AKAP12 Upregulation Associates With PDE8A to Accelerate Cardiac Dysfunction.

BACKGROUND: In heart failure, signaling downstream the ß2-adrenergic receptor is critical. Sympathetic stimulation of ß2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind ß2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. METHODS: cAMP accumulation in real time downstream of the ß2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. RESULTS: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. CONCLUSIONS: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.

A Small Molecule That In Vitro Neutralizes Infection of SARS-CoV-2 and Its Most Infectious Variants, Delta, and Omicron.

The COVID-19 pandemic has underscored the urgent need to develop highly potent and safe medications that are complementary to the role of vaccines. Specifically, it has exhibited the need for orally bioavailable broad-spectrum antivirals that are able to be quickly deployed against newly emerging viral pathogens. The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and its variants Delta and Omicron are still a major threat to patients of all ages. In this brief report, we describe that the small molecule CD04872SC was able to neutralize SARS-CoV2 infection with a half-maximal effective concentration (EC50) = 248 µM. Serendipitously, we also were able to observe that CD04872SC inhibited the infection of the SARS-CoV-2 variants; Delta (EC50 = 152 µM) and Omicron (EC50 = 308 µM). These properties may define CD04872SC as a potential broad-spectrum candidate lead for the development of treatments for COVID-19.

Reversible lysine fatty acylation of an anchoring protein mediates adipocyte adrenergic signaling.

N-myristoylation on glycine is an irreversible modification that has long been recognized to govern protein localization and function. In contrast, the biological roles of lysine myristoylation remain ill-defined. We demonstrate that the cytoplasmic scaffolding protein, gravin-α/A kinase-anchoring protein 12, is myristoylated on two lysine residues embedded in its carboxyl-terminal protein kinase A (PKA) binding domain. Histone deacetylase 11 (HDAC11) docks to an adjacent region of gravin-α and demyristoylates these sites. In brown and white adipocytes, lysine myristoylation of gravin-α is required for signaling via ß2- and ß3-adrenergic receptors (ß-ARs), which are G protein-coupled receptors (GPCRs). Lysine myristoylation of gravin-α drives ß-ARs to lipid raft membrane microdomains, which results in PKA activation and downstream signaling that culminates in protective thermogenic gene expression. These findings define reversible lysine myristoylation as a mechanism for controlling GPCR signaling and highlight the potential of inhibiting HDAC11 to manipulate adipocyte phenotypes for therapeutic purposes.

AKAP12 Signaling Complex: Impacts of Compartmentalizing cAMP-Dependent Signaling Pathways in the Heart and Various Signaling Systems.

Heart failure is a complex clinical syndrome, represented as an impairment in ventricular filling and myocardial blood ejection. As such, heart failure is one of the leading causes of death in the United States. With a mortality rate of 1 per 8 individuals and a prevalence of 6.2 million Americans, it has been projected that heart failure prevalence will increase by 46% by 2030. Cardiac remodeling (a general determinant of heart failure) is regulated by an extensive network of intertwined intracellular signaling pathways. The ability of signalosomes (molecular signaling complexes) to compartmentalize several cellular pathways has been recently established. These signalosome signaling complexes provide an additional level of specificity to general signaling pathways by regulating the association of upstream signals with downstream effector molecules. In cardiac myocytes, the AKAP12 (A-kinase anchoring protein 12) scaffolds a large signalosome that orchestrates spatiotemporal signaling through stabilizing pools of phosphatases and kinases. Predominantly upon ß-AR (ß2-adrenergic-receptor) stimulation, the AKAP12 signalosome is recruited near the plasma membrane and binds tightly to ß-AR. Thus, one major function of AKAP12 is compartmentalizing PKA (protein kinase A) signaling near the plasma membrane. In addition, it is involved in regulating desensitization, downregulation, and recycling of ß-AR. In this review, the critical roles of AKAP12 as a scaffold protein in mediating signaling downstream GPCRs (G protein-coupled receptor) are discussed with an emphasis on its reported and potential roles in cardiovascular disease initiation and progression.

The effects of hookah/waterpipe smoking on general health and the cardiovascular system.

Hookah or waterpipe smoking or use is an emerging trend in the US population, especially among the youth. The misperception of hookah being less harmful than cigarettes and the availability of different but "appealing" flavors are considered among the main reasons for this trend. Hookah users however are exposed to many of the same toxic compounds/by-products as cigarette users, but at dramatically higher levels, which might lead to more severe negative health effects. In fact, hookah users are at risks of infections, cancers, lung disease, and other medical conditions. Moreover, because of the overlapping toxicant/chemical profile to conventional cigarettes, hookah smoke effects on the cardiovascular system are thought to be comparable to those of conventional cigarettes. A major source of tobacco addiction is nicotine, whose levels in hookah are extremely variable as they depend on the type of tobacco used. Taken together, in this review of literature, we will provide insights on the negative health effects of hookah in general, with a focus on what is known regarding its impact on the cardiovascular system.

Arhgef1 Plays a Vital Role in Platelet Function and Thrombogenesis.

Background Platelets are the cellular mediators of hemostasis and thrombosis, and their function is regulated by a number of molecular mediators, such as small GTP ases. These small GTP ases are themselves regulated by guanine nucleotide exchange factors such as Arhgefs, several of which are found in platelets, including the highly expressed Arhgef1. However, the role of Arhgef1 in platelets has not yet been investigated. Methods and Results We employed mice with genetic deletion of Arhgef1 (ie, Arhgef1-/-) and investigated their platelet phenotype by employing a host of in vivo and in vitro platelet assays. Our results indicate that Arhgef1-/- mice had prolonged carotid artery occlusion and tail bleeding times. Moreover, platelets from these mice exhibited defective aggregation, dense and α granule secretion, α II bß3 integrin activation, clot retraction and spreading, in comparison to their wild-type littermates. Finally, we also found that the mechanism by which Arhgef1 regulates platelets is mediated in part by a defect in the activation of the RhoA-Rho-associated kinase axis, but not Rap1b. Conclusions Our data demonstrate, for the first time, that Arhgef1 plays a critical role in platelet function, in vitro and in vivo.

Regulator of G-Protein Signaling 16 Is a Negative Modulator of Platelet Function and Thrombosis.

Background Members of the regulator of G-protein signaling ( RGS ) family inhibit G-protein coupled receptor signaling by modulating G-protein activity. In platelets, there are 3 different RGS isoforms that are expressed at the protein level, including RGS 16. Recently, we have shown that CXCL 12 regulates platelet function via RGS 16. However, the role of RGS 16 in platelet function and thrombus formation is poorly defined. Methods and Results We used a genetic knockout mouse model approach to examine the role(s) of RGS 16 in platelet activation by using a host of in vitro and in vivo assays. We observed that agonist-induced platelet aggregation, secretion, and integrin activation were much more pronounced in platelets from the RGS 16 knockout ( Rgs16 -/-) mice relative to their wild type ( Rgs16 +/+) littermates. Furthermore, the Rgs16 -/- mice had a markedly shortened bleeding time and were more susceptible to vascular injury-associated thrombus formation than the controls. Conclusions These findings support a critical role for RGS 16 in regulating hemostatic and thrombotic functions of platelets in mice. Hence, RGS 16 represents a potential therapeutic target for modulating platelet function.

Short-Term E-Cigarette Exposure Increases the Risk of Thrombogenesis and Enhances Platelet Function in Mice.

BACKGROUND: Cardiovascular disease is the main cause of death in the United States, with smoking being the primary preventable cause of premature death, and thrombosis being the main mechanism of cardiovascular mortality in smokers. Due to the perception that electronic/e-cigarettes are "safer/less harmful" than conventional cigarettes, their usage-among a variety of ages-has increased tremendously during the past decade. Notably, there are limited studies regarding the negative effects of e-cigarettes on the cardiovascular system, which is also the subject of significant debate. METHODS AND RESULTS: We employed a passive e-VapeTM vapor inhalation system and developed an in vivo whole-body e-cigarette mouse exposure protocol that mimics real-life human exposure scenarios/conditions and investigated the effects of e-cigarettes and clean air on platelet function and thrombogenesis. Our results show that platelets from e-cigarette-exposed mice are hyperactive, with enhanced aggregation, dense and α granule secretion, activation of the αIIbß3 integrin, phosphatidylserine expression, and Akt and ERK activation, when compared with clean air-exposed platelets. E-cigarette-exposed platelets were also found to be resistant to inhibition by prostacyclin, relative to clean air. Furthermore, the e-cigarette-exposed mice exhibited a shortened thrombosis occlusion and bleeding times. CONCLUSIONS: Taken together, our data demonstrate for the first time that e-cigarettes alter physiological hemostasis and increase the risk of thrombogenic events. This is attributable, at least in part, to the hyperactive state of platelets. Thus, the negative health consequences of e-cigarette exposure should not be underestimated and warrant further investigation.

Role of IκB kinase ß in regulating the remodeling of the CARMA1-Bcl10-MALT1 complex.

The current work investigates the notion that inducible clustering of signaling mediators of the IKK pathway is important for platelet activation. Thus, while the CARMA1, Bcl10, and MALT1 (CBM) complex is essential for triggering IKK/NF-κB activation upon platelet stimulation, the signals that elicit its formation and downstream effector activation remain elusive. We demonstrate herein that IKKß is involved in membrane fusion, and serves as a critical protein kinase required for initial formation and the regulation of the CARMA1/MALT1/Bcl10/CBM complex in platelets. We also show that IKKß regulates these processes via modulation of phosphorylation of Bcl10 and IKKγ polyubiquitination. Collectively, our data demonstrate that IKKß regulates membrane fusion and the remodeling of the CBM complex formation.