Adenylyl Cyclase Type Research Articles

Overexpression of a Neuronal Type Adenylyl Cyclase (Type 8) in Sinoatrial Node Markedly Impacts Heart Rate and Rhythm

Heart rate (HR) and HR variability (HRV), predictors of over-all organism health, are widely believed to be driven by autonomic input to the sinoatrial node (SAN), with sympathetic input increasing HR and reducing HRV. However, variability in spontaneous beating intervals in isolated SAN tissue and single SAN cells, devoid of autonomic neural input, suggests that clocks intrinsic to SAN cells may also contribute to HR and HRV in vivo. We assessed contributions of both intrinsic and autonomic neuronal input mechanisms of SAN cell function on HR and HRV via in vivo, telemetric EKG recordings. This was done in both wild type (WT) mice, and those in which adenylyl cyclase type 8 (ADCY8), a main driver of intrinsic cAMP-PKA-Ca2+ mediated pacemaker function, was overexpressed exclusively in the heart (TGAC8). We hypothesized that TGAC8 mice would: (1) manifest a more coherent pattern of HRV in vivo, i.e., a reduced HRV driven by mechanisms intrinsic to SAN cells, and less so to modulation by autonomic input and (2) utilize unique adaptations to limit sympathetic input to a heart with high levels of intrinsic cAMP-Ca2+ signaling. Increased adenylyl cyclase (AC) activity in TGAC8 SAN tissue was accompanied by a marked increase in HR and a concurrent marked reduction in HRV, both in the absence or presence of dual autonomic blockade. The marked increase in intrinsic HR and coherence of HRV in TGAC8 mice occurred in the context of: (1) reduced HR and HRV responses to β-adrenergic receptor (β-AR) stimulation; (2) increased transcription of genes and expression of proteins [β-Arrestin, G Protein-Coupled Receptor Kinase 5 (GRK5) and Clathrin Adaptor Protein (Dab2)] that desensitize β-AR signaling within SAN tissue, (3) reduced transcripts or protein levels of enzymes [dopamine beta-hydorxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT)] required for catecholamine production in intrinsic cardiac adrenergic cells, and (4) substantially reduced plasma catecholamine levels. Thus, mechanisms driven by cAMP-PKA-Ca2+ signaling intrinsic to SAN cells underlie the marked coherence of TGAC8 mice HRV. Adaptations to limit additional activation of AC signaling, via decreased neuronal sympathetic input, are utilized to ensure the hearts survival and prevent Ca2+ overload.

Constitutive inhibitory G protein activity upon adenylyl cyclase-dependent cardiac contractility is limited to adenylyl cyclase type 6.

PurposeWe previously reported that inhibitory G protein (Gi) exerts intrinsic receptor-independent inhibitory activity upon adenylyl cyclase (AC) that regulates contractile force in rat ventricle. The two major subtypes of AC in the heart are AC5 and AC6. The aim of this study was to determine if this intrinsic Gi inhibition regulating contractile force is AC subtype selective.MethodsWild-type (WT), AC5 knockout (AC5KO) and AC6 knockout (AC6KO) mice were injected with pertussis toxin (PTX) to inactivate Gi or saline (control).Three days after injection, we evaluated the effect of simultaneous inhibition of phosphodiesterases (PDE) 3 and 4 with cilostamide and rolipram respectively upon in vivo and ex vivo left ventricular (LV) contractile function. Also, changes in the level of cAMP were measured in left ventricular homogenates and at the membrane surface in cardiomyocytes obtained from the same mouse strains expressing the cAMP sensor pmEPAC1 using fluorescence resonance energy transfer (FRET).ResultsSimultaneous PDE3 and PDE4 inhibition increased in vivo and ex vivo rate of LV contractility only in PTX-treated WT and AC5KO mice but not in saline-treated controls. Likewise, Simultaneous PDE3 and PDE4 inhibition elevated total cAMP levels in PTX-treated WT and AC5KO mice compared to saline-treated controls. In contrast, simultaneous PDE3 and PDE4 inhibition did not increase in vivo or ex vivo rate of LV contractility or cAMP levels in PTX-treated AC6KO mice compared to saline-treated controls. Using FRET analysis, an increase of cAMP level was detected at the membrane of cardiomyocytes after simultaneous PDE3 and PDE4 inhibition in WT and AC5KO but not AC6KO. These FRET data are consistent with the functional data indicating that AC6 activity and PTX inhibition of Gi is necessary for simultaneous inhibition of PDE3 and PDE4 to elicit an increase in contractility.ConclusionsTogether, these data suggest that AC6 is tightly regulated by intrinsic receptor-independent Gi activity, thus providing a mechanism for maintaining low basal cAMP levels in the functional compartment that regulates contractility.

1813-P: An Experimental Model to Combat the Increased Prevalence of Diabetes in Alzheimer’s Disease

Alzheimer’s disease is associated with many other disorders of healthful aging; one of the most prominent is the increased prevalence of diabetes in patients with Alzheimer’s, resulting in almost twice the number of hospitalizations compared with that of non-Alzheimer’s patients. The goal of this investigation was to discover an animal model that was protected both from Alzheimer’s Disease and diabetes. The adenylyl cyclase type 5 (AC5) knockout (KO) was selected since it not only lives a third longer than its wild type (WT), but is also a model of healthful aging, with improved metabolism, enhanced exercise capacity and protection against obesity and diabetes. Accordingly, we compared the AC5 KO with that of the J20 mouse, a model for Alzheimer’s disease. Comparisons were made among AC5 KO, J20 and WT mice for behavioral tests indicative of Alzheimer’s and diabetes protection as reflected by the glucose tolerance test. All 3 behavioral tests studied demonstrated that AC5 KO were protected compared to WT, whereas the J20 mice demonstrated behavioral alterations consistent with Alzheimer’s Disease. For example, the water maze test, used to assess spatial learning and memory, demonstrated the expected 134± 7.6% deficit, p<0.05, in the J20 mice, in contrast to the 38± 3.0% improvement, in performance, p<0.05, in AC5 KO. The glucose tolerance test (GTT) also demonstrated protection in AC5 KO and impaired GTT in the J20 mice, i.e., the area under the curve following 1g/kg IP glucose, was 47± 5.2% less, p<0.05, in AC5 KO compared to WT, whereas it was 137± 10.2% higher, p<.05, than WT in the J20 mice. Thus, the AC5 KO mice were protected both against impaired GTT and behavioral changes associated with Alzheimer’s disease, suggesting that inhibition of AC5 would be a novel therapeutic modality, not only for diabetes, but also for the diabetes associated with Alzheimer’s Disease. Disclosure D. Babici: None. J. Zhang: None. M. Oydanich: None. K. Nishimura: None. D.E. Vatner: None. M. Mouradian: None. S.F. Vatner: None. Funding National Institutes of Health (R01HL102472, R01HL106511, R01HL119464, R01HL124282, R01HL130848, R01HL137368, R01HL137405, R21AG053514)

Effect of Adenylyl Cyclase Type 6 on Localized Production of cAMP by β-2 Adrenoceptors in Human Airway Smooth-Muscle Cells.

β 2-Adrenoceptors (β 2ARs) are concentrated in caveolar lipid raft domains of the plasma membrane in airway smooth-muscle (ASM) cells, along with adenylyl cyclase type 6 (AC6). This is believed to contribute to how these receptors can selectively regulate certain types of cAMP-dependent responses in these cells. The goal of the present study was to test the hypothesis that β 2AR production of cAMP is localized to specific subcellular compartments using fluorescence resonance energy transfer-based cAMP biosensors targeted to different microdomains in human ASM cells. Epac2-MyrPalm and Epac2-CAAX biosensors were used to measure responses associated with lipid raft and nonraft regions of the plasma membrane, respectively. Activation of β 2ARs with isoproterenol produced cAMP responses that are most readily detected in lipid raft domains. Furthermore, overexpression of AC6 somewhat paradoxically inhibited β 2AR production of cAMP in lipid raft domains without affecting β 2AR responses detected in other subcellular locations or cAMP responses to EP2 prostaglandin receptor activation, which were confined primarily to nonraft domains of the plasma membrane. The inhibitory effect of overexpressing AC6 was blocked by inhibition of phosphodiesterase type 4 (PDE4) activity with rolipram, inhibition of protein kinase A (PKA) activity with H89, and inhibition of A kinase anchoring protein (AKAP) interactions with the peptide inhibitor Ht31. These results support the idea that overexpression of AC6 leads to enhanced feedback activation of PDE4 via phosphorylation by PKA that is part of an AKAP-dependent signaling complex. This provides insight into the molecular basis for localized regulation of cAMP signaling in human ASM cells.

A Novel Drug to Reduce Myocardial Infarct Size, Even When Administered After Coronary Artery Reperfusion

Despite the thousands of studies on drug therapy designed to reduce myocardial infarct size (IS), almost none have been successfully translated to the clinics. A major reason for failure is that many of these drugs are only effective when administered before coronary artery reperfusion (CAR), which limits their utility in the clinical setting, where CAR must be instituted immediately in patients after the myocardial infarction (MI) diagnosis has been made, not leaving time for drug therapy. It is also important that the drug does not increase myocardial oxygen consumption. In our prior work on the adenylyl cyclase type 5 (AC5) knock out (KO) mouse model, we found that it enhanced longevity, protected against diabetes and obesity, improved exercise performance and reduced IS after coronary artery occlusion (CAO) and CAR. Based on these studies, and since it is difficult to delete genes in humans, we developed a novel pharmacological candidate to inhibit AC5, named C90. C90 reduced AC activity and cAMP in response to forskolin more than vehicle, (12.9±1.9 vs. 14.2 ± 3.1 pmol/mg/min). However, in AC5 KO C90 no longer reduced cAMP in response to forskolin, indicating its selectivity for AC5 inhibition. The goal of this investigation was to examine the new AC5 inhibitor, C90, and compare its efficacy in reducing IS when the drug was delivered after CAR with that of adenosine, the gold standard for ischemic cardioprotection. Wild type (WT) mice were subjected to 30 min CAO followed by 24 hours CAR. C90 or vehicle was then administered at 5 min after CAR by intravenous (IV) administration at doses of 0.01, 0.03, 0.06, 0.6, and 1.2 mg/kg. The area at risk was similar in all groups, determined by triphenyltetrazolium chloride (TTC) staining. IS was reduced in a dose‐response manner by C90. IS was reduced by 32% at a dose of 0.06 mg/kg of C90, whereas the same dose of adenosine reduced IS similarly when administered before CAR but failed to reduce IS when administered after CAR. C90 further reduced IS by 64% at a dose of 0.6 mg/kg and by 71% at a dose of 1.2 mg/kg. When C90 was delivered at 10 min after CAR, it still IS by 50% and 53% at doses of 0.6 mg/kg and 1.2 mg/kg, respectively. In addition, LV ejection fraction (LV EF) was also examined at 4 weeks after myocardial infarction in mice, and was preserved in the C90 group (LVEF = 65±1.6%) vs. the vehicle group (LVEF = 52±2.9%). C90 also reduced IS by 49% in the Watanabe rabbit, model of atherosclerosis. Thus, C90, an inhibitor of AC5, is a novel target for clinical development, since it reduces myocardial IS when delivered after reperfusion, a key feature for patients coming to the emergency room, where reperfusion must be carried out before additional therapy. An additional advantageous feature of C90 and inhibition of AC5, is that it reduces beta‐adrenergic receptor signaling and myocardial oxygen consumption, which are important for patients with heart disease.Support or Funding InformationNIH R21AG053514; NIH R44 HL142390; NIH R44HL112512This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An NPY Y1 receptor antagonist unmasks latent sensitization and reveals the contribution of protein kinase A and Epac to chronic inflammatory pain.

Peripheral inflammation produces a long-lasting latent sensitization of spinal nociceptive neurons, that is, masked by tonic inhibitory controls. We explored mechanisms of latent sensitization with an established four-step approach: (1) induction of inflammation; (2) allow pain hypersensitivity to resolve; (3) interrogate latent sensitization with a channel blocker, mutant mouse, or receptor antagonist; and (4) disrupt compensatory inhibition with a receptor antagonist so as to reinstate pain hypersensitivity. We found that the neuropeptide Y Y1 receptor antagonist BIBO3304 reinstated pain hypersensitivity, indicative of an unmasking of latent sensitization. BIBO3304-evoked reinstatement was not observed in AC1 knockout mice and was prevented with intrathecal co-administration of a pharmacological blocker to the N-methyl-D-aspartate receptor (NMDAR), adenylyl cyclase type 1 (AC1), protein kinase A (PKA), transient receptor potential cation channel A1 (TRPA1), channel V1 (TRPV1), or exchange protein activated by cAMP (Epac1 or Epac2). A PKA activator evoked both pain reinstatement and touch-evoked pERK expression in dorsal horn; the former was prevented with intrathecal co-administration of a TRPA1 or TRPV1 blocker. An Epac activator also evoked pain reinstatement and pERK expression. We conclude that PKA and Epac are sufficient to maintain long-lasting latent sensitization of dorsal horn neurons that is kept in remission by the NPY-Y1 receptor system. Furthermore, we have identified and characterized 2 novel molecular signaling pathways in the dorsal horn that drive latent sensitization in the setting of chronic inflammatory pain: NMDAR→AC1→PKA→TRPA1/V1 and NMDAR→AC1→Epac1/2. New treatments for chronic inflammatory pain might either increase endogenous NPY analgesia or inhibit AC1, PKA, or Epac.

Cardiac-Directed Expression of Adenylyl Cyclase Catalytic Domain (C1C2) Attenuates Deleterious Effects of Pressure Overload.

A fusion protein (C1C2) constructed by fusing the intracellular C1 and C2 segments of adenylyl cyclase type 6 (AC6) retains beneficial effects of AC6 expression, without increasing cyclic adenosine monophosphate generation. The effects of cardiac-directed C1C2 expression in pressure overload is unknown. Left ventricular (LV) pressure overload was induced by transverse aortic constriction (TAC) in C1C2 mice and in transgene negative (TG-) mice. Four weeks after TAC, LV systolic function and diastolic function were measured, and Ca2+ handling was assessed. Four weeks after TAC, TG- animals showed reduced LV peak +dP/dt. LV peak +dP/dt in C1C2 mice was statistically indistinguishable from that of normal mice and was higher than that seen in TG- mice 4 weeks after TAC (p = 0.02), despite similar and substantial cardiac hypertrophy. In addition to higher LV peak +dP/dt in vivo, cardiac myocytes from C1C2 mice showed shorter time-to-peak Ca2+ transient amplitude (p = 0.002) and a reduced time constant of cytosolic Ca2+ decline (Tau; p = 0.003). Sarcomere shortening fraction (p < 0.03) and the rate of sarcomere shortening (p < 0.02) increased in C1C2 cardiac myocytes. Myofilament sensitivity to Ca2+ was increased in systole (p = 0.02) and diastole (p = 0.04) in C1C2 myocytes. These findings indicate enhanced Ca2+ handling associated with C1C2 expression. Favorable effects on Ca2+ handling and LV function were associated with increased LV SERCA2a protein content (p = 0.015) and reduced LV fibrosis (p = 0.008). Cardiac-directed C1C2 expression improves Ca2+ handling and increases LV contractile function in pressure overload. These data provide a rationale for further exploration of C1C2 gene transfer as a potential treatment for heart failure.

Epidermal growth factor receptor controls glycogen phosphorylase in T cells through small GTPases of the RAS family

We recently uncovered a regulatory pathway of the muscle isoform of glycogen phosphorylase (PYGM) that plays an important role in regulating immune function in T cells. Here, using various enzymatic, pulldown, and immunoprecipitation assays, we describe signaling cross-talk between the small GTPases RAS and RAP1A, member of RAS oncogene family (RAP1) in human Kit 225 lymphoid cells, which, in turn, is regulated by the epidermal growth factor receptor (EGFR). We found that this communication bridge is essential for glycogen phosphorylase (PYG) activation through the canonical pathway because this enzyme is inactive in the absence of adenylyl cyclase type 6 (ADCY6). PYG activation required stimulation of both exchange protein directly activated by cAMP 2 (EPAC2) and RAP1 via RAS and ADCY6 phosphorylation, with the latter being mediated by Raf-1 proto-oncogene, Ser/Thr kinase (RAF1). Consistent with this model, PYG activation was EGFR-dependent and may be initiated by the constitutively active form of RAS. Consequently, PYG activation in Kit 225 T cells could be blocked with specific inhibitors of RAS, EPAC, RAP1, RAF1, ADCY6, and cAMP-dependent protein kinase. Our results establish a new paradigm for the mechanism of PYG activation, which depends on the type of receptor involved.

Methylglyoxal and a spinal TRPA1-AC1-Epac cascade facilitate pain in the db/db mouse model of type 2 diabetes.

Painful diabetic neuropathy (PDN) is a devastating neurological complication of diabetes. Methylglyoxal (MG) is a reactive metabolite whose elevation in the plasma corresponds to PDN in patients and pain-like behavior in rodent models of type 1 and type 2 diabetes. Here, we addressed the MG-related spinal mechanisms of PDN in type 2 diabetes using db/db mice, an established model of type 2 diabetes, and intrathecal injection of MG in conventional C57BL/6J mice. Administration of either a MG scavenger (GERP10) or a vector overexpressing glyoxalase 1, the catabolic enzyme for MG, attenuated heat hypersensitivity in db/db mice. In C57BL/6J mice, intrathecal administration of MG produced signs of both evoked (heat and mechanical hypersensitivity) and affective (conditioned place avoidance) pain. MG-induced Ca2+ mobilization in lamina II dorsal horn neurons of C57BL/6J mice was exacerbated in db/db, suggestive of MG-evoked central sensitization. Pharmacological and/or genetic inhibition of transient receptor potential ankyrin subtype 1 (TRPA1), adenylyl cyclase type 1 (AC1), protein kinase A (PKA), or exchange protein directly activated by cyclic adenosine monophosphate (Epac) blocked MG-evoked hypersensitivity in C57BL/6J mice. Similarly, intrathecal administration of GERP10, or inhibitors of TRPA1 (HC030031), AC1 (NB001), or Epac (HJC-0197) attenuated hypersensitivity in db/db mice. We conclude that MG and sensitization of a spinal TRPA1-AC1-Epac signaling cascade facilitate PDN in db/db mice. Our results warrant clinical investigation of MG scavengers, glyoxalase inducers, and spinally-directed pharmacological inhibitors of a MG-TRPA1-AC1-Epac pathway for the treatment of PDN in type 2 diabetes.

Functional characterization of AC5 gain-of-function variants: Impact on the molecular basis of ADCY5-related dyskinesia

Adenylyl cyclases are key points for the integration of stimulatory and inhibitory G protein-coupled receptor (GPCR) signals. Adenylyl cyclase type 5 (AC5) is highly expressed in striatal medium spiny neurons (MSNs), and is known to play an important role in mediating striatal dopaminergic signaling. Dopaminergic signaling from the D1 expressing MSNs of the direct pathway, as well as the D2 expressing MSNs of the indirect pathway both function through the regulation of AC5 activity, controlling the production of the 2nd messenger cAMP, and subsequently the downstream effectors. Here, we used a newly developed cell line that used Crispr-Cas9 to eliminate the predominant adenylyl cyclase isoforms to more accurately characterize a series of AC5 gain-of-function mutations which have been identified in ADCY5-related dyskinesias. Our results demonstrate that these AC5 mutants exhibit enhanced activity to Gαs-mediated stimulation in both cell and membrane-based assays. We further show that the increased cAMP response at the membrane effectively translates into increased downstream gene transcription in a neuronal model. Subsequent analysis of inhibitory pathways show that the AC5 mutants exhibit significantly reduced inhibition following D2 dopamine receptor activation. Finally, we demonstrate that an adenylyl cyclase “P-site” inhibitor, SQ22536 may represent an effective future therapeutic mechanism by preferentially inhibiting the overactive AC5 gain-of-function mutants.

Insights into the Regulatory Properties of Human Adenylyl Cyclase Type 9

Membrane-bound adenylyl cyclase (AC) isoforms have distinct regulatory mechanisms that contribute to their signaling specificity and physiologic roles. Although insight into the physiologic relevance of AC9 has progressed, the understanding of AC9 regulation is muddled with conflicting studies. Currently, modes of AC9 regulation include stimulation by Gαs, protein kinase C (PKC) βII, or calcium-calmodulin kinase II (CaMKII) and inhibition by Gαi/o, novel PKC isoforms, or calcium-calcineurin. Conversely, the original cloning of human AC9 reported that AC9 is insensitive to Gαi inhibition. The purpose of our study was to clarify which proposed regulators of AC9 act directly or indirectly, particularly with respect to Gαi/o. The proposed regulators, including G proteins (Gαs, Gαi, Gαo, Gβγ), protein kinases (PKCβII, CaMKII), and forskolin, were systematically evaluated using classic in vitro AC assays and cell-based cAMP accumulation assays in COS-7 cells. Our studies show that AC9 is directly regulated by Gαs with weak conditional activation by forskolin; other modes of proposed regulation either occur indirectly or possibly require additional scaffolding proteins to facilitate regulation. We also show that AC9 contributes to basal cAMP production; knockdown or knockout of endogenous AC9 reduces basal AC activity in COS-7 cells and splenocytes. Importantly, although AC9 is not directly inhibited by Gαi/o, it can heterodimerize with Gαi/o-regulated isoforms, AC5 and AC6.

Optimization of a 1,3,4-oxadiazole series for inhibition of Ca2+/calmodulin-stimulated activity of adenylyl cyclases 1 and 8 for the treatment of chronic pain

Adenylyl cyclases type 1 (AC1) and 8 (AC8) are group 1 transmembrane adenylyl cyclases (AC) that are stimulated by Ca2+/calmodulin. Studies have shown that mice depleted of AC1 have attenuated inflammatory pain response, while AC1/AC8 double-knockout mice display both attenuated pain response and opioid dependence. Thus, AC1 has emerged as a promising new target for treating chronic pain and opioid abuse. We discovered that the 1,3,4-oxadiazole scaffold inhibits Ca2+/calmodulin-stimulated cyclic adenosine 3‘,5‘-monophosphate (cAMP) production in cells stably expressing either AC1 or AC8. We then carried out structure-activity relationship studies, in which we designed and synthesized 65 analogs, to modulate potency and selectivity versus each AC isoform in cells. Furthermore, molecular docking of the analogs into an AC1 homology model suggests the molecules may bind at the ATP binding site. Finally, a prioritized analog was tested in a mouse model of inflammatory pain and exhibited modest analgesic properties. In summary, our data indicate the 1,3,4-oxadiazoles represent a novel scaffold for the cellular inhibition of Ca2+/calmodulin-stimulated AC1- and AC8 cAMP and warrant further exploration as potential lead compounds for the treatment of chronic inflammatory pain.

Antinociceptive effects of the adenylyl cyclase inhibitor ST034307 on tooth-movement-induced nociception in rats

ObjectiveThis study aimed to investigate the antinociceptive effects of the selective adenylyl cyclase type 1 (AC1) inhibitor ST034307 on tooth movement nociception through orofacial nociceptive behavior tests and molecular examination. MethodsWe placed fixed nickel-titanium alloy closed-coil springs around the incisors of male Sprague-Dawley rats to induce tooth movement. We subsequently administered ST034307 (3 mg/kg), for 2 days, intraperitoneally, and then subjected the rats to a battery of behavioral tests (n = 10/group) to assess orofacial nociception. The changes in the expression of key molecules in the anterior cingulate cortex were measured by ELISA (n = 8/group) and Western blotting (n = 8/group). ResultsTooth movement increased face-grooming activities and rat grimace scale scores. Tooth movement was also associated with enhanced cyclic adenosine monophosphate (cAMP) generation as well as protein kinase A (PKA) activation. Moreover, the phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and expression of N-methyl-d-aspartate (NMDA) receptors in the anterior cingulate cortex increased during tooth movement. ST034307 significantly decreased mouth wiping and rat grimace scale scores, accompanied by reductions in cAMP generation, PKA activation, AMPA receptor phosphorylation, and NMDA receptor expression in the anterior cingulate cortex. ConclusionsThese results suggest that adenylyl cyclase type 1 plays an important role in the development of orthodontic tooth movement nociception. Furthermore, ST034307 can be used as an effective pharmacotherapy for orthodontic nociception.

Pharmacological modulation of the CO2/HCO3-/pH-, calcium-, and ATP-sensing soluble adenylyl cyclase.

Cyclic AMP (cAMP), the prototypical second messenger, has been implicated in a wide variety of (often opposing) physiological processes. It simultaneously mediates multiple, diverse processes, often within a single cell, by acting locally within independently-regulated and spatially-restricted microdomains. Within each microdomain, the level of cAMP will be dependent upon the balance between its synthesis by adenylyl cyclases and its degradation by phosphodiesterases (PDEs). In mammalian cells, there are many PDE isoforms and two types of adenylyl cyclases; the G protein regulated transmembrane adenylyl cyclases (tmACs) and the CO2/HCO3-/pH-, calcium-, and ATP-sensing soluble adenylyl cyclase (sAC). Discriminating the roles of individual cyclic nucleotide microdomains requires pharmacological modulators selective for the various PDEs and/or adenylyl cyclases. Such tools present an opportunity to develop therapeutics specifically targeted to individual cAMP dependent pathways. The pharmacological modulators of tmACs have recently been reviewed, and in this review, we describe the current status of pharmacological tools available for studying sAC.

A novel adenylyl cyclase type 5 inhibitor that reduces myocardial infarct size even when administered after coronary artery reperfusion

We developed a novel adenylyl cyclase type 5 (AC5) inhibitor, C90, that reduces myocardial infarct size even when administered after coronary reperfusion. This is key, since it is not practical to administer a drug to a patient with myocardial infarction before revascularization, and is one reason why so many prior drugs, which reduced infarct in experimental animals, failed in clinical trials. C90 is the most potent AC5 inhibitor, as exhibited by its IC50 value for AC5 inhibition, which was 5 times lower than the next most potent AC5 inhibitor. C90 reduced cAMP in response to forskolin in wild type mice by 42%, but no longer reduced cAMP in response to forskolin in mice with disruption of AC5, indicating that the mechanism of C90 was specific for AC5 inhibition. Compared with vehicle treatment, C90 reduced infarct size by 64% at a dose of 0.6 mg/kg. Thus, C90 is a novel, selective and potent AC5 inhibitor that reduces infarct size, when delivered after coronary artery reperfusion, rendering it potentially clinically useful. It also reduces beta-adrenergic receptor signaling, which will provide additional benefit to patients with coronary artery disease or heart failure.

Design of a Phase 3 trial of intracoronary administration of human adenovirus 5 encoding human adenylyl cyclase type 6 (RT-100) gene transfer in patients with heart failure with reduced left ventricular ejection fraction: The FLOURISH Clinical Trial

The prognosis of patients with HFrEF remains poor despite the use of current medical and device therapies. Preclinical studies of HFrEF using IC delivery of RT-100, a replication deficient, E1/E3-deleted human adenovirus 5 encoding human AC6 was associated with favorable effects on LV function and remodeling. A recent multicenter, double-blind, placebo-controlled, phase 2 study demonstrated the safety of IC delivery of RT-100 in HFrEF patients and potential efficacy at the higher doses. This phase 2 dose finding study, which included doses not expected to be effective, identified a potential reduction in congestive heart failure admissions in the AC6-treated group one year after randomization. The FLOURISH study is designed to investigate the prospect of reduction of heart failure hospitalization and other clinical adverse events and improvement in EF.The FLOURISH study is a double-blind, placebo-controlled, multicenter Phase 3 clinical trial that will randomize 536 patients to a one-time IC administration of RT-100 (1012 vp) or placebo in a 1:1 ratio. Subjects will be 18–80 years of age, on optimal standard of care HF therapy with LVEF ≥10% and ≤35% by echocardiogram, and will undergo IC administration of RT-100 vs. placebo on Day 1. Follow-up study visits will be performed at Weeks 1 and 4, and Months 3, 6, and 12. Patients will be followed for an additional 36 months for safety assessments with telephone contact at Months 24, 36, and 48.The primary objective is to determine the efficacy of IC RT-100 vs. placebo in reducing the event rate of all (first and repeat) HF hospitalizations occurring from baseline to 12 months. The secondary objectives are to determine the efficacy of IC RT-100 on CV death, all cause death, and all HF events and in improving NYHA functional classification. Exploratory endpoints will include echocardiographic parameters of left ventricular systolic and diastolic function, HF symptoms and physical limitations, 6-minute walking distance, Borg dyspnea score, and NT-proBNP levels.The FLOURISH study, which received fast track designation from the Food and Drug Administration in December 2017, will further investigate the role of a one-time intracoronary injection of RT-100 in reducing HF hospitalizations and will serve as a registration trial (potentially pivotal investigation) for RT-100 as a treatment for HFrEF.

Cardiac Anxiety

Constitutive Ca2+-calmodulin activated adenylyl cyclase Type 8 (AC8) is crucial for pacemaker cell automaticity (1). When human AC8 is overexpressed in mice (TGAC8), in vivo heart rate measured using telemetry devices at 3 months age was more than 30% higher throughout the 24 hour period compared to wildtype (WT)(2). In other words, the heart appears to perpetuate exercise in the absence of corporal exertion. The goal of the present study was to measure ventricular contractility and heart rate to determine how long an increased workload on the heart can be sustained and whether it is associated with reduced longevity. We performed echocardiography under light anesthesia on TGAC8 and WT littermates at 3, 10, 14, and 19 months. Linear mixed effects models assessed the changes in the repeated measurements of the cardiac markers from the echocardiogram. Tukey's post-hoc test evaluated the pair-wise comparisons of the mean values in the cardiac markers at 3 and 19 months old. The rate of change of heart rate decreases with age in TGAC8 mice, while the rate of change of heart rate increases with age in WT mice (p<0.0005). The TGAC8 heart rate is significantly higher than WT at 3 months of age (p<0.0003). This difference disappears, however, at 19 months of age. The rate of change in ejection fraction (EF) decreases with age in TGAC8 mice, while the rate of change of EF stays constant with age in WT mice (p<0.0001). The TGAC8 EF is significantly higher than WT at 3 months of age (p<0.0001). At 19 months of age, the opposite is observed, the TGAC8 EF is lower than the EF of the WT (p<0.07). The rate of change in posterior wall thickness (PWT) increases with age in TGAC8 mice and WT (p<0.005). The TGAC8 PWT is significantly higher than WT at 3 months of age (p<0.005). However, the difference was not statistically different at 19 months of age. The rate of change in end-diastolic volume (EDV) increases with age in TGAC8 mice, while the rate of change of EDV stays constant with age in WT mice (p<0.0001). The TGAC8 EDV is significantly lower than WT at 3 months (p<0.0002), but significantly higher at 19 months of age (p<0.05). The median lifespan of the TGAC8 was observed to be approximately at 19 months of age, while the WT median lifespan was approximately at 27 months of age. The longevity of the TGAC8 mouse is significantly shorter than the WT (p<0.0001). The changes observed in echocardiography reveal progressive decline in cardiac performance in TGAC8 over time that is accompanied by reduced longevity. However, until approximately 14 months cardiac performance was maintained without any excess mortality. Our study demonstrates how long-term hypercontractility and chronotropic stress leads to accelerated deterioration in cardiac function and reduced longevity. Further study will investigate the molecular mechanisms that undermine the maintenance of this chronic hyperfunctional heart while ensuring survival up to 14 months, and mechanisms that lead to reduced longevity. Support or Funding Information Research was supported by the National Institutes of Health/National Institute on Aging Intramural Research Training Program. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanisms of Gender Differences in Exercise Capacity

The examination of gender differences has been a recent priority of the NIH. It is well documented that female rodents exhibit higher exercise capacity than their male counterparts. We confirmed this in C57 wild‐type(WT) mice, where female mice exhibited greater, p&lt;0.05, maximal exercise capacity for running distance (469 ± 24 m) than age‐matched male WT mice (337 ± 14 m). The first goal was to examine whether these differences persisted with models of enhanced exercise capacity, e.g. exercise training and the adenylyl cyclase type 5 knockout (AC5 KO) mouse. When WT mice underwent chronic exercise‐training, female mice maintained their superiority over males, with females running a 32% greater distance (p&lt;0.05) than age‐matched trained males. However, AC5 KO mice, which demonstrated enhanced exercise capacity, did not exhibit a gender difference, with female AC5 KO mice (695 ± 51 m) running similar distances to male AC5 KO mice (661 ± 45 m). The next goal was to determine mechanisms mediating these differences. After ovariectomy, WT female mice no longer demonstrated enhanced exercise compared with males. Conversely, chronic administration of estrogen to male mice improved their exercise performance and eliminated the gender differences with exercise. Next we investigated nitric oxide (NO), a downstream target of estrogen. Total NO synthase (NOS) activity was higher in female mice compared with male mice in both sedentary (207 ± 14 U/mg vs. 138 ± 4 U/mg, p&lt;0.05) and exercise trained groups (338 ± 42 vs. 193 ± 15 U/mg, p&lt;0.05). Ovariectomized females exhibited nitric oxide levels similar to males (165 ± 11 U/mg). Administration of L‐NAME, resulting in NO blockade, eliminated the gender differences in exercise performance. In AC5 KO mice NOS activity was not different in male and female mice and similar exercise performance in males and females was still observed after treatment with L‐NAME. Thus, both estrogen and nitric oxide are key mechanisms mediating the enhanced exercise performance in female mice.Support or Funding InformationThis study was supported by the National Institute of Health grant R01HL106511This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elevated Vasodilatory Cyclases and Shorter Telomere Length Contribute to High-Altitude Pulmonary Edema.

High-altitude (HA) genetics is complex with respect to health and disease (HA pulmonary edema i.e., HAPE). Based on the widely recognized fact that oxidative stress is a major trigger of several physiological processes, this study was designed to establish the significance of vasodilatory cyclases and telomere length in HA physiology. The study was performed in three groups, namely HAPE-free sojourners (HAPE-f, n = 150), HAPE patients (HAPE-p, n = 150), and healthy highland natives or highlanders (HLs, n = 150). Variations in soluble guanylyl cyclase β1-subunit (GUCY1B3) and adenylyl cyclase type 6 (ADCY6) were genotyped by the SNaPshot method and/or Fluidigm SNP type genotyping. Plasma GUCY1B3 and ADCY6 levels were estimated using ELISA, and relative telomere length was estimated by qRT-PCR. The rs7638AA genotype was over-represented in HLs compared with HAPE-f and HAPE-p (p = 0.035 and p = 0.012, respectively). Similarly, the rs7638A allele was prevalent in HLs compared with both groups, but significance was attained against HAPE-p (p = 0.012). Significantly elevated plasma levels of GUCY1B3 and ADCY6 were obtained in HAPE-p compared with HAPE-f (p = 0.001 and p = 0.006, respectively) and HLs (p = 3.31E-05 and p = 0.05, respectively). Shorter telomere length was observed in HAPE-p compared with HAPE-f (p > 0.05) and HLs (p = 0.017). Elevated cyclases and shorter telomere length associate with HAPE pathophysiology.

Cardiac Overexpression of Human Adenylyl Cyclase Type 8 Elicits Wide Spread Adaptations to Limit Adrenergic Signaling in Sinoatrial Nodal Pacemaker Cells that Extend Well Beyond Desensitization of β-Adrenergic Receptors

Cardiac Overexpression of Human Adenylyl Cyclase Type 8 Elicits Wide Spread Adaptations to Limit Adrenergic Signaling in Sinoatrial Nodal Pacemaker Cells that Extend Well Beyond Desensitization of β-Adrenergic Receptors