Takashi Tsunematsu

Epac1-dependent phospholamban phosphorylation mediates the cardiac response to stresses

PKA phosphorylates multiple molecules involved in calcium (Ca2+) handling in cardiac myocytes and is considered to be the predominant regulator of β-adrenergic receptor-mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular Ca2+ storage and the magnitude of Ca2+ movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCε. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol- and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses.

Isoform-specific regulation of adenylyl cyclase: a potential target in future pharmacotherapy

Adenylyl cyclase (AC) is a target enzyme of multiple G-protein-coupled receptors (GPCRs). In the past decade, the cloning, structure and biochemical properties of nine AC isoforms were reported, and each isoform of AC shows distinct patterns of tissue distribution and biochemical/pharmacological properties. In addition to the conventional regulators of this enzyme, such as calmodulin (CaM) or PKC, novel regulators, for example, caveolin, have been identified. Most importantly, these regulators work on AC in an isoformdependent manner. Recent studies have demonstrated that certain classic AC inhibitors, i.e., P-site inhibitors, show an isoform-dependent inhibition of AC. The side chain modifications of forskolin, a diterpene extract from Coleus forskolii, markedly enhance its isoform selectivity. When taken together, these findings suggest that it is feasible to develop new pharmacotherapeutic agents that target AC isoforms to regulate various neurohormonal signals in a highly tissue-/organ-specific manner.

Disruption of type 5 adenylyl cyclase negates the developmental increase in Gαolf expression in the striatum

The two stimulatory G protein α subunits, Gαs and Gαolf, activate adenylyl cyclase in a similar way. We examined whether type 5 adenylyl cyclase knockout, the major striatal isoform, can differentially and/or developmentally change the expression of these G proteins in the striatum. Gαs and Gαolf expressions at birth were unaffected in knockouts, which, however, demonstrated a blunted developmental increase in Gαolf, but not Gαs. Adenylyl cyclase activity was unaffected at birth, but subsequently became lower in knockouts. These findings suggest that type 5 adenylyl cyclase does not contribute to striatal cAMP signaling at birth. However, it may play an important role in developmental changes in the expression of Gαolf, but not Gαs.

Type 5 adenylyl cyclase plays a major role in stabilizing heart rate in response to microgravity induced by parabolic flight

It is well known that autonomic nervous activity is altered under microgravity, leading to disturbed regulation of cardiac function, such as heart rate. Autonomic regulation of the heart is mostly determined by beta-adrenergic receptors/cAMP signal, which is produced by adenylyl cyclase, in cardiac myocytes. To examine a hypothesis that a major cardiac isoform, type 5 adenylyl cyclase (AC5), plays an important role in regulating heart rate during parabolic flights, we used transgenic mouse models with either disrupted (AC5KO) or overexpressed AC5 in the heart (AC5TG) and analyzed heart rate variability. Heart rate had a tendency to decrease gradually in later phases within one parabola in each genotype group, but the magnitude of decrease was smaller in AC5KO than that in the other groups. The inverse of heart rate, i.e., the R-R interval, was much more variable in AC5KO and less variable in AC5TG than that in wild-type controls. The standard deviation of normal R-R intervals, a marker of total autonomic variability, was significantly greater in microgravity phase in each genotype group, but the magnitude of increase was much greater in AC5KO than that in the other groups, suggesting that heart rate regulation became unstable in the absence of AC5. In all, AC5 plays a major role in stabilizing heat rate under microgravity.

Polymorphism of the type 6 adenylyl cyclase gene and cardiac hypertrophy.

Summary: We investigated whether polymorphism of the type 6 adenylyl cyclase gene influences the occurrence of left ventricular hypertrophy in a Japanese population. Type 6 adenylyl cyclase is a major cardiac adenylyl cyclase isoform and plays an important role in regulating cardiac function. We examined the type 6 adenylyl cyclase gene for single nucleotide polymorphism by heteroduplex analysis and found a mutation (T11215A) in intron 17. We genotyped the single nucleotide polymorphism (TT/TA/AA groups) by the mutagenically separated polymerase chain reaction method in 2068 subjects who underwent health screening for cardiovascular disease. Genetic variation was in the Hardy‐Weinberg equilibrium. We found no significant association between the frequency of left ventricular hypertrophy and any of the genotype groups. In the TT and the TA genotype group, however, left ventricular hypertrophy was associated with increased blood pressure, while no association with increased blood pressure was found in the AAgenotype group. It was concluded that the AA group may be at risk of developing left ventricular hypertrophy independent of increased blood pressure.

A case of heparin-induced thrombocytopenia with subacute stent thrombosis, multiple cerebral infarction, and acute limb ischemia

Heparin-induced thrombocytopenia (HIT) is an adverse immune-mediated drug reaction that is associated with thromboembolic complications. We report the case of an 82-year-old man with unstable angina pectoris who suffered from recurrent arterial thromboembolism due to HIT. Coronary angiography (CAG) was performed while we administered unfractionated heparin bolus. CAG showed triple-vessel disease without left main coronary artery. We performed elective percutaneous coronary angioplasty (PCI) to the left anterior descending coronary artery (LAD). The sudden thrombus formation in the LAD occurred during the procedure. We suspected HIT and administered argatroban. We deployed four everolimus-eluting stents in the LAD and intra-aortic balloon pumping (IABP) support was started. The platelet counts were rapidly reduced almost 50% next day after PCI and IgG-specific anti-PF4/heparin antibodies were elevated. Multiple cerebral infarctions were detected by magnetic resonance imaging after the PCI. The patient received the continuous argatroban administration and IABP support for 4 days. Subacute stent thrombosis occurred after quitting argatroban. We performed thrombus aspiration and fibrinolytic treatment. Finally we re-inserted IABP and stabilized the hemodynamic state. Right popliteal arterial thromboembolism occurred after emergency PCI. Argatroban is essential and following oral anticoagulant therapy is necessary to prevent thromboembolic complications. <Learning objective: Heparin-induced thrombocytopenia (HIT) is an adverse immune-mediated drug reaction that is associated with thromboembolic complications. The incidence of HIT in patients who received unfractionated heparin is reported to be 0.1- 1%. We should be aware of HIT when thromboembolic complications occur during the percutaneous coronary intervention procedure. Argatroban is essential and following oral anticoagulant therapy is necessary to prevent thromboembolic complications among patients with HIT.>.