Translational large animal model of hibernating myocardium: characterization by serial multimodal imaging.

Nonrevascularizable coronary artery disease is a frequent cause of hibernating myocardium leading to heart failure (HF). Currently, there is a paucity of therapeutic options for patients with this condition. There is a lack of animal models resembling clinical features of hibernating myocardium. Here we present a large animal model of hibernating myocardium characterized by serial multimodality imaging. Yucatan minipigs underwent a surgical casein ameroid implant around the proximal left anterior descending coronary artery (LAD), resulting in a progressive obstruction of the vessel. Pigs underwent serial multimodality imaging including invasive coronary angiography, cardiac magnetic resonance (CMR), and hybrid 18F-Fluorodeoxyglucose positron emission tomography-computed tomography (FDG-PET/CT). A total of 43 pigs were operated on and were followed for 120 ± 37 days with monthly multimodality imaging. 24 pigs (56%) died during the follow-up. Severe LAD luminal stenosis was documented in all survivors. In the group of 19 long-term survivors, 17 (90%) developed left ventricular systolic dysfunction [median LVEF of 35% (IQR 32.5-40.5%)]. In 17/17, at-risk territory was viable on CMR and 14 showed an increased glucose uptake in the at-risk myocardium on 18FDG-PET/CT. The present pig model resembles most of the human hibernated myocardium characteristics and associated heart failure (systolic dysfunction, viable myocardium, and metabolic switch to glucose). This human-like model might be used to test novel interventions for nonrevascularizable coronary artery disease and ischemia heart failure as a previous stage to clinical trials.

The mKATP Channels and protein-kinase C Are Involved in the Cardioprotective Effects of Genistein on Estrogen-Deficient Rat Hearts Exposed to Ischemia/Reperfusion: Energetic Study.

Estrogenic deficiency is considered a risk of coronary disease in women. The phytoestrogen genistein could be a safe preventive strategy. The first aim of this work was to validate a model of cardiac stunning in which natural estrogenic deficiency rats, ie, adult young male (YM) and aged female (AgF), are compared with young female rats (YF). The second aim was to study whether the in vivo administration of genistein prevents the stunning in estrogenic deficiency rats. The third aim was to evaluate whether in our estrogenic deficiency model exists a synergy between genistein and estradiol. The fourth aim was to characterize the underlying mechanisms of genistein. Stunning was induced by ischemia/reperfusion (I/R) in isolated hearts inside a calorimeter. The left ventricular pressure (P) and total heat rate (Ht) were simultaneously measured, while diastolic contracture and muscle economy (P/Ht) were calculated. During R, P/Ht and P recovered less in AgF and YM than in YF rat hearts. Genistein through i.p. (GST-ip) improved P and P/Ht in AgF and YM, but not in YF. In YM, the cardioprotections of GST-ip and estradiol were synergistic. After ischemia, GST-ip increased SR Ca leak causing diastolic contracture. The GST-ip cardioprotection neither was affected by blockade of PI3K-Akt, NO synthases, or phosphatases, but it was sensitive to blockade of protein-kinase C and mKATP channels. Results suggest that (1) estrogenic deficiency worsens cardiac stunning, (2) GST-ip was more cardioprotective in estrogenic deficiency and synergistic with estradiol, and (3) cardioprotection of GST-ip depends on the protein-kinase C and mKATP channel pathway activation.

Identification of new biophysical markers for pathological ventricular remodelling in tachycardia-induced dilated cardiomyopathy.

Our aim was to identify biophysical biomarkers of ventricular remodelling in tachycardia-induced dilated cardiomyopathy (DCM). Our study includes healthy controls (N = 7) and DCM pigs (N = 10). Molecular analysis showed global myocardial metabolic abnormalities, some of them related to myocardial hibernation in failing hearts, supporting the translationality of our model to study cardiac remodelling in dilated cardiomyopathy. Histological analysis showed unorganized and agglomerated collagen accumulation in the dilated ventricles and a higher percentage of fibrosis in the right (RV) than in the left (LV) ventricle (P = .016). The Fourier Transform Infrared Spectroscopy (FTIR) 1st and 2nd indicators, which are markers of the myofiber/collagen ratio, were reduced in dilated hearts, with the 1st indicator reduced by 45% and 53% in the RV and LV, respectively, and the 2nd indicator reduced by 25% in the RV. The 3rd FTIR indicator, a marker of the carbohydrate/lipid ratio, was up-regulated in the right and left dilated ventricles but to a greater extent in the RV (2.60-fold vs 1.61-fold, P = .049). Differential scanning calorimetry (DSC) showed a depression of the freezable water melting point in DCM ventricles - indicating structural changes in the tissue architecture - and lower protein stability. Our results suggest that the 1st, 2nd and 3rd FTIR indicators are useful markers of cardiac remodelling. Moreover, the 2nd and 3rd FITR indicators, which are altered to a greater extent in the right ventricle, are associated with greater fibrosis.

Taxonomy of segmental myocardial systolic dysfunction.

The terms used to describe different states of myocardial health and disease are poorly defined. Imprecision and inconsistency in nomenclature can lead to difficulty in interpreting and applying trial outcomes to clinical practice. In particular, the terms 'viable' and 'hibernating' are commonly applied interchangeably and incorrectly to myocardium that exhibits chronic contractile dysfunction in patients with ischaemic heart disease. The range of inherent differences amongst imaging modalities used to define myocardial health and disease add further challenges to consistent definitions. The results of several large trials have led to renewed discussion about the classification of dysfunctional myocardial segments. This article aims to describe the diverse myocardial pathologies that may affect the myocardium in ischaemic heart disease and cardiomyopathy, and how they may be assessed with non-invasive imaging techniques in order to provide a taxonomy of myocardial dysfunction.

Baicalein Rescues Delayed Cooling via Preservation of Akt Activation and Akt-Mediated Phospholamban Phosphorylation.

Cooling reduces the ischemia/reperfusion (I/R) injury seen in sudden cardiac arrest (SCA) by decreasing the burst of reactive oxygen species (ROS). Its cardioprotection is diminished when delay in reaching the target temperature occurs. Baicalein, a flavonoid derived from the root of ScutellariabaicalensisGeorgi, possesses antioxidant properties. Therefore, we hypothesized that baicalein can rescue cooling cardioprotection when cooling is delayed. Two murine cardiomyocyte models, an I/R model (90 min ischemia/3 h reperfusion) and stunning model (30 min ischemia/90 min reperfusion), were used to assess cell survival and contractility, respectively. Cooling (32 °C) was initiated either during ischemia or during reperfusion. Cell viability and ROS generation were measured. Cell contractility was evaluated by real-time phase-contrast imaging. Our results showed that cooling reduced cell death and ROS generation, and this effect was diminished when cooling was delayed. Baicalein (25 µM), given either at the start of reperfusion or start of cooling, resulted in a comparable reduction of cell death and ROS production. Baicalein improved phospholamban phosphorylation, contractility recovery, and cell survival. These effects were Akt-dependent. In addition, no synergistic effect was observed with the combined treatments of cooling and baicalein. Our data suggest that baicalein may serve as a novel adjunct therapeutic strategy for SCA resuscitation.

Free breathing three-dimensional late gadolinium enhancement cardiovascular magnetic resonance using outer volume suppressed projection navigators.

PURPOSE: To develop a three-dimensional, free-breathing, late gadolinium enhancement (3D FB-LGE) cardiovascular magnetic resonance (CMR) technique, and to compare it with clinically used two-dimensional breath-hold LGE (2D BH-LGE). METHODS: The proposed 3D FB-LGE method consisted of inversion preparation, inversion delay, fat saturation, outer volume suppression, one-dimensional projection navigators, and a segmented stack of spirals acquisition. The 3D FB-LGE and 2D BH-LGE scans were performed on 29 cardiac patients. Qualitative analysis and quantitative analysis (in patients with scar) were performed. RESULTS: No significant differences were noted between the 3D FB-LGE and 2D BH-LGE data sets in terms of overall image quality score (2D: 4.69 ± 0.60 versus 3D: 4.55 ± 0.51, P = 0.46) and image artifact score (2D: 1.10 ± 0.31 versus 3D: 1.17 ± 0.38; P = 0.63). The average difference in fractional scar volume between the 3D and 2D methods was 1.9% (n = 5). Acquisition time was significantly shorter for the 3D FB-LGE over 2D BH-LGE by a factor of 2.83 ± 0.77 (P < 0.0001). CONCLUSIONS: The 3D FB-LGE is a viable option for patients, particularly in acute settings or in patients who are unable to comply with breath-hold instructions. Magn Reson Med 77:1533-1543, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Clinical evaluation of three-dimensional late enhancement MRI.

PURPOSE: To assess the diagnostic value of three-dimensional late enhancement (3D-LGE) for the detection of myocardial necrosis in a routine clinical setting. 3D-LGE has been proposed as a novel magnetic resonance (MR) technique for the accurate detection of myocardial scar in both the ventricles and atria. Its performance in clinical practice has been poorly examined. MATERIALS AND METHODS: Fifty-seven patients referred for cardiac MR examination including scar imaging were prospectively enrolled. Gadolinium enhanced single breathhold 3D T1-weighted gradient-echo inversion recovery sequence and a conventional 2D-LGE sequence were performed using a 1.5 Tesla clinical MR imaging system. The presence, pattern and transmurality of LGE, diagnostic accuracy and level of diagnostic confidence as well as image quality (median quality, mean LGE signal intensity, sharpness, virtual scan time) were graded on a 4-point scale. RESULTS: Interpretable images were obtained in 52/57 2D-LGE and in 47/57 3D high-resolution exams. LGE was detected in 10 patients with ischemic pattern, 9 with nonischemic pattern, while it was absent in 28, resulting in a total of 47 complete datasets. The detection of global and segmental LGE as well as its transmural extent were similar for both techniques (P = 0.65, P = 0.305, and P = 0.15, respectively). Image quality (median quality, LGE/ myocardial and LGE/ blood pool sharpness) was similar for both techniques (P = 0.740, P = 0.34, and P = 1.00, respectively), but LGE signal intensity was higher with 2D (P = 0.020). CONCLUSION: 3D-LGE diagnostic and quality scores were comparable to 2D-LGE in a routine clinical setting. Further technical refinement is required for 3D LGE to offer a reliable alternative for high quality scar imaging. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 2 J. MAGN. RESON. IMAGING 2017;45:1675-1683.

Comparative Efficacy of Intracoronary Allogeneic Mesenchymal Stem Cells and Cardiosphere-Derived Cells in Swine with Hibernating Myocardium.

RATIONALE: Allogeneic bone marrow-derived mesenchymal stem cells (MSCs) and cardiosphere-derived cells (CDCs) have each entered clinical trials, but a direct comparison of these cell types has not been performed in a large animal model of hibernating myocardium. OBJECTIVE: Using completely blinded methodology, we compared the efficacy of global intracoronary allogeneic MSCs (icMSCs, ≈35×10(6)) and CDCs (icCDCs, ≈35×10(6)) versus vehicle in cyclosporine-immunosuppressed swine with a chronic left anterior descending coronary artery stenosis (n=26). METHODS AND RESULTS: Studies began 3 months after instrumentation when wall thickening was reduced (left anterior descending coronary artery % wall thickening [mean±SD], 38±11% versus 83±26% in remote; P<0.01) and similar among groups. Four weeks after treatment, left anterior descending coronary artery % wall thickening increased similarly after icCDCs and icMSCs, whereas it remained depressed in vehicle-treated controls (icMSCs, 51±13%; icCDCs, 51±17%; vehicle, 34±3%, treatments P<0.05 versus vehicle). There was no change in myocardial perfusion. Both icMSCs and icCDCs increased left anterior descending coronary artery myocyte nuclear density (icMSCs, 1601±279 nuclei/mm(2); icCDCs, 1569±294 nuclei/mm(2); vehicle, 973±181 nuclei/mm(2); treatments P<0.05 versus vehicle) and reduced myocyte diameter (icMSCs, 16.4±1.5 µm; icCDCs, 16.8±1.2 µm; vehicle, 20.2±3.7 µm; treatments P<0.05 versus vehicle) to the same extent. Similar changes in myocyte nuclear density and diameter were observed in the remote region of cell-treated animals. Cell fate analysis using Y-chromosome fluorescent in situ hybridization demonstrated rare cells from sex-mismatched donors. CONCLUSIONS: Allogeneic icMSCs and icCDCs exhibit comparable therapeutic efficacy in a large animal model of hibernating myocardium. Both cell types produced equivalent increases in regional function and stimulated myocyte regeneration in ischemic and remote myocardium. The activation of endogenous myocyte proliferation and regression of myocyte cellular hypertrophy support a common mechanism of cardiac repair.

Mitochondrial Bioenergetics During Ischemia and Reperfusion.

During ischemia and reperfusion (I/R) mitochondria suffer a deficiency to supply the cardiomyocyte with chemical energy, but also contribute to the cytosolic ionic alterations especially of Ca2+. Their free calcium concentration ([Ca2+]m) mainly depends on mitochondrial entrance through the uniporter (UCam) and extrusion in exchange with Na+ (mNCX) driven by the electrochemical gradient (ΔΨm). Cardiac energetic is frequently estimated by the oxygen consumption, which determines metabolism coupled to ATP production and to the maintaining of ΔΨm. Nevertheless, a better estimation of heart energy consumption is the total heat release associated to ATP hydrolysis, metabolism, and binding reactions, which is measurable either in the presence or the absence of oxygenation or perfusion. Consequently, a mechano-calorimetrical approach on isolated hearts gives a tool to evaluate muscle economy. The mitochondrial role during I/R depends on the injury degree. We investigated the role of the mitochondrial Ca2+ transporters in the energetic of hearts stunned by a model of no-flow I/R in rat hearts. This chapter explores an integrated view of previous and new results which give evidences to the mitochondrial role in cardiac stunning by ischemia o hypoxia, and the influence of thyroid alterations and cardioprotective strategies, such as cardioplegic solutions (high K-low Ca, pyruvate) and the phytoestrogen genistein in both sex. Rat ventricles were perfused in a flow-calorimeter at either 30 °C or 37 °C to continuously measure the left ventricular pressure (LVP) and total heat rate (Ht). A pharmacological treatment was done before exposing to no-flow I and R. The post-ischemic contractile (PICR as %) and energetical (Ht) recovery and muscle economy (Eco: P/Ht) were determined during stunning. The functional interaction between mitochondria (Mit) and sarcoplasmic reticulum (SR) was evaluated with selective mitochondrial inhibitors in hearts reperfused with Krebs-10 mM caffeine-36 mM Na+. The caffeine induced contracture (CIC) was due to SR Ca2+ release, while relaxation mainly depends on mitochondrial Ca2+ uptake since neither SL-NCX nor SERCA are functional under this media. The ratio of area-under-curves over ischemic values (AUC-ΔHt/AUC-ΔLVP) estimates the energetical consumption (EC) to maintain CIC. Relaxation of CIC was accelerated by inhibition of mNCX or by adding the aerobic substrate pyruvate, while both increased EC. Contrarily, relaxation was slowed by cardioplegia (high K-low Ca Krebs) and by inhibition of UCam. Thus, Mit regulate the cytosolic [Ca2+] and SR Ca2+ content. Both, hyperthyroidism (HpT) and hypothyroidism (HypoT) reduced the peak of CIC but increased EC, in spite of improving PICR. Both, CIC and PICR in HpT were also sensitive to inhibition of mNCX or UCam, suggesting that Mit contribute to regulate the SR store and Ca2+ release. The interaction between mitochondria and SR and the energetic consequences were also analyzed for the effects of genistein in hearts exposed to I/R, and for the hypoxia/reoxygenation process. Our results give evidence about the mitochondrial regulation of both PICR and energetic consumption during stunning, through the Ca2+ movement.

Sex differences in the mechano-energetic effects of genistein on stunned rat and guinea pig hearts.

Although the phytoestrogen genistein (Gen) is considered protective in cardiovascular diseases, its direct effects on stunned hearts after transient ischemia-reperfusion (I/R) are unknown. This report studied the effects of 20 µmol/L Gen on the mechano-calorimetric behaviour during I/R of rat and guinea pig hearts to evaluate the energetics of Ca(2+) homeostasis. Isolated beating hearts were perfused with control Krebs solution inside a calorimeter with or without perfusion of Gen before a transient period of I/R. Left ventricular pressure development (P) and total heat rate (Ht) were continuously measured. At 37°C, Gen did not change post-ischemic contractile recovery (PICR), but it increased the relaxation rate. However, PICR was reduced in hearts of male rats and guinea pigs at 30°C. Total muscle economy (P/Ht) showed the same behaviour as P at each temperature. Inhibition of phosphatases with orthovanadate during Gen perfusion prevented a decrease in PICR in male rat hearts, suggesting that this effect is due to tyrosine kinase inhibition. Reperfusing ischemic hearts with 10 mmol/L caffeine-36 mmol/L Na(+)-Krebs induced contracture dependent on the sarcoreticular Ca(2+) content. Contracture relaxation depends on mitochondrial Ca(2+) uptake and Gen reduced the relaxation rate. Moreover, Gen prevented the increase in Rhod-2 fluorescence (free [Ca(2+)]m) of rat cardiomyocytes. In guinea pig hearts, Gen maintained ischemic preconditioning, but was reduced by 5-hydroxydecanoate, suggesting the participation of mitochondrial adenosine triphosphate (ATP)-dependent K channels. Results suggest that Gen acts on several mechanisms that regulate myocardial calcium homeostasis and energetics during I/R, which differ in a temperature- and sex-dependent manner.

Subarachnoid Hemorrhage-Triggered Acute Hypotension Is Associated with Left Ventricular Cardiomyocyte Apoptosis in a Rat Model.

Whether hypotension that occurs due to neurogenic stunned myocardium after subarachnoid hemorrhage (SAH) is associated with cardiomyocyte apoptotic cell death remains unknown. In this study, 18 male rats were subjected to sham or the endovascular perforation model of SAH surgery. Based on the mean arterial pressure (MAP) after SAH, rats were separated into SAH with hypotension (SAH hypotension) or SAH with blood pressure preservation (SAH BP preservation) groups. All animals were euthanized 2 h after the surgical procedure. Hearts were removed and separated transversely into base and apex parts, then Western blot analyses and immunohistochemistry were performed only in the apex part. One rat died as a result of severe SAH and two rats with mild SAH were excluded. We analyzed data from 15 rats that were divided into three groups: sham, SAH hypotension, and SAH BP preservation (n = 5, each). There was a significantly higher cleaved caspase-3/caspase-3 ratio in the SAH hypotension group compared with sham and the SAH BP preservation group. Cardiomyocyte apoptosis was demonstrated in the SAH rats. This is the first experimental report that describes SAH-induced neurogenic stunned myocardium with ensuing hypotension may result from the acute apoptotic cardiomyocyte cell death in the left ventricle.

Super-resolution reconstruction of late gadolinium-enhanced MRI for improved myocardial scar assessment.

PURPOSE: To develop and validate a method for improving image resolution of late gadolinium-enhanced (LGE) magnetic resonance imaging (MRI) for accurate assessment of myocardial scar. MATERIALS AND METHODS: In a cohort of 37 postinfarction patients, LGE was performed prior to ventricular tachycardia catheter ablation therapy at 1.5T. A super-resolution reconstruction (SRR) technique was applied to the three anisotropic views: short-axis (SA), two-chamber, and four-chamber, to reconstruct a single isotropic volume. For compensation of the interscan heart motion, a joint localized gradient-correlation-based scheme was developed. Scar was identified as either core or gray zone in both the SRR and original SA volumes, and evaluated based on the clinically established bipolar voltage range of the in vivo electroanatomical voltage mapping (EAVM). RESULTS: Compared to the SA volume, the SRR method resulted in significantly (P < 0.05) reduced myocardial scar gray zone sizes (10.5 ± 8.8 g vs. 9.2 ± 8.1 g) and improved agreement of the bipolar voltage range of scar gray zone (0.99 ± 0.65 mV vs. 1.46 ± 1.15 mV). CONCLUSION: We propose an SRR method to automatically reconstruct a high-quality isotropic LGE volume from three orthogonal views. Analysis of the in vivo EAVM demonstrated improved myocardial scar assessment from the SRR volume compared with the SA LGE alone.

Regional mapping of myocardial hibernation phenotype in idiopathic end-stage dilated cardiomyopathy.

Myocardial hibernation (MH) is a well-known feature of human ischaemic cardiomyopathy (ICM), whereas its presence in human idiopathic dilated cardiomyopathy (DCM) is still controversial. We investigated the histological and molecular features of MH in left ventricle (LV) regions of failing DCM or ICM hearts. We examined failing hearts from DCM (n = 11; 41.9 ± 5.45 years; left ventricle-ejection fraction (LV-EF), 18 ± 3.16%) and ICM patients (n = 12; 58.08 ± 1.7 years; LVEF, 21.5 ± 6.08%) undergoing cardiac transplantation, and normal donor hearts (N, n = 8). LV inter-ventricular septum (IVS) and antero-lateral free wall (FW) were transmurally (i.e. sub-epicardial, mesocardial and sub-endocardial layers) analysed. LV glycogen content was shown to be increased in both DCM and ICM as compared with N hearts (P < 0.001), with a U-shaped transmural distribution (lower values in mesocardium). Capillary density was homogenously reduced in both DCM and ICM as compared with N (P < 0.05 versus N), with a lower decrease independent of the extent of fibrosis in sub-endocardial and sub-epicardial layers of DCM as compared with ICM. HIF1-α and nestin, recognized ischaemic molecular hallmarks, were similarly expressed in DCM-LV and ICM-LV myocardium. The proteomic profile was overlapping by ~50% in DCM and ICM groups. Morphological and molecular features of MH were detected in end-stage ICM as well as in end-stage DCM LV, despite epicardial coronary artery patency and lower fibrosis in DCM hearts. Unravelling the presence of MH in the absence of coronary stenosis may be helpful to design a novel approach in the clinical management of DCM.

Combined saturation/inversion recovery sequences for improved evaluation of scar and diffuse fibrosis in patients with arrhythmia or heart rate variability.

PURPOSE: To develop arrhythmia-insensitive inversion recovery sequences for improved visualization of myocardial scar and quantification of diffuse fibrosis. METHODS: A novel preparation pre-pulse, called saturation pulse prepared heart-rate-independent inversion recovery, is introduced, which consists of a combination of saturation and inversion pulses to remove the magnetization history in each heartbeat in late gadolinium enhancement (LGE) imaging and eliminate the need for rest periods in T1 mapping. The proposed LGE and T1 mapping sequences were evaluated against conventional LGE and modified Look-Locker inversion sequences using numerical simulations, phantom and imaging in healthy subjects and patients with suspected or known cardiovascular disease. RESULTS: Simulations and phantom experiments show that the saturation pulse prepared heart-rate-independent inversion recovery pre-pulse in LGE reduces ghosting artifacts and results in perfect nulling of the healthy myocardium in the presence of arrhythmia. In T1 mapping, saturation pulse prepared heart-rate-independent inversion recovery results in (a) reduced scan time (17 vs. 9 heartbeats), (b) insensitivity to heart rate for long T1, and (c) increased signal homogeneity for short T1. LGE images in a patient in atrial fibrillation during the scan show improved myocardial nulling. In vivo T1 maps demonstrate increased signal homogeneity in blood pools and myocardium. CONCLUSION: The proposed sequences are insensitive to heart rate variability, yield improved LGE images in the presence of arrhythmias, as well as T1 mapping with shorter scan times.

Left atrial late gadolinium enhancement with water-fat separation: the importance of phase-encoding order.

PURPOSE: To compare two late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR) methods: a Dixon LGE sequence with sequential phase-encoding order, reconstructed using water-fat separation, and standard fat-saturated LGE. MATERIALS AND METHODS: We implemented a dual-echo Dixon LGE method for reconstructing water-only images and compared it to fat-saturated LGE in 12 patients prior to their first pulmonary vein isolation (PVI) procedure. Images were analyzed for quality and fat-suppression. Regions of the left atrium were evaluated by a blinded observer (1 = prominent enhancement, 0 = mild or absent enhancement) on two sets of images (fat-saturated and water-only LGE) and agreement was assessed. RESULTS: Water-only LGE showed a trend toward better fat-suppression (P = 0.06), with a significantly more homogeneous blood pool signal and reduced inflow artifacts (both P < 0.01). Agreement between fat-saturated LGE and water-only methods was found in 84% of regions, significantly correlated by chi-squared test (P < 0.001). The kappa value was 0.52 (moderate). The average number of enhancing segments was higher for fat-saturated LGE than water-only LGE (4.2 ± 2.7 vs. 3.2 ± 2.9, P = 0.03). CONCLUSION: The two-point Dixon LGE technique reduces artifacts due to a centric k-space order. A similar enhancement pattern was observed irrespective of the LGE technique, with more enhancement detected by fat-saturated LGE.

Cardiomyocyte-specific overexpression of an active form of Rac predisposes the heart to increased myocardial stunning and ischemia-reperfusion injury.

The GTP-binding protein Rac regulates diverse cellular functions including activation of NADPH oxidase, a major source of superoxide production (O(2)(·-)). Rac1-mediated NADPH oxidase activation is increased after myocardial infarction (MI) and heart failure both in animals and humans; however, the impact of increased myocardial Rac on impending ischemia-reperfusion (I/R) is unknown. A novel transgenic mouse model with cardiac-specific overexpression of constitutively active mutant form of Zea maize Rac D (ZmRacD) gene has been reported with increased myocardial Rac-GTPase activity and O(2)(·-) generation. The goal of the present study was to determine signaling pathways related to increased myocardial ZmRacD and to what extent hearts with increased ZmRacD proteins are susceptible to I/R injury. The effect of myocardial I/R was examined in young adult wild-type (WT) and ZmRacD transgenic (TG) mice. In vitro reversible myocardial I/R for postischemic cardiac function and in vivo regional myocardial I/R for MI were performed. Following 20-min global ischemia and 45-min reperfusion, postischemic cardiac contractile function and heart rate were significantly reduced in TG hearts compared with WT hearts. Importantly, acute regional myocardial I/R (30-min ischemia and 24-h reperfusion) caused significantly larger MI in TG mice compared with WT mice. Western blot analysis of cardiac homogenates revealed that increased myocardial ZmRacD gene expression is associated with concomitant increased levels of NADPH oxidase subunit gp91(phox), O(2)(·-), and P(21)-activated kinase. Thus these findings provide direct evidence that increased levels of active myocardial Rac renders the heart susceptible to increased postischemic contractile dysfunction and MI following acute I/R.

Hibernating myocardium results in partial sympathetic denervation and nerve sprouting.

Hibernating myocardium due to chronic repetitive ischemia is associated with regional sympathetic nerve dysfunction and spontaneous arrhythmic death in the absence of infarction. Although inhomogeneity in regional sympathetic innervation is an acknowledged substrate for sudden death, the mechanism(s) responsible for these abnormalities in viable, dysfunctional myocardium (i.e., neural stunning vs. sympathetic denervation) and their association with nerve sprouting are unknown. Accordingly, markers of sympathetic nerve function and nerve sprouting were assessed in subendocardial tissue collected from chronically instrumented pigs with hibernating myocardium (n = 18) as well as sham-instrumented controls (n = 7). Hibernating myocardium exhibited evidence of partial sympathetic denervation compared with the normally perfused region and sham controls, with corresponding regional reductions in tyrosine hydroxylase protein (-32%, P < 0.001), norepinephrine uptake transport protein (-25%, P = 0.01), and tissue norepinephrine content (-45%, P < 0.001). Partial denervation induced nerve sprouting with regional increases in nerve growth factor precursor protein (31%, P = 0.01) and growth associated protein-43 (38%, P < 0.05). All of the changes in sympathetic nerve markers were similar in animals that developed sudden death (n = 9) compared with electively terminated pigs with hibernating myocardium (n = 9). In conclusion, sympathetic nerve dysfunction in hibernating myocardium is most consistent with partial sympathetic denervation and is associated with regional nerve sprouting. The extent of sympathetic remodeling is similar in animals that develop sudden death compared with survivors; this suggests that sympathetic remodeling in hibernating myocardium is not an independent trigger for sudden death. Nevertheless, sympathetic remodeling likely contributes to electrical instability in combination with other factors.

Quantitative magnetic resonance imaging can distinguish remodeling mechanisms after acute myocardial infarction based on the severity of ischemic insult.

The type and extent of myocardial infarction encountered clinically is primarily determined by the severity of the initial ischemic insult. The purpose of the study was to differentiate longitudinal fluctuations in remodeling mechanisms in porcine myocardium following different ischemic insult durations. Animals (N = 8) were subjected to coronary balloon occlusion for either 90 or 45 min, followed by reperfusion. Imaging was performed on a 3 T MRI scanner between day-2 and week-6 postinfarction with edema quantified by T2, hemorrhage by T2*, vasodilatory function by blood-oxygenation-level-dependent T2 alterations and infarction/microvascular obstruction by contrast-enhanced imaging. The 90-min model produced large transmural infarcts with hemorrhage and microvascular obstruction, while the 45 min produced small nontransmural and nonhemorrhagic infarction. In the 90-min group, elevation of end-diastolic-volume, reduced cardiac function, persistence of edema, and prolonged vasodilatory dysfunction were all indicative of adverse remodeling; in contrast, the 45-min group showed no signs of adverse remodeling. The 45- and 90-min porcine models seem to be ideal for representing the low- and high-risk patient groups, respectively, commonly encountered in the clinic. Such in vivo characterization will be a key in predicting functional recovery and may potentially allow evaluation of novel therapies targeted to alleviate ischemic injury and prevent microvascular obstruction/hemorrhage.

Assessment of left ventricular myocardial scar in coronary artery disease by a three-dimensional MR imaging technique.

PURPOSE: To evaluate the feasibility of free-breathing three-dimensional (3D) phase sensitive inversion recovery (PSIR) Turbo FLASH late gadolinium enhancement (LGE) magnetic resonance images (MRI) on left ventricular scar in patients with coronary artery disease (CAD) compared with clinically established breathhold two-dimensional (2D) PSIR Turbo FLASH images. MATERIALS AND METHODS: In 58 consecutive patients with confirmed CAD, LGE MRI using the two sequences have been acquired. Image quality was graded on a four-point scale according to the image appearance. Qualitative evaluation including the distribution area and the transmural extent of the scar based on the American Heart Association's (AHA's) 17-segment model was performed in both of 2D and 3D images. The scar volumes were compared quantitatively between 2D and 3D images. RESULTS: A total of 51 individuals were used for final statistical analysis. No differences were noted in image quality (P = 0.80), scar distribution area (P = 0.17), and scar transmural extent (P = 0.20) between 3D and 2D images. There was strong correlation in scar volume between the 3D and 2D results (r = 0.940; P < 0.001; Y = 0.298 + 1.251X, R(2) = 0.876). But the scar volume derived from 3D images was significantly larger than that derived from 2D images (2D versus 3D, 20.08 ± 9.41 cm(3) versus 25.41 ± 12.57 cm(3) , t = -7.60; P < 0.001). The trend toward a larger scar volume identified by 3D method was indicated through Bland-Altman analysis. CONCLUSION: Free-breathing 3D PSIR Turbo FLASH imaging is another feasible method to identify left ventricular myocardial scar in patients with CAD and detects more scar volume compared with breathhold 2D PSIR Turbo FLASH imaging.

PKA-mediated eNOS phosphorylation in the protection of ischemic preconditioning against no-reflow.

OBJECTIVE: To investigate whether ischemic preconditioning (IP) can reduce myocardial no-reflow by activating endothelial (e-) nitric oxide synthase (NOS) via the protein kinase A (PKA) pathway. METHODS AND RESULTS: In a 90-min ischemia and 3-h reperfusion model, minipigs were assigned into sham, ischemia-reperfusion (IR), IR+IP, IR+IP+L-NNA (an eNOS inhibitor, 10mg·kg(-1)), IR+IP+H-89 (a PKA inhibitor, 1.0µg·kg(-1)·min(-1)), IR+L-NNA, and IR+H-89 groups. IP pretreatment improved cardiac function and coronary blood flow, decreased the activities of creatine kinase by 36.6% after 90 min of ischemia and by 32.8% after 3 h of reperfusion (P<0.05), reduced the no-reflow areas from 49.9% to 11.0% (P<0.01), and attenuated the infarct size from 78.2% to 35.4% (P<0.01). IP stimulated myocardial PKA activities and the expression of PKA and Ser(133) phosphorylated (p-) cAMP response element-binding protein (CREB) in the reflow and no-reflow myocardium, and enhanced the activities of constitutive NOS and the phosphorylation of eNOS at Ser(1179) and Ser(635) in the no-reflow myocardium. IP suppressed the expression of tumor necrosis factor-α and P-selectin, and attenuated cardiomyocytes apoptosis by regulating the expression of Bcl-2 and caspase-3 in the reflow and no-reflow myocardium. The eNOS inhibitor L-NNA completely canceled these beneficial effects of IP without any influence on PKA activity, whereas the PKA inhibitor H-89 partially blocked the IP cardioprotective effects and eNOS phosphorylation at the same time. CONCLUSION: IP attenuates myocardial no-reflow and infarction after ischemia and reperfusion by activating the phosphorylation of eNOS at Ser(1179) and Ser(635) in a partly PKA-dependent manner.