G.J.M. Stienen

Alterations in Myofilament Function Contribute to Left Ventricular Dysfunction in Pigs Early After Myocardial Infarction

Myocardial infarction (MI) initiates cardiac remodeling, depresses pump function, and predisposes to heart failure. This study was designed to identify early alterations in Ca2+ handling and myofilament proteins, which may contribute to contractile dysfunction and reduced &bgr;-adrenergic responsiveness in postinfarct remodeled myocardium. Protein composition and contractile function of skinned cardiomyocytes were studied in remote, noninfarcted left ventricular (LV) subendocardium from pigs 3 weeks after MI caused by permanent left circumflex artery (LCx) ligation and in sham-operated pigs. LCx ligation induced a 19% increase in LV weight, a 69% increase in LV end-diastolic area, and a decrease in ejection fraction from 54±5% to 35±4% (all P<0.05), whereas cardiac responsiveness to exercise-induced increases in circulating noradrenaline levels was blunted. Endogenous protein kinase A (PKA) was significantly reduced in remote myocardium of MI animals, and a negative correlation (R=0.62; P<0.05) was found between cAMP levels and LV weight-to-body weight ratio. Furthermore, SERCA2a expression was 23% lower after MI compared with sham. Maximal isometric force generated by isolated skinned myocytes was significantly lower after MI than in sham (15.4±1.5 versus 19.2±0.9 kN/m2; P<0.05), which might be attributable to a small degree of troponin I (TnI) degradation observed in remodeled postinfarct myocardium. An increase in Ca2+ sensitivity of force (pCa50) was observed after MI compared with sham (&Dgr;pCa50=0.17), which was abolished by incubating myocytes with exogenous PKA, indicating that the increased Ca2+ sensitivity resulted from reduced TnI phosphorylation. In conclusion, remodeling of noninfarcted pig myocardium is associated with decreased SERCA2a and myofilament function, which may contribute to depressed LV function. The full text of this article is available online at http://circres.ahajournals.org.

ATP utilization for calcium uptake and force production in skinned muscle fibres of Xenopus laevis.

1. A method has been developed to discriminate between the rate of ATP hydrolysis associated with calcium uptake into the sarcoplasmic reticulum (SR) and force development of the contractile apparatus in mechanically or saponin‐skinned skeletal muscle fibres. The rate of ATP hydrolysis was determined in fibres of different types from the iliofibularis muscle of Xenopus laevis by enzymatic coupling of ATP re‐synthesis to the oxidation of NADH. 2. The ATPase activity was determined before and after exposure of the preparations for 30 min to a solution containing 0.5% Triton X‐100, which effectively abolishes the SR ATPase activity. The fibres were activated in a solution containing 5 mM caffeine to ensure that calcium uptake into the SR was maximal. 3. At saturating Ca2+ concentrations the actomyosin (AM) and SR ATPase activities in fast‐twitch fibres, at 4.3 degrees C, amounted to 1.52 +/‐ 0.07 and 0.58 +/‐ 0.10 mumol s‐1 (g dry wt)‐1, respectively (means +/‐ S.E.M.; n = 25). The SR ATPase activity was 25% of the total ATPase activity. At submaximal calcium concentrations the AM ATPase activity varied in proportion to the isometric force. 4. The calcium sensitivity of the SR ATPase was larger than that of the AM ATPase and its dependence on [Ca2+] was less steep. The AM ATPase activity was half‐maximal at a pCa of 6.11 (pCa = ‐log [Ca2+]) whereas the SR ATPase activity was half‐maximal at a pCa of 6.62. 5. In Triton X‐100‐treated fibres, at different 2,3‐butanedione monoxime (BDM) concentrations, the AM ATPase activity and isometric force varied proportionally. The SR ATPase activity determined by extrapolation of the total ATPase activity in mechanically skinned or saponin‐treated fibres to zero force, was independent of the BDM concentration in the range studied (0‐20 mM). The values obtained for the SR ATPase activity in this way were similar to those obtained with Triton X‐100 treatment. 6. The AM ATPase activity in slow‐twitch fibres amounted to 0.74 +/‐ 0.13 mumol s‐1 (g dry wt)‐1, i.e. about a factor of two smaller than in fast‐twitch fibres. The SR ATPase activity amounted to 0.47 +/‐ 0.07 mumol s‐1 (g dry wt)‐1, i.e. rather similar to the value in fast‐twitch fibres. The proportion of the total ATPase activity that was due to SR ATPase (40%) was larger than in fast‐twitch fibres. 7. The temperature dependence of the AM and SR ATPase activities in fast‐twitch fibres differed. In the temperature range 5‐10 degrees C, the relative changes in AM and SR ATPase activities for a 10 degrees C temperature change (Q10) were 3.9 +/‐ 0.3 and 7.2 +/‐ 1.5, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of inorganic phosphate and pH on ATP utilization in fast and slow skeletal muscle fibers

The influence of P(i) and pH was studied on myofibrillar ATP turnover and force development during maximally activated isometric contractions, in skinned single fibers from rabbit soleus and psoas muscle. ATP hydrolysis was coupled to the breakdown of NADH, which was monitored photometrically at 340 nm. In psoas the depression by phosphate of force is twice that of ATP turnover, but in soleus force and ATP turnover are depressed equally by P(i). Most, but not all, of the ATPase and force values observed for a combination of high P(i) and low pH could be explained by independent effects of P(i) and pH. The effects of P(i) and pH on ATP turnover can be understood by a three-state cross-bridge scheme. Mass action of phosphate on the reaction from the actomyosin(AM).ADP state to the AM.ADP.P(i) state may largely account for the phosphate dependencies of ATPase activity found. Protons affect cross-bridge detachment from the AM.ADP state and the rate of the AM.ADP.P(i)-to-AM.ADP transition. In this scheme, the effects of P(i) and pH on cross-bridge kinetics appeared to be largely independent.

Age-dependent changes in myosin composition correlate with enhanced economy of contraction in guinea-pig hearts

1 The composition of myosin heavy chains (MHCs) was investigated in young (1‐ to 8‐week‐old) and mature (9‐ to 26‐week‐old) guinea‐pigs using two monoclonal antibodies directed specifically against α‐MHC and β‐MHC. In addition, maximum force and the rate of ATP consumption during isometric contraction were measured in chemically skinned trabeculae taken from the same hearts. 2 An age‐dependent shift in the MHC composition was found. The α‐MHC fraction decreased from 0.17 ± 0.02 (mean ± s.e.m.; n= 24) in young to 0.04 ± 0.01 (n= 43) in mature hearts. This shift was correlated with a decrease in tension cost (i.e. ATP consumption per second per trabecula volume/force per cross‐sectional area) from 4.1 ± 0.2 mmol kN−1 m−1 s−1 (n= 23) in young to 2.5 ± 0.1 mmol kN−1 m−1 s−1 (n= 57) in mature hearts. 3 From the results it follows that the slow β‐MHC isoform, which predominates in hearts of mature guinea‐pigs, is about 5 times more economical than the fast α‐MHC isoform. Calcium sensitivity of force and ATP consumption decreased with age, but stabilized within a few weeks after birth. The pronounced dependence of cardiac energetics on MHC composition should be taken into account in long‐term studies of cardiac overload.

Increase in ATP consumption during shortening in skinned fibres from rabbit psoas muscle: effects of inorganic phosphate.

1. The influence of inorganic phosphate (P(i)) on the relationship between ATP consumption and mechanical performance under isometric and dynamic conditions was investigated in chemically skinned single fibres or thin bundles from rabbit psoas muscle. Myofibrillar ATPase activity was measured photometrically by enzymatic coupling of the regeneration of ATP to the oxidation of NADH. NADH absorbance at 340 nm was determined inside a miniature (4 microliters) measuring chamber. 2. ATP consumption due to isovelocity shortenings was measured in the range between 0.0625 and 1 L0 s‐1(L0: fibre length previous to shortening, corresponding to a sarcomere length of 2.64 microns), in solutions without added P(i) and with 30 mM P(i). To get an estimate of the amount of ATP utilized during the shortening phase, quick releases of various amplitudes were also performed. 3. After quick releases, sufficiently large that force dropped to zero, extra ATP was hydrolysed which was largely independent of the amplitude of the release and of the period of unloaded shortening. This extra amount, above the isometric ATP turnover, corresponded to about 0.7 and 0.5 ATP molecules per myosin head at 0 and 30 mM P(i), respectively. 4. ATP turnover during the isovelocity shortenings was higher than isometric turnover and increased with increasing shortening velocity up to about 2.7 times the isometric value. At low and moderate velocities of shortening (< 0.5 L0 s‐1), P(i) reduced ATP turnover during isovelocity shortening and isometric ATP turnover to a similar extent, i.e. a decrease to about 77% between 0 and 30 mM added P(i). 5. The extra ATP turnover above the isometric value, resulting from isovelocity shortenings studied at different speeds, was proportional to the power output of the preparation, both in the absence and presence of added [P(i)]. 6. The effect of shortening velocity and [P(i)] on energy turnover can be understood in a cross‐bridge model that consists of a detached, a non‐ or low‐force‐producing, and a force‐producing state. In this model, mass action of P(i) influences the equilibrium between the force‐producing and the non‐or‐low‐force‐producing cross‐bridges, and shortening enhances cross‐bridge detachment from both attached states.

Influence of phosphate and pH on myofibrillar ATPase activity and force in skinned cardiac trabeculae from rat.

1. The effects of inorganic phosphate (Pi) and pH on maximal calcium‐activated isometric force and MgATPase activity were studied in chemically skinned cardiac trabeculae from rat. ATP hydrolysis was coupled enzymatically to the breakdown of NADH, and its concentration was determined photometrically. Measurements were performed at 2.1 microns sarcomere length and 20 degrees C. ATPase activity and force were also determined when square‐wave‐shaped length changes were applied, with a frequency of 23 Hz and an amplitude of 2.5%. 2. At pH 7.0 without added Pi, the average isometric force (+/‐ S.E.M.) was 51 +/‐ 3 kN m‐2 (n = 23). The average isometric ATPase activity was 0.43 +/‐ 0.02 mM s‐1 (n = 23). During the changes in length ATPase activity increased to 152 +/‐ 3% of the isometric value, while the average force level decreased to 48 +/‐ 2%. 3. Isometric force gradually decreased to 31 +/‐ 2% of the control value when the Pi concentration was increased to 30 mM. Isometric ATPase activity, however, remained constant for Pi concentrations up to 5 mM and decreased to 87 +/‐ 3% at 30 mM Pi. When Pi accumulation inside the preparation due to ATP hydrolysis was taken into account, a linear relationship was found between isometric force and log [Pi]. The decrease in relative force was found to be 44 +/‐ 4% per decade. 4. During the length changes, ATPase activity and average force showed, apart from the increase in ATPase activity and decrease in average force, the same dependence on Pi as the isometric values. Stiffness, estimated from the amplitude of the force responses during the length changes, decreased in proportion to isometric force when the Pi concentration was increased. The changes in the shape of the force responses due to the repetitive changes in length as a function of the Pi concentration were relatively small. These results suggest that the effect of Pi on the transitions which influence ATP turnover is rather insensitive to changes in cross‐bridge strain. 5. Isometric force, normalized to the control value at pH 7.0, increased gradually from 54 +/‐ 1% at pH 6.2 to 143 +/‐ 10% at pH 7.5. ATPase activity remained practically constant for pH values from 6.8 to 7.2 but decreased to 80 +/‐ 1% at pH 6.2 and to 83 +/‐ 5% at pH 7.5. ATPase activity during the length changes was reduced more than the isometric ATPase activity when pH was lowered.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of pH on myofibrillar ATPase activity in fast and slow skeletal muscle fibers of the rabbit

In permeabilized single fibers of fast (psoas) and slow (soleus) muscle from the rabbit, the effect of pH on isometric myofibrillar ATPase activity and force was studied at 15 degrees C, in the pH range 6.4-7.9. ATPase activity was measured photometrically by enzymatic coupling of the regeneration of ATP to the oxidation of NADH, present in the bathing solution. NADH absorbance at 340 nm was determined inside a measuring chamber. To measure ATP turnover in single soleus fibers accurately, a new measuring chamber (volume 4 microliters) was developed that produced a sensitivity approximately 8 times higher than the system previously used. Under control conditions (pH 7.3), the isometric force was 136 and 115 kN/m2 and the ATP turnover was 0.43 and 0.056 mmol per liter fiber volume per second in psoas and soleus fibers, respectively. Over the pH range studied, isometric force increased monotonically by a factor 1.7 for psoas and 1.2 for soleus fibers. In psoas the isometric ATPase activity remained constant, whereas in soleus it slightly decreased with increasing pH. The pH dependency of relative tension cost (isometric ATPase activity divided by force) was practically identical for psoas and soleus fibers. In both cases it decreased by about a factor 0.57 as pH increased from 6.4 to 7.9. The implications of these findings are discussed in terms of cross-bridge kinetics. For both fiber types, estimates of the reaction rates and the distribution of cross-bridges and of their pH dependencies were obtained. A remarkable similarity was found between fast- and slow-twitch fibers in the effects of pH on the reaction rate constants.

Myocardial force development and structural changes associated with monocrotaline induced cardiac hypertrophy and heart failure

In this study alterations are characterized which occur, in myocardial force development morphological appearance and protein composition, during the development of cardiac hypertrophy and heart failure in monocrotaline (MCT) treated rats. The transition from cardiac hypertrophy to heart failure was studied by comparing the results from control (CON) and two MCT groups (40 and 44 mg/kg body weight). The three experimental groups consisted of at least five animals each. Parameters studied were: body weight (measured daily), lung/body weight ratio, right ventricular wall volume and thickness, and force development in thin right ventricular trabeculae at 27°C, using different extracellular calcium concentrations and pacing frequencies. MCT injection resulted in marked right ventricular hypertrophy and heart failure as evidenced by an up to 2-fold increase in lung/body weight ratio and a 1.7-fold increase in wall volume. The MCT groups showed a negative force–frequency relation and maximum force was up to 2-fold less than in the CON group. Protein analysis by means of one- and two-dimensional gel electrophoresis revealed a marked (7-fold) up-regulation of the slow myosin heavy chain isoform as well as a 4.5-fold increase in the content of the cytoskeletal protein desmin, whereas the mitochondrial protein ATP-synthase content was reduced. Hence MCT-induced cardiac hypertrophy and heart failure result in altered cellular calcium handling, depression of maximum force output, an increase in the economy of myocardial contraction and changes in cytoskeletal structure and energy supply.

Origin of concurrent ATPase activities in skinned cardiac trabeculae from rat.

1. To determine the rate of ATP turnover by the sarcoplasmic reticulum (SR) Ca2+ pump in cardiac muscle, and to assess the contributions of other ATPase activities to the overall ATP turnover rate, ATPase activity and isometric force production were studied in saponin‐skinned trabeculae from rat. ATP hydrolysis was enzymatically coupled to the oxidation of NADH; the concentration of NADH was monitored photometrically. All measurements were performed at 20 +/‐ 1 degrees C and pH 7.0. Resting sarcomere length was adjusted to 2.1 microns. All solutions contained 5 mM caffeine to ensure continuous release of Ca2+ from the SR. 2. The Ca(2+)‐independent ATPase activity, determined in relaxing solution (pCa 9), amounted to 130 +/‐ 13 microM s‐1 (mean +/‐ S.E.M., n = 7) at the beginning of an experiment. During subsequent measurements in relaxing solution, a decrease in ATPase activity was observed, indicative of loss of membrane‐bound ATPase activity. The steady‐state Ca(2+)‐independent (basal) ATPase activity was 83 +/‐ 5 microM s‐1 (n = 66). 3. Treatment of saponin‐skinned preparations with Triton X‐100 abolished 50 microM s‐1 (60%) of the basal ATPase activity. Addition of ouabain (1 mM) suppressed 14 +/‐ 5% of the basal activity, whereas 8 +/‐ 3% was suppressed by 20 microM cyclopiazonic acid (CPA). It is argued that 31 microM s‐1 of the basal ATPase activity may be associated with MgATPase from the transverse tubular system. 4. The maximal Ca(2+)‐activated ATPase activity, i.e. the total ATPase activity (determined in activating solution, pCa 4.3) corrected for basal ATPase activity, was found to be 409 +/‐ 15 microM s‐1 (n = 66). Experiments with CPA indicated that at least 9 +/‐ 6% of the maximal Ca(2+)‐activated ATPase activity originates from the sarcoplasmic Ca2+ pump. These experiments indicate that the rate of ATP consumption by the SR Ca2+ transporting ATPase amounts to at least 37 microM s‐1. 5. Treatment of preparations with Triton X‐100 abolished 15 +/‐ 3% of the maximal Ca(2+)‐activated ATPase activity, indicating that 15 +/‐ 3% of the maximal Ca(2+)‐activated ATPase activity is membrane bound. 6. Variation of free [Ca2+] indicated that apart from the actomyosin ATPase activity a second Ca(2+)‐dependent ATPase activity contributed to the overall ATP turnover rate. This activity was half‐maximal at pCa 6.21, and probably reflects the SR Ca2+ transporting ATPase. It constituted 18 +/‐ 3% of the Ca(2+)‐dependent ATPase activity, yielding an upper limit for the SR Ca2+ transporting ATPase activity of 74 microM s‐1.

Dependency of the force-velocity relationships on Mg ATP in different types of muscle fibers from Xenopus laevis

MgATP binding to the actomyosin complex is followed by the dissociation of actin and myosin. The rate of this dissociation process was determined from the relationship between the maximum velocity of shortening and the MgATP concentration. It is shown here that the overall dissociation rate is rather similar in different types of muscle fibers. The relation between MgATP concentration and the maximum shortening velocity was investigated in fast and slow fibers and bundles of myofibrils of the iliofibularis muscle of Xenopus laevis at 4 degrees C from which the sarcolemma was either removed mechanically or made permeable by means of a detergent. A small segment of each fiber was used for a histochemical determination of fiber type. At 5 mM MgATP, the fast fibers had a maximum shortening velocity (Vmax) of 1.74 +/- 0.12 Lo/s (mean +/- SEM) (Lo: segment length at a sarcomere length of 2.2 microns). For the slow fibers Vmax was 0.41 +/- 0.15 Lo/s. In both cases, the relationship between Vmax and the ATP concentration followed the hyperbolic Michaelis-Menten relation. A Km of 0.56 +/- 0.06 mM (mean +/- SD) was found for the fast fibers and of 0.16 +/- 0.03 mM for the slow fibers. Assuming that Vmax is mainly determined by the crossbridge detachment rate, the apparent second order dissociation rate for the actomyosin complex in vivo would be 3.8.10(5) M-1s-1 for the fast fibers and 2.9.10(5) M-1 s-1 for the slow fibers. Maximum power output as a function of the MgATP concentration was derived from the force-velocity relationships. At 5 mM MgATP, the maximum power output in fast fibers was (73 +/- 8) mW.g-1 dry weight and (15 +/- 5) mW.g-1 in slow fibers. The Km for MgATP for the maximum power output for the fast fibers was (0.15 +/- 0.03) mM, which is about a factor of 4 lower than the Km for Vmax. The implications of these results are discussed in terms of a kinetic scheme for crossbridge action.