Changes In Cross-bridge Kinetics Research Articles

Effect of active shortening and stretching on the rate of force re-development in rabbit psoas muscle fibres

The steady-state isometric force produced by skeletal muscle after active shortening and stretching is depressed and enhanced, respectively, compared with purely isometric force produced at corresponding final lengths and at the same level of activation. One hypothesis proposed to account for these force depression (FD) and force enhancement (FE) properties is a change in cross-bridge cycling kinetics. The rate of cross-bridge attachment (f) and/or cross-bridge detachment (g) may be altered following active shortening and active stretching, leading to FD and FE, respectively. Experiments elucidating cross-bridge kinetics in actively shortened and stretched muscle preparations and their corresponding purely isometric contractions have yet to be performed. The aim of this study was to investigate cross-bridge cycling kinetics of muscle fibres at steady-state following active shortening and stretching. This was done by determining muscle fibre stiffness and rate of active force redevelopment following a quick release-re-stretch protocol (kTR). Applying these measures to equations previously used in the literature for a two-state cross-bridge cycling model (attached/detached cross-bridges) allowed us to determine apparent f and g, the proportion of attached cross-bridges, and the force produced per cross-bridge. kTR, apparent f and g, the proportion of attached cross-bridges and the force produced per cross-bridge were significantly decreased following active shortening compared with corresponding purely isometric contractions, indicating a change in cross-bridge cycling kinetics. Additionally, we showed no change in cross-bridge cycling kinetics following active stretch compared with corresponding purely isometric contractions. These findings suggest that FD is associated with changes in cross-bridge kinetics, whereas FE is not.

Skeletal muscle contractile properties in a novel murine model for limb girdle muscular dystrophy 2i.

Limb-girdle muscular dystrophy (LGMD) 2i results from mutations in fukutin-related protein and aberrant α-dystroglycan glycosylation. Although this significantly compromises muscle function and ambulation, the comprehensive characteristics of contractile dysfunction are unknown. Therefore, we quantified the in situ contractile properties of the medial gastrocnemius in young adult P448L mice, an affected muscle of a novel model of LGMD2i. Normalized maximal twitch force, tetanic force, and power were significantly smaller in P448L mice, compared with sex-matched, wild-type mice. These differences were consistent with the replacement of contractile fibers by passive tissue. The shape of the active force-length relationships were similar in both groups, regardless of sex, consistent with an intact sarcomeric structure in P448L mice. Passive force-length curves normalized to maximal isometric force were steeper in P448L mice, and passive elements contribute disproportionately more to total contractile force in P448L mice. Sex differences were mostly noted in the force-velocity curves, as normalized values for maximal and optimal velocities were significantly slower in P448L males, compared with wild-type, but not in P448L females. This suggests that the dystrophic phenotype, which may include possible changes in cross-bridge kinetics and fiber-type proportions, progresses more quickly in P448L males. These results together indicate that active force and power generation are compromised in both sexes of P448L mice, while passive forces increase. More importantly, the results identified several functional markers of disease pathophysiology that could aid in developing and assessment of novel therapeutics for LGMD2i and possibly other dystroglycanopathies as well. NEW & NOTEWORTHY Comprehensive assessments of muscle contractile function have, until now, never been performed in an animal model for any dystroglycanopathy. This study suggests that skeletal muscle contractile properties are significantly compromised in a recently developed model for limb-girdle muscular dystrophy 2i, the P448L mouse. It further identifies novel pathological markers of muscle function that are suitable for developing therapeutics and for better understanding of disease pathogenesis.

Abnormal Cardiac Cross-Bridge Kinetics in a Troponin T ILE79ASN Transgenic Mouse Model

The missense mutation Ile79Asn in human cardiac troponin T (HcTnT-I79N) has been associated with phenotypic outcomes of familial hypertrophic cardiomyopathy, arrhythmias, and sudden cardiac death. Previous characterization of a transgenic mouse model bearing the HcTnT-I79N mutation, which expressed 52% HcTnT-I79N of total troponin T, demonstrated increased Ca2+ sensitivity of isometric force. However, little is known about the changes in cross-bridge kinetics, if any, that result from the mutation. This study investigates isometric force, sinusoidal stiffness and rate of tension redevelopment (kTR) of skinned papillary muscle fiber bundles at two sarcomere lengths (1.9 µm and 2.1 µm); cardiac muscle was isolated from the left ventricle of non-transgenic wild-type (WT) mice or transgenic mice bearing the HcTnT-I79N mutation. Sarcomere length (SL) was set at pCa8 using HeNe laser diffraction after the attachment points of each skinned fiber bundle were fixed with glutaraldehyde to minimize end compliance. Sinusoidal stiffness and kTR were obtained after recording steady-state isometric tension by oscillating the fiber bundle 0.2% of its relaxed length or by applying a ramped shortening followed by a quick re-stretch to original fiber bundle length, respectively. As previously reported, the HcTnT-I79N mutation resulted in increased Ca2+-sensitivity of isometric force although no difference in maximum force per cross-sectional area was observed. Length-dependent activation at the longer SL resulted in increased Ca2+-sensitivity of isometric force and sinusoidal stiffness, and decreased kTR for both WT and HcTnT-I79N. However, HcTnT-I79N exhibited faster kTR and greater sinusoidal stiffness at sub-maximum pCa (-log[Ca2+]) solutions compared to WT regardless of SL. These data indicate that HcTnT-I79N mutation, and SL, markedly influence not only the dynamics of individual regulatory units, but also the kinetics of cross-bridge cycling.

Myosin MgADP Release Rate Decreases as Sarcomere Length Increases in Skinned Rat Soleus Muscle Fibers

Actin-myosin cross-bridges use chemical energy from MgATP hydrolysis to generate force and shortening in striated muscle. Previous studies show that increases in sarcomere length can reduce thick-to-thin filament spacing in skinned muscle fibers, thereby increasing force production at longer sarcomere lengths. However, it is unclear how changes in sarcomere length and lattice spacing affect cross-bridge kinetics at fundamental steps of the cross-bridge cycle, such as the MgADP release rate. We hypothesize that decreased lattice spacing, achieved through increased sarcomere length or osmotic compression of the fiber via dextran T-500, could slow MgADP release rate and increase cross-bridge attachment duration. To test this, we measured cross-bridge cycling and MgADP release rates in skinned soleus fibers using stochastic length-perturbation analysis at 2.5 and 2.0 μm sarcomere lengths as pCa and [MgATP] varied. In the absence of dextran, the force-pCa relationship showed greater Ca2+ sensitivity for 2.5 vs. 2.0 μm sarcomere length fibers (pCa50 = 5.68 ± 0.01 vs. 5.60 ± 0.01). When fibers were compressed with 4% dextran, the length-dependent increase in Ca2+ sensitivity of force was attenuated, though the Ca2+ sensitivity of the force-pCa relationship at both sarcomere lengths was greater with osmotic compression via 4% dextran compared to no osmotic compression. Without dextran, the cross-bridge detachment rate slowed by ∼15% as sarcomere length increased, due to a slower MgADP release rate (11.2 ± 0.5 vs. 13.5 ± 0.7 s−1). In the presence of dextran, cross-bridge detachment was ∼20% slower at 2.5 vs. 2.0 μm sarcomere length due to a slower MgADP release rate (10.1 ± 0.6 vs. 12.9 ± 0.5 s−1). However, osmotic compression of fibers at either 2.5 or 2.0 μm sarcomere length produced only slight (and statistically insignificant) slowing in the rate of MgADP release. These data suggest that skeletal muscle exhibits sarcomere-length-dependent changes in cross-bridge kinetics and MgADP release that are separate from, or complementary to, changes in lattice spacing.

Myosin MgADP Release Rate Decreases with Greater Sarcomere Length and Reduced Thick-to-Thin Filament Spacing in Skinned Soleus Muscle Fibers from Rats

The actin-myosin cross-bridge interaction uses chemical energy from MgATP hydrolysis to generate force and shortening in striated muscle. Previous studies have shown that increases in sarcomere length can reduce thick-to-thin filament spacing in skinned muscle fibers, thereby increasing force production at longer sarcomere lengths. However, it is unclear how changes in sarcomere length and lattice spacing affect cross-bridge kinetics at fundamental steps of the cross-bridge cycle, such as the MgADP release rate. We hypothesize that decreased lattice spacing, achieved through increased sarcomere length or osmotic compression of the fiber via Dextran T-500, could reduce MgADP release rate and increase cross-bridge attachment time (ton). To test this, we measured ton in skinned soleus fibers using stochastic length-perturbation analysis at 2.5 and 2.0 µm sarcomere lengths as pCa and [MgATP] varied. In the absence of Dextran, the force-pCa relationship showed greater Ca2+-sensitivity for 2.5 vs 2.0 sarcomere length fibers (pCa50=5.68±0.01 vs 5.60±0.01; pCa 4.8, 5.0 mM MgATP, 17° C). When fibers were compressed with 4% Dextran, the force-pCa relationships exhibited even greater Ca2+-sensitivities and the length-dependent increases in Ca2+-sensitivity were preserved (pCa50=5.84±0.03 vs 5.80±0.04). Without Dextran, longer sarcomere lengths also exhibited increased ton (86±5 vs 70±2 ms), resulting from a slower MgADP release rate at 2.5 vs. 2.0 sarcomere length (11.24±0.5 vs 13.14±0.7 s−1). Osmotic compression further increased ton at both sarcomere lengths (98±7 vs 73±4 ms) due to even slower MgADP dissociation from myosin (9.57±0.5 vs 12.6±0.5 s−1), yet length-dependent kinetic differences were preserved. These data suggest that skeletal muscle exhibits length-dependent changes in cross-bridge kinetics as a function of lattice spacing, which may impact overall muscle behavior as length varies.

Effect of Active Shortening and Stretching on Lattice Spacing and Cross-Bridge Binding in Skinned Muscle Fibres

When a tetanized skeletal muscle is actively shortened or stretched the resulting steady-state force is different from the purely isometric force at the corresponding length. Active shortening reduces steady-state force (termed force depression-FD), while active lengthening increases steady-state force (termed force enhancement-FE). According to the cross-bridge theory, FD and FE can occur if myofilament lattice spacing was modified after active shortening and stretching leading to a change in the proportion of attached cross-bridges, or if the kinetics of cross-bridges were modified leading to a change in the proportion of attached cross-bridges and/or the average force per cross-bridge. Here, we determined the effect of active shortening and stretching on the myofilament lattice spacing and the proximity of the myosin heads to the thin filaments using small angle X-ray diffraction.Lattice spacing (d1,0) was calculated from the spacing between the 1,0 and 1,1 equatorial reflections. The ratio of the intensities of the 1,1 and 1,0 equatorial reflections (I1,1:I1,0) was used as an estimate of the degree of association of myosin heads with the thin filaments.d1,0 increased (38.9±0.1 versus 38.1±0.2nm, p 0.05) between the FE state and its reference state, and I1,1:I1,0 was increased (2.5±0.2 versus 1.3±0.1, p<0.05) in the FE state. These results suggest that the proportion of attached cross-bridges might be increased after active stretch and that this increase is not due to a decrease in lattice spacing but rather a change in cross-bridge kinetics.

Myosin MgADP release rate decreases at longer sarcomere length to prolong myosin attachment time in skinned rat myocardium.

Cardiac contractility increases as sarcomere length increases, suggesting that intrinsic molecular mechanisms underlie the Frank-Starling relationship to confer increased cardiac output with greater ventricular filling. The capacity of myosin to bind with actin and generate force in a muscle cell is Ca(2+) regulated by thin-filament proteins and spatially regulated by sarcomere length as thick-to-thin filament overlap varies. One mechanism underlying greater cardiac contractility as sarcomere length increases could involve longer myosin attachment time (ton) due to slowed myosin kinetics at longer sarcomere length. To test this idea, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure ton as [MgATP] varied (0.05-5 mM) at 1.9 and 2.2 μm sarcomere lengths. From this ton-MgATP relationship, we calculated cross-bridge MgADP release rate and MgATP binding rates. As MgATP increased, ton decreased for both sarcomere lengths, but ton was roughly 70% longer for 2.2 vs. 1.9 μm sarcomere length at maximally activated conditions. These ton differences were driven by a slower MgADP release rate at 2.2 μm sarcomere length (41 ± 3 vs. 74 ± 7 s(-1)), since MgATP binding rate was not different between the two sarcomere lengths. At submaximal activation levels near the pCa50 value of the tension-pCa relationship for each sarcomere length, length-dependent increases in ton were roughly 15% longer for 2.2 vs. 1.9 μm sarcomere length. These changes in cross-bridge kinetics could amplify cooperative cross-bridge contributions to force production and thin-filament activation at longer sarcomere length and suggest that length-dependent changes in myosin MgADP release rate may contribute to the Frank-Starling relationship in the heart.

Elevated rates of force development and MgATP binding in F764L and S532P myosin mutations causing dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a disease characterized by dilation of the ventricular chambers and reduced contractile function. We examined the contractile performance of chemically-skinned ventricular strips from two heterozygous murine models of DCM-causing missense mutations of myosin, F764L/+ and S532P/+, in an α-myosin heavy chain (MyHC) background. In Ca2+-activated skinned myocardial strips, the maximum developed tension in F764L/+ was only ~50% that of litter-mate controls (+/+). The F764L/+ also exhibited significantly reduced rigor stiffness, loaded shortening velocity and power output. Corresponding indices for S532P/+ strips were not different from controls. Manipulation of MgATP concentration in conjunction with measures of viscoelasticity, which provides estimates of myosin detachment rate 2πc, allowed us to probe the molecular basis of changes in crossbridge kinetics that occur with the myosin mutations. By examining the response of detachment rate to varying MgATP we found the rate of MgADP release was unaffected by the myosin mutations. However, MgATP binding rate was higher in the DCM groups compared to controls (422±109mM−1·s−1 in F764L/+, 483±74mM−1·s−1 in S532P/+ and 303±18mM−1·s−1 in +/+). In addition, the rate constant of force development, 2πb, was significantly higher in DCM groups compared to controls (at 5mM MgATP: 36.9±4.9s−1 in F764L/+, 32.9±4.5s−1 in S532P/+ and 18.2±1.7s−1 in +/+). These results suggest that elevated rates of force development and MgATP binding are features of cardiac myofilament function that underlie the development of DCM.

Cardiac Myosin Binding Protein C Insufficiency Leads to Early Onset of Mechanical Dysfunction

Decreased expression of cardiac myosin binding protein C (cMyBPC) as a result of genetic mutations may contribute to the development of hypertrophic cardiomyopathy (HCM); however, the mechanisms that link cMyBPC expression and HCM development, especially contractile dysfunction, remain unclear. We evaluated cardiac mechanical function in vitro and in vivo in young mice (8-10 weeks of age) carrying no functional cMyBPC alleles (cMyBPC(-/-)) or 1 functional cMyBPC allele (cMyBPC(±)). Skinned myocardium isolated from cMyBPC(-/-) hearts displayed significant accelerations in stretch activation cross-bridge kinetics. Cardiac MRI studies revealed severely depressed in vivo left ventricular (LV) magnitude and rates of LV wall strain and torsion compared with wild-type (WT) mice. Heterozygous cMyBPC(±) hearts expressed 23±5% less cMyBPC than WT hearts but did not display overt hypertrophy. Skinned myocardium isolated from cMyBPC(±) hearts displayed small accelerations in the rate of stretch induced cross-bridge recruitment. MRI measurements revealed reductions in LV torsion and circumferential strain, as well reduced circumferential strain rates in early systole and diastole. Modest decreases in cMyBPC expression in the mouse heart result in early-onset subtle changes in cross-bridge kinetics and in vivo LV mechanical function, which could contribute to the development of HCM later in life.

Protein kinase A changes calcium sensitivity but not crossbridge kinetics in human cardiac myofibrils

We investigated the effect of PKA treatment (1 U/ml) on the mechanical properties of isolated human cardiac myofibrils. PKA treatment was associated with significant incorporation of radiolabeled phosphate into several sarcomeric proteins including troponin I and myosin binding protein C and was also associated with a right shift in the tension-pCa relation (ΔpCa(50) = 0.2 ± 0.1). PKA treatment also caused right shifts in the pCa dependence of the rate of tension development, tension redevelopment, and the linear and exponential phases of myofibril relaxation. However, there was no change in the same measures of crossbridge turnover when expressed as a function of tension. We conclude that the changes in crossbridge kinetics as a function of calcium concentration reflect a reduced tension due to a lower calcium sensitivity and that the relationship between crossbridge kinetics and tension was unchanged, indicating no direct effect of PKA treatment on crossbridge cycling.

Active Stretch does not Affect the Rate of Force Redevelopment in Rabbit Skinned Fibres

University of Calgary, Calgary, AB, Canada. The steady state force produced by a muscle following active stretch is greater than the corresponding purely isometric force. This phenomenon is called “Residual Force Enhancement” (RFE). Most of the proposed mechanisms of RFE were either related to the development of structural non-uniformities, or with the engagement of a passive structural element upon activation. So far there have been hardly any experiments investigating the possibility for a change in cross bridge kinetics after active stretching. The aim of this study was to investigate if RFE was accompanied with changes in the rate of force redevelopment in rabbit psoas and soleus skinned fibres. The rate of force redevelopment (Ktr) was evaluated after a quick release-stretch cycle. Furthermore, stiffness and the time that the cross bridges require to start redevelop tension (Δt) were measured after a purely isometric contraction and active stretch. Ktr and stiffness were not changed after active stretch compared to a purely isometric contraction in both psoas and soleus fibres. Δt after active stretch was smaller than Δt after a purely isometric contraction in psoas and soleus fibres. Using a two-state cross bridge model, as described by Brenner (PNAS, 1987), we estimated that the apparent rate constants of cross bridge attachment and detachment were not altered in the force enhanced state. Consequently, RFE might be explained, in part, in terms of changes in cross-bridge based forces. The strain of a passive element, likely the molecular spring titin, during active stretch and then its recoil when the fibre is released might be responsible for the observed decrease in Δt after stretch.

Functional Consequences of the Human Cardiac Troponin I Hypertrophic Cardiomyopathy Mutation R145G in Transgenic Mice

In this study, we addressed the functional consequences of the human cardiac troponin I (hcTnI) hypertrophic cardiomyopathy R145G mutation in transgenic mice. Simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145G transgenic mice (Tg-R145G) versus hcTnI wild type transgenic mice (Tg-WT) showed a significant decrease in the maximal Ca(2+)-activated force without changes in the maximal ATPase activity and an increase in the Ca(2+) sensitivity of both ATPase and force development. No difference in the cross-bridge turnover rate was observed at the same level of cross-bridge attachment (activation state), showing that changes in Ca(2+) sensitivity were not due to changes in cross-bridge kinetics. Energy cost calculations demonstrated higher energy consumption in Tg-R145G fibers compared with Tg-WT fibers. The addition of 3 mm 2,3-butanedione monoxime at pCa 9.0 showed that there was approximately 2-4% of force generating cross-bridges attached in Tg-R145G fibers compared with less than 1.0% in Tg-WT fibers, suggesting that the mutation impairs the ability of the cardiac troponin complex to fully inhibit cross-bridge attachment under relaxing conditions. Prolonged force and intracellular [Ca(2+)] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared with Tg-WT. These results suggest that the phenotype of hypertrophic cardiomyopathy is most likely caused by the compensatory mechanisms in the cardiovascular system that are activated by 1) higher energy cost in the heart resulting from a significant decrease in average force per cross-bridge, 2) slowed relaxation (diastolic dysfunction) caused by prolonged [Ca(2+)] and force transients, and 3) an inability of the cardiac TnI to completely inhibit activation in the absence of Ca(2+) in Tg-R145G mice.

Phosphorylation-dependent Conformational Transition of the Cardiac Specific N-Extension of Troponin I in Cardiac Troponin

We present here the solution structure for the bisphosphorylated form of the cardiac N-extension of troponin I (cTnI1-32), a region for which there are no previous high-resolution data. Using this structure, the X-ray crystal structure of the cardiac troponin core, and uniform density models of the troponin components derived from neutron contrast variation data, we built atomic models for troponin that show the conformational transition in cardiac troponin induced by bisphosphorylation. In the absence of phosphorylation, our NMR data and sequence analyses indicate a less structured cardiac N-extension with a propensity for a helical region surrounding the phosphorylation motif, followed by a helical C-terminal region (residues 25–30). In this conformation, TnI1-32 interacts with the N-lobe of cardiac troponin C (cTnC) and thus is positioned to modulate myofilament Ca2+-sensitivity. Bisphosphorylation at Ser23/24 extends the C-terminal helix (residues 21–30) which results in weakening interactions with the N-lobe of cTnC and a re-positioning of the acidic amino terminus of cTnI1-32 for favorable interactions with basic regions, likely the inhibitory region of cTnI. An extended poly(L-proline)II helix between residues 11 and 19 serves as the rigid linker that aids in re-positioning the amino terminus of cTnI1-32 upon bisphosphorylation at Ser23/24. We propose that it is these electrostatic interactions between the acidic amino terminus of cTnI1-32 and the basic inhibitory region of troponin I that induces a bending of cTnI at the end that interacts with cTnC. This model provides a molecular mechanism for the observed changes in cross-bridge kinetics upon TnI phosphorylation.

Ischemia reperfusion dysfunction changes model-estimated kinetics of myofilament interaction due to inotropic drugs in isolated hearts

BackgroundThe phase-space relationship between simultaneously measured myoplasmic [Ca2+] and isovolumetric left ventricular pressure (LVP) in guinea pig intact hearts is altered by ischemic and inotropic interventions. Our objective was to mathematically model this phase-space relationship between [Ca2+] and LVP with a focus on the changes in cross-bridge kinetics and myofilament Ca2+ sensitivity responsible for alterations in Ca2+-contraction coupling due to inotropic drugs in the presence and absence of ischemia reperfusion (IR) injury.MethodsWe used a four state computational model to predict LVP using experimentally measured, averaged myoplasmic [Ca2+] transients from unpaced, isolated guinea pig hearts as the model input. Values of model parameters were estimated by minimizing the error between experimentally measured LVP and model-predicted LVP.ResultsWe found that IR injury resulted in reduced myofilament Ca2+ sensitivity, and decreased cross-bridge association and dissociation rates. Dopamine (8 μM) reduced myofilament Ca2+ sensitivity before, but enhanced it after ischemia while improving cross-bridge kinetics before and after IR injury. Dobutamine (4 μM) reduced myofilament Ca2+ sensitivity while improving cross-bridge kinetics before and after ischemia. Digoxin (1 μM) increased myofilament Ca2+ sensitivity and cross-bridge kinetics after but not before ischemia. Levosimendan (1 μM) enhanced myofilament Ca2+ affinity and cross-bridge kinetics only after ischemia.ConclusionEstimated model parameters reveal mechanistic changes in Ca2+-contraction coupling due to IR injury, specifically the inefficient utilization of Ca2+ for contractile function with diastolic contracture (increase in resting diastolic LVP). The model parameters also reveal drug-induced improvements in Ca2+-contraction coupling before and after IR injury.

The Essential Light Chain N-terminal Extension Alters Force and Fiber Kinetics in Mouse Cardiac Muscle

The functional significance of the actin-binding region at the N terminus of the cardiac myosin essential light chain (ELC) remains elusive. In a previous experiment, the endogenous ventricular ELC was replaced with a protein containing a 10-amino acid deletion at positions 5-14 (ELC1vDelta5-14, referred to as 1vDelta5-14), a region that interacts with actin. 1vDelta5-14 mice showed no discernable mutant phenotype in skinned ventricular strips. However, because the myofilament lattice swells upon skinning, the mutant phenotype may have been concealed by the inability of the ELC to reach the actin-binding site. Using the same mouse model, we repeated earlier measurements and performed additional experiments on skinned strips osmotically compressed to the intact lattice spacing as determined by x-ray diffraction. 1vDelta5-14 mice exhibited decreased maximum isometric tension without a change in calcium sensitivity. The decreased force was most evident in 5-6-month-old mice compared with 13-15-month-old mice and may account for the greater ventricular wall thickness in young 1vDelta5-14 mice compared with age-matched controls. No differences were observed in unloaded shortening velocity at maximum calcium activation. However, 1vDelta5-14 mice exhibited a significant difference in the frequency at which minimum complex modulus amplitude occurred, indicating a change in cross-bridge kinetics. We hypothesize that the ELC N-terminal extension interaction with actin inhibits the reversal of the power stroke, thereby increasing isometric force. Our results strongly suggest that an interaction between residues 5-14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance.

New inotropic concepts: rationale for and differences between calcium sensitizers and phosphodiesterase inhibitors.

Positive inotropic compounds may be harmful in the long-term treatment of chronic congestive heart failure because they may induce a calcium overload, unwanted changes in cross-bridge kinetics and an acceleration in heart rate. As a result of all three alterations, energy consumption would be increased. Different pharmacological modes of action may have different effects on the molecular mechanisms underlying the positive inotropic effect, and hence on myocardial energy consumption. Therefore, we studied the effects of a variety of cardiotonic agents on the heat released from small guinea pig papillary muscles contracting isometrically at an experimental temperature of 21 degrees C and a stimulation frequency of 12 per minute using rapid antimony-bismuth thermopiles. We were able to define the economy of muscle contraction, which was lowest with phosphodiesterase inhibitors and highest with calcium sensitizers. Compared with an increase in extracellular calcium concentration, beta 1-adrenoceptor stimulators and phosphodiesterase inhibitors profoundly decrease the economy of myocardial contraction, and calcium-sensitizers (pimobendan and EMD-53998) slightly increase myocardial economy, whereas ouabain and the calcium channel agonist BAY K 8644 have no effect on this parameter. In addition, we provide evidence that acceleration of heart rate may be harmful not only from an energetic point of view: an increase in heart rate may also decrease the contractility of the failing human myocardium (inverse force-frequency relationship). Taking these observations into consideration, an "optimal' positive inotropic compound should have no, or even negative, chronotropic effects, should not be mediated by increases in calcium transients, and should decelerate, rather than accelerate, cross-bridge kinetics.

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

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. 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. 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]. (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.

A New Cardiotonic Drug Reduces the Energy Cost of Active Tension in Cardiac Muscle

The novel thiadiazinone EMD 57033 (EMD) increases the calcium responsiveness of the contractile proteins in cardiac muscle. In skinned ventricular trabeculate isolated from guinea-pig heart, application of 10 μM EMD shifted the curve relating isometric tension to the applied calcium concentration to the left and increased maximal tension by 15%. In intact trabeculae, the rate of heat production, an indicator of the rate of ATP hydrolysis in the steady state, and isometric tension were measured at 37°C. Both the thiadiazinone (EMD; 2.5, 5, and 10 μM) and the cardiac glycoside dihydro-ouabain (DHO; 5, 10, and 20 μM) produced a concentration-dependent increase in contraction-related heart production (Hc) and in the tension time integral of isometric contractions (Td). In the presence of EMD the energy cost of active tension (Hc/Td) was substantially decreased as compared to control conditions. The energy cost of the positive inotropic effect of EMD (43.8 mW N-1 cm-1) was only about half as large as the energy cost of the positive inotropic effect of DHO (88.4 mW N-1 cm-1). It is concluded that EMD causes a change in cross-bridge kinetics that increases the contractility of cardiac muscle and improves the economy of chemo-mechanical energy transduction. Our results suggest that EMD 57033 represents a prototype of a new class of cardiotonic agents that might be potentially useful in the therapy of congestive heart failure.

Depression of Myocardial Force and Stiffness without Change in Crossbridge Kinetics

The authors examined the effects of nifedipine, a sarcolemmal slow Ca2+ channel blocker, on dynamic stiffness and force of rabbit right ventricular trabeculum and papillary muscle in Ba2+ contracture, in an attempt to reproduce the effects of halothane, enflurane, and isoflurane on a similar preparation as reported by Shibata et al. Once barium contracture force was established, muscle length was perturbed with small amplitude sinusoidal oscillations in the frequency range of 0.1-100 Hz. Nifedipine 1 microM was then added to the superfusate and dynamic stiffness was again measured. Additional barium was used to determine restoration of contracture force to and beyond control levels. Nifedipine produced a significant decrease in contracture force and high-frequency stiffness with no effect on the frequency (fmin) at which stiffness amplitude exhibited a minimum (P less than 0.005). Contracture force and stiffness could be restored by adding additional barium to the nifedipine-treated muscles. These results are similar to those reported by Shibata et al. using volatile anesthetics. Since nifedipine, which acts specifically at the sarcolemmal slow Ca2+ channel, affects contracture force and dynamic stiffness in this preparation in a manner similar to the volatile anesthetics, the authors suggest that the anesthetics studied by Shibata et al. may well exert a significant component of their negative inotropic activity via their action on the sarcolemmal slow Ca2+ channel.

Effects of phosphate on the contractile properties of fast and slow muscle fibres from an Antarctic fish.

Single fast myotomal fibres and small bundles of slow fibres (from the adductor pectoralis profundus muscle) were isolated from the Antarctic teleost Notothenia neglecta. Fibres were skinned by a brief detergent treatment. The effects of phosphate on the mechanical properties and ATPase activity of fast and slow fibres were studied. 20 mM-phosphate inhibited maximum isometric tension in slow fibres by 34%, but by only 11% in fast fibres. A half-maximal response was obtained at approximately 5 mM-phosphate. These concentrations are within the range measured in muscle, and the effect is probably of physiological significance. This species is of particular interest, since there is evidence that the energy supply to the fast muscle is largely based on phosphocreatine breakdown, which would result in large changes in intracellular phosphate concentration during exercise. The maximum contraction velocity of both fast and slow fibres was not affected by 10 mM-phosphate, nor was the ATPase activity of the slow fibres during isometric contraction. The phosphate-induced depression in tension in slow fibres was associated with a proportional decrease in stiffness. The rate of force recovery after rapid, small amplitude stretches and releases was increased by phosphate, as was the rate of rise of force during stretch activation. The results are discussed with reference to the different patterns of energy supply for contraction in muscle, and an attempt is made at explaining the data in terms of changes in cross-bridge kinetics.