Perfused Rabbit Hearts Research Articles

Postconditioning and Controlled Reperfusion

Postconditioning and Controlled Reperfusion

Pharmacological modifications of the stretch-induced effects on ventricular fibrillation in perfused rabbit hearts

Stretch induces modifications in myocardial electrical and mechanical activity. Besides the effects of substances that block the stretch-activated channels, other substances could modulate the effects of stretch through different mechanisms that affect Ca(2+) handling by myocytes. Thirty-six Langendorff-perfused rabbit hearts were used to analyze the effects of the Na(+)/Ca(2+) exchanger blocker KB-R7943, propranolol, and the adenosine A(2) receptor antagonist SCH-58261 on the acceleration of ventricular fibrillation (VF) produced by acute myocardial stretching. VF recordings were obtained with two epicardial multiple electrodes before, during, and after local stretching in four experimental series: control (n = 9), KB-R7943 (1 microM, n = 9), propranolol (1 microM, n = 9), and SCH-58261 (1 microM, n = 9). Both the Na(+)/Ca(2+) exchanger blocker KB-R7943 and propranolol induced a significant reduction (P < 0.001 and P < 0.05, respectively) in the dominant frequency increments produced by stretching with respect to the control and SCH-58261 series (control = 49.9%, SCH-58261 = 52.1%, KB-R7943 = 9.5%, and propranolol = 12.5%). The median of the activation intervals, the functional refractory period, and the wavelength of the activation process during VF decreased significantly under stretch in the control and SCH-58261 series, whereas no significant variations were observed in the propranolol and KB-R7943 series, with the exception of a slight but significant decrease in the median of the fibrillation intervals in the KB-R7943 series. KB-R7943 and propranolol induced a significant reduction in the activation maps complexity increment produced by stretch with respect to the control and SCH-58261 series. In conclusion, the electrophysiological effects responsible for stretch-induced VF acceleration in the rabbit heart are reduced by the Na(+)/Ca(2+) exchanger blocker KB-R7943 and by propranolol but not by the adenosine A(2) receptor antagonist SCH-58261.

Comparisons of the release of vasodilator substances from left and right cardiac chambers of the isolated perfused rabbit heart: Implications for intraventricular thrombus formation

Prostacyclin (PgI 2) and endothelium-derived nitric oxide (EDNO) are produced by the arterial and venous endothelium. In addition to their vasodilator action on vascular smooth muscle, both act together to inhibit platelet aggregation and promote platelet disaggregation. EDNO also inhibits platelet adhesion to the endothelium. EDNO and PgI 2 have been shown to be released from the cultured endocardial cells. In this study, we examined the release of vasoactive substances from the intact endocardium by using isolated rabbit hearts perfused with physiological salt solution (95% O 2/5% CO 2, T = 37 °C). The right and left cardiac chambers were perfused through separate constant-flow perfusion loops (physiological salt solution, 8 ml min −1). Effluent from left and right cardiac, separately, was bioassayed on canine coronary artery smooth muscle, which had been contracted with prostaglandin F 2α_(2 × 10 −6 M) and no change in tension was exhibit. However, addition of calcium ionophore A23187 (10 −6 M) to the cardiac chambers’ perfusion line induced vasodilation of the bioassay coronary ring, 61.4 ± 7.4% versus 70.49 ± 6.1% of initial prostaglandin F 2α contraction for the left and right cardiac chambers perfusate, respectively (mean ± SEM, n = 10, p > 0.05). Production of vasodilator was blocked totally in the left heart but, only partially blocked in the right heart by adding indomethacin (10 −5 M) to the perfusate, respectively, 95.2 ± 2.2% versus 41.5 ± 4.8% (mean ± SEM, n = 10, p < 0.05). 6-Keto prostaglandin F 1α, measured in the endocardial superfusion effluent was also higher for the left cardiac chambers than for the right at the time of stimulation with the A23187, respectively, 25385.88 ± 5495 pg/ml ( n = 8) versus 13,132.45 ± 1839.82 pg/ml ( n = 8), ( p < 0.05). These results showed that cyclooxygenase pathway plays major role in generating vasoactive substances for the left cardiac chamber endocardium; while it is not the main pathway for the right ventricular endocardium at which EDNO and PgI 2 could act together and potentiate their antithrombogenic activities in isolated perfused rabbit heart. This may be an explanation for the intraventricular thrombus mostly seen in left ventricle rather than in right ventricle as a complication of myocardial infarction.

Alchornea Cordifolia (Euphorbiaceae), the Major Constituent of Antiasthmatic Herbal Formulations in Ghana, Stimulates β- adrenoceptors.

We have investigated the scientific basis for the traditional use of Alchornea cordifolia (Euphorbiaceae), by using isolated tissue models of smooth muscles in the laboratory. The aqueous extract of the leaves of Alchornea cordifolia produced a dose-related relaxation of the isolated rabbit duodenum in a manner similar to noradrenaline, adrenaline and isoprenaline, which were antagonised by propranolol (10μg/ml) by 63% but not phentolamine, suggesting a non-specific β- receptor-mediated activity. Additionally, the extract (12mg) evoked increased rate and force of contraction similar to isoprenaline on the Langendorff's isolated perfused rabbit heart preparation by 87% by an action on β1- receptors. These effects were blocked by propranolol (0.1mg), a nonspecific β-blocker. Carbachol-induced contractions on the guinea-pig tracheal smooth muscle were reversed by the extract in a cumulative dose manner, akin to the bronchodilator actions of isoprenaline. Similarly, 4μg/ml carbachol-elicited contractions on both pregnant and non-pregnant rat uterus were antagonised by the extract (50mg/ml) by 72% and 64% respectively. All together, our data suggest that the extract stimulates smooth muscles through activation of β-adrenoceptors in a manner similar to isoprenaline, which supports its traditional use as a constituent of anti-asthmatic and cough formulations, and in the control of spontaneous abortion. Journal of the Ghana Science Association Vol. 10 (2) 2008: pp. 1-11

Reduction of myocardial infarction by postischemic administration of the calpain inhibitor A-705253 in comparison to the Na+/H+ exchange inhibitor Cariporide® in isolated perfused rabbit hearts

The calpain inhibitor A-705253 and the Na(+)/H(+)-exchange inhibitor Cariporide were studied in isolated perfused rabbit hearts subjected to 60 min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid solely or in combination at the beginning of reperfusion. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were performed. Myocardial infarct size and area at risk (transiently not perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5-triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 72.7+/-4.0% of the area at risk in untreated controls, but was significantly smaller in the presence of the inhibitors. The largest effect was seen with 10(-6) m A-705253, which reduced the infarcted area to 49.2+/-4.1% of the area at risk, corresponding to a reduction of 33.6%. Cariporide at 10(-6) m reduced the infarct size to the same extent. The combination of both inhibitors, however, did not further improve cardioprotection. No statistical difference was observed between the experimental groups in coronary perfusion, left ventricular pressure, heart rate, and in the release of lactate dehydrogenase and creatin kinase from heart muscle.

Cardiac repolarization instability during normal postnatal development

Long QT syndrome is a disease characterized by abnormal lengthening of the QT interval and by sudden cardiac death. It is a disease of development, with the incidence of a sudden event increasing during childhood. Repolarization instability during postnatal development could make the substrate susceptible to a fatal arrhythmia. Dynamic changes in repolarization that occur on a beat-to-beat basis, known as alternans, are a hallmark of electrical instability. T-wave alternans (TWA) in the electrocardiogram correlates with arrhythmia risk and long-term survival in adults. We determined TWA properties longitudinally in vivo in 7 propofol-sedated New Zealand white rabbits using transesophageal pacing weekly from 2 to 10 weeks of age. Furthermore, TWA induction after the onset of rapid pacing was characterized in vitro in 6 infant (2 weeks) and 6 adolescent (7 weeks) isolated, arterially perfused rabbit hearts. In vivo , TWA amplitude was maximum at 2 weeks and declined with age. Isoproterenol increased TWA at 8 weeks (adolescence). In vitro , large-amplitude TWA was induced with rapid pacing in both infant and adolescents but decreased to low, steady-state levels in infants. We conclude that TWA properties are age dependent in rabbit. Significant TWA is induced in rabbit at the onset of rapid pacing.

Assessment of 18F-labeled mitochondrial complex I inhibitors as PET myocardial perfusion imaging agents in rats, rabbits, and primates

Myocardial extractions of mitochondria complex I (MC-I) inhibitors were high and well correlated with flow. This study assessed the potential of MC-I inhibitors to be developed as myocardial perfusion imaging (MPI) agents. RP1003, RP1004, and RP1005 representing three classes of MC-I inhibitor were synthesized and radio-labeled with (18)F. These agents were evaluated for IC(50) values, tissue biodistribution, and cardiac PET imaging. (18)F-RP1004 was further examined for first-pass extraction and by imaging in non-human primates (NHP) and rats following coronary ligation. RP1003, RP1004, and RP1005 exhibited high MC-I inhibitory activity with IC(50) of 3.7, 16.7, and 14.4 nM. Heart uptakes in rats (percent injected dose per gram tissue) at 15 and 60 min after injection were 3.52 +/- 0.36 and 2.68 +/- 0.20 for (18)F-RP1003, 2.40 +/- 0.21 and 2.67 +/- 0.27 for (18)F-RP1004, and 2.28 +/- 0.12 and 1.81 +/- 0.17 for (18)F-RP1005. The heart to lung and liver uptake ratios were favorable for cardiac imaging with these agents. In isolated perfused rabbit hearts, the uptake of (18)F-RP1004 increased from 0.74 +/- 0.19 to 1.68 +/- 0.39 mL/min/g at flow rates of 1.66 to 5.06 mL/min/g. These values were higher than or similar to that of (99m)Tc-sestamibi. Cardiac imaging with these agents in rats and rabbits allowed visualization of the heart with minimal lung interference and rapid liver activity clearance. Imaging with (18)F-RP1004 also showed clear myocardium and marked liver activity washout in the NHP and clear detection of the perfusion-deficit area associated with left coronary artery ligation in the rat. MC-I inhibitors have the potential to be a new class of MPI agent.

Effects of Autoantibodies against M2 Muscarinic Acetylcholine Receptors on Rabbit Atria in vivo

Background: Evidence has shown that autoantibodies against M2 muscarinic acetylcholine receptors may play a role in the development of atrial fibrillation. The goal of this study was to evaluate the effects of anti-M2 receptor autoantibodies on rabbit atria in vivo. Methods: Rabbits were immunized monthly with a synthetic peptide corresponding to the M2 receptor. The atrial electrophysiology of the isolated perfused rabbit hearts was studied. Western blots and RT-PCR were performed to determine the expression of the atrial muscarinic receptor and the acetylcholine-activated potassium channel. Atrial tissue was stained with Masson’s trichrome stain for fibrosis detection. Results: Autoantibodies were persistently detected in immunized rabbits. M2 rabbits showed a significantly shorter atrial effective refractory period and a longer intra-atrial activation time than control rabbits. Electrical stimuli induced a significantly larger number of repetitive atrial responses in M2 rabbits. The protein levels of the M2 receptor and GIRK4 were upregulated in M2 rabbits. The mRNA levels of GIRK1 and GIRK4 were also upregulated. Histological examination revealed significantly increased diffuse fibrotic deposition in M2 rabbit atria compared with control rabbits. Conclusion: The M2 receptor autoantibody-positive rabbits showed altered atrial electrophysiology, overexpression of the M2 receptor-I_K,ACh pathway and atrial fibrosis, which indicates that the autoantibodies against M2 receptors may participate in the induction and perpetuation of atrial fibrillation.

Analyzing the electrophysiological effects of local epicardial temperature in experimental studies with isolated hearts

As a result of their modulating effects upon myocardial electrophysiology, both hypo- and hyperthermia can be used to study the mechanisms that generate or sustain cardiac arrhythmias. The present study describes an original electrode developed with thick-film technology and capable of controlling regional temperature variations in the epicardium while simultaneously registering its electrical activity. In this way, it is possible to measure electrophysiological parameters of the heart at different temperatures. The results obtained with this device in a study with isolated and perfused rabbit hearts are reported. An exploration has been made of the effects of local temperature changes upon the electrophysiological parameters implicated in myocardial conduction. Likewise, an analysis has been made of the influence of local temperature upon ventricular fibrillation activation frequency. It is concluded that both regional hypo- and hyperthermia exert reversible and opposite effects upon myocardial refractoriness and conduction velocity in the altered zone. The ventricular activation wavelength determined during constant pacing at 250 ms cycles is not significantly modified, however. During ventricular fibrillation, the changes in the fibrillatory frequency do not seem to be transmitted to normal temperature zones.

Ca2+ sensitizer superior to catecholamine during myocardial stunning?☆

After open-chest cardiac surgery, ventricular function remains depressed (myocardial stunning). Catecholamines (epinephrine) improve ventricular function by increasing the intracellular Ca(2+) concentration. In parallel, the oxygen consumption is increased, so that the hitherto intact myocardium can be jeopardized. In the very insufficient ventricle, epinephrine can even become ineffective. Since Ca(2+) sensitizers provide another therapeutic avenue, the effects of epinephrine and levosimendan on postischemic hemodynamics were investigated. After hemodynamic steady state, isolated, blood (erythrocyte-enriched Krebs-Henseleit solution)-perfused rabbit hearts were subjected to 25 min normothermic, no-flow ischemia and 20 min reperfusion. Heart rate (HR), cardiac output (CO), left ventricular pressure (LVP), coronary blood flow (CBF), and arterio-venous oxygen difference (AVDO(2)) were recorded during reperfusion and after administration of either epinephrine (n=16; 0.03 micromol), or levosimendan (n=11; 0.75 micromol) or epinephrine plus levosimendan (n=5). Epinephrine increased HR (19%, p=0.01) and improved hemodynamics in terms of CO (62%, p=0.0006), stroke volume SV (46%, p=0.02), stroke work W (158%, p=0.01), LVP(max) (58%, p=0.0001), maximal pressure increase dP/dt(max)(140%, p=0.0004), minimal pressure increase dP/dt(min) (104%, p=0.0002), LVP(ed) (-26%, p=0.02), and increased coronary resistance CR (31%, p=0.05). Epinephrine impaired hemodynamics in terms of AVDO(2) (+63%, p=0.003), myocardial oxygen consumption MVO(2) (+67%, p=0.0003) and MVO(2)/beat (+36%, p=0.01). External efficiency eta was increased by 92% (p=0.02). Levosimendan in postischemic hearts increased HR (32%, p=0.009) and improved hemodynamics in terms of CO (85%, p=0.01), SV (44%, p=0.03), W (115%, p=0.04), LVP(max) (95%, p=0.04), dP/dt(max) (133%, p=0.009), dP/dt(min) (121%, p=0.007), LVP(ed) (-63%, p=0.0006), and CR (-17%; n.s., p=0.1). It altered hemodynamics in terms of AVDO(2) (+7.0%; n.s., p=0.3) and MVO(2) (+32%, p=0.007) and MVO(2)/beat (+2.3%; n.s., p=0.4). External efficiency was increased by 307% (p=0.04). In five additional extremely dysfunctional rabbit hearts, epinephrine was ineffective. Additional levosimendan increased hemodynamics in terms of HR (56%; n.s., p=0.1), CO (159%, p=0.04), SV (89%, p=0.03), W (588%, p=0.02), LVP(max) (168%, p=0.03), dP/dt(max) (102%, p=0.005), dP/dt(min) (78%, p=0.006), LVP(ed) (-98%, p=0.0006), and CR (-50%, p=0.02). It altered hemodynamics in terms of AVDO(2) (-11%; n.s., p=0.05), MVO(2) (+131%, p=0.04) and MVO(2)/beat (+171%, p=0.03). External efficiency was increased by 212% (p=0.04). In contrast to epinephrine, levosimendan improves ventricular function without increasing oxygen demand, thereby considerably improving external efficiency. Even during epinephrine resistance in extremely dysfunctional hearts, levosimendan successfully improves ventricular function.

6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)quinolinone (Cilostazol), a Phosphodiesterase Type 3 Inhibitor, Reduces Infarct Size via Activation of Mitochondrial Ca2+-Activated K+ Channels in Rabbit Hearts

6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)quinolinone (cilostazol), a phosphodiesterase type 3 (PDE III) inhibitor, activates cAMP-dependent protein kinase A (PKA). The cAMP/PKA pathway potentiates the opening of mitochondrial Ca(2+)-activated K(+) (mitoK(Ca)) channels and confers cardioprotection. Although cilostazol has been reported to directly activate sarcolemmal large-conductance Ca(2+)-activated K(+) channels, it remains unclear whether cilostazol modulates the opening of mitoK(Ca) channels. Therefore, we tested the possibility that cilostazol opens mitoK(Ca) channels and protects hearts against ischemia/reperfusion injury. Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoK(Ca) channel activity. Infarct size in the isolated perfused rabbit hearts subjected to 30-min global ischemia and 120-min reperfusion was determined by triphenyltetrazolium chloride staining. Cilostazol (1, 3, 10, and 30 microM) oxidized flavoprotein in a concentration-dependent manner. The oxidative effect of cilostazol (10 microM) was antagonized by the mitoK(Ca) channel blocker paxilline (2 microM). Activation of PKA by 8-bromoadenosine 3'5'-cyclic monophosphate (0.5 mM) potentiated the cilostazol-induced flavoprotein oxidation. Treatment with cilostazol (10 microM) for 10 min before ischemia significantly reduced the infarct size from 67.2 +/- 1.3 (control) to 33.6 +/- 5.3% (p < 0.05). This infarct size-limiting effect of cilostazol was abolished by paxilline (60.3 +/- 4.9%) but not by the PKA inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]-benzodiazocine-10-carboxylic acid hexyl ester (KT5720) (200 nM, 40.5 +/- 3.5%). On the other hand, another PDE III inhibitor, milrinone (10 microM), neither oxidized flavoprotein nor reduced infarct size. Our results suggest that cilostazol exerts a cardioprotective effect via direct activation of mitoK(Ca) channels.

A Phased-Array Stimulator System for Studying Planar and Curved Cardiac Activation Wavefronts

Wavefront propagation in cardiac tissue is affected greatly by the geometry of the wavefront. We describe a computer-controlled stimulator system that creates reproducible wavefronts of a predetermined shape and orientation for the investigation of the effects of wavefront geometry. We conducted demonstration experiments on isolated perfused rabbit hearts, which were stained with the voltage-sensitive dye, di-4-ANEPPS. The wavefronts were imaged using a laser and a charge-coupled device (CCD) camera. The stimulator and imaging systems have been used to characterize the relationship between wavefront velocity and fiber orientation. This approach has potential applications in investigating curvature effects, testing numerical models of cardiac tissue, and creating complex wavefronts using one-, two-, or three-dimensional electrode arrays.

Changes in the Spectral Characteristics of Ventricular Fibrillation in Lesions Produced by Radiofrequency Ablation. An Experimental Study

Although electro- physiological databases contain information about changes in the time domain in lesions produced by radiofrequency ablation, very few data on changes in the frequency domain are available. The aim of this study was to investigate changes in the spectral characteristics of ventricular fibrillation in zones with radiofrequency lesions. Recordings of ventricular fibrillation were obtained in 11 isolated perfused rabbit heart preparations using a multiple epicardial electrode located on the left ventricular free wall. Spectral parameters derived by Fourier analysis before and after the creation of transmural radiofrequency lesions were compared. In the ablated zones, significant reductions were observed in the spectral density of the dominant (0.168+/-0.113 mV(2)/Hz vs 0.025+/-0.018 mV(2)/Hz; P< .001) and mean frequencies (0.053+/-0.057 mV(2)/Hz vs 0.012+/-0.016 mV(2)/Hz; P< .001), the normalized energy around the dominant frequency (0.860+/-0.570 vs 0.128+/-0.091; P< .001), and the standard deviation of the power spectrum (0.031+/-0.020 mV(2)/Hz vs 0.004+/-0.001 mV(2)/Hz; P< .001). There was no significant change in the dominant (16.2+/-5.6 vs 14.8+/-1.8 Hz) or mean frequency (17.7+/-3.4 vs 16.6+/-1.3 Hz). The spectral parameters that could be used in a multivariate model to identify the lesion were the standard deviation of the power spectrum and the spectral density of the mean frequency. During ventricular fibrillation, the spectral parameters associated with spectral power and spectral energy were significantly altered in zones with radiofrequency lesions and could be used to identify those zones. There was no significant change in either the dominant or mean frequency in these zones.

Abstract 995: Blockade Of Electron Transport Preserves The Contents Of Bcl-2 And Cytochrome c In Subsarcolemmal Mitochondria During Ischemia

Cardiac ischemia decreases the rate of oxidative phosphorylation (OXPHOS) and the contents of cardiolipin and cytochrome c (CYTc) in subsarcolemmal mitochondria (SSM) in the isolated rabbit heart. CYTc release first requires damage to the inner mitochondrial membrane to delocalize CYTc to the intermembrane space, followed by breach of the outer membrane. The decrease in cardiolipin content allows CYTc detachment from the inner membrane. It is still unclear how CYTc passes the outer membrane for release into cytosol. We propose that ischemia increases outer-membrane leakage by depletion of bcl-2 content, and that oxidants generated by the electron transport chain (ETC) during ischemia favor bcl-2 depletion. We used blockade of the proximal ETC at complex I during ischemia with amobarbital (AMO) to test the role of ETC during ischemia. Langendorff perfused rabbit hearts were treated with AMO (2.5 mM for 1 min) or vehicle immediately before 30 min global ischemia (37°C). Time controls were perfused for 45 min. SSM were isolated at the end of ischemia. CYTc content (reduced minus oxidized spectra), OXPHOS and bcl-2 (western blotting) were measured. Ischemia decreased OXPHOS with TMPD-ascorbate as substrate (electron donor to complex IV via CYTc) and the contents of CYTc and bcl-2. In contrast, AMO preserves OXPHOS, CYTc and bcl-2. Thus, blockade of electron transport preserves bcl-2 content during ischemia with enhanced CYTc retention by SSM. The ETC contributes to mitochondrial damage during ischemia, depleting cardiolipin in the inner membrane and bcl-2 in the outer membrane favoring the two steps required for release of CYTc from mitochondria during ischemia and reperfusion.

The herbal drug Catuama reverts and prevents ventricular fibrillation in the isolated rabbit heart

Introduction Catuama, an herbal drug very popular in Brazil, was tested on the reversion and prevention of ventricular fibrillation (VF) in the isolated rabbit heart. Materials and Methods Catuama (a mixture of Trichilia catigua, Paullinia cupana, Ptychopetalum olacoides, and Zinziber officinalis) was perfused in the isolated perfused rabbit heart. Its effects on intraventricular conduction, heart rate, and monophasic action potential (MAP) duration were evaluated, and sustained VF was induced. The effects on reversion and reinduction of arrhythmia were observed, and new measures were taken in the hearts that reverted. Results Catuama and T catigua reverted VF in all hearts, prevented reinduction, and prolonged intraventricular conduction. Catuama prolonged MAP phase 2. On the other hand, P cupana reverted VF in 3 of 5 hearts, but depressed automatism, prolonged MAP phase 3, and did not prevent reinduction. Discussion Catuama reverted and prevented VF in this model. T catigua extract is probably the main agent responsible for the beneficial actions observed. Further studies are now in progress to clarify these actions.

WITHDRAWN: Infarct size limitation by adrenomedullin: Protein kinase A but not PI3-kinase is linked to mitochondrial KCa channels

Adrenomedullin (ADM) has been shown to protect the heart against ischaemic injury, but little is known of the underlying mechanism. Mitochondrial Ca(2+)-activated K(+) (mitoK(Ca)) channels play a key role in cardioprotection. This study examined whether mitoK(Ca) channel is involved in the protection afforded by ADM. Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoK(Ca) channel activity. Infarct size in the isolated perfused rabbit hearts subjected to 30-min global ischaemia and 120-min reperfusion was determined by triphenyltetrazolium chloride staining. The mitoK(Ca) channel opener NS1619 (30 microM) partially oxidized flavoprotein. ADM (10 nM) augmented the NS1619-induced flavoprotein oxidation when applied after the effect of NS1619 had reached steady state. This potentiating effect of ADM was prevented by the protein kinase A (PKA) inhibitor KT5720 (200 nM), but not by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 (5 microM). The mitoK(Ca) channel blocker paxilline (PX, 2 microM) completely blocked the oxidative effects of NS1619 in the presence of ADM. Treatment with ADM for 10 min before ischaemia significantly reduced infarct size after ischaemia/reperfusion from 63 +/- 3% in controls to 32 +/- 4% (P < 0.01). This infarct size-limiting effect of ADM was abolished by PX (61 +/- 2%), as well as by KT5720 (62 +/- 3%). ADM treatment for the first 10 min of reperfusion significantly reduced infarct size compared with controls (42 +/- 3%, P < 0.01). This cardioprotective effect of ADM was unaffected by PX (38 +/- 4%), but was abolished by LY294002 (60 +/- 4%). ADM augments the opening of mitoK(Ca) channels by PKA activation, but not by PI3-K activation. ADM treatment prior to ischaemia reduces infarct size via PKA-mediated activation of mitoK(Ca) channels. On the other hand, ADM treatment upon reperfusion reduces infarct size via a PI3-K-mediated pathway without activating mitoK(Ca) channels.

IKs Block by HMR 1556 Lowers Ventricular Defibrillation Threshold and Reverses the Repolarization Shortening by Isoproterenol Without Rate‐Dependence in Rabbits

The slow delayed rectifier K+ current (I(Ks)) contributes little to ventricular repolarization at rest. It is unclear whether I(Ks) plays a role during ventricular fibrillation (VF) or ventricular repolarization at rapid rates during beta-adrenergic stimulation. In an in vivo rabbit model, we evaluated the effects of HMR 1556 (1 mg Kg(-1) + 1 mg kg(-1) hr(-1) i.v.), a selective I(Ks) blocker, on monophasic action potential duration at 90% repolarization (MAPD90), ventricular effective refractory period (VERP), and defibrillation threshold (DFT). In perfused rabbit hearts, the effects of HMR 1556 (10 and 100 nM) in the presence of isoproterenol (5 nM) on MAPD90 and VERP were studied at cycle lengths (CLs) 200-500 msec. In vivo, HMR 1556 prolonged MAPD90 by 6 +/- 1 msec at CL 200 msec (P < 0.01, n = 6), lowered DFT from 558 +/- 46 V to 417 +/- 31 V (P < 0.01), and decreased the coefficient of variation in the VF inter-beat deflection intervals from 8.9 +/- 0.6% to 6.5 +/- 0.4% (P < 0.05) compared with control. In perfused rabbit hearts, isoproterenol shortened MAPD90 by 5 +/- 1 msec at CL 200 msec and 11 +/- 4 msec at CL 500 msec (P < 0.05, n = 7). This shortening was reversed by HMR 1556 (P < 0.05), and both effects were rate-independent. I(Ks) block increases VF temporal organization and lowers DFT, and I(Ks) that is activated following beta-adrenergic stimulation contributes to ventricular repolarization without rate dependence.

Preshock phase singularity and the outcome of ventricular defibrillation

Phase singularity (PS) is a topological defect that serves as a source of ventricular fibrillation (VF). Whether or not the quantity of preshock PS determines defibrillation outcome is unclear. The purpose of this study was to test the hypothesis that the number of PSs at the time of shock is an important factor that determines the shock outcome. Isolated, perfused rabbit hearts (n = 7) were optically mapped with a potentiometric dye (di-4-ANNEPS). Shocks were delivered during short (10 seconds) and long (1 minute) VF, and the outcome was classified as successful type A (immediate termination), type B (postshock repetitive responses before termination), and unsuccessful. When shock strengths of 50% probability of successful defibrillation (DFT50) +/- 50 V were given in short VF, the types A and B and unsuccessful shocks were associated with a preshock PS number of 0.3 +/- 0.4, 1.4 +/- 0.3, and 1.5 +/- 0.4 (P <.01 by analysis of variance) and shock strengths of 205 +/- 77, 207 +/- 65, and 173 +/- 74 V (P <.01), respectively. When the same shocks were applied during long VF, the PS numbers were 1.7 +/- 0.5, 3.0 +/- 0.5, and 3.5 +/- 0.6, respectively (P <.01), and the shock strengths were 282 +/- 100, 283 +/- 135, and 256 +/- 126 V, respectively (P <.01). If we only analyze shocks with strength at DFT(50), the preshock PS number was still significantly different for short VF (0.6 +/- 0.5, 1.6 +/- 0.9, and 1.5 +/- 0.8; P <.05) and for long VF (1.4 +/- 0.5, 2.7 +/- 0.6, and 2.7+/-1.3; P <.05), respectively. All preshock PSs were eliminated by shocks. However, rapid repetitive activity was then reinitiated in unsuccessful and type B successful shocks but not in type A successful shocks. A low number or an absence of preshock PS was associated with type A successful defibrillation. There was no difference in preshock PS numbers between unsuccessful and type B successful defibrillation.

Three-dimensional panoramic imaging of cardiac arrhythmias in rabbit heart

Cardiac fluorescent optical imaging provides the unique opportunity to investigate the dynamics of propagating electrical waves during ventricular arrhythmias and the termination of arrhythmias by strong electric shocks. Panoramic imaging systems using charge-coupled device (CCD) cameras as the photodetector have been developed to overcome the inability to monitor electrical activity from the entire cardiac surface. Photodiode arrays (PDAs) are known to have higher temporal resolution and signal quality, but lower spatial resolution compared to CCD cameras. We construct a panoramic imaging system with three PDAs and image Langendorff perfused rabbit hearts (n=18) during normal sinus rhythm, epicardial pacing, and arrhythmias. The recorded spatiotemporal dynamics of electrical activity is texture mapped onto a reconstructed 3-D geometrical heart model specific to each heart studied. The PDA-based system provides sufficient spatial resolution (1.72 mm without interpolation) for the study of wavefront propagation in the rabbit heart. The reconstructed 3-D electrical activity provides us with a powerful tool to investigate the fundamental mechanisms of arrhythmia maintenance and termination.

Reduction and Redistribution of Gap and Adherens Junction Proteins After Ischemia and Reperfusion

Previous studies have demonstrated that alterations in myocardial structure, consistent with tissue and sarcomere disruption as well as myofibril dissociation, occur after myocardial ischemia and reperfusion. In this study we determine the onset of these structural changes and their contribution to electrical conduction. Langendorff perfused rabbit hearts (n = 47) were subjected to 0, 5, 10, 15, 20, 25, and 30 minutes global ischemia, followed by 120 minutes reperfusion. Hemodynamics were recorded and tissue samples were collected for histochemical and immunohistochemical studies. Orthogonal epicardial conduction velocities were measured, with temperature controlled, in a separate group of 10 hearts subjected to 0 or 30 minutes of global ischemia, followed by 120 minutes of reperfusion. Histochemical and quantitative light microscopy spatial analysis showed significantly increased longitudinal and transverse interfibrillar separation after 15 minutes or more of ischemia (p < 0.05 versus control). Confocal immunohistochemistry and Western blot analysis demonstrated significant reductions (p < .05 versus control) of the intercellular adherens junction protein, N-cadherin, and the active phosphorylated isoform of the principal gap junction protein, connexin 43 at more than 15 minutes of ischemia. Cellular redistribution of connexin 43 was also evidenced on immunohistochemistry. No change in integrin-beta1, an extracellular matrix attachment protein, or in epicardial conduction velocity anisotropy was observed. These data indicate that there are significant alterations in the structural integrity of the myocardium as well as gap and adherens junction protein expression with increasing global ischemia time. The changes occur coincident with previously observed significant decreases in postischemic functional recovery, but are not associated with altered expression of matrix binding proteins or electrical anisotropic conduction.