Differences in Ca(2+)-handling and sarcoplasmic reticulum Ca(2+)-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) Ca(2+)-content (using rapid cooling contractures, RCCs) in isolated ventricular muscle strips from rat and rabbit hearts at a physiological temperature of 37 degrees C. In addition, the frequency-dependent relative contribution of SR Ca(2+)-uptake and Na(+)/Ca(2+)-exchange for cytosolic Ca(2+)-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 s P<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 Ca(2+)-uptake relative to Na(+)/Ca(2+)-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 Ca(2+)-load contributes to the positive force-frequency relationship in rabbits and post-rest potentiation of twitch force in rats. Decreased SR Ca(2+)-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 Ca(2+)-release channel refractoriness may contribute importantly to the negative force-frequency relationship in rat and recovery from refractoriness may contribute to post-rest potentiation.     

Gingerol, isoproterenol and ouabain normalize impaired post-rest behavior but not force-frequency relation in failing human myocardium

OBJECTIVE: Rest- and stimulation frequency-dependent potentiation of contractile force is blunted in failing human myocardium. These alterations have been related to reduced sarcoplasmic reticulum (SR) Ca(2+)-reuptake and enhanced transsarcolemmal Ca(2+)-elimination by Na+/Ca(2+)-exchange. We investigated whether inotropic interventions that enhance SR Ca(2+)-uptake, or reduce Ca(2+)-elimination by Na+/Ca(2+)-exchange, normalize impaired post-rest and force-frequency behavior in left ventricular muscle strips from failing human hearts. METHODS: We tested the influence of [10]-gingerol which activates SR Ca(2+)-ATPase (10 mumol/l; n = 13), and isoproterenol which activates cAMP-dependent pathways (0.01, 0.1, 1 mumol/l; n = 40) on post-rest and force-frequency behavior. Ouabain which blocks Na+/K(+)-ATPase (0.03 mumol/l; n = 16) was used to test the effects of inhibiting Ca(2+)-elimination by Na+/Ca(2+)-exchange. For comparison, the effects of blocking SR Ca(2+)-uptake by thapsigargin (10 mumol/l; n = 14) were tested. In addition, Ca(2+)-uptake in myocardial homogenates was measured for gingerol (10 mumol/l; n = 6). RESULTS: Gingerol, isoproterenol (0.1, 1 mumol/l) and ouabain exerted significant positive inotropic effects under basal experimental conditions and normalized post-rest behavior. In contrast, force-frequency relation was only slightly improved by gingerol and isoproterenol (0.01 mumol/l). Ouabain and isoproterenol (1 mumol/l) further deteriorated force-frequency relation due to frequency-dependent significant increases in diastolic tension. Thapsigargin exerted negative inotropic effects and significantly deteriorated post-rest and force-frequency behavior. In addition, gingerol increased SR Ca(2+)-uptake significantly in myocardial homogenates. CONCLUSIONS: Inotropic interventions that stimulate SR Ca(2+)-ATPase or inhibit Na+/Ca(2+)-exchange normalize impaired post-rest behavior. Force-frequency behavior is only slightly improved by stimulation of SR Ca(2+)-ATPase but not by inhibition of Na+/Ca(2+)-exchange. This dissociation between post-rest and force-frequency behavior results from diastolic dysfunction at high stimulation rates.     

Disproportionate enhancement of myocardial contractility by the xanthine oxidase inhibitor oxypurinol in failing rat myocardium

OBJECTIVE: Xanthine oxidase (XO) inhibitors enhance myofilament Ca(2+) responsiveness of normal rat myocardium. We examined whether this inotropic action is preserved or magnified in failing rat myocardium and whether the magnitude of this effect correlates with tissue xanthine-oxidoreductase (XOR) activity. METHODS: Hearts of 18-20 month-old SHHF (spontaneous hypertensive/heart failure) rats with end-stage heart failure, as well as of normal control rats, were perfused with the XO inhibitor oxypurinol. Afterwards, [Ca(2+)](i) and tension were measured simultaneously in fura-2-loaded intact isolated right ventricular trabeculae. XOR activity was determined fluorometrically in myocardial homogenates. RESULTS: In failing myocardium, 100 microM oxypurinol significantly increased systolic twitch tension (by 87 and 92% at 1.0 and 1.5 mM extracellular [Ca(2+)], respectively), without altering [Ca(2+)](i) transient amplitude. Oxypurinol did not alter the midpoint or cooperativity of the steady-state tension-[Ca(2+)](i) relationship, but significantly enhanced maximum Ca(2+)-activated tension by 75% in failing myocardium. Oxypurinol also exerted a positive inotropic effect in failing myocardium, which was, however, of significantly smaller relative magnitude. Failing rat myocardium exhibited higher XOR activity than nonfailing myocardium, and this activity was largely suppressed in oxypurinol-treated preparations. CONCLUSIONS: The magnitude of functional improvement with XOR inhibitors depends on the initial level of XOR activity. Specifically, the inotropic actions of oxypurinol are more pronounced in failing rat myocardium, a tissue that exhibits enhanced XOR activity. Our findings rationalize how XO inhibitors boost cardiac contractility and improve mechanoenergetic coupling, and why the effects might be relatively 'selective' for heart failure.     

Insulin-like growth factor-1 exerts Ca2+-dependent positive inotropic effects in failing human myocardium

Myocardial generation of insulin-like growth factor-1 (IGF-1) is altered in hypertrophy and heart failure, but there are no reports on acute functional effects of IGF-1 in human cardiac muscle. We examined inotropic responses and signal transduction mechanisms of IGF-1 in human myocardium. Experiments were performed in isolated trabeculae or cardiomyocytes from 46 end-stage failing hearts. The effect of IGF-1 (0.001 to 0.2 micromol/L) on isometric twitch force (37 degrees C, 1 Hz), intracellular Ca2+ transients (aequorin method), sarcoplasmic reticulum (SR) Ca2+ content (rapid cooling contractures), L-type Ca2+ current (whole-cell voltage clamp), and cAMP concentrations was assessed. In addition, the effects of blocking IGF-1 receptors, phosphoinositide 3-kinase (PI3-kinase), protein kinase C (PKC), or transsarcolemmal Ca2+ entry were tested. IGF-1 exerted concentration-dependent positive inotropic effects (twitch force increased to maximally 133+/-4% of baseline values at 0.1 micromol/L; P<0.05). The IGF-1 receptor antibody alphaIR3 or the PI3-kinase inhibitor wortmannin prevented the functional effects. The inotropic response was paralleled by increases in Ca2+ transients and SR Ca2+ content. IGF-1 (0.1 micromol/L) increased L-type Ca2+ current amplitude by 24+/-7% (P<0.05). Blockade of SR function did not affect the inotropic response to IGF-1. In contrast, L-type Ca2+ channel blockade with diltiazem partially prevented ( approximately 50%) the inotropic response to IGF-1. Inhibition of PKC (GF109203X), Na+-H+ exchange (HOE642), or reverse-mode Na+-Ca2+ exchange (KB-R7943) reduced the response to IGF-1 by approximately 60% to 70%. IGF-1 exerts Ca2+-dependent positive inotropic effects through activation of IGF-1 receptors and a PI3-kinase-dependent pathway in failing human myocardium. The increased [Ca2+]i with IGF-1 originates from both enhanced L-type Ca2+ currents and enhanced Na+-H+ exchange-dependent reverse-mode Na+-Ca2+ exchange. These nongenomic functional effects of IGF-1 may be of clinical relevance.     

Influence of mild hypothermia on myocardial contractility and circulatory function

Myocardial contractility depends on temperature. We investigated the influence of mild hypothermia (37–31°) on isometric twitch force, sarcoplasmic reticulum (SR) Ca²⁺-content and intracellular Ca²⁺-transients in ventricular muscle strips from human and porcine myocardium, and on in vivo hemodynamic parameters in pigs. In vitro experiments: muscle strips from 5 nonfailing human and 8 pig hearts. Electrical stimulation (1 Hz), simultaneous recording of isometric force and rapid cooling contractures (RCCs) as an indicator of SR Ca²⁺-content, or intracellular Ca²⁺-transients (aequorin method). In vivo experiments: 8 pigs were monitored with Millar-Tip (left ventricular) and Swan-Ganz catheter (pulmonary artery). Hemodynamics parameters were assessed at baseline conditions (37°), and after stepwise cooling on cardiopulmonary bypass to 35, 33, and 31°C. Hypothermia increase isometric twitch force significantly by 91 ± 16% in human and by 50 ± 9% in pig myocardium (31 vs. 37°C; p < 0.05, respectively). RCCs or aequorin light emission did not change significantly. In anesthetized pigs, mild hypothermia resulted in an increase in hemodynamic paramters of myocardial contractility. While heart rate decreased from 111 ± 3 to 73 ± 1 min⁻¹, cardiac output increased from 2.4 ± 0.1 to 3.1 ± 0.3 l/min, and stroke volume increased from 21 ± 1 to 41 ± 3 ml. +dP/dt_max increased by 25 ± 8% (37 vs. 31°C; p < 0.05 for all values). Systemic and pulmonary vascular resistance did not change significantly during cooling. Mild hypothermia exerts significant positive inotropic effects in human and porcine myocardium without increasing intracellular Ca²⁺-transients or SR Ca²⁺-content. These effects translate into improved hemodynamics parameters of left ventricular function. Received: 26 June 2000, Returned for revision: 20 July 2000, Revision received: 11 October 2000, Accepted: 17 October 2000     

Functional relevance of the stretch-dependent slow force response in failing human myocardium

Stretch induces immediate and delayed inotropic effects in mammalian myocardium via distinct mechanosensitive pathways, but these effects are poorly characterized in human cardiac muscle. We tested the effects of stretch on immediate and delayed force response in failing human myocardium. Experiments were performed in muscle strips from 52 failing human hearts (37 degrees C, 1 Hz, bicarbonate buffer). Muscles were stretched from 88% of optimal length to 98% of optimal length. The resulting immediate and delayed (ie, slow force response [SFR]) increases in twitch force were assessed without and after blockade of the sarcoplasmic reticulum (SR; cyclopiazonic acid and ryanodine), stretch-activated ion channels (SACs; gadolinium, streptomycin), L-type Ca2+-channels (diltiazem), angiotensin II type-1 (AT1) receptors (candesartan), endothelin (ET) receptors (PD145065 or BQ123), Na+/H+ exchange (NHE1; HOE642), or reverse-mode Na+/Ca+ exchange (NCX; KB-R7493). We also tested the effects of stretch on SR Ca2+ load (rapid cooling contractures [RCCs]) and intracellular pH (in BCECF-loaded trabeculae). Stretch induced an immediate (<10 beats), followed by a slow (5 to 10 minutes), force response. Twitch force increased to 232+/-6% of prestretch value during the immediate phase, followed by a further increase to 279+/-8% during the SFR. RCC amplitude significantly increased, but pHi did not change during SFR. Inhibition of SACs, L-type Ca2+ channels, AT1 receptors, or ET receptors did not affect the stretch-dependent immediate or SFR. In contrast, the SFR was reduced by NHE1 inhibition and almost completely abolished by reverse-mode NCX inhibition or blockade of sarcoplasmic reticulum function. The data demonstrate the existence of a functionally relevant, SR-Ca2+-dependent SFR in failing human myocardium, which partly depends on NHE1 and reverse-mode NCX activation.     

Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins

OBJECTIVE: The alterations in contractile proteins underlying enhanced Ca(2+)-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca(2+)-responsiveness in human heart failure. METHODS: Isometric force and its Ca(2+)-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca(2+)-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting. RESULTS: Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca(2+)-sensitivity of the contractile apparatus (pCa(50)) was significantly higher in failing myocardium (deltapCa(50)=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca(2+)-responsiveness. An inverse correlation was found between pCa(50) and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca(2+)-sensitivity to a larger extent in failing (deltapCa(50)=0.20) than in donor (deltapCa(50)=0.03) myocytes, abolishing the difference in Ca(2+)-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca(2+)-responsiveness. CONCLUSIONS: The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.     

Reduced myocardial sarcoplasmic reticulum Ca(2+)-ATPase protein expression in compensated primary and secondary human cardiac hypertrophy.

Pathological intracellular calcium handling has been proposed to underlie the alterations of contractile behavior in hypertrophied myocardium. However, the myocardial protein expression of intracellular calcium transport proteins in compensated human left ventricular hypertrophy has not yet been studied. We investigated septal myocardial specimens of patients suffering from hypertrophic obstructive cardiomyopathy (n=14) or from acquired aortic valve stenosis (n=11) undergoing myectomy or aortic valve replacement, respectively. For comparison, we studied non-hypertrophied myocardium of six non-failing hearts which could not be transplanted for technical reasons. The myocardial density of the calcium release channel of the sarcoplasmic reticulum (SR) was determined by(3)H-ryanodine binding. Myocardial contents of SR Ca(2+)-ATPase, phospholamban, calsequestrin and Na(+)/Ca(2+)-exchanger were analysed by Western blot analysis. The myocardial SR calcium release channel density was not significantly different in hypertrophied and non-failing human myocardium. In both hypertrophic obstructive cardiomyopathy and in aortic valve stenosis, SR Ca(2+)-ATPase expression was reduced by about 30% compared to non-failing myocardium (P<0.05), whereas the expression of phospholamban, calsequestrin, and the Na(+)/Ca(2+)-exchanger was unchanged. The decrease of SR Ca(2+)-ATPase expression was still observable when related to its regulatory protein phospholamban or to the myosin content of the homogenates (P<0.05). Furthermore, the SR Ca(2+)-ATPase expression was inversely correlated to the septum thickness assessed by echocardiography, but not to age, cardiac index or outflow tract gradient. In primary as well as in secondary hypertrophied human myocardium, the expression of SR Ca(2+)-ATPase is reduced and inversely related to the degree of the hypertrophy. The diminished SR Ca(2+)-ATPase expression might result in reduced Ca(2+)reuptake into the SR and might contribute to altered contractile behavior in hypertrophied human myocardium.     

An inhibitor of nitric oxide synthase does not increase contraction or beta-adrenoceptor sensitivity of ventricular myocytes from failing human heart

OBJECTIVE: Nitric oxide (NO) has been implicated in the depression of cardiac function in human heart failure. Some reports have identified iNOS (inducible nitric oxide synthase) within the myocyte component of the failing human heart, and NO is known to decrease the contraction amplitude of isolated ventricular myocytes. We have treated myocytes from failing human ventricle with a NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), in an attempt to restore contractile function. METHODS AND RESULTS: Myocytes were isolated from failing and non-failing human ventricles and their contraction amplitude was measured during superfusion (32 degrees C, 1-2 mmol/l Ca2+) and electrical stimulation (0.1-2 Hz). The contraction amplitude of myocytes from failing hearts was depressed in a frequency-dependent manner. At 1 Hz, the contraction amplitude of cells from non-failing heart was 4.70 +/- 0.53% cell shortening (mean +/- SEM, n = 13 subjects), compared with 2.18 +/- 0.27% (P < 0.01, 11 patients) from patients with ischaemic heart disease (IHD) or 2.56 +/- 0.74% (P < 0.02, six patients) with dilated cardiomyopathy (DCM). Superfusion with 0.1 mmol/l L-NMMA did not increase contraction amplitude in myocytes from failing heart at either 0.2 Hz (n = 11) or 1 Hz (n = 7). Responses to beta-adrenoceptor stimulation were reduced in myocytes from failing human heart, with contraction amplitude in maximum isoprenaline 0.47 +/- 0.11 of that in high Ca2+ in the same cell (n = 6), compared to 0.99 +/- 0.07 in non-failing heart (n = 14, P < 0.01). The presence of 0.1 mmol/l L-NMMA did not increase the isoprenaline/Ca2+ ratio in myocytes from failing heart (0.40 +/- 0.09, P = NS). CONCLUSION: These results do not suggest a functional role for tonic NO production in the frequency-dependent depression of contraction or beta-adrenoceptor desensitisation in myocytes from failing human ventricle.     

In vitro motility analysis of thin filaments from failing and non-failing human heart: troponin from failing human hearts induces slower filament sliding and higher Ca(2+) sensitivity

Contractility of the myocardium is altered in end-stage heart failure. We investigated whether this was related to functional changes in troponin. We isolated troponin from 1 g samples of end-stage failing, non-failing and foetal human heart and studied its regulation of actin-tropomyosin movement over immobilised HMM by in vitro motility assay. At pCa5.4 the sliding velocity of thin filaments reconstituted with non-failing heart troponin was 52+/-4% more than actin-tropomyosin, with failing heart troponin velocity increased by 35+/-2% and with foetal heart troponin velocity increased by 11+/-4%. Thin filaments containing troponin from failing hearts were more Ca(2+)-sensitive than non-failing heart troponin. EC(50) for the fraction of filaments motile and filament velocity decreased 1.76+/-0.20 and 1.89+/-0.62-fold respectively relative to non-failing heart troponin. With foetal heart troponin the EC(50) decreased 2.16+/-0.23 and 3.50+/-1.73-fold for fraction and velocity respectively. Western blots revealed no difference in troponin T or troponin I isoform expression in troponin from failing and non-failing adult hearts but foetal isoforms of troponin I and T were observed in troponin from foetal heart. The level of PKA phosphorylation of troponin from failing and non-failing heart was not significantly different, however, complete non-specific dephosphorylation of troponin abolished most of the difference between failing and non-failing heart troponin. These findings show functional alterations in troponin in failing hearts which could account for the reduced contractile function but there is no change in troponin isoform expression or PKA phosphorylation. Differential phosphorylation by other kinases may account for altered troponin function.     

Contribution of cAMP-phosphodiesterase inhibition and sensitization of the contractile proteins for calcium to the inotropic effect of pimobendan in the failing human myocardium.

Previous studies have shown reduced effects of cAMP-dependent positive inotropic agents in the failing human myocardium; thus other cAMP-independent mechanisms of action may be useful to increase force of contraction in this condition. The purpose of this investigation was to determine whether a positive inotropic effect of the cAMP-phosphodiesterase (PDE) inhibitor pimobendan is observed in the failing human myocardium and to study whether other factors, such as an increase in the Ca2+ sensitivity of myofilaments, play a functional role in the increase in force of contraction. Pimobendan produced a positive inotropic effect in isolated preparations from nonfailing donor hearts; however, in moderately (New York Heart Association class II-III, NYHA II-III) and severely (NYHA IV) failing myocardium, this effect was reduced. In addition, in NYHA IV specimens pimobendan inhibited the crude cAMP-PDE (crude PDE) and the isoenzymes I-III (PDE I-III) in a concentration-dependent way. As judged from the IC50 values found in this tissue for the inhibition of PDE III and of crude PDE, the potency of the compound was 18.1 times greater on PDE III. Consistent with a cAMP-PDE-dependent mechanism of action, the positive inotropic effect was potentiated by isoproterenol and inhibited by adenosine in failing myocardium. In failing myocardium, pimobendan also increased the sensitivity of skinned cardiac fibers to Ca2+ and shifted the Ca(2+)-tension relation to the left. This sensitizing effect began at 0.01 mumol/l in NYHA II-III and NYHA IV and rose to about 200% at 300 mumol/l in both groups. In contrast, the demethylated metabolite UD-CG 212 Cl failed to produce positive inotropic effects in failing myocardium alone, but in the presence of isoproterenol, it exerted an increase in force of contraction. The potency of UD-CG 212 Cl for PDE III inhibition in NYHA IV was greater than that of pimobendan. The metabolite pronouncedly decreased the sensitivity of skinned cardiac fibers to Ca2+ at 30-300 mumol/l in NYHA II-III and NYHA IV. It is concluded that in the failing human heart pimobendan inhibited PDE III and sensitized contractile proteins for Ca2+. Both effects appear to be involved in the positive inotropic effect of the compound, because its metabolite, UD-CG 212 Cl, had no effect on force of contraction and on the Ca2+ sensitivity of skinned cardiac fibers but inhibited PDE III even more potently than pimobendan.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for a sustained effectiveness of sodium-channel activators in failing human myocardium.

Elevation of cytosolic sodium is thought to be correlated with an increase in force of contraction due to an activation of sodium-calcium exchange. We investigated the inotropic response mediated by the new sodium-channel activator BDF 9148 (0.01-100 mumol/l) on failing human myocardium. Force of contraction was studied using electrically driven human papillary muscle strips from moderately (NYHA II-III, mitral valve replacement) and terminally (NYHA IV, heart transplantation) failing hearts. We also investigated the effects in auricular trabeculae from non-failing hearts (aortocoronary bypass operation). Results were compared with inotropic responses to DPI 201-106 (DPI, 0.1-3 mumol/l), Ca2+ (1.8-15 mmol/l) and isoprenaline (0.001-1 mumol/l). Carbachol (100 mumol/l) and adenosine (1000 mumol/l) were examined in the presence of BDF 9148 and isoprenaline. Both sodium-channel activators, BDF 9148 and DPI 201-106, increased force of contraction in a dose-dependent manner in papillary muscle strips as well as in auricular trabeculae. BDF 9148 and DPI 201-106 were more effective (max. PIE NYHA II-III 1.6 +/- 0.2 mN, NYHA IV 5.9 +/- 0.7 mN, P less than 0.05) and more potent (EC50 (in mumol/l): NYHA IV 0.35, 0.19-0.66; NYHA II-III 1.85, 1.37-2.41) in terminally failing as compared to moderately failing left ventricular myocardium. Moreover, the positive inotropic effects of BDF 9148 were greater than those of DPI 201-106 in NYHA IV (max. PIE 2.7 +/- 0.3 mN, P less than 0.05). In NYHA IV, BDF 9148 was as effective as CA2+ (max. PIE 5.1 +/- 0.4 mN). In the same hearts, the positive inotropic effects of isoprenaline were reduced in NYHA IV (max. PIE 2.1 +/- 0.3 mN) compared to NYHA II-III (max. PIE 3.4 +/- 0.4 mN, P less than 0.05). Adenosine as well as carbachol did not affect the positive inotropic response of BDF 9148 or DPI 201-106 but reduced the effectiveness of isoprenaline (P less than 0.05). In myocardial membranes, BDF 9148 was 1000-fold less effective in competition experiments with 3H-ouabain than ouabain. We conclude that (1) sodium-channel activators may produce a significant cAMP-independent positive inotropic effect in left ventricular myocardium from failing human hearts; (2) the inotropic effect of sodium-channel activators were more potent and more effective in NYHA IV as compared to NYHA II-III. The degree of myocardial failure does not reduce the effectiveness of the sodium-channel activator BDF 9148.     

Abnormal frequency-dependent responses represent the pathophysiologic signature of contractile failure in human myocardium

BACKGROUND. - The normal increase in isometric developed force (DF) with faster pacing rates, known as the positive force-frequency response/relationship (FFR), is altered in failing myocardium, as shown by its negative response to increased pacing. The objective of this study was to determine if increasing Ca2+ influx with L-type Ca2+ channel (L-CaCh) agonists: BayK 8644 (BayK) and FPL 64176 (FPL) or increased extracellular Ca2+ could increase contractility and normalize the FFR in failing myocardium. METHODS. - Isometric DF was measured in right ventricular trabeculae from failing (n = 28) and non-failing (n = 12) human hearts at various stimulation frequencies (0.5-2.5 Hz) before and after bath application of BayK (250 nM), FPL (100 nM), or high Ca2+ (7.0 mM). Post-rest (PR) experiments were also conducted on several trabeculae. RESULTS. - In trabeculae from failing hearts, the DF decreased with an increase in pacing. Addition of L-CaCh agonists increased DF to similar levels in trabeculae from both failing and non-failing hearts at slow pacing rates, but did not alter the negative FFR in the failing group. During increased rest intervals, the amount of PR potentiation was diminished in trabeculae from failing hearts as compared to the non-failing preparations. CONCLUSION. - This study demonstrates that the abnormal FFR observed in trabeculae from failing hearts is a reliable physiologic signature of the cardiomyopathic state even when DF, at slow stimulation frequencies, is relatively high. These studies further demonstrate that the impaired FFR is not due to an inability to further increase contractility. Rather, our findings suggest that the abnormal FFR and blunted PR potentiation alike are a reflection of an altered functional balance between Ca2+ re-uptake and Ca2+ extrusion.     

Dantrolene sodium improves the force-frequency relationship and beta-adregenic responsiveness in failing human myocardium

BACKGROUND: Failing human myocardium is characterized by a negative force-frequency relationship and impaired beta-adrenergic responsiveness which have been related to alterations of the intracellular Ca2+ homeostasis. Dantrolene sodium is a clinically used drug that modulates myocardial [Ca2+]i handling in animal models. This study investigated the effects of dantrolene sodium on intracellular Ca2+ handling and contractile function in failing human myocardium. METHODS AND RESULTS: Twenty-three muscle strips from human left ventricular trabeculae were obtained from patients undergoing heart transplantation for end-stage heart failure caused by idiopathic dilated cardiomyopathy (n = 15). Isometric contraction and intracellular Ca2+ transients (Ca2+ indicator: aequorin) were recorded simultaneously. The experiments were performed in three separate groups exposed to control condition (n = 8), addition of dantrolene (10 micromol/l; n = 8), or addition of verapamil (1 micromol/l; n = 7). Isoproterenol induced a moderate positive inotropic effect in the control group with a maximal increase of developed tension from 10.8 +/- 2.9 to 23.4 +/- 4.7 mN/mm2 and a parallel rise in peak systolic [Ca2+]i to a maximum of 1.36 +/- 0.20 micromol/l. Dantrolene significantly improved (10.2 +/- 3.8 to 32.4 +/- 0.9 mN/mm2) and verapamil blunted (8.3 +/- 2.8 to 17.1 +/- 4.3 mN/mm2) the inotropic response to isoproterenol. The diastolic and systolic [Ca2+]i during isoproterenol stimulation were slightly lower in the dantrolene group but significantly depressed in the verapamil group as compared to the control group. Similarly, analyses of force-frequency relationships revealed an improvement of developed tension in dantrolene-treated as compared to control preparations whereas the peak systolic [Ca2+]i was almost identical. CONCLUSION: Dantrolene improves the negative force-frequency relationship and beta-adrenergic responsiveness in failing human myocardium. These effects are not accompanied by an additional increase in intracellular [Ca2+]i but might be related to modifications of the diastolic [Ca2+]i homeostasis.     

Sarcoplasmic reticulum Ca 2+ load in human heart failure

Excitation-contraction coupling and intracellular Ca ²⁺ homeostasis are altered in heart failure. We tested the hypothesis that these changes are related to disturbed Ca ²⁺ handling of the sarcoplasmic reticulum (SR). Isolated, electrically stimulated trabeculae were obtained from end-stage failing (NYHA IV) and nonfailing human hearts. Isometric twitch tension, intracellular Ca ²⁺ transients (aequorin method) and SR Ca ²⁺ content (rapid cooling contractures) were assessed under basal conditions (1 Hz, 37 °C) as well as after stepwise increasing rest intervals from 2 – 240 s (post-rest contractions). Protein expression of SERCA2a and phospholamban (Western blot) was assessed in a subset of failing trabeculae. In addition, the effects of SERCA1 overexpression on contractile function of isolated myocytes was tested. On average, post-rest twitch tension continuously increased with increasing rest intervals in nonfailing, but declined with rest intervals longer than 15s in failing myocardium. The rest-dependent contractile changes were accompanied by parallel changes in intracellular Ca ²⁺ transients. Failing trabeculae (n = 40) were grouped (group A: post-rest potentiation (force of contraction > pre-rest twitch force) after 120s rest interval; group B: post-rest decay (force of contraction < pre-rest twitch force) after 120 s rest interval), and post-rest contractile function was related to SERCA2a and PLB expression. While PLB protein expression was not different, SERCA2a protein expression as well as SERCA2a/PLB ratio was significantly higher in group A vs. group B. Transfection of SERCA1 increased shortening amplitude and enhanced relaxation kinetics in failing human myocytes. In conclusion, SR Ca ²⁺ handling is severely altered in human heart failure. Reduced SR Ca ²⁺ release is due to diminished SR Ca ²⁺ content directly related to a depressed expression of SERCA2a protein. Enhancing SERCA function or expression may improve SR Ca ²⁺ handling in failing human myocardium.     

High intracellular Na+ preserves myocardial function at low heart rates in isolated myocardium from failing hearts

We investigated the hypothesis that increased intracellular [Na+]i in heart failure contributes to preservation of SR Ca2+ load which may become particularly evident at slow heart rates. [Na+]i in SBFI-loaded myocytes from rabbits with pacing-induced heart failure (PHF) was significantly higher at each frequency as compared to Sham-operated animals. Furthermore, PHF rabbits demonstrated reduced SR Ca2+-ATPase protein levels (-37%, p < 0.04) but unchanged Na+/Ca2+ exchanger protein levels. At 0.25 Hz, isometric force was similar in cardiac trabeculae from PHF rabbits as compared to control (PHF, 3.6+/-1.3; Sham, 4.4+/-0.6 mN/mm2). Rapid cooling contractures (RCCs) were unchanged indicating preserved SR Ca2+ load at this frequency. In Sham, isometric twitch force increased with rising frequencies to 29.0+/-2.8 mN/mm2 at 3.0 Hz (p < 0.05) as compared to 0.25 Hz. RCCs showed a parallel increase by 186+/-47% (p < 0.01). In PHF, frequency-dependent increase in force (15.8+/-4.7 mN/mm2 at 3.0 Hz) and RCCs (increase by 70+/-40%) were significantly blunted. Thus, in PHF in rabbits SR Ca2+ load is preserved at low frequencies despite decreased SR Ca2+-ATPase expression. This may result from [Na+]i-dependent changes in Na+/Ca2+ exchanger activity.     

Regional expression and functional characterization of the L-type Ca2+-channel in myocardium from patients with end-stage heart failure and in non-failing human hearts

The purpose of the present study was to investigate the expression and functional relevance of sarcolemmal L-type Ca2+-channels in failing and non-failing human myocardium. The protein expression of sarcolemmal L-type Ca2+-channels was determined with 3H-(+)-PN 200-110-binding experiments and Western blot analysis using a specific antibody against the alpha1-subunit in membrane preparations of ventricular and atrial myocardium from both failing (n = 15) and non-failing hearts (n = 8). The gene expression of the ion conducting pore of the L-type Ca2+-channel was examined with Northern blot technique in human failing and non-failing RNA. For normalization the RNA expression of calsequestrin was used. In electrically driven ventricular papillary muscle strips and auricular trabeculae, the responses to nifedipine and Ca2+ as parameters of myocardial function were studied. The protein expression as measured by 3H-(+)-PN 200-110-binding (Bmax) and Western Blot analysis with calsequestrin as reference was similar in left ventricular failing and non-failing myocardium. However, both were reduced in atrial compared to ventricular tissue in failing and non-failing hearts. The KD remained unchanged. Calsequestrin levels were unaltered in failing and non-failing hearts. The gene expression of the alpha1-subunit was similar in human failing and non-failing hearts. The L-type Ca2+-channel antagonist nifedipine reduced force of contraction with the same potency and efficiency in ventricular failing and non-failing myocardium. In contrast, the potency of nifedipine was higher in atrial than in ventricular tissue. Consistently, atrial myocardium from patients with dilated cardiomyopathy was more sensitive towards Ca2+ than those of the control group. In conclusion, the altered Ca2+-homeostasis in failing human myocardium may be less due to changes in sarcolemmal L-type Ca2+-channel expression or function than due to an altered intracellular Ca2+-handling.     

Functional effects of protein kinase C-mediated myofilament phosphorylation in human myocardium

OBJECTIVE: In human heart failure beta-adrenergic-mediated protein kinase A (PKA) activity is down-regulated, while protein kinase C (PKC) activity is up-regulated. PKC-mediated myofilament protein phosphorylation might be detrimental for contractile function in cardiomyopathy. This study was designed to reveal the effects of PKC on myofilament function in human myocardium under basal conditions and upon modulation of protein phosphorylation by PKA and phosphatases. METHODS: Isometric force was measured at different [Ca(2+)] in single permeabilized cardiomyocytes from non-failing and failing human left ventricular tissue. Basal phosphorylation of myofilament proteins and the influence of PKC, PKA, and phosphatase treatments were analyzed by one- and two-dimensional gel electrophoresis, Western immunoblotting, and ELISA. RESULTS: Troponin I (TnI) phosphorylation at the PKA sites was decreased in failing compared to non-failing hearts and correlated well with myofilament Ca(2+) sensitivity (pCa(50)). Incubation with the catalytic domain of PKC slightly decreased maximal force under basal conditions, but not following PKA and phosphatase pretreatments. PKC reduced Ca(2+) sensitivity to a larger extent in failing (DeltapCa(50)=0.19+/-0.03) than in non-failing (DeltapCa(50)=0.08+/-0.01) cardiomyocytes. This shift was reduced, though still significant, when PKC was preceded by PKA, while PKA following PKC did not further decrease pCa(50). Protein analysis indicated that PKC phosphorylated PKA sites in human TnI and increased phosphorylation of troponin T, while myosin light chain phosphorylation remained unaltered. CONCLUSION: In human myocardium PKC-mediated myofilament protein phosphorylation only has a minor effect on maximal force development. The PKC-mediated decrease in Ca(2+) sensitivity may serve to improve diastolic function in failing human myocardium in which PKA-mediated TnI phosphorylation is decreased.     

Troponin phosphorylation and regulatory function in human heart muscle: dephosphorylation of Ser23/24 on troponin I could account for the contractile defect in end-stage heart failure

We made quantitative measurements of phosphorylation in troponin isolated from 6 non-failing donor hearts and 6 explanted hearts with end-stage heart failure in SDS-PAGE gels using Pro-Q Diamond phosphoprotein stain. The troponin T phosphorylation level was the same in troponin from failing and non-failing heart (3.1 mol Pi/mol). However, troponin I phosphorylation was significantly lower in failing (0.37+/-0.18 mol Pi/mol) compared with non-failing heart troponin (2.25+/-0.36 mol Pi/mol). Levels of troponin I PKA-dependent phosphorylation, measured with a phosphoserine 23/24-specific antibody, were also significantly lower in failing heart troponin (0.19+/-0.06 mol Pi/mol) compared to non-failing troponin (1.14+/-0.09 mol Pi/mol). We calculate that there is phosphorylation in addition to serine 23/24 of 1.11+/-0.34 mol Pi/mol in non-failing reduced to 0.18+/-0.17 mol Pi/mol in failing heart troponin, attributed to phosphorylation on the PKC sites. To test for the functional role of troponin I phosphorylation, the native troponin I from either non-failing or failing heart troponin was exchanged for a recombinant (unphosphorylated) human cardiac troponin I. Thin filament Ca(2+)-regulatory function was studied with the quantitative in vitro motility assay: thin filaments containing the replaced troponin I resulted in a failing phenotype of a 17-26% reduced sliding speed and an increased Ca(2+)-sensitivity relative to non-failing troponin (EC(50) TnI-exchanged/non-failing=0.57, p<0.001). When exchanged with troponin I phosphorylated with PKA motility parameters reverted to a pattern indistinguishable from non-failing troponin (p=0.35-0.75). We suggest that changes in troponin function can account for the contractile abnormality in failing heart muscle and that the functional changes in troponin are due to reduced phosphorylation of troponin I at the PKA sites.     

Differences in Ca-Handling and Sarcoplasmic Reticulum Ca-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.