Aging impairs the neurovascular interface in the heart.

Aging is a major risk factor for impaired cardiovascular health. Because the aging myocardium is characterized by microcirculatory dysfunction, and because nerves align with vessels, we assessed the impact of aging on the cardiac neurovascular interface. We report that aging reduces nerve density in the ventricle and dysregulates vascular-derived neuroregulatory genes. Aging down-regulates microRNA 145 (miR-145) and derepresses the neurorepulsive factor semaphorin-3A. miR-145 deletion, which increased Sema3a expression or endothelial Sema3a overexpression, reduced axon density, mimicking the aged-heart phenotype. Removal of senescent cells, which accumulated with chronological age in parallel to the decline in nerve density, rescued age-induced denervation, reversed Sema3a expression, preserved heart rate patterns, and reduced electrical instability. These data suggest that senescence-mediated regulation of nerve density contributes to age-associated cardiac dysfunction.

The Microenvironment of the Pathogenesis of Cardiac Hypertrophy.

Pathological cardiac hypertrophy is a key risk factor for the development of heart failure and predisposes individuals to cardiac arrhythmia and sudden death. While physiological cardiac hypertrophy is adaptive, hypertrophy resulting from conditions comprising hypertension, aortic stenosis, or genetic mutations, such as hypertrophic cardiomyopathy, is maladaptive. Here, we highlight the essential role and reciprocal interactions involving both cardiomyocytes and non-myocardial cells in response to pathological conditions. Prolonged cardiovascular stress causes cardiomyocytes and non-myocardial cells to enter an activated state releasing numerous pro-hypertrophic, pro-fibrotic, and pro-inflammatory mediators such as vasoactive hormones, growth factors, and cytokines, i.e., commencing signaling events that collectively cause cardiac hypertrophy. Fibrotic remodeling is mediated by cardiac fibroblasts as the central players, but also endothelial cells and resident and infiltrating immune cells enhance these processes. Many of these hypertrophic mediators are now being integrated into computational models that provide system-level insights and will help to translate our knowledge into new pharmacological targets. This perspective article summarizes the last decades' advances in cardiac hypertrophy research and discusses the herein-involved complex myocardial microenvironment and signaling components.

Sex-specific repolarization heterogeneity in mouse left ventricle: Optical mapping combined with mathematical modeling predict the contribution of specific ionic currents.

Ventricular repolarization shows notable sex-specificity, with female sex being associated with longer QT-intervals in electrocardiography irrespective of the species studied. From a clinical point of view, women are at a greater risk for drug-induced torsade de pointes and symptomatic long-QT syndrome. Here, we present an optical mapping (OM) approach to reveal sex-specific action potential (AP) heterogeneity in a slice preparation of mouse hearts. Left ventricular epicardial repolarization in female versus male mice shows longer and, interindividually, more variable AP duration (APD), yielding a less prominent transmural APD gradient. By combining OM with mathematical modeling, we suggest a significant role of IKto,f and IKur in AP broadening in females. Other transmembrane currents, including INaL , only marginally affect basal APD. As in many cardiac pathophysiologies, increasing [Ca2+ ]i poses a risk for arrhythmia, the response of AP morphology to enhanced activation of L-type calcium channels (LTCC) was assessed in a sex-selective manner. Both APD and its variation increased significantly more in female versus male mice after pharmacological LTCC activation, which we hypothesize to be due to sex-specific INaL expression based on mathematical modeling. Altogether, we demonstrate a more delayed repolarization of LV epicardium, a leveled LV transmural APD gradient, and a more pronounced epicardial APD response to Ca2+ influx in females versus males. Mathematical modeling quantifies the relative contributions of selected ionic currents to sex-specific AP morphology under normal and pathophysiological conditions.

Extracellular flux analysis in intact cardiac tissue slices-A novel tool to investigate cardiac substrate metabolism in mouse myocardium.

AIM: Cardiac pathologies are accompanied by alterations in substrate metabolism, and extracellular flux analysis is a standard tool to investigate metabolic disturbances, especially in immortalized cell lines. However, preparations of primary cells, such as adult cardiomyocytes require enzymatic dissociation and cultivation affecting metabolism. Therefore, we developed a flux analyzer-based method for the assessment of substrate metabolism in intact vibratome-sliced mouse heart tissue. METHODS: Oxygen consumption rates were determined using a Seahorse XFe24-analyzer and "islet capture plates." We demonstrate that tissue slices are suitable for extracellular flux analysis and metabolize both free fatty acids (FFA) and glucose/glutamine. Functional integrity of tissue slices was proven by optical mapping-based assessment of action potentials. In a proof-of-principle approach, the sensitivity of the method was tested by analyzing substrate metabolism in the remote myocardium after myocardial infarction (I/R). RESULTS: Here, I/R increased uncoupled OCR compared with sham animals indicating a stimulated metabolic capacity. This increase was caused by a higher glucose/glutamine metabolism, whereas FFA oxidation was unchanged. CONCLUSION: In conclusion, we describe a novel method to analyze cardiac substrate metabolism in intact cardiac tissue slices by extracellular flux analysis. The proof-of-principle experiment demonstrated that this approach has a sensitivity allowing the investigation of pathophysiologically relevant disturbances in cardiac substrate metabolism.

Selectivity Determinants of RHO GTPase Binding to IQGAPs.

IQ motif-containing GTPase-activating proteins (IQGAPs) modulate a wide range of cellular processes by acting as scaffolds and driving protein components into distinct signaling networks. Their functional states have been proposed to be controlled by members of the RHO family of GTPases, among other regulators. In this study, we show that IQGAP1 and IQGAP2 can associate with CDC42 and RAC1-like proteins but not with RIF, RHOD, or RHO-like proteins, including RHOA. This seems to be based on the distribution of charged surface residues, which varies significantly among RHO GTPases despite their high sequence homology. Although effector proteins bind first to the highly flexible switch regions of RHO GTPases, additional contacts outside are required for effector activation. Sequence alignment and structural, mutational, and competitive biochemical analyses revealed that RHO GTPases possess paralog-specific residues outside the two highly conserved switch regions that essentially determine the selectivity of RHO GTPase binding to IQGAPs. Amino acid substitution of these specific residues in RHOA to the corresponding residues in RAC1 resulted in RHOA association with IQGAP1. Thus, electrostatics most likely plays a decisive role in these interactions.

Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice.

The tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b/PEX5R) is an interaction partner and auxiliary subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are key for rhythm generation in the brain and in the heart. Since TRIP8b is expressed in central neurons but not in cardiomyocytes, the TRIP8b-HCN interaction has been studied intensely in the brain, but is deemed irrelevant in the cardiac conduction system. Still, to date, TRIP8b has not been studied in the intrinsic cardiac nervous system (ICNS), a neuronal network located within epicardial fat pads. In vitro electrophysiological studies revealed that TRIP8b-deficient mouse hearts exhibit increased atrial refractory and atrioventricular nodal refractory periods, compared to hearts of wild-type littermates. Meanwhile, heart rate, sino-nodal recovery time, and ventricular refractory period did not differ between genotypes. Trip8b mRNA was detected in the ICNS by quantitative polymerase chain reaction. RNAscope in situ hybridization confirmed Trip8b localization in neuronal somata and nerve fibers. Additionally, we found a very low amount of mRNAs in the sinus node and atrioventricular node, most likely attributable to the delicate fibers innervating the conduction system. In contrast, TRIP8b protein was not detectable. Our data suggest that TRIP8b in the ICNS may play a role in the modulation of atrial electrophysiology beyond HCN-mediated sino-nodal control of the heart.

A comprehensive analysis of RAS-effector interactions reveals interaction hotspots and new binding partners.

RAS effectors specifically interact with GTP-bound RAS proteins to link extracellular signals to downstream signaling pathways. These interactions rely on two types of domains, called RAS-binding (RB) and RAS association (RA) domains, which share common structural characteristics. Although the molecular nature of RAS-effector interactions is well-studied for some proteins, most of the RA/RB-domain-containing proteins remain largely uncharacterized. Here, we searched through human proteome databases, extracting 41 RA domains in 39 proteins and 16 RB domains in 14 proteins, each of which can specifically select at least one of the 25 members in the RAS family. We next comprehensively investigated the sequence-structure-function relationship between different representatives of the RAS family, including HRAS, RRAS, RALA, RAP1B, RAP2A, RHEB1, and RIT1, with all members of RA domain family proteins (RASSFs) and the RB-domain-containing CRAF. The binding affinity for RAS-effector interactions, determined using fluorescence polarization, broadly ranged between high (0.3 µM) and very low (500 µM) affinities, raising interesting questions about the consequence of these variable binding affinities in the regulation of signaling events. Sequence and structural alignments pointed to two interaction hotspots in the RA/RB domains, consisting of an average of 19 RAS-binding residues. Moreover, we found novel interactions between RRAS1, RIT1, and RALA and RASSF7, RASSF9, and RASSF1, respectively, which were systematically explored in sequence-structure-property relationship analysis, and validated by mutational analysis. These data provide a set of distinct functional properties and putative biological roles that should now be investigated in the cellular context.

Functional Dysregulation of CDC42 Causes Diverse Developmental Phenotypes.

Exome sequencing has markedly enhanced the discovery of genes implicated in Mendelian disorders, particularly for individuals in whom a known clinical entity could not be assigned. This has led to the recognition that phenotypic heterogeneity resulting from allelic mutations occurs more commonly than previously appreciated. Here, we report that missense variants in CDC42, a gene encoding a small GTPase functioning as an intracellular signaling node, underlie a clinically heterogeneous group of phenotypes characterized by variable growth dysregulation, facial dysmorphism, and neurodevelopmental, immunological, and hematological anomalies, including a phenotype resembling Noonan syndrome, a developmental disorder caused by dysregulated RAS signaling. In silico, in vitro, and in vivo analyses demonstrate that mutations variably perturb CDC42 function by altering the switch between the active and inactive states of the GTPase and/or affecting CDC42 interaction with effectors, and differentially disturb cellular and developmental processes. These findings reveal the remarkably variable impact that dominantly acting CDC42 mutations have on cell function and development, creating challenges in syndrome definition, and exemplify the importance of functional profiling for syndrome recognition and delineation.

The RAS-Effector Interface: Isoform-Specific Differences in the Effector Binding Regions.

RAS effectors specifically interact with the GTP-bound form of RAS in response to extracellular signals and link them to downstream signaling pathways. The molecular nature of effector interaction by RAS is well-studied but yet still incompletely understood in a comprehensive and systematic way. Here, structure-function relationships in the interaction between different RAS proteins and various effectors were investigated in detail by combining our in vitro data with in silico data. Equilibrium dissociation constants were determined for the binding of HRAS, KRAS, NRAS, RRAS1 and RRAS2 to both the RAS binding (RB) domain of CRAF and PI3Kα, and the RAS association (RA) domain of RASSF5, RALGDS and PLCε, respectively, using fluorescence polarization. An interaction matrix, constructed on the basis of available crystal structures, allowed identification of hotspots as critical determinants for RAS-effector interaction. New insights provided by this study are the dissection of the identified hotspots in five distinct regions (R1 to R5) in spite of high sequence variability not only between, but also within, RB/RA domain-containing effectors proteins. Finally, we propose that intermolecular ß-sheet interaction in R1 is a central recognition region while R3 may determine specific contacts of RAS versus RRAS isoforms with effectors.

IQGAP1 Interaction with RHO Family Proteins Revisited: KINETIC AND EQUILIBRIUM EVIDENCE FOR MULTIPLE DISTINCT BINDING SITES.

IQ motif-containing GTPase activating protein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity, and transmigration. The RHO family proteins CDC42 and RAC1 have been shown to mainly interact with the GAP-related domain (GRD) of IQGAP1. However, the role of its RASGAP C-terminal (RGCT) and C-terminal domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underlie a multiple-step binding mechanism: (i) a high affinity, GTP-dependent binding of RGCT to the switch regions of CDC42 or RAC1 and (ii) a very low affinity binding of GRD and a C terminus adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of IQGAP1-mediated cellular responses.

Deciphering the Molecular and Functional Basis of RHOGAP Family Proteins: A SYSTEMATIC APPROACH TOWARD SELECTIVE INACTIVATION OF RHO FAMILY PROTEINS.

RHO GTPase-activating proteins (RHOGAPs) are one of the major classes of regulators of the RHO-related protein family that are crucial in many cellular processes, motility, contractility, growth, differentiation, and development. Using database searches, we extracted 66 distinct human RHOGAPs, from which 57 have a common catalytic domain capable of terminating RHO protein signaling by stimulating the slow intrinsic GTP hydrolysis (GTPase) reaction. The specificity of the majority of the members of RHOGAP family is largely uncharacterized. Here, we comprehensively investigated the sequence-structure-function relationship between RHOGAPs and RHO proteins by combining our in vitro data with in silico data. The activity of 14 representatives of the RHOGAP family toward 12 RHO family proteins was determined in real time. We identified and structurally verified hot spots in the interface between RHOGAPs and RHO proteins as critical determinants for binding and catalysis. We have found that the RHOGAP domain itself is nonselective and in some cases rather inefficient under cell-free conditions. Thus, we propose that other domains of RHOGAPs confer substrate specificity and fine-tune their catalytic efficiency in cells.

The anticancer phytochemical rocaglamide inhibits Rho GTPase activity and cancer cell migration.

Chemotherapy is one of the pillars of anti-cancer therapy. Although chemotherapeutics cause regression of the primary tumor, many chemotherapeutics are often shown to induce or accelerate metastasis formation. Moreover, metastatic tumors are largely resistant against chemotherapy. As more than 90% of cancer patients die due to metastases and not due to primary tumor formation, novel drugs are needed to overcome these shortcomings. In this study, we identified the anticancer phytochemical Rocaglamide (Roc-A) to be an inhibitor of cancer cell migration, a crucial event in metastasis formation. We show that Roc-A inhibits cellular migration and invasion independently of its anti-proliferative and cytotoxic effects in different types of human cancer cells. Mechanistically, Roc-A treatment induces F-actin-based morphological changes in membrane protrusions. Further investigation of the molecular mechanisms revealed that Roc-A inhibits the activities of the small GTPases RhoA, Rac1 and Cdc42, the master regulators of cellular migration. Taken together, our results provide evidence that Roc-A may be a lead candidate for a new class of anticancer drugs that inhibit metastasis formation.

Biophysical Characterization of Nucleophosmin Interactions with Human Immunodeficiency Virus Rev and Herpes Simplex Virus US11.

Nucleophosmin (NPM1, also known as B23, numatrin or NO38) is a pentameric RNA-binding protein with RNA and protein chaperon functions. NPM1 has increasingly emerged as a potential cellular factor that directly associates with viral proteins; however, the significance of these interactions in each case is still not clear. In this study, we have investigated the physical interaction of NPM1 with both human immunodeficiency virus type 1 (HIV-1) Rev and Herpes Simplex virus type 1 (HSV-1) US11, two functionally homologous proteins. Both viral proteins show, in mechanistically different modes, high affinity for a binding site on the N-terminal oligomerization domain of NPM1. Rev, additionally, exhibits low-affinity for the central histone-binding domain of NPM1. We also showed that the proapoptotic cyclic peptide CIGB-300 specifically binds to NPM1 oligomerization domain and blocks its association with Rev and US11. Moreover, HIV-1 virus production was significantly reduced in the cells treated with CIGB-300. Results of this study suggest that targeting NPM1 may represent a useful approach for antiviral intervention.

Functional cross-talk between ras and rho pathways: a Ras-specific GTPase-activating protein (p120RasGAP) competitively inhibits the RhoGAP activity of deleted in liver cancer (DLC) tumor suppressor by masking the catalytic arginine finger.

The three deleted in liver cancer genes (DLC1-3) encode Rho-specific GTPase-activating proteins (RhoGAPs). Their expression is frequently silenced in a variety of cancers. The RhoGAP activity, which is required for full DLC-dependent tumor suppressor activity, can be inhibited by the Src homology 3 (SH3) domain of a Ras-specific GAP (p120RasGAP). Here, we comprehensively investigated the molecular mechanism underlying cross-talk between two distinct regulators of small GTP-binding proteins using structural and biochemical methods. We demonstrate that only the SH3 domain of p120 selectively inhibits the RhoGAP activity of all three DLC isoforms as compared with a large set of other representative SH3 or RhoGAP proteins. Structural and mutational analyses provide new insights into a putative interaction mode of the p120 SH3 domain with the DLC1 RhoGAP domain that is atypical and does not follow the classical PXXP-directed interaction. Hence, p120 associates with the DLC1 RhoGAP domain by targeting the catalytic arginine finger and thus by competitively and very potently inhibiting RhoGAP activity. The novel findings of this study shed light on the molecular mechanisms underlying the DLC inhibitory effects of p120 and suggest a functional cross-talk between Ras and Rho proteins at the level of regulatory proteins.

Rho-kinase: regulation, (dys)function, and inhibition.

In a variety of normal and pathological cell types, Rho-kinases I and II (ROCKI/II) play a pivotal role in the organization of the nonmuscle and smooth muscle cytoskeleton and adhesion plaques as well as in the regulation of transcription factors. Thus, ROCKI/II activity regulates cellular contraction, motility, morphology, polarity, cell division, and gene expression. Emerging evidence suggests that dysregulation of the Rho-ROCK pathways at different stages is linked to cardiovascular, metabolic, and neurodegenerative diseases as well as cancer. This review focuses on the current status of understanding the multiple functions of Rho-ROCK signaling pathways and various modes of regulation of Rho-ROCK activity, thereby orchestrating a concerted functional response.

Evaluation of p-phenylene-bis and phenyl dithiocarbamate sodium salts as inhibitors of mushroom tyrosinase.

Two structurally related compounds, phenyl dithiocarbamate sodium salt (I) and p-phenylene-bis (dithiocarbamate) sodium salt (II) were prepared by reaction of the parent aniline and p-phenylenediamine with CS2 in the presence of sodium hydroxide. These water soluble compounds were characterized by spectroscopic techniques, IR, ¹H NMR and elemental analysis. The inhibitory effects of both compounds on both activities of mushroom tyrosinase (MT) from Agricus bisporus were studied at two temperatures, 27°C and 37°C. L-3, 4-dihydroxyphenylalanine (L-DOPA), and l-tyrosine were used as natural substrates for the catecholase and cresolase enzyme reactions, respectively. Kinetic analysis confirmed noncompetitive inhibition mode of I and mixed type of II on both activities of MT; I and II inhibit MT with inhibition constants (K(I)) of 300 µM and 4 µM, respectively. Analysis of thermodynamic parameters indicated predominant involvement of hydrophobic interactions in binding of I and electrostatic ones in binding of II to MT. It seems that II is a more potent MT inhibitor due to its two charged head groups able to chelate copper ions in the enzyme active site. Intrinsic fluorescence studies as a function of concentrations of both compounds showed unexpectedly quenching of emission intensity without any shift of emission maximum. Extrinsic ANS-fluorescence indicated that only binding of I induces limited changes in the tertiary structure of MT, in agreement with the postulated hydrophobic nature of the binding mechanism.