Mechanism and potential treatment of the "no reflow" phenomenon after acute myocardial infarction: role of pericytes and GPR39.

The "no reflow" phenomenon, where the coronary artery is patent after treatment of acute myocardial infarction (AMI) but tissue perfusion is not restored, is associated with worse outcome. The mechanism of no reflow is unknown. We hypothesized that pericytes contraction, in an attempt to maintain a constant capillary hydrostatic pressure during reduced coronary perfusion pressure, causes capillary constriction leading to no reflow and that this effect is mediated through the orphan receptor, GPR39, present in pericytes. We created AMI (coronary occlusion followed by reperfusion) in GPR39 knock out mice and littermate controls. In a separate set of experiments, we treated wild-type mice undergoing coronary occlusion with vehicle or VC43, a specific inhibitor of GPR39, before reperfusion. We found that no reflow zones were significantly smaller in the GPR39 knockouts compared with controls. Both no reflow and infarct size were also markedly smaller in animals treated with VC43 compared with vehicle. Immunohistochemistry revealed greater capillary density and larger capillary diameter at pericyte locations in the GPR39-knockout and VC43-treated mice compared with controls. We conclude that GPR39-mediated pericyte contraction during reduced coronary perfusion pressure causes capillary constriction resulting in no reflow during AMI and that smaller no reflow zones in GPR39-knockout and VC43-treated animals are associated with smaller infarct sizes. These results elucidate the mechanism of no reflow in AMI, as well as providing a therapeutic pathway for the condition.NEW & NOTEWORTHY The mechanism of "no reflow" phenomenon, where the coronary artery is patent after treatment of acute myocardial infarction but tissue perfusion is not restored, is unknown. This condition is associated with worse outcome. Here, we show that GPR39-mediated pericyte contraction during reduced coronary perfusion pressure causes capillary constriction resulting in no reflow. Smaller no-reflow zones in GPR39-knockout animals and those treated with a GPR39 inhibitor are associated with smaller infarct size. These results could have important therapeutic implications.

Pericyte constriction underlies capillary derecruitment during hyperemia in the setting of arterial stenosis.

Capillary derecruitment distal to a coronary stenosis is implicated as the mechanism of reversible perfusion defect and potential myocardial ischemia during coronary hyperemia; however, the underlying mechanisms are not defined. We tested whether pericyte constriction underlies capillary derecruitment during hyperemia under conditions of stenosis. In vivo two-photon microscopy (2PM) and optical microangiography (OMAG) were used to measure hyperemia-induced changes in capillary diameter and perfusion in wild-type and pericyte-depleted mice with femoral artery stenosis. OMAG demonstrated that hyperemic challenge under stenosis produced capillary derecruitment associated with decreased RBC flux. 2PM demonstrated that hyperemia under control conditions induces 26 ± 5% of capillaries to dilate and 19 ± 3% to constrict. After stenosis, the proportion of capillaries dilating to hyperemia decreased to 14 ± 4% (P = 0.05), whereas proportion of constricting capillaries increased to 32 ± 4% (P = 0.05). Hyperemia-induced changes in capillary diameter occurred preferentially in capillary segments invested with pericytes. In a transgenic mouse model featuring partial pericyte depletion, only 14 ± 3% of capillaries constricted to hyperemic challenge after stenosis, a significant reduction from 33 ± 4% in wild-type littermate controls (P = 0.04). These results provide for the first time direct visualization of hyperemia-induced capillary derecruitment distal to arterial stenosis and demonstrate that pericyte constriction underlies this phenomenon in vivo. These results could have important therapeutic implications in the treatment of exercise-induced ischemia. NEW & NOTEWORTHY In the setting of coronary arterial stenosis, hyperemia produces a reversible perfusion defect resulting from capillary derecruitment that is believed to underlie cardiac ischemia under hyperemic conditions. We use optical microangiography and in vivo two-photon microscopy to visualize capillary derecruitment distal to a femoral arterial stenosis with cellular resolution. We demonstrate that capillary constriction in response to hyperemia in the setting of stenosis is dependent on pericytes, contractile mural cells investing the microcirculation.

Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.

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.

Using exomarkers to assess mitochondrial reactive species in vivo.

BACKGROUND: 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. SCOPE OF REVIEW: 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. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: 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.

Combining MRI with PET for partial volume correction improves image-derived input functions in mice.

Accurate kinetic modelling using dynamic PET requires knowledge of the tracer concentration in plasma, known as the arterial input function (AIF). AIFs are usually determined by invasive blood sampling, but this is prohibitive in murine studies due to low total blood volumes. As a result of the low spatial resolution of PET, image-derived input functions (IDIFs) must be extracted from left ventricular blood pool (LVBP) ROIs of the mouse heart. This is challenging because of partial volume and spillover effects between the LVBP and myocardium, contaminating IDIFs with tissue signal. We have applied the geometric transfer matrix (GTM) method of partial volume correction (PVC) to 12 mice injected with 18F-FDG affected by a Myocardial Infarction (MI), of which 6 were treated with a drug which reduced infarction size [1]. We utilised high resolution MRI to assist in segmenting mouse hearts into 5 classes: LVBP, infarcted myocardium, healthy myocardium, lungs/body and background. The signal contribution from these 5 classes was convolved with the point spread function (PSF) of the Cambridge split magnet PET scanner and a non-linear fit was performed on the 5 measured signal components. The corrected IDIF was taken as the fitted LVBP component. It was found that the GTM PVC method could recover an IDIF with less contamination from spillover than an IDIF extracted from PET data alone. More realistic values of Ki were achieved using GTM IDIFs, which were shown to be significantly different (p<0.05) between the treated and untreated groups.

Functional assessment of the mouse heart by MRI with a 1-min acquisition.

In vivo assessment of heart function in mice is important for basic and translational research in cardiology. MRI is an accurate tool for the investigation of the anatomy and function in the preclinical setting; however, the long scan duration limits its usage. We aimed to reduce the acquisition time of cine MRI to 1 min. We employed spatiotemporal compressed sensing and parallel imaging to accelerate retrospectively gated cine MRI. We compared the functional parameters derived from full and undersampled data in Cartesian and radial MRI by means of Bland-Altman plots. We found that the scan time for the whole heart could be reduced to 2 min with Cartesian sampling and to 1 min with radial sampling. Despite a reduction in the signal-to-noise ratio, the accuracy in the estimation of left and right ventricular volumes was preserved for all tested subjects. This method can be used to perform accurate functional MRI examinations in mice for high-throughput phenotyping or translational studies.

Protection through postconditioning or a mitochondria-targeted S-nitrosothiol is unaffected by cardiomyocyte-selective ablation of protein kinase G.

Protein kinase G type I (PKGI) plays a critical role in survival signaling of pre- and postconditioning downstream of cardiac cGMP. However, it is unclear whether PKGI exerts its protective effects in the cardiomyocyte or if other cardiac cell types are involved, and whether nitric oxide (NO) metabolism can target cardiomyocyte mitochondria independently of cGMP/PKGI. We tested whether protection against reperfusion injury by ischemic postconditioning (IPost), soluble guanylyl cyclase (sGC) activation and inhibition, adenosine A(2B) receptor (A(2B)AR) agonist, phosphodiesterase type-5 (PDE-5) inhibitor, or mitochondria-targeted S-nitrosothiol (MitoSNO) was affected by a cardiomyocyte-specific ablation of the PKGI gene in the mouse (CMG-KO). In situ hearts underwent 30 min of regional ischemia followed by 2 h of reperfusion. As expected, in CMG-CTRs all interventions at early reperfusion lead to profound infarct size reduction: IPost (six cycles of 10-s reperfusion and 10-s coronary occlusion) with or without treatment with the sGC inhibitor ODQ, treatment with the specific sGC activator BAY58-2667 (BAY58), the selective A(2B)AR agonist BAY60-6583 (BAY60), PDE-5 inhibitor sildenafil, and MitoSNO. MitoSNO accumulates within mitochondria, driven by the membrane potential, where it generates NO· and S-nitrosates thiol proteins. In contrast, the hearts of CMG-KO animals were not protected by BAY58 and sildenafil, whereas the protective effects of IPost, IPost with ODQ, BAY60, and MitoSNO were unaffected by the lack of PKGI. Taken together, PKGI is important for the protection against ischemia reperfusion injury afforded by sGC activation or PDE-5 inhibition. However, the beneficial effects of IPost, activation of the A(2B)AR, as well as the direct effects via mitochondrial S-nitrosation do not depend on PKGI in cardiomyocytes.

A fast protocol for infarct quantification in mice.

PURPOSE: To demonstrate and validate a late gadolinium enhancement (LGE) imaging protocol, optimized for tissue viability assessment in interventional mouse models of myocardial infarction. MATERIALS AND METHODS: The method uses an efficient sampling scheme using multiple slices in a single heartbeat interleaving slice packages between alternate TRs. Sampling multiple slices instead of multiple echoes or multiple k-lines achieves higher SNR efficiency, and images covering the whole heart can be obtained in 3 min. Saturation effects are exploited for an optimum compromise between contrast and speed. RESULTS: The images obtained show high hyperenhancement with good contrast-to-noise. It is shown that inversion time optimization can be reliably omitted with this scheme, and that measured infarct sizes correlate well with histological measures. CONCLUSION: Our protocol offers a new efficient tool for the measurement of infarct size in mouse models of heart disease.

Mechanisms of the "No-Reflow" Phenomenon After Acute Myocardial Infarction: Potential Role of Pericytes.

Pericytes contract during myocardial ischemia resulting in capillary constriction and no reflow. Reversing pericyte contraction pharmacologically reduces no reflow and infarct size. These findings open up an entire new venue of research aimed at altering pericyte function in myocardial ischemia and infarction.

Low-Intensity Ultrasound Reduces Brain Infarct Size by Upregulating Phosphorylated Endothelial Nitric Oxide in Mouse Model of Middle Cerebral Artery Occlusion.

OBJECTIVE: There have been attempts to use therapeutic ultrasound (US) for the treatment of both experimental and clinical stroke. We hypothesized that low-intensity US has direct beneficial effects on the brain independent of cerebral blood flow (CBF) during middle cerebral artery occlusion (MCAO). METHODS: Three groups of mice were studied. Group I included 84 mice with MCAO undergoing US treatment/no treatment at two US frequencies (0.25 and 1.05 MHz) with three different acoustic pressures at each frequency in which infarct size (IS) was measured 24 h later. Group II included 11 mice undergoing treatment based on best US results from group I animals in which the IS/risk area (RA) ratio was measured 24 h later. Group III included 38 normal mice undergoing US treatment/no treatment for assessment of CBF, tissue metabolite and protein expression and histopathology. DISCUSSION: Ultrasound at both frequencies and most acoustic pressures resulted in reduction in IS in group I animals, with the best results obtained with 0.25 MHz at 2.0 MPa: IS was reduced 4-fold in the cerebral cortex, 1.5-fold in the caudate putamen and 3.5-fold in the cerebral hemisphere compared with control. US application in group III animals elicited only a marginal increase in CBF despite a 2.6-fold increase in phosphorylated endothelial nitric oxide synthase (p-eNOS)-S1177 and a corresponding decrease in p-eNOS-T494. Histopathology revealed no evidence of hemorrhage, inflammation or necrosis. CONCLUSION: Low-intensity US at specific frequencies and acoustic pressures results in marked neuroprotection in a mouse model of stroke by modulation of p-eNOS independent of its effect on CBF.

Evidence for an intracellular localization of the adenosine A2B receptor in rat cardiomyocytes.

Protection achieved by ischemic preconditioning is dependent on A(2B) adenosine receptors (A(2B)AR) in rabbit and mouse hearts and, predictably, an A(2B)AR agonist protects them. But it is controversial whether cardiomyocytes themselves actually express A(2B)AR. The present study tested whether A(2B)AR could be demonstrated on rat cardiomyocytes. Isolated rat hearts experienced 30 min of ischemia and 120 min of reperfusion. The highly selective, cell-permeant A(2B)AR agonist BAY60-6583 (500 nM) infused at reperfusion reduced infarct size from 40.4 ± 2.0% of the risk zone in control hearts to 19.9 ± 2.8% indicating that A(2B)AR are protective in rat heart as well. Furthermore, BAY60-6583 reduced calcium-induced mitochondrial permeability transition in isolated rat cardiomyocytes. A(2B)AR protein could be demonstrated in isolated cardiomyocytes by western blotting. In addition, message for A(2B)AR was found in individual cardiomyocytes using quantitative RT-PCR. Surprisingly, immunofluorescence microscopy did not show A(2B)AR on the cardiomyocyte's sarcolemma but rather at intracellular sites. Co-staining with MitoTracker Red in isolated cardiomyocytes revealed A(2B)AR are localized to mitochondria. Western blot analysis of a mitochondrial fraction from either rat heart biopsies or isolated cardiomyocytes revealed a strong A(2B)AR band. Thus, the present study demonstrates that activation of A(2B)AR is strongly cardioprotective in rat heart and suppresses transition pores in isolated cardiomyocytes, and A(2B)AR are expressed in individual cardiomyocytes. However, surprisingly, A(2B)AR are present in or near mitochondria rather than on the sarcolemma as are other adenosine receptors. Because A(2B)AR signaling is thought to result in inhibition of mitochondrial transition pores, this convenient location may be important.

Both A2a and A2b adenosine receptors at reperfusion are necessary to reduce infarct size in mouse hearts.

Pre- and postconditioning depend on the activation of adenosine receptors (ARs) at the end of the index ischemia. The aim of this study was to determine which receptor subtypes must be activated. In situ mouse hearts underwent 30 min of regional ischemia, followed by 2 h of reperfusion. As expected, either ischemic postconditioning (6 cycles of 10 s of reperfusion and 10 s of coronary occlusion) or infusion of the selective A(2b) adenosine receptor (A(2b)AR) agonist BAY60-6583 (BAY60) for 60 min, starting 5 min before reperfusion reduced infarct size in wild-type C57Bl/6N mice. Protection from either was abolished by the selective A(2b)AR antagonist MRS-1754, confirming a role for A(2b)AR. Additionally, the coadministration of ischemic postconditioning and a selective A(2a)AR antagonist led to the loss of protection as well. 5'-Ectonucleotidase (CD73) is thought to be necessary for the production of adenosine during ischemia. As predicted, ischemic postconditioning did not protect CD73 knockout mice. Selective agonists of either A(2b)AR (BAY60) or A(2a)AR (CGS-21680), as well as the coadministration of ischemic postconditioning and BAY60, also failed to protect hearts of the CD73 knockout mice. But the nonselective A(1)/A(2)AR agonist 5'-(N-ethylcarboxamido)adenosine (NECA) was protective, suggesting that the activation of multiple AR subtypes might be required. The coadministration of CGS-21680 and BAY60 also elicited profound protection, indicating that two AR subtypes, A(2a) and A(2b), must be simultaneously activated for protection to occur.

BAY 58-2667, a nitric oxide-independent guanylyl cyclase activator, pharmacologically post-conditions rabbit and rat hearts.

AIMS: BAY 58-2667 (BAY-58) directly activates soluble guanylyl cyclase without tolerance in a nitric oxide (NO)-independent manner, and its haemodynamic effect is similar to that of nitroglycerin. We tested whether BAY-58 could make both rabbit and rat hearts resistant to infarction when given at the end of an ischaemic insult. METHODS AND RESULTS: All hearts were exposed to 30 min regional ischaemia followed by 120-(isolated hearts) or 180-(in situ hearts) min reperfusion. BAY-58 (1-50 nM) infused for 60 min starting 5 min before reperfusion significantly reduced infarction from 33.0 +/- 3.2% in control isolated rabbit hearts to 9.5-12.7% (P < 0.05). In a more clinically relevant in situ rabbit model, infarct size was similarly reduced with a loading dose of 53.6 microg/kg followed by a 60 min infusion of 1.25 microg/kg/min (41.1 +/- 3.1% infarction in control hearts to 16.0 +/- 4.4% in treated hearts, P < 0.05). BAY-58 similarly decreased infarction in the isolated rat heart, and protection was abolished by co-treatment with a protein kinase G (PKG) antagonist, or a mitochondrial K(ATP) channel antagonist. Conversely, N(omega)-nitro-L-arginine-methyl-ester-hydrochloride, a NO-synthase inhibitor, failed to block BAY-58's ability to decrease infarction, consistent with the latter's putative NO-independent activation of PKG. Finally, BAY-58 increased myocardial cGMP content in reperfused hearts while cAMP was unchanged. CONCLUSION: When applied at reperfusion, BAY-58 is an effective cardioprotective agent with a mechanism similar to that of ischaemic pre-conditioning and, hence, should be a candidate for treatment of acute myocardial infarction in man.

The role of pericytes in hyperemia-induced capillary de-recruitment following stenosis.

PURPOSE: The microvascular capillary network is ensheathed by cells called pericytes - a heterogeneous population of mural cells derived from multiple lineages. Pericytes play a multifaceted role in the body, including in vascular structure and permeability, regulation of local blood flow, immune and wound healing functions, induction of angiogenesis, and generation of various progenitor cells. Here, we consider the role of pericytes in capillary de-recruitment, a pathophysiologic phenomenon that is observed following hyperemic stimuli in the presence of a stenosis and attenuates the hyperemic response. RECENT FINDINGS: We discuss recent observations that conclusively demonstrate pericytes to be the cellular structures that contract in response to hyperemic stimuli when an upstream arterial stenosis is present. This response constricts capillaries, which is likely aimed at maintaining capillary hydrostatic pressure, an important factor in tissue homeostasis. Nonetheless, the ensuing attenuation of the hyperemic response can lead to a decrease in energy supply and negatively impact tissue health. SUMMARY: Therapeutics aimed at preventing pericyte-mediated capillary de-recruitment may prove beneficial in conditions such as coronary stenosis and peripheral arterial disease by reducing restriction in hyperemic flow. Identification of the pericyte subtypes involved in this de-recruitment and the underlying molecular mechanisms regulating this process will greatly assist this purpose.

Trajectory correction for free-breathing radial cine MRI.

Radial acquisitions can suffer from trajectory errors leading to reduced image quality. Here we present a new method of trajectory correction that uses all spokes of a radial acquisition and compare it to an existing method that uses a two-spoke pre-scan calibration. For both methods, estimates of the necessary shifts were made using magnitude or phase data and the performances were compared. The additional effect of B0 correction was considered in all cases. Mouse cardiac scans were used for the comparisons and we also compared the quality of navigator signals obtained from the radial data with each technique. The proposed method gave improved image quality over the existing method, as assessed by visual inspection and quantitative evaluation of artifacts. The typical shading artifacts seen in radial scans were significantly reduced with both approaches, with phase-based corrections generally outperforming magnitude-based methods. B0 correction gave further improvements in each case. Furthermore, modulation of navigator signals due to the acquisition angle was significantly reduced with the new technique. We show that our proposed method works well to reduce artifacts seen in mouse cardiac imaging that can make faster imaging feasible.

PET/MRI assessment of the infarcted mouse heart.

Heart failure originating from myocardial infarction (MI) is a leading cause of death worldwide. Mouse models of ischaemia and reperfusion injury (I/R) are used to study the effects of novel treatment strategies targeting MI, however staging disease and treatment efficacy is a challenge. Damage and recovery can be assessed on the cellular, tissue or whole-organ scale but these are rarely measured in concert. Here, for the first time, we present data showing measures of injury in infarcted mice using complementary techniques for multi-modal characterisation of the heart. We use in vivo magnetic resonance imaging (MRI) to assess heart function with cine-MRI, hindered perfusion with late gadolinium enhancement imaging and muscular function with displacement encoded with stimulated echoes (DENSE) MRI. These measures are followed by positron emission tomography (PET) with 18-F-fluorodeoxyglucose to assess cellular metabolism. We demonstrate a protocol combining each of these measures for the same animal in the same imaging session and compare how the different markers can be used to quantify cardiac recovery on different scales following injury.

Mitochondria selective S-nitrosation by mitochondria-targeted S-nitrosothiol protects against post-infarct heart failure in mouse hearts.

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.

Complex I deficiency due to selective loss of Ndufs4 in the mouse heart results in severe hypertrophic cardiomyopathy.

Mitochondrial complex I, the primary entry point for electrons into the mitochondrial respiratory chain, is both critical for aerobic respiration and a major source of reactive oxygen species. In the heart, chronic dysfunction driving cardiomyopathy is frequently associated with decreased complex I activity, from both genetic and environmental causes. To examine the functional relationship between complex I disruption and cardiac dysfunction we used an established mouse model of mild and chronic complex I inhibition through heart-specific Ndufs4 gene ablation. Heart-specific Ndufs4-null mice had a decrease of ∼ 50% in complex I activity within the heart, and developed severe hypertrophic cardiomyopathy as assessed by magnetic resonance imaging. The decrease in complex I activity, and associated cardiac dysfunction, occurred absent an increase in mitochondrial hydrogen peroxide levels in vivo, accumulation of markers of oxidative damage, induction of apoptosis, or tissue fibrosis. Taken together, these results indicate that diminished complex I activity in the heart alone is sufficient to drive hypertrophic cardiomyopathy independently of alterations in levels of mitochondrial hydrogen peroxide or oxidative damage.

A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes.

The glycation of protein and nucleic acids that occurs as a consequence of hyperglycemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs after the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal, and disruption of mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear owing to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells, and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography-tandem mass spectrometry, enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycemia both in cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging.