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).

Functional integrity of the SH1 site in myosin from hypertrophied myocardium.

The hypothesis that an alteration in the SH1 site of hypertrophy myosin is reponsible for the reduced Ca2+-stimulated ATPase activity is examined.The functional integrity of the SH1 site was evaluated by measurement of the (K+)-EDTA-stimulated and Mg2+-inhibited ATPase activities. Neither activity differed from control although the Ca2+-stimulated ATPase of the same preparations was significantly reduced. The reduction in Ca2+-activated ATPase was independent of ionic strength. Titration with N-ethylmaleimide elevated the Ca2+-stimulated ATPase of hypertrophy myosin to the same peak activity as control. Actin-stimulated ATPase activity of hypertrophy myosin was also reduced. The results indicate that the SH1 of hypertrophy myosin is functionally intact for (K+)EDTA-stimulated ATPase and Mg2+ inhibition, but functionally deficient with regard to Ca2+-stimulated and actin-activated ATPase activities. This implies a partition of the functional aspects of SH1.

The ATPase activity of subfragment-1 from the hypertrophied heart.

Myosin and subfragment-1 were prepared from rabbit hearts hypertrophied secondary to pulmonary artery constriction. The Ca2+ -stimulated ATPase activity was reduced while the potassium/EDTA-stimulated ATPase activity was unchanged in both the myosin and subfragment 1 (S-1) from hypertrophied hearts. When hypertrophy myosin was mixed with an equal quantity of control myosin, the ATPase activity of the mixed protein fell halfway between control and hypertrophy values. Similar results were obtained with control and hypertrophy S-1. The actin-stimulated ATPase activity of hypertrophy S-1 was slightly depressed but unlike hypertrophy myosin this depression was not significant when compared to normal S-1. This suggests that papain cleavage may have removed part of the conformational difference that exists between control and hypertrophy myosins.

Purification of cardiac myosin. Application to hypertrophied myocardium.

A method is described for the preparation of high purity myosin from small amounts of cardiac muscle. The method employs homogenization and prolonged extraction of the cardiac tissue. Purification is achieved through three successive precipitation-dissolution cycles and without the use of column chromatographic techniques. Purity of the myosin preparation is assessed at various stages of the purification procedure by sodium dodecylsulfate-acrylamide gel electrophoresis and by measurement of RNA and nucleoprotein content. With 1.5-2.0 g of rabbit right ventricle as the starting tissue, this method yields 4-6 mg myosin per g wet tissue. The method is also shown to give similar results with rabbit right ventricles hypertrophied by pulmonary stenosis.

The effect of shortening on the time-course of active state decay.

The active state describes the force developed in a muscle when the contractile elements are neither lengthening nor shortening. Recently it was suggested that perturbations used to measure the active state also alter the time-course of the active state. The present research was undertaken to assess quantitatively the effect of two such perturbations, isotonic shortening and quick release, on the active state in frog sartorius muscle. Methods were developed which allowed the determination of active state points following periods of controlled isotonic shortening or quick release early in the contraction cycle. All experiments were carried out within the plateau region of the length-tension curve. Both isotonic shortening and quick release altered the active state decay. The active state force decreased as the extent of shortening or release was increased. For each 0.1 mm of isotonic shortening there was a 2% decrease in active state force. Quick release produced a larger decrement. From this data we conclude that the time-course of active state can be measured only in relative terms because it is altered by the motion which takes place in the contractile machine while the active state is being measured. This finding helps to resolve paradoxes in the literature relating to the time-course of the active state, calculated and experimentally determined isometric tetanic myograms, and the heat of shortening.

Isometric force development, isotonic shortening, and elasticity measurements from Ca2+-activated ventricular muscle of the guinea pig.

Isometric tension and isotonic shortening were measured at constant levels of calcium activation of varying magnitude in mechanically disrupted EGTA-treated ventricular bundles from guinea pigs. The results were as follows: (a) The effect of creatine phosphate (CP) on peak tension and rate of shortening saturated at a CP concentration more than 10 mM; below that level tension was increased and shortening velocity decreased. We interpreted this to mean that CP above 10 mM was sufficient to buffer MgATP(2-) intracellularly. (b) The activated bundles exhibited an exponential stress-strain relationship and the series elastic properties did not vary appreciably with degree of activation or creatine phosphate level. (c) At a muscle length 20 percent beyond just taut, peak tension increased with Ca(2+) concentration over the range slightly below 10(-6) to slightly above 10(-4)M. (d) By releasing the muscle length-active tension curves were constructed. Force declined to 20 percent peak tension with a decrease in muscle length (after the recoil) of only 11 percent at 10(-4)M Ca(2+) and 6 percent at 4x10(-6)M Ca(2+). (e) The rate of shortening after a release was greater at lower loads. At identical loads (relative to maximum force at a given Ca(2+) level), velocity at a given time after the release was less at lower Ca(2+) concentrations; at 10 M(-5), velocity was 72 percent of that at 10(-4)M, and at 4x10(-6)M, active shortening was usually delayed and was 40 percent of the velocity at 10(-4) M. Thus, under the conditions of these experiments, both velocity and peak tension depend on the level of Ca(2+) activation over a similar range of Ca(2+) concentration.

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.

The inhomogeneity and appropriateness of the myocardial response to stress.

Myocardial hypertrophy, with high morbidity and mortality, is a natural outcome of hypertensive heart disease. The increase in myocardial mass is associated with a cellular and subcellular reorganization of the myocytes. The following study uses rapid myothermal techniques to assess the contribution of the major intracellular changes to the adaptive hypertrophic process in various heart models. Pressure overload and thyrotoxic hypertrophy were produced in the rabbit. In the rat, hypertrophy was produced by constricting the renal artery (Goldblatt hypertensive rat) or by using the spontaneously hypertensive rat strain. Atrophy was produced by administration of propylthiouracil in the drinking water. The V1/V3 myosin isoenzyme ratio was decreased in the pressure overload, Goldblatt, and propylthiouracil animals. This was associated with a decrease in total activity-related heat, initial heat, and tension-dependent heat per tension time integral. The tension-independent heat was decreased in the pressure overload, while the time to peak tension was increased. The economy of the metabolic recovery process was unchanged in the pressure overload and Goldblatt preparations. In the propylthiouracil preparation the recovery processes became uneconomical. The spontaneously hypertensive rat exhibited mild cardiac hypertrophy but in all other respects the heart was unchanged from the normal animals. The thyrotoxic hearts had a high V1/V3 myosin isoenzyme ratio, which was associated with a high total activity-related heat, initial heat, and tension-dependent heat per tension time integral. The tension-independent heat was reduced in the thyrotoxic preparations. The appropriateness of each of the intracellular changes is evaluated in terms of the demands made on the heart.

Cloning and characterization of the gene encoding rabbit cardiac calsequestrin.

A cDNA encoding rabbit cardiac calsequestrin was isolated and characterized. The deduced nascent cardiac calsequestrin contains 409 amino acids of which 26% are acidic residues, and had 93% and 67% aa identity with canine cardiac calsequestrin and rabbit fast-twitch skeletal muscle calsequestrin, respectively. RNA blot analyses indicate that this mRNA is expressed in atrium, ventricle and to a lesser amount in slow-twitch skeletal muscle. This mRNA transcript is not expressed in adult fast-twitch skeletal muscle, smooth muscle, or nonmuscle tissues. Analysis of in vitro skeletal muscle myogenesis using a mouse myoblast cell line C2C12, demonstrates that both cardiac and skeletal calsequestrin isoforms are coproduced during muscle differentiation.

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.

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.

Functional consequences of altered cardiac myosin isoenzymes.

This is a review of work dealing with the contribution of myocardial myosin isoenzymes to the performance of the heart muscle. Isoenzyme composition is altered in rabbit hearts by banding the pulmonary artery (increase in %V3) and thyroid hormone injection (increase in V1). Both treatments result in myocardial hypertrophy. The performance of the hearts rich in V3 or V1 myosin isoenzyme is assessed by simultaneous analysis of the myothermal and mechanical output of papillary muscles isolated from the right ventricle. Initial and tension dependent heart per tension time integral are decreased in the pressure overload and increased in the thyrotoxic hypertrophied hearts. Thus, the economy of isometric contraction is high in the hearts with the V3 myosin and low in the hearts with the V1 myosin. This change in performance is explained in terms of alterations in the cycling frequency and tension time integral of the myosin cross-bridge. In the pressure overload hearts the cross-bridge cycling frequency is decreased, while the tension time integral is increased. Conversely, in the thyrotoxic preparations the cycling frequency is increased and the tension time integral is decreased.

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.

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.

Excitation-contraction coupling and contractile protein function in failing and nonfailing human myocardium.

Isometric force, heat output, and aequorin light emission were measured in isolated muscle strips from nonfailing human hearts and from hearts with endstage failing dilated cardiomyopathy (37 degrees C; 30-180 beats per minute (bpm)). In nonfailing myocardium, peak twitch tension increased with higher rates of stimulation, whereas the force-frequency relation was inverse in the failing myocardium. At 60 bpm and at higher rates of stimulation, peak twitch tension was reduced significantly in the failing myocardium. Myothermal measurements, performed at 60 bpm, indicated that the number of crossbridge interactions and the amount of calcium cycling are reduced significantly in the failing myocardium. Furthermore, aequorin light transients indicated that the inverse force-frequency relation in failing myocardium results from altered calcium cycling; with increasing rates of stimulation aequorin light emission increased continuously in the nonfailing and decreased continuously in the failing myocardium. The data suggest that impaired myocardial performance in failing human myocardium may result primarily from disturbed excitation-contraction coupling processes with a reduced amount of calcium cycling and, thus, a decreased activation of contractile proteins.

Time course of mechanical efficiency during afterloaded contractions in isolated cardiac muscle.

The time course of mechanical efficiency during working contractions in rabbit papillary muscle is presented. Efficiency is found to remain relatively constant during the working portion of the twitch, when the muscle is contracting against a constant load. As afterload was decreased, efficiency increased to 65 +/- 11% (mean +/- SE, n = 3) at 10% developed force at maximum length. This is in contrast to muscle work, which reached a peak of 3.0 +/- 0.3 (n = 6) mJ/g at 50% developed force at maximum length.

The effects of isoproterenol, UDCG 115, and caffeine on the heart related to excitation-contraction coupling in heart muscle.

A myothermal technique was used to measure initial heat and tension independent heat from isometrically contracting papillary muscles taken from the right ventricle of rabbits. Tension independent heat produced by the muscle at Lo was isolated with a 2,3-butanedione monoxime (diacetyl monoxime)--hyperosmotic Krebs solution. The effects of the inotropic drugs isoproterenol (1 X 10(-7) M), UDCG 115 (2 X 10(-4) M), and caffeine (2 X 10(-3) M) on heat and mechanical output were measured. We tested the hypothesis that these drugs alter peak twitch tension by increasing the total amount of Ca2+ cycled during the twitch, assuming that net tension independent heat is proportional to total Ca2+ cycled. The hypothesis was rejected for each drug as the positive inotropic effects of isoproterenol and UDCG 115 on twitch tension were not accompanied by increases in net tension independent heat. Net tension independent heat was actually depressed by UDCG 115. The negative inotropic effect of caffeine on twitch tension was accompanied by an increase in tension independent heat at times between the end of mechanical relaxation and the next stimulus. Possible mechanisms to account for these results are discussed.

Determinants of energy utilization in the activated myocardium.

This is a review of work dealing with the effect of pressure overload and thryotoxic hypertrophy of rabbit hearts on the production of total activity related (TA) and initial (I) heats during isometric contraction. Pressure overload hypertrophy is produced by constricting the pulmonary artery with a spiral monel metal clip. Thyrotoxic hypertrophy is produced by 14 daily i.m. injections of 0.2 mg L-thyroxine per kilogram. Heat output is measured with Hill-type planar vacuum deposited bismuth and antimony thermopiles, and force is measured with a capacitance strain gauge. The pressure overload results in a depressed velocity of unloaded shortening, a depressed rate of isometric force development, and an increased time-to-peak tension. These changes are associated with a decreased myosin ATPase, a heart with no V1 myosin isoenzyme, and an increase in the economy of isometric force development (integral of Pdt/TA, integral of Pdt/I). The thyrotoxic hearts exhibit an increased velocity of shortening and rate of force development, and a decrease in time-to-peak tension. These changes are associated with an increase in myosin ATPase activity, a heart with increase in the V1 isoenzyme composition (88% V1), and a decrease in the economy of isometric force development (integral of Pdt/TA, integral of Pdt/I). The changes in the two types of hypertrophied hearts are interpreted in terms of altered cross-bridge cycling rates and changes in cross-bridge tension time integral as well as excitation contraction coupling phenomena. In the thyrotoxic hearts there is an increase in the economy of the recovery processes. Both types of hypertrophy are considered to be adaptive and involve the coordinated restructuring of the excitation-contraction, contractile, and recovery systems.

The response of the heart to stress: a biological view of myocardial adaptation and failure.

The response of the myocardium to persistent stress involves an increase in mass and a restructuring of the cellular and subcellular elements. The experiments described in this article are designed to test the hypothesis that the restructuring of the various systems (contractile, excitation-contraction coupling, recovery, etc.) that occurs in adaptive hypertrophy is a coordinated (matched) process. When the restructuring of the systems in response to stress occurs in an uncoordinated fashion, congestive heart failure results. In addition to controls, three heart models with normal pump performance are used (control, C; pressure overload, P; thyrotoxic, T; and pressure overload plus thyrotoxic, PT4) and one with inadequate pump performance (pressure overload plus thyrotoxic, PT2). In this analysis the contractile and excitation-contraction coupling systems are evaluated. The former is assessed by sensitive myothermal measurement of tension dependent heat (TDH) normalized for the isometric tension time integral (integral of Pdt). The latter is assessed from measurement of the time to peak isometric tension (TPT). The TDH/integral of Pdt (mu cal/g.cm.s) and TPT (ms) for the C, P, T, PT4, and PT2 hearts are 2.4, 1.8, 5.2, 5.1, and 0.1, mu cal/g.cm.s and 627, 816, 352, 484, and 465 ms, respectively. According to the coordination or matching hypothesis, if TDH/integral of Pdt is low, then TPT should be increased, or if TDH/integral of Pdt is high, then TPT should be decreased. Relative to control hearts, matched restructuring of the contractile and excitation-contraction coupling systems occurred for the P, T, and PT4 preparations. In these animals the hypertrophy has been adaptive and the pump performance is adequate.(ABSTRACT TRUNCATED AT 250 WORDS)