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

Abstract

Phosphodiesterase 5 (PDE5) inhibitors limit myocardial injury caused by stresses, including doxorubicin chemotherapy. cGMP binding to PKG I␣ 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 I␣ disulfide formation. To investigate the role of PKG I␣ disulfide dimerization in the development of apoptosis, doxorubicin-induced cardiomyopathy was compared in male wild type (WT) or disulfide-resistant C42S PKG I␣ 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 I␣ 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 coadministered tadalafil. PKG I␣ 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.

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

“…S1). It was recently reported that PKGI␣ Cys 43 disulfide bond formation increased in vitro PKGI␣ activity toward histone H1 but not RhoA (16). However, in contrast to those results, we found that Cys 43 oxidation did not increase PKGI␣ activity toward histone H1 (Fig.…”

“…6A in Ref. 16). In our experience, it is very difficult to fully remove cAMP during PKG purification, especially from PKGI␣ (10,20,22), and in general, Western blotting is semi-quantitative and is not an accurate method to measure kinase activity.…”

“…This suggests that the kinase was proteolytically degraded, leading to a largely cGMP-independent kinase activity, and the poor quality of the enzyme may have affected the biochemical assays. In a subsequent paper by Prysyazhna et al (16), PKGI␣ was purified using a cAMP-agarose affinity column, followed by cAMP elution and dialysis to remove cAMP. This kinase could be stimulated ϳ8-fold by cGMP as determined by Western blotting performed with phospho-specific antibodies to detect RhoA and histone H1 phosphorylation (see Fig.…”

Sevoflurane ameliorates doxorubicin-induced myocardial injury by affecting the phosphorylation states of proteins in PI3K/Akt/mTOR signaling pathway

“…Two rats which died were from LYG, and thus DOX and LY294002 may have a synergistic function for causing myocardial injury. Cell death was reported when PI3K/Akt pathway was blocked by the PI3K inhibitor LY294002 [1]. The optimization is going to be performed in future, including one-time dosage, interval time and accumulated dosages.…”

“…The anthracycline doxorubicin (DOX) is widely used for tumor therapy, but its wide use often leads to heart failure [1].…”