Atrial natriuretic peptide regulates Ca channel in early developmental cardiomyocytes

Abstract

Background: Cardiomyocytes derived from murine embryonic stem (ES) cells possess various membrane currents and signaling cascades link to that of embryonic hearts. The role of atrial natriuretic peptide (ANP) in regulation of membrane potentials and Ca(2+) currents has not been investigated in developmental cardiomyocytes.

Methodology/principal findings: We investigated the role of ANP in regulating L-type Ca(2+) channel current (I(CaL)) in different developmental stages of cardiomyocytes derived from ES cells. ANP decreased the frequency of action potentials (APs) in early developmental stage (EDS) cardiomyocytes, embryonic bodies (EB) as well as whole embryo hearts. ANP exerted an inhibitory effect on basal I(CaL) in about 70% EDS cardiomyocytes tested but only in about 30% late developmental stage (LDS) cells. However, after stimulation of I(CaL) by isoproterenol (ISO) in LDS cells, ANP inhibited the response in about 70% cells. The depression of I(CaL) induced by ANP was not affected by either Nomega, Nitro-L-Arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, or KT5823, a cGMP-dependent protein kinase (PKG) selective inhibitor, in either EDS and LDS cells; whereas depression of I(CaL) by ANP was entirely abolished by erythro-9-(2-Hydroxy-3-nonyl) adenine (EHNA), a selective inhibitor of type 2 phosphodiesterase(PDE2) in most cells tested. CONCLUSION/SIGNIFICANCES: Taken together, these results indicate that ANP induced depression of action potentials and I(CaL) is due to activation of particulate guanylyl cyclase (GC), cGMP production and cGMP-activation of PDE2 mediated depression of adenosine 3', 5'-cyclic monophophate (cAMP)-cAMP-dependent protein kinase (PKA) in early cardiomyogenesis.

Figure 1. Expression of ANP in mouse embryonic cardiomyocytes.

A: double staining of ANP (left, green) and α-MHC (right, red) in E8.5d (a), E12.5d (b), and E15.5d (c) cardiomyocytes. B: PCR product of ANP. Lanes indicate 1 marker, 2 E8.5d, 3 E12.5d, and 4 E15.5d, respectively. The scale bar is 10 micron.

Figure 2. Inhibition of spontaneous action potentials by ANP.

A: Action potentials recorded from a spontaneously contracting EDS cell (upper) and an E8.5d cardiomyocyte (lower) in the current clamp mode. The frequency of action potentials was decreased by application of ANP in a reversible manner. B: action potentials recorded from an early developmental stage EB (upper) and an embryonic 8.5 day heart (lower). As seen in single EDS and E8.5d cells, application of ANP exerted a negative chronotropic effect that was washable.

Figure 3. ANP depresses basal I_CaL in EDS cardiomyocytes.

A, Typical recording of I_CaL from a single cell. Upon application of 20 nM ANP, rat 3–28, basal I_CaL was decreased. This effect could be partially reversed by washout. The inset represents currents recorded as indicated by the mumbers in the time course diagram. B, I/V relationship: ANP decreased peak I_CaL without a shift in the I/V relationship. C, percentage of cells responding to ANP. D, density of peak I_CaL.

Figure 4. Effect of ANP on basal and ISO stimulated I_CaL in LDS cells.

Aa & Ba, time courses of peak I_CaL recorded from single LDS cells. Both basal and ISO pre-stimulated I_CaL were strongly depressed by application of ANP. Ab & Bb, percentage of cells responding to ANP. Note that the percentage of responding cell in EDS is much lower compared to LDS cells. Ac & Bc, current density of peak I_CaL. *P<0.05, **P<0.01 compared with control, respectively.

Figure 5. Effect of ATP-γ-S plus forskolin on ANP induced I_CaL inhibition.

Aa, time course shows that ANP depressed forskolin pre-stimulated I_CaL, however the depressed effect of ANP on forskolin pre-stimulated I_CaL was abolished in the presence of ATP-γ-S (Ba). The insets are traces recorded in the same cells as time courses. Ab & Bb, percentage of cells with or without responding to ANP. Ac & Bc, density of peak I_CaL. *P<0.05 compared with control.

Figure 6. PTX did not abolish ANP induced I_CaL inhibition.

Aa, time course demonstrates that in PTX pre-treated cardiomyocytes derived from ES cells ANP still inhibited I_CaL. Ba, time course of PTX positive control experiment which indicates PTX abolished CCh caused inhibition on I_CaL. Ab and Bb,percentage of cells with or without responding to ANP(Ab) or CCh (Bb). Ac and Bc, density of peak I_CaL. *P<0.05 compared with control.

Figure 7. PDE2-cAMP-PKA pathway of ANP depression on I_CaL.

A, time course of I_CaL (a) shows ANP still inhibits the current in KT5823, a PKG selective inhibitor, pre-treated myocytes in about 70% cells tested (b); Ac, density of peak I_CaL. B, time course (a) demonstrates that ANP fails to depress I_CaL in the presence of EHNA, a PDE2 selective inhibitor, in almost all cells tested (b). Bc, density of peak I_CaL. *P<0.05 compared with control.