Abstract
Natriuretic peptides (NPs) are important hormones that regulate multiple cellular functions including cardiovascular physiology. In the heart, two natriuretic peptide receptors NPR1 and NPR2 act as membrane guanylyl cyclases to produce 3′,5′-cyclic guanosine monophosphate (cGMP). Although both receptors protect from cardiac hypertrophy, their effects on contractility are markedly different, from little effect (NPR1) to pronounced negative inotropic and positive lusitropic responses (NPR2) with unclear underlying mechanisms. Here we use a scanning ion conductance microscopy (SICM) approach combined with Förster resonance energy transfer (FRET)-based cGMP biosensors to show that whereas NPR2 is uniformly localised on the cardiomyocyte membrane, functional NPR1 receptors are found exclusively in membrane invaginations called transverse (T)-tubules. This leads to far-reaching CNP/NPR2/cGMP signals, whereas ANP/NPR1/cGMP signals are highly confined to T-tubular microdomains by local pools of phosphodiesterase 2. This provides a previously unrecognised molecular basis for clearly distinct functional effects engaged by different cGMP producing membrane receptors.
Highlights
Natriuretic peptides (NPs) are important hormones that regulate multiple cellular functions including cardiovascular physiology
To localise NP receptors on cell membrane, we scanned ventricular myocytes (VMs) freshly isolated from transgenic mice expressing the highly sensitive cytosolic cyclic guanosine monophosphate (cGMP) biosensor[19] red cGES-DE5 and stimulated them with atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) locally applied onto clearly defined membrane structures from a precisely positioned scanning nanopipette
The simulations showed that when applying from a pipette filled with 100 μM ANP or CNP under bath perfusion, a maximum concentration of ~700 nM can be observed at the membrane with a relatively steep gradient which allows for a ~5–10-time concentration drop at a distance of 2 μm from the activated spot (Supplementary Fig. 3)
Summary
Natriuretic peptides (NPs) are important hormones that regulate multiple cellular functions including cardiovascular physiology. Two natriuretic peptide receptors NPR1 and NPR2 act as membrane guanylyl cyclases to produce 3′,5′-cyclic guanosine monophosphate (cGMP) Both receptors protect from cardiac hypertrophy, their effects on contractility are markedly different, from little effect (NPR1) to pronounced negative inotropic and positive lusitropic responses (NPR2) with unclear underlying mechanisms. We use a scanning ion conductance microscopy (SICM) approach combined with Förster resonance energy transfer (FRET)-based cGMP biosensors to show that whereas NPR2 is uniformly localised on the cardiomyocyte membrane, functional NPR1 receptors are found exclusively in membrane invaginations called transverse (T)-tubules This leads to far-reaching CNP/ NPR2/cGMP signals, whereas ANP/NPR1/cGMP signals are highly confined to T-tubular microdomains by local pools of phosphodiesterase 2. We show that NPR2 is rather evenly distributed across VM membrane and produces far-reaching, diffusible cGMP signals, whereas NPR1 is exclusively found in Ttubules where it creates a microdomain with restricted cGMP diffusion locally confined by PDE2
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