Alterations of myocardial dynamic stiffness implicating abnormal crossbridge function in human mitral regurgitation heart failure.

Mitral regurgitation (MR) causes ventricular dilation, a blunted myocardial force-frequency relation, and increased crossbridge force-time integral (FTI). The mechanism of FTI increase was investigated using sinusoidal length perturbation analysis to compare crossbridge function in skinned left ventricular (LV) epicardial muscle strips from 5 MR and 5 nonfailing (NF) control hearts. Myocardial dynamic stiffness was modeled as 3 parallel viscoelastic processes. Two processes characterize intermediate crossbridge cycle transitions, B (work producing) and C (work absorbing) with Q(10)s of 4 to 5. No significant differences in moduli or kinetic constants of these processes were observed between MR and NF. The third process, A, characterizes a nonenzymatic (Q(10)=0.9) work-absorbing viscoelasticity, whose modulus increases sigmoidally with [Ca(2+)]. Effects of temperature, crossbridge inhibition, or variation in [MgATP] support associating the calcium-dependent portion of A with the structural "backbone" of the myosin crossbridge. Extension of the conventional sinusoidal length perturbation analysis allowed using the A modulus to index the lifetime of the prerigor, AMADP crossbridge. This index was 75% greater in MR than in NF (P=0.02), suggesting a mechanism for the previously observed increase in crossbridge FTI. Notably, the A-process modulus was inversely correlated (r(2)=0.84, P=0.03) with in vivo LV ejection fraction in MR patients. The longer prerigor dwell time in MR may be clinically relevant not only for its potential role as a compensatory mechanism (increased economy of tension maintenance and increased resistance to ventricular dilation) but also for a potentially deleterious effect (reduced elastance and ejection fraction).

Altered cross-bridge characteristics following haemodynamic overload in rabbit hearts expressing V3 myosin.

1. Our goal in this study was to evaluate the effect of haemodynamic overload on cross-bridge (XBr) kinetics in the rabbit heart independently of myosin heavy chain (MHC) isoforms, which are known to modulate kinetics in small mammals. We applied a myothermal-mechanical protocol to isometrically contracting papillary muscles from two rabbit heart populations: (1) surgically induced right ventricular pressure overload (PO), and (2) sustained treatment with propylthiouracil (PTU). Both treatments resulted in a 100 % V3 MHC profile. 2. XBr force-time integral (FTI), evaluated during the peak of the twitch from muscle FTI and tension-dependent heat, was greater in the PO hearts (0.80 +/- 0.10 versus 0.45 +/- 0.05 pN s, means +/- S.E.M., P = 0.01). 3. Within the framework of a two-state XBr model, the PO XBr developed more force while attached (5.8 +/- 0.9 versus 2.7 +/- 0.3 pN), with a lower cycling rate (0.89 +/- 0.10 versus 1.50 +/- 0.14 s(-1)) and duty cycle (0.14 +/- 0.03 versus 0.24 +/- 0.02). 4. Only the ventricular isoforms of myosin light chain 1 and 2 and cardiac troponin I (cTnI) were expressed, with no difference in cTnI phosphorylation between the PO and PTU samples. The troponin T (TnT) isoform compositions in the PO and PTU samples were significantly different (P = 0.001), with TnT2 comprising 2.29 +/- 0.03 % in PO hearts versus 0.98 +/- 0.01 % in PTU hearts of total TnT. 5. This study demonstrates that MHC does not mediate dramatic alterations in XBr function induced by haemodynamic overload. Our findings support the likelihood that differences among other thick and thin filament proteins underlie these XBr alterations.

A mechanistic analysis of reduced mechanical performance in human heart failure.

In failing human hearts (FHH) (NYHA IV) the cardiac output is inadequate to meet the metabolic needs of the peripheral systems. By means of thermo-mechanical analysis we have shown that epicardial strips from FHH (37 degrees C) have a depressed tension independent heat (TIH) and tension independent heat rate (dTIH / dt) liberation that correlates with depression in peak isometric force and the rate of relaxation. Furthermore, in response to a change in frequency of stimulation, FHH shows a severe blunting of the force-frequency relationship resulting in a decrease in myocardial reserve and in the frequency at which optimum force is obtained. We used ventricular ANF as an index of the severity of myocardial disease and demonstrated an inverse relationship between ANF mRNA and the sarcoplasmic reticulum (SR) calcium cycling proteins (SERCA 2, Phospholamban, Ryanodine Receptor) while these latter proteins all had a positive correlation with each other. At the same time there was an increase in sarcolemmal sodium calcium exchange protein. The decrease in SR pump proteins correlates with the decrease in myocardial reserve and optimum frequency of contraction. The latter mechanical changes are explainable in terms of a frequency dependent decrease in calcium concentration (aequorin light) in FHH.

R403Q and L908V mutant beta-cardiac myosin from patients with familial hypertrophic cardiomyopathy exhibit enhanced mechanical performance at the single molecule level.

Familial hypertrophic cardiomyopathy (FHC) is a disease of the sarcomere. In the beta-myosin heavy chain gene, which codes for the mechanical enzyme myosin, greater than 40 point mutations have been found that are causal for this disease. We have studied the effect of two mutations, the R403Q and L908V, on myosin molecular mechanics. In the in vitro motility assay, the mutant myosins produced a 30% greater velocity of actin filament movement (v(actin)). At the single molecule level, v(actin) approximately d/t(on), where d is the myosin unitary step displacement and t(on) is the step duration. Laser trap studies were performed at 10 microM MgATP to estimate d and t(on) for the normal and mutant myosin molecules. The increase in v(actin) can be explained by a significant decrease in the average t(on)'s in both the R403Q and L908V mutants (approximately 30 ms) compared to controls (approximately 40 ms), while d was not different for all myosins tested (approximately 7 nm). Thus the mutations affect the kinetics of the cross-bridge cycle without any effect on myosin's inherent motion and force generating capacity. Based on these studies, the primary signal for the hypertrophic response appears to be an apparent gain in function of the individual mutant myosin molecules.

Kinetic differences at the single molecule level account for the functional diversity of rabbit cardiac myosin isoforms.

1. Cardiac V3 myosin generates slower actin filament velocities and higher average isometric forces (in an in vitro motility assay) when compared with the V1 isoform. 2. To account for differences in V1 and V3 force and motion generation at the molecular level, we characterized the mechanics and kinetics of single V1 and V3 myosin molecules using a dual laser trap setup. 3. No differences in either unitary displacement (approximately 7 nm) or force (approximately 0.8 pN) were observed between isoforms; however, the duration of unitary displacement events was significantly longer for the V3 isoform at MgATP concentrations > 10 microM. 4. Our results were interpreted on the basis of a cross-bridge model in which displacement event durations were determined by the rates of MgADP release from, and MgATP binding to, myosin. 5. We propose that the release rate of MgADP from V3 myosin is half that of V1 myosin without any difference in their rates of MgATP binding; thus, kinetic differences between the two cardiac myosin isoforms are sufficient to account for their functional diversity.

A mechanistic analysis of the force-frequency relation in non-failing and progressively failing human myocardium.

This review focuses on the role of the myocardial force-frequency relation (FFR) in human ventricular performance and how changes in the FFR can reduce cardiac output and, ultimately, can contribute to altering the stability of the in-vivo cardiovascular system in a way that contributes to the progression of heart failure. Changes in the amplitude, shape, and position of the myocardial FFR occurring in various forms of heart failure are characterized in terms of maximal isometric twitch tension, slope of the ascending limb (myocardial reserve), and position of the peak of the FFR on the frequency axis (optimum stimulation frequency). All three of these parameters decline according to severity of myocardial disease in the following order: non-failing atrial septal defect, non-failing coronary artery disease, non-failing coronary artery disease with diabetes mellitus, failing mitral regurgitation, failing viral myocarditis, failing idiopathic dilated cardiomyopathy. Evidence is presented supporting a sarcoplasmic reticulum Ca-pump based mechanism for this progressive depression of the FFR. Intracellular calcium cycling and concentration and Ca-pump content all diminish in proportion to degree of depression of the FFR. Additional evidence from myocyte culture studies suggests a cause of diminished Ca-pump content is sustained, elevated levels of plasma norepinephrine. A hypothesis is presented to explain the mechanism of myocardial failure and its progression in terms of changes in the cardiovascular feedback control system that are triggered by reduced myocardial reserve. Sustained elevation of plasma norepinephrine levels depresses expression of sarcoplasmic reticulum Ca-pump protein causing depression of the FFR and this causes a compensatory further increase in norepinephrine levels and a further depression of Ca-pump protein.

Cross-bridge dynamics in the contracting heart.

The mechanical characteristics of the myosin motor is one of the key determinants of ventricular function. In small mammals there are two myosin isoforms, V1 and V3, with profoundly different performance characteristics. We used myothermal and mechanical analysis of intact papillary muscles from thryoxine (V1) and popylthiouracil (V3) treated rabbit hearts to assess the mechanical attributes of the myosin cross-bridge cycle. The average cross-bridge force time integral for V1 papillary muscles is 0.15 +/- 0.02 pNs for the entire isometric twitch and 0.19 +/- 0.03 pNs for the portion of the isometric twitch between 0.9 peak isometric force for the rising and declining portions of the twitch. The ratio of V1/V3 for the cross-bridge force time integral for the entire twitch and at the peak of the twitch is 0.5 (p < 0.05) and 0.4 (p < 0.05), respectively. Since the peak of the twitch measurements minimize internal shortening only these will be presented below. The average unitary force and attachment time during the peak of the twitch for V1 hearts was 1.55 +/- 0.37 pN and 140 +/- 20 msec, respectively. The ratios of V1/V3 for these parameters were 0.6 (p < 0.05) and 0.8 (ns). The cycling rate and duty cycle for V1 were 4.37 +/- 0.81 cycles per head-second and 0.66 +/- 0.22. The ratios of V1/V3 for cycling rate and duty cycle were 2.8 (p < 0.05) and 2.7 (ns). These measurements are consistent with and help explain the energetic and mechanical function of the intact heart.

Role of cAMP in modulating relaxation kinetics and the force-frequency relation in mitral regurgitation heart failure.

The report is a discussion of previously published and newly analyzed results concerning the association between heart diseases and alterations in the force-frequency relation (FFR). The optimum stimulation frequency of the FFR is measured and compared in isolated left ventricular myocardium from non-failing hearts with atrial septal defect, coronary artery disease (without and with insulin dependent diabetes mellitus) and from failing hearts with mitral regurgitation, or idiopathic dilated cardiomyopathy. Specifically, we examine the role of altered control of the excitation-contraction coupling system in blunting the force-frequency relation. We use the percent slope of the FFR as a measure of changes in the frequency sensitivity of this control. Our finding of a linear, direct relation between optimum stimulation frequency and % slope across all disease types suggests both parameters are coupled to the same underlying mechanism. To investigate the possible role of altered control of the calcium pump in this mechanism, we analyzed the detailed relation between isometric twitch relaxation kinetics and stimulation frequency in mitral regurgitation myocardium (MR). In the presence of 0.5 microM forskolin the depressed slope and optimum frequency of the FFR and the prolonged half-time of twitch relaxation were all restored to values found in non-failing myocardium. We use the kinetics of isometric twitch relaxation as an index of changes in pumping rate that occur in response to changes in stimulation frequency or in intracellular cyclic adenosine monophosphate concentration. A mathematical model based on the Hill relations for calcium pump uptake rate and for isometric tension as a function of intracellular pCa is developed to simulate isometric twitch relaxation in MR and non-failing myocardium. The success of this model in simulating non-failing and failing twitch relaxation supports a proposed mechanism for the prolonged relaxation time and depressed FFR in MR involving depressed protein kinase-A activity (due to lowered cAMP or to a defect in the Ser16 site of phospholamban) as a mechanism of altered control of the calcium pump in MR heart disease.

Human heart failure: determinants of ventricular dysfunction.

Thin muscle strips were obtained from non-failing (NF) and failing (dilated cardiomyopathy (DCM)) hearts, using a new harvesting and dissection technique. The strips were used to carry out a myothermal and mechanical analysis so that contractile and excitation coupling phenomena in the NF and failing (DCM-F) preparations can be compared. Peak isometric force and rate of relaxation in DCM-F were reduced 46% (p < 0.02) while time to peak tension was increased 14% (p < 0.03). Initial, tension dependent, tension independent and the rate of tension independent heat liberation were reduced 62-70% in DCM-F (p < 0.03). The crossbridge force-time integral (FTIXBr) was calculated from these measurements and was shown to increase 40% while the amount and rate of calcium cycled per beat was reduced 70%. As a result of these changes in the contractile and excitation-contraction coupling systems in DCM-F, the force-frequency relationship was significantly blunted while the power output was markedly reduced. These fundamental alterations account for the substantial ventricular dysfunction found in the dilated cardiomyopathic failing heart.

Influence of isoproterenol and ouabain on excitation-contraction coupling, cross-bridge function, and energetics in failing human myocardium.

BACKGROUND: In patients with heart failure, long-term treatment with catecholamines and phosphodiesterase inhibitors, both of which increase cyclic AMP levels, may be associated with increased mortality, whereas mortality may not be increased with glycoside treatment. Differences in clinical benefit between cyclic AMP-dependent inotropic agents and cardiac glycosides may be related to differences of these drugs on calcium cycling and myocardial energetics. METHODS AND RESULTS: Isometric heat and force measurements were used to investigate the effects of isoproterenol and ouabain on myocardial performance, cross-bridge function, excitation-contraction coupling, and energetics in myocardium from end-stage failing human hearts. Isoproterenol (1 mumol/L) increased peak twitch tension by 55% and decreased time to peak tension and relaxation time by 30% and 26%, respectively (P < .005). Ouabain (0.38 +/- 0.11 mumol/L) increased peak twitch tension and relaxation time by 41% and 20%, respectively, and decreased time to peak tension by 12% (P < .05). With isoproterenol, the amount of excitation-contraction coupling-related heat evolution (tension-independent heat) increased by 246% (P < .05) and the economy of excitation-contraction coupling decreased by 61% (P < .05). Ouabain increased tension-independent heat by only 61% (P < .05) and did not significantly influence economy of excitation-contraction coupling. The effects of isoproterenol on excitation-contraction coupling resulted in a 21% (P < .005) decrease of overall contraction economy, which was not significantly changed with ouabain. Neither isoproterenol nor ouabain influenced energetics of cross-bridge cycling or recovery metabolism. CONCLUSIONS: Major differences between the effects of isoproterenol and ouabain in failing human myocardium are related to calcium cycling with secondary effects on myocardial energetics.

Molecular motor mechanics in the contracting heart. V1 versus V3 myosin heavy chain.

The amount of iron in the low molecular weight pool (LMW) increases during no-flow ischemia and is thought to be essential to oxygen radical-derived damage upon reperfusion. Applying three short ischemic periods (5 min) preconditioning before 15 min ischemia results in an improved contractility compared to a direct 15 min ischemic insult. This raises the question whether preconditioning leads to a decrease in hte LMW iron pool. We therefore investigated the change in in hte LMW iron pool during ischemic insult after applying preconditioning. It is assumed that an increase in LMW iron is dependent on the accumulation of reduction equivalents derived from the anaerobic glycolysis. Therefore the glycogen content was also reduced by administration by anoxia and glucagon administration to study the effect on the LMW iron pool.

Maximal actomyosin ATPase activity and in vitro myosin motility are unaltered in human mitral regurgitation heart failure.

Myofibrillar but not actomyosin ATPase is depressed in failing myocardium from patients with dilated cardiomyopathy. Since there is a similar depression of myofibrillar ATPase in mitral regurgitation myocardium, we investigated whether or not the hydrolytic and mechanical performances of myosin are altered by comparing the maximal actomyosin ATPase activity and the in vitro myosin motility of myocardial myosin from patients with mitral regurgitation heart failure with that of patients with normal ventricular function. The results show that there is no significant difference (P > .05) between nonfailing and failing values for either the maximal actomyosin ATPase activity (0.3 s-1.head-1) or the myosin motility (1 micron/s). These observations suggest that changes, other than in the myosin heavy chain, contribute to the altered myocardial performance in mitral regurgitation myocardium.

Cardiac V1 and V3 myosins differ in their hydrolytic and mechanical activities in vitro.

The two mammalian cardiac myosin heavy chain isoforms, alpha and beta, have 93% amino acid homology, but hearts expressing these myosins exhibit marked differences in their mechanical activities. To further understand the function of these cardiac myosins as molecular motors, we compared the ability of these myosins to hydrolyze ATP and to both translocate actin filaments and generate force in an in vitro motility assay. V1 myosin has twice the actin-activated ATPase activity and three times the actin filament sliding velocity when compared with V3 myosin. In contrast, the force-generating ability of these myosins is quite different when the total force produced by a small population of myosin molecules (> 50) is examined. V1 myosin produces only one half the average cross-bridge force of V3 myosin. With discrete areas of primary structural heterogeneity known to exist between alpha and beta heavy chains, the differences we report in the hydrolytic and mechanical activities of the motors are explored in the context of potential structural and kinetic differences between the V1 and V3 myosins.

Myocyte reorganization in hypertrophied and failing hearts.

In hypertrophied and failing hearts there are major changes in the overall contractile performance. We present a review of our previous work relating the alterations in myocardial force, work, power and relaxation, that lead to changes in overall ventricular performance, to changes in the actin-myosin cross-bridge cycle characteristics along with the degree of activation and inactivation (calcium cycling). Tissues from hypertrophied rabbit and failing human (volume overload, dilated cardiomyopathy) heart were used in these studies. Myocardial peak twitch tension (mN.mm-2) was reduced in dilated cardiomyopathy (human) (25.9 +/- 3.9 vs 13.9 +/- 2.0, 37 degrees C), volume overload (human) (44.0 +/- 11.7 vs 19.9 +/- 3.7, 21 degrees C) and pressure overload (rabbit) (46.1 +/- 2.6 vs 41.7 +/- 5.0, 21 degrees C). We used myothermal and mechanical data to analyse the average cross-bridge force time integral and the amount of calcium cycled per gram per beat. Tension-dependent Heat (mJ.g-1) (TDH) (cross-bridge cycling) and tension-independent heat (mJ.g-1) (TIH) were reduced in all of the experimental preparations (dilated cardiomyopathy, human, 37 degrees C: TDH, 3.39 +/- 0.59 vs 1.34 +/- 0.22; TIH 1.51 +/- 0.02 vs 0.16 +/- 0.03) (volume overload, human 21 degrees C: TDH, 7.23 +/- 2.22 vs 1.92 +/- 0.25; TIH, 0.75 +/- 0.19 vs 0.39 +/- 0.04) (pressure overload, rabbit, 21 degrees C: TDH, 6.60 +/- 0.75 vs 3.05 +/- 0.46; TIH, 1.00 +/- 0.17 vs 0.41 +/- 0.08).(ABSTRACT TRUNCATED AT 250 WORDS)

Sarcoplasmic reticulum gene expression in pressure overload-induced cardiac hypertrophy in rabbit.

Pressure overload (PO)-induced cardiac hypertrophy in rabbits has been utilized extensively to study alterations in systolic and diastolic functions of the heart. In earlier studies we showed that the levels of mRNA encoding two important sarcoplasmic reticulum (SR) proteins, the cardiac/slow-twitch muscle Ca(2+)-ATPase (SERCA2a) and phospholamban, were decreased in PO rabbit hearts. In this study, we analyzed the expression of the Ca(2+)-release channel (ryanodine receptor), calsequestrin, SERCA2a, and phospholamban in PO-induced cardiac hypertrophy after 2, 4, 8, and 16 days of pulmonary artery banding. Northern blot and slot blot analyses showed that the steady-state level of mRNA encoding the cardiac ryanodine receptor, SERCA2a, and phospholamban was decreased significantly as early as 2 days after PO. In 16-day PO hearts, SERCA2a mRNA was reduced to 7.9 +/- 3.4% (P < 0.05), phospholamban mRNA was reduced to 15.9 +/- 6.5% (P < 0.05), and ryanodine receptor mRNA was reduced to 49.2 +/- 23.6% (P < 0.05). In this study, calsequestrin mRNA levels were also reduced to 29.9 +/- 15.2% by day 16 (P < 0.05). ATP-dependent Ca2+ uptake was reduced to 78% (P < 0.05); in contrast, the steady-state formation of ATPase phosphoenzyme was reduced to 81% of control (P < 0.05) and Ca(2+)-ATPase protein was reduced to 78% of control (P < 0.05) in crude SR vesicles or total muscle homogenate obtained from 16-day PO hearts. On the basis of these data, we propose that decreases in the expression of SR proteins may contribute to dysfunctions seen in systolic and diastolic properties of the hypertrophied myocardium.

Effects of calcium sensitizers on intracellular calcium handling and myocardial energetics.

Calcium sensitizers may influence myocardial energetics by their action on calcium turnover and on crossbridge behavior. Using a myothermal method, the effects of the Ca2+ sensitizer EMD-53998 on calcium cycling, crossbridge behavior, and myocardial energy turnover were compared with the effects of an increase in extracellular calcium from 1.25 to 7.5 mM and with the effects of the catecholamine isoproterenol. All three inotropic interventions increased isometric force development in right ventricular rabbit papillary muscles. Relaxation time was decreased with isoproterenol, unchanged with high calcium, and increased with EMD 53998. Calcium cycling-related energy consumption, as measured by tension-independent heat, increased by 234% with high calcium, by 439% with isoproterenol, and by 77% with EMD 53998. In contrast to high calcium and isoproterenol, EMD 53998 increased economy of crossbridge cycling by increasing the force-time integral of the individual crossbridge cycle. The data indicate that EMD 53998 acts by phosphodiesterase inhibition and myofilament calcium sensitization. The latter effect is in part mediated by alteration of crossbridge behavior. Because of its effects on calcium cycling and crossbridge function myocardial energy turnover was reduced significantly with EMD 53998, whereas energy turnover was unchanged with high calcium and was increased with isoproterenol. The new calcium sensitizer levosimendan was investigated in isolated failing human myocardium. Levosimendan dose-dependently increased isometric tension. The inotropic effect was associated with increased rate of relaxation and reduced relaxation time. Measurements of intracellular calcium using the photoprotein aequorin suggest that levosimendan acts by increasing myofilament calcium sensitivity and by increasing cAMP due to phosphodiesterase inhibition. However, the contribution of the cAMP system to the action of levosimendan appears to be rather small. Therefore, the finding of a positive lusitropic effect of levosimendan may be consistent with the notion that levosimendan binds to troponin-C and increases calcium sensitivity only at high (systolic) intracellular calcium concentrations.

Influence of isoproterenol on contractile protein function, excitation-contraction coupling, and energy turnover of isolated nonfailing human myocardium.

Previous animal experiments indicated that the effects of catecholamines on myocardial function and subcellular systems vary considerably depending on the species and type of myocardium investigated. In the present study, we used isometric force and heat measurements to investigate the influence of isoproterenol on energetics of excitation-contraction coupling and contractile proteins in isolated nonfailing human myocardium. Isoproterenol, in an average concentration of 0.8 +/- 0.3 microM, resulted in a significant increase in peak twitch tension, maximum rate of tension rise and maximum rate of tension fall by 46% (P < 0.025), 126% (P < 0.001) and 137% (P < 0.005), respectively (37 degrees C, 60 beat/min). The amount and rate of excitation-contraction coupling-related heat evolution (tension-independent heat) increased by 116% (P < 0.03) and 176% (P < 0.02), respectively. Furthermore, the relationship of tension-independent heat to isometric tension or tension-time integral increased by 47% (P < 0.05) and 91% (P < 0.01), respectively. That is, the energy used in calcium cycling increased by a greater proportion than did mechanical output. Isoproterenol increased the rate of the acto-myosin crossbridge high-energy phosphate hydrolysis (tension-dependent heat rate) by 61% (P < 0.006) and decreased the force-time integral (consistent with decrease in the attachment time) of the individual crossbridge cycle by 21% (P < 0.025). Decreased crossbridge force-time integral with isoproterenol indicates decreased economy of isometric force production at the level of the contractile proteins. Increased energy turnover of excitation-contraction coupling processes and reduced force-time integral generation by the individual crossbridge cycle resulted in increased myocardial energy turnover as indicated by a 41% increase in the ratio of total activity related heat per tension-time integral (P < 0.02). The efficiency of the metabolic recovery process as assessed by the ratio of initial heat to total activity related heat, was similar with and without isoproterenol (0.52 +/- 0.05 v 0.49 +/- 0.03; P > 0.05). Thus, isoproterenol significantly influences excitation-contraction coupling processes and crossbridge cycling, thereby increasing myocardial energy turnover per unit of isometric force production in the human myocardium.

Positive inotropism and myocardial energetics: influence of beta receptor agonist stimulation, phosphodiesterase inhibition, and ouabain.

OBJECTIVE: The aim was to study the effect of three positive inotropic interventions on myocardial force development and heat production in guinea pig papillary muscles in order to investigate the energetic consequences. METHODS: The positive inotropic agents used were epinine (beta adrenoceptor stimulation), E-1020 (phosphodiesterase inhibition), and ouabain (sodium-potassium ATPase inhibition). Heat measurements were accomplished using antimony-bismuth thermopiles, and initial heat was separated into tension dependent and tension independent heat using the butanedione-monoxime (BDM) and the shortening methods. RESULTS: Optimal concentrations of epinine, E-1020, and ouabain increased peak developed force from 20.0(SD 6.6) to 55.5(9.3) (n = 5; p < 0.01), from 20.9(9.1) to 27.2(7.2) (n = 6; p < 0.05), and from 23.4(9.2) to 44.9(18.0) mN.mm-2 (n = 6; p < 0.01), respectively. Epinine and E-1020 decreased the tension-time integral per unit initial heat, ie, the economy of isometric contraction, from 5.5(1.4) to 3.6(0.5) (p < 0.01) and from 5.5(1.4) to 3.1(0.9) N.m.s.J-1 (p < 0.01), respectively; no significant change was observed with ouabain [6.7(1.4) to 8.3(0.5) N.m.s.J-1]. The tension independent heat (calcium turnover) was measured in two different ways using BDM or shortening to abolish force production. It was increased significantly by epinine (by 141-243%), E-1020 (by 77-114%), and ouabain (by 23-38%). The first measurement in brackets is the BDM estimate, the second is the shortening estimate. From the tension-time integral and the tension dependent heat the crossbridge force-time integral was analysed: epinine and E-1020 decreased the crossbridge force-time integral from 0.46(0.16) to 0.31(0.06) pN.s (p < 0.01) and from 0.50(0.19) to 0.31(0.08) pN.s (p < 0.01), respectively, while ouabain left the force-time integral unchanged [0.59(0.27) to 0.63(0.20) pN.s]. CONCLUSIONS: (1) The inotropic effect of ouabain results from an increase in muscle activation with no change in crossbridge kinetics; (2) epinine and E-1020 increase the tension independent heat and decrease the crossbridge force-time integral, both effects reducing the overall economy; and (3) the shortening and BDM methods for measuring the tension independent heat give qualitatively similar but quantitatively different results.

Smooth, cardiac and skeletal muscle myosin force and motion generation assessed by cross-bridge mechanical interactions in vitro.

Differences in the mechanical properties of mammalian smooth, skeletal, and cardiac muscle have led to the proposal that the myosin isozymes expressed by these tissues may differ in their molecular mechanics. To test this hypothesis, mixtures of fast skeletal, V1 cardiac, V3 cardiac and smooth muscle (phosphorylated and unphosphorylated) myosin were studied in an in vitro motility assay in which fluorescently-labelled actin filaments are observed moving over a myosin coated surface. Pure populations of each myosin produced actin filament velocities proportional to their actin-activated ATPase rates. Mixtures of two myosin species produced actin filament velocities between those of the faster and slower myosin alone. However, the shapes of the myosin mixture curves depended upon the types of myosins present. Analysis of myosin mixtures data suggest that: (1) the two myosins in the mixture interact mechanically and (2) the same force-velocity relationship describes a myosin's ability to operate over both positive and negative forces. These data also allow us to rank order the myosins by their average force per cross-bridge and ability to resist motion (phosphorylated smooth > skeletal = V3 cardiac > V1 cardiac). The results of our study may reflect the mechanical consequence of multiple myosin isozyme expression in a single muscle cell.

Myocardial force-frequency defect in mitral regurgitation heart failure is reversed by forskolin.

BACKGROUND: Postoperative ejection phase parameters and patient survival rates for mitral valve replacement surgery are considerably lower than for similar aortic valve surgery. While chordal transection probably is the major contributor to the lowered values, there is also evidence for decreased preoperative myocardial contractile reserve in mitral regurgitation patients. This study characterizes abnormalities in the force-frequency relation that may underlie impaired function of myocardium isolated from mitral regurgitation patients with New York Heart Association class II-III heart failure. METHODS AND RESULTS: Left ventricular epicardial myocardium was obtained by surgical biopsy during mitral valve replacement surgery in patients with mitral regurgitation heart failure (left ventricular ejection fraction, 0.64 +/- 0.05) and during coronary artery bypass surgery in patients with normal ventricular function. The steady-state twitch tension versus frequency relation was measured in myocardial strip preparations (37 degree C, 12 to 228 min-1) in the absence and presence of forskolin. Relative to normal function, peak isometric twitch tension in mitral regurgitation is depressed by 50% (P < .02) and 74% (P < .003) at contraction frequencies of 60 min-1 and 168 min-1, respectively. The slope of the tension-frequency curve is blunted and its peak is shifted to a lower frequency (mitral regurgitation: 134 min-1; normal function: 173 min-1; P < .02). The myosin heavy chain concentration did not differ between mitral regurgitation and normal function strips (53 +/- 4 versus 54 +/- 4 nmol/g blotted wt). Forskolin (0.5 mumol/L) completely reversed the tension depression, blunting, and the lowered peak frequency in the mitral regurgitation preparations. CONCLUSIONS: Preoperatively, myocardial tension generation in mitral regurgitation patients is severely depressed, and the force-frequency curve is blunted and has a negative slope in the exercise range of heart rates. The reversal of these defects by forskolin suggests that abnormal excitation-contraction coupling may underlie the decreased contractile reserve in mitral regurgitation patients.