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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.

Arachidonic acid metabolites epoxyeicosatrienoates (EETs) and hydroxyeicosatetraenoates (HETEs) are important regulators of myocardial blood flow and coronary vascular resistance (CVR), but their mechanisms of action are not fully understood. We identified G protein-coupled receptor 39 (GPR39) as a microvascular smooth muscle cell (mVSMC) receptor antagonistically regulated by two endogenous eicosanoids: 15-HETE, which stimulates GPR39 to increase mVSMC intracellular calcium and augment microvascular CVR, and 14,15-EET, which inhibits these actions. Furthermore, zinc ion acts as an allosteric modulator of GPR39 to potentiate the efficacy of the two ligands. Finally, GPR39 knockout mice are protected from myocardial ischemia compared to wild-type littermates. Our findings will have a major impact on understanding the roles of eicosanoids in cardiovascular physiology and disease, and provide an opportunity for the development of novel GPR39-targeting therapies for cardiovascular disease. One Sentence Summary: GPR39 is a microvascular smooth muscle cell receptor regulated by two vasoactive eicosanoids with opposing actions.

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.

Aim Stimulation of the nitric oxide (NO) – soluble guanylate (sGC) - protein kinase G (PKG) pathway confers protection against acute ischaemia/reperfusion injury, but more chronic effects in reducing post-myocardial infarction (MI) heart failure are less defined. The aim of this study was to not only determine whether the sGC stimulator riociguat reduces infarct size but also whether it protects against the development of post-MI heart failure. Methods and Results Mice were subjected to 30 min ischaemia via ligation of the left main coronary artery to induce MI and either placebo or riociguat (1.2 µmol/l) were given as a bolus 5 min before and 5 min after onset of reperfusion. After 24 hours, both, late gadolinium-enhanced magnetic resonance imaging (LGE-MRI) and 18F-FDG-positron emission tomography (PET) were performed to determine infarct size. In the riociguat-treated mice, the resulting infarct size was smaller (8.5±2.5% of total LV mass vs. 21.8%±1.7%. in controls, p = 0.005) and LV systolic function analysed by MRI was better preserved (60.1%±3.4% of preischaemic vs. 44.2%±3.1% in controls, p = 0.005). After 28 days, LV systolic function by echocardiography treated group was still better preserved (63.5%±3.2% vs. 48.2%±2.2% in control, p = 0.004). Conclusion Taken together, mice treated acutely at the onset of reperfusion with the sGC stimulator riociguat have smaller infarct size and better long-term preservation of LV systolic function. These findings suggest that sGC stimulation during reperfusion therapy may be a powerful therapeutic treatment strategy for preventing post-MI heart failure.

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.

Differential DNA methylation exists in the epigenome of end-stage failing human hearts but whether it contributes to disease progression is presently unknown. Here, we report that cardiac specific deletion of Dnmt3b, the predominant DNA methyltransferase in adult mouse hearts, leads to an accelerated progression to severe systolic insufficiency and myocardial thinning without a preceding hypertrophic response. This was accompanied by widespread myocardial interstitial fibrosis and myo-sarcomeric disarray. By targeted candidate gene quantitative RT-PCR, we discovered an over-activity of cryptic splice sites in the sarcomeric gene Myh7, resulting in a transcript with 8 exons missing. Moreover, a region of differential methylation overlies the splice site locus in the hearts of the cardiac-specific conditional knockout (CKO) mice. Although abundant and complex forms of alternative splice variants have been reported in diseased hearts and the contribution of each remains to be understood in further detail, our results demonstrate for the first time that a link may exist between alternative splicing and the cardiac epigenome. In particular, this gives the novel evidence whereby the loss of an epigenome modifier promotes the development and progression of heart disease.

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.

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. Copyright © 2014 John Wiley & Sons, Ltd.

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., Highlights • A mitochondria-targeted mass spectrometric probe, MitoG, has been developed to measure glyoxal and methylglyoxal. • Using MitoG we show that mitochondrial glyoxal and methylglyoxal can be measured in hyperglycemic cells. • MitoG can also be used in vivo to infer mitochondrial glyoxal and methylglyoxal production in a mouse model of type I diabetes. • These findings suggest that the accumulation of glyoxal and methylglyoxal within mitochondria may contribute to mitochondrial dysfunction in diabetes.

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. J. Magn. Reson. Imaging 2013;38:468–473. © 2012 Wiley Periodicals, Inc.

Aim: The endogenous autacoid bradykinin (BK) reportedly reduces myocardial infarct size when given exogenously at reperfusion. Muscarinic and opioid G-protein-coupled receptors are equally protective and have been shown to couple through a matrix metalloproteinase (MMP)-dependent transactivation of the epidermal growth factor receptor (EGFR). Here we test whether BK protects the rat heart through the EGFR by an MMP-dependent pathway. Methods: Infarct size was measured in isolated perfused rat hearts undergoing 30 min regional ischaemia followed by 120 min reperfusion. In additional studies HL-1 cardiomyocytes were loaded with tetramethylrhodamine ethyl to measure their mitochondrial membrane potential (Ψm). Adding the calcium ionophore calcimycin, causes Ψm-collapse presumably due to calcium-induced mitochondrial permeability transition. Results: As expected, BK (100 nmol L−1) started 5 min prior to reperfusion reduced infarct size from 38.9 ± 2.0% of the ischaemic zone in control hearts to 22.2 ± 3.3% (P

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 µg/kg followed by a 60 min infusion of 1.25 µg/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 KATP channel antagonist. Conversely, Nω-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.

Kinetic modelling in PET requires the arterial input function (AIF), defined as the time-activity curve (TAC) in plasma. This measure is challenging to obtain in mice due to low blood volumes, resulting in a reliance on image-based methods for AIF derivation. We present a comparison of PET- and MR-based region-of-interest (ROI) analysis to obtain image-derived AIFs from the left ventricle (LV) of a mouse model. ROI-based partial volume correction (PVC) was performed to improve quantification. MRI and dynamic PET images were obtained from a recent study investigating treatment effects in 12 mice following myocardial infarction [1], where half the mice received a new treatment and half did not. Prospectively gated MRI (4.7T Bruker BioSpec, FLASH TR/TE 400/3ms, spatial resolution 140μm in 1mm slices) were acquired prior to PET acquisition (approx. 25MBq 18F-FDG bolus, 45 minute emission listmode acquisition reconstructed with 3DRP in four cardiac frames) on a split-magnet PET camera [2]. Images were co-registered using SPMMouse [3] (see Figure ​Figure11). Figure 1 Mouse heart MR (left) and fused with 18F-FDG static PET (right). Arrow indicates infarcted region. AIF extraction AIFs were obtained by taking mean time courses from LV Lumen ROIs, shown in Figure ​Figure2.2. The regional geometric transfer matrix (GTM) method was applied for PVC [4], using ROIs drawn on either the co-registered MR images or directly onto the last dynamic frame PET images. ROIs covered LV lumen, myocardium, lungs/body and background. Patlak [5] analysis was performed to evaluate glucose metabolism. Figure 2 AIFs and TACs derived for single subject using (A) MR ROIs, (B) PET ROIs with PVC and (C) MR ROIs with PVC. Insets detail first 500s Uncorrected AIFs and myocardial TACs produced by manual ROI delineation displayed contamination with myocardial signal. AIFs and myocardial curves became distinguishable if GTM PVC was applied. Only MR-based PVC produced significant differences (p

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.

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.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies. In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment. In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

Chronic heart failure, as a result of acute myocardial infarction, is a leading cause of death worldwide. Combining diagnostic imaging modalities may aid the direct assessment of experimental treatments targeting heart failure in vivo. Here we present preliminary data using the Cambridge combined PET/MRI imaging system in a mouse model of acute myocardial infarction. The split-magnet design can deliver uncompromised MRI and PET performance, for better assessment of disease and treatment in a preclinical environment.

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.

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.

Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear1,2,3,4,5,6. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.

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.

Distinct epigenomic patterns of DNA methylation exist in important elements of the cardiac genome in human end-stage cardiomyopathy but whether these contribute to disease progression is unknown. We examined and found that the main DNA methyltransferase expressed in the adult mammalian heart is DNMT3B. We therefore generated a conditional inducible Dnmt3b knockout mouse to test the effect of altered DNA methylation in the heart. Upon induction in the adult mouse, Dnmt3b knockout (CKO) led rapidly to severe systolic insufficiency and myocardial thinning. Examination of myocyte nuclei reflected a loss of homogeniety and re-distribution of heterochromatin. This was accompanied by widespread myocardial interstitial fibrosis and myofibrillary disarray. We examined the myocardial transcriptome of CKO mice by RNA-seq and discovered significant changes in levels of transcript splicing isoforms especially those of sarcomeric genes. When subjected to pressure overload, CKO mice failed to mount a compensatory hypertrophic response and instead developed accelerated fulminant dilated cardiomyopathy. Our results demonstrate that DNA methylation has a distinct role with regulating transcript isoform splicing apart from gene expression. In particular, we have highlighted a mechanism by which the epigenome may mediate the development and progression of a complex disease.

Objective We developed mice with a cardiomyocyte-specific ablation of the cGKI gene (CMG-KO) and tested whether protection against reperfusion injury by ischemic postconditioning (IPost), soluble guanylyl cyclase (sGC) activation, the adenosine A2B receptor (A2BAR), or the mitochondria-targeted S-nitrosothiol (MitoSNO) was affected. MitoSNO accumulates within mitochondria, driven by the membrane potential, where it generates NO and S-nitrosated thiol proteins [1].

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 A2b adenosine receptor (A2bAR) 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 A2bAR antagonist MRS-1754, confirming a role for A2bAR. Additionally, the coadministration of ischemic postconditioning and a selective A2aAR 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 A2bAR (BAY60) or A2aAR (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 A1/A2AR 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, A2a and A2b, must be simultaneously activated for protection to occur.

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.

Oxidative damage from elevated production of reactive oxygen species contributes to ischemia-reperfusion injury (IRI) in myocardial infarction (MI) and stroke. We recently determined a critical mechanism for protection from acute IRI by S-nitrosation using a mitochondria-selective S-nitrosating agent, MitoSNO. Protection by MitoSNO is afforded by inhibition of complex I through selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischaemia (1). Here, we determine that MitoSNO action acutely at reperfusion can also prevent the development of post-MI heart failure in mice. Cardiac function and morphology was assessed 24 h post-infarction by late gadolinium-enhanced magnetic resonance imaging. The resulting infarct size was significantly smaller in the group treated with MitoSNO during early reperfusion (bolus of 100 ng/kg, followed by 2 h infusion of 1 ng/kg/min). Furthermore, left ventricular ejection fraction (EF) was increased with MitoSNO treatment from 51.3 ± 2.1% in controls to 64.4% ± 1.9, p = 0.001, and this increase was still present when the hearts were reassessed via cardiac MRI after 28 days. In addition, MitoSNO-treated hearts showed significantly less tissue fibrosis after 28 days. Taken together, reversible S-nitrosation of complex I ND3 Cys39 results in significant protection from acute and long-term effects of IRI. In demonstrating amelioration of clinically relevant end points of post-MI heart failure, we provide evidence for MitoSNO to be a promising rational therapeutic strategy for translation to use in humans. Reference Chouchani et al . (2013) Nature Medicine , 19-753-759.

Introduction Activation of the nitric oxide-protein kinase G pathway confers protection against acute ischemia/reperfusion injury, but more chronic effects in reducing post-myocardial infarction (MI) heart failure are less defined. The aim of this study was to determine whether riociguat, a selective soluble guanylate cyclase (sGC) stimulator, not only reduces infarct size but also protects against the development of post-MI heart failure. Methods Mice were subjected to 30 min ischemia via ligation of the left main coronary artery to induce MI and either placebo or riociguat (1.2 µmol/l) were given as a bolus 5 min before and 5 min after onset of reperfusion. After 24 hours, both, late gadolinium-enhanced magnetic resonance imaging (LGE-MRI) and 18F-FDG-positron emission tomography (PET) were performed to determine infarct size and cardiac function. Furthermore riociguats effect on blood pressure was tested by either the non-invasive tail cuff assessment or the LV catheterization via the right carotid artery. Results In the riociguat-treated mice, the resulting infarct size was smaller (21.8%±1.7% vs. 8.5±2.5% of total LV mass, p=0.005), and LV systolic function was better preserved (44.2%±3.1% vs. 60.1%±3.4%, p=0.005) compared to control animals. After 28 days, echocardiography showed markedly preserved LV systolic function (48.2%±2.2 vs. 63.5%±3.2%, p=0.004) in the group treated with riociguat compared to control. Riociguat showed no significant effect on blood pressure. Conclusion Taken together, mice treated acutely at the onset of reperfusion with the sGC stimulator riociguat have smaller infarct size, better preserved LV systolic function and develop less adverse LV remodeling. These findings suggest that sGC stimulation after an acute MI may be a powerful therapeutic treatment strategy for post-MI heart failure.

Background Chronic heart failure (CHF), as a result of acute myocardial infarction (AMI), is currently a leading cause of death worldwide. The direct assessment of novel treatments targeting CHF in vivo however remains difficult. While MRI can reveal cardiac morphology and tissue perfusion through the use of gadolinium (Gd) contrast agents, PET provides information on myocardial function at the molecular level. Here we present preliminary data using a combined 18FDG PET/MRI imaging system in an AMI mouse model with the aim of acquiring simultaneous morphological and functional data. Methods An open-chest mouse (C57Bl/6J) heart model was utilised in which the left anterior descending coronary artery was occluded for 30 minutes to induce an ischaemic insult. A cardiac MRI with a 4.7T Bruker BioSpec system was performed 24 h after surgery. At 4 weeks post-AMI a second MRI was performed followed by a combined 18FDG PET-MRI investigation using the Cambridge split-magnet system. Results Gd-enhanced MRI at 24 hr reperfusion revealed hyperenhancement of the infarcted region which corresponded to a signal void in the PET image obtained at 4 weeks post-MI. Furthermore, this was associated with a decrease in global ejection fraction as well as left ventricular wall thinning, consistent with the onset of heart failure. Conclusion These preliminary findings demonstrate that the combined PET/MRI imaging system is a powerful tool in studying the pathophysiology of CHF allowing a better understanding of the progressive changes that occur and the direct evaluation of novel therapeutic treatments.

Background:\ud 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.\ud \ud Scope of review:\ud 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.\ud \ud Major conclusions and general significance:\ud 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.\ud \ud Abbreviations:\ud EPR, electron paramagnetic resonance; GFP, green fluorescent protein; 4-HNE, 4-hydroxynonenal; MitoB, 3-(dihydroxyboronyl)benzyltriphenylphosphonium bromide; MitoP, (3-hydroxybenzyl)triphenylphosphonium bromide; ROS, reactive oxygen species; SOD, superoxide dismutase; TPMP, methyltriphenylphosphonium; TPP, triphenylphosphonium cation