S‐glutathiolation impairs phosphoregulation and function of cardiac myosin‐binding protein C in human heart failure

Abstract

Cardiac myosin-binding protein C (cMyBP-C) regulates actin-myosin interaction and thereby cardiac myocyte contraction and relaxation. This physiologic function is regulated by cMyBP-C phosphorylation. In our study, reduced site-specific cMyBP-C phosphorylation coincided with increased S-glutathiolation in ventricular tissue from patients with dilated or ischemic cardiomyopathy compared to nonfailing donors. We used redox proteomics, to identify constitutive and disease-specific Sglutathiolation sites in cMyBP-C in donor and patient samples, respectively. Among those, a cysteine cluster in the vicinity of the regulatory phosphorylation sites within the myosin S2 interaction domain C1-M-C2 was identified and showed enhanced S-glutathiolation in patients. In vitro S-glutathiolation of recombinant cMyBP-C C1-M-C2 occurred predominantly at Cys 249 , which attenuated phosphorylation by protein kinases. Exposure to glutathione disulfide induced cMyBP-C S-glutathiolation, which functionally decelerated the kinetics of Ca 2+ -activated force development in ventricular myocytes from wild-type, but not those from Mybpc3-targeted knockout mice. These oxidation events abrogate protein kinase-mediated phosphorylation of cMyBP-C and therefore potentially contribute to the reduction of its phosphorylation and the contractile dysfunction observed in human heart

Oxidant sensor in the cGMP-binding pocket of PKGIα regulates nitroxyl-mediated kinase activity

“…PKGIα activity was expressed as pmol phosphate incorporated into PKGIα substrate per minute. On the other hand, activity of human WT, Cys42Ser, Cys117/195Ser, or Cys42/117/195Ser PKGIα was assessed by phosphorylation of recombinantly expressed His 6 -tagged cardiac myosin-binding protein C (amino acid residues 153-450) 41 , which was previously described as a PKGIα substrate 42 , in the presence of radiolabeled γ 32 P-ATP (GE Healthcare). HEK-293 cells were transfected as described before and exposed to AS (500 µmol/L, 15 min), NCA (100 µmol/L, 30 min) or vehicle (NaOH for AS; DMSO for NCA).…”

Stress-induced protein S-glutathionylation and phosphorylation crosstalk in cardiac sarcomeric proteins - Impact on heart function

“…Secondly, S-glutathionylation itself could prevent phosphorylations on specific sites. Previous publications showed that several cysteines of cMyBP-C can be S-glutathionylated in vitro [15,16].…”

“…Stathopoulou et al [16] showed that in vitro S-glutathionylation of Cys249, localized near the phosphorylation sites, attenuated phosphorylations by protein kinases (PKA and CaMKII). This may explain why after HSE, PKA-mediated phosphorylation of TnI and cMyBP-C was similar in control and HSE hearts despite higher PKA activity ( Fig.…”

“…Later a study showed clearly in vitro that titin can be S-glutathionylated [20]. Interestingly, an interaction between phosphorylation and Sglutathionylation of cMyBP-C was explicitly demonstrated in vitro [16]. Reduced site-specific cMyBP-C phosphorylation is accompanied by increased protein S-glutathionylation in ventricular tissue from patients with heart failure.…”

“…The ROS associated with the disease induced S-glutathionylation of cMyBP-C, which slowed cross-bridge kinetics and increased Ca 2+ sensitivity. Several cysteines of cMyBP-C can be Sglutathionylated in vitro and lead to an increase in myofilament Ca 2+sensitivity [15,16]. Previous experiments have found that every cardiac myofilament protein, except TnT and MLC2 has a cysteine that can be modified by oxidants [17,18].…”