Edward M. Blanchard

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.

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.

Targeted Ablation of the Murine α-Tropomyosin Gene

Abstract We created a mouse that lacks a functional α-tropomyosin gene using gene targeting in embryonic stem cells and blastocyst-mediated transgenesis. Homozygous α-tropomyosin “knockout” mice die between embryonic day 9.5 and 13.5 and lack α-tropomyosin mRNA. Heterozygous α-tropomyosin knockout mice have ≈50% as much cardiac α-tropomyosin mRNA as wild-type littermates but similar α-tropomyosin protein levels. Cardiac gross morphology, histology, and function (assessed by working heart preparations) of heterozygous α-tropomyosin knockout and wild-type mice were indistinguishable. Mechanical performance of skinned papillary muscle strips derived from mutant and wild-type hearts also revealed no differences. We conclude that haploinsufficiency of the α-tropomyosin gene produces little or no change in cardiac function or structure, whereas total α-tropomyosin deficiency is incompatible with life. These findings imply that in heterozygotes there is a regulatory mechanism that maintains the level of myofibri...

Altered Crossbridge Kinetics in the αMHC403/+ Mouse Model of Familial Hypertrophic Cardiomyopathy

A mutation in the cardiac beta-myosin heavy chain, Arg403Gln (R403Q), causes a severe form of familial hypertrophic cardiomyopathy (FHC) in humans. We used small-amplitude (0.25%) length-perturbation analysis to examine the mechanical properties of skinned left ventricular papillary muscle strips from mouse hearts bearing the R403Q mutation in the alpha-myosin heavy chain (alphaMHC403/+). Myofibrillar disarray with variable penetrance occurred in the left ventricular free wall of the alphaMHC403/+ hearts. In resting strips (pCa 8), dynamic stiffness was approximately 40% greater than in wild-type strips, consistent with elevated diastolic stiffness reported for murine hearts with FHC. At pCa 6 (submaximal activation), strip isometric tension was approximately 3 times higher than for wild-type strips, whereas at pCa 5 (maximal activation), tension was marginally lower. At submaximal calcium activation the characteristic frequencies of the work-producing (b) and work-absorbing (c) steps of the crossbridge were less in alphaMHC403/+ strips than in wild-type strips (b=11+/-1 versus 15+/-1 Hz; c= 58+/-3 versus 66+/-3 Hz; 27 degrees C). At maximal calcium activation, strip oscillatory power was reduced (0. 53+/-0.25 versus 1.03+/-0.18 mW/mm3; 27 degrees C), which is partly attributable to the reduced frequency b, at which crossbridge work is maximum. The results are consistent with the hypothesis that the R403Q mutation reduces the strong binding affinity of myosin for actin. Myosin heads may accumulate in a preforce state that promotes cooperative activation of the thin filament at submaximal calcium but blunts maximal tension and oscillatory power output at maximal calcium. The calcium-dependent effect of the mutation (whether facilitating or debilitating), together with a variable degree of fibrosis and myofibrillar disorder, may contribute to the diversity of clinical symptoms observed in murine FHC.

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 on myocardial energetics. Experimental and clinical investigations

SummaryThe influence of isoproterenol on myocardial performance and energetics was investigated in normal guinea pig myocardium and in patients with normal left ventricular function.The in vitro experiments were performed by simultaneous isometric force and heat measurements using sensitive antimony-bismuth thermopiles. Following the application of isoproterenol (10−8 M) isometric peak twitch tension and tension-time integral increased significantly by 185% and 142%, respectively. Tension-independent heat which reflects high energy phosphate hydrolysis of excitation-contraction coupling increased by 183%. Tension-dependent heat reflecting the high energy phosphate hydrolysis of the crossbridges increased by 417%. The ratio of tension-dependent heat to tension-time integral increased by 131%. The recovery/initial heat ratio, reflecting the efficiency of the recovery metabolism, and the resting metabolism did not significantly change.In the patients the effect of isoproterenol on myocardial energetics was evaluated in terms of myocardial oxygen consumption per left ventricular systolic stress-time integral and external myocardial efficiency. Following isoproterenol administration, left ventricular systolic stress-time integral decreased by 49% due to reductions in end-diastolic pressure, end-diastolic volume and duration of systole. Pressure-volume work remained unchanged. Myocardial oxygen consumption per minute increased in proportion to heart rate. The ratio of myocardial oxygen consumption per beat to left ventricular systolic stress-time integral increased significantly by 95%. External myocardial efficiency was unaltered.Thus, isoproterenol increases the energy turnover of excitation-contraction coupling and increases the energy consumption of the crossbridges disproportionately to developed tension-time integral in the guinea pig heart. Likewise, in the working human heart, the increase in oxygen consumption per left ventricular systolic stress-time integral is considered to represent the isoproterenol induced changes in excitation contraction coupling and crossbridge energetics.

Myothermal economy of rat myocardium, chronic adaptation versus acute inotropism.

By means of rapid planar Hill type antimony-bismuth thermophiles the initial heat liberated by papillary muscles was measured synchronously with developed tension for control (C), pressure-overload (GOP), and hypothyrotic (PTU) rat myocardium (chronic experiments) and after application of 10(-6) M isoproterenol or 200 10(-6) M UDCG-115. Economy of force production was analyzed by the ratio of initial heat versus developed tension-time integral. This ratio was found to be reduced by 34% in GOP and by 43% in PTU myocardium (P less than 0.01, respectively) indicating increased economy of force production. In contrast, isoproterenol increased initial heat versus tension-time integral by 70% (P less than 0.01) indicating reduced economy of force production. No change in this ratio was found for UDCG-115. The presented data indicates that long and short term modulation of myocardial energetic costs of force generation is possible. The basic mechanisms for these myocardial alterations are discussed.

The effects of acute and chronic inotropic interventions on tension independent heat of rabbit papillary muscle

We have used the myothermal method to noninvasively monitor the amount of calcium cycled during a single isometric twitch of rabbit papillary muscle. Experiments were designed to test the working hypothesis that changes in peak twitch tension caused by pharmacological agents or changing haemodynamic conditions are accompanied by parallel changes in the tension independent heat (TIH) signal associated with Ca2+ cycling. We isolated the TIH signal by eliminating the tension dependent component of initial heat with a hyperosmotic Krebs solution containing 2,3-butanedione monoxime. Contrary to the working hypothesis, positive or negative inotropic effects on twitch tension caused by pressure overload hypertrophy, thyrotoxic hypertrophy, isoproterenol, and UDCG115 were not accompanied by parallel changes in TIH. Alternative explanations for the relation between peak twitch tension and TIH are explored.

Dynamic calcium requirements for activation of rabbit papillary muscle calculated from tension-independent heat

The heat generated by right ventricular papillary muscles of rabbits was measured after adenosine triphosphate (ATP) splitting by the contractile proteins was chemically inhibited. This tension-independent heat (TIH) (1 mJ/g wet weight) was used to calculate the total calcium (Ca) cycled in a muscle twitch by assuming that 87% of TIH was due to Ca2+ transport by the sarcoplasmic reticulum with a coupling ratio of 2 Ca2+/ATP split; the enthalpy of creatine phosphate hydrolysis buffering ATP was taken as -34 KJ/mol. The estimated Ca turnover per muscle twitch at 21 degrees C, 0.2 Hz pacing rate, and 2.5 mM Ca in the Krebs solution was approximately equal to 50 nmol/g wet weight. There was a tight positive correlation between TIH and mechanical activation during steady-state measurements but no correlation during the sharp increase in mechanical activation (treppe) when stimulation was resumed after a rest period. It is suggested that while total Ca cycling remains unchanged during the initial period of tension treppe, the free Ca2+ transient and mechanical activation increase sharply due to resaturation of high affinity Ca2+ buffers, other than troponin C, depleted of Ca2+ during the rest period.

Dynamic calcium requirements for activation of human ventricular muscle calculated from tension-independent heat

The heat and tension generated by strips of human left ventricle taken from nonfailing hearts were measured at 30 C before and after partial inhibition of ATP splitting by the contractile proteins. We used 2, 3-butanedione monoxime (BDM) (4mM) as the chemical inhibition agent and alterations in solution calcium concentration and stimulus frequency to estimate the heat associated with calcium cycling for a wide range of activation levels. Tension-independent heat (TIH) was used to calculate the total calcium cycled per twitch by assuming that two-thirds of TIH was due to ATP splitting by the sarcoplasmic reticulum CA2+ ATPase with a coupling ratio of 2 Ca2+/ATP split and that one-third of TIH was due to ATP splitting by the sarcolemmal Na+ -K+ ATPase supporting the Na+ -Ca2+ exchanger (1 Ca2+/ATP). The enthalpy of creatine phosphate hydrolysis buffering ATP was taken as -34 KJ/mol. There was a highly positive correlation between TIH and mechanical activation during steady-state and nonsteady-state stimulation. The estimated total calcium turnover per twitch at 39% activation (0.3 Hz pacing rate and 2.5 mM Calcium) was approximately 0.17 nmol/g wet weight. This estimate is less than that calculated from biochemical data describing the cellular content and Ca2+ affinity of major Ca2+ buffers, but is similar to values calculated from recent electron probe microanalysis experiments.