Abstract 1581: Extension of Titin as a Function of Filling Pressure and Sarcomere Length in the Porcine Left Ventricle
The giant sarcomeric protein titin is a molecular spring that is the chief source of cardiomyocyte passive tension and a major determinant of myocardial stiffness. The spring portion is located in the I band and consists of PEVK, Ig repeat and N2B and N2A elements. Titin occurs as two isoforms. N2B is a smaller and stiffer isoform that contains only the N2B element and predominates in the left ventricle (LV) of rodents. N2BA titin contains both N2A and N2B elements. N2B and N2BA are co-expressed in the sarcomere of large mammals (~60:40 ratio). As a result, passive cardiomyocyte and myocardial stiffness in large mammals is less than in rodents. Details of titin extension as a function of sarcomere length (SL) have been elucidated in rodents but not in large mammals, where the presence of both isoforms would be expected to modify extension and passive tension. Accordingly, we studied titin extension in miniswine. We first established the relation between filling pressure and SL in the anterior LV wall of the in situ, freshly arrested (KCl) heart. SL was determined over a range of filling pressures using a light microscopic method that minimizes shrinkage. At equilibrium volume (transmural pressure 0 mmHg), SL was between 2.00–2.10 μm, longer than slack length of ~1.85 μm in muscle strips. SL reached a maximum of ~2.50 βm when the LV was over-distended (filling pressure >40 mmHg). We then examined extension of titin in myocardial strips using electron microscopy and immuno-labeling of selected epitopes. The chief difference between isoforms was that the N2B-Us epitope segment in N2B titin lengthened ~four times more than the N2B-Us segment in N2BA titin over SLs from ~1.80 to ~2.50 μm. This difference remained large over the SL range present in the in situ LV. Linear fits of the measured end-to-end length of N2B-Us segments were used to estimate the force-SL relation of single N2B and N2BA molecules. This analysis predicted a much steeper relation for N2B titin. Thus, over the range of SLs present in the in situ LV the most prominent difference in extension of N2B and N2BA titin is greater lengthening of the N2B segment of N2B titin. This predicts a much greater in situ stiffness for N2B titin and demonstrates how passive stiffness can be exquisitely controlled by varying isoform expression.