Total Mechanical Unloading Minimizes Metabolic Demand of Left Ventricle and Dramatically Reduces Infarct Size in Myocardial Infarction

BackgroundLeft ventricular assist device (LVAD) mechanically unloads the left ventricle (LV). Theoretical analysis indicates that partial LVAD support (p-LVAD), where LV remains ejecting, reduces LV preload while increases afterload resulting from the elevation of total cardiac output and mean aortic pressure, and consequently does not markedly decrease myocardial oxygen consumption (MVO₂). In contrast, total LVAD support (t-LVAD), where LV no longer ejects, markedly decreases LV preload volume and afterload pressure, thereby strikingly reduces MVO₂. Since an imbalance in oxygen supply and demand is the fundamental pathophysiology of myocardial infarction (MI), we hypothesized that t-LVAD minimizes MVO₂ and reduces infarct size in MI. The purpose of this study was to evaluate the differential impact of the support level of LVAD on MVO₂ and infarct size in a canine model of ischemia-reperfusion.MethodsIn 5 normal mongrel dogs, we examined the impact of LVAD on MVO₂ at 3 support levels: Control (no LVAD support), p-LVAD and t-LVAD. In another 16 dogs, ischemia was induced by occluding major branches of the left anterior descending coronary artery (90 min) followed by reperfusion (300 min). We activated LVAD from the beginning of ischemia until 300 min of reperfusion, and compared the infarct size among 3 different levels of LVAD support.Resultst-LVAD markedly reduced MVO₂ (% reduction against Control: -56 ± 9%, p<0.01) whereas p-LVAD did less (-21 ± 14%, p<0.05). t-LVAD markedly reduced infarct size compared to p-LVAD (infarct area/area at risk: Control; 41.8 ± 6.4, p-LVAD; 29.1 ± 5.6 and t-LVAD; 5.0 ± 3.1%, p<0.01). Changes in creatine kinase-MB paralleled those in infarct size.ConclusionsTotal LVAD support that minimizes metabolic demand maximizes the benefit of LVAD in the treatment of acute myocardial infarction.     

Three-Dimensional Phase-Sensitive Inversion-Recovery Turbo FLASH Sequence for the Assessment of Left Ventricular Myocardial Scar in Swine

Objectives

We sought to evaluate the feasibility and accuracy of free-breathing three-dimensional (3D) phase-sensitive inversion-recovery (PSIR) Turbo FLASH sequence for noninvasive assessment of left ventricular myocardial scar in swine models.

Materials and Methods

Nine Chinese minipigs with experimentally induced acute myocardial infarction were studied. At 1 week and the study endpoint 4 weeks after myocardial infarction surgery, the 3D and 2D contrasted cardiac magnetic resonance (CMR) imaging were performed randomly by using a 1.5T clinical MR imaging system. Comparisons of myocardial scar volume (in cubic centimeters), scar transmurality (on a 5 points scale) and image quality (on a 4 points Likert scale) were performed by using the Pearson correlation and Bland-Altman analysis (for myocardial scar volume) or κ statistics (for transmurality) or Wilcoxon signed rank test (for image quality).

Results

In 6 of the 9 pigs, all procedures were successfully completed. In these pigs, a total of 48 segments with myocardial scars were detected by both 3D and 2D sequences, and there was good agreement for classification of scar transmurality (κ=0.930). The scar volume determined by triphenyltetrazolium chloride (TTC) staining (3.52±1.40cm³) showed a good correlation with both 3D (3.54±1.36cm³, r=0.957, P=0.003) and 2D sequence (3.53±1.26cm³, r=0.942, P=0.005) at 4 weeks. And there were good correlation between scar volumes obtained from 3D and 2D techniques (r=0.859, P<0.001) at both time points. Both 3D and 2D images detected a small reduction of scar volume from week 1 to week 4 by a factor of 1.179 and 1.176, respectively. Although slightly more artifacts were observed on 2D PSIR images, the overall image quality was not significantly different between the two sequences (3.17±0.83 for 2D vs. 3.25±0.75 for 3D, P =0.655).

Conclusions

The free-breathing 3D PSIR Turbo FLASH sequence enables accurate assessment of left ventricular myocardial scar.     

Temporal Evolution of Ischemic Lesions in Nonhuman Primates: A Diffusion and Perfusion MRI Study

MethodsPermanent MCA occlusion was induced with silk sutures through an interventional approach via the femoral artery in adult rhesus monkeys (n = 8, 10–21 years old). The stroke lesions were examined with high-resolution DWI and perfusion MRI, and T2-weighted imaging (T2W) on a clinical 3T scanner at 1–6, 48, and 96 hours post occlusion and validated with H&E staining.ResultsThe stroke infarct evolved via a natural logarithmic pattern with the mean infarct growth rate = 1.38 ± 1.32 ml per logarithmic time scale (hours) (n = 7) in the hyperacute phase (1–6 hours). The mean infarct volume after 6 hours post occlusion was 3.6±2.8 ml (n = 7, by DWI) and increased to 3.9±2.9 ml (n = 5, by T2W) after 48 hours, and to 4.7±2.2ml (n = 3, by T2W) after 96 hours post occlusion. The infarct volumes predicted by the natural logarithmic function were correlated significantly with the T2W-derived lesion volumes (n = 5, r = 0.92, p = 0.01) at 48 hours post occlusion. The final infarct volumes derived from T2W were correlated significantly with those from H&E staining (r = 0.999, p < 0.0001, n = 4). In addition, the diffusion-perfusion mismatch was visible generally at 6 hours but nearly diminished at 48 hours post occlusion.ConclusionThe infarct evolution follows a natural logarithmic pattern in the hyperacute phase of stroke. The logarithmic pattern of evolution could last up to 48 hours after stroke onset and may be used to predict the infarct volume growth during the acute phase of ischemic stroke. The nonhuman primate model, MRI protocols, and post data processing strategy may provide an excellent platform for characterizing the evolution of acute stroke lesion in mechanistic studies and therapeutic interventions of stroke disease.     

Macroscopic Fluorescence Imaging: A Novel Technique to Monitor Retention and Distribution of Injected Microspheres in an Experimental Model of Ischemic Heart Failure

Background

The limited effectiveness of cardiac cell therapy has generated concern regarding its clinical relevance. Experimental studies show that cell retention and engraftment are low after injection into ischemic myocardium, which may restrict therapy effectiveness significantly. Surgical aspects and mechanical loss are suspected to be the main culprits behind this phenomenon. As current techniques of monitoring intramyocardial injections are complex and time-consuming, the aim of the study was to develop a fast and simple model to study cardiac retention and distribution following intramyocardial injections. For this purpose, our main hypothesis was that macroscopic fluorescence imaging could adequately serve as a detection method for intramyocardial injections.

Methods and Results

A total of 20 mice underwent ligation of the left anterior descending artery (LAD) for myocardial infarction. Fluorescent microspheres with cellular dimensions were used as cell surrogates. Particles (5×10⁵) were injected into the infarcted area of explanted resting hearts (Ex vivo myocardial injetions EVMI, n = 10) and in vivo into beating hearts (In vivo myocardial injections IVMI, n = 10). Microsphere quantification was performed by fluorescence imaging of explanted organs. Measurements were repeated after a reduction to homogenate dilutions. Cardiac microsphere retention was 2.78×10⁵±0.31×10⁵ in the EVMI group. In the IVMI group, cardiac retention of microspheres was significantly lower (0.74×10⁵±0.18×10⁵; p<0.05). Direct fluorescence imaging revealed venous drainage through the coronary sinus, resulting in a microsphere accumulation in the left (0.90×10⁵±0.20×10⁵) and the right (1.07×10⁵±0.17×10⁵) lung. Processing to homogenates involved further particle loss (p<0.05) in both groups.

Conclusions

We developed a fast and simple direct fluorescence imaging method for biodistribution analysis which enabled the quantification of fluorescent microspheres after intramyocardial delivery using macroscopic fluorescence imaging. This new technique showed massive early particle loss and venous drainage into the right atrium leading to substantial accumulation of graft particles in both lungs.     

Intravascular Ultrasound Observation of the Mechanism of No-Reflow Phenomenon in Acute Myocardial Infarction

Objective

To study the mechanism of the no-reflow phenomenon using coronary angiography (CAG) and intravascular ultrasound (IVUS).

Methods

A total of 120 patients with acute myocardial infarction (AMI) who successfully underwent indwelling intracoronary stent placement by percutaneous coronary intervention (PCI). All patients underwent pre- and post-PCI CAG and pre-IVUS. No-reflow was defined as post-PCI thrombolysis in myocardial infarction (TIMI) grade 0, 1, or 2 flow in the absence of mechanical obstruction. Normal reflow was defined as TIMI grade 3 flow. The pre-operation reference vascular area, minimal luminal cross-sectional area, plaque cross-sectional area, lesion length, plaque volume and plaque traits were measured by IVUS.

Results

The no-reflow group was observed in 14 cases (11.6%) and normal blood-flow group in 106 cases (89.4%) based on CAG results. There was no statistically significant difference in the patients’ medical history, reference vascular area (no-flow vs. normal-flow; 15.5 ± 3.2 vs. 16.2 ± 3.3, p> 0.05) and lesion length (21.9 ± 5.1 vs. 19.5 ± 4.8, p> 0.05) between the two groups. No-reflow patients had a longer symptom onset to reperfusion time compared to normal blood-flow group [(6.6 ± 3.1) h vs (4.3 ± 2.7) h; p< 0.05] and higher incidence of TIMI flow grade< 3 (71.4% vs 49.0%, p< 0.05). By IVUS examination, the no-reflow group had a significantly increased coronary plaque area and plaque volume compared to normal blood-flow group [(13.7 ± 3.0) mm² vs (10.2 ± 2.9) mm²; (285.4 ± 99.8) mm³ vs (189.7 ± 86.4) mm³; p< 0.01]. The presence of IVUS-detected soft plaque (57.1% vs. 24.0%, p< 0.01), eccentric plaque (64.2% vs. 33.7%, p< 0.05), plaque rupture (50.0% vs. 21.2%, p< 0.01), and thrombosis (42.8% vs. 15.3%) were significantly more common in no-reflow group.

Conclusion

There was no obvious relationship between the coronary risk factors and no-reflow phenomenon. The symptom onset to reperfusion time, TIMI flow grade before stent deployment, plaque area, soft plaques, eccentric plaques, plaque rupture and thrombosis may be risk factors for the no-reflow phenomenon after PCI.     

Protein Kinase G1 α Overexpression Increases Stem Cell Survival and Cardiac Function after Myocardial Infarction

Background

We hypothesized that overexpression of cGMP-dependent protein kinase type 1α (PKG1α) could mimic the effect of tadalafil on the survival of bone marrow derived mesenchymal stem cells (MSCs) contributing to regeneration of the ischemic heart.

Methods and Results

MSCs from male rats were transduced with adenoviral vector encoding for PKG1α (^PKG1αMSCs).Controls included native MSCs (^NatMSCs) and MSCs transduced with an empty vector (^NullMSCs). PKG1α activity was increased approximately 20, 5 and 16 fold respectively in ^PKG1αMSCs. ^PKG1αMSCs showed improved survival under oxygen and glucose deprivation (OGD) which was evidenced by lower LDH release, caspase-3/7 activity and number of positive TUNEL cells. Anti-apoptotic proteins pAkt, pGSK3β, and Bcl-2 were significantly increased in ^PKG1αMSCs compared to ^NatMSCs and ^NullMSCs. Higher release of multiple prosurvival and angiogenic factors such as HGF, bFGF, SDF-1 and Ang-1 was observed in ^PKG1αMSCs before and after OGD. In a female rat model of acute myocardial infarction, ^PKG1αMSCs group showed higher survival compared with ^NullMSCs group at 3 and 7 days after transplantation as determined by TUNEL staining and sry-gene quantitation by real-time PCR. Increased anti-apoptotic proteins and paracrine factors in vitro were also identified. Immunostaining for cardiac troponin I combined with GFP showed increased myogenic differentiation of ^PKG1αMSCs. At 4 weeks after transplantation, compared to DMEM group and ^NullMSCs group, ^PKG1αMSCs group showed increased blood vessel density in infarct and peri-infarct areas (62.5±7.7; 68.8±7.3 per microscopic view, p<0.05) and attenuated infarct size (27.2±2.5%, p<0.01). Heart function indices including ejection fraction (52.1±2.2%, p<0.01) and fractional shortening (24.8%±1.3%, p<0.01) were improved significantly in ^PKG1αMSCs group.

Conclusion

Overexpression of PKG1α transgene could be a powerful approach to improve MSCs survival and their angiomyogenic potential in the infarcted heart.     

Temporal Evolution of Parotid Volume and Parotid Apparent Diffusion Coefficient in Nasopharyngeal Carcinoma Patients Treated by Intensity-Modulated Radiotherapy Investigated by Magnetic Resonance Imaging: A Pilot Study

Purpose

To concurrently quantify the radiation-induced changes and temporal evolutions of parotid volume and parotid apparent diffusion coefficient (ADC) in nasopharyngeal carcinoma (NPC) patients treated by intensity-modulated radiotherapy by using magnetic resonance imaging (MRI).

Materials and Methods

A total of 11 NPC patients (9 men and 2 women; 48.7 ± 11.7 years, 22 parotid glands) were enrolled. Radiation dose, parotid sparing volume, severity of xerostomia, and radiation-to-MR interval (RMI) was recorded. MRI studies were acquired four times, including one before and three after radiotherapy. The parotid volume and the parotid ADC were measured. Statistical analysis was performed using SPSS and MedCalc. Bonferroni correction was applied for multiple comparisons. A P value less than 0.05 was considered as statistically significant.

Results

The parotid volume was 26.2 ± 8.0 cm³ before radiotherapy. The parotid ADC was 0.8 ± 0.15 × 10⁻³ mm²/sec before radiotherapy. The parotid glands received a radiation dose of 28.7 ± 4.1 Gy and a PSV of 44.1 ± 12.6%. The parotid volume was significantly smaller at MR stage 1 and stage 2 as compared to pre-RT stage (P < .005). The volume reduction ratio was 31.2 ± 13.0%, 26.1 ± 13.5%, and 17.1 ± 16.6% at stage 1, 2, and 3, respectively. The parotid ADC was significantly higher at all post-RT stages as compared to pre-RT stage reciprocally (P < .005 at stage 1 and 2, P < .05 at stage 3). The ADC increase ratio was 35.7 ± 17.4%, 27.0 ± 12.8%, and 20.2 ± 16.6% at stage 1, 2, and 3, respectively. The parotid ADC was negatively correlated to the parotid volume (R = -0.509; P < .001). The parotid ADC was positively associated with the radiation dose significantly (R² = 0.212; P = .0001) and was negatively associated with RMI significantly (R² = 0.203; P = .00096) significantly. Multiple regression analysis further showed that the post-RT parotid ADC was related to the radiation dose and RMI significantly (R² = 0.3580; P < .0001). At MR stage 3, the parotid volume was negatively associated with the dry mouth grade significantly (R² = 0.473; P < .0001), while the parotid ADC was positively associated with the dry mouth grade significantly (R² = 0.288; P = .015).

Conclusion

Our pilot study successfully demonstrates the concurrent changes and temporal evolution of parotid volume and parotid ADC quantitatively in NPC patients treated by IMRT. Our results suggest that the reduction of parotid volume and increase of parotid ADC are dominated by the effect of acinar loss rather than edema at early to intermediate phases and the following recovery of parotid volume and ADC toward the baseline values might reflect the acinar regeneration of parotid glands.     

Inhibition of Propofol Anesthesia on Functional Connectivity between LFPs in PFC during Rat Working Memory Task

Working memory (WM) refers to the temporary storage and manipulation of information necessary for performance of complex cognitive tasks. There is a growing interest in whether and how propofol anesthesia inhibits WM function. The aim of this study is to investigate the possible inhibition mechanism of propofol anesthesia based on the functional connections of multi-local field potentials (LFPs) and behavior during WM tasks. Adult SD rats were randomly divided into 3 groups: pro group (0.5 mg·kg⁻¹·min⁻¹,2 h), PRO group (0.9 mg·kg⁻¹·min⁻¹, 2 h) and control group. The experimental data were 16-channel LFPs obtained at prefrontal cortex with implanted microelectrode array in SD rats during WM tasks in Y-maze at 24, 48, 72, 96, 120 hours (day 1-day 5) after propofol anesthesia, and the behavior results of WM were recoded at the same time. Directed transfer function (DTF) method was applied to analyze the connections among LFPs directly. Furthermore, the causal networks were identified by DTF. The clustering coefficient (C), network density (D) and global efficiency (E_global) were selected to describe the functional connectivity quantitatively. The results show that: comparing with the control group, the LFPs functional connectivity in pro group were no significantly difference (p>0.05); the connectivity in PRO group were significantly decreased (p<0.05 at 24 hours, p<0.05 at 48 hours), while no significant difference at 72, 96 and 120 hours for rats (p>0.05), which were consistent with the behavior results. These findings could lead to improved understanding the mechanism of inhibition of anesthesia on WM functions from the view of connections among LFPs.     

Effect of ATP on the Calcium Efflux in Dialyzed Squid Giant Axons

Dialysis perfusion technique makes it possible to control the internal composition of squid giant axons. Calcium efflux has been studied in the presence and in the virtual absence (<5 µM) of ATP. The mean calcium efflux from axons dialyzed with 0.3 µM ionized calcium, [ATP]_i > 1,000 µM, and bathed in artificial seawater (ASW) was 0.24 ± 0.02 pmol·cm^-2·s^-1 (P/CS) (n = 8) at 22°C. With [ATP]_i < 5 µM the mean efflux was 0.11 ± 0.01 P/CS (n = 15). The curve relating calcium efflux to [ATP]_i shows a constant residual calcium efflux in the range of 1–100 µM [ATP]_i. An increase of the calcium efflux is observed when [ATP]_i is >100 µM and saturates at [ATP]_i > 1,000 µM. The magnitude of the ATP-dependent fraction of the calcium efflux varies with external concentrations of Na⁺, Ca⁺⁺, and Mg⁺⁺. These results suggest that internal ATP changes the affinity of the calcium transport system for external cations.     

Obesity Is Associated with Lower Coronary Microvascular Density

Background

Obesity is associated with diastolic dysfunction, lower maximal myocardial blood flow, impaired myocardial metabolism and increased risk of heart failure. We examined the association between obesity, left ventricular filling pressure and myocardial structure.

Methods

We performed histological analysis of non-ischemic myocardium from 57 patients (46 men and 11 women) undergoing coronary artery bypass graft surgery who did not have previous cardiac surgery, myocardial infarction, heart failure, atrial fibrillation or loop diuretic therapy.

Results

Non-obese (body mass index, BMI, ≤30 kg/m², n=33) and obese patients (BMI >30 kg/m², n=24) did not differ with respect to myocardial total, interstitial or perivascular fibrosis, arteriolar dimensions, or cardiomyocyte width. Obese patients had lower capillary length density (1145±239, mean±SD, vs. 1371±333 mm/mm³, P=0.007) and higher diffusion radius (16.9±1.5 vs. 15.6±2.0 μm, P=0.012), in comparison with non-obese patients. However, the diffusion radius/cardiomyocyte width ratio of obese patients (0.73±0.11 μm/μm) was not significantly different from that of non-obese patients (0.71±0.11 μm/μm), suggesting that differences in cardiomyocyte width explained in part the differences in capillary length density and diffusion radius between non-obese and obese patients. Increased BMI was associated with increased pulmonary capillary wedge pressure (PCWP, P<0.0001), and lower capillary length density was associated with both increased BMI (P=0.043) and increased PCWP (P=0.016).

Conclusions

Obesity and its accompanying increase in left ventricular filling pressure were associated with lower coronary microvascular density, which may contribute to the lower maximal myocardial blood flow, impaired myocardial metabolism, diastolic dysfunction and higher risk of heart failure in obese individuals.     

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.     

Pesticidal and pest repellency activities of a plant derived triterpenoid 2α,3β,21β,23,28-penta hydroxyl 12-oleanene against Tribolium castaneum

Background

Tribolium castaneum (Herbst) is a major pest of stored grain-based products, and cause severe damage to cereal grains throughout the world. The present investigation was aimed to determine the pesticidal and pest repellent activities of 2α,3β,21β,23,28-penta hydroxyl 12-oleanene against T. castaneum. The compound 2α,3β,21β,23,28-penta hydroxyl 12-oleanene is a triterpenoid which was isolated from the roots of Laportea crenulata Gaud. Surface film technique was used for pesticidal screening, whereas, pest repellency property of the triterpenoid was determined by filter paper disc method.

Results

At 24 hours of exposure duration, significant mortality records (80% and 86%) were observed at doses 0.88 and 1.77 mg/cm². No significant change in mortality records was observed when duration of exposure was increased up to 48 hours. The triterpenoid showed significant repellency activity at doses 0.47 and 0.94 mg/cm².

Conclusion

These data suggest that the triterpenoid 2α,3β,21β,23,28-penta hydroxyl 12-oleanene possess both pesticidal and pest repellency activities against T. castaneum and can be used in controlling the pest of grain-based products.

Electronic supplementary material

The online version of this article (doi:10.1186/0717-6287-47-68) contains supplementary material, which is available to authorized users.     

DJ-1 protects against cell death following acute cardiac ischemia–reperfusion injury

Novel therapeutic targets are required to protect the heart against cell death from acute ischemia–reperfusion injury (IRI). Mutations in the DJ-1 (PARK7) gene in dopaminergic neurons induce mitochondrial dysfunction and a genetic form of Parkinson''s disease. Genetic ablation of DJ-1 renders the brain more susceptible to cell death following ischemia–reperfusion in a model of stroke. Although DJ-1 is present in the heart, its role there is currently unclear. We sought to investigate whether mitochondrial DJ-1 may protect the heart against cell death from acute IRI by preventing mitochondrial dysfunction. Overexpression of DJ-1 in HL-1 cardiac cells conferred the following beneficial effects: reduced cell death following simulated IRI (30.4±4.7% with DJ-1 versus 52.9±4.7% in control; n=5, P<0.05); delayed mitochondrial permeability transition pore (MPTP) opening (a critical mediator of cell death) (260±33 s with DJ-1 versus 121±12 s in control; n=6, P<0.05); and induction of mitochondrial elongation (81.3±2.5% with DJ-1 versus 62.0±2.8% in control; n=6 cells, P<0.05). These beneficial effects of DJ-1 were absent in cells expressing the non-functional DJ-1^L166P and DJ-1^Cys106A mutants. Adult mice devoid of DJ-1 (KO) were found to be more susceptible to cell death from in vivo IRI with larger myocardial infarct sizes (50.9±3.5% DJ-1 KO versus 41.1±2.5% in DJ-1 WT; n≥7, P<0.05) and resistant to cardioprotection by ischemic preconditioning. DJ-1 KO hearts showed increased mitochondrial fragmentation on electron microscopy, although there were no differences in calcium-induced MPTP opening, mitochondrial respiratory function or myocardial ATP levels. We demonstrate that loss of DJ-1 protects the heart from acute IRI cell death by preventing mitochondrial dysfunction. We propose that DJ-1 may represent a novel therapeutic target for cardioprotection.     

Extracellular Adenosine Formation by Ecto-5’-Nucleotidase (CD73) Is No Essential Trigger for Early Phase Ischemic Preconditioning

Background

Adenosine is a powerful trigger for ischemic preconditioning (IPC). Myocardial ischemia induces intracellular and extracellular ATP degradation to adenosine, which then activates adenosine receptors and elicits cardioprotection. Conventionally extracellular adenosine formation by ecto-5’-nucleotidase (CD73) during ischemia was thought to be negligible compared to the massive intracellular production, but controversial reports in the past demand further evaluation. In this study we evaluated the relevance of ecto-5’-nucleotidase (CD73) for infarct size reduction by ischemic preconditioning in in vitro and in vivo mouse models of myocardial infarction, comparing CD73^-/- and wild type (WT) mice.

Methods and Results

3x5 minutes of IPC induced equal cardioprotection in isolated saline perfused hearts of wild type (WT) and CD73^-/- mice, reducing control infarct sizes after 20 minutes of ischemia and 90 minutes of reperfusion from 46 ± 6.3% (WT) and 56.1 ± 7.6% (CD73^-/-) to 26.8 ± 4.7% (WT) and 25.6 ± 4.7% (CD73^-/-). Coronary venous adenosine levels measured after IPC stimuli by high-pressure liquid chromatography showed no differences between WT and CD73^-/- hearts. Pharmacological preconditioning of WT hearts with adenosine, given at the measured venous concentration, was evenly cardioprotective as conventional IPC. In vivo, 4x5 minutes of IPC reduced control infarct sizes of 45.3 ± 8.9% (WT) and 40.5 ± 8% (CD73^-/-) to 26.3 ± 8% (WT) and 22.6 ± 6.6% (CD73^-/-) respectively, eliciting again equal cardioprotection. The extent of IPC-induced cardioprotection in male and female mice was identical.

Conclusion

The infarct size limiting effects of IPC in the mouse heart in vitro and in vivo are not significantly affected by genetic inactivation of CD73. The ecto-5’-nucleotidase derived extracellular formation of adenosine does not contribute substantially to adenosine’s well known cardioprotective effect in early phase ischemic preconditioning.     

Longitudinal Strain Is a Marker of Microvascular Obstruction and Infarct Size in Patients with Acute ST-Segment Elevation Myocardial Infarction

Objectives

We assessed the value of speckle tracking imaging performed early after a first ST-segment elevation myocardial infarction (STEMI) in order to predict infarct size and functional recovery at 3-month follow-up.

Methods

44 patients with STEMI who underwent revascularization within 12 h of symptom onset were prospectively enrolled. Echocardiography was performed 3.9±1.2 days after myocardial reperfusion, assessing circumferential (CGS), radial (RGS), and longitudinal global (GLS) strains. Late gadolinium-enhanced cardiac magnetic imaging (CMR), for assessing cardiac function, infarct size, and microvascular obstruction (MVO), was conducted 5.6±2.5 days and 99.4±4.6 days after myocardial reperfusion.

Results

GLS was evaluable in 97% of the patients, while CGS and RGS could be assessed in 85%. Infarct size significantly correlated with GLS (R = 0.601, p<0.001), RGS (R = −0.405, p = 0.010), CGS (R = 0.526, p = 0.001), ejection fraction (R = −0.699, p<0.001), wall motion score index (WMSI) (R = 0.539, p = 0.001), and left atrial volume (R = 0.510, p<0.001). Baseline ejection fraction and GLS were independent predictors of 3-month infarct size. MVO mass significantly correlated with GLS (R = 0.376, p = 0.010), WMSI (R = 0.387, p = 0.011), and ejection fraction (R = −0.389, p = 0.011). In multivariate analysis, GLS was the only independent predictor of MVO mass (p = 0.015). Longitudinal strain >−6.0% within the infarcted area exhibited 96% specificity and 61% sensitivity for predicting the persistence of akinesia (≥3 segments) at 3-month follow-up.

Conclusions

Speckle-tracking strain imaging performed early after a STEMI is easy-to-use as a marker for persistent akinetic territories at 3 months. In addition, GLS correlated significantly with MVO and final infarct size, both parameters being relevant post-MI prognostic factors, usually obtained via CMR.     

Determination of the Pressure Drop Across an Arterial Stenosis Utilizing Angiocinedensitometry

A method is presented for an evaluation of the hemodynamic significance of a stenotic lesion in the arterial tree.Twenty-three patients were examined with arteriosclerosis obliterans and intermittent claudication of the same severity. Flow velocity data obtained by angiodensitometry and viscosity values calculated from the hematocrit were inserted into the Poiseuille''s flow formula to obtain the pressure drop across a stenotic lesion in the left external iliac artery. By the same way, the pressure gradient was calculated in 33 “normal” subjects.The normal pressure gradient along the external iliac artery varied between 23 to 110 dynes/cm² (52 ± 24 dynes/cm² for mean and S.d), and the normal resistance to flow was 6.08 ± 4.1 dyne sec/cm⁵).Stenotic lesions of similar dimensions gave widely varying pressure drops (114-4,736 dynes/cm²) (mean and S.d 1,309 ± 1,224 dynes/cm²) indicating a difference in the hemodynamic significance of the various lesions. These values were significantly different (p(t) < 0.001) from the normal values. The resistance across these stenotic lesions ranged between 21 to 768 dyne sec/cm⁵ (196 ± 192 dyne sec/cm⁵) for the mean and S.d and this was significantly different from the normal group; p(t) < 0.001.Direct measurement of blood viscosity coupled with angiocinedensitometry at rest and after forced vasodilatation should provide an accurate means of determining the relative significance of a stenotic lesion and distal vessel disease in a given patient on blood flow to the leg.     

The Novel Mas agonist,CGEN-856S,Attenuates Isoproterenol-Induced Cardiac Remodeling and Myocardial Infarction Injury in Rats

CGEN-856S is a novel Mas agonist. Herein, we examined the effects of this peptide on isoproterenol (ISO)-induced cardiac remodeling and myocardial infarction (MI) injury. We also sought to determine whether CGEN-856S activates the underlying mechanisms related to Mas receptor activation. Heart hypertrophy and fibrosis were induced by ISO (2 mg·kg⁻¹·day⁻¹) in Wistar rats. After a 7-day treatment period with CGEN-856S (90 µg·kg⁻¹·day⁻¹) or vehicle, the cardiomyocyte diameter was evaluated in left ventricular sections stained with hematoxylin and eosin, and immunofluorescence labeling and quantitative confocal microscopy were used to quantify the deposition of type I and III collagen and fibronectin in the left ventricles. MI was induced by coronary artery ligation, and CGEN-856S (90 µg·kg⁻¹·day⁻¹) or saline was administered for 14 days. The Langendorff technique was used to evaluate cardiac function, and left ventricular sections were stained with Masson’s trichrome dye to quantify the infarct area. Using Chinese hamster ovary cells stably transfected with Mas cDNA, we evaluated whether CGEN-856S alters AKT and endothelial nitric oxide synthase (eNOS) phosphorylation. CGEN-856S reduced the degree of ISO-induced hypertrophy (13.91±0.17 µm vs. 12.41±0.16 µm in the ISO+CGEN-856S group). In addition, the Mas agonist attenuated the ISO-induced increase in collagen I, collagen III, and fibronectin deposition. CGEN-856S markedly attenuated the MI-induced decrease in systolic tension, as well as in +dT/dt and -dT/dt. Furthermore, CGEN-856S administration significantly decreased the infarct area (23.68±2.78% vs. 13.95±4.37% in the MI+CGEN-856S group). These effects likely involved the participation of AKT and NO, as CGEN-856S administration increased the levels of p-AKT and p-eNOS. Thus, our results indicate that CGEN-856S exerts cardioprotective effects on ISO-induced cardiac remodeling and MI-mediated heart failure in rats through a mechanism likely involving the eNOS/AKT pathway.     

Subdose of human chorionic gonadotropin applied at the Hou Hai acupoint on follicular dynamics and luteal development in donkeys

The objective of this study was to evaluate the effects of an hCG subdose applied at the Hou Hai acupoint as an ovulation inducer in donkeys. Eleven donkeys were distributed in randomized blocks in T1 = application of 1,500 IU of hCG intravenous (IV); T2 = 450 IU of hCG applied at the false acupoint (IV), and T3 = 450 IU of hCG applied at the Hou Hai acupoint. There was no difference (P > 0.05) between the treatments regarding the mean diameter of the pre-ovulatory follicle (34.5 ± 1.3 mm), the ovulation rate (96.97%), the interval between induction and ovulation (58.07 ± 16.82 h), the mean diameter of the CL (D0 = 23.0 ± 0.6; D2 = 27.7 ± 1.9 and D8 = 28.2 ± 0.8mm), and serum P₄ concentrations (10.50 ± 2.99 ng.mL^-1). The application of 450 IU of hCG at the Hou Hai acupoint increased ovulation rate (72.73%) more than 48 h after induction (P = 0.03) and a larger diameter of the CL on D4 (30.7 ± 5.1 mm) (P = 0.04). The vascularization area of the CL on D8, obtained by minimum number of colored pixel (NCP), was greater (P < 0.05) in the donkeys that received 1,500 IU of IV hCG (T1, 41.91 ± 1.17), and we found a positive correlation (P < 0.05) between mean NCP and P₄ concentration in the donkeys that received 450 IU of hCG IV at the false acupoint (T2) or at the Hou Hai acupoint (T3). The application of 450 IU of hCG by IV route at the false acupoint or the Hou Hai acupoint was sufficient to induce ovulation in donkeys, demonstrating that the average dosage commonly used for this species is too high.     

Blockade of Thrombopoietin Reduces Organ Damage in Experimental Endotoxemia and Polymicrobial Sepsis

Background and Purpose

Thrombopoietin (TPO), a growth factor primarily involved in thrombopoiesis may also have a role in the pathophysiology of sepsis. In patients with sepsis, indeed, TPO levels are markedly increased, with disease severity being the major independent determinant of TPO concentrations. Moreover, TPO increases and correlates with ex vivo indices of platelet activation in patients with burn injury upon sepsis development, and may contribute to depress cardiac contractility in septic shock. Still, the role of TPO in sepsis pathophysiology remains controversial, given the protective role of TPO in other experimental disease models, for instance in doxorubicin-induced cardiotoxicity and myocardial ischemia/reperfusion injury. The aim of our study was to define the contribution of TPO in the development of organ damage induced by endotoxemia or sepsis, and to investigate the effects of inhibiting TPO in these conditions.

Methods

We synthesized a chimeric protein able to inhibit TPO, mTPOR-MBP, and studied its effect in two murine experimental models, acute endotoxemia and cecal ligation and puncture (CLP) model.

Results

In both models, TPO levels markedly increased, from 289.80±27.87 pg/mL to 465.60±45.92 pg/mL at 3 hours in the LPS model (P<0.01), and from 265.00±26.02 pg/mL to 373.70±26.20 pg/mL in the CLP model (P<0.05), respectively. Paralleling TPO levels, also platelet-monocyte aggregates increased, from 32.86±2.48% to 46.13±1.39% at 3 hours in the LPS model (P<0.01), and from 43.68±1.69% to 56.52±4.66% in the CLP model (P<0.05). Blockade of TPO by mTPOR-MBP administration reduced histological damage in target organs, namely lung, liver, and gut. In particular, neutrophil infiltration and lung septal thickening were reduced from a score of 1.86±0.34 to 0.60±0.27 (P<0.01) and from 1.43±0.37 to 0.40±0.16 (P<0.05), respectively, in the LPS model at 3 hours, and from a score of 1.75±0.37 to 0.38±0.18 (P<0.01) and from 1.25±0.31 to 0.13±0.13 (P<0.001), respectively, in the CLP model. Similarly, the number of hepatic microabscesses was decreased from 14.14±1.41 to 3.64±0.56 in the LPS model at 3 hours (P<0.001), and from 1.71±0.29 to 0.13±0.13 in the CLP model (P<0.001). Finally, the diameter of intestinal villi decreased from 90.69±3.95 μm to 70.74±3.60 μm in the LPS model at 3 hours (P<0.01), and from 74.29±4.29 μm to 57.50±1.89 μm in the CLP model (P<0.01). This protective effect was associated with the blunting of the increase in platelet-monocyte adhesion, and, on the contrary, with increased platelet-neutrophil aggregates in the circulation, which may be related to decreased neutrophil sequestration into the inflamed tissues. Conversely, circulating cytokine levels were not significantly changed, in both models, by mTPOR-MBP administration.

Conclusion

Our results demonstrate that TPO participates in the development of organ damage induced by experimental endotoxemia or polymicrobial sepsis via a mechanism involving increased platelet-leukocyte adhesion, but not cytokine release, and suggest that blocking TPO may be useful in preventing organ damage in patients affected by systemic inflammatory response or sepsis.