Differential activation of natriuretic peptide receptors modulates cardiomyocyte proliferation during development

Abstract

Organ development is a highly regulated process involving the coordinated proliferation and differentiation of diverse cellular populations. The pathways regulating cell proliferation and their effects on organ growth are complex and for many organs incompletely understood. In all vertebrate species, the cardiac natriuretic peptides (ANP and BNP) are produced by cardiomyocytes in the developing heart. However, their role during cardiogenesis is not defined. Using the embryonic zebrafish and neonatal mammalian cardiomyocytes we explored the natriuretic peptide signaling network during myocardial development. We observed that the cardiac natriuretic peptides ANP and BNP and the guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2 are functionally redundant during early cardiovascular development. In addition, we demonstrate that low levels of the natriuretic peptides preferentially activate Npr3, a receptor with Gi activator sequences, and increase cardiomyocyte proliferation through inhibition of adenylate cyclase. Conversely, high concentrations of natriuretic peptides reduce cardiomyocyte proliferation through activation of the particulate guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2, and activation of protein kinase G. These data link the cardiac natriuretic peptides in a complex hierarchy modulating cardiomyocyte numbers during development through opposing effects on cardiomyocyte proliferation mediated through distinct cyclic nucleotide signaling pathways.

Keywords: ANP; BNP; Cardiomyocyte proliferation; Heart development; Natriuretic peptides; Npr3.

Fig. 1.

Developmental induction of cardiac natriuretic peptides peaks at 48 hpf in the embryonic zebrafish. (A) Whole-mount in situ hybridization of nppa and nppb zebrafish embryos. A, atrium; V, ventricle. (B) Quantitative RT-PCR measurement of nppa and nppb during different developmental time points. Data are expressed as mean + s.e.m. ^*P<0.05, relative to 24 hpf values.

Fig. 2.

Altered cardiac natriuretic expression changes heart growth in vivo. (A) Morpholino knockdown of Nppa or Nppb in isolation or simultaneously. Heart enlargement was seen in the Nppa/Nppb double knockdown embryos but was absent in single knockdown morphants (72 hpf). (B) Inducible Gal4/UAS transactivator system to overexpress nppb in vivo. (C) The nppb overexpression embryos (HS/nppb) had a large reduction of pericardial fluid volume (magnified panel, red line details normal pericardial border) but overall embryonic growth was minimally altered (72 hpf). (D) The cmlc2/CFP fluorescent marker was utilized to visualize the chamber dimensions better in control (HS/-) and nppb overexpression (HS/nppb) embryos (48 hpf). The ventricular and atrial sizes were both reduced in nppb overexpression embryos. Red fluorescent marker in lens denotes UAS/nppb carrier status. A, atrium; V, ventricle. Scale bars: 200 μm.

Fig. 3.

Manipulation of cardiac natriuretic peptide expression alters total cardiomyocyte numbers in the embryonic heart. (A) Total cardiomyocytes at 48 hpf were increased in the Nppa/Nppb double knockdown (nppa/nppb MO) embryos, but were decreased in the nppb overexpression embryos (HS/nppb). ^*P<0.02 compared with control. (B) Representative images from cmlc2:CFP (pseudo-colored green) and DAPI (pseudo-colored red) stained hearts. (C) The total number of apoptotic heart cells was not increased in the nppb overexpression embryos. P>0.05 compared with control. (D) Knockdown of Nppa/Nppb and overexpression of nppb were able to significantly alter cardiomyocyte proliferation at 52 hpf. ^*P<0.03 compared with control. (E) Representative images of BrdU-labeled (red) cmlc2:CFP (green)-positive heart tissue. White arrowhead indicates BrdU-positive cardiomyocytes. Intracavitary BrdU+ cells were considered to be endothelial in origin and were not counted. All data are expressed as mean + s.e.m. Scale bars: 100 μm.

Fig. 4.

Manipulating natriuretic peptide concentrations can modulate mammalian cardiomyocyte proliferation. (A) Example of rat neonatal cardiomyocytes undergoing karyokinesis using Ki-67 marker (red) and sarcomeric alpha actinin (green). White arrow denotes area of sarcomere disassembly in preparation for cell division. (B) Measurement of cell proliferation in P1 to P8 rat ventricular cardiomyocytes shows an early hyperplastic phase of cellular growth followed by a cessation of cell proliferation independent of initial plating density. Data are mean ± s.d. (C) Proliferating NRVMs show a concentration-dependent effect of ANP on cardiomyocyte proliferation over 48 hours. ^*P<0.0001 compared with control. Data are expressed as mean + s.d. (D,E) Low or high concentration ANP does not increase NRVM apoptosis (annexin V or TUNEL labeling). P>0.05 (N.S., not significant). (F) NRVM proliferation measured by EdU incorporation confirmed that low concentration ANP (10 nM) enhanced NRVM proliferation whereas high concentration ANP (10 μM) suppressed NRVM proliferation. P<0.05. Data expressed as mean + s.e.m. Scale bar: 50 μm.

Fig. 5.

The cardiomyocyte proliferation effects of the cardiac natriuretic peptides are coordinated by the action of multiple natriuretic peptide receptors in vivo. (A) Lateral views of 48 hpf cmlc2:CFP embryos show significantly altered atrial and ventricular chamber sizes in Nppa/Nppb double knockdown and nppb overexpression embryos. Likewise, double knockdown of both natriuretic peptide guanylate cyclase-linked receptors, Npr1 and Npr2, caused a similar phenotype as the Nppa/Nppb double knockdown and could block the effect of nppb overexpression (npr1/npr2 MO + HS/nppb). Finally, the knockdown of Npr3 caused a significant reduction in both atrial and ventricular chamber sizes that was similar to that observed after overexpression of nppb. (B) Corresponding heart surface areas of all treatment groups shown in A. ^*P<0.05 compared with control heart size, ^#P<0.05 compared with HS/nppb alone. (C) Cardiomyocyte proliferation was increased in the Npr1 and Npr2 knockdown embryos, whereas Npr3 knockdown embryos had reduced cardiomyocyte proliferation at 52 hpf as assessed by BrdU labeling of cardiomyocytes. ^*P<0.01 compared with control. (D) Differentiation of ventricular cardiomyocytes between 30 and 48 hours of heart development was not altered by knockdown of the natriuretic peptide receptors. White arrowhead denotes arterial pole, areas of green tissue were added after 30 hpf (non-photoconverted). Yellow tissue is a mixture of photoconverted red tissue and non-photoconverted green tissue. All data are expressed as mean + s.e.m. Scale bars: 100 μm.

Fig. 6.

The concentration-dependent effect of cardiac natriuretic peptides on cardiomyocyte proliferation is controlled by both cGMP and cAMP signaling pathways. (A) The reduced cardiomyocyte proliferation seen with high concentration ANP (10 μM) can be blocked by inhibiting PKG using either Rp-8-pCPT-cGMP (Rp) or KT5823 (KT). ^*P<0.01 compared with 10 μM ANP alone. (B) The addition of cell-permeable cGMP showed a concentration-dependent reduction of cardiomyocyte proliferation. ^*P<0.02 compared with control (0 nM). (C) The Npr3-specific antagonist AP-811 could completely abolish the enhanced cardiomyocyte proliferation seen with low concentration ANP (10 nM). Antagonizing Npr3 had no effect on the reduced cardiomyocyte proliferation seen with high concentration ANP (10 μM). ^*P<0.01 compared with ANP 10 nM alone. (D) Addition of cAMP could abolish the enhanced proliferation seen with low concentration ANP (10 nM). Direct inhibition of adenylyl cyclase with SQ 22536 (SQ) can mimic the enhanced proliferation seen with low concentration ANP (10 nM). Combination of low concentration ANP (10 nM) and 0.5 μM SQ showed an additive effect on proliferation. However, adding low concentration ANP to 5.0 μM SQ showed no increased benefit. ^*P<0.01 compared with ANP 10 nM alone; ^#P<0.01 compared with control; ^**P<0.02 compared with SQ 0.5 μM. Data are expressed as mean + s.e.m.

Fig. 7.

Knockdown of Npr3 in proliferating neonatal mammalian cardiomyocytes. (A) Npr3 expression was completely abolished by targeted siRNA. (B) The increased cardiomyocyte proliferation caused by low concentration ANP (10 nM) exposure could be completely abolished by siRNA knockdown of Npr3. (C) EdU labeling of sarcomeric alpha actinin-positive cells confirms that Npr3 knockdown could block the effect of low concentration ANP on cell proliferation. ^*P<0.03 compared with control. (D) Representative images of EdU-labeled proliferating cardiomyocytes exposed to low concentration ANP with normal Npr3 expression (top) or lacking Npr3 expression (bottom). Data are expressed as mean + s.e.m. Scale bar: 100 μm.

Fig. 8.

Concentration-dependent effect of cardiac natriuretic peptides on cAMP and cGMP signaling pathways regulate cardiomyocyte proliferation. (A) The enhanced cardiomyocyte proliferation seen with low concentrations of cardiac natriuretic peptides is primarily mediated through activation of Npr3 and the inhibition of adenylyl cyclase. (B) The inhibition of cardiomyocyte proliferation seen with high concentrations of natriuretic peptides is primarily mediated through Npr1 and Npr2 and the activation of PKG.