This report describes studies designed to study roles of PDE3A and PDE3B in regulation of myocardial function, cardioprotection, and energy homeostasis. Myocardial function: PDE3A In human and mouse heart, cAMP stimulates myocardial contractility by increasing PKA-induced phosphorylation of membrane-bound substrates involved in intracellular Ca++ cycling and excitation/contraction coupling. Using PDE3A and PDE3B KO mice, we found that PDE3A, not PDE3B, regulates basal contractility, and that inhibition of PDE3A, not PDE3B, mediates inotropic effects of the PDE3 inhibitor, milrinone (Circ Res 112:289-97, 2013). In human heart, immunohistochemical staining of normal myocardium indicated that PDE3A co-localized in myocyte Z-bands with desmin, SERCA2, PLB, and AKAP18. During gel- filtration chromatography of solubilized microsomal membranes, PDE3 activity was recovered in distinct high (HMW) and low (LMW) molecular weight peaks. HMW peaks contained PDE3A1 and PDE3A2 isoforms. Membrane LMW fractions contained PDE3A1, PDE3A2 and PDE3A3. Recombinant PKA catalytic subunit (rPKAc) phosphorylated endogenous PDE3A1 and PDE3A2, not PDE3A3, and increased PDE3 activity. Incubation of pooled LMW membrane fractions with rPKAc moved PDE3A1 and PDE3A2 to HMW fractions. rPKAc also increased phosphorylation and co-immunoprecipitation (co-IP) of PDE3A with SERCA2 and other components of SERCA2/AKAP18 signalosomes, including cav3, PKARII, PP2A, and AKAP18. Similarly, rPKAc increased co-IP of rPDE3A with rSERCA2 and rAKAP18. Deletion of rPDE3A N-terminus, but not C-terminal catalytic region, blocked co-IP with rSERCA2, indicating that PDE3A N-terminus regulated these phosphorylation- dependent interactions. In human SR fractions, cAMP or rPKAc increased PLB phosphorylation, SERCA2 activity, and Ca++ uptake. PDE3 inhibition, not PDE4 inhibition, potentiated these effects. Taken together, our results suggest that, upon phosphorylation, PDE3A isoforms are incorporated into SERCA2/AKAP18 signalosomes in human and murine cardiomyocytes, where PDE3A regulates a discrete cAMP pool that controls contractility by modulating phosphorylation- dependent protein/protein interactions, PLB phosphorylation, SERCA2 activation, and Ca++ uptake into the SR. Cardioprotection: PDE3B. Although inhibition of PDE3 has been reported to protect rodent heart against ischemia/reperfusion (I/R) injury, neither the specific PDE3 isoform involved nor the underlying mechanisms have been identified. Targeted disruption of PDE3B, not PDE3A, protected mouse heart from I/R injury in vivo and in vitro, with reduced infarct size and improved cardiac function. The cardioprotective effect in PDE3B-/- heart was reversed by blocking PKA, and by paxilline, an inhibitor of mitoKCa channels, the opening of which is potentiated by cAMP/PKA signaling. Compared to WT mitochondria, PDE3B-/- mitochondria were enriched in anti-apoptotic Bcl-2, produced less ROS, and more frequently contacted T-tubules, where PDE3B was localized with caveolin-3. Moreover, a PDE3B-/- mitochondrial fraction containing connexin 43 and caveolin-3 was more resistant to Ca2+-induced opening of the mitochondrial permeability transition pore. Proteomics analyses indicated that PDE3B-/- heart mitochondria fractions were enriched in buoyant ischemia-induced caveolin-3-enriched fractions (ICEF), which contained cardioprotective proteins. Accumulation of proteins into ICEF was PKA-dependent and achieved by ischemic preconditioning or treatment of WT heart with the PDE3 inhibitor cilostamide. Taken together, PDE3B deletion confers cardioprotective effects due to cAMP/PKA-induced preconditioning, which is associated with accumulation of proteins with cardioprotective function in ICEF. Our study is the first to define a role for PDE3B in cardioprotection against I/R injury and suggests PDE3B as a target for cardiovascular therapies. PDE3B regulates energy homeostasis: PDE3B regulates energy metabolism (J Clin Invest 116:3240-3251, 2006), and, in PDE3B KO mice (C57Bl6 background), white adipose tissue (WAT) assumes phenotypic characteristics of brown adipose tissue (BAT) (Endocrinology 154:3152-67, 2013). Since white adipocytes and classical brown adipocytes (e.g., in supraclavicular brown fat pads) develop from different precursors and genetic lineages, and since classical brown adipocytes and ectopic brown adipocytes (arising in white adipose tissue depots) exhibit distinct but overlapping patterns of gene expression , the inducible ectopic brown-like cells are referred to as beige or brite adipocytes. The BAT phenotypic conversion in C57Bl6 PDE3B KO EWAT was markedly enhanced by the Beta3 receptor agonist CL316243 (CL), and mediated, perhaps, by cAMP-induced differentiation of prostaglandin-responsive progenitor cells in KO EWAT stromal vascular fractions into functional beige adipocytes. In SvJ129 PDE3B KO mice, EWAT also assumes characteristics of BAT, without administration of CL and without induction of COX-2, suggesting critical influences of genetic background on the phenotypic switch from white to beige adipocytes. In SvJ129 PDE3B KO EWAT, cAMP/PKA- and AMP-activated protein kinase (AMPK)-signaling pathways were activated, resulting in morphological alterations and increased expression of genes, such as PGC-1alpha, PDRM16, LRP130, and Elovl3, which are critical for mitochondrial biogenesis, induction of the thermogenic program, and differentiation/recruitment of beige adipocytes. UCP-1, a marker for BAT usually not present in EWAT, is markedly elevated in KO EWAT. These findings contribute to several phenotypic characteristics of PDE3B KO mice, including a smaller increase in body weight in response to high fat diets, smaller gonadal fat deposits and adipocytes, uncoupled EWAT mitochondrial respiration, increased oxygen consumption in vivo in response to CL stimulation, increased oxygen consumption in isolated BAT and EWAT fragments, increased fatty acid oxidation in PDE3B KO adipocytes, and increased treadmill endurance. In cultured 3T3 L1 adipocytes, cilostamide (specific PDE3 inhibitor) activated PKA, AMPK, and PGC-1alpha, and cilostamide or siRNA knockdown of PDE3B markedly potentiated induction of UCP-1 by CL. In SvJ129 PDE3B KO WAT expression of pro-inflammatory markers was reduced, compared to WT, as were components of the NLRP3 inflammasome (activation of NLRP3 inflammasomes may be related to insulin resistance and obesity-related inflammation). Taken together, these results suggest that PDE3B may regulate a cAMP-sensitive switch for browning of EWAT, regulating downstream effects of cAMP on cAMP/PKA- and AMPK-signaling, mitochondrial biogenesis and function, energy dissipation, and inhibition of inflammation. Understanding mechanisms for these changes in KO EWAT is important, since conversion of EWAT from fat-storing to fat-burning, with reduced inflammation, represents a potential strategy in treatment of obesity and diabetes.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
U.S. National Heart Lung and Blood Inst
Zip Code
Zhang, Li; Pacheco-Rodriguez, Gustavo; Steagall, Wendy K et al. (2015) Tuberous sclerosis complex 2 loss of heterozygosity in patients with lung disease and cancer. Am J Respir Crit Care Med 191:352-5
Barochia, Amisha V; Kaler, Maryann; Cuento, Rosemarie A et al. (2015) Serum apolipoprotein A-I and large high-density lipoprotein particles are positively correlated with FEV1 in atopic asthma. Am J Respir Crit Care Med 191:990-1000
DiPilato, Lisa M; Ahmad, Faiyaz; Harms, Matthew et al. (2015) The Role of PDE3B Phosphorylation in the Inhibition of Lipolysis by Insulin. Mol Cell Biol 35:2752-60
Ahmad, F; Murata, T; Shimizu, K et al. (2015) Cyclic nucleotide phosphodiesterases: important signaling modulators and therapeutic targets. Oral Dis 21:e25-50
Chung, Youn Wook; Lagranha, Claudia; Chen, Yong et al. (2015) Targeted disruption of PDE3B, but not PDE3A, protects murine heart from ischemia/reperfusion injury. Proc Natl Acad Sci U S A 112:E2253-62
Ahmad, Faiyaz; Shen, Weixing; Vandeput, Fabrice et al. (2015) Regulation of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) activity by phosphodiesterase 3A (PDE3A) in human myocardium: phosphorylation-dependent interaction of PDE3A1 with SERCA2. J Biol Chem 290:6763-76
Andrade, Bruno B; Pavan Kumar, Nathella; Amaral, Eduardo P et al. (2015) Heme Oxygenase-1 Regulation of Matrix Metalloproteinase-1 Expression Underlies Distinct Disease Profiles in Tuberculosis. J Immunol 195:2763-73
Ke, Bilun; Zhao, Zhiyun; Ye, Xin et al. (2015) Inactivation of NF-?B p65 (RelA) in Liver Improves Insulin Sensitivity and Inhibits cAMP/PKA Pathway. Diabetes 64:3355-62
Azevedo, Monalisa F; Faucz, Fabio R; Bimpaki, Eirini et al. (2014) Clinical and molecular genetics of the phosphodiesterases (PDEs). Endocr Rev 35:195-233
Hiramoto, Kenichi; Murata, Taku; Shimizu, Kasumi et al. (2014) Role of phosphodiesterase 2 in growth and invasion of human malignant melanoma cells. Cell Signal 26:1807-17

Showing the most recent 10 out of 32 publications