Examining a role for PKG Iα oxidation in the pathogenesis of cardiovascular dysfunction during diet-induced obesity

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Protein kinase G (PKG) Iα is the end-effector kinase that mediates nitric oxide (NO)-dependent and oxidant-dependent vasorelaxation to maintain blood pressure during health. A hallmark of cardiovascular disease is attenuated NO production, which in part is caused by NO Synthase (NOS) uncoupling, which in turn increases oxidative stress because of superoxide generation. NOS uncoupling promotes PKG Iα oxidation to the interprotein disulfide state, likely mediated by superoxide-derived hydrogen peroxide, and because the NO-cyclic guanosine monophosphate (cGMP) pathway otherwise negatively regulates oxidation of the kinase to its active disulfide dimeric state. Diet-induced obesity is associated with NOS uncoupling, which may in part contribute to the associated cardiovascular dysfunction due to exacerbated PKG Iα disulfide oxidation to the disulfide state. This is a rational hypothesis because PKG Iα oxidation is known to significantly contribute to heart failure that arises from chronic myocardial oxidative stress.

Methods and results

Bovine arterial endothelial cells (BAECs) or smooth muscle cells (SMCs) were exposed to drugs that uncouple NOS. These included 1,3-bis(2-chloroethyl)−1-nitrosourea (BCNU) which promotes its S-glutathiolation, 4-diamino-6-hydroxy-pyrimidine (DAHP) which inhibits guanosine-5′-triphosphate-cyclohydrolase 2 to prevent BH4 synthesis or methotrexate (MTX) which inhibits the regeneration of BH4 from BH2 by dihydrofolate reductase. While all the drugs mentioned above induced robust PKG Iα disulfide dimerization in cells, exposure of BAECs to NOS inhibitor L-NMMA did not. Increased PKG Iα disulfide formation occurred in hearts and aortae from mice treated in vivo with DAHP (10 mM in a drinking water for 3 weeks). Redox-dead C42S PKG Iα knock-in (KI) mice developed less pronounced cardiac posterior wall hypertrophy and did not develop cardiac dysfunction, assessed by echocardiography, compared to the wild-type (WT) mice after chronic DAHP treatment. WT or KI mice were then subjected to a diet-induced obesity protocol by feeding them with a high fat Western-type diet (RM 60% AFE) for 27 weeks, which increased body mass, adiposity, plasma leptin, resistin and glucagon levels comparably in each genotype. Obesity-induced hypertension, assessed by radiotelemetry, was mild and transient in the WT, while the basally hypertensive KI mice were resistant to further increases in blood pressure following high fat feeding. Although the obesogenic diet caused mild cardiac dysfunction in the WT but not the KI mice, gross changes in myocardial structure monitored by echocardiography were not apparent in either genotype. The level of cyclic guanosine monophosphate (cGMP) was decreased in the aortae of WT and KI mice following high fat feeding. PKG Iα oxidation was not evident in the hearts of WT mice fed a high fat diet.


Despite robust evidence for PKG Iα oxidation during NOS uncoupling in cell models, it is unlikely that PKG Iα oxidation occurs to a significant extent in vivo during diet-induced obesity and so is unlikely to mediate the associated cardiovascular dysfunction.

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