Thursday, December 26, 2024
12/26/2024
Project Summary Increased oxidative stress is associated with cardiac cell dysfunction in heart disease. An unbalanced redox state leads to an increase in the post-translational modification of S-Glutathione, which modifies cysteine residues on key myofilament proteins, such as cardiac myosin binding protein-C (cMyBP-C). cMyBP-C regulates contraction and relaxation of the sarcomere. The phosphorylation of cMyBP-C by Protein Kinase A (PKA) is cardioprotective. Yet, in the failing heart, phosphorylation levels of cMyBP-C are reduced, contrary to an increase in S-glutathionylated cMyBP-C. When cardiomyocytes were incubated with oxidized glutathione (GSSG), myofilament calcium sensitivity increased and cross-bridge kinetics slowed. Prior experiments were unable to isolate the specific effects of S-glutathionylated cMyBP-C (i.e., without the effects of other S- glutathionylated proteins) nor the specific sites responsible for the functional change. Phosphorylation and S- glutathionylation of cMyBP-C may have antagonistic effects. Incubating three N’-terminal domains of cMyBP-C with GSSG led to a significant increase in S-glutathionylated cMyBP-C and a downregulation in cMyBP-C phosphorylation. These results indicate that the decrease in cMyBP-C phosphorylation and consequent loss of the cardioprotective effect of phosphorylated cMyBP-C seen in the failing heart could be due to an increase in S-glutathionylated cMyBP-C. In addition, the anti-ischemic drug, ranolazine, has been shown to improve diastolic function to sham levels, which correlated with S-glutathionylated cMyBP-C. These data indicate that a currently available cardiac therapy might be useful in moderating the levels of cMyBP-C S-glutathionylation. Thus, this proposal will identify how the interaction between phosphorylation and S-glutathionylation affects cardiomyocyte function under normal, elevated, and therapeutically treated conditions using our novel “cut and paste” SpyC3 mouse model. Protein domains C0C7 of cMyBP-C will be “cut” from the sarcomere using the tobacco etch viral protease and, after washing steps, recombinant C0C7sc protein with and without modified cysteine residues will replace its location within its endogenous location in the sarcomere. Site-directed mutagenesis will be used to generate cysteine substitution constructs preventing S-glutathionylation at specific residues and the functional effects of each construct will be measured using the “cut and paste” model. Dual incubation of PKA and GSSG with and without ranolazine treatment will determine the functional effects of this interaction. A combination of ProQ Diamond staining, immunoblotting, Phos-tag gels, and mass spectrometry will be used to measure total modification levels and identify the site-specific modifications. Results from this proposal will be the first to identify the functional effects of individual cMyBP-C S-glutathionylated residues and of phosphorylation and S-glutathionylation cMyBP-C crosstalk. This research proposal will lead to a better understanding of the effects of oxidative stress on cMyBP-C function, its potential to affect phosphorylation in the heart, and if a currently available therapeutic might benefit hearts affected by oxidative stress.
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