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

Doxorubicin-induced chronic dilated cardiomyopathy—the apoptosis hypothesis revisited

The chemotherapeutic agent doxorubicin (DOX) has significantly increased survival rates of pediatric and adult cancer patients. However, 10% of pediatric cancer survivors will 10–20 years later develop severe dilated cardiomyopathy (DCM), whereby the exact molecular mechanisms of disease progression after this long latency time remain puzzling. We here revisit the hypothesis that elevated apoptosis signaling or its increased likelihood after DOX exposure can lead to an impairment of cardiac function and cause a cardiac dilation. Based on recent literature evidence, we first argue why a dilated phenotype can occur when little apoptosis is detected. We then review findings suggesting that mature cardiomyocytes are protected against DOX-induced apoptosis downstream, but not upstream of mitochondrial outer membrane permeabilisation (MOMP). This lack of MOMP induction is proposed to alter the metabolic phenotype, induce hypertrophic remodeling, and lead to functional cardiac impairment even in the absence of cardiomyocyte apoptosis. We discuss findings that DOX exposure can lead to increased sensitivity to further cardiomyocyte apoptosis, which may cause a gradual loss in cardiomyocytes over time and a compensatory hypertrophic remodeling after treatment, potentially explaining the long lag time in disease onset. We finally note similarities between DOX-exposed cardiomyocytes and apoptosis-primed cancer cells and propose computational system biology as a tool to predict patient individual DOX doses. In conclusion, combining recent findings in rodent hearts and cardiomyocytes exposed to DOX with insights from apoptosis signal transduction allowed us to obtain a molecularly deeper insight in this delayed and still enigmatic pathology of DC

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

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

Increased cardiac myocyte PDE5 levels in human and murine pressure overload hypertrophy cntribute 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 (PDE5TG) to TAC. LV hypertrophy and fibrosis were similar as in WT, but PDE5-TG had increased cardiac dimensions, and decreased dP/dt(max) and dP/dt(min) 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

MiR-425 and miR-155 produced an additive decrease in cGMP levels relative to either miRNA alone.

<p>Cardiomyocytes were transfected with 1 nM (A) or 5 nM (B) of negative control miRNA (NC), miR-425, miR-155, or the combination of miR-425 and miR-155, each at half the concentration of the other miRs. Cyclic GMP levels were measured in NPR1-expressing cells that were incubated for 2 hours with media collected from the cardiomyocytes. Cyclic GMP levels were expressed as picomoles of cGMP per mg of protein and were shown relative to levels in cells exposed to media from cardiomyocytes transfected with negative control miRNA. *<i>P</i><0.01 and **<i>P</i><1x10^-6 versus cells transfected with negative control miRNA. N = 3 experiments (4–12 replicate wells for each condition) for (A) and n = 8 experiments (4 replicate wells per condition) for (B).</p

MiR-425 and miR-155 had an additive repressive effect on cardiomyocyte <i>NPPA</i> expression.

<p><i>NPPA</i> mRNA levels in hESC-CMs that were transfected with either miR-425 or miR-155 alone or with a combination of miR-425 and miR-155. Negative control miRNA (NC) was used as needed to make the total concentration of miRNA constant in each condition. <i>NPPA</i> expression is shown relative to that in cells transfected with negative control miRNA. *<i>P</i><0.01 and **<i>P</i><1x10^-6 versus cells transfected with negative control miRNA. N = 3 experiments (6–12 replicate wells per condition).</p