Morphine-Induced Preconditioning: Involvement of Protein Kinase A and Mitochondrial Permeability Transition Pore

Background

Morphine induces myocardial preconditioning (M-PC) via activation of mitochondrial large conductance Ca²⁺-sensitive potassium (mK_Ca) channels. An upstream regulator of mK_Ca channels is protein kinase A (PKA). Furthermore, mK_Ca channel activation regulates mitochondrial bioenergetics and thereby prevents opening of the mitochondrial permeability transition pore (mPTP). Here, we investigated in the rat heart in vivo whether 1) M-PC is mediated by activation of PKA, and 2) pharmacological opening of the mPTP abolishes the cardioprotective effect of M-PC and 3) M-PC is critically dependent on STAT3 activation, which is located upstream of mPTP within the signalling pathway.

Methods

Male Wistar rats were randomised to six groups (each n = 6). All animals underwent 25 minutes of regional myocardial ischemia and 120 minutes of reperfusion. Control animals (Con) were not further treated. Morphine preconditioning was initiated by intravenous administration of 0.3 mg/kg morphine (M-PC). The PKA blocker H-89 (10 μg/kg) was investigated with and without morphine (H-89+M-PC, H-89). We determined the effect of mPTP opening with atractyloside (5 mg/kg) with and without morphine (Atr+M-PC, Atr). Furthermore, the effect of morphine on PKA activity was tested in isolated adult rat cardiomyocytes. In further experiments in isolated hearts we tested the protective properties of morphine in the presence of STAT3 inhibition, and whether pharmacological prevention of the mPTP-opening by cyclosporine A (CsA) is cardioprotective in the presence of STAT3 inhibition.

Results

Morphine reduced infarct size from 64±5% to 39±9% (P<0.05 vs. Con). H-89 completely blocked preconditioning by morphine (64±9%; P<0.05 vs. M-PC), but H-89 itself had not effect on infarct size (61±10%; P>0.05 vs. Con). Also, atractyloside abolished infarct size reduction of morphine completely (65±9%; P<0.05 vs. M-PC) but had no influence on infarct size itself (64±5%; P>0.05 vs. Con). In isolated hearts STAT3 inhibitor Stattic completely abolished morphine-induced preconditioning. Administration of Stattic and mPTP inhibitor cyclosporine A reduced infarct size to 31±6% (Stat+CsA, P<0.05 vs. Con). Cyclosporine A alone reduced infarct size to 26±7% (CsA P<0.05 vs. Con). In cardiomyocytes, PKA activity was increased by morphine.

Conclusion

Our data suggest that morphine-induced cardioprotection is mediated by STAT3-activation and inhibition of mPTP, with STA3 located upstream of mPTP. There is some evidence that protein kinase A is involved within the signalling pathway.     

Temporal Changes in Sequential Quantitative Thallium-201 Imaging Following Myocardial Infarction in Dogs: Comparison of Four and Twenty-Four Hour Infarct Images

Thallium-201 (²⁰¹T1) myocardial perfusion imaging allows definition of zones of myocardial infarction and ischemia. The temporal changes in sequential quantitative ²⁰¹T1 infarct imaging was studied 4 and 24 hours in dogs subjected to closed-chest anterior wall myocardial infarction. A temporal decrease in ²⁰¹T1 imaged infarct areas was noted in 10 of 13 animals. In no animal did the infarct area increase. The imaged infarct area decreased by an average of 30% from 12.9 ± 6.2 cm² at 4 hours to 9.1 ± 5.1 cm² at 24 hours (p < 0.001), and involved 34 ± 16% of the total ²⁰¹T1 left ventricular distribution at 4 hours and 22 ± 14% at 24 hours (p < 0.001). The magnitude of temporal change in imaged infarct area was not predicted by initial image defect or final histopathologic infarct size. Thus, the results of ²⁰¹T1 infarct imaging in the early period of infarction are clearly dependent upon the time at which the procedure is performed.     

Left ventricular assist device for end-stage heart failure: results of the first LVAD destination program in the Netherlands

PurposeMechanical circulatory support with a continuous-flow left ventricular assist device (LVAD) may be a valuable treatment in end-stage heart failure patients for an extended period of time. The purpose of this study was to evaluate the safety and efficacy of implantation of a continuous-flow LVAD in end-stage heart failure patients within the first destination program in the Netherlands.MethodsA third-generation LVAD was implanted in 16 heart failure patients (age 61 ± 8; 81 % male; left ventricular ejection fraction 20 ± 6 %) as destination therapy. All patients were ineligible for heart transplant. At baseline, 3 and 6 months, New York Heart Association (NYHA) functional class, quality-of-life and exercise capacity were assessed. Clinical adverse events were registered.ResultsSurvival at 30 days and 6 months was 88 and 75 %, respectively. In the postoperative phase, 6 (38 %) patients required continuous veno-venous haemofiltration for renal failure and 2 (13 %) patients required extracorporeal membrane oxygenation because of severe right ventricular failure. During follow-up, NYHA functional class and quality-of-life improved from 3.7 ± 0.1 to 2.3 ± 0.1 and 57 ± 5 to 23 ± 3 at 6 months (P < 0.001), respectively. The 6 min walking distance improved from 168 ± 42 m to 291 ± 29 m at 6 months (P = 0.001).ConclusionContinuous-flow LVAD therapy is a promising treatment for patients with end-stage heart failure ineligible for heart transplant.     

Impact of Acute Changes of Left Ventricular Contractility on the Transvalvular Impedance: Validation Study by Pressure-Volume Loop Analysis in Healthy Pigs

Background

The real-time and continuous assessment of left ventricular (LV) myocardial contractility through an implanted device is a clinically relevant goal. Transvalvular impedance (TVI) is an impedentiometric signal detected in the right cardiac chambers that changes during stroke volume fluctuations in patients. However, the relationship between TVI signals and LV contractility has not been proven. We investigated whether TVI signals predict changes of LV inotropic state during clinically relevant loading and inotropic conditions in swine normal heart.

Methods

The assessment of RVTVI signals was performed in anesthetized adult healthy anesthetized pigs (n = 6) instrumented for measurement of aortic and LV pressure, dP/dt_max and LV volumes. Myocardial contractility was assessed with the slope (Ees) of the LV end systolic pressure-volume relationship. Effective arterial elastance (Ea) and stroke work (SW) were determined from the LV pressure-volume loops. Pigs were studied at rest (baseline), after transient mechanical preload reduction and afterload increase, after 10-min of low dose dobutamine infusion (LDDS, 10 ug/kg/min, i.v), and esmolol administration (ESMO, bolus of 500 µg and continuous infusion of 100 µg·kg−1·min−1).

Results

We detected a significant relationship between ESTVI and dP/dtmax during LDDS and ESMO administration. In addition, the fluctuations of ESTVI were significantly related to changes of the Ees during afterload increase, LDDS and ESMO infusion.

Conclusions

ESTVI signal detected in right cardiac chamber is significantly affected by acute changes in cardiac mechanical activity and is able to predict acute changes of LV inotropic state in normal heart.     

Cardiac Effects of Attenuating Gsα - Dependent Signaling

Aims

Inhibition of β-adrenergic signalling plays a key role in treatment of heart failure. Gsα is essential for β-adrenergic signal transduction. In order to reduce side-effects of beta-adrenergic inhibition diminishing β-adrenergic signalling in the heart at the level of Gsα is a promising option.

Methods and Results

We analyzed the influence of Gsα on regulation of myocardial function and development of cardiac hypertrophy, using a transgenic mouse model (C57BL6/J mice) overexpressing a dominant negative Gsα-mutant under control of the α-MHC-promotor. Cardiac phenotype was characterized in vivo and in vitro and under acute and chronic β-adrenergic stimulation. At rest, Gsα-DN-mice showed bradycardia (602 ± 13 vs. 660 ± 17 bpm, p<0.05) and decreased dp/dt_max (5037 ± 546- vs. 6835 ± 505 mmHg/s, p = 0.02). No significant differences were found regarding ejection fraction, heart weight and cardiomyocyte size. β-blockade by propranolol revealed no baseline differences of hemodynamic parameters between wildtype and Gsα-DN-mice. Acute adrenergic stimulation resulted in decreased β-adrenergic responsiveness in Gsα-DN-mice. Under chronic adrenergic stimulation, wildtype mice developed myocardial hypertrophy associated with increase of LV/BW-ratio by 23% (4.4 ± 0.2 vs. 3.5 ± 0.1 mg/g, p<0.01) and cardiac myocyte size by 24% (14927 ± 442 px vs. 12013 ± 583 px, p<0.001). In contrast, both parameters were unchanged in Gsα-DN-mice after chronic isoproterenol stimulation.

Conclusion

Overexpression of a dominant negative mutant of Gsα leads to decreased β-adrenergic responsiveness and is protective against isoproterenol-induced hypertrophy. Thus, Gsα-DN-mice provide novel insights into β-adrenergic signal transduction and its modulation in myocardial overload and failure.     

Comparison of NOGA Endocardial Mapping and Cardiac Magnetic Resonance Imaging for Determining Infarct Size and Infarct Transmurality for Intramyocardial Injection Therapy Using Experimental Data

Objectives

We compared the accuracy of NOGA endocardial mapping for delineating transmural and non-transmural infarction to the results of cardiac magnetic resonance imaging (cMRI) with late gadolinium enhancement (LE) for guiding intramyocardial reparative substance delivery using data from experimental myocardial infarction studies.

Methods

Sixty domestic pigs underwent diagnostic NOGA endocardial mapping and cMRI-LE 60 days after induction of closed-chest reperfused myocardial infarction. The infarct size was determined by LE of cMRI and by delineation of the infarct core on the unipolar voltage polar map. The sizes of the transmural and non-transmural infarctions were calculated from the cMRI transmurality map using signal intensity (SI) cut-offs of>75% and>25% and from NOGA bipolar maps using bipolar voltage cut-off values of <0.8 mV and <1.9 mV. Linear regression analysis and Bland-Altman plots were used to determine correlations and systematic differences between the two images. The overlapping ratios of the transmural and non-transmural infarcted areas were calculated.

Results

Infarct size as determined by 2D NOGA unipolar voltage polar mapping correlated with the 3D cMRI-LE findings (r = 0.504, p<0.001) with a mean difference of 2.82% in the left ventricular (LV) surface between the two images. Polar maps of transmural cMRI and bipolar maps of NOGA showed significant association for determining of the extent of transmural infarction (r = 0.727, p<0.001, overlap ratio of 81.6±11.1%) and non-transmural infarction (r = 0.555, p<0.001, overlap ratio of 70.6±18.5%). NOGA overestimated the transmural scar size (6.81% of the LV surface) but slightly underestimated the size of the non-transmural infarction (−3.04% of the LV surface).

Conclusions

By combining unipolar and bipolar voltage maps, NOGA endocardial mapping is useful for accurate delineation of the targeted zone for intramyocardial therapy and is comparable to cMRI-LE. This may be useful in patients with contraindications for cMRI who require targeted intramyocardial regenerative therapy.     

Impact of Mitochondrial Ca2+-Sensitive Potassium (mBKCa) Channels in Sildenafil-Induced Cardioprotection in Rats

Background

Mitochondrial large-conductance Ca²⁺-sensitive potassium (mBK_Ca) channels are involved in myocardial ischemic preconditioning. Their role in sildenafil-induced cardioprotection is unknown. We investigated whether sildenafil-induced acute cardioprotection is mediated by activation of mBK_Ca channels in the rat heart in vitro.

Methods

Male Wistar rats (n = 8 per group) were randomized and anesthetized with pentobarbital (90 mg/kg). Hearts were isolated, mounted on a Langendorff system and perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. Hearts underwent 30 min of global ischemia followed by 60 min of reperfusion. At the end of the experiments infarct size was determined by TTC staining. In the control group rats were not further treated. Sildenafil (3 μM) was administered over 10 min before the beginning of ischemia. The mBK_Ca channel inhibitor paxilline (1 μM) was administered with and without sildenafil before the onset of ischemia. The pathway underlying sildenafil-induced cardioprotection was further investigated with the protein kinase G blocker KT5823 (1 μM). Myocardial cGMP concentration was measured by ELISA. Data (mean±SD) were analysed with a one and two-way analysis of variance as appropriate.

Results

In control animals infarct size was 52±8%. Sildenafil increased cGMP concentration and reduced infarct size to 35±6% (P<0.05 vs. control). Paxilline and KT5823 completely blocked sildenafil-induced cardioprotection (paxilline+sildenafil: 50±8%, KT5823+sildenafil: 45±8%; both P<0.05 vs. sildenafil). Functional heart parameters and coronary flow were not different between the study groups.

Conclusion

This study shows that in male rats protein kinase G-dependent opening of mBK_Ca channels plays a pivotal role in sildenafil-induced cardioprotection.     

Pronounced Effects of HERG-Blockers E-4031 and Erythromycin on APD,Spatial APD Dispersion and Triangulation in Transgenic Long-QT Type 1 Rabbits

Background

Prolongation of action potential duration (APD), increased spatial APD dispersion, and triangulation are major factors promoting drug-induced ventricular arrhythmia. Preclinical identification of HERG/I_Kr-blocking drugs and their pro-arrhythmic potential, however, remains a challenge. We hypothesize that transgenic long-QT type 1 (LQT1) rabbits lacking repolarizing I_Ks current may help to sensitively detect HERG/I_Kr-blocking properties of drugs.

Methods

Hearts of adult female transgenic LQT1 and wild type littermate control (LMC) rabbits were Langendorff-perfused with increasing concentrations of HERG/I_Kr-blockers E-4031 (0.001–0.1 µM, n = 9/7) or erythromycin (1–300 µM, n = 9/7) and APD, APD dispersion, and triangulation were analyzed.

Results

At baseline, APD was longer in LQT1 than in LMC rabbits in LV apex and RV mid. Erythromycin and E-4031 prolonged APD in LQT1 and LMC rabbits in all positions. However, erythromycin-induced percentaged APD prolongation related to baseline (%APD) was more pronounced in LQT1 at LV base-lateral and RV mid positions (100 µM, LQT1, +40.6±9.7% vs. LMC, +24.1±10.0%, p<0.05) and E-4031-induced %APD prolongation was more pronounced in LQT1 at LV base-lateral (0.01 µM, LQT1, +29.6±10.6% vs. LMC, +19.1±3.8%, p<0.05) and LV base-septal positions. Moreover, erythromycin significantly increased spatial APD dispersion only in LQT1 and increased triangulation only in LQT1 in LV base-septal and RV mid positions. Similarly, E-4031 increased triangulation only in LQT1 in LV apex and base-septal positions.

Conclusions

E-4031 and erythromycin prolonged APD and increased triangulation more pronouncedly in LQT1 than in LMC rabbits. Moreover, erythromycin increased APD dispersion only in LQT1, indicating that transgenic LQT1 rabbits could serve as sensitive model to detect HERG/I_Kr-blocking properties of drugs.     

Myocardial Integrated Backscatter in Obese Adolescents: Associations with Measures of Adiposity and Left Ventricular Deformation

Background

Myocardial fibrosis has been proposed to play an important pathogenetic role in left ventricular (LV) dysfunction in obesity. This study tested the hypothesis that calibrated integrated backscatter (cIB) as a marker of myocardial fibrosis is altered in obese adolescents and explored its associations with adiposity, LV myocardial deformation, and metabolic parameters.

Methods/Principal Findings

Fifty-two obese adolescents and 38 non-obese controls were studied with conventional and speckle tracking echocardiography. The average cIB of ventricular septum and LV posterior wall was measured. In obese subjects, insulin resistance as estimated by homeostasis model assessment (HOMA-IR) and glucose tolerance were determined. Compared with controls, obese subjects had significantly greater cIB of ventricular septum (-16.8±7.8 dB vs -23.2±7.8 dB, p<0.001), LV posterior wall (-20.5±5.6 dBvs -25.0±5.1 dB, p<0.001) and their average (-18.7±5.7 dB vs -24.1±5.0 dB, p<0.001). For myocardial deformation, obese subjects had significantly reduced LV longitudinal systolic strain rate (SR) (p = 0.045) and early diastolic SR (p = 0.015), and LV circumferential systolic strain (p = 0.008), but greater LV longitudinal late diastolic SR (p<0.001), and radial early (p = 0.037) and late (p = 0.002) diastolic SR than controls. For the entire cohort, myocardial cIB correlated positively with body mass index (r = 0.45, p<0.001) and waist circumference (r = 0.45, p<0.001), but negatively with LV circumferential systolic strain (r = -0.23, p = 0.03) and systolic SR (r = -0.25, p = 0.016). Among obese subjects, cIB tended to correlate with HOMA-IR (r = 0.26, p = 0.07).

Conclusion

Obese adolescents already exhibit evidence of increased myocardial fibrosis, which is associated with measures of adiposity and impaired LV circumferential myocardial deformation.     

Decrease in Plasma Cyclophilin A Concentration at 1 Month after Myocardial Infarction Predicts Better Left Ventricular Performance and Synchronicity at 6 Months: A Pilot Study in Patients with ST Elevation Myocardial Infarction

Background: Cyclophilin A (CyPA) concentration increases in acute coronary syndrome. In an animal model of acute myocardial infarction, administration of angiotensin-converting-enzyme inhibitor was associated with lower left ventricular (LV) CyPA concentration and improved LV performance. This study investigated the relationships between changes in plasma CyPA concentrations and LV remodeling in patients with ST-elevation myocardial infarction (STEMI).Methods and Results: We enrolled 55 patients who underwent percutaneous coronary intervention for acute STEMI. Plasma CyPA, matrix metalloproteinase (MMP), interleukin-6 and high-sensitivity C-reactive protein concentrations were measured at baseline and at one-month follow-up. Echocardiography was performed at baseline and at one-, three-, and six-month follow-up. Patients with a decrease in baseline CyPA concentration at one-month follow-up (n = 28) had a significant increase in LV ejection fraction (LVEF) (from 60.2 ± 11.5% to 64.6 ± 9.9%, p < 0. 001) and preserved LV synchrony at six months. Patients without a decrease in CyPA concentration at one month (n = 27) did not show improvement in LVEF and had a significantly increased systolic dyssynchrony index (SDI) (from 1.170 ± 0.510% to 1.637 ± 1.299%, p = 0.042) at six months. Multiple linear regression analysis showed a significant association between one-month CyPA concentration and six-month LVEF. The one-month MMP-2 concentration was positively correlated with one-month CyPA concentration and LV SDI.Conclusions: Decreased CyPA concentration at one-month follow-up after STEMI was associated with better LVEF and SDI at six months. Changes in CyPA, therefore, may be a prognosticator of patient outcome.     

Low tidal volume protects pulmonary vasomotor function from “second-hit” injury in acute lung injury rats

Background

Sepsis could induce indirect acute lung injury(ALI), and pulmonary vasomotor dysfunction. While low tidal volume is advocated for treatment of ALI patients. However, there is no evidence for low tidal volume that it could mitigate pulmonary vasomotor dysfunction in indirect ALI. Our study is to evaluate whether low tidal volume ventilation could protect the pulmonary vascular function in indirect lipopolysaccharide (LPS) induced acute lung injury rats.

Methods

An indirect ALI rat model was induced by intravenous infusion of LPS. Thirty rats (n = 6 in each group) were randomly divided into (1)Control group; (2) ALI group; (3) LV group (tidal volume of 6mL/kg); (4) MV group (tidal volume of 12mL/kg); (5)VLV group (tidal volume of 3mL/kg). Mean arterial pressure and blood gas analysis were monitored every 2 hours throughout the experiment. Lung tissues and pulmonary artery rings were immediately harvested after the rats were bled to be killed to detect the contents of endothelin-1 (ET-1), endothelial nitric oxide synthase (eNOS) and TNF-α. Acetylcholine (Ache)-induced endothelium-dependent and sodium nitroprusside (SNP)-induced endothelium-independent relaxation of isolated pulmonary artery rings were measured by tensiometry.

Results

There was no difference within groups concerning blood pressure, PaCO₂ and SNP-induced endothelium-independent relaxation of pulmonary artery rings. Compared with MV group, LV group significantly reduced LPS-induced expression of ET-1 level (113.79 ± 7.33pg/mL vs. 152.52 ± 12.75pg/mL, P < 0.05) and TNF-α (3305.09 ± 334.29pg/mL vs.4144.07 ± 608.21pg/mL, P < 0.05), increased the expression of eNOS (IOD: 15032.05 ± 5925.07 vs. 11454.32 ± 6035.47, P < 0.05). While Ache (10^-7mol/L-10^-4mol/L)-induced vasodilatation was ameliorated 30% more in LV group than in MV group.

Conclusions

Low tidal volume could protect the pulmonary vasodilative function during indirect ALI by decreasing vasoconstrictor factors, increasing expressions of vasodilator factors in pulmonary endothelial cells, and inhibiting inflammation injuries.     

Riociguat Reduces Infarct Size and Post-Infarct Heart Failure in Mouse Hearts: Insights from MRI/PET Imaging

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.     

Cardiac Restricted Overexpression of Membrane Type-1 Matrix Metalloproteinase Causes Adverse Myocardial Remodeling following Myocardial Infarction

The membrane type-1 matrix metalloproteinase (MT1-MMP) is a unique member of the MMP family, but induction patterns and consequences of MT1-MMP overexpression (MT1-MMPexp), in a left ventricular (LV) remodeling process such as myocardial infarction (MI), have not been explored. MT1-MMP promoter activity (murine luciferase reporter) increased 20-fold at 3 days and 50-fold at 14 days post-MI. MI was then induced in mice with cardiac restricted MT1-MMPexp (n = 58) and wild type (WT, n = 60). Post-MI survival was reduced (67% versus 46%, p < 0.05), and LV ejection fraction was lower in the post-MI MT1-MMPexp mice compared with WT (41 ± 2 versus 32 ± 2%,p < 0.05). In the post-MI MT1-MMPexp mice, LV myocardial MMP activity, as assessed by radiotracer uptake, and MT1-MMP-specific proteolytic activity using a specific fluorogenic assay were both increased by 2-fold. LV collagen content was increased by nearly 2-fold in the post-MI MT1-MMPexp compared with WT. Using a validated fluorogenic construct, it was discovered that MT1-MMP proteolytically processed the pro-fibrotic molecule, latency-associated transforming growth factor-1 binding protein (LTBP-1), and MT1-MMP-specific LTBP-1 proteolytic activity was increased by 4-fold in the post-MI MT1-MMPexp group. Early and persistent MT1-MMP promoter activity occurred post-MI, and increased myocardial MT1-MMP levels resulted in poor survival, worsening of LV function, and significant fibrosis. A molecular mechanism for the adverse LV matrix remodeling with MT1-MMP induction is increased processing of pro-fibrotic signaling molecules. Thus, a proteolytically diverse portfolio exists for MT1-MMP within the myocardium and likely plays a mechanistic role in adverse LV remodeling.     

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.     

Task-specific performance fatigability and the bilateral deficit during isokinetic leg extensions

Objectives:The purpose of the present study was to compare the fatigue-induced changes in performance fatigability, bilateral deficit, and patterns of responses for the electromyographic (EMG) and mechanomyographic (MMG) amplitude (AMP) and mean power frequency (MPF), during unilateral and bilateral maximal, fatiguing leg extensions.Methods:Nine men (Mean±SD; age =21.9±2.4 yrs; height =181.8±11.9 cm; body mass =85.8±6.2 kg) volunteered to perform 50 consecutive maximal, bilateral (BL), unilateral dominant (DL), and unilateral non-dominant (NL) isokinetic leg extensions at 180°·s^-1, on 3 separate days. Electromyographic and MMG signals from both vastus lateralis (VL) muscles were recorded. Repeated measures ANOVAs were utilized to examine mean differences in normalized force, EMG AMP, EMG MPF, MMG AMP, MMG MPF and the bilateral deficit.Results:The results demonstrated a Condition × Repetition interaction for normalized force (p=0.004, η²_p=0.222) and EMG MPF (p=0.034, η²_p=0.214) and main effects for Repetition for EMG AMP (p=0.019, η²_p=0.231), MMG AMP (p<0.001, η²_p=0.8550), MMG MPF (p=0.009, η²_p=0.252), and the bilateral deficit (p<0.001, η²_p=0.366).Conclusions:The findings demonstrated less performance fatigability during the BL than the unilateral tasks, likely due to a reduced relative intensity via interhemispheric inhibition that attenuated the development of excitation-contraction coupling failure during the BL task.     

Association between Myocardial Triglyceride Content and Cardiac Function in Healthy Subjects and Endurance Athletes

Ectopic fat accumulation plays important roles in various metabolic disorders and cardiovascular diseases. Recent studies reported that myocardial triglyceride (TG) content measured by proton magnetic resonance spectroscopy (¹H-MRS) is associated with aging, diabetes mellitus, and cardiac dysfunction. However, myocardial TG content in athletes has not yet been investigated. We performed ¹H-MRS and cardiac magnetic resonance imaging in 10 male endurance athletes and 15 healthy male controls. Serum markers and other clinical parameters including arterial stiffness were measured. Cardiopulmonary exercise testing was also performed. There were no significant differences in clinical characteristics including age, anthropometric parameters, blood test results, or arterial stiffness between the two groups. Peak oxygen uptakes, end–diastolic volume (EDV), end–systolic volume (ESV), left ventricular (LV) mass, peak ejection rates and peak filling rates were significantly higher in the athlete group than in the control group (all P<0.02). Myocardial TG content was significantly lower in the athlete group than in the control group (0.60±0.20 vs. 0.89±0.41%, P<0.05). Myocardial TG content was negatively correlated with EDV (r = −0.47), ESV (r = −0.64), LV mass (r = −0.44), and epicardial fat volume (r = 0.47) (all P<0.05). In conclusion, lower levels of myocardial TG content were observed in endurance athletes and were associated with morphological changes related to physiological LV alteration in athletes, suggesting that metabolic imaging for measurement of myocardial TG content by ¹H-MRS may be a useful technique for noninvasively assessing the “athlete’s heart”.     

Inactivation of Enteric Adenovirus and Feline Calicivirus by Chlorine Dioxide

Chlorine dioxide (ClO₂) inactivation experiments were conducted with adenovirus type 40 (AD40) and feline calicivirus (FCV). Experiments were carried out in buffered, disinfectant demand-free water under high- and low-pH and -temperature conditions. Ct values (the concentration of ClO₂ multiplied by contact time with the virus) were calculated directly from bench-scale experiments and from application of the efficiency factor Hom (EFH) model. AD40 Ct ranges for 4-log inactivation (Ct_99.99%) at 5°C were >0.77 to <1.53 mg/liter × min and >0.80 to <1.59 mg/liter × min for pH 6 and 8, respectively. For 15°C AD40 experiments, >0.49 to <0.74 mg/liter × min and <0.12 mg/liter × min Ct_99.99% ranges were observed for pH 6 and 8, respectively. FCV Ct_99.99% ranges for 5°C experiments were >20.20 to <30.30 mg/liter × min and >0.68 mg/liter × min for pH 6 and 8, respectively. For 15°C FCV experiments, Ct_99.99% ranges were >4.20 to <6.72 and <0.18 mg/liter × min for pH 6 and 8, respectively. Viral inactivation was higher at pH 8 than at pH 6 and at 15°C than at 5°C. Comparison of Ct values and inactivation curves demonstrated that the EFH model described bench-scale experiment data very well. Observed bench-scale Ct_99.99% ranges and EFH model Ct_99.99% values demonstrated that FCV is more resistant to ClO₂ than AD40 for the conditions studied. U.S. Environmental Protection Agency guidance manual Ct_99.99% values are higher than Ct_99.99% values calculated from bench-scale experiments and from EFH model application.     

Cardiac Mechanics and Ventricular Twist by Three-Dimensional Strain Analysis in Relation to B-Type Natriuretic Peptide as a Clinical Prognosticator for Heart Failure Patients

BackgroundThree dimensional (3D) echocardiography-derived measurements of myocardial deformation and twist have recently advanced as novel clinical tools. However, with the exception of left ventricular ejection fraction and mass quantifications in hypertension and heart failure populations, the prognostic value of such imaging techniques remains largely unexplored.MethodsWe studied 200 subjects (mean age: 60.2±16 years, 54% female, female n = 107) with known hypertension (n = 51), diastolic heart failure (n = 61), or systolic heart failure (n = 30), recruited from heart failure outpatient clinics. Fifty-eight healthy volunteers were used as a control group. All participants underwent 3D-based myocardial deformation and twist analysis (Artida, Toshiba Medical Systems, Tokyo, Japan). We further investigated associations between these measures and brain natriuretic peptide levels and clinical outcomes.ResultsThe global 3D strain measurements of the healthy, hypertension, diastolic heart failure, and systolic heart failure groups were 28.03%, 24.43%, 19.70%, and 11.95%, respectively (all p<0.001). Global twist measurements were estimated to be 9.49°, 9.77°, 8.32°, and 4.56°, respectively. We observed significant differences regarding 3D-derived longitudinal, radial, and global 3D strains between the different disease categories (p<0.05), even when age, gender, BMI and heart rate were matched. In addition, 3D-derived longitudinal, circumferential, and 3D strains were all highly correlated with brain natriuretic peptide levels (p<0.001). At a mean 567.7 days follow-up (25^th–75^th IQR: 197–909 days), poorer 3D-derived longitudinal, radial, and global 3D strain measurements remained independently associated with a higher risk of cardiovascular related death or hospitalization due to heart failure, after adjusting for age, gender, and left ventricular ejection fraction (all p<0.05).Conclusions3D-based strain analysis may be a feasible and useful diagnostic tool for discriminating the extent of myocardial dysfunction. Furthermore, it is able to provide a prognostic value beyond traditional echocardiographic parameters in terms of ejection fraction.     

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.     

Anti-Inflammatory and Cardioprotective Effects of Tadalafil in Diabetic Mice

Background

Insulin resistance impairs nitric oxide (NO) bioavailability and obesity promotes a state of chronic inflammation and damages the vascular endothelium. Phosphodiesterase-5 inhibitors restore NO signaling and may reduce circulating inflammatory markers, and improve metabolic parameters through a number of mechanisms. We hypothesized that daily administration of the PDE-5 inhibitor, tadalafil (TAD) will attenuate inflammation, improve fasting plasma glucose and triglyceride levels, body weight, and reduce infarct size after ischemia/reperfusion injury in obese, diabetic mice.

Methods

Twenty leptin receptor null (db/db) mice underwent treatment with TAD (1 mg/Kg) or 10% DMSO for 28 days. Body weight and fasting plasma glucose levels were determined weekly. Upon completion, hearts were isolated and subjected to 30 min global ischemia followed by 60 min reperfusion in a Langendorff model. Plasma samples were taken for cytokine analysis and fasting triglyceride levels. Infarct size was measured using computer morphometry of tetrazolium stained sections. Additionally, ventricular cardiomyocytes were isolated and subjected to 40 min of simulated ischemia and reoxygenation. Necrosis was determined using trypan blue exclusion and LDH release assay and apoptosis was assessed by TUNEL assay after 1 h or 18 h of reoxygenation, respectively.

Results

Treatment with TAD caused a reduction in infarct size in the diabetic heart (23.2±1.5 vs. 47.8±3.7%, p<0.01, n = 6/group), reduced fasting glucose levels (292±31.8 vs. 511±19.3 mg/dL, p<0.001) and fasting triglycerides (43.3±21 vs. 129.7±29 mg/dL, p<0.05) as compared to DMSO, however body weight was not significantly reduced. Circulating tumor necrosis factor-α and interleukin-1β were reduced after treatment compared to control (257±16.51 vs. 402.3±17.26 and 150.8±12.55 vs. 264±31.85 pg/mL, respectively; P<0.001) Isolated cardiomyocytes from TAD-treated mice showed reduced apoptosis and necrosis.

Conclusion

We have provided the first evidence that TAD therapy ameliorates circulating inflammatory cytokines and chemokines in a diabetic animal model while improving fasting glucose levels and reducing infarct size following ischemia-reperfusion injury in the heart.