No abstract

SUMMARY. To investigate the extent to which the properties of the cardiac myofibrils contribute to the length-force relation of cardiac muscle, we determined the sarcomere length-force relations for rat ventricular trabeculae both before and after the muscles were skinned with the detergent Triton X-100 Sarcomere length was measured continuously by laser diffraction. In the unskinned trabeculae stimulated at 0.2 Hz, the relation between active force and sarcomere length at an extracellular calcium concentration of 1.5 HIM was curved away from the sarcomere length axis, with zero force at sarcomere length of 1.5-1.6 firm. At 0 3 rriM calcium, the sarcomere length-force relation was curved toward the sarcomere length axis Chemical skinning of the muscle with 1% Triton X-100 in a "relaxing solution" caused an increase in intensity and decrease in dispersion of the first order diffraction beam, indicating an increased uniformity of sarcomere length in the relaxed muscle During calcium-regulated contractures in the skinned muscles, the central sarcomeres shortened by up to 20%. As the calcium concentration was increased over the range 1-50 /IM, the relation between steady calcium-regulated force and sarcomere length shifted to higher force values and changed in shape in a manner similar to that observed for changes in extracellular calcium concentration before skinning. The sarcomere length-force relations for the intact muscles at an extracellular calcium concentration of 1 5 ITIM were similar to the curves at calcium concentration of 8 9 ^M in the skinned preparations, whereas the curves at an extracellular calcium concentration of 0 3 ITIM in intact muscles fell between the relations at calcium concentrations of 2.7 and 4.3 ^M in the skinned preparations A factor contributing to the shape of the curves in the skinned muscle at submaximal calcium concentrations was that the calcium sensitivity of force production increased with increasing sarcomere length. The calcium concentration required for 50% activation decreased from 7 71 ± 0.52 /IM to 3.77 ± 0.33 MM for an increase of sarcomere length from 1 75 to 2 15 fim. The slope of the force-calcium concentration relation increased from 2.82 to 4.54 with sarcomere length between 1.75 and 2.15 ^m. This change in calcium sensitivity was seen over the entire range of sarcomere lengths corresponding to the ascending limb of the cardiac length-force relation. It is concluded that the properties of the cardiac contractile machinery (including the length-dependence of calcium sensitivity) can account for much of the shape of the ascending limb in intact cardiac muscle (Circ Res 58: 755-768, 1986)

Abstract-Phosphorylation of cardiac myofibrils by cAMP-dependent protein kinase (PKA) can increase the intrinsic rate of myofibrillar relaxation, which may contribute to the shortening of the cardiac twitch during ␤-adrenoceptor stimulation. However, it is not known whether the acceleration of myofibrillar relaxation is due to phosphorylation of troponin I (TnI) or of myosin binding protein-C (MyBP-C). To distinguish between these possibilities, we used transgenic mice that overexpress the nonphosphorylatable, slow skeletal isoform of TnI in the myocardium and do not express the normal, phosphorylatable cardiac TnI. The intrinsic rate of relaxation of myofibrils from wild-type and transgenic mice was measured using flash photolysis of diazo-2 to rapidly decrease the [Ca 2ϩ ] within skinned muscles from the mouse ventricles. Incubation with PKA nearly doubled the intrinsic rate of myofibrillar relaxation in muscles from wild-type mice (relaxation half-time fell from Ϸ150 to Ϸ90 ms at 22°C) but had no effect on the relaxation rate of muscles from the transgenic mice. In parallel studies with intact muscles, we assessed crossbridge kinetics indirectly by determining f min (the frequency for minimum dynamic stiffness) during tetanic contractions. Stimulation of ␤-adrenoceptors with isoproterenol increased f min from 1.9 to 3.1 Hz in muscles from wild-type mice but had no effect on f min in muscles from transgenic mice. We conclude that the acceleration of myofibrillar relaxation rate by PKA is due to phosphorylation of TnI, rather than MyBP-C, and that this may be due, at least in part, to faster crossbridge cycle kinetics. . This increase of relaxation rate is important for proper pump function, because it allows adequate time for diastolic filling of the ventricles despite the raised heart rate during sympathetic stimulation. The activation of ␤ 1 -adrenoceptors stimulates the cAMP/protein kinase A (PKA) pathway, and the faster relaxation of the myocardial cells is partly due to an enhanced reuptake of Ca 2ϩ into the sarcoplasmic reticulum (SR) as a result of phosphorylation of phospholamban by PKA. 1 In addition, PKA phosphorylates the cardiac myofibrils during ␤-stimulation. [2][3][4] This may lead to an acceleration of the intrinsic rate of myofibrillar relaxation, thereby contributing to the abbreviation of the twitch. Using flash photolysis of the caged chelator of Ca 2ϩ , diazo-2, to rapidly decrease Ca 2ϩ concentration inside skinned fibers, Zhang et al 5 reported that PKA accelerated relaxation in pig skinned muscles. However, a later study by Johns et al 6 found no effect in similar experiments using guinea pig skinned muscles. Recent work with intact mouse muscles has suggested that ␤-stimulation can produce an SR-independent, presumably myofibril-mediated, acceleration of relaxation that is seen in isometric but not isotonic contractions. 4 Assuming that phosphorylation does increase the relaxation rate of cardiac myofibrils, how might this be produced? It is known that phosphorylation of troponin I (...

SUMMARY1. Thin ventricular trabeculae from rat hearts were skinned with the non-ionic detergent Triton X-100. The isometric force development of these muscles was investigated over a range of Ca2+ concentrations (0-2-200 gM) in the presence of various concentrations of creatine phosphate (CP), creatine and inorganic phosphate (Pi). Creatine (0-30 mM) was without effect on the skinned muscles.5. The inhibitory effects of Pi suggest that a net hydrolysis of CP to Pi and creatine in the myoplasm of intact cardiac cells could reduce force development at a given myoplasmic [Ca2+], especially if the latter was below the level needed to fully activate the myofibrils. This suggestion is discussed in relation to the CP hydrolysis and decrease in force development that are observed in cardiac muscle during hypoxia or ischaemia.

Changes in cytosolic [Ca2+] ([Ca2+]i) were measured in isolated rat trabeculae that had been micro‐injected with fura‐2 salt, in order to investigate the mechanism by which twitch force changes following an alteration of muscle length. A step increase in length of the muscle produced a rapid potentiation of twitch force but not of the Ca2+ transient. The rapid rise of force was unaffected by inhibiting the sarcoplasmic reticulum (SR) with ryanodine and cyclopiazonic acid. The force‐[Ca2+]i relationship of the myofibrils in situ, determined from twitches and tetanic contractions in SR‐inhibited muscles, showed that the rapid rise of force was due primarily to an increase in myofibrillar Ca2+ sensitivity, with a contribution from an increase in the maximum force production of the myofibrils. After stretch of the muscle there was a further, slow increase of twitch force which was due entirely to a slow increase of the Ca2+ transient, since there was no change in the myofibrillar force‐[Ca2+]i relationship. SR inhibition slowed down, but did not alter the magnitude of, the slow force response. During the slow rise of force there was no slow increase of diastolic [Ca2+]i, whether or not the SR was inhibited. The same was true in unstimulated muscles. We conclude that the rapid increase in twitch force after muscle stretch is due to the length‐ dependent properties of the myofibrils. The slow force increase is not explained by length dependence of the myofibrils or the SR, or by a rise in diastolic [Ca2+]i. Evidence from tetani suggests the slow force responses result from increased Ca2+ loading of the cell during the action potential.