PKG1α Cysteine-42 Redox State Controls mTORC1 Activation in Pathological Cardiac Hypertrophy.

RATIONALE: Stimulated PKG1α (protein kinase G-1α) phosphorylates TSC2 (tuberous sclerosis complex 2) at serine 1365, potently suppressing mTORC1 (mechanistic [mammalian] target of rapamycin complex 1) activation by neurohormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine-42 is also induced by these stressors, which blunts its cardioprotective effects. OBJECTIVE: We tested the dependence of mTORC1 activation on PKG1α C42 oxidation and its capacity to suppress such activation by soluble GC-1 (guanylyl cyclase 1) activation. METHODS AND RESULTS: Cardiomyocytes expressing wild-type (WT) PKG1α (PKG1αWT) or cysteine-42 to serine mutation redox-dead (PKG1αCS/CS) were exposed to ET-1 (endothelin 1). Cells expressing PKG1αWT exhibited substantial mTORC1 activation (p70 S6K [p70 S6 kinase], 4EBP1 [elF4E binding protein-1], and Ulk1 [Unc-51-like kinase 1] phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1αCS/CS expression. Mice with global knock-in of PKG1αCS/CS subjected to pressure overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1αWT mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2-S1365 phosphorylation increased in PKG1αCS/CS more than PKG1αWT myocardium following PO. TSC2S1365A/S1365A (TSC2 S1365 phospho-null, created by a serine to alanine mutation) knock-in mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1αCS/CS mice, protection against PO-induced mTORC1 activation, cardiodepression, and mortality in PKG1αCS/CS mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1αWT and PKG1αCS/CS after PO and blocked ET-1 stimulated mTORC1 in TSC2S1365A-expressing myocytes. CONCLUSIONS: Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.

Self-regulation and the foraging gene (PRKG1) in humans.

Foraging is a goal-directed behavior that balances the need to explore the environment for resources with the need to exploit those resources. In Drosophila melanogaster, distinct phenotypes have been observed in relation to the foraging gene (for), labeled the rover and sitter. Adult rovers explore their environs more extensively than do adult sitters. We explored whether this distinction would be conserved in humans. We made use of a distinction from regulatory mode theory between those who "get on with it," so-called locomotors, and those who prefer to ensure they "do the right thing," so-called assessors. In this logic, rovers and locomotors share similarities in goal pursuit, as do sitters and assessors. We showed that genetic variation in PRKG1, the human ortholog of for, is associated with preferential adoption of a specific regulatory mode. Next, participants performed a foraging task to see whether genetic differences associated with distinct regulatory modes would be associated with distinct goal pursuit patterns. Assessors tended to hug the boundary of the foraging environment, much like behaviors seen in Drosophila adult sitters. In a patchy foraging environment, assessors adopted more cautious search strategies maximizing exploitation. These results show that distinct patterns of goal pursuit are associated with particular genotypes of PRKG1, the human ortholog of for.

A substitution in cGMP-dependent protein kinase 1 associated with aortic disease induces an active conformation in the absence of cGMP.

Type 1 cGMP-dependent protein kinases (PKGs) play important roles in human cardiovascular physiology, regulating vascular tone and smooth-muscle cell phenotype. A mutation in the human PRKG1 gene encoding cGMP-dependent protein kinase 1 (PKG1) leads to thoracic aortic aneurysms and dissections. The mutation causes an arginine-to-glutamine (RQ) substitution within the first cGMP-binding pocket in PKG1. This substitution disrupts cGMP binding to the pocket, but it also unexpectedly causes PKG1 to have high activity in the absence of cGMP via an unknown mechanism. Here, we identified the molecular mechanism whereby the RQ mutation increases basal kinase activity in the human PKG1α and PKG1ß isoforms. Although we found that the RQ substitution (R177Q in PKG1α and R192Q in PKG1ß) increases PKG1α and PKG1ß autophosphorylation in vitro, we did not detect increased autophosphorylation of the PKG1α or PKG1ß RQ variant isolated from transiently transfected 293T cells, indicating that increased basal activity of the RQ variants in cells was not driven by PKG1 autophosphorylation. Replacement of Arg-177 in PKG1α with alanine or methionine also increased basal activity. PKG1 exists as a parallel homodimer linked by an N-terminal leucine zipper, and we show that the WT chain in WT-RQ heterodimers partly reduces basal activity of the RQ chain. Using hydrogen/deuterium-exchange MS, we found that the RQ substitution causes PKG1ß to adopt an active conformation in the absence of cGMP, similar to that of cGMP-bound WT enzyme. We conclude that the RQ substitution in PKG1 increases its basal activity by disrupting the formation of an inactive conformation.

The PKGIα-Rac1 pathway is a novel regulator of insulin-dependent glucose uptake in cultured rat podocytes.

Insulin plays a major role in regulating glucose homeostasis in podocytes. Protein kinase G type Iα (PKGIα) plays an important role in regulating glucose uptake in these cells. Rac1 signaling plays an essential role in the reorganization of the actin cytoskeleton and is also essential for insulin-stimulated glucose transport. The experiments were conducted using primary rat podocytes. We performed western blot analysis, evaluated small GTPases activity assays, measured radioactive glucose uptake, and performed immunofluorescence imaging to analyze the role of PKGIα-Rac1 signaling in regulating podocyte function. We also utilized a small-interfering RNA-mediated approach to determine the role of PKGIα and Rac1 in regulating glucose uptake in podocytes. The present study investigated the influence of the PKGI pathway on the insulin-dependent regulation of activity and cellular localization of small guanosine triphosphatases in podocytes. We found that the PKGIα-dependent activation of Rac1 signaling induced activation of the PAK/cofilin pathway and increased insulin-mediated glucose uptake in podocytes. The downregulation of PKGIα or Rac1 expression abolished this effect. Rac1 silencing prevented actin remodeling and GLUT4 translocation close to the cell membrane. These data provide evidence that PKGIα-dependent activation of the Rac1 signaling pathways is a novel regulator of insulin-mediated glucose uptake in cultured rat podocytes.

Expression profiles of PRKG1, SDF2L1 and PPP1R12A are predictive and prognostic factors for therapy response and survival in high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGS-EOCs) is generally sensitive to front-line platinum (Pt)-based chemotherapy although most patients at an advanced stage relapse with progressive resistant disease. Clinical or molecular data to identify primary resistant cases at diagnosis are not yet available. HGS-EOC biopsies from 105 Pt-sensitive (Pt-s) and 89 Pt-resistant (Pt-r) patients were retrospectively selected from two independent tumor tissue collections. Pathway analysis was done integrating miRNA and mRNA expression profiles. Signatures were further validated in silico on a cohort of 838 HGS-EOC cases from a published dataset. In all, 131 mRNAs and 5 miRNAs belonging to different functionally related molecular pathways distinguish Pt-s from Pt-r cases. Then, 17 out of 23 selected elements were validated by orthogonal approaches (SI signature). As resistance to Pt is associated with a short progression-free survival (PFS) and overall survival (OS), the prognostic role of the SI signature was assessed, and 14 genes associated with PFS and OS, in multivariate analyses (SII signature). The prognostic value of the SII signature was validated in a third extensive cohort. The expression profiles of SDF2L1, PPP1R12A and PRKG1 genes (SIII signature) served as independent prognostic biomarkers of Pt-response and survival. The study identified a prognostic molecular signature based on the combined expression profile of three genes which had never been associated with the clinical outcome of HGS-EOC. This may lead to early identification, at the time of diagnosis, of patients who would not greatly benefit from standard chemotherapy and are thus eligible for novel investigational approaches.

TNF-α elicits phenotypic and functional alterations of vascular smooth muscle cells by miR-155-5p-dependent down-regulation of cGMP-dependent kinase 1.

cGMP-dependent protein kinase 1 (PKG1) plays an important role in nitric oxide (NO)/cGMP-mediated maintenance of vascular smooth muscle cell (VSMC) phenotype and vasorelaxation. Inflammatory cytokines, including tumor necrosis factor-α (TNFα), have long been understood to mediate several inflammatory vascular diseases. However, the underlying mechanism of TNFα-dependent inflammatory vascular disease is unclear. Here, we found that TNFα treatment decreased PKG1 expression in cultured VSMCs, which correlated with NF-κB-dependent biogenesis of miR-155-5p that targeted the 3'-UTR of PKG1 mRNA. TNFα induced VSMC phenotypic switching from a contractile to a synthetic state through the down-regulation of VSMC marker genes, suppression of actin polymerization, alteration of cell morphology, and elevation of cell proliferation and migration. All of these events were blocked by treatment with an inhibitor of miR-155-5p or PKG1, whereas transfection with miR-155-5p mimic or PKG1 siRNA promoted phenotypic modulation, similar to the response to TNFα. In addition, TNFα-induced miR-155-5p inhibited the vasorelaxant response of de-endothelialized mouse aortic vessels to 8-Br-cGMP by suppressing phosphorylation of myosin phosphatase and myosin light chain, both of which are downstream signal modulators of PKG1. Moreover, TNFα-induced VSMC phenotypic alteration and vasodilatory dysfunction were blocked by NF-κB inhibition. These results suggest that TNFα impairs NO/cGMP-mediated maintenance of the VSMC contractile phenotype and vascular relaxation by down-regulating PKG1 through NF-κB-dependent biogenesis of miR-155-5p. Thus, the NF-κB/miR-155-5p/PKG1 axis may be crucial in the pathogenesis of inflammatory vascular diseases, such as atherosclerotic intimal hyperplasia and preeclamptic hypertension.

The natural history of type B aortic dissection in patients with PRKG1 mutation c.530G>A (p.Arg177Gln).

OBJECTIVE: The c.530G>A (p.Arg177Gln) mutation in PRKG1 has been shown to be associated with thoracic aortic aneurysms and dissections. This rare mutation accounts for an estimated 1% of nonsyndromic heritable thoracic aortic disease. We sought to describe the clinical presentation of type B aortic dissection (TBAD), management, and outcomes in patients with this mutation. METHODS: This is a descriptive multi-institutional retrospective study of patients from six families with the PRKG1 mutation. Patients with TBAD were selected for analysis. Demographics, family histories, TBAD management, and outcomes were reviewed. RESULTS: Of the 29 individuals diagnosed with the PRKG1 mutation, 12 (41.3%) had TBAD (50% male, TBAD median age: 31 years [range, 16-58 years], median follow-up: 6 years [range, 3-15 years] after TBAD). All had a family history of aortic dissections and none had features of Marfan syndrome. The median size of the descending thoracic aorta (DTA) at TBAD was 4.1 cm (range, 3.8-5 cm). Most cases (9 acute TBAD, 1 incidental TBAD diagnosis during screening) were managed medically. One case had open DTA repair the acute phase. Repair for dissection-related aneurysmal degeneration was performed in seven cases (58.3%) in the chronic phase at a median of 2 years (range, 1-8 years) after TBAD. In four cases (33.3%), the DTA remained stable in size over a range of 1 to 7 years after TBAD. Type A aortic dissection subsequent to TBAD occurred in three cases (25%). There were four (33.3%) deaths in the series, all aortic related at a median age of 24 years (range, 19-43 years). CONCLUSIONS: The PRKG1 (p.Arg177Gln) mutation although rare is associated with nonsyndromic TBAD in young and middle-aged patients. Workup for this gene mutation should be included as part of the workup for TBAD etiology in relatively young patients and those with familial history of aortic dissections. Once diagnosed, testing of first-degree family members is warranted. In all individuals with a PRKG1 mutation, close follow-up for aortic root dilatation and hypertension control is essential to reduce the risk of type A or type B aortic dissection, and in cases of TBAD, to decrease the risk of dissection-related aneurysmal degeneration.

H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iß pathway activation.

Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H- ras gene deletion produces mice hypotension via a soluble guanylate cyclase-protein kinase G (PKG)-dependent mechanism. In this study, we analyzed the consequences of H- ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H -ras-/-) and control wild-type (H -ras+/+) mice, as were extracellular matrix protein expression. Increased cardiac PKG-Iß protein expression in H -ras-/- mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H -ras-/- mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3ß-dependent activation of the transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iß overexpression in H -ras-/- mouse embryonic fibroblasts. This study demonstrates that H- ras deletion protects against AngII-induced cardiac remodeling, possibly via a mechanism in which PKG-Iß overexpression could play a partial role, and points to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.-Martín-Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López-Olañeta, M., Chiloeches, A., Lara-Pezzi, E., de Frutos, S., Rodríguez-Puyol, M., Calleros, L., Rodríguez-Puyol, D. H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iß pathway activation.

A post-GWAS confirming effects of PRKG1 gene on milk fatty acids in a Chinese Holstein dairy population.

BACKGROUND: We previously conducted a genome-wide association study (GWAS) strategy for milk fatty acids in Chinese Holstein, and identified 83 genome-wide significant single nucleotide polymorphisms (SNPs) and 314 suggestive significant SNPs. Among them, two SNPs, BTB-01077939 and BTA-11275-no-rs associated with C10:0, C12:0, and C14 index (P = 0.000014 ~ 0.000024), were within and close to (0.85 Mb) protein kinase, cGMP-dependent, type І (PRKG1) gene on BTA26, respectively. PRKG1 gene plays a key role in lipolysis to release fatty acids and glycerol through the hydrolysis of triacyglycerol in adipocytes. We herein considered it as a promising candidate for milk fatty acids. The purpose of this study was to investigate whether PRKG1 had effects on milk fatty acids. RESULTS: By direct sequencing the PCR products of pooled DNA, we identified a total of six SNPs, including one in 5' flanking region, four in 3' untranslated region (UTR), and one in 3' flanking region. The single-locus association analysis was carried out, and showed that the six SNPs mainly had significant associations with C6:0, C8:0 and C17:1 (P < 0.0001 ~ 0.0035). In addition, we observed a haplotype block formed by g.6903810G > A and g.6904047G > T with Haploview 4.1, and it was strongly associated with C8:0, C10:0, C16:1, C17:1, C20:0 and C16 index (P = < 0.0001 ~ 0.0123). The SNP, g.8344262A > T, was predicted to alter the binding site (BS) of transcription factor (TF) GAGA box with Genomatix software, and the subsequent luciferase assay verified that it really changed the transcriptional activity of PRKG1 gene (P = 0.0009). CONCLUSION: In conclusion, to our best of knowledge, we are the first who identified the significant effects of PRKG1 on milk fatty acids in dairy cattle.

A genome-wide gene-by-trauma interaction study of alcohol misuse in two independent cohorts identifies PRKG1 as a risk locus.

Traumatic life experiences are associated with alcohol use problems, an association that is likely to be moderated by genetic predisposition. To understand these interactions, we conducted a gene-by-environment genome-wide interaction study (GEWIS) of alcohol use problems in two independent samples, the Army STARRS (STARRS, N=16 361) and the Yale-Penn (N=8084) cohorts. Because the two cohorts were assessed using different instruments, we derived separate dimensional alcohol misuse scales and applied a proxy-phenotype study design. In African-American subjects, we identified an interaction of PRKG1 rs1729578 with trauma exposure in the STARRS cohort and replicated its interaction with trauma exposure in the Yale-Penn cohort (discovery-replication meta-analysis: z=5.64, P=1.69 × 10-8). PRKG1 encodes cyclic GMP-dependent protein kinase 1, which is involved in learning, memory and circadian rhythm regulation. Considering the loci identified in stage-1 that showed same effect directions in stage-2, the gene ontology (GO) enrichment analysis showed several significant results, including calcium-activated potassium channels (GO:0016286; P=2.30 × 10-5), cognition (GO:0050890; P=1.90 × 10-6), locomotion (GO:0040011; P=6.70 × 10-5) and Stat3 protein regulation (GO:0042517; P=6.4 × 10-5). To our knowledge, this is the largest GEWIS performed in psychiatric genetics, and the first GEWIS examining risk for alcohol misuse. Our results add to a growing body of literature highlighting the dynamic impact of experience on individual genetic risk.

Results of next-generation sequencing gene panel diagnostics including copy-number variation analysis in 810 patients suspected of heritable thoracic aortic disorders.

Simultaneous analysis of multiple genes using next-generation sequencing (NGS) technology has become widely available. Copy-number variations (CNVs) in disease-associated genes have emerged as a cause for several hereditary disorders. CNVs are, however, not routinely detected using NGS analysis. The aim of this study was to assess the diagnostic yield and the prevalence of CNVs using our panel of Hereditary Thoracic Aortic Disease (H-TAD)-associated genes. Eight hundred ten patients suspected of H-TAD were analyzed by targeted NGS analysis of 21 H-TAD associated genes. In addition, the eXome hidden Markov model (XHMM; an algorithm to identify CNVs in targeted NGS data) was used to detect CNVs in these genes. A pathogenic or likely pathogenic variant was found in 66 of 810 patients (8.1%). Of these 66 pathogenic or likely pathogenic variants, six (9.1%) were CNVs not detectable by routine NGS analysis. These CNVs were four intragenic (multi-)exon deletions in MYLK, TGFB2, SMAD3, and PRKG1, respectively. In addition, a large duplication including NOTCH1 and a large deletion encompassing SCARF2 were detected. As confirmed by additional analyses, both CNVs indicated larger chromosomal abnormalities, which could explain the phenotype in both patients. Given the clinical relevance of the identification of a genetic cause, CNV analysis using a method such as XHMM should be incorporated into the clinical diagnostic care for H-TAD patients.

The activity of cGMP-dependent protein kinase Iα is not directly regulated by oxidation-induced disulfide formation at cysteine 43.

The type I cGMP-dependent protein kinases (PKGs) are key regulators of smooth muscle tone, cardiac hypertrophy, and other physiological processes. The two isoforms PKGIα and PKGIß are thought to have unique functions because of their tissue-specific expression, different cGMP affinities, and isoform-specific protein-protein interactions. Recently, a non-canonical pathway of PKGIα activation has been proposed, in which PKGIα is activated in a cGMP-independent fashion via oxidation of Cys43, resulting in disulfide formation within the PKGIα N-terminal dimerization domain. A "redox-dead" knock-in mouse containing a C43S mutation exhibits phenotypes consistent with decreased PKGIα signaling, but the detailed mechanism of oxidation-induced PKGIα activation is unknown. Therefore, we examined oxidation-induced activation of PKGIα, and in contrast to previous findings, we observed that disulfide formation at Cys43 does not directly activate PKGIα in vitro or in intact cells. In transfected cells, phosphorylation of Ras homolog gene family member A (RhoA) and vasodilator-stimulated phosphoprotein was increased in response to 8-CPT-cGMP treatment, but not when disulfide formation in PKGIα was induced by H2O2 Using purified enzymes, we found that the Cys43 oxidation had no effect on basal kinase activity or Km and Vmax values; however, PKGIα containing the C43S mutation was less responsive to cGMP-induced activation. This reduction in cGMP affinity may in part explain the PKGIα loss-of-function phenotype of the C43S knock-in mouse. In conclusion, disulfide formation at Cys43 does not directly activate PKGIα, and the C43S-mutant PKGIα has a higher Ka for cGMP. Our results highlight that mutant enzymes should be carefully biochemically characterized before making in vivo inferences.

Two isoforms of cyclic GMP-dependent kinase-I exhibit distinct expression patterns in the adult mouse dorsal root ganglion.

cGMP-dependent kinase-I (cGKI) is known to regulate spinal pain processing. This enzyme consists of two isoforms (cGKIα and cGKIß) that show distinct substrate specificity and tissue distribution. It has long been believed that the α isoform is exclusively expressed in the adult dorsal root ganglion. The aim of the present study was to reexamine the expression of cGKI isoforms in the adult mouse dorsal root ganglion using isoform-specific cGKI antibodies whose specificities had been validated in the previous studies. Immunoblot and immunohistochemical analyses revealed the presence of both isoforms in the dorsal root ganglion. Moreover, cGKIα was found to be mainly expressed within the cytoplasm of small- to medium-sized peptidergic and nonpeptidegic C-fibers, whereas cGKIß was located within the nuclei of a wide range of dorsal root ganglion neurons. In addition, glutamine synthetase-positive satellite glial cells expressed both isoforms to varying degrees. Finally, using an experimental model for neuropathic pain produced by L5 spinal nerve transection, we found that cGKIα expression was downregulated in the injured, but not in the uninjured, dorsal root ganglion. In contrast, cGKIß expression was upregulated in both the injured and uninjured dorsal root ganglions. Also, injury-induced cGKIß upregulation was found to occur in small-to-medium-diameter dorsal root ganglion neurons. These data thus demonstrate the existence of two differently distributed cGKI isoforms in the dorsal root ganglion, and may provide insight into the cellular and molecular mechanisms of pain.

Exome sequencing reveals a de novo PRKG1 mutation in a sporadic patient with aortic dissection.

BACKGROUND: Thoracic aortic aneurysm and dissection (TAAD) is a common condition associated with high mortality. It is predominantly inherited in an autosomal dominant manner with reduced penetrance and variable expression. The genetic basis of the majority of TAAD cases remains unknown. CASE PRESENTATION: We described a 53 years old male presented with abdominal aortic dissection as well as aortic tortuosity. To investigate the genetic basis of the clinical presentation, whole-exome sequencing was performed. Exome sequencing identified a de novo heterozygous undescribed mutation in the PRKG1 gene (NM_001098512.2: c.1108 G > A), predicted to cause the missense change p.Gly370Ser in the ATP binding motif of the protein. This mutation was not reported in the dbSNP, 1000 Genome Project, and Exome sequencing databases. Furthermore, the Glycine370 residue of PRKG1 is highly conserved among various species and it is predicted to be damaging by multiple in silico programs, suggesting that this substitution may cause a major disruption of protein function. To our knowledge, this is the second reported mutation locus of PRKG1 accounting for the disease. CONCLUSIONS: Our study expands the mutation spectrum of PRKG1 and clinical phenotype of mutation-carriers. Screening for PRKG1 mutations should be considered in patients with unexplained aortic disease, and identification of the causative gene will aid in individualized, gene-tailored management.

Altered Smooth Muscle Cell Force Generation as a Driver of Thoracic Aortic Aneurysms and Dissections.

The importance of maintaining contractile function in aortic smooth muscle cells (SMCs) is evident by the fact that heterozygous mutations in the major structural proteins or kinases controlling contraction lead to the formation of aneurysms of the ascending thoracic aorta that predispose to life-threatening aortic dissections. Force generation by SMC requires ATP-dependent cyclic interactions between filaments composed of SMC-specific isoforms of α-actin (encoded by ACTA2) and myosin heavy chain (MYH11). ACTA2 and MYH11 mutations are predicted or have been shown to disrupt this cyclic interaction predispose to thoracic aortic disease. Movement of the myosin motor domain is controlled by phosphorylation of the regulatory light chain on the myosin filament, and loss-of-function mutations in the dedicated kinase for this phosphorylation, myosin light chain kinase (MYLK) also predispose to thoracic aortic disease. Finally, a mutation in the cGMP-activated protein kinase (PRKG1) results in constitutive activation of the kinase in the absence of cGMP, thus driving SMC relaxation in part through increased dephosphorylation of the regulatory light chain and predisposes to thoracic aortic disease. Furthermore, SMCs cannot generate force without connections to the extracellular matrix through focal adhesions, and mutations in the major protein in the extracellular matrix, fibrillin-1, linking SMCs to the matrix also cause thoracic aortic disease in individuals with Marfan syndrome. Thus, disruption of the ability of the aortic SMC to generate force through the elastin-contractile units in response to pulsatile blood flow may be a primary driver for thoracic aortic aneurysms and dissections.

Replication of the Interaction of PRKG1 and Trauma Exposure on Alcohol Misuse in an Independent African American Sample.

In the present study, we sought to replicate recent findings of Polimanti et al. (2017), who conducted a genome-wide gene-by-environment interaction study (GEWIS) and identified a gene-by-trauma interaction that predicts alcohol misuse among African Americans.  Consistent with the findings published by Polimanti and colleagues, results of the current study demonstrated an interaction effect, b = 0.41, of trauma exposure and rs1729578 in the intron of PRKG1 on alcohol misuse in a subsample of ancestral African Americans. The minor allele (rs1729578*C) was positively associated with increased alcohol use disorder symptoms in trauma-exposed subjects and negatively associated in non-trauma-exposed subjects.  This effect, however, was only significant for one out of three alcohol outcome measures we investigated, suggesting the interaction may be most salient when predicting higher severity of alcohol misuse. Additionally, the effect did not remain significant after we accounted for testing the effect on three different outcome variables. Also in line with the original study, the gene-by-environment effect was not demonstrated among the ancestral European subsample.  The findings suggest this gene variant may increase an individual's susceptibility to environmental influences, both adverse and supportive.

Neural Activity-Dependent Regulation of Radial Glial Filopodial Motility Is Mediated by Glial cGMP-Dependent Protein Kinase 1 and Contributes to Synapse Maturation in the Developing Visual System.

UNLABELLED: Radial glia in the developing optic tectum extend highly dynamic filopodial protrusions within the tectal neuropil, the motility of which has previously been shown to be sensitive to neural activity and nitric oxide (NO) release. Using in vivo two-photon microscopy, we performed time-lapse imaging of radial glial cells and measured filopodial motility in the intact albino Xenopus laevis tadpole. Application of MK801 to block neuronal NMDA receptor (NMDAR) currents confirmed a significant reduction in radial glial filopodial motility. This reduction did not occur in glial cells expressing a dominant-negative form of cGMP-dependent protein kinase 1 (PKG1), and was prevented by elevation of cGMP levels with the phosphodiesterase type 5 inhibitor sildenafil. These results suggest that neuronal NMDAR activation results in the release of NO, which in turn modulates PKG1 activation in glial cells to control filopodial motility. We further showed that interfering with the function of the small GTPases Rac1 or RhoA, known to be regulated by PKG1 phosphorylation, decreased motility or eliminated filopodial processes respectively. These manipulations led to profound defects in excitatory synaptic development and maturation of neighboring neurons. SIGNIFICANCE STATEMENT: Radial glia in the developing brain extend motile filopodia from their primary stalk. Neuronal NMDA receptor activity controls glial motility through intercellular activation of cGMP-dependent protein kinase 1 (PKG1) signaling in glial cells. Manipulating PKG1, Rac1, or RhoA signaling in radial glia in vivo to eliminate glial filopodia or impair glial motility profoundly impacted synaptogenesis and circuit maturation.

Phosphodiesterase 5 Inhibition Limits Doxorubicin-induced Heart Failure by Attenuating Protein Kinase G Iα Oxidation.

Phosphodiesterase 5 (PDE5) inhibitors limit myocardial injury caused by stresses, including doxorubicin chemotherapy. cGMP binding to PKG Iα attenuates oxidant-induced disulfide formation. Because PDE5 inhibition elevates cGMP and protects from doxorubicin-induced injury, we reasoned that this may be because it limits PKG Iα disulfide formation. To investigate the role of PKG Iα disulfide dimerization in the development of apoptosis, doxorubicin-induced cardiomyopathy was compared in male wild type (WT) or disulfide-resistant C42S PKG Iα knock-in (KI) mice. Echocardiography showed that doxorubicin treatment caused loss of myocardial tissue and depressed left ventricular function in WT mice. Doxorubicin also reduced pro-survival signaling and increased apoptosis in WT hearts. In contrast, KI mice were markedly resistant to the dysfunction induced by doxorubicin in WTs. In follow-on experiments the influence of the PDE5 inhibitor tadalafil on the development of doxorubicin-induced cardiomyopathy in WT and KI mice was investigated. In WT mice, co-administration of tadalafil with doxorubicin reduced PKG Iα oxidation caused by doxorubicin and also protected against cardiac injury and loss of function. KI mice were again innately resistant to doxorubicin-induced cardiotoxicity, and therefore tadalafil afforded no additional protection. Doxorubicin decreased phosphorylation of RhoA (Ser-188), stimulating its GTPase activity to activate Rho-associated protein kinase (ROCK) in WTs. These pro-apoptotic events were absent in KI mice and were attenuated in WTs co-administered tadalafil. PKG Iα disulfide formation triggers cardiac injury, and this initiation of maladaptive signaling can be blocked by pharmacological therapies that elevate cGMP, which binds kinase to limit its oxidation.

Suppression of Npr1, not Npr2 gene function induces hypertrophic growth in H9c2 cells in vitro.

Npr1 gene (coding for NPR-A) and Npr2 gene (coding for NPR-B) are identified as intrinsic anti-hypertrophic genes that opposes abnormal cardiac remodeling. However, the functional role of Npr1 and Npr2 genes during cardiac hypertrophic growth is not well understood. Hence, the present investigation was aimed to study the effect of Npr1 and Npr2 gene silencing, respectively on ß-AR activation induced cardiac hypertrophic growth in H9c2 cells in vitro. The control, Npr1, and Npr2 gene suppressed H9c2 cells, respectively were treated with ISO (10-5 M) for 48 h. The mRNA and protein expression profile of NPR-A, NPR-B, PKG-I and cGMP were analyzed by qPCR, Western blotting, ELISA, and immunofluorescence methods, respectively. A marked increase in cell size (30.10 ± 0.51 µm vs 61.83 ± 0.43 µm, 2-fold) accompanied by elevated hypertrophic marker genes (α-sk and ß-MHC 3-fold, respectively) expression was observed in Npr1 gene suppressed H9c2 cells as compared with control cells. In contrast, the Npr2 gene suppression in H9c2 cells neither altered the cell size nor the level of hypertrophic marker genes expression. Upon exposure to Isoproterenol, the Npr1 suppressed H9c2 cells exhibited further increase in cell size (1.5 fold), whereas, no significant increase in cell size or marker genes expression was noticed in Npr2 suppressed cells. Moreover, the intracellular cGMP level was down-regulated by 2-fold in Npr1 suppressed cells, while, no significant change was observed in Npr2 suppressed cells. Together, these results suggest that Npr1, not Npr2 gene function is positively associated with the initiation of cardiac fetal gene program and development of cardiac hypertrophic growth.

Tumor necrosis factor (TNF)-α-induced repression of GKAP42 protein levels through cGMP-dependent kinase (cGK)-Iα causes insulin resistance in 3T3-L1 adipocytes.

Insulin receptor substrates (IRSs) have been shown to be major mediators of insulin signaling. Recently, we found that IRSs form high-molecular weight complexes, and here, we identify by yeast two-hybrid screening a novel IRS-1-associated protein: a 42-kDa cGMP-dependent protein kinase-anchoring protein (GKAP42). GKAP42 knockdown in 3T3-L1 adipocytes suppressed insulin-dependent IRS-1 tyrosine phosphorylation and downstream signaling, resulting in suppression of GLUT4 translocation to plasma membrane induced by insulin. In addition, GLUT4 translocation was also suppressed in cells overexpressing GKAP42-N (the IRS-1 binding region of GKAP42), which competed with GKAP42 for IRS-1, indicating that GKAP42 binding to IRS-1 is required for insulin-induced GLUT4 translocation. Long term treatment of 3T3-L1 adipocytes with TNF-α, which induced insulin resistance, significantly decreased the GKAP42 protein level. We then investigated the roles of cGMP-dependent kinase (cGK)-Iα, which bound to GKAP42, in these changes. cGK-Iα knockdown partially rescued TNF-α-induced decrease in GKAP42 and impairment of insulin signals. These data indicated that TNF-α-induced repression of GKAP42 via cGK-Iα caused reduction of insulin-induced IRS-1 tyrosine phosphorylation at least in part. The present study describes analysis of the novel TNF-α-induced pathway, cGK-Iα-GKAP42, which regulates insulin-dependent signals and GLUT4 translocation.