does not alter β1AR function. To validate that antibodies against the ECL1 of human β1AR allosterically modulates metoprolol signaling, the cells were also pre-treated with competing doses of ECL1 peptide followed by metoprolol treatment. Consistent with our hypothesis, incubation with ECL1 peptide remarkably blocked cAMP generation in response to metoprolol. This shows that antibody binding to the ECL1 of human β1AR mediates this unique signaling in response to metoprolol wherein, a traditionally cAMP blocking β-blocker metoprolol now mediates cAMP generation. Conclusions These observations suggest that the allosteric binding of β1AR ECL1 by the antibodies mediates unexpected downstream signal that could underlie the benefits observed in the patients harboring IgG3(+) AAbs. Mechanisms underlying this unique allosteric modulation will be discussed in our presentation.

Background: Impaired beta-adrenergic receptor (β1 and β2AR) function following hypoxia underlies ischemic heart failure/stroke. Activation of PI3Kγ (phosphoinositide 3-kinase γ) by beta-adrenergic receptor leads to feedback regulation of the receptor by hindering beta-adrenergic receptor dephosphorylation through inhibition of PP2A (protein phosphatase 2A). However, little is known about PI3Kγ feedback mechanism in regulating hypoxia-mediated β1 and β2AR dysfunction and cardiac remodeling. Methods: Human embryonic kidney 293 cells or mouse adult cardiomyocytes and C57BL/6 (WT) or PI3Kγ knockout (KO) mice were subjected to hypoxia. Cardiac plasma membranes and endosomes were isolated and evaluated for β1 and β2AR density and function, PI3Kγ activity and β1 and β2AR-associated PP2A activity. Metabolic labeling was performed to assess β1 and β2AR phosphorylation and epinephrine/norepinephrine levels measured post-hypoxia. Results: Hypoxia increased β1 and β2AR phosphorylation, reduced cAMP, and led to endosomal accumulation of phosphorylated β2ARs in human embryonic kidney 293 cells and WT cardiomyocytes. Acute hypoxia in WT mice resulted in cardiac remodeling and loss of adenylyl cyclase activity associated with increased β1 and β2AR phosphorylation. This was agonist-independent as plasma and cardiac epinephrine and norepinephrine levels were unaltered. Unexpectedly, PI3Kγ activity was selectively increased in the endosomes of human embryonic kidney 293 cells and WT hearts post-hypoxia. Endosomal β1- and β2AR-associated PP2A activity was inhibited upon hypoxia in human embryonic kidney 293 cells and WT hearts showing regulation of beta-adrenergic receptors by PI3Kγ. This was accompanied with phosphorylation of endogenous inhibitor of protein phosphatase 2A whose phosphorylation by PI3Kγ inhibits PP2A. Increased β1 and β2AR-associated PP2A activity, decreased beta-adrenergic receptor phosphorylation, and normalized cardiac function was observed in PI3Kγ KO mice despite hypoxia. Compared to WT, PI3Kγ KO mice had preserved cardiac response to challenge with β1AR-selective agonist dobutamine post-hypoxia. Conclusions: Agonist-independent activation of PI3Kγ underlies hypoxia sensing as its ablation leads to reduction in β1- and β2AR phosphorylation and amelioration of cardiac dysfunction.

Although phosphoinositide 3-kinase (PI3Kγ) null mice (PI3Kγ -/- ) show increased ventricular rate/contraction, it is unknown whether PI3Kγ regulates calcium recycling machinery underlying this phenotype. Primary adult cardiomyocytes from PI3Kγ -/- mice show reduced calcium reuptake by sarcoendoplasmic reticulum (SR) following caffeine. Unexpectedly, PI3Kγ -/- cardiomyocytes showed significant reduction in phosphorylation of phospholamban (PLN) at Thr17, a key regulator of SR calcium re-uptake. Furthermore, loss in PLN phosphorylation in PI3Kγ -/- cardiomyocytes was associated with augmented interaction with SR calcium ATPase (SERCA). Surprisingly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ inact ) in global PI3Kγ -/- mice (PI3Kγ inact /PI3Kγ -/- ) normalized caffeine-induced calcium re-uptake, PLN phosphorylation at Thr17 and decreased PLN-SERCA interaction. These data suggested kinase-independent function of PI3Kγ in regulation of SR calcium load and PLN phosphorylation. Since phosphorylation of Thr17 of PLN is carried out by Ca 2+ /Calmodulin dependent protein kinase (CamKII) we probed for the role of PI3Kγ in regulation of CamKII in PLN phosphorylation. Mechanistically, PI3Kγ exhibits scaffolding function in recruitment of CamKII to PLN at SR to mediate PLN phosphorylation. The study unravels a yet to be recognized kinase-independent role of PI3Kγ in regulating PLN with implications in cardiac function.

Insulin (INS) treatment results in impaired response to β-agonist isoproterenol (ISO) associated with phosphorylated β2-adrenergic receptor (β2AR) and impaired cAMP response. Although INS does not mediate dissociation of G-protein Gβγ subunits, yet surprisingly recruits phosphoinositide 3-kinase γ (PI3Kγ) to the plasma membrane. Correspondingly, knock-down of PI3Kγ (PI3Kγ KD) in HEK 293 or ablation in primary adult cardiomyocytes and fibroblasts (isolated from PI3Kγ knockout mice) abrogated β2AR phosphorylation reflecting a key role for PI3Kγ in retaining β2AR phosphorylation. Also, adult cardiomyocytes isolated from C57Bl6 mice showed significant loss of in vitro ISO-stimulated cardiomyocyte contraction upon INS pre-treatment which was remarkably preserved in the PI3Kγ knockout cardiomyocytes despite INS. As PI3Kγ is traditionally recruited to the βAR complex by the Gβγ subunits, we hypothesized that INS-mediates Gβγ-independent recruitment of PI3Kγ to the βAR complex underlying its dysfunction. INS stimulation is known to recruit insulin receptor substrate (IRS 1 & 2) to the receptor complex, and therefore, we tested whether PI3Kγ interacts with IRS 1/2 leading to Gβγ-independent recruitment of PI3Kγ to the β2AR complex. Immunoprecipitation PI3Kγ following INS showed that PI3Kγ interacts with IRS2 recruiting PI3Kγ to the β2AR complex bypassing the traditional Gβγ-dependent pathway. Since PI3Kγ KD resulted in loss of INS-mediated β2AR phosphorylation, we tested whether PI3Kγ inhibits protein phosphatase 2A (PP2A) function impairing β2AR de-phosphorylation. INS treatment resulted in significant loss of β2AR-associated PP2A activity which was rescued in PI3Kγ KD cells showing that PI3Kγ impairs PP2A function. Furthermore, our studies show that PI3Kγ phosphorylates the endogenous inhibitor of PP2A, I2PP2A which then robustly binds and inhibits PP2A activity impairing β2AR dephosphorylation. Consistently, CRISPR ablation of I2PP2A relieves this inhibition on PP2A leading to increased PP2A activity. This reduces accumulation of phosphorylated βARs despite the presence of PI3Kγ showing an underappreciated regulation of PP2A by insulin through non-canonical recruitment of PI3Kγ that impairs β2AR function.

Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ -/- ) results in increased cAMP levels and enhanced ventricular rate/contractility. We investigated whether PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling. Caffeine treatment of adult cardiomyocytes from PI3Kγ -/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) indicating that PI3Kγ locally regulates SR function. This resulted in elevated levels of intracellular calcium for prolonged period following caffeine. Our findings show that delayed re-uptake of calcium was caused by changes in phosphorylation of phospholamban (PLN), a major regulator of SR calcium reuptake. PI3Kγ -/- cardiomyocytes showed significantly reduced PLN phosphorylation due to increase in PLN-associated protein phosphatase (PP) activity as reflected by decreased demethylated-PP2A. Abrogation of PLN phosphorylation in the PI3Kγ -/- cardiomyocytes shows that the loss in the steady-state phosphorylation of PLN leads to increased inhibition of SERCA. This inhibition is reflected by the slow reuptake of calcium by the SR in the PI3Kγ -/- cardiomyocytes. Concomitantly, significant interaction was observed between SERCA and PLN in the PI3Kγ -/- hearts compared to the controls. Consistently, the altered calcium regulation in the cardiomyocytes of PI3Kγ -/- can be restored by inhibition of PP by okadaic acid. Unexpectedly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ inact ) in the global PI3Kγ -/- cardiomyocytes normalized caffeine induced calcium reuptake, restored PLN phosphorylation, and decreased PLN-associated PP activity reflected by increased demethylated-PP2A. These studies bring-to-fore an unrecognized kinase-independent regulation of PLN by PI3Kγ through PP2A with implications in deleterious cardiac remodeling as PI3Kγ is significantly upregulated following cardiac stress.

Although microRNA-7 (miRNA-7) is known to regulate proliferation of cancer cells by targeting Epidermal growth factor receptor (EGFR/ERBB) family, less is known about its role in cardiac physiology. Transgenic (Tg) mouse with cardiomyocyte-specific overexpression of miRNA-7 was generated to determine its role in cardiac physiology and pathology. Echocardiography on the miRNA-7 Tg mice showed cardiac dilation instead of age-associated physiological cardiac hypertrophy observed in non-Tg control mice. Subjecting miRNA-7 Tg mice to transverse aortic constriction (TAC) resulted in cardiac dilation associated with increased fibrosis bypassing the adaptive cardiac hypertrophic response to TAC. miRNA-7 expression in cardiomyocytes resulted in significant loss of ERBB2 expression with no changes in ERBB1 (EGFR). Cardiac proteomics in the miRNA-7 Tg mice showed significant reduction in mitochondrial membrane structural proteins compared to NTg reflecting role of miRNA-7 beyond the regulation of EGFR/ERRB in mediating cardiac dilation. Consistently, electron microscopy showed that miRNA-7 Tg hearts had disorganized rounded mitochondria that was associated with mitochondrial dysfunction. These findings show that expression of miRNA-7 in the cardiomyocytes results in cardiac dilation instead of adaptive hypertrophic response during aging or to TAC providing insights on yet to be understood role of miRNA-7 in cardiac function.

Rationale Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ −/− ) results in increased cAMP levels and enhanced ventricular rate/contractility. Whether PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling is not known. Objective To understand the mechanism of PI3Kγ mediated regulation of cardiac contractility. Methods and Results Caffeine treatment of adult cardiomyocytes from PI3Kγ −/− mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) indicating that PI3Kγ locally regulates SR function. This resulted in elevated levels of intracellular calcium for prolonged period following caffeine. Our findings show that delayed re-uptake of calcium was caused by changes in phosphorylation of phospholamban (PLN), a major regulator of SR calcium reuptake. PI3Kγ −/− cardiomyocytes show significantly reduced PLN phosphorylation due to increase in PLN-associated protein phosphatase (PP) activity as reflected by decreased demethylated-PP2A. Consistently, the altered calcium regulation in the cardiomyocytes of PI3Kγ −/− can be restored by inhibition of PP by okadaic acid. Unexpectedly, cardiomyocyate-specific overexpression of kinase-dead PI3Kγ PI3Kγ inact ) in the global PI3Kγ −/− cardiomyocytes normalized caffeine induced calcium reuptake, restored PLN phosphorylation, and decreased PLN-associated PP activity reflected by increased demethylated-PP2A. Conclusions These studies bring-to-fore an unrecognized regulation of PLN by PI3Kγ through PP2A with implications in deleterious cardiac remodeling as PI3Kγ is significantly upregulated following cardiac stress.

Background and Aims Despite the high clinical significance of sarcopenia in alcohol‐associated cirrhosis, there are currently no effective therapies because the underlying mechanisms are poorly understood. We determined the mechanisms of ethanol‐induced impaired phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) and adenosine monophosphate–activated protein kinase (AMPK) with consequent dysregulated skeletal muscle protein homeostasis (balance between protein synthesis and breakdown). Approach and Results Differentiated murine myotubes, gastrocnemius muscle from mice with loss and gain of function of regulatory genes following ethanol treatment, and skeletal muscle from patients with alcohol‐associated cirrhosis were used. Ethanol increases skeletal muscle autophagy by dephosphorylating mTORC1, circumventing the classical kinase regulation by protein kinase B (Akt). Concurrently and paradoxically, ethanol exposure results in dephosphorylation and inhibition of AMPK, an activator of autophagy and inhibitor of mTORC1 signaling. However, AMPK remains inactive with ethanol exposure despite lower cellular and tissue adenosine triphosphate, indicating a “pseudofed” state. We identified protein phosphatase (PP) 2A as a key mediator of ethanol‐induced signaling and functional perturbations using loss and gain of function studies. Ethanol impairs binding of endogenous inhibitor of PP2A to PP2A, resulting in methylation and targeting of PP2A to cause dephosphorylation of mTORC1 and AMPK. Activity of phosphoinositide 3‐kinase‐γ (PI3Kγ), a negative regulator of PP2A, was decreased in response to ethanol. Ethanol‐induced molecular and phenotypic perturbations in wild‐type mice were observed in PI3Kγ−/− mice even at baseline. Importantly, overexpressing kinase‐active PI3Kγ but not the kinase‐dead mutant reversed ethanol‐induced molecular perturbations. Conclusions Our study describes the mechanistic underpinnings for ethanol‐mediated dysregulation of protein homeostasis by PP2A that leads to sarcopenia with a potential for therapeutic approaches by targeting the PI3Kγ‐PP2A axis.