Effects of Nitrosylation on Cardiac Myofilament Proteins

Abstract

In the heart, protein nitrosylation has been described as cardio-protective against oxidative stress, yet the mechanisms are largely unknown. We hypothesize that direct nitrosylation of myofilament proteins at specific cysteine residues could contribute to this cardio-protective effect. We previously described how the nitrosonium donor, CysNO, could affect adult cardiac myocyte contractility by direct nitrosylation of the myofilament regulatory proteins cardiac troponin I (cTnI) and troponin C (cTnC). In addition, we found that nitrosylation of cTnI could affect its phosphorylation at serines 23 and 24. In the current study, we further examined how nitrosylation could affect myofilament proteins, focusing on cTnI and cTnC. First, we found endogenous nitrosylated myofilament proteins, including troponin I and C, that could be de-nitrosylated by the addition of ascorbate as a reductant. Using cysteine knockout mutants, we determined the rate and efficiency of individual cysteine residues to trans-nitrosation by CysNO. Next, we found that direct nitrosylation of cTnI and cTnC affected its subsequent protein phosphorylation. Conversely, we found that a pseudo-phosphorylated cTnI at serines 23 and 24 also affected its nitrosylation. Finally, direct nitrosylation of myofilament proteins with CysNO reduced its degradation by H2O2. Together, our results provide evidence that direct nitrosylation of myofilament proteins is site-specific and could be protective against oxidative damage.