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1.
FASEB J ; 31(4): 1620-1638, 2017 04.
Article in English | MEDLINE | ID: mdl-28138039

ABSTRACT

LIM domain proteins have been identified as essential modulators of cardiac biology and pathology; however, it is unclear which role the cysteine-rich LIM-only protein (CRP)4 plays in these processes. In studying CRP4 mutant mice, we found that their hearts developed normally, but lack of CRP4 exaggerated multiple parameters of the cardiac stress response to the neurohormone angiotensin II (Ang II). Aiming to dissect the molecular details, we found a link between CRP4 and the cardioprotective cGMP pathway, as well as a multiprotein complex comprising well-known hypertrophy-associated factors. Significant enrichment of the cysteine-rich intestinal protein (CRIP)1 in murine hearts lacking CRP4, as well as severe cardiac defects and premature death of CRIP1 and CRP4 morphant zebrafish embryos, further support the notion that depleting CRP4 is incompatible with a proper cardiac development and function. Together, amplified Ang II signaling identified CRP4 as a novel antiremodeling factor regulated, at least to some extent, by cardiac cGMP.-Straubinger, J., Boldt, K., Kuret, A., Deng, L., Krattenmacher, D., Bork, N., Desch, M., Feil, R., Feil, S., Nemer, M., Ueffing, M., Ruth, P., Just, S., Lukowski, R. Amplified pathogenic actions of angiotensin II in cysteine-rich LIM-only protein 4 negative mouse hearts.


Subject(s)
Angiotensin II/metabolism , Cardiomegaly/metabolism , alpha-Defensins/genetics , Angiotensin II/pharmacology , Animals , Cardiomegaly/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cells, Cultured , Cyclic GMP/metabolism , Heart/drug effects , Heart/growth & development , LIM Domain Proteins/genetics , LIM Domain Proteins/metabolism , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Zebrafish , alpha-Defensins/metabolism
3.
Proc Natl Acad Sci U S A ; 111(35): 12925-9, 2014 Sep 02.
Article in English | MEDLINE | ID: mdl-25139994

ABSTRACT

Conflicting results have been reported for the roles of cGMP and cGMP-dependent protein kinase I (cGKI) in various pathological conditions leading to cardiac hypertrophy and fibrosis. A cardioprotective effect of cGMP/cGKI has been reported in whole animals and isolated cardiomyocytes, but recent evidence from a mouse model expressing cGKIß only in smooth muscle (ßRM) but not in cardiomyocytes, endothelial cells, or fibroblasts has forced a reevaluation of the requirement for cGKI activity in the cardiomyocyte antihypertrophic effects of cGMP. In particular, ßRM mice developed the same hypertrophy as WT controls when subjected to thoracic aortic constriction or isoproterenol infusion. Here, we challenged ßRM and WT (Ctr) littermate control mice with angiotensin II (AII) infusion (7 d; 2 mg ⋅ kg(-1) ⋅ d(-1)) to induce hypertrophy. Both genotypes developed cardiac hypertrophy, which was more pronounced in Ctr animals. Cardiomyocyte size and interstitial fibrosis were increased equally in both genotypes. Addition of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, in the drinking water had a small effect in reducing myocyte hypertrophy in WT mice and no effect in ßRM mice. However, sildenafil substantially blocked the increase in collagen I, fibronectin 1, TGFß, and CTGF mRNA in Ctr but not in ßRM hearts. These data indicate that, for the initial phase of AII-induced cardiac hypertrophy, lack of cardiomyocyte cGKI activity does not worsen hypertrophic growth. However, expression of cGKI in one or more cell types other than smooth muscle is necessary to allow the antifibrotic effect of sildenafil.


Subject(s)
Angiotensin II/pharmacology , Cardiomegaly/metabolism , Cyclic GMP-Dependent Protein Kinase Type I/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5/metabolism , Hypertension/metabolism , Animals , Cardiomegaly/chemically induced , Cyclic GMP/metabolism , Fibrosis/chemically induced , Fibrosis/metabolism , Genetic Markers , Hypertension/chemically induced , Mice , Muscle, Smooth/metabolism , Myocardial Contraction/drug effects , Myocardial Contraction/physiology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Nitric Oxide/metabolism , Phosphodiesterase 5 Inhibitors/pharmacology , Piperazines/pharmacology , Purines/pharmacology , Sildenafil Citrate , Sulfones/pharmacology , Vasoconstrictor Agents/pharmacology
4.
Am J Physiol Heart Circ Physiol ; 301(3): H672-82, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21666108

ABSTRACT

Signaling by nitric oxide (NO) determines several cardiovascular functions including blood pressure regulation, cardiac and smooth muscle hypertrophy, and platelet function. NO stimulates the synthesis of cGMP by soluble guanylyl cyclases and thereby activates cGMP-dependent protein kinases (PKGs), mediating most of the cGMP functions. Hence, an elucidation of the PKG signaling cascade is essential for the understanding of the (patho)physiological aspects of NO. Several PKG signaling pathways were identified, meanwhile regulating the intracellular calcium concentration, mediating calcium desensitization or cytoskeletal rearrangement. During the last decade it emerged that the inositol trisphosphate receptor-associated cGMP-kinase substrate (IRAG), an endoplasmic reticulum-anchored 125-kDa membrane protein, is a main signal transducer of PKG activity in the cardiovascular system. IRAG interacts specifically in a trimeric complex with the PKG1ß isoform and the inositol 1,4,5-trisphosphate receptor I and, upon phosphorylation, reduces the intracellular calcium release from the intracellular stores. IRAG motifs for phosphorylation and for targeting to PKG1ß and 1,4,5-trisphosphate receptor I were identified by several approaches. The (patho)physiological functions for the regulation of smooth muscle contractility and the inhibition of platelet activation were perceived. In this review, the IRAG recognition, targeting, and function are summarized compared with PKG and several PKG substrates in the cardiovascular system.


Subject(s)
Cardiovascular Agents/therapeutic use , Cardiovascular Diseases/drug therapy , Cardiovascular System/drug effects , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Protein Kinase Inhibitors/therapeutic use , Signal Transduction/drug effects , Amino Acid Sequence , Animals , Cardiovascular Diseases/enzymology , Cardiovascular Diseases/physiopathology , Cardiovascular System/enzymology , Cardiovascular System/physiopathology , Cyclic GMP/metabolism , Cyclic GMP-Dependent Protein Kinases/metabolism , Humans , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Molecular Sequence Data , Molecular Targeted Therapy , Nitric Oxide/metabolism , Phosphorylation
5.
FEBS Lett ; 584(18): 3979-84, 2010 Sep 24.
Article in English | MEDLINE | ID: mdl-20691687

ABSTRACT

We analysed the function and intracellular signalling of the cyclic pyrimidinic nucleotide cCMP. The membrane-permeable cCMP analogue dibutyryl-cCMP mediated mouse aorta relaxation. cCMP activated purified cGMP-dependent protein kinase (cGK) Iα and Iß and stimulated cGK in aorta lysates. cCMP-induced relaxation was abolished in cGKI-knockout tissue. Additionally, deletion of inositol-trisphosphate receptor associated cGKI substrate (IRAG) suppressed cCMP-mediated relaxation. Signalling of cCMP via cGKI/IRAG appears to be of broader physiological importance because cCMP-mediated inhibition of platelet aggregation was absent in cGKI- and IRAG-deficient platelets. These results demonstrate that cCMP acts as intracellular messenger molecule, most unexpectedly utilizing the cGMP signal transduction pathway.


Subject(s)
Cyclic CMP/metabolism , Cyclic GMP-Dependent Protein Kinases/metabolism , Muscle Relaxation , Muscle, Smooth/physiology , Animals , Aorta/drug effects , Aorta/enzymology , Aorta/physiology , Cyclic CMP/pharmacology , Cyclic GMP-Dependent Protein Kinases/genetics , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Mice , Mice, Knockout , Muscle, Smooth/drug effects , Muscle, Smooth/enzymology , Platelet Aggregation/drug effects , Platelet Aggregation/physiology , Signal Transduction
6.
Cardiovasc Res ; 86(3): 496-505, 2010 Jun 01.
Article in English | MEDLINE | ID: mdl-20080989

ABSTRACT

AIMS: Nitric oxide (NO) and atrial natriuretic peptide (ANP) signalling via cGMP controls smooth muscle tone. One important signalling pathway of cGMP-dependent protein kinase type I (cGKI) is mediated by IRAG (IP(3) receptor associated cGKI substrate) which is highly expressed in smooth muscle tissues. To elucidate the role of IRAG for NO- and ANP-mediated smooth muscle tone regulation, cGKI localization, and for its possible function in blood pressure adjustment, we generated IRAG-knockout mice by targeted deletion of exon 3. METHODS AND RESULTS: IRAG deletion prevented stable interaction of IP(3) receptor type I (IP(3)RI) with cGKIbeta determined by cGMP affinity chromatography. Confocal microscopy in vascular smooth muscle cells (VSMCs) showed that localization of cGKIbeta and cGKIalpha did not change in absence of IRAG. NO-, ANP-, and cGMP-dependent relaxation of hormone-contracted aortic vessels and colon was significantly affected in IRAG-knockout mice. The suppression of cGMP-induced relaxation was not rescued by selective expression of cGKIbeta in smooth muscle from cGKIbeta-transgenic mice. NO-, ANP-, and cGMP-mediated inhibition of the hormone-induced increase in intracellular calcium concentration measured by Fura2 was suppressed in IRAG-deficient VSMC. Telemetric measurements revealed that IRAG-deficient animals exhibited normal basal tone, but were resistant to blood pressure reduction induced by lipopolysaccharide-treatment. CONCLUSION: These findings indicate that signalling of cGKIbeta via IRAG is an essential functional part for regulation of smooth muscle tone and of intracellular calcium by NO (exogenously applicated or endogenously synthesized) and by ANP. IRAG signalling does not modulate basal tone but might be important for blood pressure regulation under pathophysiological conditions.


Subject(s)
Atrial Natriuretic Factor/metabolism , Muscle Relaxation , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth/metabolism , Nitric Oxide/metabolism , Phosphoproteins/metabolism , Vasodilation , Animals , Aorta/metabolism , Blood Pressure , COS Cells , Calcium/metabolism , Chlorocebus aethiops , Chromatography, Affinity , Colon/metabolism , Cyclic GMP-Dependent Protein Kinase Type I , Cyclic GMP-Dependent Protein Kinases/genetics , Cyclic GMP-Dependent Protein Kinases/metabolism , Exons , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Intracellular Signaling Peptides and Proteins , Membrane Proteins , Mice , Mice, Knockout , Mice, Transgenic , Microscopy, Confocal , Phosphoproteins/deficiency , Phosphoproteins/genetics , Signal Transduction , Transfection
7.
Handb Exp Pharmacol ; (191): 163-93, 2009.
Article in English | MEDLINE | ID: mdl-19089330

ABSTRACT

Signalling of cGK (cGMP-dependent protein kinases) are mediated through phosphorylation of specific substrates. Several substrates of cGKI and cGKII were identified meanwhile. Some cGKI substrates are specifically regulated by the cGKIalpha or the cGKIbeta isozyme. In various cells and tissues, different cGK substrates exist that are essential for the regulation of diverse functions comprising tissue contractility, cell motility, cell contact, cellular secretion, cell proliferation, and cell differentiation. On the molecular level, cGKI substrates fulfill various cellular functions regulating e.g. the intracellular calcium and potassium concentration, the calcium sensitivity, and the organisation of the intracellular cytoskeleton. cGKII substrates are involved e.g. in chloride transport, sodium/proton transport and transcriptional regulation. The understanding of cGK signalling and function depends strongly on the identification of further specific substrates. In the last years, diverse approaches ranging from biochemistry to genetic deletion lead to the identification and establishment of several substrates, which raised new insights in the molecular mechanisms of cGK functions and elucidated new cellular cGK functions. However, the analysis of the dynamic signalling of cGK in tissues and cells will be necessary to discover new signalling pathways and substrates.


Subject(s)
Cyclic GMP-Dependent Protein Kinases/metabolism , Signal Transduction , Animals , Cyclic GMP-Dependent Protein Kinase Type I , Cyclic GMP-Dependent Protein Kinase Type II , Humans , Peptides/metabolism , Phosphorylation/physiology , Proteins/metabolism , Substrate Specificity
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