Guinea-pig Langendorff Heart Research Articles

Calcium Overload or Underload? The Effects of Doxorubicin on the Calcium Dynamics in Guinea Pig Hearts

The severe doxorubicin (DOXO) side effect of cardiomyopathy limits it clinical application as an effective anticancer drug. Although Ca2+ overload was postulated as one of the mechanisms for this toxicity, its role was, however, disputable in terms of the contractile dysfunction. In this work, the dynamics of the intracellular Ca2+ signal were optically mapped in a Langendorff guinea pig heart. We found that DOXO treatment: (1) Delayed the activation of the Ca2+ signal. With the reference time set at the peak of the action potential (AP), the time lag between the peak of the Ca2+ signal and AP (Ca-AP-Lag) was significantly prolonged. (2) Slowed down the intracellular Ca2+ releasing and sequestering process. Both the maximum rising (MRV) and falling (MFV) velocity of the Ca2+ signal were decreased. (3) Shortened the duration of the Ca2+ signal in one cycle of Ca2+ oscillation. The duration of the Ca2+ signal at 50% amplitude (CaD50) was significantly shortened. These results suggested a reduced level of intracellular Ca2+ after DOXO treatment. Furthermore, we found that the effect of tachypacing was similar to that of DOXO, and, interestingly, DOXO exerted contradictory effects on the tachypaced hearts: it shortened the Ca-AP-Lag, accelerated the MRV and MFV, and prolonged the CaD50. We, therefore, concluded that DOXO had a different effect on intracellular Ca2+. It caused Ca2+ underload in hearts with sinus rhythm; this might relate to the contractile dysfunction in DOXO cardiomyopathy. It led to Ca2+ overload in the tachypaced hearts, which might contribute to the Ca2+-overload-related toxicity.

Cardiotoxicity assessment using 3D vascularized cardiac tissue consisting of human iPSC-derived cardiomyocytes and fibroblasts

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for cardiac safety assessment but have limitations for the evaluation of drug-induced contractility. Three-dimensional (3D) cardiac tissues are similar to native tissue and valuable for the assessment of contractility. However, a longer time and specialized equipment are required to generate 3D tissues. We previously developed a simple method to generate 3D tissue in a short period by coating the cell surfaces with extracellular matrix proteins. We hypothesized that this 3D cardiac tissue could be used for simultaneous evaluation of drug-induced repolarization and contractility. In the present work, we examined the effects of several compounds with different mechanisms of action by cell motion imaging. Consequently, human ether-a-go-go-related gene (HERG) channel blockers with high arrhythmogenic risk caused prolongation of contraction-relaxation duration and arrhythmia-like waveforms. Positive inotropic drugs, which increase intracellular Ca2+ levels or myocardial Ca2+ sensitivity, caused an increase in maximum contraction speed (MCS) or average deformation distance (ADD) (ouabain, 138% for MCS at 300 nM; pimobendane, 132% for ADD at 3 μM). For negative inotropic drugs, verapamil reduced both MCS and ADD (61% at 100 nM). Thus, this 3D cardiac tissue detected the expected effects of various cardiovascular drugs, suggesting its usefulness for cardiotoxicity evaluation.

Cardiac safety assessment with motion field imaging analysis of human iPS cell-derived cardiomyocytes is improved by an integrated evaluation with cardiac ion channel profiling.

We validated a motion field imaging (MFI) assay with human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) as a model to assess multiple cardiac liabilities by comparing the guinea-pig Langendorff heart with hiPS-CMs using 4 reference compounds and 9 internal compounds. We investigated repolarization duration, beating rate (BR), conduction speed, contractility, and inhibitory profile of three cardiac ion channels: hERG, Cav1.2, and Nav1.5. For repolarization, the contraction-relaxation duration (CRDc) of hiPS-CMs was generally consistent with the QTc interval of Langendorff heart. However, 2 internal compounds shortened CRDc despite QTc prolongation in Langendorff heart. Cardiac ion channel profiling revealed that hiPS-CMs could not be used to detect QTc prolongation when the value of Cav1.2 IC50 / hERG IC50 for a compound was between 1 and 10, whereas hiPS-CMs showed responses largely consistent with Langendorff heart when Cav1.2 IC50 / hERG IC50 was below 1 or above 10. The accuracy of hiPS-CMs for the BR was not high, mainly because the BR of hiPS-CMs was increased by an inhibition of Cav1.2. The hiPS-CMs were highly sensitive to conduction speed and contractility, able to detect QRS widening caused by Nav1.5-inhibition, as well as decreased LVdP/dtmax caused by the inhibition of Cav1.2 and/or Nav1.5. In conclusion, the MFI assay with hiPS-CMs would be useful for evaluating multiple cardiac liabilities. The ion channel profile helps to interpret the results of MFI assay and correctly evaluate cardiac risks. Therefore, an integrated cardiac safety assessment with MFI and ion channel profiling is recommended.

Attenuation of Conduction Slowing during Global Ischemia in Guinea Pig Hearts through Increased Extracellular Calcium

Cardiac ischemia is associated with decreased conduction velocity (CV) which can lead to conduction block and reentrant arrhythmias. Previously we have demonstrated that physiologically relevant changes in extracellular [K+] and [Ca2+] can modulate CV. The aim of the current study was to test whether changes in perfusate [K+] or [Ca2+] could alter loss of conduction during global ischemia and reperfusion. Guinea pig Langendorff hearts were perfused with a modified Tyrode's solution containing 153mM Na+, 4.6 or 6.9mM K+, and 1.25 or 2.0mM Ca2+, and loaded with the voltage-sensitive dye di-4-ANEPPS. Hearts were paced at a cycle length of 300ms at baseline and every 2 minutes during no flow ischemia to assess changes in transverse CV (CVT) and the time to conduction block. In separate studies, hearts were perfused with Tyrode's solution containing 146mM Na+, 4.6mM K+, and 1.25mM Ca2+, then subjected to 15 minutes of no flow ischemia before reperfusion with the test solutions for 15 minutes. At baseline (preischemia), there were no significant differences in CVT among the test solutions (153Na+/4.6K+/1.25Ca2+: 19.3±1.7 cm/s, n=6; 153Na+/4.6K+/2.0Ca2+: 16.4±1.9 cm/s, n=7; 153Na+/6.9K+/1.25Ca2+: 20.8±1.1 cm/s, n=6; p=NS). By 4 minutes of ischemia, CVT decreased significantly in the 153Na+/4.6K+/1.25Ca2+ (95% of baseline, p<0.05) and 153Na+/6.9K+/1.25Ca2+ (84% of baseline, p<0.05) solutions. In contrast, increasing [Ca2+] preserved CVT (153Na+/4.6K+/2.0Ca2+: 111% of baseline, p=NS). This preservation of CVT persisted through 12 minutes of ischemia (98% of baseline, p=NS), whereas at the same time point CVT in the 153Na+/4.6K+/1.25Ca2+ solution had decreased by 40% (p<0.01) and all hearts with 153Na+/6.9K+/1.25Ca2+ exhibited conduction block by 8 minutes. Despite the Ca2+-mediated differences in ischemia-induced changes in CV, the median time to conduction block was not significantly different between the 153Na+/4.6K+/1.25Ca2+ (16 minutes) and 153Na+/4.6K+/2.0Ca2+ (12 minutes, p=NS) solutions. However, increasing [K+] significantly shortened the median time to block (153Na+/6.9K+/1.25Ca2+: 7 minutes) relative to either solution containing 4.6mM K+ (p<0.01). Upon reperfusion, there were no significant differences in CVT among the test solutions, with a restoration of preischemia values within 3 minutes for each solution. In conclusion, increased extracellular Ca2+ improved conduction during global no flow ischemia, whereas decreased extracellular K+ prolonged the time to conduction block. Therefore, altering plasma ionic milieu during acute ischemia, in order to preserve cardiac conduction, may be a novel mechanism for preventing reentrant arrhythmias.

Effects of monocarboxylate transporter 1 (MCT1) inhibition on cardiac function under ischaemic conditions: A comparison between rat and guinea-pig Langendorff hearts

The capacity of the brain to metabolize non-glucose substrates under hypoglycemic state maintains its energy requirements. We hypothesized that exercise-induced increase in capacity for brain utilization of lactate by up regulation of the monocarboxylate transporters (MCTs) may contribute metabolic substrates during hypoglycemia in diabetic rats induced by streptozotocin. The induced diabetes increased MCT1 and MCT2 expression in the cortex and the hippocampus in the sedentary diabetic animals. There were exercise-induced increases in MCT1 in the cortex and the hippocampus and MCT2 expression in the cortex in trained diabetic animals; whereas, no changes were found in the healthy trained animals. Both diabetic and healthy trained animals showed higher values for brain lactate uptake during insulin-induced hypoglycemia when animals were intraperitoneally injected by L(+)-lactic acid. However, the response of counterregulatory hormones during hypoglycemia were blunted in the diabetic trained animals which indicates to carefully monitoring of glycemic targets both during and following prolonged exercise.

CORONARY VASOCONSTRICTION INDUCED BY POLYMERS OF ANG II IS PROPORTIONAL TO LUMINAL ENDOTHELIAL AT1 RECEPTORS DENSITY

We have shown that intravascular AngII does not diffuse across the coronary vessel wall and acts solely in coronary endothelial luminal membrane AngII receptors (CELM‐AT1R) and its sustained intracoronary infusion causes transient vasoconstriction effects (+V). This contrasted with the sustained +V by a large size, non‐diffusible polymer of AngII (15,000 kDa, AngIIPol) that unquestionably activate solely CELM‐AT1R. We also show that AngII transient +V paralleled a decrease of CELM‐AT1R density (AT1RD), but, for AngIIPol the AT1RD remained constant. HYPOTHESIS; transiently of +V effects depends directly on AT1RD both are inversely determined by the molecular weight (MW) of AT1R agonist. To test this hypothesis we synthesized AngIIPol's (included AngII) of various MW (kDa): 1.3, 2.7, 11, 47, 527, 3270 and 15000. In isolated constant flow (8ml/min) perfused Langendorff guinea pig hearts AII‐Pol's were separately intracoronary given for 16 min and coronary perfusion pressure measure as an index V. All AngIIPol's caused the same maximal +V (+Vmax) that decayed with time at a rate and to a stable level of +V, both inversely proportional to MW. During AngIIpol's infusion AT1RD decayed parallel to +V; the relationship +V‐‐AT1RD was linear. Further dependence of +V on AT1RD was demonstrated by two successive administrations of AngIIPol's, the primary dose caused a +Vmax but the +Vmax to the secondary dose was depressed; a tachyphylaxis which was inversely proportional to the logarithm of MW. These results support our hypothesis and indicate that AT1R internalization is dependent on MW.

Proadrenergic effects of cardiac natriuretic peptides: inhibition by histamine H3‐receptor (H3R) activation

Natriuretic peptides (NP) are generally viewed as cardioprotective. Yet, NP were recently found to increase myocardial infarct size in mice, while their deletion decreased it. Also, mice lacking NP receptor-A are resistant to endotoxic shock. Further, a recombinant NP (nesiritide) was shown to increase death risk in heart failure patients. Excessive norepinephrine (NE) release may have been pivotal in these unexpected events. Langendorff guinea-pig hearts were perfused with NP; this resulted in a ≈ 73% increase in NE overflow. NP treatment of guinea-pig cardiac synaptosomes and NGF-differentiated PC12 cells also increased NE and dopamine release (≈ 34% and ≈ 48% increase, respectively). Thus, we investigated the mechanisms of the pro-adrenergic effect of NP. We found that NP markedly increased PKG activity, cAMP levels and PKA activity in differentiated PC12 cells; these effects derived from a cGMP/PKG-mediated prevention of cAMP hydrolysis by PDE3. Because of the antiexocytotic properties of H3R, we next questioned whether H3R activation might inhibit the proadrenergic effects of NP. Activation of H3R, constitutively expressed in heart synaptosomes and permanently transfected in PC12 cells, inhibited NP-induced catecholamine release and prevented the increases in cAMP level and PKA activity. Since the clinical use of NP in congestive heart failure (CHF) remains controversial, our findings may foster the design of selective H3R agonists to enable a safe and effective treatment of CHF with NP. This work was supported by NIH Grant HL 034215 and by a PhRMA Pre-doctoral fellowship.

The Electrophysiological Effects of Cardiac Glycosides in Human iPSC-derived Cardiomyocytes and in Guinea Pig Isolated Hearts

Background/aims: Monitoring changes in the field potential (FP) of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) following compound administration has been proposed as a novel screening tool to evaluate cardiac ion channel interactions and QT liability. Here we extended the use of FP to evaluate the pharmacological and toxicological properties of cardiac glycosides. Methods: FPs were recorded using microelectrode arrays (MEAs) in spontaneously beating hiPSC-CMs. The in vitro effects of ouabain and digoxin on FPs were compared with data generated on hemodynamic and ECG parameters in guinea pig Langendorff hearts. Results: In hiPSC-CMs, ouabain and digoxin reduced Na⁺-spike amplitude, shortened FP duration (FPD), increased Ca²⁺-wave amplitude, and dose-dependently induced arrhythmic beats. The ouabain-induced changes observed in hiPSC-CMs correlated well with the effects seen in isolated hearts which revealed QT shortening, enhancement of contractility, and arrhythmogenesis. Nifedipine, an L-type Ca²⁺ channel blocker, reduced Ca²⁺-wave amplitude and FPD in hiPSC-CMs, and led to parallel effects of decreased ventricular contractility and shortened QT interval in isolated hearts. Further, nifedipine attenuated the Ca²⁺-peak amplitude and proarrhythmic effect of both glycosides. These results suggested that FPD and Ca²⁺-wave amplitude are comparable surrogates of QT interval and contractility of intact hearts, respectively. Conclusion: hiPSC-CMs reflect similar cardiac pharmacology as seen in isolated cardiac preparations and thus are a suitable model in study of the pharmacology and toxicology of cardioactive ion channel and transporter modulators.

Accurate detection of drug-induced delayed ventricular repolarization with a suitable correction formula in Langendorff guinea pig heart

The aims of this study were to determine a suitable method to correct the ventricular repolarization period against the RR interval in isolated perfused Langendorff guinea pig heart and to clarify the reliability of this model using several drugs. QT and RR intervals from an electrocardiogram and the epicardial monophasic action potential duration (MAP(90)) were measured. Two drugs clinically known to be QT-prolonging (E-4031, moxifloxacin) and two known to be non-QT-prolonging (verapamil, zatebradine) were used for the study. To determine a method of correcting the ventricular repolarization period against RR interval, heart rates were slowed with 0.3 µM zatebradine, a specific bradycardiac agent, and then accelerated with atrial pacing to obtain a wide range of MAP(90)/RR relationships. An exponential rate-correction model elicited the most appropriate algorithm for the relationship among the four models tested. Based on linear regression analysis, the exponential showed superior dissociation of corrected MAP(90)s against RR intervals than generic Bazett's and Fridericia's formulae. E-4031 and moxifloxacin prolonged the corrected QT (QTc) intervals and MAP(90) under atrial pacing at a cycle length of 0.25 sec (MAP(90(pacing))) dose-dependently; verapamil and zatebradine failed to prolong them, indicating that the reliability of this model was excellent. MAP(90(pacing)) prolongation by moxifloxacin, the positive compound in the clinical "Thorough QT/QTc Study", was seen at around QTc-prolonging concentrations in clinic, suggesting that the sensitivity would be appropriate for QT evaluation. We therefore concluded that the isolated guinea pig heart model is sufficiently sensitive and useful for assessing the potential QT prolongation of drugs.

Validation of a guinea pig Langendorff heart model for assessing potential cardiovascular liability of drug candidates

IntroductionCardiac liabilities represent a major cause of recent withdrawal of marketed drugs and also for the high attrition rate evidenced during late stage drug development. To identify molecules with potential cardiovascular risks early in drug development, a screening model of ex vivo Langendorff hearts has been validated with 26 reference compounds of various chemical and therapeutic classes. MethodsThe hearts of adult guinea pigs were maintained by retrograde perfusion in Langendorff mode, beating spontaneously at sinus rhythm or paced via the right atrium at 200 and 300 beats per minute. Multiple parameters consisting of hemodynamic function (coronary and left ventricle pressure), cardiac electrophysiology (electrocardiogram and monophasic action potential) and indices of arrhythmogenesis (triangulation, reverse-use dependence, repolarization dispersion and beat-to-beat instability), together with overt arrhythmia were evaluated simultaneously. Ascending concentrations up to either 100-fold of the determined hERG IC50 or nominally 100 µM were routinely tested utilizing 4–6 hearts per compound. ResultsEach compound exhibited a unique cardiovascular profile: (i) the majority displayed concentration and heart rate-dependent mixed-ion channel or multiple-target effects that frequently resulted in bradycardia, atrioventricular block, negative inotropy, coronary vasodilatation, and QRS widening. (ii) Compounds associated with high arrhythmogenic risk in the clinic exhibited more “positive signals” at concentrations within 30-fold of their maximal therapeutic free plasma concentration than those with less arrhythmogenic potential. (iii) For several potent torsadogens, proarrhythmic indices other than the prolongation of QT/QTc and MAP duration appeared more sensitive in representing proarrhythmic liability. (iv) A scoring system was developed to assist in the rank-ordering of potential cardiotoxicants. DiscussionThe cardiovascular action of reference compounds profiled by this isolated heart model was generally consistent with their known mechanisms and, except for the sinus heart rate, correlated well with that observed in the clinic. Further, the overall cardiac liability estimated by the scoring system matched the clinical documentation, suggesting this model could serve as a valuable tool in early cardiovascular drug safety assessment.

Adenosine enhances cytosolic phosphorylation potential and ventricular contractility in stunned guinea pig heart: receptor-mediated and metabolic protection

Mechanisms of adenosine (ADO) protection of reperfused myocardium are not fully understood. We tested the hypothesis that ADO (0.1 mM) alleviates ventricular stunning by ADO A(1)-receptor stimulation combined with purine metabolic enhancements. Langendorff guinea pig hearts were stunned at constant left ventricular end-diastolic pressure by low-flow ischemia. Myocardial phosphate metabolites were measured by (31)P-NMR, with phosphorylation potential {[ATP]/([ADP].[P(i)]), where brackets indicate concentration} estimated from creatine kinase equilibrium. Creatine and IMP, glycolytic intermediates, were measured enzymatically and glycolytic flux and extracellular spaces were measured by radiotracers. All treatment interventions started after a 10-min normoxic stabilization period. At 30 min reperfusion, ventricular contractility (dP/dt, left ventricular pressure) was reduced 17-26%, ventricular power (rate-pressure product) by 37%, and [ATP]/([ADP].[P(i)]) by 53%. The selective A(1) agonist 2-chloro-N(6)-cyclo-pentyladenosine marginally preserved [ATP]/([ADP].[P(i)]) and ventricular contractility but not rate-pressure product. Purine salvage precursor inosine (0.1 mM) substantially raised [ATP]/([ADP].[P(i)]) but weakly affected contractility. The ATP-sensitive potassium channel blocker glibenclamide (50 microM) abolished ADO protection of [ATP]/([ADP].[P(i)]) and contractility. ADO raised myocardial IMP and glucose-6-phosphate, demonstrating increased purine salvage and pentose phosphate pathway flux potential. Coronary hyperemia alone (papaverine) was not cardioprotective. We found that ADO protected energy metabolism and contractility in stunned myocardium more effectively than both the A(1)-receptor agonist 2-chloro-N(6)-cyclo-pentyladenosine and the purine salvage precursor inosine. Because ADO failed to stimulate glycolytic flux, the enhancement of reperfusion, [ATP]/([ADP].[P(i)]), indicates protection of mitochondrial function. Reduced ventricular dysfunction at enhanced [ATP]/([ADP].[P(i)]) argues against opening of mitochondrial ATP-sensitive potassium channel. The results establish a multifactorial mechanism of ADO antistunning, which appears to combine ADO A(1)-receptor signaling with metabolic adenylate and antioxidant enhancements.

Celecoxib dilates guinea-pig coronaries and rat aortic rings and amplifies NO/cGMP signaling by PDE5 inhibition

Celecoxib carries a smaller cardiovascular risk for myocardial infarction and hypertension than other cyclooxygenase-2 (COX-2)-selective non-steroidal anti-inflammatory drugs NSAIDs ("coxibs") and may ameliorate endothelial dysfunction. We aimed to determine which mechanism possibly accounts for the beneficial effect by investigating its vascular action in different in vitro preparations in comparison with other coxibs and reference phosphodiesterase-5 (PDE5) inhibitors. To uncover potential effects on coronary flow, the effects of celecoxib in comparison with other NSAIDs and the PDE5 inhibitors, sildenafil and zaprinast, were investigated in guinea-pig Langendorff heart. This was supported by studies for vasorelaxation, interaction with the NO/cGMP pathway, and measurement of cyclic nucleotide amounts released from rat aortic rings, and inhibition of human PDE5 as well as PDE4 activity. Bolus injections of sildenafil, celecoxib, and zaprinast (at 100 nmol) into the Langendorff heart increased coronary flow by approximately 100, 65, and 25%, respectively, while rofecoxib, lumiracoxib, parecoxib, and diclofenac, except valdecoxib (>100 nmol), failed to increase coronary flow up to 300 nmol. In rat aorta, sildenafil, celecoxib and zaprinast caused endothelium-dependent relaxation with -log[EC(50)]M values of 8.90, 6.66 and 5.56, respectively; their rank order of potency corresponds to their coronary dilatory effect. Celecoxib-induced relaxation of aorta was attenuated by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) and by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 10(-5) M). In aortic rings, celecoxib (3x10(-5) M) caused a fivefold increase in the cGMP level and potentiated that induced by sodium nitroprusside (5x10(-7) M). Celecoxib and valdecoxib inhibited human PDE5A1 with an IC(50) of 1.6x10(-5) and 1x10(-4) M, respectively, whereas other coxibs were without inhibitory effect. Celecoxib caused coronary vasodilatation in guinea-pig hearts and relaxation of rat aorta and had a potentiating effect on the NO/cGMP signaling pathway in rat aorta through specific blockade of PDE5. These unexpected findings clearly support the notion that celecoxib possesses an as yet undisclosed molecule-specific property that possibly compensates a decrease of prostacyclin-dependent cAMP generation by concomitantly increasing cGMP levels resulting from inhibition of PDE5.

Protective effects of EGCg or GCg, a green tea catechin epimer, against postischemic myocardial dysfunction in guinea-pig hearts

The protective effects of (−)-epigallocatechin-3-gallate (EGCg) or the C-2 epimer, (−)-gallocatechin-3-gallate (GCg), afforded by their antioxidative activity among green tea catechins were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. The recovery (%) of the left ventricular developed pressure from ischemia by reperfusion was 34.4% in the control, while in the presence of EGCg (3 × 10 − 5 M) or GCg (3 × 10 − 6 M, a more diluted concentration than that of EGCg), it led to a maximal increase of 78.4% or 76.2%, consistent with a significant preservative effect on the tissue level of ATP at the end of ischemia or reperfusion. In the perfused preparation of mitochondria, EGCg (10 − 5 M) inhibited mitochondrial Ca 2+ elevation by changes in the Ca 2+ content or the acidification of perfusate, similarly to findings with cyclosporin A, a well known inhibitor of the mitochondrial permeability transition pore. By in vitro electron paramagnetic resonance (EPR), EGCg or GCg was found to directly quench the activity of active oxygen radicals, with the strongest activity in tea catechins. EGCg or GCg decreased the caspase-3 activity induced apoptosis. Therefore, it is concluded that the beneficial effects of EGCg or GCg play an important role in ischemia–reperfusion hearts in close relation with nitric oxide (NO), active oxygen radicals and biological redox systems in mitochondria.

Different effects of optical isomers of the 5-HT 1A receptor antagonist pyrapyridolol against postischemic guinea-pig myocardial dysfunction and apoptosis through the mitochondrial permeability transition pore

The recovery (%) of the left ventricular developed pressure by ( S)-(−)-pyrapyridolol (5 × 10 − 8 M) (90.7%), an optical isomer of a new 5-HT 1A receptor antagonist, was greater than that by ( R)-(+)-pyrapyridolol (66.2%, control: 34.4%) against ischemia–reperfusion injury in perfused Langendorff guinea-pig hearts. In the perfused mitochondrial preparation, ( S)-(−)-pyrapyridolol inhibited the mitochondrial Ca 2+ (Ca m) elevation that was brought about by the change of Ca 2+ content or pH of perfusate, similar to findings with cyclosporin A, well known to be an inhibitor of the mitochondrial permeability transition pore (MPTP). The mitochondrial K ATP channel opener, diazoxide, also inhibited the Ca m elevation, but the mitochondrial K ATP channel antagonist, 5-hydroxydecanoic acid, attenuated it. There were significantly fewer numbers of TUNEL-positive cells in these ( S)-(−)-pyrapyridolol-treated hearts than the control or ( R)-(+)-pyrapyridolol, with decreases of the caspase-3 activity. Therefore, these results suggest that ( S)-(−)-pyrapyridolol likely inhibits the opening of the MPTP by preventing the Ca m overload induced apoptosis related to endogenous 5-HT accumulation in ischemia–reperfusion hearts.

The kinetics of spontaneous calcium oscillations and arrhythmogenesis in the in vivo heart during ischemia/reperfusion

The correlation between spontaneous calcium oscillations (S-CaOs) and arrhythmogenesis has been investigated in a number of theoretical and experimental in vitro models. There is an obvious lack of studies that directly investigate how the kinetics of S-CaOs correlates with a specific arrhythmia in the in vivo heart. The purpose of the study is to investigate the correlation between the kinetics of S-CaOs and arrhythmogenesis in the intact heart using an experimental model of ischemia/reperfusion (I/R). Perfused Langendorff guinea pig (GP) hearts were subjected to global I/R (10-15 minutes/10-15 minutes). The heart was stained with a voltage-sensitive dye (RH237) and loaded with a Ca2+ indicator (Rhod-2 AM). Membrane voltage (Vm) and intracellular calcium transient (Ca(i)T) were simultaneously recorded with an optical mapping system of two 16 x 16 photodiode arrays. S-CaOs were considered to arise from a localized focal site within the mapped surface when these preceded the associated membrane depolarizations by 2-15 ms. In 135 episodes of ventricular arrhythmias from 28 different GP experiments, 23 were linked to S-CaOs that were considered to arise from or close to the mapped epicardial window. Self-limited or sustained S-CaOs had a cycle length of 130-430 ms and could trigger propagated ventricular depolarizations. Self-limited S-CaOs that followed the basic beat action potential (AP)/Ca(i)T closely resembled phase 3 early afterdepolarizations. Fast S-CaOs could remain confined to a localized site (concealed) or exhibit varying conduction patterns. This could manifest as (1) an isolated premature beat (PB), bigeminal, or trigeminal rhythm; (2) ventricular tachycardia (VT) when a regular 2:1 conduction from the focal site develops; or (3) ventricular fibrillation (VF) when a complex conduction pattern results in wave break and reentrant excitation. The study examined, for the first time in the intact heart, the correlation between the kinetics of focal S-CaOs during I/R and arrhythmogenesis. S-CaOs may remain concealed or manifest as PBs, VT, or VF. A "benign looking" PB during I/R may represent "the tip of the iceberg" of an underlying potentially serious arrhythmic mechanism.

Relationship between prolongation of QTc and prolongation of the peak of T (Tp) to the end of T (Te)

Introduction Lengthening of ventricular repolarization is known to be a risk factor for development of torsade de pointes, a form of ventricular tachycardia thought to be initiated by an early after depolarization and to be sustained by a novel reentrant mechanism precipitated, putatively, by heterogeneity of repolarization among ventricular myocytes. While prolongation of QT and QTc are good predictors of torsdogenic potential, the duration from the peak to the end of the T wave (Tp–Te) is thought to be a more accurate reflection of heterogeneity of ventricular repolarization. This study, conducted on Langendorff guinea pig hearts, was designed to compare lengthening of QTc with lengthening of Tp–Te for 16 test articles known to be and 7 known not to be torsadogenic. Methods Bipolar, transventricular electrograms, recorded from 83 guinea pig hearts perfused according to methods of Langendorff, were exposed to escalating concentrations of test articles. RR, QT, QTc and Tp–Te were measured. QTc was calculated by the method of Fridericia. Data was analyzed using a mixed model ANOVA where least squared means ( t-test) was used for comparing males vs. females and for concentration levels for each parameter studied. Results QTc lengthened in 16 of 16 test articles known to be torsadogenic and did not lengthen in 7 of 7 test articles known not to be torsadogenic—sensitivity and specificity of 1.0. In 9 out of 16 torsadogenic test compounds, the Tp–Te interval increased parallel with QTc. In 7 test compounds known not to be torsadogenic, two test compounds increased Tp–Te. Discussion It is clear that QTc prolongation is a more robust predictor of torsadogenicity than Tp–Te in the male guinea pig heart.

Synchronization of spontaneous calcium oscillations in the intact heart. An experimental/theoretical correlative study

We have recently reported that spontaneous calcium oscillations (S-CaOs) triggering propagated membrane depolarizations can contribute to arrhythmogenesis in the setting of ischemia/reperfusion (I/R). In a perfused Langendorff guinea pig heart preparation subjected to global I/R, simultaneous optical mapping of intracellular calcium transient and membrane voltage showed, in a majority of experiments, that once S-CaOs developed they were either followed every basic beat resulting in a bigeminal rhythm or became sustained resulting in a tachyarrhythmia. In some experiments, S-CaOs seemed to occur sporadically following some but not all basic beats. To investigate the mechanism of synchronization of S-CaOs in the intact heart resulting in a localized dominant focal activity we developed a theoretical model of pacemaker cells coupled to excitable cells. An array of excitable cells coupled to a small number of oscillatory cells were modeled using modified FHN equations. Small changes in both the intrinsic period of the oscillatory cells and the diffusion coefficient resulted in transitions similar to those seen in experiments. The behavior of the model strongly suggests that intermittent synchronization of S-CaOs in the intact heart can result in sporadic focal propagated responses.

Protective effects of sarpogrelate, a 5-HT2A antagonist, against postischemic myocardial dysfunction in guinea-pig hearts

The protective effects of sarpogrelate (SG), a 5-HT2A antagonist, were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Changes in cellular levels of high phosphorous energy, NO and Ca2+ in the heart together with simultaneous recordings of left ventricular developed pressure (LVDP) were monitored using an nitric oxide (NO) electrode, fluorometry and 31P-NMR. The recovery of LVDP from ischemia by reperfusion was 30.1% in the control, while the treatment with SG (5 x 10(-7) M) in pre- and post-ischemia hearts produced a gradual increase to 73.1 and 53.6%, respectively. At the final stage of ischemia, the intracellular concentration of Ca2+ ([Ca2+]i) and release of NO increased with no twitching and remained at a high steady level. The addition of SG increased the transient NO signal (TNO) level at the end of ischemia compared with the control, but [Ca2+]i during ischemia decreased. Meanwhile, mitochondrial Ca2+ uptake on acidification or Ca2+ content changes of the perfusate was suppressed by pre-treatment with SG or the KATP channel opener diazoxide, but not the KATP channel blocker 5-HD. The myocardial NO elevated with 5-HT in normal Langendorff hearts was suppressed by the treatment with SG. Therefore, the existence of the 5HT2A receptor in a Langendorff heart was anticipated. By in vitro EPR, SG was found to directly quench the hydroxy radical. Thus, these findings suggested that the 5-HT2A receptor induced in ischemia-reperfusion plays an important role in the mitochondrial KATP channel of hearts in close relation with NO and active oxygen radicals.

Differences in the effects of Na +–H + exchange inhibitors on cardiac function and apoptosis in guinea-pig ischemia-reperfused hearts

The protective effects of the Na +–H + exchange (NHE) inhibitors SM-198110 (2-[[(aminoiminomethyl) amino] carbonyl]-4-chloro-1 H-indole-1-propanesulfonic acid monohydrate) and SM-197378 ( N-(aminoiminomethyl)-1-methyl-7-(sulfooxy)-4-(trifluoromethyl)-1 H-indole-2-carboxamide monohydrate) were investigated in perfused Langendorff guinea-pig hearts subjected to ischemia (40 min) and reperfusion (40 min). The recovery of left ventricular developed pressure (LVDP) from ischemia by reperfusion was 39.0% in the control, while in the hearts pretreated with SM-198110 or SM-197378 (10 −7 M), it was about 100%. The ATP level, monitored simultaneously by 31P-nuclear magnetic resonance spectrometry, was already higher than the control value at the end of the ischemic period, and the elevation in Na + or Ca 2+ fluorometric signals induced during ischemia was suppressed. In post-treated hearts, the LVDP recovery rate was significantly higher with SM-198110 than with SM-197378. By in vitro electron paramagnetic resonance spectrometry, SM-197378 was found to directly quench the active oxygen radical, whereas SM-198110 had no effect. Numbers of apoptotic cardiomyocytes after ischemia (1 h) followed by reperfusion (5 h) were significantly lower in SM-197378-treated than in SM-198110-treated hearts, consistent with the level of activity of caspase-3. These results suggest that the antioxidant effects of NHE inhibitors have an important role in apoptosis during ischemia–reperfusion, but apoptosis is not a major manifestation of cardiac function during postischemic recovery, and that NHE-sensitive mechanisms of reperfusion injury promote both necrotic and apoptotic processes death.

Contrasting effects of ischemia on the kinetics of membrane voltage and intracellular calcium transient underlie electrical alternans.

Repolarization alternans has been considered a strong marker of electrical instability. The objective of this study was to investigate the hypothesis that ischemia-induced contrasting effects on the kinetics of membrane voltage and intracellular calcium transient (Ca(i)T) can explain the vulnerability of the ischemic heart to repolarization alternans. Ischemia-induced changes in action potential (AP) and Ca(i)T resulting in alternans were investigated in perfused Langendorff guinea pig hearts subjected to 10-15 min of global no-flow ischemia followed by 10-15 min of reperfusion. The heart was stained with 100 microl of rhod-2 AM and 25 microl of RH-237, and AP and Ca(i)T were simultaneously recorded with an optical mapping system of two 16 x 16 photodiode arrays. Ischemia was associated with shortening of AP duration (D) but delayed upstroke, broadening of peak, and slowed decay of Ca(i)T resulting in a significant increase of Ca(i)T-D. The changes in APD were spatially heterogeneous in contrast to a more spatially homogeneous lengthening of Ca(i)T-D. Ca(i)T alternans could be consistently induced with the introduction of a shorter cycle when the upstroke of the AP occurred before complete relaxation of the previous Ca(i)T and generated a reduced Ca(i)T. However, alternans of Ca(i)T was not necessarily associated with alternans of APD, and this was correlated with the degree of spatially heterogeneous shortening of APD. Sites with less shortening of APD developed alternans of both Ca(i)T and APD, whereas sites with greater shortening of APD could develop a similar degree of Ca(i)T alternans but slight or no APD alternans. This resulted in significant spatial dispersion of APD. The study shows that the contrasting effects of ischemia on the duration of AP and Ca(i)T and, in particular, on their spatial distribution explain the vulnerability of ischemic heart to alternans and the increased dispersion of repolarization during alternans.