Our results strongly support the conclusion that a unique RFRP-1-specific signaling pathway exists in mammalian ventricular cardiac myocytes. Detailed SAR analysis laid the foundation for the design of a functional receptor antagonist to a novel hRFRP-1-specific cardiac signaling pathway. Cellular and animal studies showed a first generation NPFF2
R antagonist, LPLAF-NH2
, inhibited the ability of hRFRP-1 to diminish both cellular and integrated cardiac performance. Additionally, gene expression experiments provided evidence RFRP-1 and NPFF2
R are present in myocytes and heart, which play important roles in regulating cardiac contractility. Our studies also provided additional support that PKC signaling is activated by hRFRP-1. Finally, we determined myofilaments are targets for downstream phosphorylation in response to hRFRP-1. Collectively, these data are consistent with RFRP-1 acting as a novel peptide involved in modulating in vivo
cardiac contractile function. Heart disease is a leading cause of death in the United States (24
). Identification of hRFRP-1 as a negative modulator of contractile function may provide an opportunity to develop therapeutic strategies targeting its transduction pathway in patients with heart failure.
Prior to this study, there were no published RFRP-1 SAR data or functional NPFF2R antagonist described in cardiovascular physiology. The SAR data confirmed the active core was present in the C terminus; additional observations were also made. The lesser magnitude influences of [A7] hRFRP-1 and [A9] hRFRP-1 reinforced the importance of the C-terminal hexamer, and suggested hRFRP-1 contains a conformation(s) required for activity which may be perturbed by substitution of a specific amino acid, but can be stabilized by the presence of the remaining naturally-occurring residues. However, an alternative explanation cannot be ruled out, that is, an analog may take on a distinct, yet active conformation which is different in effectiveness.
Our detailed studies on the full-length peptide resulted in several novel findings; frequently, the focus of SAR RF-NH2
analysis is directed toward the C terminus because of its high degree of structure conservation among species (25
). The alanine scan revealed the enhanced ability of [A3
] hRFRP-1 to decrease cardiac myocyte performance (, ). Its 23% greater reduction in shortening rate compared to hRFRP-1 may result from a more flexible conformation which occurs when H3
is replaced in the alanyl-substituted analog. Considerable differences in length, aromaticity, and hydrophilicity between histidine and alanine side chains are expected to contribute to an overall increase in conformational flexibility. This result suggests the potency of the parent peptide may be increased through the design of analogs to the N-terminal extension.
N-terminal truncated analogs analysis demonstrated removal of F5 resulted in ANLPLRF-NH2, an analog with significantly attenuated ability to modify contractile function (, ). Loss of the bulky, aromatic side chain in F5 may have diminished peptide function because it is directly involved in ligand receptor interaction. The remaining small, hydrophobic side chain in the adjacent A6 may be unable to compensate for the loss of F5. However, this result is in contrast to the outcome observed with the alanyl-substituted analog data, where the F5A substitution did not significantly reduce activity. Thus, a more likely explanation may be the N-terminal A6 in hRFRP-1(6-12) increased flexibility in the peptide and disrupted the conformation required for activity. The restoration of an active peptide conformation when A6 was removed to form NLPLRF-NH2 supports the argument that increased mobility contributed to the functional results observed with hRFRP-1(6-12).
G protein-coupled receptors are associated with RF-NH2
peptide signal transduction pathways (26
). RFRP-1 binds to expressed GPCR proteins NPFF1
R and NPFF2
). RFRP-1 and NPFF2
R are present in the brain and NPFF2
R is known to exist in heart (2
), however, no previous report identified hRFRP-1 in the myocardium or the cellular location of NPFF2
R within the heart (2
). In this study, transcripts for RFRP and NPFF2
R were detected in rat isolated cardiac myocytes and heart, and human myocardium (, ). Although investigated NPFF1
R transcript was not amplified from rat cardiomyocyte mRNA; NPFF1
R was detected in brain. The RFRP and NPFFR(s) transcript expression data and ability of hRFRP-1 to influence contractility in isolated cardiac myocytes supports the conclusion hRFRP-1 acts through NPFF2
R. Further, the observation that hRFRP-3 fails to influence contractility in isolated cardiomyocytes is consistent with the signaling pathway being hRFRP-1 specific. Additionally, the inability of RF9 to antagonize hRFRP-1 effects suggests a difference exists between the receptor protein present in cardiomyocytes and the central nucleus of amygdala (14
). These data also argue a difference exists between the expressed receptor proteins and cardiomyocyte NPFF2
R to explain the selectivity of hRFRP-1 versus hRFRP 3 and RF9.
The unexpected maintenance of the Ca2+
transient when hRFRP-1 impaired contractile shortening indicated the myofilament is a primary downstream target for this peptide (). The ability of Bis-1 to block the Ca2+
transient to hRFRP-1 independently supported our previous work showing PKC inhibition attenuated the influence of hRFRP-1 on isolated cardiac myocytes (; 9
). This conclusion was further supported by the enhanced myofilament protein phosphorylation detected after hRFRP-1 treatment compared to controls, and the absence of a phosphorylation response in myocytes treated with the receptor antagonist LPLAF-NH2
(). In future studies, it will be important to address whether hRFRP-1 and/or its receptor are associated with pleiotropic activation via G-dependent and G-independent mechanisms found with other peptides such as angiotensin II (29
In summary, SAR, transcript, Ca2+ transient, kinase inhibitor, and phospholabeling data presented here provided a roadmap to identify a first generation receptor antagonist of hRFRP-1 activity on cardiac performance and gain insight into a novel signaling pathway, which may be used for drug development. These results provide strong evidence a hRFRP-1 signaling pathway modulates contractile function in mammalian myocardium.