Iyer, S. R. | Search bioRxiv

bioRxiv2023

EVIDENCE FOR ANGIOTENSIN II AS A NATURALLY EXISTING SUPPRESSOR FOR THE NATRIURETIC PEPTIDE SYSTEM

BackgroundNatriuretic peptide system (NPS) and renin angiotensin aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date support this notion. ObjectivesThis study was designed to systematically investigate ANGII-NPS interaction in humans, in vivo, and in vitro for translational insights. MethodsCirculating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in rat model to determine influence of ANGII on ANP actions. Multiple engineered HEK293 cells and surface plasmon resonance (SPR) technology were leveraged for mechanistic exploration. ResultsIn humans, ANGII showed inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and interaction term between ANGII and natriuretic peptide increased predicting accuracy of base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed positive association between cGMP with ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at physiological dose attenuated blood pressure reduction and cGMP generation triggered by ANP infusion. In vitro, we showed that the suppression effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), which can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using SPR, we showed ANGII has low affinity for particulate guanylyl cyclase A (GC-A) receptor binding compared to ANP or BNP. ConclusionsOur study reveals ANGII as a natural suppressor for cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular disease. STRUCTURED GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/525806v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@6febe8org.highwire.dtl.DTLVardef@1e8e21corg.highwire.dtl.DTLVardef@54a98eorg.highwire.dtl.DTLVardef@47ade7_HPS_FORMAT_FIGEXP M_FIG C_FIG CLINICAL PERSPECTIVESAccumulating evidence continues to support the NPS as a promising therapeutic target via the function of the GC-A receptor and production of the second messenger cGMP for heart failure, hypertension, and other cardiovascular diseases. Improving our mechanistic understanding on GC-A/cGMP pathway regulation may further advance the development of novel NPS enhancing therapies. Here we report evidence from multiple avenues supporting a fundamental, yet previously underappreciated mechanism involving a negative action of ANGII in suppressing GC-A receptor-mediated cGMP production via an AT1 receptor-dependent manner. This study also provides a solid rationale for the superiority of combinatory neurohormonal therapies such as sacubitril/valsartan in treating cardiovascular disease, and further highlights a promising therapeutic avenue of dual targeting both the NPS and RAAS to maximize protection.

molecular biology↗

bioRxiv2022

Discovery of oncogenic ROS1 missense mutations with sensitivity to tyrosine kinase inhibitors

Chromosomal rearrangements of ROS1 generate ROS1 tyrosine kinase fusion proteins that are established oncogenes predicting effectiveness of tyrosine kinase inhibitors (TKI) treatment. The cancer genome reveals nonsynonymous missense mutations in ROS1, however, their oncogenic potential remains unknown. We nominated thirty-four tumor-associated missense mutations in ROS1 kinase domain for functional interrogation. Immunoblotting revealed diverse impact of the mutations on the kinase, ranging from loss of function to significant increase in catalytic activity. Notably, Asn and Gly substitutions at the Asp-2113 position in ROS1 kinase domain were TKI- sensitive hyper-activating mutations, and transformative oncogenes in independent cell models. Molecular modeling revealed drastic alterations in the activation loop of ROS1D2113N compared to wildtype kinase. Proteomics studies showed that ROS1D2113N increases phosphorylation of known effectors akin to ROS1 fusions, and upregulates pathways not previously linked to ROS1, including mTORC2, JNK1/2, AP-1, TGFB1 and CCN1/2. In vivo, ROS1D2113N drove tumor formation that was sensitive to inhibition by crizotinib and lorlatinib. Taken together, these data show that select point mutations within ROS1 RTK are oncogenic, and maybe therapeutically targetable with FDA-approved TKI.

cancer biology↗

bioRxiv2018

Optical recording of action potential initiation and propagation in mouse skeletal muscle fibers

Individual skeletal muscle fibers have been used to examine a wide variety of cellular functions and pathologies. Among other parameters, skeletal muscle action potential propagation has been measured to assess the integrity and function of skeletal muscle. In this paper, we utilize Di-8-ANEPPS, a potentiometric dye and mag-fluo-4, a low-affinity intracellular calcium indicator to non-invasively and reliably measure action potential conduction velocity in skeletal muscle. We used an extracellular bipolar electrode to generate an electric field that will initiate an action potential at one end of the fiber or the other. Using enzymatically dissociated flexor digitorum brevis (FDB) fibers, we demonstrate the strength and applicability of this technique. Using high-speed line scans, we estimate the conduction velocity to be approximately 0.4 m/s. In addition to measuring the conduction velocity, we can also measure the passive electrotonic potentials elicited by pulses by either applying tetrodotoxin (TTX) or reducing the bath sodium levels. We applied these methodologies to FDB fibers under elevated extracellular potassium conditions, and found that the conduction velocity is significantly reduced compared to our control concentration. Lastly, we have constructed a circuit model of a skeletal muscle in order to predict passive polarization of the fiber by the field stimuli. Our predictions from the model fiber closely resemble the recordings acquired from in vitro assays. With these techniques, we can examine how many different pathologies and mutations affect skeletal muscle action potential propagation. Our work demonstrates the utility of using Di-8-ANEPPS or mag-fluo-4 to non-invasively measure action potential conduction velocity.

biophysics↗