Depressed unloaded sarcomere shortening velocity in acute murine coxsackievirus myocarditis: myocardial remodelling in the absence of necrosis or hypertrophy.

We used right ventricular papillary muscles to study cellular dysfunction in acute murine coxsackievirus myocarditis. We measured unloaded sarcomere shortening velocity (V0) with laser diffraction (HeNe, lambda = 623.8 nm) 7 days after coxsackievirus infection (M) (n = 7) and after infection + monoclonal antibodies to eliminate T cells (T) (n = 4) and in normals (N) (n = 8). A servomotor rapidly shortened a muscle until slack early in contraction and V0 was measured at the onset of zero force. V0 in N was 4.14 +/- 0.84 microns.s-1 at Sl = 2.08 +/- 0.09 microns, 1.70 +/- 0.33 microns.s-1 at 2.06 +/- 0.08 microns in M and, in preliminary experiments, 4.75 +/- 0.96 microns.s-1 at 2.06 +/- 0.07 microns in T. Resting force and stiffness were normal in M. Ventriculor weights in M and T were the same as N. There was an increase in mononuclear cells in M papillary muscles, but no fibrosis or necrosis. Thus, V0 was markedly reduced in acute viral myocarditis in the absence of tissue disruption or hypertrophy, but not if T cells were absent. Pyrophosphate gel electrophoresis showed a shift from predominantly fast in N to a slow myosin isoform in M. Myosin remodelling and reduced unloaded sarcomere shortening velocity occur early in acute coxsackievirus myocarditis and are dependent on immune responses to the virus, but are not a result of histopathological changes.

Reduced unloaded sarcomere shortening velocity and a shift to a slower myosin isoform in acute murine coxsackievirus myocarditis.

We developed a mouse myocardial preparation to study cellular dysfunction in acute coxsackievirus myocarditis. Thin right ventricular papillary muscles from normal mice (n = 8) were compared with muscles from mice 7 days after coxsackievirus infection (n = 7). Sarcomere shortening was studied with laser diffraction (HeNe, lambda = 623.8 nm). A servomotor was used to shorten a muscle until slack early in isometric contraction. Unloaded sarcomere shortening velocity (Vo) was measured at the start of zero force at slack length. Vo was independent of the extent of slack release and was the same as that estimated with an isotonic force-sarcomere shortening velocity relation. Resting muscle stiffness was calculated from shortening perturbations in resting muscles. The histology of some papillary muscles (normal, n = 4; infected, n = 3) was studied. There was no ventricular hypertrophy. Resting sarcomere length (SL) in infected preparations (2.11 +/- 0.07 micron) (mean +/- 1 SD) was the same as in normal preparations (2.11 +/- 0.08 micron). In isometric twitches in normal and infected muscles, total peak force (4.31 +/- 1.07 and 3.77 +/- 1.86 g/mm2, respectively) resting force (0.81 +/- 0.37 and 0.81 +/- 0.35 g/mm2, respectively), and time to peak force (129.5 +/- 20.3 and 125.2 +/- 13.0 milliseconds, respectively) were not significantly different. Vo was 4.14 +/- 0.84 micron/s in normal muscles at an SL of 2.08 +/- 0.09 micron and 1.70 +/- 0.33 micron/s in infected muscles at an SL of 2.06 +/- 0.08 micron. Resting stiffness was the same for normal and infected muscles. There was inflammation but no fibrosis or necrosis. Thus, Vo was depressed early in acute viral myocarditis without hypertrophy, myocyte necrosis, fibrosis, or altered resting stiffness. Pyrophosphate gel electrophoresis showed a shift from predominantly fast to slow myosin isoforms. Apparently, there is remodeling of the contractile apparatus early in acute coxsackievirus myocarditis that is caused either by the direct effects of the virus or the immune response.

Sarcomere shortening velocity in pressure overload hypertrophied rabbit right ventricular myocardium at physiological sarcomere lengths.

Earlier we noted the importance of internal loads for sarcomere shortening in heart muscle. Internal loading may change with hypertrophy and influence sarcomere shortening velocity. Therefore, we force clamped trabeculae from normal rabbit right ventricles and those hypertrophied due to pulmonary artery constriction. Diffraction of a laser (lambda = 632.8 nm) by a trabecula was used to measure sarcomere length (SL) in isotonic twitches. Resting SL was set at 2.36 +/- 0.17 microns in the normal (n = 5) and 2.23 +/- 0.08 microns in the hypertrophied (n = 5) (+/- S.E.M.) muscles. The relationship of total stress with log10 sarcomere shortening velocity was linear. In the normals, log(SL/s) = 0.39-1.32 (P(total)/P(isom)), and in hypertrophy, log(SL/s) = -0.16-0.57 (P(total)/P(isom)). SL/s is sarcomere shortening velocity in microns/s divided by SL at the onset of constant force. P(total) is isotonic plus resting stress and P(isom) is peak total isometric stress. The hypertrophy (P(total)/P(isom))-(SL/s) relationship is depressed below normal at low loads (P < 0.001). Estimated unloaded SL/s in the normals was 2.47 and 0.69 in hypertrophy. Paired stimulation had no effect on normal SL/s. In hypertrophy, paired stimulation increased SL/s at low loads to normal levels. Thus, depressed sarcomere shortening velocity at low loads in hypertrophy at physiological sarcomere lengths disappeared when myoplasmic [Ca2+] increased. The results suggested that internal loading was greater than normal during shortening of hypertrophied myocardium. Increased myoplasmic [Ca2+] increased the number of crossbridges and reduced the load per crossbridge. This was a likely mechanism for the increase of sarcomere shortening velocity to normal levels.

Reduced isotonic sarcomere shortening in rabbit right ventricular pressure overload hypertrophy.

We found previously that sarcomere shortening was reduced in hypertrophied rabbit right ventricular (RV) trabeculae, even when total isometric force and damaged end compliance were the same as normal. Here we studied isotonic shortening of similar preparations force-clamped with a servomotor. A force clamp holds the length of damaged end compliance constant. Sarcomere length (SL) was measured with laser diffraction in twitches with single and optimally paired stimuli. delta SL was sarcomere shortening divided by SL at the onset of shortening. Muscle shortening divided by unloaded muscle length (ML) at the onset of shortening was delta ML. RV hypertrophy was produced with pulmonary artery constriction in 11 rabbits and there were eight normal rabbits. delta SL was smaller than normal in hypertrophy, but delta ML was unchanged from normal. delta SL/delta ML in hypertrophy, 0.90 +/- 0.02, was significantly less than normal, 2.40 +/- 0.07 (mean +/- S.E.M.) (P less than 0.01). delta SL/delta ML did not depend on sarcomere shortening, load, time during shortening or stimulus pattern. Therefore, the reduced delta SL in hypertrophy was independent of contractile state parameters. The ratio was also independent of resting SL (normal = 2.29 +/- 0.07 microns; hypertrophy = 2.23 +/- 0.03 microns; P greater than 0.05) or where diffraction was sampled along central muscle length. One explanation for the findings includes reduced compliance of series viscoelastic elements within the central undamaged region of a hypertrophied muscle. This explanation is consistent with changes from normal in myocardial mechanics and connective tissue in cardiac hypertrophy. Ventricular function remains adequate in hypertrophy without heart failure perhaps because reduced delta SL/delta ML in hypertrophy results in less sarcomere work at any level of muscle work.

Isotonic muscle and sarcomere shortening in rabbit right ventricular preparations.

Cardiac muscle fibers are suspended within and attached to an elaborate connective tissue matrix that includes numerous compliant interconnections. Myocardial muscle fibers are not branched, but connect at small angles to each other to form a branched array. Therefore, fiber shortening occurs as a vector within a connective tissue framework and individual fiber work may exceed external muscle work. To evaluate the latter we measured isotonic muscle shortening simultaneous with sarcomere shortening. The hearts were obtained from rabbits (n = 4) anesthetized with intravenous pentobarbital sodium. We isolated right ventricular trabeculae or free wall papillary muscles in Krebs-Ringer's solution (2.5 mM Ca2+, 28 degrees C). Cross-sectional area was 0.038 +/- 0.003 mm2 (+/- SE throughout) and resting sarcomere length was 2.33 +/- 0.12 microns. Sarcomere length was measured with laser diffraction (He-Ne, lambda = 632.8 nm) during force clamps in single- and paired-stimulation twitches. Relative sarcomere shortening (delta SL) was isotonic sarcomere shortening divided by sarcomere length at the onset of isotonic shortening. Relative muscle shortening (delta ML) was isotonic muscle shortening divided by muscle length at zero load; the latter was estimated from the stress-strain relation of elastic recoil at the onset of load clamps. Average delta SL/delta ML at peak shortening was 3.38 +/- 0.16 and was independent of stimulus pattern, isotonic load, amount of shortening, time during a twitch or laser beam position along a muscle. Therefore, the ratio greater than 1 was neither a function of activation nor heterogeneous sarcomere length change.(ABSTRACT TRUNCATED AT 250 WORDS)

Active force during hypoxia of hypertrophied rabbit right ventricular trabeculae.

Hypertrophy is often accompanied by increased myocardial oxygen demand, but any unique effects of hypoxia on contraction in hypertrophy are unknown. Trabeculae from normal [n = 9; 0.119 +/- 0.014 mm2 (means +/- SE) cross-sectional area] and hypertrophied (pulmonary artery constriction; n = 7; 0.108 +/- 0.028 mm2) rabbit right ventricles were subjected to graded hypoxia (Krebs-Ringer solution, 28 degrees C, 1 Hz stimulus frequency). During normoxia, peak active isometric (Pmax) and resting stress (Prest) at optimum length and peak rate of stress development (dP/dt) in hypertrophy were the same as normal and time to peak stress was longer than normal. Time to peak stress and dP/dt decreased with hypoxia, but time to peak stress remained longer than normal in hypertrophy; Prest was unchanged. The ratio of peak active stress (P) during hypoxia to Pmax decreased linearly with superfusate PO2, but the hypertrophy relationship (y = 4.00 X 10(-3) x + 0.084) is the same as normal (y = 3.70 X 10(-3) x + 0.154; p greater than 0.05). Therefore, a normal level of P was preserved in hypertrophied myocardium and prolonged time to peak stress might have been important for that preservation.

Sarcomere shortening in pressure overload hypertrophy.

Sarcomere shortening during contraction was measured by using laser diffraction, in thin, rabbit right ventricular (RV) trabeculae from normal hearts (N) (n = 5) and from hearts subjected to RV pressure overload by pulmonary banding (H) (n = 5). Banding resulted in substantial RV hypertrophy after 2 wk. Hypertrophied preparations had the same resting muscle length (H = 3.15 +/- 0.29 mm) and resting sarcomere lengths (H = 2.16 +/- 0.005 micron) as the normal preparations (3.10 +/- 0.37 mm, 2.16 +/- 0.008 micron, respectively). Total tension at the peak of isometric twitches was the same as normal in the hypertrophied muscles (N = 8.06 +/- 1.20, H = 8.51 +/- 1.95 g/mm2). However, the amount of auxotonic sarcomere shortening was much less than normal in the hypertrophied preparations (N = 0.39 +/- 0.028, H = 0.19 +/- 0.034 micron; P less than 0.001). In isotonic contractions in which the ratio of muscle shortening to resting muscle length was the same in both the normal and hypertrophied muscles (ratio of 0.05 in both groups), the extent of sarcomere shortening relative to resting sarcomere length was less in the hypertrophied muscles than in the normal preparations (N = 0.14 +/- 0.01), H = 0.07 +/- 0.01; P less than 0.01). Series elasticity was the same as normal in the hypertrophied muscle P less than 0.05). Less auxotonic sarcomere shortening for a given level of isometric tension development and less isotonic sarcomere shortening per unit muscle shortening indicate that there is less than normal work per sarcomere during contraction in hypertrophied myocardium. These findings may have important implications for intracellular compensatory adaptation in pressure overload cardiac hypertrophy.

Increased active elastic stiffness in tetanized papillary muscles from hypertrophied rabbit hearts.

Studies of skeletal muscle suggest that the ratio of stiffness to tension will increase in the presence of a slower rate of crossbridge head rotation from the attached perpendicular state (non-force generating) to the attached 45 degree angle state (force generating). Maximum shortening velocity is depressed proportionate with adenosinetriphosphatase activity in pressure overload cardiac hypertrophy. The maximum rate of isometric force generation also is less than normal but active isometric force levels are normal. The myosin isoenzymes of hypertrophied heart muscle are shifted to predominantly slower than normal types. Among a number of possibilities, the overall rate of crossbridge cycling may be less than normal and crossbridge head rotation may be slower. We reasoned that a greater than normal ratio of active elastic stiffness to total tension development in hypertrophy would be suggestive of an alteration from normal in crossbridge dynamics. We studied right ventricular septal papillary muscles from normal rabbits and from rabbits with hypertrophy induced by pulmonary artery constriction. A high level of mechanical activation was obtained by tetanizing the muscles in solutions containing caffeine. Small (less than or equal to 2% muscle length) and rapid (0.8 ms) length perturbations were applied to the preparations with a servo-controlled motor. Active elastic stiffness was estimated from the linear relationship of minimum (for releases) or maximum (for stretches) tension reached during a length change with muscle length change (strain). Although total tetanic tension development was normal in the hypertrophied muscles (p greater than 0.1), active elastic stiffness was greater than normal in hypertrophy (p less than 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

Myocyte morphology of free wall trabeculae in right ventricular pressure overload hypertrophy in rabbits.

Right ventricular (RV) hypertrophy and changes in mechanical properties develop in response to sustained pulmonary artery construction in rabbits. We use basilar RV free wall trabeculae from rabbits for measurements of force, shortening and sarcomere length (diffraction and/or photomicrography). With enzymes we dispersed calcium tolerant myocytes from trabeculae similar to those used for the above mechanical studies. The average weight of the normal (N) rabbits (n = 16) was 2.21 +/- 0.16(1) kg and was 2.11 +/- 0.10 kg for the rabbits with RV hypertrophy (H; n = 16). The ratio of RV free wall to total ventricular weight was 0.17 +/- 0.01 in the N and 0.31 +/- 0.02 in H hearts (P less than 0.01). Average length and width were determined from digitized measures of the projected image of 42 +/- 3 Ca2+ tolerant myocytes from each N heart and 41 +/- 3 from each H heart. Average myocyte length increased from 102.9 +/- 0.9 in N to 109.8 +/- 1.0 micron in H (6.7% above N; P less than 0.05) and average width from 15.4 +/- 0.2 to 20.0 +/- 0.2 micron (29.9% above N; P less than 0.01). Sarcomere length in these quiescent myocytes was 1.92 +/- 0.003 micron in the N and 1.90 +/- 0.004 in H (P greater than 0.05); consequently, the restoring forces in the myocytes were the same as N in H. The greater addition of parallel myofibrils than of series sarcomeres in H is important for tension generation in the presence of the increased pressure load of pulmonary artery constriction. The addition of sarcomeres in series may be important to sustain muscle shortening in H and is consistent with our measures of sarcomere shortening in N and H trabeculae.

Decreased auxotonic sarcomere shortening in hypertrophied rabbit myocardium.

Auxotonic sarcomere length change occurs during isometric twitches of isolated cardiac muscle preparations. To assess the amount of internal work in hypertrophied myocardium, we measured auxotonic sarcomere length change during isometric tension development over a range of initial muscle and sarcomere lengths. Hypertrophy was produced by banding the pulmonary artery, which resulted in an increase in the ratio of right ventricular free wall weight to total ventricular weight (normal 0.19 +/- 0.004; hypertrophy 0.35 +/- 0.008; P less than 0.001). Right ventricular free wall trabeculae and papillary muscles were studied with optical and mechanical instrumentation, including a helium-neon laser, to measure sarcomere length and isometric twitch parameters. The resting sarcomere length-resting tension relationship was shifted to the left of normal in the hypertrophied preparations (P less than 0.001). The relationship of sarcomere length at the peak of the twitch with total tension at the same instant was shifted downward and to the right of normal in hypertrophy (P less than 0.01). For the same amount of total tension development there was less than normal sarcomere shortening in the hypertrophied preparations (P less than 0.001). Consequently, there is less than normal work per sarcomere during auxotonic sarcomere shortening in hypertrophied heart muscle. Less sarcomere work for a particular functional state is important to consider in the assessment of the basis of myocardial function in compensated pressure overload hypertrophy.

Heart muscle mechanics.

The goal implicit in the research reviewed above is to describe the contractile behavior of heart muscle in terms of crossbridge and filament behavior. It is necessary to elucidate these details in cardiac muscle because of the distinct biochemical differences between skeletal and cardiac myosin. As is evident in this review, significant advances have been made toward describing unique mechanical properties of cardiac muscle crossbridges. Several major problems now require attention: (a) Activation parameters are labile, making mechanical measurements sensitive to measurement perturbation; (b) significant structural inhomogeneities at the cellular and sarcomere level prevent precise assignment of externally measured force to internal structures (force generators, passive elements) within whole cardiac muscle and individual cells; (c) high resting stiffness and forces of poorly understood origin and properties confound attempts to interpret force measurements and dynamics. The differences between heart and skeletal muscle myosin may provide the means for identifying structural counterparts of the Huxley-Simmons model (33); they may also be useful in evaluating the electrostatic and quantum-mechanical models.

The relationship of mechanical Vmax to myosin ATPase activity in rabbit and marmot ventricular muscle.

Papillary muscle mechanics and ventricular myosin calcium-activated ATPase activity were measured in the same heart as a function of temperature (8--28 degrees) in rabbits and marmots, in order to examine further the hypothesis that the velocity of cardiac muscle shortening at zero load (Vmax) is correlated with myosin ATPase activity. There was a similar Q10 for Vmax in each muscle type, as measured with isotonic afterloaded quick-releases at 30--33% time-to-peak tension; the calcium activated ATPase of myosin in the two muscle types also was similar. The least squares linear regression of rabbit Vmax on calcium-activated myosin ATPase activity was the same as in the marmot, so all the data were pooled to yield a linear regression (Y = 0.47 +/- 3.82X) with a high correlation between the two variables [r = 0.95, P less than 0.01 (ANOV)]. Furthermore, the correlation proved to be predictive of cardiac Vmax and myosin ATPase activity levels in other experiments where these two measurements decreased below normal as a result of hypertrophic growth. Consequently, the quantitative relationship between Vmax and myosin ATPase defined here may prove to be predictive of the ability of cardiac muscle to release bond energy.

The mechanical characteristics of hypertrophied rabbit cardiac muscle in the absence of congestive heart failure: the contractile and series elastic elements.

Right ventricular papillary muscles from normal rabbits and rabbits with sustained pulmonary artery constriction (67% decrease in external diameter) were studied at several resting muscle lengths and at an early instant in the isometric twitch. Instantaneous force-velocity data were obtained at 30-38% of time to peak tension (TPT) and at 96%, 98%, and 100% of the resting muscle length at which active twitch tension was maximal. Unloaded shortening velocity (Vmax) was estimated with a linearized form of the Hill hyperbolic formula, and was depressed in hypertrophy to 36% less than normal. We found that Vmax did not change with muscle length in the normal or hypertrophied muscles; therefore there was a length- and time-independent depression of contractile element shortening capacity that was consistent with previous work from this laboratory which demonstrated a depression of myosin and actomyosin ATPase activity in hypertrophy.

A stable, sensitive, low-compliance capacitance force transducer.

The measurement of active and passive force levels in heart muscle requires short-and long-term base-line stability. The capacitance force transducer described here represents an optimization of the relationship between sensitivity, compliance, and frequency response in a design that minimizes long-term base-line drift related to thermal gradients within the apparatus. Thermal stability of the instrument is obtained with the use of quartz and Invar in the construction of the variable capacitor, the maintenance of internal transducer temperature at a constant level well above ambient, and the use of thermally insulating air gaps. Sensitivity ranges from 1.0 to 2.0 V/g wt in the several instruments tested, the output is linear, compliance is negligible with static loads up to 6 g wt, hysteresis is not significant with transient loading with 20 g wt, and long-term drift is greater than or equal to 0.050 g wt. These instruments are designed for use with myocardial preparations but can be adapted for skeletal muscle experiments.