Adenylyl Cyclase 6 Research Articles

Adenylyl cyclase 6 plays a minor role in the mouse inner ear and retina

Adenylyl cyclase 6 (AC6) synthesizes second messenger cAMP in G protein-coupled receptor (GPCR) signaling. In cochlear hair cells, AC6 distribution relies on an adhesion GPCR, ADGRV1, which is associated with Usher syndrome (USH), a condition of combined hearing and vision loss. ADGRV1 is a component of the USH type 2 (USH2) protein complex in hair cells and photoreceptors. However, the role of AC6 in the inner ear and retina has not been explored. Here, we found that AC6 distribution in hair cells depends on the USH2 protein complex integrity. Several known AC6 regulators and effectors, which were previously reported to participate in ADGRV1 signaling in vitro, are localized to the stereociliary compartments that overlap with AC6 distribution in hair cells. In young AC6 knockout (Adcy6−/−) mice, the activity of cAMP-dependent protein kinase, but not Akt kinase, is altered in cochleas, while both kinases are normal in vestibular organs. Adult Adcy6−/− mice however exhibit normal hearing function. AC6 is expressed in mouse retinas but rarely in photoreceptors. Adcy6−/− mice have slightly enhanced photopic but normal scotopic vision. Therefore, AC6 may participate in the ADGRV1 signaling in hair cells but AC6 is not essential for cochlear and retinal development and maintenance.

Anti-inflammatory effects of α7-nicotinic ACh receptors are exerted through interactions with adenylyl cyclase-6.

Nicotinic ACh receptors containing the α7 sub-unit (α7-nAChRs) suppress inflammation through a wide range of pathways in immune cells. These receptors are thus potentially involved in a number of inflammatory diseases. However, the detailed mechanisms underlying the anti-inflammatory effects of α7-nAChRs remain to be described. Anti-inflammatory effects of α7-nAChR agonists were assessed in both murine macrophages (RAW 264.7) and bone marrow-derived macrophages (BMDM), stimulated with LPS, using immunoblotting, RT-PCR and luciferase reporter assays. The role of adenylyl cyclase-6 in the degradation of Toll-like receptor 4 (TLR4) following endocytosis, was explored via overexpression and knockdown. A mouse model of chronic obstructive pulmonary disease (COPD) induced by porcine pancreatic elastase was used to confirm key findings. Anti-inflammatory effects of α7-nAChRs were largely dependent on adenylyl cyclase-6 activation, as knockdown of adenylyl cyclase-6 considerably reduced the effects of α7-nAChR agonists while adenylyl cyclase-6 overexpression promoted them. We found that α7-nAChRs and adenylyl cyclase-6 are co-localized in lipid rafts of macrophages and directly interact. Activation of adenylyl cyclase-6 led to increased degradation of TLR4. Administration of the α7-nAChR agonist PNU-282987 attenuated pathological and inflammatory end points in a mouse model of COPD. The α7-nAChRs inhibit inflammation through activating adenylyl cyclase-6 and promoting degradation of TLR4. The use of α7-nAChR agonists may represent a novel therapeutic approach for treating COPD and possibly other inflammatory diseases.

Primary cilium-mediated MSC mechanotransduction is dependent on Gpr161 regulation of hedgehog signalling.

The benefits of physical loading to skeletal mass are well known. The primary cilium has emerged as an important organelle in bone mechanobiology/mechanotransduction, particularly in mesenchymal stem/stromal cells, yet the molecular mechanisms of cilium mechanotransduction are poorly understood. In this study, we demonstrate that Gpr161 is a mechanoresponsive GPCR, that localises to the cilium, and is involved in fluid shear-induced cAMP signalling and downstream osteogenesis. This Gpr161-mediated mechanotransduction is dependent on IFT88/cilium and may act through adenylyl cyclase 6 (AC6) to regulate cAMP and MSC osteogenesis. Moreover, we demonstrate that Hh signalling is positively associated with osteogenesis and that Hh gene expression is mechanically regulated and required for loading-induced osteogenic differentiation through a mechanism that involves IFT88, Gpr161, AC6, and cAMP. Therefore, we have delineated a molecular mechanism of MSC mechanotransduction which likely occurs at the cilium, leading to MSC osteogenesis, highlighting novel mechanotherapeutic targets to enhance osteogenesis.

Loss of Adenylyl Cyclase 6 in Leptin Receptor-Expressing Stromal Cells Attenuates Loading-Induced Endosteal Bone Formation.

ABSTRACTBone marrow stromal/stem cells represent a quiescent cell population that replenish the osteoblast bone‐forming cell pool with age and in response to injury, maintaining bone mass and repair. A potent mediator of stromal/stem cell differentiation in vitro and bone formation in vivo is physical loading, yet it still remains unclear whether loading‐induced bone formation requires the osteogenic differentiation of these resident stromal/stem cells. Therefore, in this study, we utilized the leptin receptor (LepR) to identify and trace the contribution of bone marrow stromal cells to mechanoadaptation of bone in vivo. Twelve‐week‐old Lepr‐cre;tdTomato mice were subjected to compressive tibia loading with an 11 N peak load for 40 cycles, every other day for 2 weeks. Histological analysis revealed that Lepr‐cre;tdTomato+ cells arise perinatally around blood vessels and populate bone surfaces as lining cells or osteoblasts before a percentage undergo osteocytogenesis. Lepr‐cre;tdTomato+ stromal cells within the marrow increase in abundance with age, but not following the application of tibial compressive loading. Mechanical loading induces an increase in bone mass and bone formation parameters, yet does not evoke an increase in Lepr‐cre;tdTomato+ osteoblasts or osteocytes. To investigate whether adenylyl cyclase‐6 (AC6) in LepR cells contributes to this mechanoadaptive response, Lepr‐cre;tdTomato mice were further crossed with AC6fl/fl mice to generate a LepR+ cell‐specific knockout of AC6. These Lepr‐cre;tdTomato;AC6 fl/fl animals have an attenuated response to compressive tibia loading, characterized by a deficient load‐induced osteogenic response on the endosteal bone surface. This, therefore, shows that Lepr‐cre;tdTomato+ cells contribute to short‐term bone mechanoadaptation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Inhibition of Endothelin system during the postnatal nephrogenic period in the rat. Its relationship with hypertension and renal disease in adulthood.

The aim of this work was to study the effect of a high sodium (HS) diet on blood pressure and renal function in male adult rats that have been treated with a dual Endothelin receptor antagonist (ERA) during their early postnatal period (day 1 to 20 of life). Male Sprague-Dawley rats were divided in four groups: CNS: control rats with normosodic diet; ERANS: ERA-treated rats with normosodic diet; CHS: control rats with high sodium diet; ERAHS: ERA-treated rats with HS diet. Systolic blood pressure (SBP) was recorded before and after the diet and 24-hour metabolic cage studies were performed. AQP2 and α-ENac expressions were measured by western blot and real time PCR in the renal medulla. Vasopressin (AVP) pathway was evaluated by measuring V2 receptor and adenylyl cyclase 6 (AC6) expression and cAMP production in the renal medulla. Pre-pro ET-1mRNA was also evaluated in the renal medulla. Only rats that had been treated with an ERA during their postnatal period increased their SBP after consumption of a HS diet, showing an impaired capacity to excrete sodium and water, i.e. developing salt sensitivity. This salt sensitivity would be mediated by an increase in renomedullary expression and activity of AQP2 and α-ENaC as a consequence of increased AC6 expression and cAMP production and/or a decreased ET-1 production in the renal medulla. The knowledge of the molecular mechanisms underlying the perinatal programming of salt sensitive hypertension will allow the development of reprogramming strategies in order to avoid this pathology.

Adenylyl Cyclase 6 Expression Is Essential for Cholera Toxin-Induced Diarrhea.

Cholera toxin (CT)-induced diarrhea is mediated by cyclic adenosine monophosphate (cAMP)-mediated active Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR). Although the constitutive activation of adenylyl cyclase (AC) in response to CT is due to adenosine diphosphate ribosylation of the small G protein α-subunit activating CFTR with consequent secretory diarrhea, the AC isoform(s) involved remain unknown. We generated intestinal epithelial cell-specific adenylyl cyclase 6 (AC6) knockout mice to study its role in CT-induced diarrhea. AC6 messenger RNA levels were the highest of all 9 membrane-bound AC isoforms in mouse intestinal epithelial cells. Intestinal epithelial-specific AC6 knockout mice (AC6loxloxVillinCre) had undetectable AC6 levels in small intestinal and colonic epithelial cells. No significant differences in fluid and food intake, plasma electrolytes, intestinal/colon anatomy and morphology, or fecal water content were observed between genotypes. Nevertheless, CT-induced fluid accumulation in vivo was completely absent in AC6loxloxVillinCre mice, associated with a lack of forskolin- and CT-induced changes in the short-circuit current (ISC) of the intestinal mucosa, impaired cAMP generation in acutely isolated small intestinal epithelial cells, and significantly impaired apical CFTR levels in response to forskolin. AC6 is a novel target for the treatment of CT-induced diarrhea.

Adenylyl Cyclase 6 Mediates Inhibition of TNF in the Inflammatory Reflex.

Macrophage cytokine production is regulated by neural signals, for example in the inflammatory reflex. Signals in the vagus and splenic nerves are relayed by choline acetyltransferase+ T cells that release acetylcholine, the cognate ligand for alpha7 nicotinic acetylcholine subunit-containing receptors (α7nAChR), and suppress TNF release in macrophages. Here, we observed that electrical vagus nerve stimulation with a duration of 0.1–60 s significantly reduced systemic TNF release in experimental endotoxemia. This suppression of TNF was sustained for more than 24 h, but abolished in mice deficient in the α7nAChR subunit. Exposure of primary human macrophages and murine RAW 264.7 macrophage-like cells to selective ligands for α7nAChR for 1 h in vitro attenuated TNF production for up to 24 h in response to endotoxin. Pharmacological inhibition of adenylyl cyclase (AC) and knockdown of adenylyl cyclase 6 (AC6) or c-FOS abolished cholinergic suppression of endotoxin-induced TNF release. These findings indicate that action potentials in the inflammatory reflex trigger a change in macrophage behavior that requires AC and phosphorylation of the cAMP response element binding protein (CREB). These observations further our mechanistic understanding of neural regulation of inflammation and may have implications for development of bioelectronic medicine treatment of inflammatory diseases.

Adenylyl cyclase 6 in acid-base balance – adding complexity

Systemic acid-base balance is tightly controlled within a narrow range of pH. Disturbances in systemic acid-base homeostasis are associated with diverse detrimental effects. The kidney is a key regulator of acid-base balance, capable of excreting HCO3- or H+, and chronic kidney disease invariably leads to acidosis. However, the regulatory pathways underlying the fine-tuned acid-base sensing and regulatory mechanisms are still incompletely understood. In the article published recently in Clinical Science (vol 132 (16) 1779-1796), Poulson and colleagues investigated the role of adenylyl cyclase 6 (AC6) in acid-base homeostasis. They uncovered a complex role of AC6, specifically affecting acid-base balance during HCO3- load, which causes pronounced alkalosis in AC6-deficient mice. However, the phenotype of AC6-deficient mice appears much more complex, involving systemic effects associated with increased energy expenditure. These observations remind us that there is much to be learned about the intricate signaling pathways involved in renal control of acid-base balance and the complex ramifications of acid-base regulation.

STIM1 activation of adenylyl cyclase 6 connects Ca2+ and cAMP signaling during melanogenesis.

Endoplasmic reticulum (ER)-plasma membrane (PM) junctions form functionally active microdomains that connect intracellular and extracellular environments. While the key role of these interfaces in maintenance of intracellular Ca2+ levels has been uncovered in recent years, the functional significance of ER-PM junctions in non-excitable cells has remained unclear. Here, we show that the ER calcium sensor protein STIM1 (stromal interaction molecule 1) interacts with the plasma membrane-localized adenylyl cyclase 6 (ADCY6) to govern melanogenesis. The physiological stimulus α-melanocyte-stimulating hormone (αMSH) depletes ER Ca2+ stores, thus recruiting STIM1 to ER-PM junctions, which in turn activates ADCY6. Using zebrafish as a model system, we further established STIM1's significance in regulating pigmentation invivo STIM1 domain deletion studies reveal the importance of Ser/Pro-rich C-terminal region in this interaction. This mechanism of cAMP generation creates a positive feedback loop, controlling the output of the classical αMSH-cAMP-MITF axis in melanocytes. Our study thus delineates a signaling module that couples two fundamental secondary messengers to drive pigmentation. Given the central role of calcium and cAMP signaling pathways, this module may be operative during various other physiological processes and pathological conditions.

A cullin 4B-RING E3 ligase complex fine-tunes pancreatic δ cell paracrine interactions.

Somatostatin secreted by pancreatic δ cells mediates important paracrine interactions in Langerhans islets, including maintenance of glucose metabolism through the control of reciprocal insulin and glucagon secretion. Disruption of this circuit contributes to the development of diabetes. However, the precise mechanisms that control somatostatin secretion from islets remain elusive. Here, we found that a super-complex comprising the cullin 4B-RING E3 ligase (CRL4B) and polycomb repressive complex 2 (PRC2) epigenetically regulates somatostatin secretion in islets. Constitutive ablation of CUL4B, the core component of the CRL4B-PRC2 complex, in δ cells impaired glucose tolerance and decreased insulin secretion through enhanced somatostatin release. Moreover, mechanistic studies showed that the CRL4B-PRC2 complex, under the control of the δ cell-specific transcription factor hematopoietically expressed homeobox (HHEX), determines the levels of intracellular calcium and cAMP through histone posttranslational modifications, thereby altering expression of the Cav1.2 calcium channel and adenylyl cyclase 6 (AC6) and modulating somatostatin secretion. In response to high glucose levels or urocortin 3 (UCN3) stimulation, increased expression of cullin 4B (CUL4B) and the PRC2 subunit histone-lysine N-methyltransferase EZH2 and reciprocal decreases in Cav1.2 and AC6 expression were found to regulate somatostatin secretion. Our results reveal an epigenetic regulatory mechanism of δ cell paracrine interactions in which CRL4B-PRC2 complexes, Cav1.2, and AC6 expression fine-tune somatostatin secretion and facilitate glucose homeostasis in pancreatic islets.

Role of HSF1-upregulated AC6 in ameliorating heart failure in mice.

Our previous studies discovered that Heat shock factor 1(HSF1) can alleviate pressure overload induced heart failure in mice. However, its molecular mechanisms are yet to be further explained. Many studies have already verified that Adenylyl Cyclase 6 (AC6) can ameliorate heart failure, but it is still unknown whether or not the pathway HSF1 is involved in the process. Our preliminary experiment showed that the expression level of AC6 is positively associated with HSF1. Therefore, in the present study, we aimed to explore whether HSF1 can play its role in ameliorating heart failure by regulating AC6, and how the potential internal mechanisms work. We applied the Transverse Aortic Constriction (TAC) for 4 weeks to develop the C57BL/6 mice pressure overload induced heart failure model. First, the mice were divided into TAC group and SHAM group. Changes in the cardiac function and morphology of the mice were observed by an ultrasonic device and Masson staining slices, expressions of AC6 mRNA were observed by RT-QPCR, expressions of HSF1 and proteinkinase A (PKA) were examined by Western Blotting, and the levels of cyclic adenosine monophosphate (cAMP) from aortic blood were measured by ELISA. Second, the TAC group were further divided into subgroups of HSF1 transgene mice, HSF1 knockout mice and wild type mice, followed by the aforesaid observations. In the SHAM group, no obvious variations of cardiac function, AC6 mRNAHSF1, PKA, cAMP and other test results were found among each of the subgroups. Compared to the SHAM group, the TAC group presented clearly weakened heart functions, while, expressions of AC6 mRNA, HSF1, PKA and cAMP all recorded obvious increases. In the TAC group, compared to the WT subgroup, the HSF1 KO subgroup presented decreases in expressions of AC6 mRNA, HSF1, PKA and cAMP, and at the same time, the heart functions were weaker, while, the HSF1 TG subgroup recorded the contrary results. In the pressure overload heart failure model, HSF1 can ameliorate heart failure by positively regulating the pathway of AC6/cAMP/PKA.

Gene Transfer Improves Heart Function in Patients With Heart Failure

Intracoronary adenylyl cyclase 6 (AC6) gene transfer in patients with heart failure safely increased left ventricular function beyond standard heart failure therapy, found a randomized, placebo-controlled, phase 2 clinical trial. Preclinical studies had previously shown benefits of increased AC6 on cardiac myocytes and the heart. Novel therapies are urgently needed for a disease that has seen little improvement in hospitalization rates and mortality in the past decade, despite advances in drug and device therapy (Hammond HK et al. JAMA Cardiol. doi:10.1001/jamacardio.2016.0008 [published online March 30, 2016]).

Association of adenylyl cyclase 6 rs3730070 polymorphism and hemolytic level in patients with sickle cell anemia

A recent study suggested that adenosine signaling pathway could promote hemolysis in patients with sickle cell anemia (SCA). This signaling pathway involves several gene coding enzymes for which variants have been described. In this study, we analyzed the genotype–phenotype relationships between functional polymorphisms or polymorphisms associated with altered expression of adenosine pathway genes, namely adenosine deaminase (ada; rs73598374), adenosine A2b receptor (adora2b; rs7208480), adenylyl cyclase6 (adcy6; rs3730071, rs3730070, rs7300155), and hemolytic rate in SCA patients. One hundred and fifty SCA patients were genotyped for adcy6, ada, and adora2b variants as well as alpha-globin gene, a genetic factor known to modulate hemolytic rate. Hematological and biochemical data were obtained at steady-state. Lactate dehydrogenase, aspartate aminotransferase, reticulocytes and total bilirubin were used to calculate a hemolytic index. Genotype–phenotype relationships were investigated using parametric tests and multivariate analysis. SCA patients carrying at least one allele of adcy6 rs3730070-G exhibited lower hemolytic rate than non-carriers in univariate analysis (p=0.006). The presence of adcy6 rs3730070-G variant was associated with a decreased hemolytic rate in adjusted model for age and alpha-thalassemia (p=0.032). Our results support a protective effect of adcy6 rs3730070-G variant on hemolysis in SCA patients.

Tolvaptan plus pasireotide shows enhanced efficacy in a PKD1 model.

Autosomal dominant polycystic kidney disease (ADPKD) is a leading cause of ESRD. A central defect associated with ADPKD pathology is elevated levels of 3', 5'-cyclic AMP (cAMP). Compounds such as tolvaptan and pasireotide, which indirectly reduce adenylyl cyclase 6 (AC6) activity, have hence proven effective in slowing cyst progression. Here, we tested the efficacy of these compounds individually and in combination in a hypomorphic PKD1 model, Pkd1(R3277C/R3277C) (Pkd1(RC/RC)), in a 5-month preclinical trial. Initially, the Pkd1(RC/RC) model was inbred into the C57BL/6 background, minimizing disease variability, and the pathogenic effect of elevating cAMP was confirmed by treatment with the AC6 stimulant desmopressin. Treatment with tolvaptan or pasireotide alone markedly reduced cyst progression and in combination showed a clear additive effect. Furthermore, combination treatment significantly reduced cystic and fibrotic volume and decreased cAMP to wild-type levels. We also showed that Pkd1(RC/RC) mice experience hepatic hypertrophy that can be corrected by pasireotide. The observed additive effect reinforces the central role of AC6 and cAMP in ADPKD pathogenesis and highlights the likely benefit of combination therapy for patients with ADPKD.

Adenylyl cyclase 6 mediates loading‐induced bone adaptationin vivo

Primary cilia are single, nonmotile, antenna-like structures extending from the apical membrane of most mammalian cells. They may mediate mechanotransduction, the conversion of external mechanical stimuli into biochemical intracellular signals. Previously we demonstrated that adenylyl cyclase 6 (AC6), a membrane-bound enzyme enriched in primary cilia of MLO-Y4 osteocyte-like cells, may play a role in a primary cilium-dependent mechanism of osteocyte mechanotransduction in vitro. In this study, we determined whether AC6 deletion impairs loading-induced bone formation in vivo. Skeletally mature mice with a global knockout of AC6 exhibited normal bone morphology and responded to osteogenic chemical stimuli similar to wild-type mice. Following ulnar loading over 3 consecutive days, bone formation parameters were assessed using dynamic histomorphometry. Mice lacking AC6 formed significantly less bone than control animals (41% lower bone formation rate). Furthermore, there was an attenuated flow-induced increase in COX-2 mRNA expression levels in primary bone cells isolated from AC6 knockout mice compared to controls (1.3±0.1- vs. 2.6±0.2-fold increase). Collectively, these data indicate that AC6 plays a role in loading-induced bone adaptation, and these findings are consistent with our previous studies implicating primary cilia and AC6 in a novel mechanism of osteocyte mechanotransduction.

Molecular Strategy to Reduce In Vivo Collagen Barrier Promotes Entry of NCX1 Positive Inducible Pluripotent Stem Cells (iPSCNCX1+) into Ischemic (or Injured) Myocardium

ObjectiveThe purpose of this study was to assess the effect of collagen composition on engraftment of progenitor cells within infarcted myocardium.BackgroundWe previously reported that intramyocardial penetration of stem/progenitor cells in epicardial patches was enhanced when collagen was reduced in hearts overexpressing adenylyl cyclase-6 (AC6). In this study we hypothesized an alternative strategy wherein overexpression of microRNA-29b (miR-29b), inhibiting mRNAs that encode cardiac fibroblast proteins involved in fibrosis, would similarly facilitate progenitor cell migration into infarcted rat myocardium.Methods In vitro: A tri-cell patch (Tri-P) consisting of cardiac sodium-calcium exchanger-1 (NCX1) positive iPSC (iPSCNCX1+), endothelial cells (EC), and mouse embryonic fibroblasts (MEF) was created, co-cultured, and seeded on isolated peritoneum. The expression of fibrosis-related genes was analyzed in cardiac fibroblasts (CFb) by qPCR and Western blot. In vivo: Nude rat hearts were administered mimic miRNA-29b (miR-29b), miRNA-29b inhibitor (Anti-29b), or negative mimic (Ctrl) before creation of an ischemically induced regional myocardial infarction (MI). The Tri-P was placed over the infarcted region 7 days later. Angiomyogenesis was analyzed by micro-CT imaging and immunofluorescent staining. Echocardiography was performed weekly.ResultsThe number of green fluorescent protein positive (GFP+) cells, capillary density, and heart function were significantly increased in hearts overexpressing miR-29b as compared with Ctrl and Anti-29b groups. Conversely, down-regulation of miR-29b with anti-29b in vitro and in vivo induced interstitial fibrosis and cardiac remodeling.ConclusionOverexpression of miR-29b significantly reduced scar formation after MI and facilitated iPSCNCX1+ penetration from the cell patch into the infarcted area, resulting in restoration of heart function after MI.

PTH‐stimulated urinary phosphate and cAMP excretion are absent in mice lacking adenylyl cyclase 6

Parathyroid hormone (PTH) activates an unidentified isoform of adenylyl cyclase (AC) thereby retrieving Na+/Pi cotransporters from the proximal tubule membrane and increasing Pi excretion. To test for a role adenylyl cyclase 6 (AC6) we studied wild‐type (WT, n=8) and AC6 knockout (AC6−/−, n=6) mice in clearance experiments under anesthesia. Bolus application of vehicle (Veh; 0.85% NaCl, 0.5 μl/g bw) or PTH (1, 3, 10, 30 or 100 μg/kg bw) was followed by a 5 min distribution phase and a 15 min quantitative urine collection. After Veh application absolute (AE Pi) as well as fractional urinary Pi excretion (FE Pi) were significantly higher in AC6−/− vs WT mice (4.3±1.2 vs 0.8±0.3 nmol/min/g bw, and 23±6 vs 4±1 %, P<0.05 vs AC6−/−, respectively), and urinary cAMP excretion (AE cAMP) tended to be lower in AC6−/− vs WT mice (0.8±0.2 vs 1.5±0.4 nmol/min/g bw), while glomerular filtration rate was modestly lower in AC6−/− vs WT mice (8.9±1 vs 11.8±1 μl/min/g bw, P<0.05 vs AC6−/−). PTH dose‐dependently increased AE Pi, FE Pi and AE cAMP in WT mice (max responses of 678±205% vs Veh, 23±4%, and 624±136% vs Veh, P<0.05 vs Veh, respectively). In contrast to WT mice, PTH decreased AE Pi (max response −81±3%, P<0.05 vs Veh), did not change FE Pi excretion and AE cAMP (all doses not significant from Veh). Lack of PTH‐induced urinary Pi and cAMP excretion in AC6−/− mice is consistent with AC6 in the proximal tubule mediating these responses.

Reduced Collagen Deposition in Infarcted Myocardium Facilitates Induced Pluripotent Stem Cell Engraftment and Angiomyogenesis for Improvement of Left Ventricular Function

The purpose of this study was to assess the effect of scar tissue composition on engraftment of progenitor cells into infarcted myocardium. Scar tissue formation after myocardial infarction creates a barrier that severely compromises tissue regeneration, limiting potential functional recovery. In vitro: A tricell patch (Tri-P) was created from peritoneum seeded and cultured with induced pluripotent stem cell-derived cardiomyocytes, endothelial cells, and mouse embryonic fibroblasts. The expression of fibrosis-related molecules from mouse embryonic fibroblasts and infarcted heart was measured by Western blot and quantitative reverse transcriptase polymerase chain reaction. In vivo: A Tri-P was affixed over the entire infarcted area 7 days after myocardial infarction in mice overexpressing adenylyl cyclase 6 (AC6). Engraftment efficiency of progenitor cells in hearts of AC6 mice was compared with that of control wild-type (WT) mice using a combination of in vivo bioluminescence imaging, post-mortem ex vivo tissue analysis, and the number of green fluorescent protein-positive cells. Echocardiography of left ventricular (LV) function was performed weekly. Hearts were harvested for analysis 4 weeks after Tri-P application. Mouse embryonic fibroblasts were stimulated with forskolin before an anoxia/reoxygenation protocol. Fibrosis-related molecules were analyzed. In AC6 mice, infarcted hearts treated with Tri-P showed significantly higher bioluminescence imaging intensity and numbers of green fluorescent protein-positive cells than in WT mice. LV function improved progressively in AC6 mice from weeks 2 to 4 and was associated with reduced LV fibrosis. Application of a Tri-P in AC6 mice resulted in significantly higher induced pluripotent stem cell engraftment accompanied by angiomyogenesis in the infarcted area and improvement in LV function.

Adenylyl Cyclase 6 Improves Calcium Uptake and Left Ventricular Function in Aged Hearts

This study tested the hypothesis that activation of adenylyl cyclase 6 (AC6) expression in cardiac myocytes improves calcium uptake and left ventricular (LV) function in aging mice. Aging hearts exhibit impaired β-adrenergic receptor signaling and LV dysfunction. Twenty-month-old mice with cardiac-directed and regulated AC6 expression were randomized into 2 groups, and AC6 expression was activated in 1 group (AC6-On) but not the other (AC6-Off). One month later, LV function and sarcoplasmic reticulum calcium uptake were assessed. AC6 expression was associated with increased LV contractility, as reflected by ejection fraction (p = 0.02), rate of pressure development (p = 0.002), and slope of the LV end-systolic pressure-volume relationship (p = 0.04). No changes in LV weight to tibial length ratio, LV fibrosis, and expression of fetal genes (atrial natriuretic factor, α-skeletal muscle actin, and β-myosin heavy chain) and collagens were observed between AC6-On and AC6-Off groups. However, LV samples from AC6-On mice showed increases in: isoproterenol-stimulated cAMP production (p = 0.04), cAMP-dependent protein kinase activity (p < 0.0004), phosphorylation of phospholamban (at Ser16 site; p = 0.04) and cardiac troponin I (at Ser23/24 sites; p = 0.01), velocity of sarcoplasmic reticulum calcium uptake (p < 0.0001), and sarcoplasmic reticulum calcium-ATPase2a (SERCA2a) affinity for calcium (p < 0.0001). Finally, we found that AC6 expression increased sarcoplasmic reticulum calcium storage in cardiac myocytes isolated from 23-month-old rats. In contrast, AC6 expression in 7-month-old mice did not change LV function and calcium uptake. These results indicate that activation of cardiac AC6 expression improves impaired function of aged hearts through improved calcium uptake.

On the Horizon From the ORS.

On the Horizon From the ORS.