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As described before, RXPF1 activation by RLN induces an increased production of second messengers such as cAMP, cGMP and NO within the cell [213]. As a result, mRNA expression and protein phosphorylation patterns are altered depending on the cell type being stimulated. RLN has been extensively studied in endothelial cells and fibroblasts, as the current widely believed hypothesis, is that RLN has mainly vasodilatory, anti-fibrotic and angiogenic properties [57, 195, 202, 210-212, 214]. On the other hand, the inotropic effects of RLN discovered in atrial cardiomyocytes by Dschietzig et al. never gained momentum for further investigation.

In this study, the inotropic pathway of RXFP1 activation by RLN was elucidated in

vitro. Different signaling proteins of the RXFP1-RLN signaling pathway were inhibited in

RXFP1 transduced NRVCMs before RLN stimulation. Change in P-PLB(S16) levels was used as an endpoint in this study in order to assess which protein played an important role in the signaling pathway.

Gs: When the Gs subunit of the heterotrimeric g-protein was inhibited, RXFP1 activation

by RLN could not produce any detectable P-PLB(S16), compared to control cells, where P- PLB(S16) was abundant. Gs is the component that initiates a signaling cascades in many GPCRs [215]. However, some GPCRs might signal through other subunits of heterotrimeric

Discussion

G-protein [78, 216]. Many studies, including this one, have shown that RLN signals through Gs in order to increase cAMP production, which leads to further responses from the cell [68, 88]. When compared to strong inotropic agents, such as ISO, RXFP1 activation produces a noticeably lower cAMP and P-PLB signal. However, this could be attributed to a loose coupling between RXFP1 and Gs subunit [77]. To conclude, the Gs subunit of the heterotrimeric G-protein is essential for RXFP1-RLN signaling pathway, and it is needed for all downstream signaling.

AC: When AC was inhibited, RXFP1 activation from RLN stimulation resulted in a

moderate reduction in P-PLB(S16) signal (20% lower than the original signal). As described before, in different cell types other than cardiomyocytes, Gs stimulates AC in order to increase cAMP production after RXFP1 activation. This increase in secondary messenger production leads to alterations in gene expression and protein phosphorylation. However, in this study, RLN seems to signal through other specific ACs presented in cardiomyocytes. Current understanding of RXFP1 signaling specifies various isoforms of AC as being responsible for cAMP production, depending on the type of cell being stimulated [217, 218]. However, most of these studies were done in fibroblast or endothelia which have different AC expression profiles than cardiomyocytes. There are two primary isoforms of AC in cardiomyocytes, AC5 and AC6 [219-221]. AC5 is primarily expressed in adult cardiomyocytes and AC6 is primarily expressed in neonatal cardiomyocytes [222]. Other isoforms of AC are also present in the heart, but in much lower quantities [223, 224]. This difference in expression profile can explain why the expected decrease in P-PLB(S16) was dampened. In addition, the AC inhibitor displays different affinities toward different isoforms of AC [219], resulting in an insufficient blockage of the relevant isoforms. In this experiment, increasing the amount of inhibitor to block the dominant isoforms was not possible, without having a detrimental effect on the cells. Thus, the moderate inhibition of P-PLB(S16) could be attributed to the insufficient inhibition of the AC isoform (e.g. AC5 and AC6), through which RLN induces cAMP production and positive inotropic response.

PKA: In this study, inhibition of PKA led to a substantial decrease in P-PLB(S16) levels.

PKA is a signaling protein downstream of cAMP [225]. Activation of PKA by cAMP lead to phosphorylation of many proteins and ion channels [225, 226]. PKA is known to directly phosphorylate PLB at serine 16, thus disinhibiting/activating SERCA2a activity [200, 227]. SERCA2a is a Ca2+-ATPase, which transports Ca2+ from the cytosol into the lumen of the SR

Discussion

increases, resulting in myocardial relaxation and better calcium handling in the cardiomyocytes [229]. Improved calcium cycling contributes to a tightly spatiotemporally- regulated elevated level of intracellular Ca2+ ([Ca2+]i) and stronger contractions of cardiac muscle without increased resting [Ca2+]i, which leads to diastolic dysfunction and HF [230].

PI3K: In this study, inhibition of PI3K with wortmannin resulted in a marked decrease in

cAMP production and P-PLB(S16). PI3K was described to be important for maintaining cAMP production after RLN stimulation [77]. Activation of RXFP1 triggers the activation of PI3K, independent of the Gs activation. Activation of PI3K leads to PIP3 production, which activates PKC , resulting in activation of AC for further production of cAMP [77, 217, 218]. This fact would explain how RXPF1-RLN signaling maintains the amount of cAMP needed for downstream signaling activation.

G: Inhibition of G led to an increase in P-PLB(S16) signal. Kern et al. have described

that RXFP1 is not easily deactivated and may behave differently than other GPCRs [231]. However, the results from the G inhibition could not confirm these finding. G functions by recruiting different GRKs to the membrane in order to phosphorylate GPCRs, thereby resulting in a desensitization of the receptor [232, 233]. Furthermore, G also plays a role in the inhibition of PI3K, which could further decrease GPCRs signal [184, 234, 235]. Thus, it is reasonable to assume that RXPF1 might not behave like other GPRCs, when being stimulated for a prolonged period of time.

Lastly, it should be noted that the RXFP1-RLN pathway was studied in healthy cardiomyocytes. It would be interesting to further investigate the RXFP-RLN pathway in failing cardiomyocytes. The molecular alterations in these cells could lead to a better understanding of the RXFP1-RLN signaling pathway.