Targeted disruption of PDE3B,but not PDE3A,protects murine heart from ischemia/reperfusion injury

Although inhibition of cyclic nucleotide phosphodiesterase type 3 (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 PDE3 subfamily B (PDE3B), but not of PDE3 subfamily A (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 cAMP-dependent PKA and by paxilline, an inhibitor of mitochondrial calcium-activated K channels, the opening of which is potentiated by cAMP/PKA signaling. Compared with WT mitochondria, PDE3B^−/− mitochondria were enriched in antiapoptotic Bcl-2, produced less reactive oxygen species, and more frequently contacted transverse 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 (ICEFs) containing cardioprotective proteins. Accumulation of proteins into ICEFs was PKA dependent and was achieved by ischemic preconditioning or treatment of WT heart with the PDE3 inhibitor cilostamide. Taken together, these findings indicate that PDE3B deletion confers cardioprotective effects because of cAMP/PKA-induced preconditioning, which is associated with the accumulation of proteins with cardioprotective function in ICEFs. To our knowledge, 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.The two cyclic nucleotide phosphodiesterase type 3 (PDE3) subfamilies PDE3A and PDE3B are products of separate but homologous genes. PDE3 isoforms hydrolyze both cAMP and cGMP with high affinity (K_m <1 μM) in a mutually competitive manner and are important regulators of cyclic nucleotide signaling pathways and responses in cardiomyocytes and vascular smooth muscle (1). PDE3A and PDE3B exhibit different patterns of expression. PDE3A is more abundant in platelets, airway and vascular smooth muscle, and cardiovascular tissues, whereas PDE3B is relatively more highly expressed in tissues that are important in regulating energy metabolism, including liver, pancreatic β cells, brown adipose tissue (BAT), and white adipose tissue (WAT) (2). Little is known about their differential localization and functions when PDE3A and PDE3B are present in the same cell. To gain further insight into specific PDE3A and PDE3B functions in physiological contexts, we have generated and studied PDE3A^−/− and PDE3B^−/− mice (3, 4).PDE3 inhibitors, e.g., milrinone, are thought to enhance myocardial inotropic responses via cAMP/PKA regulation of Ca²⁺ cycling in the sarcoplasmic reticulum (SR) (1, 5). The PDE3 inhibitor cilostazol (6–9) and the PDE5 inhibitor sildenafil (10, 11) have been reported to protect hearts against ischemia/reperfusion (I/R) injury in various species. Fukasawa et al. (8) have suggested that cilostazol exerts its cardioprotective effect by activating mitochondrial Ca²⁺-activated K⁺ (mitoK_Ca) channels, whose opening protects hearts against infarction (12). Furthermore, studies have shown that the opening of mitoK_Ca channels is potentiated by cAMP-dependent PKA signaling (13), whereas PKC potentiates mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channel activation (14). Kukreja and his associates have suggested that the cardioprotective effects of sildenafil are mediated by activation of both mitoK_ATP (10) and mitoK_Ca channels (11).Ischemic preconditioning (PreC), a process in which brief intermittent episodes of ischemia and reperfusion protect the heart from subsequent prolonged ischemic injury (15), initiates a number of cardioprotective signaling pathways at the plasma membrane, which are transduced to mitochondria (16). According to the “signalosome” hypothesis, cardioprotective [e.g., G protein-coupled receptor (GPCR)-induced or ouabain-induced] signals are delivered to mitochondria by specialized caveolae-derived vesicular structures, signalosomes, which contain a wide variety of receptors (e.g., GPCRs) and signaling molecules (e.g., Akt, Src, eNOS, and PKCε) that are assembled in lipid rafts and caveolae (17). In recent years, the role of lipid rafts and caveolae in cardiovascular signaling has attracted much attention (18), and adenylyl cyclases and PDEs have emerged as key players in shaping and organizing intracellular signaling microdomains (19–21).Accumulating evidence implicates the mitochondrial permeability transition (MPT) pore as a key effector of cardioprotection against I/R injury, and reperfusion-induced elevation of reactive oxygen species (ROS) can trigger the opening of the MPT pore, resulting in ischemic injury, apoptosis, and cell death (16). A wide range of cardioprotective signaling pathways converge on glycogen synthase kinase-3β (GSK-3β), and its inhibition directly and/or indirectly regulates MPT pore-regulatory factors (e.g., cyclophilin D and voltage-dependent anion channels) and antiapoptotic Bcl-2 family members (22). Physical association between mitochondria and the endoplasmic reticulum (ER) [via mitochondria-associated ER membranes (MAMs)] (23) or the SR (24) also may reduce reperfusion-induced mitochondrial Ca²⁺ overload and consequent oxidative stress and thus block MPT pore opening (25).In this study, we report that, 24 h after in vivo coronary artery ligation, I/R or, in a Langendorff cardiac I/R model system, infarct size is reduced in PDE3B^−/− heart, but not in PDE3A^−/− heart, compared with WT heart. This protective effect is most likely caused by reduced production of ROS and reduced Ca²⁺-induced MPT pore opening in PDE3B^−/− mitochondria. The mechanism(s) for cardioprotection in PDE3B^−/− mice may be related to cAMP/PKA-induced opening of mitoK_Ca channels and assembly of ischemia-induced caveolin-3–enriched fraction (ICEF) signalosomes in which various cardioprotective molecules accumulate, resulting in functional cardiac preconditioning. Our results also suggest that the increased physical interaction between mitochondria and transverse tubules (T-tubules) (indirectly via the SR at dyads or directly) in PDE3B^−/− heart may be involved in ICEF/signalosome delivery of cardioprotective molecules to mitochondria, leading to reduced ROS generation and increased resistance to Ca²⁺-induced MPT pore opening in PDE3B^−/− mitochondria. Although PDE3A is more highly expressed than PDE3B in cardiovascular tissues, our findings of cardioprotection against I/R injury in PDE3B^−/− mice but not in PDE3A^−/− mice and the different subcellular locations of PDE3A and PDE3B in cardiomyocytes [PDE3A colocalizes with sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) on SR membranes, and PDE3B localizes with caveolin-3 in T-tubules along Z-lines] may reflect an important example of individual PDEs at distinct subcellular sites regulating the compartmentalization of specific cAMP/PKA-signaling pathways (19, 21). In this case, PDE3B, located in regions where cardiomyocyte mitochondria, T-tubules, and SR may be in close proximity, may regulate stress responses and/or the assembly of ICEF signalosomes or other specific cardioprotective pathways.