We investigated the effect of protein kinase A (PKA) on passive force in skinned cardiac tissues that express different isoforms of titin, i.e., stiff (N2B) and more compliant (N2BA) titins, at different levels. We used rat ventricular (RV), bovine left ventricular (BLV), and bovine left atrial (BLA) muscles (passive force: RV > BLV > BLA, with the ratio of N2B to N2BA titin, ∼90:10, ∼40:60, and ∼10:90%, respectively) and found that N2B and N2BA isoforms can both be phosphorylated by PKA. Under the relaxed condition, sarcomere length was increased and then held constant for 30 min and the peak passive force, stress-relaxation, and steady-state passive force were determined. Following PKA treatment, passive force was significantly decreased in all muscle types with the effect greatest in RV, lowest in BLA, and intermediate in BLV. Fitting the stress-relaxation data to the sum of three exponential decay functions revealed that PKA blunts the magnitude of stress-relaxation and accelerates its time constants. To investigate whether or not PKA-induced decreases in passive force result from possible alteration of titin–thin filament interaction (e.g., via troponin I phosphorylation), we conducted the same experiments using RV preparations that had been treated with gelsolin to extract thin filaments. PKA decreased passive force in gelsolin-treated RV preparations with a magnitude similar to that observed in control preparations. PKA was also found to decrease restoring force in skinned ventricular myocytes of the rat that had been shortened to below the slack length. Finally, we investigated the effect of the β-adrenergic receptor agonist isoprenaline on diastolic force in intact rat ventricular trabeculae. We found that isoprenaline phosphorylated titin and that it reduced diastolic force to a degree similar to that found in skinned RV preparations. Taken together, these results suggest that during β-adrenergic stimulation, PKA increases ventricular compliance in a titin isoform-dependent manner.

We have explored the role of the giant elastic protein titin in the Frank‐Starling mechanism of the heart by measuring the sarcomere length (SL) dependence of activation in skinned cardiac muscles with different titin‐based passive stiffness characteristics. We studied muscle from the bovine left ventricle (BLV), which expresses a high level of a stiff titin isoform, and muscle from the bovine left atrium (BLA), which expresses more compliant titin isoforms. Passive tension was also varied in each muscle type by manipulating the pre‐history of stretch prior to activation. We found that the SL‐dependent increases in Ca2+ sensitivity and maximal Ca2+‐activated tension were markedly more pronounced when titin‐based passive tension was high. Small‐angle X‐ray diffraction experiments revealed that the SL dependence of reduction of interfilament lattice spacing is greater in BLV than in BLA and that the lattice spacing is coupled with titin‐based passive tension. These results support the notion that titin‐based passive tension promotes actomyosin interaction by reducing the lattice spacing. This work indicates that titin may be a factor involved in the Frank‐Starling mechanism of the heart by promoting actomyosin interaction in response to stretch.

No abstract

Abstract-The effect of MgADP on the sarcomere length (SL) dependence of tension generation was investigated using skinned rat ventricular trabeculae. Increasing SL from 1.9 to 2.3 m decreased the muscle width by Ϸ11% and shifted the midpoint of the pCa-tension relationship (pCa 50 ) leftward by about 0.2 pCa units. MgADP (0.1, 1, and 5 mmol/L) augmented maximal and submaximal Ca 2ϩ -activated tension and concomitantly diminished the SL-dependent shift of pCa 50 in a concentration-dependent manner. In contrast, pimobendan, a Ca 2ϩ sensitizer, which promotes Ca 2ϩ binding to troponin C (TnC), exhibited no effect on the SL-dependent shift of pCa 50 , suggesting that TnC does not participate in the modulation of SL-dependent tension generation by MgADP. At a SL of 1.9 m, osmotic compression, produced by 5% wt/vol dextran (molecular weight Ϸ464 000), reduced the muscle width by Ϸ13% and shifted pCa 50 leftward to a similar degree as that observed when increasing SL to 2.3 m. This favors the idea that a decrease in the interfilament lattice spacing is the primary mechanism for SL-dependent tension generation. MgADP (5 mmol/L) markedly attenuated the dextran-induced shift of pCa 50 , and the degree of attenuation was similar to that observed in a study of varying SL. The actomyosin-ADP complex (AM.ADP) induced by exogenous MgADP has been reported to cooperatively promote myosin attachment to the thin filament. We hereby conclude that the increase in the number of force-generating crossbridges on a decrease in the lattice spacing is masked by the cooperative effect of AM.ADP, resulting in depressed SL-dependent tension generation. The full text of this article is available at http://www.circresaha.org. This intrinsic ability of the heart to alter cardiac output forms the basis for the Frank-Starling law of the heart. It is well established that twitch tension and Ca 2ϩ responsiveness in cardiac muscle preparations are enhanced as muscle length (ie, sarcomere length [SL]) is increased within the normal physiological range (SL from Ϸ1.8 to Ϸ2.3 m). 1-5 Although a number of studies have been conducted to account for the SL dependence of tension generation in living myocardium, its mechanism has not been completely elucidated. 6 However, at the myofilament level, there is an increasing amount of evidence suggesting that the SL dependence is primarily due to a change in the interfilament lattice spacing that accompanies the SL change. 7-9 A possible consequence of the decreased lattice spacing is an increase in the probability of myosin attachment to the thin filament, resulting in an increase in the number of force-generating crossbridges. 7,10,11 Ishiwata and Oosawa 12 proposed a model based on the Ca 2ϩ -dependent flexibility of the thin filament, in which they assumed that (1) the muscle volume (ie, the lattice volume) remains constant on a change in SL and that (2) there is a critical distance between the thick and thin filaments for tension generation. This model quantitatively explains both the stretch-induced increas...

We investigated the molecular mechanism by which troponin (Tn) regulates the Frank-Starling mechanism of the heart. Quasi-complete reconstitution of thin filaments with rabbit fast skeletal Tn (sTn) attenuated length-dependent activation in skinned porcine left ventricular muscle, to a magnitude similar to that observed in rabbit fast skeletal muscle. The rate of force redevelopment increased upon sTn reconstitution at submaximal levels, coupled with an increase in Ca2+ sensitivity of force, suggesting the acceleration of cross-bridge formation and, accordingly, a reduction in the fraction of resting cross-bridges that can potentially produce additional active force. An increase in titin-based passive force, induced by manipulating the prehistory of stretch, enhanced length-dependent activation, in both control and sTn-reconstituted muscles. Furthermore, reconstitution of rabbit fast skeletal muscle with porcine left ventricular Tn enhanced length-dependent activation, accompanied by a decrease in Ca2+ sensitivity of force. These findings demonstrate that Tn plays an important role in the Frank-Starling mechanism of the heart via on–off switching of the thin filament state, in concert with titin-based regulation.

¡Vive la différence! In cardiac contraction, the reduction in sarcomere length—rather than length itself—determines contractile force.

Background-At the basis of the Frank-Starling mechanism is the intrinsic ability of cardiac muscle to produce active tension in response to stretch. Titin, a giant filamentous molecule involved in passive tension development, is intimately associated with the thick filament in the sarcomere. Titin may therefore contribute to active tension development by modulating the thick filament structure when the muscle is elongated. Methods and Results-Rat skinned right ventricular trabeculae were used. Passive tension at a sarcomere length (SL) of 2.0 to 2.4 m was decreased after treatment of the preparation with trypsin (0.25 g/mL) for 13 minutes in the relaxed state at 20°C. This mild trypsin treatment degraded titin without affecting other major contractile proteins. The sarcomere structure was little affected by brief contractions in the trypsin-treated preparations. When SL was adjusted to the slack SL (1.9 m), active tension was unaffected by trypsin under partial (pCa 5.55) and maximal (pCa 4.8) activation. At longer SLs, however, active tension was significantly (PϽ0.01) decreased after trypsin treatment at either pCa. The increase in active tension on reduction of interfilament lattice spacing, produced by dextran T-500 (molecular weight Ϸ500 000), was not influenced by trypsin (SL 1.9 m). In trypsin-treated preparations, the increase in active tension as a function of muscle diameter was nearly the same for lengthening and osmotic compression at the slack SL. Conclusions-The length-dependent activation in cardiac muscle, an underlying mechanism of the Frank-Starling law of the heart, is at the myofilament level, predominantly modulated by titin and interfilament lattice spacing changes.

In our continuous work on the enhancement of the antibacterial activity of beta-lactam antibiotics against the cells of methicillin-resistant Staphylococcus aureus (MRSA) strains by Keggin-structural polyoxotungstates and their lacunary species, Wells-Dawson, double-Keggin, and Keggin-sandwich polyoxotungstates are also found to be synergistic but highly cytotoxic. The coexistence of polylysine or protamine sulphate decreased the synergistic potency of the polyoxotungstates, due to their electrostatic interaction with negatively charged polyoxotungstates. Inductively coupled plasma atomic emission spectrometry (ICP) analysis of the polyoxotungstate-treated cells indicated that the polyoxotungstates uptaken in the cell are preferentially located at the membrane fraction with intact composition. The polyoxotungstates depressed not only the production of PBP2', but also the production of beta-lactamase which hydrolyzes beta-lactam antibiotics on the membrane. This leads to the synergistic effect of polyoxotungstates against the MRSA cells in the coexistence of beta-lactam antibiotics which have high affinities to PBPs 1-4. MRSA cells which were modified to be susceptible to beta-lactam antibiotics during incubation in the presence of polyoxotungstates recovered their resistance to beta-lactam antibiotics when they were subcultured in the absence of the polyoxotungstate.