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Nitroxyl-mediated disulfide bond formation between cardiac myofilament cysteines enhances contractile function.

ABSTRACT: In the myocardium, redox/cysteine modification of proteins regulating Ca(2+) cycling can affect contraction and may have therapeutic value. Nitroxyl (HNO), the one-electron-reduced form of nitric oxide, enhances cardiac function in a manner that suggests reversible cysteine modifications of the contractile machinery.To determine the effects of HNO modification in cardiac myofilament proteins.The HNO-donor, 1-nitrosocyclohexyl acetate, was found to act directly on the myofilament proteins, increasing maximum force (F(max)) and reducing the concentration of Ca(2+) for 50% activation (Ca(50)) in intact and skinned cardiac muscles. The effects of 1-nitrosocyclohexyl acetate are reversible by reducing agents and distinct from those of another HNO donor, Angeli salt, which was previously reported to increase F(max) without affecting Ca50. Using a new mass spectrometry capture technique based on the biotin switch assay, we identified and characterized the formation by HNO of a disulfide-linked actin-tropomyosin and myosin heavy chain-myosin light chain 1. Comparison of the 1-nitrosocyclohexyl acetate and Angeli salt effects with the modifications induced by each donor indicated the actin-tropomyosin and myosin heavy chain-myosin light chain 1 interactions independently correlated with increased Ca(2+) sensitivity and force generation, respectively.HNO exerts a direct effect on cardiac myofilament proteins increasing myofilament Ca(2+) responsiveness by promoting disulfide bond formation between critical cysteine residues. These findings indicate a novel, redox-based modulation of the contractile apparatus, which positively impacts myocardial function, providing further mechanistic insight for HNO as a therapeutic agent.

SUBMITTER: Gao WD

PROVIDER: S-EPMC3470471 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Nitroxyl-mediated disulfide bond formation between cardiac myofilament cysteines enhances contractile function.

Gao Wei Dong WD Murray Christopher I CI Tian Ye Y Zhong Xin X DuMond Jenna F JF Shen Xiaoxu X Stanley Brian A BA Foster D Brian DB Wink David A DA King S Bruce SB Van Eyk Jennifer E JE Paolocci Nazareno N

Circulation research 20120731 8

<h4>Rationale</h4>In the myocardium, redox/cysteine modification of proteins regulating Ca(2+) cycling can affect contraction and may have therapeutic value. Nitroxyl (HNO), the one-electron-reduced form of nitric oxide, enhances cardiac function in a manner that suggests reversible cysteine modifications of the contractile machinery.<h4>Objective</h4>To determine the effects of HNO modification in cardiac myofilament proteins.<h4>Methods and results</h4>The HNO-donor, 1-nitrosocyclohexyl acetat ...[more]

PMID: 22851540

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Project description:The protein secretory pathway must maintain homoeostasis while producing a wide assortment of proteins in different conditions. It is also used extensively to produce many useful proteins in biotechnology. As such, secretory pathway dysfunction can be highly detrimental to the cell, resulting in the molecular basis for many human diseases, and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. To better understand some of these dysfunctions, we measured multiple systems-level states of the cell (physiology, transcriptome, metabolism) while secreting a small protein (insulin precursor) or a large protein (?-amylase). This was carried out in the presence and absence of HAC1, a key transcription factor in maintaining secretory homeostasis. Clear trends in cellular stress were apparent across multiple data resulting from our perturbations. In particular, processes involving (1) degradation of protein / recycling amino acids, (2) overall transcription/translation repression, and (3) oxidative stress. Apparent runaway oxidative radical production was explained by a thermodynamic model that we put forward for disulfide formation in the endoplasmic reticulum. This model predicts that balancing the relative rates of protein folding and disulfide bond formation are key to easing oxidative stress. These predictions have direct implications in how to engineer a broad range of recombinant proteins for secretion and provide potential hypotheses for the root causes of several secretory-associated diseases. Yeast strains were constructed that produce and secrete (a) IP or (b) ?-amylase and were compared to yeast strains containing (c) an empty vector in both wild-type and HAC1 deletion backgrounds. These strains are named WN (WT with empty vector), WI (WT secreting IP), WA (WT secreting ?-amylase), dN (?hac1 with empty vector), dI (?hac1 secreting IP), and dA (?hac1 secreting ?-amylase). Strains were characterized in batch fermentation and samples were taken in mid-exponential phase. Triplicate fermentations were carried out for each strain.

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Nitroxyl-mediated disulfide bond formation between cardiac myofilament cysteines enhances contractile function.

Publications

Nitroxyl-mediated disulfide bond formation between cardiac myofilament cysteines enhances contractile function.

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