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.

Titin is a giant elastic protein that is responsible for the majority of passive force generated by the myocardium. Titin's force is derived from its extensible I-band region, which, in the cardiac isoform, comprises three main extensible elements: tandem Ig segments, the PEVK domain, and the N2B unique sequence (N2B-Us). Using atomic force microscopy, we characterized the single molecule force-extension curves of the PEVK and N2B-Us spring elements, which together are responsible for physiological levels of passive force in moderately to highly stretched myocardium. Stretch-release force-extension curves of both the PEVK domain and N2B-Us displayed little hysteresis: the stretch and release data nearly overlapped. The force-extension curves closely followed worm-like chain behavior. Histograms of persistence length (measure of chain bending rigidity) indicated that the single molecule persistence lengths are ϳ1.4 and ϳ0.65 nm for the PEVK domain and N2B-Us, respectively. Using these mechanical characteristics and those determined earlier for the tandem Ig segment (assuming folded Ig domains), we modeled the cardiac titin extensible region in the sarcomere and calculated the extension of the various spring elements and the forces generated by titin, both as a function of sarcomere length. In the physiological sarcomere length range, predicted values and those obtained experimentally were indistinguishable.Titin forms a striated muscle-specific myofilament that develops passive force in response to sarcomere stretch (for a recent review with original citations, see Ref. 1). Titin's force is generated by serially linked and mechanically distinct spring elements (2). Tandem Ig segments (tandemly arranged Ig-like domains) and the PEVK domain (rich in proline, glutamate, valine, and lysine residues) are spring elements found in both cardiac and skeletal muscle titins (3, 4) that vary in length in different isoforms of titin. For example, in human, the PEVK domain varies from 188 residues in the cardiac-specific N2B isoform to 2181 residues in skeletal soleus muscle (2). The cardiac-specific N2B unique sequence (N2B-Us) 1 forms a third spring element in cardiac titins and provides extensibility at the upper range of physiological sarcomere lengths in the heart (5-7). The three-spring system of cardiac titin results in a unique force-extension curve that underlies the majority of the physiological passive tensions of the myocardium, and the variable-length tandem Ig and PEVK elements allows passive tension to be adjusted so that it matches the mechanical demands placed on normal and diseased myocardium (8, 9).Immunoelectron microscopy has shown that in slack sarcomeres (no external force), the tandem Ig segments are in a "contracted" state. When the sarcomeres are stretched, the segments greatly extend (4) due to unbending of linkers between folded Ig domains (4) and possibly to limited domain unfolding (10). The tandem Ig segments exhibit worm-like chain (WLC) behavior with a persistence length (measure of chain bendi...

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Titin/connectin is the main determinant of physiological levels of passive muscle force. This force is generated by the extensible I-band region of the molecule, which is composed of serially-linked immunoglobulin (Ig)-like domains and several unique sequence elements. Here we address the role of titin/connectin in sarcomeres shortened to below the slack length (length attained by an un-activated cell in absence of external forces). Such shortened cells develop so-called restoring forces that re-extend the cells upon relaxation. The experiments that we present are based on a high throughput method with a rapid solution switching system which allows unattached single cardiac myocytes to be activated (resulting in shortening below the slack length) and then to be rapidly relaxed while their maximal re-lengthening velocity is measured at the sarcomere level (dSL/dtmax), with high-resolution imaging techniques. Experiments were carried out on myocytes that express different isoforms of titin/connectin. We measured the relation between dSL/dtmax and the minimal SL during contraction (SLmin) and determined the slope of this relation as a measure of 'restoring stiffness.' We found that the restoring stiffness correlates with the isoform expression profile with myocytes that express high levels of the stiff isoform (N2B) having the highest restoring stiffness. These results support the notion that titin/connectin is a bi-directional spring that develops passive force when stretched above the slack length and restoring force when shortened to below this length. We also discuss in detail the mechanisms that underlie titin/connectin's restoring force development and focus on whether or not unfolding of Ig domains plays a role.

The aim of this work was to analyse the comparative effects of the antibacterial properties of partially purified lectins from the seeds of Artocarpus heterophyllus (jack fruit), Canavalia ensiformis (jack bean), Lens culinaris (lentil) and Pisum sativum (pea) against the bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. The lectins were isolated by partial purification using ammonium sulphate precipitation and dialysis. The antimicrobial activity was studied using agar well diffusion method. The results showed that the Jack fruit lectin had a potent anti-bacterial activity against S. aureus, B. subtilis, E. coli and P. aeruginosa whereas Pea and jack bean lectin were found to be effective bacteriostatic agents which reduced the growth of bacteria and lentil lectin showed the least antibacterial activity. A comparison of the antibacterial activity of phytolectins with conventional antibiotics namely ampicillin and tetracycline was also carried out. Studies revealed that the antibacterial activities of the conventional antibiotics are higher than that of the plant extracts at the same concentration in accordance to literature.DOI: http://dx.doi.org/10.3329/icpj.v2i2.13192 International Current Pharmaceutical Journal 2013, 2(2): 18-22