Negative Force-frequency Relationship Research Articles

Angiotensin-converting enzyme inhibitor improves force and Ca2+-frequency relationships in myocytes from rats with heart failure

Objectives — The objectives were to study the effects of an ACE inhibitor on the force-frequency relationship (FFR) and its possible mechanism in isolated failing myocytes.Methods and results — Male Wistar rats were randomized into a heart failure group treated with perindopril (CHF-T, 3mg.kg-1.d-1), a heart failure group without treatment (CHF-C) and a sham-operated group (PS). Heart failure was induced by constriction of the abdominal aorta. All groups were further followed up for 12weeks. Left ventricular myocytes were isolated. Cell-shortening fraction (FS) and intracellular calcium transients were measured at different frequency field stimulations. A negative force-frequency relationship (FFR) was found in the CHF-C group compared with the positive-negative biphasic FFR in the PS group, and a flat FFR in the CHF-T group. Intracellular Ca2+ frequency relationships (CaFRs) were positive-negative biphasic in both the PS and CHF-T groups, whereas a negative CaFR was found in the CHF-C group. Regardless of the stimulation frequency, FS significantly correlated with [Ca2+]imax in the PS or CHF-C groups. Compared to the PS group, protein levels of SERCA2 significantly decreased and NCX1 increased in the CHF-C group. In the CHF-T group, these changes were reduced.Conclusion —The ACE inhibitor could improve the impaired FFR of isolated failing myocytes.This effect was possibly mediated via ameliorating the disturbance of CaFR.

Effect of intracellular Ca2+ and action potential duration on L-type Ca2+ channel inactivation and recovery from inactivation in rabbit cardiac myocytes

Ca(2+) current (I(Ca)) recovery from inactivation is necessary for normal cardiac excitation-contraction coupling. In normal hearts, increased stimulation frequency increases force, but in heart failure (HF) this force-frequency relationship (FFR) is often flattened or reversed. Although reduced sarcoplasmic reticulum Ca(2+)-ATPase function may be involved, decreased I(Ca) availability may also contribute. Longer action potential duration (APD), slower intracellular Ca(2+) concentration ([Ca(2+)](i)) decline, and higher diastolic [Ca(2+)](i) in HF could all slow I(Ca) recovery from inactivation, thereby decreasing I(Ca) availability. We measured the effect of different diastolic [Ca(2+)](i) on I(Ca) inactivation and recovery from inactivation in rabbit cardiac myocytes. Both I(Ca) and Ba(2+) current (I(Ba)) were measured. I(Ca) decay was accelerated only at high diastolic [Ca(2+)](i) (600 nM). I(Ba) inactivation was slower but insensitive to [Ca(2+)](i). Membrane potential dependence of I(Ca) or I(Ba) availability was not affected by [Ca(2+)](i) <600 nM. Recovery from inactivation was slowed by both depolarization and high [Ca(2+)](i). We also used perforated patch with action potential (AP)-clamp and normal Ca(2+) transients, using various APDs as conditioning pulses for different frequencies (and to simulate HF APD). Recovery of I(Ca) following longer APD was increasingly incomplete, decreasing I(Ca) availability. Trains of long APs caused a larger I(Ca) decrease than short APD at the same frequency. This effect on I(Ca) availability was exacerbated by slowing twitch [Ca(2+)](i) decline by approximately 50%. We conclude that long APD and slower [Ca(2+)](i) decline lead to cumulative inactivation limiting I(Ca) at high heart rates and might contribute to the negative FFR in HF, independent of altered Ca(2+) channel properties.

Multiple Alterations in Ca2+ Handling Determine the Negative Staircase in a Cellular Heart Failure Model

The flat or negative force frequency relationship (FFR) is a hallmark of the failing heart. Either decreases in SERCA2a expression, increases in Na(+)/Ca(2+) exchanger (NCX) expression or elevated Na(+)(i) have been independently proposed as mediators of the negative FFR. To determine whether each one of these mechanisms is sufficient to account for the negative FFR of the failing heart or on the contrary, various mechanisms, acting in concert are required. SERCA2a was pharmacologically inhibited with thapsigargin (TG) or cyclopiazonic acid (CPA) or by using siRNA technology; Na(+)(i) was increased with either ouabain (Oua) or monensin and NCX protein was overexpressed by gene transfer (Ad.NCX), to mimic in nonfailing cat myocytes the phenotype of the failing heart and examine their effect on the FFR. The positive FFR of healthy myocytes remained unaffected after either SERCA2a inhibition, Na(+)(i) elevation, or NCX overexpression. However, the combination of TG + Oua, Oua + Ad.NCX, or TG + Ad.NCX, converted the positive FFR to negative. Moreover, the FFR became negative at lower frequencies, when the 3 interventions were combined. Ca(2+) handling has to be altered at several levels to explain the negative FFR of the failing heart. These anomalies in Ca(2+) homeostasis acting in synergy have additive effects.

Neonatal Murine Heart Slices. A Robust Model to Study Ventricular Isometric Contractions

Background/Aims: Cardiac function is increasingly studied using murine models. However, current multicellular preparations to investigate contractile properties have substantial technical and biological limitations and are especially difficult to apply to the developing murine heart. Methods: Newborn murine hearts were cut with a vibratome into viable tissue slices. The structural and functional integrity of the tissue was shown by histology, ATP content and sharp electrode recordings. Results: Within the first 48 hours after slicing structure remained intact without induction of apoptosis. ATP concentrations and action potential parameters were comparable to those of physiological tissue. Isometric force measurements demonstrated a physiological force-frequency relationship with a ‘primary-phase’ negative force-frequency relationship up to 1-2 Hz and a ‘secondary-phase’ positive force-frequency relationship up to 8 Hz. (-)-Isoproterenol (10^-6 mol/l) increased active force to 251±35% (n=15) of baseline values and shortened relaxation times indicating a preserved beta-adrenergic regulation of contraction. Changes of the force-frequency relationship after application of ryanodine and nifedipine indicated functionality of calcium release from the sarcoplasmic reticulum and of L-type calcium channels. Conclusion: Generation of viable, physiological intact ventricular slices from neonatal hearts is feasible and provides a robust model to study loaded contractions.

Myocardial dysfunction and neurohumoral activation without remodeling in left ventricle of monocrotaline-induced pulmonary hypertensive rats

In monocrotaline (MCT)-induced pulmonary hypertension (PH), only the right ventricle (RV) endures overload, but both ventricles are exposed to enhanced neuroendocrine stimulation. To assess whether in long-standing PH the left ventricular (LV) myocardium molecular/contractile phenotype can be disturbed, we evaluated myocardial function, histology, and gene expression of autocrine/paracrine systems in rats with severe PH 6 wk after subcutaneous injection of 60 mg/kg MCT. The overloaded RV underwent myocardial hypertrophy (P < 0.001) and fibrosis (P = 0.014) as well as increased expression of angiotensin-converting enzyme (ACE) (8-fold; P < 0.001), endothelin-1 (ET-1) (6-fold; P < 0.001), and type B natriuretic peptide (BNP) (15-fold; P < 0.001). Despite the similar upregulation of ET-1 (8-fold; P < 0.001) and overexpression of ACE (4-fold; P < 0.001) without BNP elevation, the nonoverloaded LV myocardium was neither hypertrophic nor fibrotic. LV indexes of contractility (P < 0.001) and relaxation (P = 0.03) were abnormal, however, and LV muscle strips from MCT-treated compared with sham rats presented negative (P = 0.003) force-frequency relationships (FFR). Despite higher ET-1 production, BQ-123 (ET(A) antagonist) did not alter LV MCT-treated muscle strip contractility distinctly (P = 0.005) from the negative inotropic effect exerted on shams. Chronic daily therapy with 250 mg/kg bosentan (dual endothelin receptor antagonist) after MCT injection not only attenuated RV hypertrophy and local neuroendocrine activation but also completely reverted FFR of LV muscle strips to positive values. In conclusion, the LV myocardium is altered in advanced MCT-induced PH, undergoing neuroendocrine activation and contractile dysfunction in the absence of hypertrophy or fibrosis. Neuroendocrine mediators, particularly ET-1, may participate in this functional deterioration.

The effect of direct autonomic nerve stimulation on left ventricular force in the isolated innervated Langendorff perfused rabbit heart

The relative contribution of the chronotropic effects of stimulating sympathetic and vagus nerves on cardiac inotropic changes in the isolated Langendorff perfused rabbit heart with intact dual autonomic nerves was studied. The force–frequency relationship was investigated, in addition to sympathetic nerve stimulation (SS) at 2 Hz (low), 5 Hz (med) and 10 Hz (high), and left and right vagus nerve stimulation (VS) studied at 2 Hz (low), 5 Hz (med) and 7 Hz (high) with and without right ventricular pacing. It was shown that a biphasic force–frequency relationship is present with a positive relationship at low heart rates and a negative force–frequency relationship at higher heart rates. There was a trend for left- and right-VS to decrease left ventricular pressure with a decrease in heart rate, whilst SS had the opposing effects in a frequency-dependent manner. When heart rate was kept constant, there was no effect from left- or right-VS, while SS increased left ventricular pressure in a frequency-dependent manner. Together these results suggest that SS, left- and right-VS alter left ventricular force by two different mechanisms. Left- and right-VS decrease left ventricular pressure predominantly via chronotropic effects whilst SS increases force predominantly by direct changes in contractility.

Frequency‐dependent acceleration of relaxation in mammalian heart: a property not relying on phospholamban and SERCA2a phosphorylation

An increase in stimulation frequency causes an acceleration of myocardial relaxation (FDAR). Several mechanisms have been postulated to explain this effect, among which is the Ca(2+)-calmodulin-dependent protein kinase (CaMKII)-dependent phosphorylation of the Thr(17) site of phospholamban (PLN). To gain further insights into the mechanisms of FDAR, we studied the FDAR and the phosphorylation of PLN residues in perfused rat hearts, cat papillary muscles and isolated cat myocytes. This allowed us to sweep over a wide range of frequencies, in species with either positive or negative force-frequency relationships, as well as to explore the FDAR under isometric (or isovolumic) and isotonic conditions. Results were compared with those produced by isoprenaline, an intervention known to accelerate relaxation (IDAR) via PLN phosphorylation. While IDAR occurs tightly associated with a significant increase in the phosphorylation of Ser(16) and Thr(17) of PLN, FDAR occurs without significant changes in the phosphorylation of PLN residues in the intact heart and cat papillary muscles. Moreover, in intact hearts, FDAR was not associated with any significant change in the CaMKII-dependent phosphorylation of sarcoplasmic/endoplasmic Ca(2+) ATPase (SERCA2a), and was not affected by the presence of the CaMKII inhibitor, KN-93. In isolated myocytes, FDAR occurred associated with an increase in Thr(17) phosphorylation. However, for a similar relaxant effect produced by isoprenaline, the phosphorylation of PLN (Ser(16) and Thr(17)) was significantly higher in the presence of the beta-agonist. Moreover, the time course of Thr(17) phosphorylation was significantly delayed with respect to the onset of FDAR. In contrast, the time course of Ser(16) phosphorylation, the first residue that becomes phosphorylated with isoprenaline, was temporally associated with IDAR. Furthermore, KN-93 significantly decreased the phosphorylation of Thr(17) that was evoked by increasing the stimulation frequency, but failed to affect FDAR. Taken together, the results provide direct evidence indicating that CaMKII phosphorylation pathways are not involved in FDAR and that FDAR and IDAR do not share a common underlying mechanism. More likely, a CaMKII-independent mechanism could be involved, whereby increasing stimulation frequency would disrupt the SERCA2a-PLN interaction, leading to an increase in SR Ca(2+) uptake and myocardial relaxation.

Reduced oxygen supply explains the negative force-frequency relation and the positive inotropic effect of adenosine in buffer-perfused hearts

In isolated rat hearts perfused with HEPES and red blood cell-enriched buffers, we examined changes in left ventricular pressure induced by increases in heart rate or infusion of adenosine to investigate whether the negative force-frequency relation and the positive inotropic effect of adenosine are related to an inadequate oxygen supply provided by crystalloid perfusates. Hearts perfused with HEPES buffer at a constant flow demonstrated a negative force-frequency relation, whereas hearts perfused with red blood cell-enriched buffer exhibited a positive force-frequency relation. In contrast, HEPES buffer-perfused hearts showed a concentration-dependent increase in left ventricular systolic pressure [EC50 = 7.0 +/- 1.2 nM, maximal effect (Emax) = 104 +/- 2 and 84 +/- 2 mmHg at 0.1 microM and baseline, respectively] in response to adenosine, whereas hearts perfused with red blood cell-enriched buffer showed no change in left ventricular pressure. The positive inotropic effect of adenosine correlated with the simultaneous reduction in heart rate (r = 0.67, P < 0.01; EC50 = 3.8 +/- 1.4 nM, baseline 228 +/- 21 beats/min to a minimum of 183 +/- 22 beats/min at 0.1 microM) and was abolished in isolated hearts paced to suppress the adenosine-induced bradycardia. In conclusion, these results indicate that the negative force-frequency relation and the positive inotropic effect of adenosine in the isolated rat heart are related to myocardial hypoxia, rather than functional peculiarities of the rat heart.

Altered myocardial Ca2+cycling after left ventricular assist device support in the failing human heart

The objective of the present study was to determine whether improved contractility after left ventricular assist device (LVAD) support reflects altered myocyte calcium cycling and changes in calcium-handling proteins. Previous reports demonstrate that LVAD support induces sustained unloading of the heart with regression of pathologic hypertrophy and improvements in contractile performance. In the human myocardium of subjects with heart failure (HF), with non-failing hearts (NF), and with LVAD-supported failing hearts (HF-LVAD), intracellular calcium ([Ca(2+)](i)) transients were measured in isolated myocytes at 0.5 Hz, and frequency-dependent force generation was measured in multicellular preparations (trabeculae). Abundance of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA), Na(+)/Ca(2+) exchanger (NCX), and phospholamban was assessed by Western analysis. Compared with NF myocytes, HF myocytes exhibited a slowed terminal decay of the Ca(2+) transient (DT(terminal), 376 +/- 18 ms vs. 270 +/- 21 ms, HF vs. NF, p < 0.0008), and HF-LVAD myocytes exhibited a DT(terminal) that was much shorter than that observed in HF myocytes (278 +/- 10 ms, HF vs. HF-LVAD, p < 0.0001). Trabeculae from HF showed a negative force-frequency relationship, compared with a positive relationship in NF, whereas a neutral relationship was observed in HF-LVAD. Although decreased SERCA abundance in HF was not altered by LVAD support, improvements in [Ca(2+)](i) transients and frequency-dependent contractile function were associated with a significant decrease in NCX abundance and activity from HF to HF-LVAD. Improvement in rate-dependent contractility in LVAD-supported failing human hearts is associated with a faster decay of the myocyte calcium transient. These improvements reflect decreases in NCX abundance and transport capacity without significant changes in SERCA after LVAD support. Our results suggest that reverse remodeling may involve selective, rather than global, normalization of the pathologic patterns associated with the failing heart.

Effects of temperature and calcium availability on ventricular myocardium from the neotropical teleost Piaractus mesopotamicus (Holmberg 1887—Teleostei, Serrasalmidae)

This study analyzed the effects of temperature on cardiac function of Piaractus mesopotamicus. The data suggest that the heart performance during temperature elevations is regulated mainly by chronotropic adjustments and that increased extracellular Ca 2+ concentrations and high adrenaline can improve inotropism at high temperatures. When stimulation frequency was increased, a negative force–frequency relationship was observed at both 25°C and 35°C concomitant with increases in resting tension, indicating that relaxation is a limiting step. The post-rest potentiation was strongly inhibited by ryanodine, suggesting that, irrespective of temperature, the sarcoplasmic reticulum may contribute to excitation–contraction coupling.

Mechanical load-dependent regulation of gene expression in monocrotaline-induced right ventricular hypertrophy in the rat.

Rats treated with monocrotaline (MCT) develop pulmonary hypertension. Their right ventricles (RVs) exhibit severe pressure overload-induced hypertrophy, whereas the left ventricles (LVs) are normally loaded. In contrast, enhanced neuroendocrine stimulation during the transition to heart failure affects both ventricles. We assessed gene expression levels of Ca2+-regulating proteins in RVs and LVs of control and MCT rats in transition to heart failure to identify biomechanical load-regulated genes. In MCT RVs, both mRNA and protein levels of the Ca2+-ATPase of the sarcoplasmic/endoplasmic reticulum (SERCA2a) were reduced by 36% (P=0.001) and 17% (P=0.016), respectively, compared with control RVs. Phospholamban and ryanodine receptor mRNA levels likewise were reduced (by 27% [P=0.05] and 21% [P=0.011], respectively) in MCT RVs, whereas sarcolemmal Na+-Ca2+ exchanger expression was not altered. MCT LVs exhibited no significant expression changes compared with control LVs. Isometrically contracting MCT intact RV trabeculae showed enhanced baseline force development. Although control RV preparations exhibited a positive force-frequency relationship, MCT RVs showed a negative force-frequency relationship and blunted postrest potentiation. Contractile function of MCT LV trabeculae was normal. Maximum Ca2+-activated tension was enhanced by 64% in permeabilized RV MCT preparations (P=0.013). beta-Myosin heavy chain protein was upregulated in MCT RVs (P<0.001) but unaltered in MCT LVs. Degradation of troponin T was prominent in MCT RVs, a phenomenon not observed in the LV. Enhanced biomechanical load is necessary to induce the gene expression changes associated with the hypertrophic phenotype of the pressure-overloaded RV. Neuroendocrine factors, which equally affect both chambers, are not sufficient to alter the expression of Ca2+-cycling proteins.

Decreased contractility of the left ventricle is induced by the neurotransmitter acetylcholine, but not by vagal stimulation in rats.

There is still controversy with respect to how an increase in vagal tone changes left ventricular (LV) contractility. It is possible that a difference in LV vagal innervation density may affect the inotropic effect. To test this, we examined the effects of vagal stimulation and acetylcholine (ACh) infusion on the rat ventricle, in which LV vagal innervation density is sparse and a negative force-frequency relationship is uniquely observed. To evaluate LV contractility, we developed an in situ Langendorff preparation, in which the effects of changes in afterload, preload, and coronary flow during an intervention were minimized. Both vagal stimulation and ACh infusion significantly increased LV systolic pressure (34 +/- 16%: 36 +/- 22%. respectively) and its maximum positive first derivative with slowing of heart rate (51 +/- 17%: 46 +/- 18%). These effects of vagal stimulation were abolished by pretreatment with atropine. During a fixed heart rate, LV systolic pressure was not changed by vagal stimulation, however, it was decreased slightly but significantly (11 +/- 8%) by ACh infusion. In conclusion, LV contractility changes due to ACh release during vagal stimulation were negligibly small, presumably due to a sparse vagal innervation density in rats, and therefore, a bradycardia-dependent indirect positive inotropic effect may be dominant compared to a direct negative inotropic action during vagal stimulation. Thus, the integrated effect of vagal nerve stimulation on LV contractility is different among species, because it is determined by a direct negative inotropic effect, which depends on the vagal innervation density in the left ventricle, as well as by bradycardia-dependent indirect inotropic changes.

Targeting phospholamban by gene transfer in human heart failure.

Myocardial cells from failing human hearts are characterized by abnormal calcium handling, a negative force-frequency relationship, and decreased sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) activity. In this study, we tested whether contractile function can be improved by decreasing the inhibitory effects of phospholamban on SERCA2a with adenoviral gene transfer of antisense phospholamban (asPL). Myocardial cells isolated from 9 patients with end-stage heart failure and 18 donor nonfailing hearts were infected with adenoviruses encoding for either the antisense of phospholamban (Ad.asPL), the SERCA2a gene (Ad.SERCA2a), or the reporter genes beta-galactosidase and green fluorescent protein (Ad.betagal-GFP). Adenoviral gene transfer with Ad.asPL decreased phospholamban expression over 48 hours, increasing the velocity of both contraction and relaxation. Compared with cardiomyocytes infected with Ad.asPL (n=13), human myocytes infected with Ad.betagal-GFP (n=8) had enhanced contraction velocity (20.3 +/- 3.9% versus 8.7 +/- 2.6% shortening/second; P<0.01) and relaxation velocity (26.0 +/- 6.2% versus 8.6 +/- 4.3% shortening/second; P<0.01). The improvement in contraction and relaxation velocities was comparable to cardiomyocytes infected with Ad.SERCA2a. Failing human cardiomyocytes had decreased contraction and Ca2+ release with increasing frequency (0.1 to 2 Hz). Phospholamban ablation restored the frequency response in the failing cardiomyocytes to normal; increasing frequency resulted in enhanced sarcoplasmic reticulum Ca2+ release and contraction. These results show that gene transfer of asPL can improve the contractile function in failing human myocardium. Targeting phospholamban may provide therapeutic benefits in human heart failure.

Myocardial force development and structural changes associated with monocrotaline induced cardiac hypertrophy and heart failure.

In this study alterations are characterized which occur, in myocardial force development morphological appearance and protein composition, during the development of cardiac hypertrophy and heart failure in monocrotaline (MCT) treated rats. The transition from cardiac hypertrophy to heart failure was studied by comparing the results from control (CON) and two MCT groups (40 and 44 mg/kg body weight). The three experimental groups consisted of at least five animals each. Parameters studied were: body weight (measured daily), lung/body weight ratio, right ventricular wall volume and thickness, and force development in thin right ventricular trabeculae at 27 degrees C, using different extracellular calcium concentrations and pacing frequencies. MCT injection resulted in marked right ventricular hypertrophy and heart failure as evidenced by an up to 2-fold increase in lung/body weight ratio and a 1.7-fold increase in wall volume. The MCT groups showed a negative force-frequency relation and maximum force was up to 2-fold less than in the CON group. Protein analysis by means of one- and two-dimensional gel electrophoresis revealed a marked (7-fold) up-regulation of the slow myosin heavy chain isoform as well as a 4.5-fold increase in the content of the cytoskeletal protein desmin, whereas the mitochondrial protein ATP-synthase content was reduced. Hence MCT-induced cardiac hypertrophy and heart failure result in altered cellular calcium handling, depression of maximum force output, an increase in the economy of myocardial contraction and changes in cytoskeletal structure and energy supply.

Force/shortening–frequency relationship in multicellular muscle strips and single cardiomyocytes of human failing and nonfailing hearts

Background: Force of contraction (FOC) frequency-dependently increases in multicellular muscle strip preparations of human nonfailing myocardium, whereas FOC declines in human failing myocardium with increasing stimulation frequency. We investigated whether these characteristics can be observed in single isolated myocytes. Methods and Results: Isolated multicellular muscle strip preparations and single isolated cardiomyocytes of failing (heart transplants, dilative cardiomyopathy; n = 11) and nonfailing (donor hearts; n = 11) human hearts were studied. The changes in contraction amplitude (cell shortening in micrometers) at increasing frequency of stimulation (0.5–2 Hz) were continuously recorded with a 1-dimensional high-speed camera that detected the cell edges and measured their distance during contraction. The increase in stimulation frequency was associated with a significant decrease in FOC (2 v 0.5 Hz; 68% basal) and a decrease in cell shortening of human left ventricular cardiomyocytes from failing hearts (2 v 0.5 Hz; 65% basal). In contrast, in human nonfailing myocardium, contraction increased at increasing stimulation frequencies (2 v 0.5 Hz; FOC, 180% basal; cell shortening, 129% basal). Conclusions: The negative force-frequency relationship measured in multicellular preparations of failing human myocardium results from alterations at the single cell level.

Role of endothelial cells in modulation of contractility induced by hexarelin in rat ventricle

The synthetic growth hormone (GH) secretagogue hexarelin has important cardiac effects, that include a reduction of dysfunction in ischemic-reperfused hearts from GH-deficient rats after a chronic treatment and an increase of ejection fraction in acutely treated men. To investigate the mechanisms of its cardiac activity, we studied the effects of hexarelin (1–10 μM) on contractility of rat papillary muscles. We observed, in hexarelin treated papillary muscles, an improved recovery of contractility after anoxia. Hexarelin induced time- and frequency-dependent inotropic effects on papillary muscle. These effects were a transient increase in contractile force, abolished by propranolol (0.2 μM), followed by a reduction at low (60–240/min), but not at high (400–600/min) beating frequencies. The typical negative force-frequency relationship present in rat papillary muscles was therefore modified, and a minor increase in diastolic tension occurred after a sudden increase in stimulus frequency. Blockade of NO synthesis with 1 mM l-NAME, partially altered the response to hexarelin. MK-677 (1 μM), a non peptidyl GH secretagogue, reduced contractility, but did not alter the force-frequency relationship. The remaining effects of hexarelin were absent in papillary muscles pre-treated with indomethacin (1 μM), or after removal of endocardial endothelium with 0.5% triton X-100. The release of the prostacyclin metabolite 6-keto-PGF 1α was increased during reoxygenation after a period of anoxia in hexarelin treated papillary muscles. Hexarelin had no significant effect on calcium transients and on I Ca measured in isolated ventricular cells. These findings suggest that the effects of hexarelin are mainly due to endothelium-released PGI 2.

Comparison of the Electromechanical Responsiveness of Alpha-1-Adrenoceptor Stimulation in Ventricles of Normal and Cardiomyopathic Hamsters

Alterations in α₁-adrenoceptor (α₁AR) density and related signal transduction proteins were reported in cardiomyopathic hearts in the failing stage. The electromechanical modification of α₁-adrenergic stimulation in the failing heart is unclear. The present study compares the α₁AR-stimulated electromechanical response in failing ventricles of genetically cardiomyopathic BIO 14.6 hamsters (280–320 days old) with that in age-matched normal Syrian hamsters. The action potential was recorded with a conventional microelectrode technique, and twitch force was measured with a transducer. In the presence of propranolol, phenylephrine increased the contraction and prolonged the action potential duration (APD) to similar values in ventricles of both strains, despite a prolonged basal APD in cardiomyopathic ventricles. The positive inotropism stimulated by phenylephrine was inhibited by staurosporine, and was potentiated by 4β-phorbol-12,13-dibutyrate (PDBu) in both strains. The maximum positive inotropic effect of phenylephrine in PDBu-treated ventricles of normal hamsters was significantly greater than that in BIO 14.6 hamsters. The effects of phenylephrine on the ventricular force-frequency relationship and on the mechanical restitution in both normal and BIO 14.6 strain hamsters were examined. The uniform negative force-frequency relationship and the altered mechanical restitution reveal a defect of intracellular Ca²⁺ handling in cardiomyopathic BIO 14.6 hamsters. α₁-Adrenergic modulation cannot convert the defective properties in the model of the failing heart. Nevertheless, phenylephrine decreased postrest potentiation in short rest periods, and enhanced postrest decay after longer resting periods. The results indicate that α₁-adrenergic action enhances a gradual loss of Ca²⁺ from the sarcoplasmic reticulum, although its action in prolonging the APD can indirectly increase the influx of Ca²⁺.

Differences in Ca2+-Handling and Sarcoplasmic Reticulum Ca2+-Content in Isolated Rat and Rabbit Myocardium

We made novel measurements of the influence of rest intervals and stimulation frequency on twitch contractions and on sarcoplasmic reticulum (SR) Ca2+-content (using rapid cooling contractures, RCCs) in isolated ventricular muscle strips from rat and rabbit hearts at a physiological temperature of 37 °C. In addition, the frequency-dependent relative contribution of SR Ca2+-uptake and Na+/Ca2+-exchange for cytosolic Ca2+-removal was assessed by paired RCCs. With increasing rest intervals (1–240 s) post-rest twitch force and RCC amplitude decreased monotonically in rabbit myocardium (after 240 s by 45±10% and 61±11%, respectively P<0.05, n=14). In contrast, rat myocardium (n=11) exhibited a parallel increase in post-rest twitch force (by 67±16% at 240 sP <0.05) and RCC amplitude (by 20±14%P<0.05). In rabbit myocardium (n=11), increasing stimulation frequency from 0.25 to 3 Hz increased twitch force by 295±50% (P<0.05) and RCC amplitude by 305±80% (P<0.05). In contrast, in rat myocardium (n=6), twitch force declined by 43±7% (P<0.05), while RCC amplitude decreased only insignificantly (by 16±7%). The SR Ca2+-uptake relative to Na+/Ca2+-exchange (based on paired RCCs) increased progressively with frequency in rabbit, but not in rat myocardium (66±2% at all frequencies). We conclude that increased SR Ca2+-load contributes to the positive force–frequency relationship in rabbits and post-rest potentiation of twitch force in rats. Decreased SR Ca2+-load contributes to post-rest decay of twitch force in rabbits, but may play only a minor role in the negative force–frequency relationship in rats. SR Ca2+-release channel refractoriness may contribute importantly to the negative force-frequency relationship in rat and recovery from refractoriness may contribute to post-rest potentiation.

Levosimendan improves diastolic and systolic function in failing human myocardium

Ca 2+-sensitizers increase myocardial contractility, but may worsen diastolic dysfunction. Levosimendan, through its unique troponin-C interaction, may preserve diastolic function. We investigated the effects of levosimendan (10 −7–10 −5 M) on diastolic and systolic function in multicellular cardiac muscle preparations from end-stage failing human hearts (1 and 2.5 Hz, 37°C, 1.25 mM [Ca 2+], pH 7.4). Levosimendan improved systolic function: at 1 Hz, developed force ( F dev) increased from 13.84±3.27 to 16.40±3.57 (10 −7 M, P<0.05), while diastolic force ( F dia) decreased from 5.32±0.67 to 4.94±0.61 mN/mm 2 ( P<0.05). Under control conditions, the increase in stimulation frequency from 1 to 2.5 Hz resulted in a decrease in F dev of −0.51±1.80 mN/mm 2 (negative force–frequency relationship). Levosimendan improved this relationship: at 10 −7 M, this change became positive (+1.81±2.06 mN/mm 2, P<0.05). Diastolic function was markedly improved in the presence of levosimendan; the increase in F dia of 1.56±0.42 mN/mm 2 (control) was attenuated to 0.70±0.19 mN/mm 2 ( P<0.05). To allow for a more detailed analysis, preparations were sometimes divided into two groups, based on their force–frequency behavior. Twitch timing parameters were accelerated by levosimendan in preparations with a negative force–frequency relationship. Levosimendan improves both systolic and diastolic function in failing human myocardium. Effects are even more pronounced at higher heart rates and under prevailing diastolic dysfunction.

Three-dimensional engineered heart tissue from neonatal rat cardiac myocytesThis work is part of the doctoral thesis of W. H. Z. at the University of Hamburg.

A technique is presented that allows neonatal rat cardiac myocytes to form spontaneously and coherently beating 3-dimensional engineered heart tissue (EHT) in vitro, either as a plane biconcaval matrix anchored at both sides on Velcro-coated silicone tubes or as a ring. Contractile activity was monitored in standard organ baths or continuously in a CO2 incubator for up to 18 days (=26 days after casting). Long-term measurements showed an increase in force between days 8 and 18 after casting and stable forces thereafter. At day 10, the twitch amplitude (TA) of electrically paced EHTs (average length × width × thickness, 11 × 6 × 0.4 mm) was 0.51 mN at length of maximal force development (Lmax) and a maximally effective calcium concentration. EHTs showed typical features of neonatal rat heart: a positive force–length and a negative force–frequency relation, high sensitivity to calcium (EC50 0.24 mM), modest positive inotropic (increase in TA by 46%) and pronounced positive lusitropic effect of isoprenaline (decrease in twitch duration by 21%). Both effects of isoprenaline were sensitive to the muscarinic receptor agonist carbachol in a pertussis toxin-sensitive manner. Adenovirus-mediated gene transfer of β-galactosidase into EHTs reached 100% efficiency. In summary, EHTs retain many of the physiological characteristics of rat cardiac tissue and allow efficient gene transfer with subsequent force measurement. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 106–114, 2000.