Judith Krall

Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte.

Mammalian oocytes are arrested in meiotic prophase by an inhibitory signal from the surrounding somatic cells in the ovarian follicle. In response to luteinizing hormone (LH), which binds to receptors on the somatic cells, the oocyte proceeds to second metaphase, where it can be fertilized. Here we investigate how the somatic cells regulate the prophase-to-metaphase transition in the oocyte, and show that the inhibitory signal from the somatic cells is cGMP. Using FRET-based cyclic nucleotide sensors in follicle-enclosed mouse oocytes, we find that cGMP passes through gap junctions into the oocyte, where it inhibits the hydrolysis of cAMP by the phosphodiesterase PDE3A. This inhibition maintains a high concentration of cAMP and thus blocks meiotic progression. LH reverses the inhibitory signal by lowering cGMP levels in the somatic cells (from ∼2 μM to ∼80 nM at 1 hour after LH stimulation) and by closing gap junctions between the somatic cells. The resulting decrease in oocyte cGMP (from ∼1 μM to ∼40 nM) relieves the inhibition of PDE3A, increasing its activity by ∼5-fold. This causes a decrease in oocyte cAMP (from ∼700 nM to ∼140 nM), leading to the resumption of meiosis.

Ca2+ uptake by cardiac sarcoplasmic reticulum from patients with idiopathic dilated cardiomyopathy.

We measured Ca2+ uptake by sarcoplasmic reticulum prepared from left ventricular myocardium obtained from six nonfailing human hearts and nine excised hearts from patients with class IV idiopathic dilated cardiomyopathy. Ca2+ uptake had a Vmax of 593 +/- 82 nmol/mg-min, a K0.5 of 0.68 +/- 0.07 microM, and an nHill of 1.7 +/- 0.1 in the nonfailing hearts. The corresponding values in the excised failing hearts were 593 +/- 36 nmol/mg-min, 0.63 +/- 0.03 microM, and 1.6 +/- 0.1. The beta-receptor density in crude sarcolemma prepared from left ventricular myocardium was 110.0 +/- 15.3 fmol/mg in the unmatched donors and 52.1 +/- 4.5 fmol/mg in the excised failing hearts. These results suggest that abnormal Ca2+ handling in idiopathic dilated cardiomyopathy in humans is not the result of any intrinsic alteration of Ca2+ uptake by sarcoplasmic reticulum.

Isoforms of Cyclic Nucleotide Phosphodiesterase PDE3 and Their Contribution to cAMP Hydrolytic Activity in Subcellular Fractions of Human Myocardium

Three isoforms of PDE3 (cGMP-inhibited) cyclic nucleotide phosphodiesterase regulate cAMP content in different intracellular compartments of cardiac myocytes in response to different signals. We characterized the catalytic activity and inhibitor sensitivity of these isoforms by using recombinant proteins. We determined their contribution to cAMP hydrolysis in cytosolic and microsomal fractions of human myocardium at 0.1 and 1.0 μm cAMP in the absence and presence of Ca2+/calmodulin. We examined the effects of cGMP on cAMP hydrolysis in these fractions. PDE3A-136, PDE3A-118, and PDE3A-94 have similar Km and kcat values for cAMP and are equal in their sensitivities to inhibition by cGMP and cilostazol. In microsomes, PDE3A-136, PDE3A-118, and PDE3A-94 comprise the majority of cAMP hydrolytic activity under all conditions. In cytosolic fractions, PDE3A-118 and PDE3A-94 comprise >50% of the cAMP hydrolytic activity at 0.1 μm cAMP, in the absence of Ca2+/calmodulin. At 1.0 μm cAMP, in the presence of Ca2+/calmodulin, activation of Ca2+/calmodulin-activated (PDE1) and other non-PDE3 phosphodiesterases reduces their contribution to <20% of cAMP hydrolytic activity. cGMP inhibits cAMP hydrolysis in microsomal fractions by inhibiting PDE3 and in cytosolic fractions by inhibiting both PDE3 and PDE1. These findings indicate that the contribution of PDE3 isoforms to the regulation of cAMP hydrolysis in intracellular compartments of human myocardium and the effects of PDE3 inhibition on cAMP hydrolysis in these compartments are highly dependent on intracellular [Ca2+] and [cAMP], which are lower in failing hearts than in normal hearts. cGMP may amplify cAMP-mediated signaling in intracellular compartments of human myocardium by PDE3-dependent and PDE3-independent mechanisms.

Sarcoplasmic reticulum-associated cyclic adenosine 5'-monophosphate phosphodiesterase activity in normal and failing human hearts.

Sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was examined in microsomes prepared from the left ventricular myocardium of eight heart transplant recipients with end-stage idiopathic dilated cardiomyopathy and six unmatched organ donors with normal cardiac function. At cAMP concentrations less than or equal to 1.0 microM, sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was functionally homogeneous. cAMP phosphodiesterase activity was inhibited competitively by cGMP (Ki = 0.031 +/- 0.008 microM) and the cilostamide derivative OPC 3911 (Ki = 0.018 +/- 0.004 microM), but was essentially insensitive to rolipram. Vmax and Km were 781.7 +/- 109.2 nmol/mg per min and 0.188 +/- 0.031 microM, respectively, in microsomes prepared from nonfailing hearts and 793.9 +/- 68.9 nmol/mg per min and 0.150 +/- 0.027 microM in microsomes prepared from failing hearts. Microsomes prepared from nonfailing and failing hearts did not differ with respect to either the ratio of cAMP phosphodiesterase activity to ATP-dependent Ca2+ accumulation activity or the sensitivity of cAMP phosphodiesterase activity to inhibition by OPC 3911. These data suggest that the diminished inotropic efficacy of phosphodiesterase inhibitors in failing human hearts does not result from changes in the level, kinetic properties, or pharmacologic sensitivity of sarcoplasmic reticulum-associated cAMP phosphodiesterase activity.

Cyclic Nucleotide Phosphodiesterase PDE1C1 in Human Cardiac Myocytes

Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to comprise a significant portion of the cGMP-inhibited cAMP hydrolytic activity in human hearts. We examined the expression of PDE1 isoforms in human myocardium, characterized their catalytic activity, and quantified their contribution to cAMP hydrolytic and cGMP hydrolytic activity in subcellular fractions of this tissue. Western blotting with isoform-selective anti-PDE1 monoclonal antibodies showed PDE1C1 to be the principal isoform expressed in human myocardium. Immunohistochemical analysis showed that PDE1C1 is distributed along the Z-lines and M-lines of cardiac myocytes in a striated pattern that differs from that of the other major dual-specificity cyclic nucleotide phosphodiesterase in human myocardium, PDE3A. Most of the PDE1C1 activity was recovered in soluble fractions of human myocardium. It binds both cAMP and cGMP with Km values of ∼1 μm and hydrolyzes both substrates with similar catalytic rates. PDE1C1 activity in subcellular fractions was quantified using a new PDE1-selective inhibitor, IC295. At substrate concentrations of 0.1 μm, PDE1C1 constitutes the great majority of cAMP hydrolytic and cGMP hydrolytic activity in soluble fractions and the majority of cGMP hydrolytic activity in microsomal fractions, whereas PDE3 constitutes the majority of cAMP hydrolytic activity in microsomal fractions. These results indicate that PDE1C1 is expressed at high levels in human cardiac myocytes with an intracellular distribution distinct from that of PDE3A and that it may have a role in the integration of cGMP-, cAMP- and Ca2+-mediated signaling in these cells.

Conserved expression and functions of PDE4 in rodent and human heart

PDE4 isoenzymes are critical in the control of cAMP signaling in rodent cardiac myocytes. Ablation of PDE4 affects multiple key players in excitation–contraction coupling and predisposes mice to the development of heart failure. As little is known about PDE4 in human heart, we explored to what extent cardiac expression and functions of PDE4 are conserved between rodents and humans. We find considerable similarities including comparable amounts of PDE4 activity expressed, expression of the same PDE4 subtypes and splicing variants, anchoring of PDE4 to the same subcellular compartments and macromolecular signaling complexes, and downregulation of PDE4 activity and protein in heart failure. The major difference between the species is a fivefold higher amount of non-PDE4 activity in human hearts compared to rodents. As a consequence, the effect of PDE4 inactivation is different in rodents and humans. PDE4 inhibition leads to increased phosphorylation of virtually all PKA substrates in mouse cardiomyocytes, but increased phosphorylation of only a restricted number of proteins in human cardiomyocytes. Our findings suggest that PDE4s have a similar role in the local regulation of cAMP signaling in rodent and human heart. However, inhibition of PDE4 has ‘global’ effects on cAMP signaling only in rodent hearts, as PDE4 comprises a large fraction of the total cardiac PDE activity in rodents but not in humans. These differences may explain the distinct pharmacological effects of PDE4 inhibition in rodent and human hearts.

Phospholamban-mediated stimulation of Ca2+ uptake in sarcoplasmic reticulum from normal and failing hearts.

Studies in animal models have suggested that alterations affecting phospholamban-mediated stimulation of Ca2+ uptake by sarcoplasmic reticulum are involved in the pathophysiology of heart disease. A monoclonal antibody that binds to phospholamban and stimulates Ca2+ uptake was used to characterize phospholamban-mediated effects in human cardiac sarcoplasmic reticulum and to compare these effects in tissue from normal and failing hearts. Stimulation of Ca2+ uptake by anti-phospholamban monoclonal antibody simulated the effect of phosphorylation of phospholamban by cAMP-dependent protein kinase. Binding of anti-phospholamban antibody reduced the K0.5 of the Ca2(+)-transporting ATPase from 0.53 microM [( Ca2+]) to 0.29 microM [( Ca2+]), without affecting Vmax or nHill. At 0.2 microM Ca2+, stimulation was 1.93-fold in sarcoplasmic reticulum prepared from normal human left ventricular myocardium and 1.94-fold in sarcoplasmic reticulum prepared from the left ventricular myocardium of patients with heart failure resulting from idiopathic dilated cardiomyopathy. Stimulation of Ca2+ uptake in canine cardiac sarcoplasmic reticulum under identical conditions was 1.89-fold. Phospholamban-mediated stimulation of Ca2+ uptake in human cardiac sarcoplasmic reticulum is thus comparable in magnitude to that observed in other species and results from an increase in the apparent affinity of the Ca2(+)-transporting ATPase for Ca2+. The pathogenesis of heart failure in idiopathic dilated cardiomyopathy does not, however, appear to involve intrinsic alterations of this mechanism.

cGMP-Hydrolytic Activity and Its Inhibition by Sildenafil in Normal and Failing Human and Mouse Myocardium

In mouse models of cardiac disease, the type 5 (PDE5)-selective cyclic nucleotide phosphodiesterase inhibitor sildenafil has antihypertrophic and cardioprotective effects attributable to the inhibition of cGMP hydrolysis. To investigate the relevance of these findings to humans, we quantified cGMP-hydrolytic activity and its inhibition by sildenafil in cytosolic and microsomal preparations from the left ventricular myocardium of normal and failing human hearts. The vast majority of cGMP-hydrolytic activity was attributable to PDE1 and PDE3. Sildenafil had no measurable effect on cGMP hydrolysis at 10 nM, at which it is selective for PDE5, but it had a marked effect on cGMP and cAMP hydrolysis at 1 μM, at which it inhibits PDE1. In contrast, in preparations from the left ventricles of normal mice and mice with heart failure resulting from coronary artery ligation, the effects of sildenafil on cGMP hydrolysis were attributable to inhibition of both PDE5 and PDE1; PDE5 comprised ∼22 and ∼43% of the cytosolic cGMP-hydrolytic activity in preparations from normal and failing mouse hearts, respectively. These differences in PDE5 activities in human and mouse hearts call into question the extent to which the effects of sildenafil in mouse models are likely to be applicable in humans and raise the possibility of PDE1 as an alternative therapeutic target.

Effect of Ryanodine on Sarcoplasmic Reticulum Ca2+ Accumulation in Nonfailing and Failing Human Myocardium

BACKGROUND The purpose of this study was to determine whether abnormal Ca2+ release through ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum might contribute to the abnormal [Ca2+]i homeostasis that has been described in failing human myocardium. METHODS AND RESULTS Occupancy of low-affinity ryanodine binding sites on ryanodine-sensitive Ca2+ channels stimulates oxalate-supported, ATP-dependent Ca2+ accumulation in sarcoplasmic reticulum-derived microsomes by inhibiting concurrent Ca2+ efflux through these channels. We examined the effects of 0.5 mmol/L ryanodine on 45Ca2+ accumulation in microsomes prepared from nonfailing (n = 8) and failing (n = 10) human left ventricular myocardium. In the absence of ryanodine, 45Ca2+ accumulation reached similar levels in microsomes from nonfailing and failing hearts. Incubation with 0.5 mmol/L ryanodine caused a 52.2 +/- 6.5% increase in peak 45Ca2+ accumulation in microsomes from nonfailing hearts and a 24.3 +/- 4.1% increase in microsomes from failing hearts. The density of high-affinity ryanodine binding sites and the inhibition of [3H]ryanodine dissociation from these sites by 0.1 mmol/L ryanodine were similar in microsomes from nonfailing and failing hearts. CONCLUSIONS These results, which demonstrate a diminished stimulation of Ca2+ accumulation by ryanodine in sarcoplasmic reticulum-derived microsomes from failing human myocardium that could be explained by an uncoupling of the occupancy of low-affinity ryanodine binding sites from the reduction in the open probability of these channels or by concurrent Ca2+ efflux through a ryanodine-insensitive mechanism, are evidence that increased efflux of Ca2+ from the sarcoplasmic reticulum may contribute to the abnormal [Ca2+]i homeostasis described in failing human myocardium.

Regulation of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) activity by phosphodiesterase 3A (PDE3A) in human myocardium: phosphorylation-dependent interaction of PDE3A1 with SERCA2.

Background: PDE3A is part of a SERCA2 signaling complex in cardiac myocytes. Results: PDE3, not PDE4, regulates the activation of SERCA2 by PKA in human myocardium; phosphorylation of PDE3A1 at Ser-292/Ser-293 promotes its integration into the SERCA2 signaling complex. Conclusion: PDE3A1 regulates cAMP-mediated control of SERCA2 through its phosphorylation-dependent interaction with SERCA2. Significance: Targeted disruption of the PDE3A1-SERCA2 interaction may provide a new therapeutic approach for heart failure. Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity.