Resting Tension Research Articles

Abstract 18778: Patient-Derived Stem Cell Models of Pediatric Restrictive Cardiomyopathy Exhibit Increased Resting Tension and Cardiomyocyte Calcium Sensitivity

Introduction: Pediatric Restrictive Cardiomyopathy (RCM) is a genetic cardiomyopathy resulting from profoundly increased stiffness of the wall of the cardiac ventricle with normal or low normal contractility. Treatment options are limited, with most patients necessitating heart transplant. Therapies that can improve diastolic function are needed for this severe disease. However, development of novel therapies has been limited by a paucity of models of this rare disease. Here, we develop two novel models of pediatric RCM and use novel high-throughput functional analysis to measure diastolic dysfunction and defects in calcium handling. Hypothesis: Patient-derived human induced pluripotent stem cell (hiPSC) models of pediatric RCM will exhibit increased resting tension indicative of diastolic dysfunction, as well as increased calcium sensitivity. Methods: Cardiomyocytes were differentiated from hiPSCs derived from two pediatric patients with RCM, as well as a paired isogenic CRISPR-corrected lines. Cells were replated onto polyacrylamide gels and stained with calcium sensitive dyes, and contractility, resting tension, and calcium sensitivity were measured using a novel high-throughput functional imaging platform. Results: RCM cells from both patients showed elevated contractility, resting tension, and calcium sensitivity compared to their isogenic controls. Conclusions: Patient-derived RCM cells show alterations in calcium sensitivity and resting tension that recapitulate and explain the diastolic dysfunction seen in patients. These lines, combined with the high-throughput measurement platform described here, will provide a powerful platform for exploring novel therapeutic strategies for this severe disease.

Importance of SERCA2a on early isolated diastolic dysfunction induced by supravalvular aortic stenosis in rats

Cardiac remodeling is defined as changes in shape and function of the heart in response to aggression (pressure overload). The sarcoplasmic reticulum calcium ATPase cardiac isoform 2a (SERCA2a) is a known factor that influences function. A wide spectrum of studies report a decrease in SERCA2a in heart failure, but none evaluate it's the role in early isolated diastolic dysfunction in supravalvular aortic stenosis (AoS). Our hypothesis was that SERCA2a participates in such dysfunction. Thirty-day-old male Wistar rats (60-80 g) were divided into AoS and Sham groups, which were submitted to surgery with or without aorta clipping, respectively. After 6 weeks, the animals were submitted to echocardiogram and functional analysis by isolated papillary muscle (IPM) in basal condition, hypoxia, and SERCA2a blockage with cyclopiazonic acid at calcium concentrations of 0.5, 1.5, and 2.5 mM. Western-blot analyses were used for SERCA2a and phospholamban detection. Data analysis was carried out with Student's t-test and ANOVA. AoS enhanced left atrium and E and A wave ratio, with preserved ejection fraction. Basal condition in IPM showed similar increases in developed tension (DT) and resting tension (RT) in AoS, and hypoxia was similar between groups. After cyclopiazonic acid blockage, final DT was equally decreased and RT was similar between groups, but the speed of relaxation was decreased in the AoS group. Western-blot was uniform in all evaluations. The hypothesis was confirmed, since functional parameters regarding SERCA2a were changed in the AoS group.

Increased resting tension abolishes endothelial anti-contractile effect induced by phenylephrine in renal hypertensive rat aorta

The role of mechanical stress for vascular contraction is still unclear in hypertensive animals. This study aimed to evaluate the role of the rest tension of 1.5 g and 3.0 g on the endothelial nitric oxide synthase (eNOS) activity for contraction induced by the α1-adrenergic agonist phenylephrine (PE) in isolated intactendothelium aorta (E+) from renal hypertensive rat (2 K-1C) as compared to normotensive rat (2 K). Concentration-effect curves for PE were constructed in E+ under resting tension of 1.5 g or 3.0 g, in the absence or after incubation for 30 min with the NOS inhibitor (L-NAME 100 μM). The potency (pD2) and efficacy (ME) of PE were evaluated. eNOS residue Ser phosphorylation activates its enzyme. Phosphorylated Ser (P-Ser) expression was evaluated by Western Blot. This study was approved by the Ethics Committee of the University of Sao Paulo (156/2009). ME induced by PE on tension of 1.5 g was lower in 2 K-1C (1.2 ± 0.2 g, n = 7; p b 0.001) than in 2 K (2.2 ± 0.1 g; pD2:7.44 ± 0.03; n = 5), which was normalized by L-NAME. PE induced higher P-Ser-eNOS in 2 K-1C than in 2 K aorta. But under tension of 3.0 g, PE-induced contraction was similar in 2 K-1C and 2 K aorta. Therefore, these results indicate that the anti-contractile effect induced by PE in 2 K-1C E+ aorta under tension of 1.5 g is due to NO through eNOSSer phosphorylation that is abolished by the increased tension to 3.0 g. Supported by FAPESP, CNPq and CAPES.

ASSESSMENT OF SPASMOLYTIC ACTIVITY OF ALCOHOLIC EXTRACT OF ACILLEA MILLEFOLIUM, RUBIA CORDIFOLIA AND SAUSSUREA LAPPA IN WISTAR ALBINO RATS

The present study was carried out to e valuate the potential of alcoholic extract of anti - inflammatory plants viz. Achillea millefolium , Rubia cordifolia and Saussurea la ppa for spasmolytic activity in rat model using radnoti organ bath . The relaxant effect of all extracts on pre - contracted rat tracheal chain by carbachol (30 µM) had been screened. Isometric contractions of isolated rat tracheas were recorded at 1.4 g rest ing tension and carbachol dose - response curves were performed. EC 50 values (27.12, 13.13 and 7.32 µM, respectively) were identified by plotting cumulative concentration response curve and pD 2 values (4.48 ± 0.99, 6.06 ± 1.03 and 7.41 ± 0.97, respectively) were calculated for individual alcoholic extract of plant . All extracts were able to relax carbachol pre contracted tracheas significantly in a concentration dependent manner. Our results suggested potential role of Achillea millefolium , Rubia cordifolia a nd Saussurea lappa in asthma for further potential therapeutic and clinical uses.

Hypophosphorylation of the Stiff N2B Titin Isoform Raises Cardiomyocyte Resting Tension in Failing Human Myocardium

High diastolic stiffness of failing myocardium results from interstitial fibrosis and elevated resting tension (F(passive)) of cardiomyocytes. A shift in titin isoform expression from N2BA to N2B isoform, lower overall phosphorylation of titin, and a shift in titin phosphorylation from N2B to N2BA isoform can raise F(passive) of cardiomyocytes. In left ventricular biopsies of heart failure (HF) patients, aortic stenosis (AS) patients, and controls (CON), we therefore related F(passive) of isolated cardiomyocytes to expression of titin isoforms and to phosphorylation of titin and titin isoforms. Biopsies were procured by transvascular technique (44 HF, 3 CON), perioperatively (25 AS, 4 CON), or from explanted hearts (4 HF, 8 CON). None had coronary artery disease. Isolated, permeabilized cardiomyocytes were stretched to 2.2-microm sarcomere length to measure F(passive). Expression and phosphorylation of titin isoforms were analyzed using gel electrophoresis with ProQ Diamond and SYPRO Ruby stains and reported as ratio of titin (N2BA/N2B) or of phosphorylated titin (P-N2BA/P-N2B) isoforms. F(passive) was higher in HF (6.1+/-0.4 kN/m(2)) than in CON (2.3+/-0.3 kN/m(2); P<0.01) or in AS (2.2+/-0.2 kN/m(2); P<0.001). Titin isoform expression differed between HF (N2BA/N2B=0.73+/-0.06) and CON (N2BA/N2B=0.39+/-0.05; P<0.001) and was comparable in HF and AS (N2BA/N2B=0.59+/-0.06). Overall titin phosphorylation was also comparable in HF and AS, but relative phosphorylation of the stiff N2B titin isoform was significantly lower in HF (P-N2BA/P-N2B=0.77+/-0.05) than in AS (P-N2BA/P-N2B=0.54+/-0.05; P<0.01). Relative hypophosphorylation of the stiff N2B titin isoform is a novel mechanism responsible for raised F(passive) of human HF cardiomyocytes.

Abstract 1574: Low Myocardial Protein Kinase G Activity Raises Cardiomyocyte Resting Tension in Diastolic Heart Failure

Cardiomyocytes (CM) of diastolic heart failure (DHF) patients (pts) are hypertrophied and have high diastolic resting tension (RT), which elevates left ventricular (LV) diastolic stiffness. In rodent LV pressure overload models, development of pathological CM hypertrophy has been related to low myocardial protein kinase G (PKG) activity. Myocardial PKG activity and in-vitro effect of PKG on RT of isolated CM were therefore determined using LV endomyocardial biopsies procured from DHF pts. All DHF pts (n=36) had been admitted to hospital for worsening heart failure (NYHA 3– 4), were free of coronary artery disease, had a LV ejection fraction &gt; 50%, a LV end-diastolic volume index &lt; 97 ml/m 2 , a LV end-diastolic pressure &gt; 16 mmHg and no histological evidence in the biopsies of myocardial infiltration or inflammation. Myocardial PKG activity was assessed by immunohistochemistry using antibodies against the specific PKG substrate vasodilator stimulated phosphoprotein (VASP) and against phosphorylated VASP ((P)-VASP). Myocardial PKG activity corresponded to the ratio of (P)-VASP/VASP. CM were isolated from the biopsies, treated with Triton X-100 to remove all membranes, attached to a force transducer and stretched to 2.2 μm to measure RT. In DHF pts, (P)-VASP/VASP ratio (0.67±0.15) was lower than in a control group (n=22) (0.98±0.19; p&lt;0.0001). CM isolated from LV myocardium of DHF pts had higher RT (7.2±0.5 kN/m 2 ) than CM of a control group (4.8±0.2 kN/m 2 ; p&lt;0.0001). In-vitro administration of PKG lowered RT of CM of DHF pts to levels similar to the control group (PKG: 4.0±0.7 kN/m 2 ; p&lt;0.01). Subsequent treatment with PKA did not further decrease RT (3.8±0.9 kN/m 2 ). DHF patients had low myocardial PKG activity. This low myocardial PKG activity could have elevated RT of CM as in-vitro administration of PKG corrected the high RT. Absence of an additional in-vitro effect of PKA after PKG suggested that high RT results from hypophosphorylation of myofilamentary proteins, whose phosphorylation sites are targets for both PKA and PKG. In pts with DHF, phosphodiesterase 5A inhibition could raise myocardial PKG activity and thereby correct their high CM RT, which is an important determinant of their high diastolic LV stiffness.

Response to Letter Regarding Article, “Diastolic Stiffness of the Failing Diabetic Heart: Importance of Fibrosis, Advanced Glycation End Products, and Myocyte Resting Tension”

The authors appreciated the thoughtful comment of Connelly et al on their study,1 which indeed did not imply that fibrosis was no determinant of diastolic left ventricular (LV) dysfunction in patients with heart failure and normal LV ejection fraction (HFNEF). A previous study from our group identified both collagen volume fraction (CVF) and cardiomyocyte resting tension to be …

Letter by Connelly et al Regarding Article, “Diastolic Stiffness of the Failing Diabetic Heart: Importance of Fibrosis, Advanced Glycation End Products, and Myocyte Resting Tension”

To the Editor: We would like to congratulate van Heerebeek and colleagues on the recent article “Diastolic Stiffness of the Failing Diabetic heart.”1 The authors provide evidence that increased myocyte stiffness is the principal abnormality in diabetic subjects with heart failure and a normal ejection fraction and that fibrosis and advanced glycation …

The significance of variable passive compliance in smooth muscle

anyone contemplating dynamic roentgenograms of hollow viscera must be impressed that the length changes in the smooth muscles that comprise their walls are larger than the threefold range produced by skeletal muscle having a fixed filament array ([1][1]), and studies of muscles isolated from these

Resting Tension Affects eNOS Activity in a Calcium-Dependent Way in Airways

The alteration of resting tension (RT) from 0.5 g to 2.5 g increased significantly airway smooth muscle contractions induced by acetylcholine (ACh) in rabbit trachea. The decrease in extracellular calcium concentration [Ca2+]o from 2 mM to 0.2 mM reduced ACh-induced contractions only at 2.5 g RT with no effect at 0.5 g RT. The nonselective inhibitor of nitric oxide synthase (NOS), NG-nitro-L-arginine methyl ester (L-NAME) increased ACh-induced contractions at 2.5 g RT. The inhibitor of inducible NOS, S-methylsothiourea or neuronal NOS, 7-nitroindazole had no effect. At 2.5 g RT, the reduction of [Ca2+]o from 2 mM to 0.2 mM abolished the effect of L-NAME on ACh-induced contractions. The NO precursor L-arginine or the tyrosine kinase inhibitors erbstatin A and genistein had no effect on ACh-induced contractions obtained at 2.5 g RT. Our results suggest that in airways, RT affects ACh-induced contractions by modulating the activity of epithelial NOS in a calcium-dependent, tyrosine-phosphorylation-independent way.

AN IN VITRO CONTRACTILE STUDY IN PEDIATRIC HUMAN HEARTS WITH CONGENITAL HEART DISEASE

Contractile compensatory mechanisms exploited in mature (older pediatric or adult) hearts may not be developed in younger pediatric hearts. With congenital heart disease (CHD), these patients are dependent on treatments specific to their cellular maturity. We hypothesized that younger CHD hearts may not comply with the contractile phenotype (ex: + force/frequency relationship (FFR), low resting tension (RT): developed tension (DT) ratio, and post-rest potentiation (PRP)) associated with mature cardiac function. We used tissue from pediatric hearts undergoing surgery; 24 with CHD and 7 with heart failure (HF), age ranged from 2mon-12yrs. Baseline contractility and FFR were assessed. In addition, PRP experiments were performed to measure sarcoplasmic reticulum (SR) function. Positive FFR was evident between 0.5-1.5Hz. DT and DT rate was lower in CHD vs HF pediatric hearts. RT was about 3x the DT in CHD vs <2x the DT in published adult human heart data. There was an absence of the PRP response in younger CHD (n=10; age 0.3-1.5yrs) vs older HF hearts (n=6; age 1.5-12yrs). A significantly lower DT, higher RT/DT ratio, and the absence of a PRP response suggest that these muscles may not be as dependent on SR function as older pediatric and adult hearts. Hence these data supports the need for more studies using younger patients where contractile regulation needs further elucidation.

A função mecânica do miocárdio remanescente a um infarto do miocárdio é normal durante o período de cicatrização, embora exista insuficiência cardíaca

The temporal relation between ventricular dysfunction (VD) after myocardial infarction (MI) and remanent myocardium mechanics is not yet clear. The present work investigated--through Doppler echocardiography (ECHO)--ventricular function in rats with extensive MI, as well as the mechanical function of papillary muscles (PM) at the end of the healing period. ECHO and PM of 9 Wistar rats (MI) were studied against 9 controls (C) three weeks after LV myocardial infarction. The following were determined: developed tension (DT) and its first negative and positive derivative, time-to-peak tension (TPT), resting tension (RT), and relaxation time at 50% of DT at 0.5, 1.0, 1.5, 2.0 and 2.5 calcium concentrations (mM). Tetanic contractions were carried out after ryanodine administration at 1.5, 2.5 and 5.0 calcium concentrations. VD was characterized by ECHO, with marked abnormality of diastolic volume and LV and ejection fraction in addition to clear restrictive pattern of blood flow through the mitral valve. No significant difference was found in myocardial mechanics data either for MI or for C rats. The heart failure (HF) reported by MI rats with > 40% MI at the end of the healing period is not myocardial function dependent. Chamber structural changes and lower population of myocites should base VD and HF.

Isometric force measurement in mouse cerebral arteries: Establishing reference values and characterizing functional consequences of endothelial nitric oxide synthase knock-out in the basilar artery

Genetically modified mice are increasingly important tools in biomedical research. The increasing number of transgenic mouse strains available has raised the need to develop adequate tools for characterizing the functional consequences of overexpressing, knocking out, or inserting a gene. We have, therefore, adapted the methodology of isometric force measurement to study the vasoreactivity of mouse cerebral arteries in vitro. Ring segments (approximately 1 mm in length) of the basilar artery (BA) were obtained from NMRI, C57/black6 (C57B6), endothelial nitric oxide synthetase knock-out (eNOS-/-) mice and the corresponding wild-type line (eNOS+/+). The segments were transferred into organ bathes filled with a modified Krebs-Henseleit solution maintained at 37 C, and continuously gassed with a mixture of 77%/17.5%/5.5% N2/O2/CO2. Contractile responses were elicited by membrane depolarisation (equimolar substitution of NaCl by KCl), or cumulative application of U46619 (a thromboxane A2-analogue) or endothelin-1 (ET-1). Relaxation was induced by applying increasing concentrations of Na-nitroprusside (SNP) or 8-bromo dibutryl-cyclic GMP (8br-cGMP) on top of stable precontraction with U46619 (1E-7 M). Contraction is given in absolute terms (mN force). Relaxation is calculated in % decrease of precontraction. Values are given as meanSD. In NMRI mice, different levels of resting tension (RT) upon membrane depolarisation (by increasing the K+ concentration)-induced contraction were studied. In segments with a RT >1.75 mN (2.00.2 mN), depolarisation-induced contraction was 1.90.6, somewhat bigger than with lower RT (1.40.2 mN; contraction, 1.70.4 mN). Thus, RT was set to 2 mN in further experiments. Contraction induced by high K+ Krebs solution was 2.30.6 in NMRI, 2.30.6 in C57B6, 2.60.8 in eNOS+/+, and in eNOS-/- 3.31.0 (p<0.05 vs all other strains). Additional results are listed in the table 1 below with †p<0.1 and *p<0.05 vs. eNOS+/+. The study presents for the first time data on vascular reactivity of mouse cerebral arteries employing the isometric force measurement technique. The results indicate pronounced differences in depolaristion- and receptor-induced contraction in different mouse strains. Furthermore, SNP enhanced relaxation in eNOS-/- mice significantly, probably indicating a hypersensitivity of the sGC due to the lack of NO release from the endothelial cells. Thus, this technique allows a powerful method to study in great detail the functional consequences of a given type of genetic engineering in mouse cerebral arteries.

Resting tension effect on airway smooth muscle: the involvement of epithelium

We studied the influence of resting tension (RT) on rabbit tracheal smooth muscle (TSM) contractions induced by acetylcholine or KCl as well as the role of epithelium and the endogenously produced nitric oxide, prostanoids and endothelin on these responses. The alteration of RT from 0.5 to 2.5 g increased the responsiveness of TSM to KCl. The presence of atropine decreased KCl-induced contractions obtained only at 2.5 g RT. The removal of epithelium increased acetylcholine-induced contractions, only at 2.5 g RT. At 0.5 g RT, the presence of L-NAME had no effect on acetylcholine-induced contractions while indomethacin decreased contractions induced by 10(-3) M acetylcholine. At 2.5 g RT, the presence of L-NAME increased acetylcholine-induced contractions while indomethacin, BQ-123 and BQ-788 had no effect. These results demonstrate that RT affects the responsiveness of TSM differentially, depending on the agonist or integrity of the epithelium. Airway epithelium modulates acetylcholine-induced contractions, only at 2.5 g RT partly via NO release. At 0.5 g RT, the endogenous production of prostanoids by sources other than epithelium modulates the contractility of TSM to acetylcholine.

Human motor compensations for thixotropy-dependent changes in resting wrist joint position after large joint movements.

Resting tension of relaxed skeletal muscle fibres held at a given length varies with the immediate previous history of length changes and contractions. The primary aim of this study was to explore the motor control consequences of this history-dependency in healthy subjects. Angular position and passive torque were recorded from the intact wrist joint. Integrated surface electromyography (IEMG) was recorded from wrist extensor and flexor muscles. In relaxed subjects, wrist joint position was displaced towards dorsiflexion after a single high-amplitude dorsiflexion movement combined with a strong flexor/extensor co-contraction (dorsiflexion conditioning), whereas after volarflexion conditioning there was a shift towards volarflexion. These after-effects could be abruptly cancelled by short periods ( approximately 5 s) of rapid flapping hand movements or forceful isometric co-contractions, findings indicative of muscle thixotropy. The IEMG-evaluated motor after-effects were as follows. A slowly subsiding wrist flexor contraction was needed to restore and maintain the original resting wrist position after dorsiflexion conditioning whereas a slowly subsiding extensor contraction was needed for the same goal after volarflexion conditioning. Furthermore, ongoing wrist extensor IEMG activity required to actively hold the wrist in a moderate dorsiflexed position or to resist a constant volar torque at resting position was temporarily reduced after dorsiflexion conditioning and enhanced (not significantly) after volarflexion conditioning. The results provide evidence that during voluntary maintenance of a desired wrist joint position the motor commands to the position-holding muscles are unconsciously adjusted to compensate for thixotropy-dependent variations in the resting tension of the muscles.

ET-1 increases distensibility of acutely loaded myocardium: a novel ETA and Na+/H+ exchanger-mediated effect.

This study investigated, in rabbit papillary muscles (n = 61) and human auricular strips (n = 7), effects of endothelin-1 (ET-1; 0.1-10 nM) on diastolic myocardial properties. ET-1 (1 nM) was also given in the presence of selective ET(A) or ET(B) antagonism, nonselective ET(A)/ET(B) antagonism, and Na(+)/H(+) exchanger inhibition. Effects of 6.3 mM Ca(2+) were also studied. ET-1 dose dependently increased inotropism. In contrast to baseline, in the presence of ET-1, resting tension (RT) decreased, after an isometric twitch, 3.4 +/- 1.4, 6.9 +/- 1.5, and 12.5 +/- 3.1% with 0.1, 1, and 10 nM, respectively, reflecting an increase in myocardial distensibility. ET-1 effects were abolished with selective ET(A) as well as with nonselective ET(A)/ET(B) antagonism, whereas they were still present with ET(B) antagonism. Na(+)/H(+) exchanger inhibition abolished ET-1 effects on distensibility, whereas it only partially inhibited positive inotropic effect. Ca(2+) increased inotropism to a similar extent to ET-1 (1 nM) but did not affect distensibility. ET-1 therefore increased diastolic distensibility of acutely loaded human and nonhuman myocardium. This effect is mediated by ET(A) receptors, requires Na(+)/H(+) exchanger activation, and cannot be elicited by Ca(2+).

Comparison of the protective actions of Na+/H+ and Na+/Ca2+ exchange inhibitors in ischemic/reperfused rat hearts

The protective effects of the Na + /H + exchange inhibitors amiloride, EIPA (5-(N-ethyl-N-sopropyl)-amiloride), and HOE 694 (3-methylsulfonyl-4-(1-piperidino) benzoyl-guanidine) and the Na + / Ca 2+ exchange inhibitor, DCB (3,4-Dichlorobenzamil) on ischemia (30 min) / reperfusion (30 min) injury were studied using Langendorff perfused rat hearts. EIPA and HOE 694 given before ischemia protected the heart during reperfusion from mechanical and metabolic disturbances. A weak protective effect was observed with amiloride, but not with DCB. The cardioprotective efficacies of these compounds correlated with their potencies as Na + /H + exchange inhibitors as assessed by the NH 4 Cl prepulse method. None of the inhibitors was effective when given at reperfusion. EIPA and HOE 694 decreased myocardial rigidity as assessed by the resting tension (RT) which elevated during reperfusion. EIPA led to a more marked attenuation of RT elevation during reperfusion rather than ischemia, whereas diltiazem, a Ca 2+ channel blocker, suppressed RT elevation during ischemia but did not cause a further attenuation of RT during reperfusion. Treatment with EIPA as well as diltiazem before ischemia showed a direct negative chronotropic effect. Cardioprotective effects were also observed with diltiazem. These results suggest that Na + /H + exchange plays a more important role in ischemia-reperfusion-induced myocardial injury than does Na + /Ca 2+ exchange. The cardioprotective effects of EIPA appear to be produced by Ca 2+ channel blockade during ischemia and by Na + /H + exchange inhibition during reperfusion.

Comparison of the protective actions of Na/H and Na/Ca2 exchange inhibitors in ischemic/reperfused rat hearts

The protective effects of the Na+/H+ exchange inhibitors amiloride, EIPA (5-(N-ethyl-N-isopropyl)-amiloride), and HOE 694 (3-methylsulfonyl-4-(1-piperidino) benzoyl-guanidine) and the Na+/Ca2+ exchange inhibitor, DCB (3,4-Dichlorobenzamil) on ischemia (30 min) / reperfusion (30 min) injury were studied using Langendorff perfused rat hearts. EIPA and HOE 694 given before ischemia protected the heart during reperfusion from mechanical and metabolic disturbances. A weak protective effect was observed with amiloride, but not with DCB. The cardioprotective efficacies of these compounds correlated with their potencies as Na+/H+ exchange inhibitors as assessed by the NH4Cl prepulse method. None of the inhibitors was effective when given at reperfusion. EIPA and HOE 694 decreased myocardial rigidity as assessed by the resting tension (RT) which elevated during reperfusion. EIPA led to a more marked attenuation of RT elevation during reperfusion rather than ischemia, whereas diltiazem, a Ca2+ channel blocker, suppressed RT elevation during ischemia but did not cause a further attenuation of RT during reperfusion. Treatment with EIPA as well as diltiazem before ischemia showed a direct negative chronotropic effect. Cardioprotective effects were also observed with diltiazem. These results suggest that Na+/H+ exchange plays a more important role in ischemia-reperfusion-induced myocardial injury than does Na+/Ca2+ exchange. The cardioprotective effects of EIPA appear to be produced by Ca2+ channel blockade during ischemia and by Na+/H+ exchange inhibition during reperfusion. Drug Dev. Res. 48:160–170, 1999. © 1999 Wiley-Liss, Inc.

Protection against myocardial ischemic/reperfusion injury by inhibitors of two separate pathways of Na+ entry.

Previous work has demonstrated that drugs which inhibit Na+ entry through voltage-sensitive Na+ channels, or via Na(+)-H+ exchange protect the heart from ischemic reperfusion damage. The purpose of our study was to determine whether these drugs in combination will have an additive protective effect in Langendorff-perfused hearts. During reperfusion following 30 min of ischemia, developed tension and resting tension were 24 +/- 3 and 162 +/- 5%, respectively, of pre-ischemic values in non-treated ischemic hearts. The administration of HOE-642 to inhibit Na+/H+ exchange increased active developed tension (DT) to 58 +/- 2% of pre-ischemic levels and decreased resting tension (RT) to 111 +/- 3% of pre-ischemic levels. The administration of tetrodotoxin (TTX) to block the Na+ channel increased DT to 56 +/- 3% of the pre-ischemic level and reduced the RT to 126 +/- 12% of the pre-ischemic level. Together, HOE-642 and TTX increased recovery of DT to 63 +/- 2% of pre-ischemic levels and improved RT to 116 +/- 4% of pre-ischemic levels after 30 min of reperfusion. All drug treatment protocols significantly lowered the creatine phosphokinase activity measured in the coronary effluent in comparison to that observed in the non-treated hearts. These data demonstrate that inhibition of Na+ entry through either Na(+)-H+ exchange or the Na+ channel protects the heart from ischemic injury, but there is no additional benefit of blocking both routes of Na+ entry simultaneously. This suggests that a threshold level of Na+i may be a critical factor in ischemic cardioprotection.

Cardioprotective effects of NIP-121, a novel ATP-sensitive potassium channel opener, during ischemia and reperfusion in coronary perfused guinea pig myocardium.

We investigated the effect of NIP-121, a novel ATP-sensitive K+ channel opener, on myocardial damage during ischemia/reperfusion. The action potential and contractile force of coronary-perfused guinea pig right ventricular walls were recorded. The preparations were subjected to 30-min no-flow ischemia with or without NIP-121 or glibenclamide, followed by 60-min reperfusion. In untreated tissues, decreases in action potential duration (APD) and contractile force and an increase in resting tension were observed during the no-flow period. On reperfusion, transient arrhythmias were observed and resting or contractile force returned to <50% of preischemic values. NIP-121, at 0.3 microM, a concentration showing only a slight negative inotropic effect, caused a faster decrease in APD and contractile force but abolished the increase in resting tension (RT) during the no-flow period. On reperfusion, no arrhythmia was observed in NIP-121-treated preparations, and contractile force recovered to approximately 80% of the preischemic value. Glibenclamide 1 microM attenuated the decrease in APD but affected neither the decrease in contractile force nor the increase in RT during the no-flow period. On reperfusion, the incidence of arrhythmia was increased in glibenclamide-treated preparations, and the recovery of basal tension and contractile force was inhibited: Contractile force recovered to only approximately 15% of the preischemic value. NIP-121 was also shown to attenuate the decrease in tissue ATP during ischemia and reperfusion. We demonstrated that NIP-121 may have protective effects against myocardial injury during ischemia and reperfusion. Activation of ATP-sensitive K+ current may be an adaptive mechanism for cardioprotection under compromised blood flow.