Antagonism of Forkhead Box Subclass O Transcription Factors Elicits Loss of Soluble Guanylyl Cyclase Expression.

Nitric oxide (NO) stimulates soluble guanylyl cyclase (sGC) activity, leading to elevated intracellular cyclic guanosine 3',5'-monophosphate (cGMP) and subsequent vascular smooth muscle relaxation. It is known that downregulation of sGC expression attenuates vascular dilation and contributes to the pathogenesis of cardiovascular disease. However, it is not well understood how sGC transcription is regulated. Here, we demonstrate that pharmacological inhibition of Forkhead box subclass O (FoxO) transcription factors using the small-molecule inhibitor AS1842856 significantly blunts sGC α and ß mRNA expression by more than 90%. These effects are concentration-dependent and concomitant with greater than 90% reduced expression of the known FoxO transcriptional targets, glucose-6-phosphatase and growth arrest and DNA damage protein 45 α (Gadd45α). Similarly, sGC α and sGC ß protein expression showed a concentration-dependent downregulation. Consistent with the loss of sGC α and ß mRNA and protein expression, pretreatment of vascular smooth muscle cells with the FoxO inhibitor decreased sGC activity measured by cGMP production following stimulation with an NO donor. To determine if FoxO inhibition resulted in a functional impairment in vascular relaxation, we cultured mouse thoracic aortas with the FoxO inhibitor and conducted ex vivo two-pin myography studies. Results showed that aortas have significantly blunted sodium nitroprusside-induced (NO-dependent) vasorelaxation and a 42% decrease in sGC expression after 48-hour FoxO inhibitor treatment. Taken together, these data are the first to identify that FoxO transcription factor activity is necessary for sGC expression and NO-dependent relaxation.

Cardioprotection by ischemic postconditioning and cyclic guanosine monophosphate-elevating agents involves cardiomyocyte nitric oxide-sensitive guanylyl cyclase.

Aims: It has been suggested that the nitric oxide-sensitive guanylyl cyclase (NO-GC)/cyclic guanosine monophosphate (cGMP)-dependent signalling pathway affords protection against cardiac damage during acute myocardial infarction (AMI). It is, however, not clear whether the NO-GC/cGMP system confers its favourable effects through a mechanism located in cardiomyocytes (CMs). The aim of this study was to evaluate the infarct-limiting effects of the endogenous NO-GC in CMs in vivo. Methods and results: Ischemia/reperfusion (I/R) injury was evaluated in mice with a CM-specific deletion of NO-GC (CM NO-GC KO) and in control siblings (CM NO-GC CTR) subjected to an in vivo model of AMI. Lack of CM NO-GC resulted in a mild increase in blood pressure but did not affect basal infarct sizes after I/R. Ischemic postconditioning (iPost), administration of the phosphodiesterase-5 inhibitors sildenafil and tadalafil as well as the NO-GC activator cinaciguat significantly reduced the amount of infarction in control mice but not in CM NO-GC KO littermates. Interestingly, NS11021, an opener of the large-conductance and Ca2+-activated potassium channel (BK), an important downstream effector of cGMP/cGKI in the cardiovascular system, protects I/R-exposed hearts of CM NO-GC proficient and deficient mice. Conclusions: These findings demonstrate an important role of CM NO-GC for the cardioprotective signalling following AMI in vivo. CM NO-GC function is essential for the beneficial effects on infarct size elicited by iPost and pharmacological elevation of cGMP; however, lack of CM NO-GC does not seem to disrupt the cardioprotection mediated by the BK opener NS11021.

Decreased Soluble Guanylate Cyclase Contributes to Cardiac Dysfunction Induced by Chronic Doxorubicin Treatment in Mice.

AIMS: The use of doxorubicin, a potent chemotherapeutic agent, is limited by cardiotoxicity. We tested the hypothesis that decreased soluble guanylate cyclase (sGC) enzyme activity contributes to the development of doxorubicin-induced cardiotoxicity. RESULTS: Doxorubicin administration (20 mg/kg, intraperitoneally [IP]) reduced cardiac sGC activity in wild-type (WT) mice. To investigate whether decreased sGC activity contributes to doxorubicin-induced cardiotoxicity, we studied mice with cardiomyocyte-specific deficiency of the sGC α1-subunit (mice with cardiomyocyte-specific deletion of exon 6 of the sGCα1 allele [sGCα1-/-CM]). After 12 weeks of doxorubicin administration (2 mg/kg/week IP), left ventricular (LV) systolic dysfunction was greater in sGCα1-/-CM than WT mice. To further assess whether reduced sGC activity plays a pathogenic role in doxorubicin-induced cardiotoxicity, we studied a mouse model in which decreased cardiac sGC activity was induced by cardiomyocyte-specific expression of a dominant negative sGCα1 mutant (DNsGCα1) upon doxycycline removal (Tet-off). After 8 weeks of doxorubicin administration, DNsGCα1tg/+, but not WT, mice displayed LV systolic dysfunction and dilatation. The difference in cardiac function and remodeling between DNsGCα1tg/+ and WT mice was even more pronounced after 12 weeks of treatment. Further impairment of cardiac function was attenuated when DNsGCα1 gene expression was inhibited (beginning at 8 weeks of doxorubicin treatment) by administering doxycycline. Furthermore, doxorubicin-associated reactive oxygen species generation was higher in sGCα1-deficient than WT hearts. Innovation and Conclusion: These data demonstrate that a reduction in cardiac sGC activity worsens doxorubicin-induced cardiotoxicity in mice and identify sGC as a potential therapeutic target. Various pharmacological sGC agonists are in clinical development or use and may represent a promising approach to limit doxorubicin-associated cardiotoxicity. Antioxid. Redox Signal. 26, 153-164.

Paracrine nitric oxide induces expression of cardiac sarcomeric proteins in adult progenitor cells through soluble guanylyl cyclase/cyclic-guanosine monophosphate and Wnt/ß-catenin inhibition.

AIM: Cardiac progenitor cells (CPC) from adult hearts can differentiate to several cell types composing the myocardium but the underlying molecular pathways are poorly characterized. We examined the role of paracrine nitric oxide (NO) in the specification of CPC to the cardiac lineage, particularly through its inhibition of the canonical Wnt/ß-catenin pathway, a critical step preceding cardiac differentiation. METHODS AND RESULTS: Sca1 + CPC from adult mouse hearts were isolated by magnetic-activated cell sorting and clonally expanded. Pharmacologic NO donors increased their expression of cardiac myocyte-specific sarcomeric proteins in a concentration and time-dependent manner. The optimal time window for NO efficacy coincided with up-regulation of CPC expression of Gucy1a3 (coding the alpha1 subunit of guanylyl cyclase). The effect of paracrine NO was reproduced in vitro upon co-culture of CPC with cardiac myocytes expressing a transgenic NOS3 (endothelial nitric oxide synthase) and in vivo upon injection of CPC in infarcted hearts from cardiac-specific NOS3 transgenic mice. In mono- and co-cultures, this effect was abrogated upon inhibition of soluble guanylyl cyclase or nitric oxide synthase, and was lost in CPC genetically deficient in Gucy1a3. Mechanistically, NO inhibits the constitutive activity of the canonical Wnt/ß-catenin in CPC and in cell reporter assays in a guanylyl cyclase-dependent fashion. This was paralleled with decreased expression of ß-catenin and down-regulation of Wnt target genes in CPC and abrogated in CPC with a stabilized, non-inhibitable ß-catenin. CONCLUSIONS: Exogenous or paracrine sources of NO promote the specification towards the myocyte lineage and expression of cardiac sarcomeric proteins of adult CPC. This is contingent upon the expression and activity of the alpha1 subunit of guanylyl cyclase in CPC that is necessary for NO-mediated inhibition of the canonical Wnt/ß-catenin pathway.