Tong Tang

Intracoronary Adenovirus Encoding Adenylyl Cyclase VI Increases Left Ventricular Function in Heart Failure

Background—We tested the hypothesis that intracoronary delivery of an adenovirus encoding adenylyl cyclase type VI (Ad.ACVI) would be associated with increased left ventricular (LV) function in pigs with congestive heart failure. Methods and Results—Pigs (52±6 kg; n=16) underwent placement of pacemakers, LV pressure transducers, and left atrial and aortic catheters. Physiological and echocardiographic studies were obtained from conscious animals 13 days later, and pacing was initiated (220 bpm). Seven days later, measures of LV function were reduced, documenting severe LV dysfunction and dilation. Pigs then received intracoronary Ad.ACVI (1.4×1012 vp; n=7) or saline (PBS; n=9) (randomized, blinded), with concomitant infusion of nitroprusside (50 μg/min, 6.4 minutes) to increase gene transfer. Pacing was continued for 14 days, and final studies were obtained. The a priori key end point was change in LV dP/dt during isoproterenol infusion (pre-Ad.ACVI value minus value after 21 days of pacing). Pigs receiving Ad.ACVI showed a smaller decrease in both LV +dP/dt (P =0.0014) and LV −dP/dt (P =0.0008). Serial echocardiography showed that Ad.ACVI treatment was associated with increased LV function and reduced LV dilation and that end-systolic wall stress was reduced. AC-stimulated cAMP production was increased 1.7-fold in LV samples from Ad.ACVI-treated pigs (P =0.006), and B-type natriuretic peptide was reduced (0.035). Gene transfer was confirmed by polymerase chain reaction. Conclusions—ACVI gene transfer increases LV function and attenuates deleterious LV remodeling in congestive heart failure.

Natriuretic effect by exendin-4, but not the DPP-4 inhibitor alogliptin, is mediated via the GLP-1 receptor and preserved in obese type 2 diabetic mice.

Activation of the glucagon-like peptide (GLP)-1 receptor (GLP-1R) and inhibition of dipeptidyl peptidase-4 (DPP-4) are new antidiabetic strategies. The GLP-1R and DPP-4 are also expressed in the renal proximal tubular brush border, where they may regulate Na(+) reabsorption. Exendin-4 (EX4) is a naturally occurring antidiabetic polypeptide (from the saliva of the lizard Heloderma suspectum) and GLP-1R agonist; however, part of its nonglucoregulatory effects are through GLP-1R-independent mechanisms. DPP-4 cleaves and inactivates GLP-1; thus the natriuretic effect of DPP-4 inhibition may be mediated by the GLP-1R. We report that parenteral application of EX4 in wild-type mice induced a diuresis and natriuresis associated with increases in glomerular filtration rate, fractional urinary fluid and Na(+) excretion, and renal membrane expression of the Na(+)/H(+) exchanger NHE3 phosphorylated at S552 and S605, established consensus sites for cAMP-dependent PKA. These effects were absent in mice lacking the GLP-1R and independent of adenylyl cyclase 6. In comparison, parenteral application of the DPP-4 inhibitor alogliptin reduced plasma DPP-4 activity by 95% and induced a diuresis and natriuresis independent of the presence of the GLP-1R or changes in phosphorylated NHE3. The inhibitory effect on renal fluid and Na(+) reabsorption of EX4, but not alogliptin, was preserved in diabetic db/db mice and associated with a modest reduction in blood pressure. These results reveal mechanistic differences in how EX4 vs. DPP-4 inhibition induces diuresis and natriuresis under normal states, with preservation of GLP-1R-mediated, but not DPP-4 inhibitor-dependent, natriuretic mechanisms in a mouse model of obese type 2 diabetes.

Adenylyl Cyclase Type 6 Deletion Decreases Left Ventricular Function via Impaired Calcium Handling

Background— Adenylyl cyclases (ACs) are a family of effector molecules for G-protein–coupled receptors. The 2 ACs most abundantly expressed in cardiac myocytes are types 5 (AC5) and 6 (AC6), which have 65% amino acid homology. It has been speculated that coexpression of 2 AC types in cardiac myocytes represents redundancy, but the specific role of AC6 in cardiac physiology and its differences from AC5 remain to be defined. Methods and Results— We generated transgenic mice with targeted deletion of AC6. Deletion of AC6 was associated with reduced left ventricular contractile function (P=0.026) and relaxation (P=0.041). The absence of AC6 was associated with a 48% decay in &bgr;-adrenergic receptor–stimulated cAMP production in cardiac myocytes (P=0.003) and reduced protein kinase A activity (P=0.015). In addition, phospholamban phosphorylation was reduced (P=0.015), sarcoplasmic reticulum Ca2+-ATPase activity was impaired (P<0.0001), and cardiac myocytes showed marked abnormalities in calcium transient formation (P=0.001). Conclusions— The combination of impaired cardiac cAMP generation and calcium handling that result from AC6 deletion underlies abnormalities in left ventricular function. The biochemical and physiological consequences of AC6 deletion reveal it to be an important effector molecule in the adult heart, serving unique biological functions not replicated by AC5.

Increased Cardiac Adenylyl Cyclase Expression Is Associated With Increased Survival After Myocardial Infarction

Background— Cardiac-directed expression of adenylyl cyclase type VI (ACVI) in mice results in structurally normal hearts with normal basal heart rate and function but increased responses to catecholamine stimulation. We tested the hypothesis that increased left ventricular (LV) ACVI content would increase mortality after acute myocardial infarction (MI). Methods and Results— Transgenic mice with cardiac-directed ACVI expression and their transgene-negative littermates (control) underwent coronary ligation, and survival, infarct size, and LV size and function were assessed 1 to 7 days after MI. Mice with increased ACVI expression had increased survival (control 41%, ACVI 74%; P=0.004). Infarct size and myocardial apoptotic rates were similar in ACVI and control mice; however, ACVI mice had less LV dilation (P<0.001) and increased ejection fractions (P<0.03). Three days after MI, studies in isolated perfused hearts showed that basal LV +dP/dt was similar, but graded dobutamine infusion was associated with a more robust LV contractile response in ACVI mice (P<0.05). Increased LV function was associated with increases in cAMP generation (P=0.0002), phospholamban phosphorylation (P<0.04), sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) affinity for calcium (P<0.015), and reduced AV block (P=0.04). Conclusions— In acute MI, increased cardiac ACVI content attenuates adverse LV remodeling, preserves LV contractile function, and reduces mortality.

Adenylate Cyclase 6 Determines cAMP Formation and Aquaporin-2 Phosphorylation and Trafficking in Inner Medulla

Arginine vasopressin (AVP) enhances water reabsorption in the renal collecting duct by vasopressin V₂ receptor (V₂R)-mediated activation of adenylyl cyclase (AC), cAMP-promoted phosphorylation of aquaporin-2 (AQP2), and increased abundance of AQP2 on the apical membrane. Multiple isoforms of adenylate cyclase exist, and the roles of individual AC isoforms in water homeostasis are not well understood. Here, we found that levels of AC6 mRNA, the most highly expressed AC isoform in the inner medulla, inversely correlate with fluid intake. Moreover, mice lacking AC6 had lower levels of inner medullary cAMP, reduced abundance of phosphorylated AQP2 (at both serine-256 and serine-269), and lower urine osmolality than wild-type mice. Water deprivation or administration of the V₂R agonist dDAVP did not increase urine osmolality of AC6-deficient mice to the levels of wild-type mice. Furthermore, AC6-deficient mice lacked dDAVP-promoted inner medullary cAMP formation and phosphorylation of serine-269 and had attenuated increases in both phosphorylation of serine-256 and apical membrane AQP2 trafficking. In summary, AC6 expression determines inner medullary cAMP formation and AQP2 phosphorylation and trafficking, the absence of which causes nephrogenic diabetes insipidus.

Renal protection in chronic kidney disease: hypoxia-inducible factor activation vs. angiotensin II blockade

The 5/6(th) nephrectomy or ablation/infarction (A/I) preparation has been used as a classic model of chronic kidney disease (CKD). We observed increased kidney oxygen consumption (Q(O2)) and altered renal hemodynamics in the A/I kidney that were normalized after combined angiotensin II (ANG II) blockade. Studies suggest hypoxia inducible factor as a protective influence in A/I. We induced hypoxia-inducible factor (HIF) and HIF target proteins by two different methods, cobalt chloride (CoCl(2)) and dimethyloxalyglycine (DMOG), for the first week after creation of A/I and compared the metabolic and renal hemodynamic outcomes to combined ANG II blockade. We also examined the HIF target proteins expressed by using Western blots and real-time PCR. Treatment with DMOG, CoCl(2), and ANG II blockade normalized kidney oxygen consumption factored by Na reabsorption and increased both renal blood flow and glomerular filtration rate. At 1 wk, CoCl(2) and DMOG increased kidney expression of HIF by Western blot. In the untreated A/I kidney, VEGF, heme oxygenase-1, and GLUT1 were all modestly increased. Both ANG II blockade and CoCl(2) therapy increased VEGF and GLUT1 but the cobalt markedly so. ANG II blockade decreased heme oxygenase-1 expression while CoCl(2) increased it. By real-time PCR, erythropoietin and GLUT1 were only increased by CoCl(2) therapy. Cell proliferation was modestly increased by ANG II blockade but markedly after cobalt therapy. Metabolic and hemodynamic abnormalities were corrected equally by ANG II blockade and HIF therapies. However, the molecular patterns differed significantly between ANG II blockade and cobalt therapy. HIF induction may prove to be protective in this model of CKD.

Activation of cardiac adenylyl cyclase expression increases function of the failing ischemic heart in mice.

OBJECTIVESnThis study sought to evaluate whether increased left ventricular (LV) adenylyl cyclase VI (AC(VI)) expression, at a time when severe congestive heart failure (CHF) was present, would increase function of the actively failing heart.nnnBACKGROUNDnIncreased LV AC(VI) content markedly reduces mortality and increases LV function after acute myocardial infarction (MI) in mice. However, the effects of increased cardiac AC(VI) content in the setting of severe heart failure caused by ischemic cardiomyopathy are unknown.nnnMETHODSnMice with cardiac-directed and regulated expression of AC(VI) underwent coronary artery ligation to induce severe CHF 5 weeks later. AC(VI) expression was then activated in 1 group (AC-On) but not the other (AC-Off). Multiple measures of LV systolic and diastolic function were obtained 5 weeks later, and LV samples were assessed for alterations in calcium and beta-adrenergic receptor signaling, apoptosis, and cardiac troponin I phosphorylation.nnnRESULTSnThe LV systolic and diastolic function was increased 5 weeks after activation of AC(VI) expression. Improved LV function was associated with normalization of cardiac troponin I phosphorylation and reduced apoptosis.nnnCONCLUSIONSnActivation of cardiac AC(VI) expression in mice with ischemic cardiomyopathy and severe CHF improves function of the failing heart.

Adenylyl Cyclase 6 Enhances NKCC2 Expression and Mediates Vasopressin-Induced Phosphorylation of NKCC2 and NCC

Arginine vasopressin (AVP) affects kidney function via vasopressin V2 receptors that are linked to activation of adenylyl cyclase (AC) and an increase in cyclic adenosine monophosphate formation. AVP/cyclic adenosine monophosphate enhance the phosphorylation of the Na-K-2Cl cotransporter (NKCC2) at serine residue 126 (pS126 NKCC2) and of the Na-Cl cotransporter (NCC) at threonine 58 (pT58 NCC). The isoform(s) of AC involved in these responses, however, were unknown. Phosphorylation of S126 NKCC2 and T58 NCC, induced by the V2 receptor agonist (1-desamino-8-D-arginine vasopressin) in wild-type mice, is lacking in knockout mice for AC isoform 6 (AC6). With regard to NKCC2 phosphorylation, the stimulatory effect of 1-desamino-8-D-AVP and the defect in AC6(-/-) mice seem to be restricted to the medullary portion of the thick ascending limb. AC6 is also a stimulator of total renal NKCC2 protein abundance in medullary and cortical thick ascending limb. Consequently, mice lacking AC6 have lower NKCC2 expression and a mild Bartter syndrome-like phenotype, including lower plasma concentrations of K+ and H+ and compensatory upregulation of NCC. Increased AC6-independent phosphorylation of NKCC2 at S126 might help to stabilize NKCC2 activity in the absence of AC6. Renal AC6 determines total NKCC2 expression and mediates vasopressin-induced NKCC2/NCC phosphorylation. These regulatory mechanisms, which are defective in AC knockout mice, are likely responsible for the observed mild Bartter syndrome.

Regulation of oxygen utilization by angiotensin II in chronic kidney disease

Angiotensin II blockade delays progression of chronic kidney disease by modifying intrarenal hemodynamics, but the effects on metabolic adaptations are unknown. Using the remnant kidney model of chronic kidney disease in rats, we measured the effects of combined angiotensin II blockade with captopril and losartan on renal oxygen consumption (QO(2)) and factors influencing QO(2). Remnant kidneys had proteinuria and reductions in the glomerular filtration rate (GFR), renal blood flow (RBF) and nitric oxide synthase-1 protein expression while QO(2), factored by sodium reabsorption (QO(2)/TNa), was markedly increased. Combined blockade treatment normalized these parameters while increasing sodium reabsorption but, since QO(2) was unchanged, QO(2)/TNa also normalized. Triple antihypertensive therapy, to control blood pressure, and treatment with lysine, to increase GFR and RBF, did not normalize QO(2)/TNa, suggesting a specific effect of angiotensin II in elevating QO(2)/TNa. Inhibition of nitric oxide synthase increased QO(2) in the kidney of sham-operated rats but not in the remnant kidney of untreated rats. Our study shows that combined captopril and losartan treatment normalized QO(2)/TNa and functional nitric oxide activity in the remnant kidney independent of blood pressure and GFR effects, suggesting that other mechanisms in addition to hemodynamics underlie the benefits of angiotensin II blockade.

Adenylyl cyclase subtype-specific compartmentalization: differential regulation of L-type Ca2+ current in ventricular myocytes.

Rationale: Adenylyl cyclase (AC) represents one of the principal molecules in the &bgr;-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules. Objective: We take advantage of ACV and ACVI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes. Methods and Results: We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for &bgr;1-adrenergic receptor signaling–mediated enhancement of the L-type Ca2+ current (ICa,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on ICa,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling. Conclusions: Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of ICa,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of &bgr;- adrenergic receptor and AC compartmentalization may also be important for &bgr;-adrenergic receptor signaling in other cell types.Rationale: Adenylyl cyclase (AC) represents one of the principal molecules in the β-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules.nnObjective: We take advantage of AC V and AC VI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes.nnMethods and Results: We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for β1-adrenergic receptor signaling–mediated enhancement of the L-type Ca2+ current ( I Ca,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on I Ca,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling.nnConclusions: Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of I Ca,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of β- adrenergic receptor and AC compartmentalization may also be important for β-adrenergic receptor signaling in other cell types.nn# Novelty and Significance {#article-title-41}