Single Molecule Force Spectroscopy of the Cardiac Titin N2B Element

Abstract

The small heat shock protein ␣B-crystallin interacts with N2B-Us, a large unique sequence found in the N2B element of cardiac titin. Using single molecule force spectroscopy, we studied the effect of ␣B-crystallin on the N2B-Us and its flanking Ig-like domains. Ig domains from the proximal tandem Ig segment of titin were also studied. The effect of wild type ␣B-crystallin on the single molecule force-extension curve was determined as well as that of mutant ␣B-crystallins harboring the dilated cardiomyopathy missense mutation, R157H, or the desmin-related myopathy mutation, R120G. Results revealed that wild type ␣B-crystallin decreased the persistence length of the N2B-Us (from ϳ0.7 to ϳ0.2 nm) but did not alter its contour length. ␣B-crystallin also increased the unfolding force of the Ig domains that flank the N2B-Us (by 51 ؎ 3 piconewtons); the rate constant of unfolding at zero force was estimated to be ϳ17-fold lower in the presence of ␣B-crystallin (1.4 ؋ 10 ؊4 s ؊1 versus 2.4 ؋ 10 ؊3 s ؊1 ). We also found that ␣B-crystallin increased the unfolding force of Ig domains from the proximal tandem Ig segment by 28 ؎ 6 piconewtons. The effects of ␣B-crystallin were attenuated by the R157H mutation (but were still significant) and were absent when using the R120G mutant. We conclude that ␣B-crystallin protects titin from damage by lowering the persistence length of the N2B-Us and reducing the Ig domain unfolding probability. Our finding that this effect is either attenuated (R157H) or lost (R120G) in disease causing ␣B-crystallin mutations suggests that the interaction between ␣B-crystallin and titin is important for normal heart function.␣B-crystallin is a member of the small heat shock protein family that by inhibiting denaturation and aggregation of proteins functions as a molecular chaperone (1). Although ␣B-crystallin has been most intensively studied in the vertebrate eye lens, it is also found in many other tissues (2) with cardiac muscle expressing ␣B-crystallin at 3-5% of the total soluble protein (3). Up-regulation of ␣B-crystallin occurs in a number of cardiac disorders, including familial cardiac hypertrophy, and overexpression appears to protect the cardiac cell from ischemia reperfusion injury (for a review see Ref. 4). An important binding partner of ␣B-crystallin in cardiac muscle is titin (5, 6). Titin is a large filamentous protein that forms a continuous filament along the myofibril, with single titin molecules spanning from the edge to the middle of the sarcomere, a distance of ϳ1 m (7). The I-band region of titin is extensible and functions as a molecular spring that, when extended, develops force (8, 9). This force is an important determinant of the passive stiffness of the heart that determines the filling characteristics during the diastolic part of the heart cycle (10). The interaction between ␣B-crystallin and titin could be important for maintaining heart function, especially when stressed, such as during ischemia (5), warranting studies of the effect of ␣B-crystallin on the biomechanica...

α-B Crystallin Reverses High Diastolic Stiffness of Failing Human Cardiomyocytes

“…15 The protective role of α-B crystallin on titin distensibility was also evident from earlier studies in which α-B crystallin lowered the persistence length of the N2B-Us segment and reduced the unfolding probability of the immunoglobulin domains flanking the N2B-Us segment. 16 The relative importance of deranged phosphorylation and structural damage of titin for diastolic stiffness of failing human cardiomyocytes is currently unclear and therefore is also addressed in the present study.…”

“…16,32 Recently, the small heat shock proteins HSP27 and α-B crystallin were shown to protect cardiomyocytes against stretch-induced damage in acidic pH. 15 In these experiments, administration of small heat shock proteins to donor cardiomyocytes corrected the combined effects of prestretch and acidic pH on the diastolic F passive -SL relation.…”

The Giant Protein Titin: A Regulatory Node That Integrates Myocyte Signaling Pathways

“…Missense mutations in ␣B-crystallin were identified in patients with dilated cardiomyopathy or desminrelated myopathy, and some mutations were shown to decrease the binding affinity to N2-Bus (98). Interaction with ␣B-crystallin increased the mechanical stability of titin Ig domains (96) and lowered the persistence length of N2-Bus (99), an effect that was lost with disease-causing ␣B-crystallin mutations (99). Another small heat shock protein, HSP27, binds to titin in heatshocked zebrafish cardiomyocytes, but the exact binding site is unknown (36).…”

Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

“…Although B-crystallin increased the mechanical stability of proximal and distal Ig domains as well as the N2-Bus, according to single-molecule atomic force spectroscopy experiments (Bullard et al, 2004;Zhu et al, 2009), isolated human cardiomyocytes did not become stiffer in the presence of sHSPs. Instead, under conditions promoting titin aggregation (prestretch and acidic pH), the passive tension was abnormally high and B-crystallin/HSP27 suppressed this increase in stiffness.…”