Negative Inotropy Research Articles

Thyronamines—Past, Present, and Future

Thyronamines (TAMs) are a newly identified class of endogenous signaling compounds. Their structure is identical to that of thyroid hormone and deiodinated thyroid hormone derivatives, except that TAMs do not possess a carboxylate group. Despite some initial publications dating back to the 1950s, TAMs did not develop into an independent area of research until 2004, when they were rediscovered as potential ligands to a class of G protein-coupled receptors called trace-amine associated receptors. Since this discovery, two representatives of TAMs, namely 3-iodothyronamine (3-T(1)AM) and thyronamine (T(0)AM), have been detected in vivo. Intraperitoneal or central injection of 3-T(1)AM or T(0)AM into mice, rats, or Djungarian hamsters caused various prompt effects, such as metabolic depression, hypothermia, negative chronotropy, negative inotropy, hyperglycemia, reduction of the respiratory quotient, ketonuria, and reduction of fat mass. Although their physiological function remains elusive, 3-T(1)AM and T(0)AM have already revealed promising therapeutic potential because they represent the only endogenous compounds inducing hypothermia as a prophylactic or acute treatment of stroke and might thus be expected to cause fewer side effects than synthetic compounds. This review article summarizes the still somewhat scattered data on TAMs obtained both recently and more than 20 yr ago to yield a complete and updated picture of the current state of TAM research.

Physiological evidence for β3-adrenoceptor in frog ( Rana esculenta) heart

β3-Adrenergic receptors (ARs) have been recently identified in mammalian hearts where, unlike β1- and β2-ARs, induce cardio-suppressive effects. The aim of this study was to describe β3-AR role in the frog ( Rana esculenta) heart and to examine its signal transduction pathway. The presence of β3-AR, by using Western blotting analysis, has been also identified. BRL 37344 , a selective β3-AR agonist, induced a dose-dependent negative inotropic effect at concentrations from 10 −12 to 10 −6 M. This effect was not modified by nadolol (β1/β2-AR antagonist) and by phentolamine (α-AR antagonist), but it was suppressed by the β3-AR-specific antagonist SR 59230 and by exposure to the Gi/o proteins inhibitor Pertussis Toxin. In addition, the involvement of EE-NOS-cGMP-PKG/PDE2 pathway in the negative inotropism of BRL 37344 has been assessed. BRL 37344 treatment induced eNOS and Akt phosphorylation as well as an increase of cGMP levels. β3-ARs activation induce a non-competitive antagonism against ISO stimulation which disappeared in presence of PKG and PDE2 inhibition. Taken together our findings provide, for the first time in the frog, a role for β3-ARs in the cardiac performance modulation which involves Gi/o protein and occurs via an EE-NO-cGMP-PKG/PDE2 cascade.

Negative Inotropy by Angiotensin II Is Mediated via Phosphoinositide 3-Kinase Alpha–Protein Kinase C-Coupled Signaling Pathway

Elevated plasma levels of angiotensin II have been identified as a key determinant of cardiac hypertrophy and congestive heart failure. Angiotensin II signaling mediates its biological actions in part by altering intracellular sarcoplasmic reticulum/endoplasmic reticulum calcium stores and voltage-dependent L-type Ca2+ channels.1 Calcium concentration is directly involved in cardiac excitation–contraction coupling and plays a central role in intracellular signaling pathways. Because calcium is the physiological activator of the contractile proteins, chronic elevations of cardiac angiotensin II-altered calcium handling have been postulated to underlie the calcium overload and contractile dysfunction after myocardial infarction.2 Kamp and Hell3 have suggested involvement of protein kinase A (PKA) and protein kinase C (PKC) pathways in the modulation of intracellular calcium stores and contractility. This is consistent with the reported ability of angiotensin II to promote cardiac hypertrophy via the phosphoinositide 3-kinase (PI3K)/Akt-dependent pathway.4,5 In this regard, PI3K transduces extracellular signals into cellular function by regulating activity of several genes downstream of angiotensin II type 1 receptor (AT1R).6 Based on sequence homology and substrate preference, 8 members of the PI3K family have been identified. The most widely studied isoforms of the PI3K family include PI3Kα and PI3Kγ, which regulate a variety of biological actions, including L-type calcium channel activity7 and genes associated with cell growth and survival.5 Although chronic angiotensin II stimulation reportedly leads to cardiac remodeling,8 metabolic alterations,9 cardiac fibrosis,10 and hypertrophy,5 little is known of the acute phase effects of angiotensin II on cardiomyocyte contractility. By definition, inotropy relates to alterations in contractile force generation independent of sarcomere length. It is mainly dictated by changes in activator calcium …

Modulation of L-type Ca2+ channels by beta3-adrenoceptor activation and the involvement of nitric oxide.

Both beta1- and beta2-adrenoceptors (AR) in cardiac tissues are responsible for the excitatory effect of catecholamines. Recent evidence demonstrated the presence of another subtype of beta-AR (beta3-AR) in cardiac ventricular preparation. Activation of beta3-AR elicited a depressant response on ventricular contraction. The underlying mechanism(s) for the negative inotropism is relatively unknown. We investigated the effects of beta3-AR activation on basal voltage-dependent Ca2+ channel (I(CaL)) amplitude of the guinea pig enzyme-dissociated single ventricular myocytes using amphotericin B (200 microg/mL) perforated-patch whole-cell patch-clamp techniques (approximately 22 degrees C). Application of (-)-isoprenaline ((-)-ISO) (100 nM, a nonselective beta-AR agonist) increased the basal I(CaL) amplitude (approximately 210% of control) (n = 8). However, in the presence of nadolol (1 degreesM, a beta1-/beta2-AR antagonist), the stimulatory effect of (-)-ISO on I(CaL )was abolished and a slowly developed inhibition of the basal I(CaL) was recorded (approximately 80% of control) (n = 9). A smaller degree of inhibition was observed with BRL 37344 (100 nM, a selective beta3-AR agonist) (58% of control) (n = 7). The inhibitory effect of (-)-ISO (with nadolol) and BRL 37344 persisted after washout. Pretreating the ventricular myocytes with L-NAME (0.3 microM, a nitric oxide synthase inhibitor), but not D-NAME (0.3 microM), abolished the inhibitory effect of (-)-ISO and BRL 37344 (n = 7-9). The results suggest that beta3-ARs are present in ventricular myocytes. Activation of the beta3-AR resulted in an inhibition of the basal I(CaL) amplitude probably due to the formation of nitric oxide.

Ventricular Dilation and Electrical Dyssynchrony Synergistically Increase Regional Mechanical Nonuniformity But Not Mechanical Dyssynchrony

Background— Heart failure (HF) in combination with mechanical dyssynchrony is associated with a high mortality rate. To quantify contractile dysfunction in patients with HF, investigators have proposed several indices of mechanical dyssynchrony, including percentile range of time to peak shortening (WTpeak), circumferential uniformity ratio estimate (CURE), and internal stretch fraction (ISF). The goal of this study was to compare the sensitivity of these indices to 4 major abnormalities responsible for cardiac dysfunction in dyssynchronous HF: dilation, negative inotropy, negative lusitropy, and dyssynchronous activation. Methods and Results— All combinations of these 4 major abnormalities were included in 3D computational models of ventricular electromechanics. Compared with a nonfailing heart model, ventricles were dilated, inotropy was reduced, twitch duration was prolonged, and activation sequence was changed from normal to left bundle branch block. In the nonfailing heart, CURE, ISF, and WTpeak were 0.97±0.004, 0.010±0.002, and 78±1 milliseconds, respectively. With dilation alone, CURE decreased 2.0±0.07%, ISF increased 58±47%, and WTpeak increased 31±3%. With dyssynchronous activation alone, CURE decreased 15±0.6%, ISF increased 14-fold (±3), and WTpeak increased 121±4%. With the combination of dilation and dyssynchronous activation, CURE decreased 23±0.8%, ISF increased 20-fold (±5), and WTpeak increased 147±5%. Conclusions— Dilation and left bundle branch block combined synergistically decreased regional cardiac function. CURE and ISF were sensitive to this combination, but WTpeak was not. CURE and ISF also reflected the relative nonuniform distribution of regional work better than WTpeak. These findings might explain why CURE and ISF are better predictors of reverse remodeling in cardiac resynchronization therapy.

Esmolol cardioplegia: the cellular mechanism of diastolic arrest

Esmolol, an ultra-short-acting beta-blocker, acts as a cardioplegic agent at millimolar concentrations. We investigated the mechanism by which esmolol induces diastolic ventricular arrest. In unpaced Langendorff-perfused rat hearts, esmolol (0.03-3 mmol/L) had a profound negative inotropic effect resulting in diastolic arrest at 1 mmol/L and above. This inhibition of contraction was maintained during ventricular pacing. At 3 mmol/L, esmolol also abolished action potential conduction. To determine the cellular mechanism for the negative inotropism, we measured contraction (sarcomere shortening) and the calcium transient (fura-2 fluorescence ratio; Ca(tr)) in electrically-stimulated rat ventricular myocytes at 23 and 34 degrees C. The decrease in contraction (by 72% at 23 degrees C, from 0.16 +/- 0.01 to 0.04 +/- 0.01 microm, P < 0.001) was similar to that of isolated hearts and was caused by a large decrease in Ca(tr) (from 0.13 +/- 0.02 to 0.07 +/- 0.02, P < 0.001). There was no additional effect on myofilament Ca(2+) sensitivity. Esmolol's effects on contraction and Ca(tr) were not shared or altered by the beta-blocker, atenolol (1 mmol/L). Sarcoplasmic reticulum inhibition with thapsigargin did not alter the inhibitory effects of esmolol. Whole-cell voltage-clamp experiments revealed that esmolol inhibited the L-type calcium current (I(Ca,L)) and the fast sodium current (I(Na)), with IC(50) values of 0.45 +/- 0.05 and 0.17 +/- 0.025 mmol/L, respectively. Esmolol at millimolar concentrations causes diastolic ventricular arrest by two mechanisms: at 1 mmol/L (and below), the pronounced negative inotropic effect is due largely to inhibition of L-type Ca(2+) channels; additionally, higher concentrations prevent action potential conduction, probably due to the inhibition of fast Na(+) channels.

Distinct signalling mechanisms are involved in the dissimilar myocardial and coronary effects elicited by quercetin and myricetin, two red wine flavonols

Background and Aims Moderate red wine consumption associates with lower incidence of cardiovascular diseases. Attention to the source of this cardioprotection was focused on flavonoids, the non-alcoholic component of the red wine, whose intake inversely correlates with adverse cardiovascular events. We analysed whether two red wine flavonoids, quercetin and myricetin, affect mammalian basal myocardial and coronary function. Methods and results Quercetin and myricetin effects were evaluated on isolated and Langendorff perfused rat hearts under both basal conditions and α- and β-adrenergic stimulation. The intracellular signalling involved in the effects of these flavonoids was analysed on perfused hearts and by western blotting on cardiac and HUVEC extracts. Quercetin induced biphasic inotropic and lusitropic effects, positive at lower concentrations and negative at higher concentrations. Contrarily, Myricetin elicits coronary dilation, without affecting contractility and relaxation. Simultaneous administration of the two flavonoids only induced vasodilation. Quercetin-elicited positive inotropism and lusitropism depend on β1/β2-adrenergic receptors and associate with increased intracellular cAMP, while the negative inotropism and lusitropism observed at higher concentrations were α-adrenergic-dependent. NOS inhibition abolished Myricetin-elicited vasodilation, also inducing Akt, ERK1/2 and eNOS phosphorylation in both ventricles and HUVEC. Myricetin-dependent vasodilation increases intracellular cGMP and is abolished by triton X-100. Conclusions The cardiomodulation elicited on basal mechanical performance by quercetin and the selective vasodilation induced by myricetin point to these flavonoids as potent cardioactive principles, able to protect the heart in the presence of cardiovascular diseases.

Accelerated whole-heart 3D CSPAMM reveals impact of beta-blocker therapy on myocardial architecture

Results In six volunteers a shift of shortening curves towards earlier heart phases (left-shift) and a decrease in the areaunder-the-curve (work) was noted. In three volunteers no significant change in phase but decrease in amplitude and work was observed. One proband showed a significant right-shift (negative inotropy) from the start. This proband was investigated a second time starting at a very low dose of 5 μg/kg/min resulting in a left-shift up to 25 μg/kg/min and a reproducible right-shift from 50 to 150 μg/kg/min. Vital parameters remained stable at all dosages in all volunteers.

Total β-adrenoceptor deficiency results in cardiac hypotrophy and negative inotropy

The present study investigated cardiac function in hearts of mice with total deficiency of the beta1-, beta2- and beta3-adrenoceptors (TKO) in comparison to wildtype mice (WT). We investigated cardiac morphology and echocardiographic function, measured protein expression of Ca2+-regulatory proteins, SERCA 2a activity, myofibrillar function, and performed running wheel tests. Heart weight and heart-to-body weight ratio were significantly smaller in TKO as compared to WT. This was accompanied by a decrease in the size of the cardiomyocytes in TKO. Heart rate and ejection fraction were significantly diminished in TKO as compared to WT. Protein expressions of SERCA 2a, ryanodine receptor and Na+/Ca2)-exchanger were similar in TKO and WT mice, but phospholamban protein expression was increased. PKA-dependent phosphorylation of phospholamban at serine 16 was absent and CaMKII-dependent phosphorylation at threonine 17 was decreased in TKO. All alterations were paralleled by a decrease in SERCA 2a-activity. A similar maximal calcium-dependent tension but an increased myofibrillar calcium-sensitivity was measured in TKO as compared to WT. We did not observe relevant functional impairments of TKO in running wheel tests. In the absence of beta-agonistic stimulation, SERCA 2a activity is mainly regulated by alterations of phospholamban expression and phosphorylation. The decreased SERCA 2a activity following beta-adrenoceptor deficiency may be partly compensated by an increased myofibrillar calcium-sensitivity.

Cannabinoid (CB) Receptors are not Involved in A-955840 Induced Negative Inotropic Effects in Isolated Cardiac Myocytes

A-955840, a selective CB2 agonist, has been shown to elicit concentration-dependent decreases in cardiac contractility in the anesthetized dog (decreased maximal velocity of left ventricular pressure development [LV dP/dt max]). However, it is unknown whether this represents a direct effect or a response dependent on other factors (such as autonomic tone and neurohumoral factors) present in vivo. This study examined if A-955840 had a direct effect on contractility of isolated cardiac myocytes, and if so to determine the potential involvement of CB1 and CB2 receptors. Contractility was assessed in vitro using percent changes in maximal shortening velocity of sarcomeres (dL/dt max) and fractional shortening of sarcomere length (FS) in rabbit left ventricular myocytes. A-955840 reduced dL/dt max and FS in a reversible and concentration-dependent manner with an IC50 of 11.4 μg/mL (based on dL/dt max) which is similar to the IC50 value of 5.5 μg/mL based on the effects of A-955840 on LV dP/dt max in anesthetized dogs. A-955840 (4.0 μg/mL) reduced myocyte contractility (%FS) to a similar extent in the absence and presence of a CB2 antagonist, SR-2 (24.0 ± 3.4 vs. 23.1 ± 3.0 %, n=5) or a CB1 antagonist, rimonabant (18.8 ± 2.3 vs. 19.8 ± 2.7 %, n=5). A-955840 (4.0 μg/mL) also reduced L-type calcium current of rabbit ventricular myocytes (1.05 ± 0.11 vs. 0.70 ± 0.12 nA, n=5, P<0.01). These results suggest that A-955840 exerts direct negative inotropic effects on isolated rabbit ventricular myocytes, which is mediated by neither CB1 nor CB2 receptors, and consistent with off-target negative inotropy mediated by inhibition of the cardiac L-type calcium current.

The Effect of Levosimendan on Bupivacaine-Induced Severe Myocardial Depression in Anesthetized Pigs

Levosimendan, an inodilator without proarrhythmogenic properties, has been shown to reverse ropivacaine-induced negative inotropy in isolated heart preparations. In this randomized and blinded study, we investigated whether levosimendan is able to reverse rapidly bupivacaine-induced myocardial depression in pigs. Twenty invasively monitored pigs anesthetized with isoflurane 1% received bupivacaine 2 mg/kg per minute into a central vein until mean arterial pressure decreased to 55% of baseline. Thereafter, levosimendan 80 microg/kg for 10 mins, followed by 0.7 microg/kg per minute during the next 50 mins (L-SIM) or corresponding amounts of placebo were administered intravenously. Simultaneously, Ringer's acetate was infused intravenously, 20 mL/kg for 10 mins, followed by 20 mL/kg for 50 mins. Two pigs in each group developed cardiac arrest immediately after bupivacaine and could not be resuscitated. Bupivacaine induced widening of the QRS complex in the electrocardiogram and bradycardia.In the remaining 16 pigs, 3 (2 in L-SIM group and 1 in placebo group) needed short-lasting manual cardiac compression and 1 dose of epinephrine. Cardiac output, ejection fraction, and stroke power/end-diastolic volume recovered initially very rapidly in the L-SIM group.However, there was no time x group effect difference in the overall recovery in the various parameters between the 2 groups, except in heart rate which was higher (P G 0.05) when levosimendan was administered.During the 50-min levosimendan infusion, mean arterial pressure and systemic vascular resistance stayed slightly lower in comparison with placebo infusion, but the difference was not statistically significant. Levosimendan together with the infusion of Ringer's solution rapidly reversed the cardiac depression, but there was no difference in overall cardiovascular recovery in comparison to treatment with Ringer's solution alone. Levosimendan-induced increase in heart rate possibly facilitated the recovery from bupivacaine intoxication.

Cytoskeleton mediates negative inotropism and lusitropism of chromogranin A-derived peptides (human vasostatin1-78 and rat CgA 1-64) in the rat heart

Cytoskeleton scaffold in cardiac myocytes provides structural support and compartmentalization of intracellular components. It is implicated in cardiac pathologies including hypertrophy and failure, playing a key role in the determinism of contractile and diastolic dysfunctions. Chromogranin A (CgA) and its derived peptides have revealed themselves as novel cardiovascular modulators. In humans, normal CgA levels considerably increase in several pathologies, including heart failure. Recent data have shown on the unstimulated rat heart that human recombinant Vasostatin-1 (hrVS-1) and rat chromogranin A 1-64 (rCgA 1-64) induce negative inotropic and lusitropic effects counteracting the β-adrenergic-dependent positive inotropism with a functional non-competitive antagonism. This study investigates, on the isolated Langendorff perfused rat heart, whether cardiac cytoskeleton is involved in the modulation of contractility and relaxation exerted by hrVS-1 and rCgA 1-64. Cytoskeleton impairment by either cytochalasin-D (actin polymerization inhibitor), BDM (myosin ATP-ase antagonist) or wortmannin (inhibitor of PI3-K/Akt transduction cascade), or W-7 (calcium–calmodulin antagonist) abolished hrVS-1 and rCgA 1-64-mediated inotropism and lusitropism. Using fluorescent phalloidin, we showed on rat cardiac H9C2 cells that hrVS-1 (10 nM ÷ 10 µM) stimulates actin polymerization. Taken together these data indicate that in the rat heart, the actin cytoskeletal network strongly contributes to the cardiotropic action of CgA-derived peptides.

Left Ventricular and Myocardial Function in Mice Expressing Constitutively Pseudophosphorylated Cardiac Troponin I

Protein kinase (PK)C-induced phosphorylation of cardiac troponin (cTn)I has been shown to regulate cardiac contraction. Characterize functional effects of increased PKC-induced cTnI phosphorylation and identify underlying mechanisms using a transgenic mouse model (cTnI(PKC-P)) expressing mutant cTnI (S43E, S45E, T144E). Two-dimensional gel analysis showed 7.2+/-0.5% replacement of endogenous cTnI with the mutant form. Experiments included: mechanical measurements (perfused isolated hearts, isolated papillary muscles, and skinned fiber preparations), biochemical and molecular biological measurements, and a mathematical model-based analysis for integrative interpretation. Compared to wild-type mice, cTnI(PKC-P) mice exhibited negative inotropy in isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, F(max)). Additionally, cTnI(PKC-P) mice exhibited slowed relaxation in both isolated hearts and intact papillary muscles. The cTnI(PKC-P) mice showed no differences in calcium sensitivity, cooperativity, steady-state force-MgATPase relationship, calcium transient (amplitude and relaxation), or baseline phosphorylation of other myofilamental proteins. The model-based analysis revealed that experimental observations in cTnI(PKC-P) mice could be reproduced by 2 simultaneous perturbations: a decrease in the rate of cross-bridge formation and an increase in calcium-independent persistence of the myofilament active state. A modest increase in PKC-induced cTnI phosphorylation ( approximately 7%) can significantly alter cardiac muscle contraction: negative inotropy via decreased cross-bridge formation and negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 versus T144).

Levosimendan as a Rescue Drug in Experimental Propranolol-Induced Myocardial Depression: A Randomized Study

Severe beta-blocker intoxication remains a clinical challenge despite a variety of treatment options. Because of its unique mechanism of action, the new calcium sensitizer levosimendan may provide more prominent cardiac support compared with current medications used to reverse negative inotropy. We hypothesize that levosimendan could reverse propranolol-induced severe negative inotropy in a porcine model of beta-blocker intoxication. Twenty-four pigs were anesthetized and monitored. After severe propranolol intoxication was completed, animals were randomized into 3 groups. With a double-blind procedure, 9 animals received a 1.25-mg levosimendan bolus, followed by saline solution infusion, 9 animals received mean arterial pressure-targeted dobutamine infusion after saline solution bolus, and 6 animals received a saline solution bolus followed by saline solution infusion. Hemodynamic and laboratory data were collected during a follow-up period of 120 minutes. All 9 pigs in the levosimendan group survived. In contrast, 4 of 6 (67%) and 7 of 9 (78%) pigs died during the experiment in the placebo and the dobutamine groups, respectively. The levosimendan group showed improved change in the maximum positive slope of the left ventricular pressure, cardiac output, stroke volume, and mean arterial pressure compared with the dobutamine and the placebo groups. Levosimendan improved hemodynamic function and survival in this animal model of severe propranolol intoxication. The potential clinical application of levosimendan in propranolol intoxication warrants further investigation.

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.

QA interval as an indirect measure of cardiac contractility in the conscious telemeterised rat: Model optimisation and evaluation

IntroductionConscious telemeterised rats are used for early assessment of cardiovascular drug safety. This paper details the surgery required to allow key cardiovascular parameters to be determined and describes how QA interval (QAI), a surrogate measure of cardiac contractility, is related to a more direct measure of cardiac contractility, dP/dtmax (LVdP/dtmax). Experimental studies examining the effect of both positive and negative inotropes are described. MethodsEight rats were surgically implanted with telemetry probes for measuring blood pressure, heart rate, activity, body temperature and ECG parameters. Initial studies focussed on evaluating surgical procedures aimed at improving the ECG signal to allow QAI quantification. Once achieved, experimental studies were undertaken with verapamil, salmeterol, milrinone or vehicle. Following treatment, data was collected for ~20 h from pair-housed animals to establish effects on QAI or LVdP/dtmax, which was measured directly from the left ventricle in a separate group of rats. ResultsThe modified ECG lead placement produced both a qualitative and quantitative improvement in ECGs. Increases in cardiac contractility (measured directly) were paralleled by a decrease in QAI (measured indirectly). Salmeterol and milrinone increased (all p<0.05) LVdP/dtmax (1942±452 and 2333±506 mmHg/s, respectively) and decreased QAI (3.5±0.54 and 2.9±0.43 ms, respectively). In contrast, the negative inotrope verapamil decreased LVdP/dtmax (–2119±300 mmHg/s) and increased QAI (4.9±0.46 ms). A statistically significant (p<0.001) linear relationship was identified between log10(QAI) and log10(LVdP/dtmax). DiscussionImprovements in ECG signal quality, resulting from refinements in surgical techniques allowed for the reliable quantification of ECG parameters. The linear relationship demonstrated between log10(QAI) and log10(LVdP/dtmax) indicates that QAI may be a useful index of cardiac contractility in routine rat cardiovascular safety studies. These limited studies support the use of QAI as an indirect measure of cardiac contractility in the rat.

The cardioprotective and inotropic components of the postconditioning effects of GLP-1 and GLP-1(9–36)a in an isolated rat heart

GLP-1 and its metabolite GLP-1(9–36)a have been shown to exert cardiotropic effects, and were demonstrated to be cardioprotective agents in isolated, postischemic rat or mouse hearts. An agent's total effect on myocardial performance in a postconditioning paradigm is a sum of its myocyte-preserving (cardioprotective) and contractility-affecting (negative or positive inotropic) action components. These components may not always be explicitly separated by the experimental protocol. We propose an analytical approach to identify and quantify the cardioprotective and inotropic components in a postconditioning protocol, as exemplified by use of GLP-1 and GLP-1(9–36)a following a global ischemia in isolated rat hearts. Peptides were administered during the first 15 min of 120 min reperfusion. GLP-1 0.3 nM reduced infarct size from 23.2 ± 2.4% to 14.1 ± 2.3% of area-at-risk ( n = 15, P = 0.0223), an effect abolished by the GLP-1 receptor antagonist, exendin(9–39) 5 nM. GLP-1 showed only a small, non-significant tendency to increase mechanical performance (increase of LVDP by 26.7%, P = 0.1621; RPP 33.5%, P = 0.0858; d P/d t max 28.5%, P = 0.1609). This could be accounted for by the cardioprotective component of GLP-1 action, rather than any true inotropic effect. In contrast, GLP-1(9–36)a did not reduce infarct size significantly, but acted as a strong negative inotrope in postischemic hearts, causing a contractility deficit (LVDP 58.8%, P = 0.0004; RPP 58.2%, P = 0.0007; d P/d t max = 58.2%, P = 0.0012), quantifiable by an analysis of infarct size-mechanical performance plots. These results help resolve certain apparent discrepancies between some of the published effects of GLP-1 and GLP-1(9–36)a.

Recent in vitro findings of negative inotropy of pantoprazole did not translate into clinically relevant effects on left ventricular function in healthy volunteers.

PurposeReports on cardiac problems with oral proton pump inhibitors have caused extensive safety reviews by the US Food and Drug Administration. We provide additional data on acute cardiac effects of an intravenous application.MethodsEchocardiography was performed in 18 healthy volunteers after administration of a common high-dose regimen of pantoprazole (80 mg i.v. bolus followed by 8 mg/h for 1 h) or placebo.DesignThe design included a randomized, double-blind, placebo-controlled cross-over trial.ResultsEjection fraction (%, mean ± SE) in the treatment group (placebo group) was 60.7 ± 1.1 (61.2 ± 1.7) at baseline, and 62.6 ± 1.1 (62.1 ± 1.9), 64.7 ± 1.6 (63.5 ± 1.3), 62.6 ± 1.6 (61.0 ± 1.6) and 63.0 ± 1.4 (61.8 ± 1.5) at 7.5, 15, 30 and 60 min after bolus application, respectively (p = n.s.). Similarly, no significant changes were found for cardiac output, cardiac index, blood pressure and heart rate. In contrast, gastric pH that was used as a treatment control was significantly increased 60 min after the application of pantoprazole as compared to baseline and to placebo.ConclusionsPantoprazole as injection is safe in healthy subjects with respect to cardiac contractile function. However, in view of recent reports of negative inotropy of the drug, further studies in heart failure patients are required.

Nitrite modulates contractility of teleost (Anguilla anguilla and Chionodraco hamatus, i.e. the Antarctic hemoglobinless icefish) and frog (Rana esculenta) hearts

Being the largest form of intravascular and tissue storage of nitric oxide (NO) and a signalling molecule itself, the nitrite anion (NO2−) has emerged as a key player in many biological processes. Since the heart is under an important NO-mediated autocrine–paracrine control, in mammals the cardiac effects of nitrite are under intensive investigation. In contrast, nothing is known in non-mammalian vertebrates. We evaluated nitrite influence on cardiac performance in the perfused beating heart of three different cold-blooded vertebrates, i.e. two teleost fishes, the temperate red-blooded Anguilla anguilla, the Antarctic stenotherm, hemoglobinless Chionodraco hamatus (icefish), and the frog Rana esculenta. We showed that, under basal conditions, in all animals nitrite influences cardiac mechanical performance, inducing negative inotropism in eel and frog, while being a positive inotrope in C. hamatus. In all species, these responses parallel the inotropic effects of authentic NO. We also demonstrated that the nitrite-dependent inotropic effects are i) dependent from NO synthase (NOS) activity in fish; ii) sensitive to NO scavenging in frog; iii) cGMP/PKG-dependent in both eel and frog. Results suggest that nitrite is an integral physiological source of NO and acts as a signalling molecule in lower vertebrate hearts, exerting relevant inotropic actions through different species-specific mechanisms.

Post-conditioning restores pre-ischaemic receptor coupling in rat isolated hearts.

Ischaemic preconditioning (IPC) and ischaemic post-conditioning (IPoC) activate signal transduction pathways that are also involved in receptor de- and re-sensitization such as phosphatidylinositol (PI) 3-kinase. Therefore, IPC and IPoC may affect post-infarct receptor coupling. Rat isolated hearts (Langendorff mode, constant flow) were exposed to 45 min flow arrest followed by 120 min reperfusion, including IPC or IPoC. Control hearts were perfused without a 45 min flow arrest. Left ventricular developed pressure (LVdevP) was determined. Thirty min after reperfusion, hearts were exposed to parathyroid hormone-related peptide (PTHrP) or isoprenaline for 10 min to monitor receptor responsiveness. Reperfusion injury was quantified by enzyme release. IPC and IPoC significantly reduced enzyme release compared with ischaemia and reperfusion alone by 75% and 62% respectively. Wortmannin or chelerythrine inhibiting either PI 3-kinase or protein kinase C, respectively, attenuated protection. Application of PTHrP 30 min after reperfusion did not change LVdevP in hearts exposed to ischaemia (+1 +/- 11%), but IPoC restored the normal and non-ischaemic response to PTHrP characterized by a negative inotropism (-8.3 +/- 3.9% and -12.9 +/- 6.1%). IPC restored a small negative inotropic effect (-4.4 +/- 4.7%). Application of a PTHrP receptor antagonist during the 45 min flow arrest attenuated receptor desensitization (DeltaLVdevP: -6.1 +/- 1.7%). Wortmannin but not chelerythrine attenuated the re-sensitizing effect of IPoC on post-ischaemic receptor coupling (DeltaLVdevP: +6.2 +/- 10.5 and -15.0 +/- 7.7%). As observed with PTHrP receptors, IPoC restored beta-adrenoceptors (DeltaLVdevP: +9.3 +/- 11.8% vs. 62.3 +/- 15.8%). IPoC restores PTHrP receptor coupling in a PI 3-kinase-dependent way. A similar mechanism may allow beta-adrenoceptor re-sensitization.