Ca2+/calmodulin-activated phosphodiesterase 1A is highly expressed in rabbit cardiac sinoatrial nodal cells and regulates pacemaker function

Abstract

Constitutive Ca2+/calmodulin (CaM)-activation of adenylyl cyclases (ACs) types 1 and 8 in sinoatrial nodal cells (SANC) generates cAMP within lipid-raft-rich microdomains to initiate cAMP–protein kinase A (PKA) signaling, that regulates basal state rhythmic action potential firing of these cells. Mounting evidence in other cell types points to a balance between Ca2+-activated counteracting enzymes, ACs and phosphodiesterases (PDEs) within these cells. We hypothesized that the expression and activity of Ca2+/CaM-activated PDE Type 1A is higher in SANC than in other cardiac cell types. We found that PDE1A protein expression was 5-fold higher in sinoatrial nodal tissue than in left ventricle, and its mRNA expression was 12-fold greater in the corresponding isolated cells. PDE1 activity (nimodipine-sensitive) accounted for 39% of the total PDE activity in SANC lysates, compared to only 4% in left ventricular cardiomyocytes (LVC). Additionally, total PDE activity in SANC lysates was lowest (10%) in lipid-raft-rich and highest (76%) in lipid-raft-poor fractions (equilibrium sedimentation on a sucrose density gradient). In intact cells PDE1A immunolabeling was not localized to the cell surface membrane (structured illumination microscopy imaging), but located approximately within about 150 nm inside of immunolabeling of hyperpolarization-activated cyclic nucleotide-gated potassium channels (HCN4), which reside within lipid-raft-rich microenvironments. In permeabilized SANC, in which surface membrane ion channels are not functional, nimodipine increased spontaneous SR Ca2+ cycling. PDE1A mRNA silencing in HL-1 cells increased the spontaneous beating rate, reduced the cAMP, and increased cGMP levels in response to IBMX, a broad spectrum PDE inhibitor (detected via fluorescence resonance energy transfer microscopy). We conclude that signaling via cAMP generated by Ca2+/CaM-activated AC in SANC lipid raft domains is limited by cAMP degradation by Ca2+/CaM-activated PDE1A in non-lipid raft domains. This suggests that local gradients of [Ca2+]–CaM or different AC and PDE1A affinity regulate both cAMP production and its degradation, and this balance determines the intensity of Ca2+-AC-cAMP-PKA signaling that drives SANC pacemaker function.

Generation and phenotypic characterization of Pde1a mutant mice

“…Reports on its importance relative to other PDE families and the relative expression of the three PDE1 subfamilies in different animal species have not been consistent (Table A in S1 File )[ 62 – 74 ]. Overall, our findings agree with some but not all published studies: i) PDE1 accounts for a large proportion of cAMP-PDE activity in the heart [ 65 – 67 , 70 ]; ii) The protein expression of PDE1C is much higher than that of PDE1A in the myocardium [ 63 , 64 , 67 , 72 – 74 ]; iii) Therefore, PDE1C is likely more important for cardiac hypertrophy than PDE1A; iv) The protein expression of PDE1A is much higher in adult aorta [ 32 , 47 , 75 ]; v) Consistent with this pattern of expression, Pde1a knockout mice in our study had lower blood pressures and a hyperdynamic circulation with an increased heart rate and ejection fraction. Interestingly, a large gene-centric meta-analysis in 87,736 individuals of European ancestry has identified an association between the PDE1A locus and diastolic and mean arterial pressures [ 76 ], and mutations of PDE3A resulting in increased PKA–mediated PDE3A phosphorylation and gain of function have been found in six families with hypertension and brachydactily syndrome [ 77 ].…”

Neurohumoral Control of Sinoatrial Node Activity and Heart Rate: Insight From Experimental Models and Findings From Humans

“…Basal PDE degradation of cAMP in rabbit SAN myocytes has been shown to modulate and compartmentalize Ca 2+ -clock mechanisms such as RyR and PLB, as PDE inhibition increases the rate and amplitude of local Ca 2+ releases (Vinogradova et al, 2008). This has been confirmed specifically for PDE1A in rabbit SAN myocytes, facilitated by its higher expression in the SAN compared to other regions of the heart (Lukyanenko et al, 2016). Although inhibition of single PDEs only moderately increases SAN myocyte firing, recently it has been shown that dual PDE3 and PDE4 inhibition synergistically increases basal SAN firing rate by ∼50% (Vinogradova et al, 2018a).…”

Cardiac Cyclic Nucleotide Phosphodiesterases: Roles and Therapeutic Potential in Heart Failure

“…In cardiomyocytes, PDE1C shows a predominantly cytosolic distribution, localising to the M-and Z-lines of the sarcomere, and is present in microsomal fractions [55]. PDE1A protein is abundant in rabbit sinoatrial (SA) node cells where it is purported to moderate pacemaker activity [59], but whether it functions in an analogous capacity in human hearts is currently unknown. Similarly, whilst PDE1A appears to regulate cell death in vascular smooth muscle cells (VSMCs) [60], a corresponding cardiac-specific role is not established.…”