Nitrate (NO3−) and nitrite (NO2−) are known to be cardioprotective and to alter energy metabolism in vivo. NO3− action results from its conversion to NO2− by salivary bacteria, but the mechanism(s) ...by which NO2− affects metabolism remains obscure. NO2− may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2−-dependent S-nitrosation of proteins thiols in vivo. Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2− under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2− in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2− on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2−, combined with the lack of S-nitrosation during anoxia alone or by NO2− during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2− exposure.
Abstract
By the time cardiotoxicity-associated cardiac dysfunction is detectable by echocardiography it is often beyond meaningful intervention.
99m
Tc-sestamibi is used clinically to image cardiac ...perfusion by single photon emission computed tomography (SPECT) imaging, but as a lipophilic cation its distribution is also governed by mitochondrial membrane potential (ΔΨ
m
). Correcting scans for variations in perfusion (using a ΔΨ
m
-independent perfusion tracer such as (bis(N-ethoxy-N-ethyldithiocarbamato)nitrido
99m
Tc(V)) (
99m
Tc-NOET) could allow
99m
Tc-sestamibi to be repurposed to specifically report on ΔΨ
m
as a readout of evolving cardiotoxicity. Isolated rat hearts were perfused within a γ-detection apparatus to characterize the pharmacokinetics of
99m
Tc-sestamibi and
99m
Tc-NOET in response to mitochondrial perturbation by hypoxia, ionophore (CCCP) or doxorubicin. All interventions induced
99m
Tc-sestamibi washout; hypoxia from 24.9 ± 2.6% ID to 0.4 ± 6.2%, CCCP from 22.8 ± 2.5% ID to −3.5 ± 3.1%, and doxorubicin from 23.0 ± 2.2% ID to 17.8 ± 0.7, p < 0.05. Cardiac
99m
Tc-NOET retention (34.0 ± 8.0% ID) was unaffected in all cases. Translating to an i
n vivo
rat model, 2 weeks after bolus doxorubicin injection, there was a dose-dependent loss of cardiac
99m
Tc-sestamibi retention (from 2.3 ± 0.3 to 0.9 ± 0.2 ID/g with 10 mg/kg (p < 0.05)), while
99m
Tc-NOET retention (0.93 ± 0.16 ID/g) was unaffected.
99m
Tc-NOET therefore traps in myocardium independently of the mitochondrial perturbations that induce
99m
Tc-sestamibi washout, demonstrating proof-of-concept for an imaging approach to detect evolving cardiotoxicity.
Copper-64-Diacetyl-bis(N
-methylthiosemicarbazone)
CuCu(ATSM) is a hypoxia-targeting PET tracer with applications in oncology and cardiology. Upon entering a hypoxic cell,
CuCu(II)(ATSM) is reduced ...to a putative
CuCu(I)(ATSM)
species which dissociates to deposit radiocopper, thereby providing hypoxic contrast. This process may be dependent upon protonation arising from intracellular acidosis. Since acidosis is a hallmark of ischemic tissue and tumors, the hypoxia specificity of
CuCu(ATSM) may be confounded by changes in intracellular pH. We have therefore determined the influence of intracellular pH on
CuCu(ATSM) pharmacokinetics. Using isolated perfused rat hearts, acidosis was induced using an ammonium pre-pulse method, with and without hypoxic buffer perfusion. Cardiac
CuCu(ATSM) pharmacokinetics were determined using NaI detectors, with intracellular pH and cardiac energetics monitored in parallel by
P NMR. To distinguish direct acidotic effects on tracer pharmacokinetics from acidosis-induced hypocontractility, parallel studies used lidocaine perfusion to abolish cardiac contraction. Hypoxic myocardium trapped
CuCu(ATSM) despite no evidence of it being acidotic when characterised by
P NMR. Independent induction of tissue acidosis had no direct effect on
CuCu(ATSM) pharmacokinetics in either normoxic or hypoxic hearts, beyond decreasing cardiac oxygen consumption to alleviate hypoxia and decrease tracer retention, leading us to conclude that tissue acidosis does not mediate the hypoxia selectivity of
CuCu(ATSM).
Aims
Recently it has been shown that the mitochondria‐targeted S‐nitrosothiol MitoSNO protects against acute ischaemia/reperfusion (IR) injury by inhibiting the reactivation of mitochondrial complex ...I in the first minutes of reperfusion of ischaemic tissue, thereby preventing free radical formation that underlies IR injury. However, it remains unclear how this transient inhibition of mitochondrial complex I‐mediated free radicals at reperfusion affects the long‐term recovery of the heart following IR injury. Here we determined whether the acute protection by MitoSNO at reperfusion prevented the subsequent development of post‐myocardial infarction heart failure.
Methods and results
Mice were subjected to 30 min left coronary artery occlusion followed by reperfusion and recovery over 28 days. MitoSNO (100 ng/kg) was applied 5 min before the onset of reperfusion followed by 20 min infusion (1 ng/kg/min). Infarct size and cardiac function were measured by magnetic resonance imaging (MRI) 24 h after infarction. MitoSNO‐treated mice exhibited reduced infarct size and preserved function. In addition, MitoSNO at reperfusion improved outcome measures 28 days post‐IR, including preserved systolic function (63.7 ±1.8% LVEF vs. 53.7 ± 2.1% in controls, P = 0.01) and tissue fibrosis.
Conclusions
MitoSNO action acutely at reperfusion reduces infarct size and protects from post‐myocardial infarction heart failure. Therefore, targeted inhibition of mitochondrial complex I in the first minutes of reperfusion by MitoSNO is a rational therapeutic strategy for preventing subsequent heart failure in patients undergoing IR injury.
The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range ...of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging.
One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules.
Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
•Exomarkers can be used to assess reactive species in vivo.•MitoB is a mitochondria-targeted ROS probe.•Tandem mass spectrometry can be used to assess ROS in vivo.
Purpose of Review
In this review, we outline the potential for hypoxia imaging as a diagnostic and prognostic tool in cardiology. We describe the lead hypoxia PET radiotracers currently in ...development and propose a rationale for how they should most appropriately be screened and validated.
Recent Findings
While the majority of hypoxia imaging agents has been developed for oncology, the requirements for hypoxia imaging in cardiology are different. Recent work suggests that the bis(thiosemicarbazone) family of compounds may be capable of detecting the subtle degrees of hypoxia associated with cardiovascular syndromes, and that they have the potential to be “tuned” to provide different tracers for different applications.
Summary
New tracers currently in development show significant promise for imaging evolving cardiovascular disease. Fundamental to their exploitation is their careful, considered validation and characterization so that the information they provide delivers the greatest prognostic insight achievable.
To determine the pharmacology of ETA- and ETB-mediated β-arrestin recruitment and compare this to established human pharmacology of these receptors to identify evidence for endothelin receptor biased ...signalling and pathway specific blockade by antagonists.
The ability of ET-1, ET-2, ET-3, sarafotoxin 6b and sarafotoxin 6c to activate ETA and ETB-mediated β-arrestin recruitment was determined in CHO-K1 cells. Affinities were obtained for ETA selective (BQ123, sitaxentan, ambrisentan), ETB selective (BQ788) and mixed (bosentan) antagonists using ET-1 and compared to affinities obtained in competition experiments in human heart and by Schild analysis in human saphenous vein. Agonist dependence of affinities was compared for BQ123 and BQ788 in the ETA and ETB β-arrestin assays respectively.
For β-arrestin recruitment, order of potency was as expected for the ETA (ET-1≥ET-2>>ET-3) and ETB (ET-1=ET-2=ET-3) receptors. However, at the ETA receptor sarafotoxin 6b and ET-3 were partial agonists. Antagonism of ET peptides by selective and mixed antagonists appeared non-competitive. BQ123, but not BQ788, exhibited agonist-dependent affinities. Bosentan was significantly more effective an inhibitor of β-arrestin recruitment mediated by ETA compared to the ETB receptor. In the ETA vasoconstrictor assay, ET-1, ET-2 and S6b were equipotent, full agonists and antagonists tested behaved in a competitive manner, although affinities were lower than predicted from the competition binding experiments in left ventricle.
These data suggest that the pharmacology of ETA and ETB receptors linked to G-protein- and β-arrestin mediated responses was different and bosentan appeared to show bias, preferentially blocking ETA mediated β-arrestin recruitment.
By the time cardiotoxicity-associated cardiac dysfunction is detectable by echocardiography it is often beyond meaningful intervention.
Tc-sestamibi is used clinically to image cardiac perfusion by ...single photon emission computed tomography (SPECT) imaging, but as a lipophilic cation its distribution is also governed by mitochondrial membrane potential (ΔΨ
). Correcting scans for variations in perfusion (using a ΔΨ
-independent perfusion tracer such as (bis(N-ethoxy-N-ethyldithiocarbamato)nitrido
Tc(V)) (
Tc-NOET) could allow
Tc-sestamibi to be repurposed to specifically report on ΔΨ
as a readout of evolving cardiotoxicity. Isolated rat hearts were perfused within a γ-detection apparatus to characterize the pharmacokinetics of
Tc-sestamibi and
Tc-NOET in response to mitochondrial perturbation by hypoxia, ionophore (CCCP) or doxorubicin. All interventions induced
Tc-sestamibi washout; hypoxia from 24.9 ± 2.6% ID to 0.4 ± 6.2%, CCCP from 22.8 ± 2.5% ID to -3.5 ± 3.1%, and doxorubicin from 23.0 ± 2.2% ID to 17.8 ± 0.7, p < 0.05. Cardiac
Tc-NOET retention (34.0 ± 8.0% ID) was unaffected in all cases. Translating to an in vivo rat model, 2 weeks after bolus doxorubicin injection, there was a dose-dependent loss of cardiac
Tc-sestamibi retention (from 2.3 ± 0.3 to 0.9 ± 0.2 ID/g with 10 mg/kg (p < 0.05)), while
Tc-NOET retention (0.93 ± 0.16 ID/g) was unaffected.
Tc-NOET therefore traps in myocardium independently of the mitochondrial perturbations that induce
Tc-sestamibi washout, demonstrating proof-of-concept for an imaging approach to detect evolving cardiotoxicity.
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