Titin, a giant sarcomeric protein, is the primary determinant of passive stiffness in the myocardium. Two major classes of titin isoform co-exist in the myocardium, a long N2BA and a shorter N2B isoform. Although changes in myofilament passive tension have been attributed to disease-related alterations in titin isoform expression, the relative stiffness of native molecules isolated from healthy and failing hearts has not been directly quantified. In this study we characterised titin isoform composition and mechanical strength in healthy and diseased myocardium and related these molecular changes to the mechanical function of myofilaments within isolated myocytes. Heart failure was induced in adult male ferrets by ascending aortic coarctation under anaesthesia (2% isoflurane in oxygen). Perioperative analgesia was provided with meloxicam (0.3mg/kg). Titin isoforms were separated by SDS PAGE and isoform ratios determined by densitometry. Titin molecules were isolated from the left ventricle, aligned and stretched by a receding liquid meniscus (which applied a tensile force of ~60pN)1 prior to visualisation by atomic force microscopy. Passive force of skinned left ventricular myocytes was measured by stretching the cell from 1.8 to 2.2 μm. Isometric force was measured at various calcium concentrations at 15°C and sarcomere length of 2.2 μm. Data are presented as mean ± SEM and statistical significance determined using a students t-test. The mean ratio of the major titin isoforms N2BA:N2B increased from 0.3 in control to 0.5 in failing hearts (n=6, p<0.01). Whilst most combed titin molecules exhibited a straightened and beaded appearance, the mean molecular diameter of titin decreased in failing hearts compared to control (0.26 ± 0.001nm vs 0.33 ± 0.001nm, p<0.001, n=104-130 molecules, 3 animals per group). Furthermore, the mean distance between beads was increased in failing hearts (49.3 ± 1.5nm vs 126.8 ± 4.5nm, p<0.001, n=370-429, 3 animals per group). Myofilament function in permeabilised failing myocytes was compared with sham control cells (n=6) and revealed a significant reduction in passive tension at each sarcomere length (p<0.05), a decrease in maximal force (35.7 ± 5.7 KN/m2 vs 58.8 ± 6.3 KN/m2 p<0.05) and an increase in calcium sensitivity (pCa50 5.72 ± 0.01 vs 5.67 ± 0.01, p<0.05). These observations demonstrate that shifts in titin isoform expression occur alongside decreased titin tensile strength in a model of heart failure and furthermore that these molecular effects are correlated with profound changes in myocyte passive tension.