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 alpha 1-subunit (mice with cardiomyocyte-specific deletion of exon 6 of the sGC alpha 1 allele [sGC alpha 1(-/-CM)]). After 12 weeks of doxorubicin administration (2 mg/kg/week IP), left ventricular (LV) systolic dysfunction was greater in sGC alpha 1(-/-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 sGC alpha 1 mutant (DNsGC alpha 1) upon doxycycline removal (Tet-off). After 8 weeks of doxorubicin administration, DNsGC alpha 1(tg/+), but not WT, mice displayed LV systolic dysfunction and dilatation. The difference in cardiac function and remodeling between DNsGC alpha 1(tg/+) and WT mice was even more pronounced after 12 weeks of treatment. Further impairment of cardiac function was attenuated when DNsGC alpha 1 gene expression was inhibited (beginning at 8 weeks of doxorubicin treatment) by administering doxycycline. Furthermore, doxorubicin-associated reactive oxygen species generation was higher in sGC alpha 1-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

Increased Cardiac Myocyte PDE5 Levels in Human and Murine Pressure Overload Hypertrophy Contribute to Adverse LV Remodeling

Background: The intracellular second messenger cGMP protects the heart under pathological conditions. We examined expression of phosphodiesterase 5 (PDE5), an enzyme that hydrolyzes cGMP, in human and mouse hearts subjected to sustained left ventricular (LV) pressure overload. We also determined the role of cardiac myocyte-specific PDE5 expression in adverse LV remodeling in mice after transverse aortic constriction (TAC). Methodology/Principal Findings: In patients with severe aortic stenosis (AS) undergoing valve replacement, we detected greater myocardial PDE5 expression than in control hearts. We observed robust expression in scattered cardiac myocytes of those AS patients with higher LV filling pressures and BNP serum levels. Following TAC, we detected similar, focal PDE5 expression in cardiac myocytes of C57BL/6NTac mice exhibiting the most pronounced LV remodeling. To examine the effect of cell-specific PDE5 expression, we subjected transgenic mice with cardiac myocyte-specific PDE5 overexpression (PDE5-TG) to TAC. LV hypertrophy and fibrosis were similar as in WT, but PDE5-TG had increased cardiac dimensions, and decreased dP/dtmax and dP/dtmin with prolonged tau (P<0.05 for all). Greater cardiac dysfunction in PDE5-TG was associated with reduced myocardial cGMP and SERCA2 levels, and higher passive force in cardiac myocytes in vitro. Conclusions/Significance: Myocardial PDE5 expression is increased in the hearts of humans and mice with chronic pressure overload. Increased cardiac myocyte-specific PDE5 expression is a molecular hallmark in hypertrophic hearts with contractile failure, and represents an important therapeutic target

Atrial natriuretic peptide is negatively regulated by microRNA-425

Numerous common genetic variants have been linked to blood pressure, but no underlying mechanism has been elucidated. Population studies have revealed that the variant rs5068 (A/G) in the 3′ untranslated region of NPPA, the gene encoding atrial natriuretic peptide (ANP), is associated with blood pressure. We selected individuals on the basis of rs5068 genotype (AG vs. AA) and fed them a low- or high-salt diet for 1 week, after which they were challenged with an intravenous saline infusion. On both diets, before and after saline administration, ANP levels were up to 50% higher in AG individuals than in AA individuals, a difference comparable to the changes induced by high-salt diet or saline infusion. In contrast, B-type natriuretic peptide levels did not differ by rs5068 genotype. We identified a microRNA, miR-425, that is expressed in human atria and ventricles and is predicted to bind the sequence spanning rs5068 for the A, but not the G, allele. miR-425 silenced NPPA mRNA in an allele-specific manner, with the G allele conferring resistance to miR-425. This study identifies miR-425 as a regulator of ANP production, raising the possibility that miR-425 antagonists could be used to treat disorders of salt overload, including hypertension and heart failure

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

cGMP Signal Transduction in Hypertrophic and Toxic Cardiomyopathy

Heart failure is a syndrome in which the heart can no longer provide sufficient blood flow to meet the body s demands. This condition often follows a chronic ominous course, leading to severe incapacitation and compromised survival. In the past quarter century, much progress has been made in understanding the molecular and cellular processes that contributeto heart failure, which has led to a considerable change in the scope of available therapies. However, chronic heart failure remains a major cause of morbidity and mortality, with a continuously increasing worldwideprevalence. Thus, the development of novel treatments targeting the underlying pathological mechanisms are warranted to halt and reverse the devastating consequences of this disease.Growing evidence suggestsa cardioprotective role of cyclic guanosine 3',5'-monophosphate (cGMP) in the pathophysiology of the heart. In the experimental studies presented in this thesis, we observed elevated myocardial expression of phosphodiesterase 5 (PDE5), an enzyme catalysing cGMP breakdown, both in patients with severe aortic stenosis and in mice with thoracic aortic constriction-induced chronic left ventricular (LV) pressure overload. Intriguingly, we detected a strikingly similar PDE5 expression pattern in cardiac myocytes when human and murine hearts were subjected to pronounced hypertrophic stress resulting in marked adverse LV remodelling. Aggravated LVdysfunction and dilatation after chronic pressure overload in mice witha transgenic cardiac myocyte-specific PDE5 overexpression, suggests that increased PDE5 levels in cardiac myocytes contribute to deleterious LVremodelling in pressure overloaded hearts. These transgenic mice displayed a blunted myocardial cGMP response to chronic pressure overload, lower cardiac levels of the sarcoplasmic reticulum Ca2+-ATPase SERCA2, and elevated myocardial passive stiffness.In a second transgenic mouse model with reduced cGMP-synthesising soluble guanylate cyclase(sGC) activity in cardiac myocytes, doxorubicin-induced cardiotoxicity was exacerbated, as evidenced by greater LV dysfunction and dilatation. Transgenic hearts revealed increased lipid peroxidation and activation of the extrinsic apoptotic pathway after chronic doxorubicin treatment. A hypothesised increase in doxorubicin-induced mitochondrial dysfunction in these transgenic hearts is currently under investigation.Taken together, these data suggest that reduced cGMP bioavailability in cardiac myocytes represents a molecular hallmark heralding adverse LV remodelling and dysfunction during hypertrophic and toxic stress. Therefore, pharmacological modulation of cGMP signalling, via administration of PDE5 inhibitors or sGC stimulators/activators, may represent a promising therapeutic approach for various cardiac pathologies resulting in heart failure, including chronicpressure overload-induced LV remodelling and anthracycline-induced cardiotoxicity.status: publishe

MicroRNA-425 and microRNA-155 cooperatively regulate atrial natriuretic peptide expression and cGMP production

Aims Atrial natriuretic peptide (ANP), secreted primarily by atrial cardiomyocytes, decreases blood pressure by raising cyclic 3’,5’-guanosine monophosphate (cGMP) levels and inducing vasorelaxation, natriuresis, and diuresis. Raising the level of ANP has been shown to be an effective treatment for hypertension. To advance the future development of an anti-microRNA (miR) approach to increasing expression of ANP, we investigated the regulation of NPPA expression by two miRs: miR-425 and miR-155. We examined whether miR-425 and miR-155 have an additive effect on the expression and function of ANP. Methods and results Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were transfected with miR-425, miR-155, or a combination of the two miRs. Two days later, NPPA expression was measured using real time qPCR. Each of the miRs decreased NPPA expression over a wide range of concentrations, with a significant reduction at concentrations as low as 1 nM. The combination of miR-425 and miR-155 reduced NPPA expression to a greater extent than either miR-425 or miR-155 alone. An in vitro assay was developed to study the potential biological significance of the miR-induced decrease in NPPA expression. The cooperative effect of miR-425 and miR-155 on NPPA expression was associated with a significant decrease in cGMP levels. Conclusions: These data demonstrate that miR-425 and miR-155 regulate NPPA expression in a cooperative manner. Targeting both miRNAs with anti-miRs (possibly at submaximal concentrations) might prove to be a more effective strategy to modulate ANP levels, and thus blood pressure, than targeting either miRNA alone

Blood Pressure-Associated Genetic Variants in the Natriuretic Peptide Receptor 1 Gene Modulate Guanylate Cyclase Activity

BACKGROUND: Human genetic variation in the NPR1 (natriuretic peptide receptor 1 gene, encoding NPR-A, atrial natriuretic peptide receptor 1) was recently shown to affect blood pressure (BP). NPR-A catalyzes the intracellular conversion of guanosine triphosphate to cGMP (cyclic 3',5'-guanosine monophosphate) on binding of ANP, BNP (atrial or brain natriuretic peptide). Increased levels of cGMP decrease BP by inducing natriuresis, diuresis, and vasodilation. METHODS: We performed a meta-analysis of low-frequency and rare NPR1 variants for BP association in up to 491 584 unrelated individuals. To examine whether the identified BP-associated variants affect NPR-A function, the cGMP response to ANP and BNP was measured in cells expressing wild-type NPR1 and cells expressing the NPR1 variants. RESULTS: In this study, we identified BP associations of 3 amino acid altering variants of NPR1. The minor alleles of rs35479618 (p.E967K, gnomAD non-Finnish European allele frequency 0.017) and rs116245325 (p.L1034F, allele frequency 0.0007) were associated with higher BP (P=4.0×10-25 and P=9.9×10-8, respectively), while the minor allele of rs61757359 (p.G541S, allele frequency 0.003) was associated with lower BP (P=1.8×10-9). Cells transiently expressing 967K or 1034F NPR-A displayed decreased cGMP production in response to ANP and BNP (all P<10-6), while cells expressing 541S NPR-A produced more cGMP compared with cells expressing wild-type NPR-A (P≤4.13×10-5 for ANP and P≤4.24×10-3 for BNP). CONCLUSIONS: In summary, the loss or gain of guanylate cyclase activity for these NPR1 allelic variants could explain the higher or lower BP observed for carriers in large population-based studies

Placental growth factor increases regional myocardial blood flow and function in a new porcine model of chronic myocardial ischemia

Liu X., Caluwe E., Reyns G., Verhamme P., Pokreisz P., Vandenwijngaert S., Dubois C., Zalewski J., Ghysels S., Maes F., Gillijns H., Pellens M., Vanden Driessche N., Patel A., Van de Werf F., Verbeken E., Bogaert J., Janssens S., ''Placental growth factor increases regional myocardial blood flow and function in a new porcine model of chronic myocardial ischemia'', Circulation, vol. 120, no. 18, suppl., pp. S837, November 2009 (American Heart Association scientific sessions 2009, November 14-18, 2009, Orlando, Florida, USA).status: publishe