Louis A. Mulieri

Altered myocardial force-frequency relation in human heart failure.

BackgroundIn congestive heart failure (idiopathic dilated cardiomyopathy), exercise is accompanied by a smaller-than-normal decrease in end-diastolic left ventricular volume, depressed peak rates of left ventricular pressure rise and fall, and depressed heart-rate-dependent potentiation of contractility (bowditch treppe). We studied contractile function of isolated left ventricular myocardium from New York Heart Association class IV-failing and nonfailing hearts at physiological temperature and heart rates in order to identify and quantitate abnormalities in myocardial function that underlie abnormal ventricular function. Methods and ResultsThe isometric tension-generating ability of isolated left ventricular strips from nonfailing and failing human hearts was investigated at 37°C and contraction frequencies ranging from 12 to 240 per minute (min−1). Strips were dissected using a new method of protection against cutting injury with 2,3-butanedione monoxime (BDM) as a cardioplegic agent. In nonfailing myocardium the twitch tension-frequency relation is bell-shaped developing 25±2 mN/mm2 at a contraction frequency of 72 min−1 and peaking at 44±3.7 mN/mm2 at a contraction frequency of 174±4 min−1. In failing myocardium the peak of the curve occurs at lower frequencies between 6 and 120 min−1 averaging 81±22 min−1, and it develops 48% (p < 0.001) and 80% (p < 0.001) less tension than in nonfailing myocardium at 72 and 174 min−1, respectively. Between 60 and 150 min−1 tension increases by 107% in nonfailing myocardium, but it does not change significantly in failing myocardium. Peak rates of rise and fall of isometric twitch tension vary in parallel with twitch tension as stimulation frequency rises in nonfailing myocardium but not in failing myocardium. ConclusionsThe quantitative agreement between these results from isolated myocardium and those from catheterization laboratory measurements on intact humans suggest that alterations of myocardial origin, independent of systemic factors, may contribute to the above mentioned abnormalities in left ventricular function seen in dilated cardiomyopathy.

Protection of human left ventricular myocardium from cutting injury with 2,3-butanedione monoxime.

To prevent dissection injury when cutting strip preparations from human left ventricular papillary muscle tissue, dissections were carried out with 2,3-butanedione monoxime (30 mM) added to Krebs-Ringer solution and followed by washout with normal solution. Eleven muscle strip preparations were dissected from left ventricular papillary muscle tissue of five patients undergoing mitral valve replacement surgery. The average muscle strip length was 6.8 ± 1.4 mm, and cross-section area was 0.49 ± 0.16 mm2. Peak twitch tension was 2.02 ± 1.33 g/mm2 and ranged from 0.67 to 5.5 g/mm2 at an extracellular calcium concentration of 2.5 mM (21° C, 0.16 Hz). In one muscle strip, which was stored in Krebs-Ringer plus 2,3-butanedione monoxime solution for 20 hours, peak twitch tension in normal Krebs-Ringer solution was 1.85 g/mm2. When temperature was increased from 21° C, there was a continuous increase in peak twitch tension (by 38%) up to about 28° C; then peak twitch tension decreased so that at 37° C (n =3) average peak twitch tension was lower than at 21° C by 47%. The force-frequency relation exhibited a broad force plateau between 40 and 120 beats/min at 37° C. The plateau was markedly narrowed at 30° C and 24° C. Thermopile heat measurements revealed appropriate waveform characteristics in high-resolution single-beat heat records indicating minimal surface cell damage. Thus, cardioplegia with 2,3-butanedione monoxime protects human left ventricular myocardium from dissection injury facilitating dissection and preservation of strip preparations with extraordinarily low cross-sectional areas and high peak twitch tensions. These preparations are suitable for myothermal and mechanical measurements.

The economy of isometric force development, myosin isoenzyme pattern and myofibrillar ATPase activity in normal and hypothyroid rat myocardium.

Hypothyroidism was induced in Wistar-Kyoto rats by adding propylthiouracil to the drinking water (0.8 mg/ml). Initial heat, total activity-related heat, and resting heat rate were measured in left ventricular papillary muscle preparations of propylthiouracil-treated and control rats contracting isometrically at 12 beats/min (21°C), using Hill type, planar vacuum-deposited bismuth and antimony thermopiles. In the propylthiouracil preparations, relative to control, time-to-peak tension increased from 288 ± 27 (mean ± SD) to 411 ± 25 msec (P < 0.001), dp/dtmax decreased from 38.3 ± 9.5 to 20.4 ± 3.5 g-mm−2/sec (P < 0.001), and peak developed tension decreased from 6.11 ± 1.75 to 4.64 ± 0.89 g-mm−2 (P < 0.05). In the propylthiouracil preparations, initial heat was significantly (P < 0.001) reduced by 27 or 43% when normalized to peak twitch tension or tension-time integral, respectively. In experiments where the papillary muscles were tetanized, the. slope of the linear function of total activity-related heat versus tension-time integral was decreased by 43% (P < 0.001) in the propylthiouracil preparations, indicating an improved economy of isometric tension maintenance. The predominant myosin isoenzyme of the left ventricular wall, as well as the papillary muscle myocardium, was the V3 variety in the propylthiouracil animals, in contrast to V1 in the controls. Myofibrillar actomyosin calcium-magnesium-stimulated adenosine triphosphatase activity was significantly (P < 0.02) decreased from 55 + 18 (control) to 31 ± 8 nmol inorganic phosphate ion/mg-min (propylthiouracil). Correspondingly, the myofibrillar myosin calcium-stimulated adenosine triphosphatase activity was also significantly (P < 0.01) decreased from 294 ± 98 (control) to 85 ± 25 nmol inorganic phosphate ion/mg-min (propylthiouracil). The results give evidence of (1) increased economy of force generation and maintenance in propylthiouracil myocardium, which is paralleled by (2) structural changes of myosin (shifts in the isoenzyme pattern), and (3) associated changes of myofibrillar adenosine triphosphatase activity. We conclude that the observed myothermal data reflect slowed crossbridge cycling in propylthiouracil myocardium, which must not necessarily be interpreted as being detrimental, in view of the increased economy of force generation.

Alteration of contractile function and excitation-contraction coupling in dilated cardiomyopathy.

Myocardial failure in dilated cardiomyopathy may result from subcellular alterations in contractile protein function, excitation-contraction coupling processes, or recovery metabolism. We used isometric force and heat measurements to quantitatively investigate these subcellular systems in intact left ventricular muscle strips from nonfailing human hearts (n = 14) and from hearts with end-stage failing dilated cardiomyopathy (n = 13). In the failing myocardium, peak isometric twitch tension, maximum rate of tension rise, and maximum rate of relaxation were reduced by 46% (p = 0.013), 51% (p = 0.003), and 46% (p = 0.018), respectively (37 degrees C, 60 beats per minute). Tension-dependent heat, reflecting the number of crossbridge interactions during the isometric twitch, was reduced by 61% in the failing myocardium (p = 0.006). In terms of the individual crossbridge cycle, the average crossbridge force-time integral was increased by 33% (p = 0.04) in the failing myocardium. In the nonfailing myocardium, the crossbridge force-time integral was positively correlated with the patients age (r = 0.86, p less than 0.02), whereas there was no significant correlation with age in the failing group. The amount and rate of excitation-contraction coupling-related heat evolution (tension-independent heat) were reduced by 69% (p = 0.24) and 71% (p = 0.028), respectively, in the failing myocardium, reflecting a considerable decrease in the amount of calcium released and in the rate of calcium removal. The efficiency of the metabolic recovery process, as assessed by the ratio of initial heat to total activity-related heat, was similar in failing and nonfailing myocardium (0.54 +/- 0.03 versus 0.50 +/- 0.02, p = 0.23).(ABSTRACT TRUNCATED AT 250 WORDS)

Energetics of isometric force development in control and volume-overload human myocardium. Comparison with animal species.

Alteration in crossbridge behavior and myocardial performance have been associated with myosin isoenzyme composition in animal models of myocardial hypertrophy or atrophy. In the hypertrophied human heart, myocardial performance is altered without significant changes in myosin isoenzymes. To better understand this discrepancy, isometric heat and force measurements were carried out in 1) control and volume-overload human myocardium, 2) control, pressure-overload, and hyperthyroid rabbit myocardium, and 3) control and hypothyroid rat myocardium. In control human myocardium, peak isometric twitch tension was 44.0 +/- 11.7 mN/mm2, and maximum rate of tension rise was 69.2 +/- 21.0 mN/sec.mm2. In volume-overload human myocardium, peak twitch tension and maximum rate of tension rise were reduced by 55% (p less than 0.05) and 65% (p less than 0.05), respectively. The average force-time integral of the individual crossbridge cycle, calculated by myothermal techniques, was increased by 85% (p less than 0.005) in volume-overload human myocardium. In control and hormonally altered myocardium, both across and within species (control human, control rat, control rabbit, hypothyroid rat, and hyperthyroid rabbit), there was a close relation between the crossbridge force-time integral and the percentage of V3-type myosin isoenzyme in the myocardium. However, hemodynamically altered (volume-overload human and pressure-overload rabbit) myocardium did not follow this relation. Across and within species, there were significant correlations between maximum rate of tension rise and average tension-dependent heat rate (r = 0.97, p less than 0.001) and between maximum rate of tension fall and average tension-independent heat rate (r = 0.82; p less than 0.025). Furthermore, there were close inverse relations between these heat rates and the crossbridge force-time integral. In addition, there was an inverse relation between tension-independent heat and the crossbridge force-time integral. Across and within species total myocardial energy turnover was significantly correlated with the crossbridge force-time integral (relative total heat, r = -0.84, p less than 0.02; relative total-activity related heat, r = -0.88, p less than 0.01). The present findings indicate that 1) factors separate from myosin isoenzymes account for the altered crossbridge cycle in volume-overload human and pressure-overload rabbit myocardium, 2) changes in excitation-contraction coupling processes accompany changes in the crossbridge cycle within and across species, and 3) the force-time integral of the crossbridge cycle is a major determinant of total myocardial energy turnover.

Functional significance of altered myosin adenosine triphosphatase activity in enlarged hearts

Abstract Enlarged hearts secondary to significant pressure overload have a depressed myosin adenosine triphosphatase (ATPase) activity. With mild stress the ATPase activity may be normal or slightly elevated. Enlargement of the heart after exercise or thyroid stress results in an increased ATPase activity. Rapid myothermal techniques, in particular measurements of tension-dependent heat, were used to evaluate: (1) the relation of in vitro measurements of actin-activated ATPase activity to the in vivo behavior of myosin, and (2) the contribution of these changes to the economy of tension development and the time course of crossbridge cycling. Experiments were carried out in animals whose hearts were enlarged secondary to pressure overload (by pulmonary arterial banding) or thyrotoxic stress. In vitro actin-activated ATPase activity levels were 70 and 175 percent of normal for the pressure-overloaded and thyrotoxic hearts, respectively, while the tension-dependent heat per unit tension for the same preparations was, respectively, 78 and 154 percent of normal. Thus there is a reasonable correlation between the in vivo and in vitro measurements of contractile protein ATPase activity, which indicates that the economy of tension development is inversely related to tensiondependent heat per unit tension or actin-activated myosin ATPase activity. Analysis of these data in terms of the kinetics of ATPase activity, crossbridge behavior and tension development leads to the conclusion that in pressure overload hypertrophy the adaptation involves a decrease in the crossbridge cycling rate, an increase in crossbridge off-time and an increase in the on-time of the crossbridge. For thyrotoxic hypertrophy the adaptation involves an increase in the cycling rate, a decrease in the off-time and a decrease in the on-time. The former is adapted for slow, economical tension development and the latter for rapid, less economical tension development.

Tension‐independent heat in rabbit papillary muscle.

1. Heat and force were measured from isometrically contracting (0.2 Hz) rabbit papillary muscles at 21 degrees C during a single contraction‐relaxation cycle using antimony‐bismuth thermopiles and a capacitance force transducer. 2. Tension‐independent heat (TIH) associated with excitation‐contraction coupling was isolated from the initial heat by eliminating tension and tension‐dependent heat with a Krebs‐Ringer solution containing 2,3‐butanedione monoxime (BDM) and mannitol. 3. A strategy for testing the validity of this new method for measuring TIH in heart muscle is described and the test confirms that the BDM‐hypertonic solution partitioning method properly estimates the magnitude of the TIH component of initial heat. 4. TIH at the time of complete mechanical relaxation is 1.00 +/‐ 0.17 mJ/g wet weight and the data suggest that calcium cycling is complete by this time. Conversion of TIH to calcium cycled, assuming that 87% of TIH is due to calcium pumping by the sarcoplasmic reticulum, indicates that approximately 52 nmol calcium/g wet weight are required to support a single cycle of mechanical activity (0.2 Hz, 21 degrees C). 5. The length and frequency dependence of excitation‐contraction coupling were demonstrated. TIH is reduced by shortening muscle length and by increasing the interval between stimuli. These steady‐state data suggest that only a portion (approximately 40%) of TIH is directly related to activation of the contractile apparatus. 6. TIH in the first twitch following a 45 min rest period is significantly reduced by approximately 30%. 7. With subsequent twitches in the positive treppe following the rest period, TIH does not increase as steeply as expected suggesting that tension rise in twitches 1‐10 may be modulated by competitive binding of calcium rather than increased calcium delivery.

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.

Heat production during hypoxic contracture of rat myocardium.

Contracture due to hypoxia, to both oxygen and glucose deficiency, and to potassium chloride was induced in rat left ventricular papillary muscle preparations. Under contracture conditions, the sum of resting heat plus contracture heat was measured using Hill-type, planar vacuum-deposited thermopiles. On the basis of the measured total and initial heat output and the corresponding tension-time integral during single twitches under control conditions (Lmax, 21 degrees C, stimulus frequency 12/min), the expected heat output during contracture was calculated, assuming that the contracture tension is maintained by the same calcium-induced cross-bridge cycling as occurs in the single twitch response. With potassium chloride, the contracture tension was 1.33 +/- 0.27 g/mm2, a value which is similar to those found in hypoxic contracture and in contracture due to both oxygen and glucose deficiency. There was no significant difference between measured and calculated values for resting heat plus contracture heat (8.40 +/- 2.84 mW/g measured, 8.55 +/- 2.50 mW/g calculated); there was a linear correlation (r = 0.99) between predicted and measured values (P less than 0.05). The measured value for resting plus contracture heat in hypoxic contracture was 1.88 +/- 0.37 mW/g, whereas a value of 4.80 +/- 1.09 mW/g (P less than 0.005) was calculated on the basis of the twitch heat per tension-time integral and contracture tension (1.09 +/- 0.31 g/mm2). Contracture tension was 1.80 +/- 0.78 g/mm2 in contracture due to oxygen and glucose deficiency, whereas the value for resting plus contracture eat was 1.61 +/- 0.56 mW/g. The calculated resting plus contracture heat value for this preparation was significantly higher (7.45 +/- 3.75 mW/g; P less than 0.05). There was no significant regression between predicted and measured resting heat plus contracture heat in the hypoxic contracture preparations (slope not different from zero). In contracture due to oxygen and glucose deficiency, the linear regression had a slope of 6.06 (P less than 0.05). The results suggest that the potassium chloride contracture relies on cross-bridge cycling as in a twitch contraction, whereas hypoxic contracture and that due to oxygen and glucose deficiency may be explained by cross-bridge formations with no, or very low, heat production, i.e., contracture tensions due to hypoxia and to oxygen and glucose deficiency are maintained by rigor-like cross-bridge formation or by slowly cycling cross-bridges with a long time of cross-bridge attachment.

The failing human heart

Heart failure remains a significant public health problem with an unacceptably high morbidity and mortality affecting about three persons per thousand per year [1]. The percent survival following diagnosis decreases significantly as a function of the severity of the disease with survival rates for NYHA (New York Heart Association) I–II being 65% at the end of 4 years, for NYHA III it is 50% at the 4-year period and for NYHA IV it is 50% after only 1 year [2,3],1. In the patients with heart failure, there is a significant correlation between survivability and ejection fraction [4]. The deficits in ventricular function in failing human hearts led us to examine the contributions of the contractile (acto–myosin interaction) and excitation–contraction–coupling (EC) (calcium cycling) systems to that insufficiency. ### 2.1 Myofibrillar ATPase activity and velocity of shortening The first demonstration of a molecular alteration in the contractile system of human failing hearts was found in studies carried out on myofibrils from failing hearts (secondary to hypertensive heart disease) and non failing hearts (accident victims) [5]. The myofibrillar ATPase activity in the failing human hearts was reduced from non failing values (mean±S.D.)2 of 0.99±0.05 to 0.69±0.04 μmole Pi/mg myofibrillar protein/15 min ( P <0.001). The functional consequences of the depressed ATPase activity were not completely clear at the time these experiments were carried out although we believed the depressed activity was associated with a decrease in contractility of the heart muscle. This view was supported by a decrease in the contractility of actomyosin strands [6] and glycerinated fibers [7] from failing heart muscle. From the perspective of ventricular function, the decreased contractility involves contractile force, velocity of shortening or a combination of the two. The relationship between force and velocity of shortening was thoroughly described by Hill [8] where the hyperbolic relationship was believed to be … * Corresponding author. Fax: +1-807-656-747