Calcium sensitivity and the Frank–Starling mechanism of the heart are increased in titin N2B region-deficient mice

Abstract

Previous work suggests that titin-based passive tension is a factor in the Frank-Starling mechanism of the heart, by increasing length-dependent activation (LDA) through an increase in calcium sensitivity at long sarcomere length. We tested this hypothesis in a mouse model (N2B KO model) in which titin-based passive tension is elevated as a result of the excision of the N2B element, one of cardiac titin's spring elements. LDA was assessed by measuring the active tension-pCa (−log[Ca 2+ ]) relationship at sarcomere length (SLs) of 1.95, 2.10 and 2.30 µm in WT and N2B KO skinned myocardium. LDA was positively correlated with titin-based passive tension, due to an increase in calcium sensitivity at the longer SLs in the KO. For example, at pCa 6.0 the KO:WT tension ratio was 1.28 ± 0.07 and 1.42 ± 0.04 at SLs of 2.1 and 2.3 µm, respectively. There was no difference in protein expression or phosphorylation of sarcomeric proteins. We also measured the calcium sensitivity after PKA treating the skinned muscle and found that titin-based passive tension was also now correlated with LDA, with a slope that was significantly increased compared to no PKA treatment. Finally, we performed isolated heart experiments and measured the Frank-Starling relation (slope of developed wall stress-LV volume relation) as well as diastolic stiffness (slope of diastolic wall stress -volume relation). The FSM was more pronounced in the N2B KO hearts and the slope of the FSM correlated with diastolic stiffness. These findings support that titin-based passive tension triggers an increase in calcium sensitivity at long sarcomere length, thereby playing an important role in the Frank-Starling mechanism of the heart.

Deletion of the titin N2B region accelerates myofibrillar force development but does not alter relaxation kinetics

“…In our experimental system, we did not detect significant differences in the Ca 2+ sensitivity of force generation between RESEARCH ARTICLE Journal of Cell Science (2014Science ( ) 127, 3666-3674 doi:10.1242 myofibrils from wild-type and knockout mice -unlike the previously published higher Ca 2+ sensitivity (+0.07 pCa) of skinned knockout papillary muscles compared with wild-type papillary muscles (Lee et al, 2013;Lee et al, 2010). Thus, it is unlikely that the faster cross-bridge kinetics of myofibrils from knockout compared with wild-type mice results from an increased Ca 2+ sensitivity or LDA.…”

“…Thus, it is unlikely that the faster cross-bridge kinetics of myofibrils from knockout compared with wild-type mice results from an increased Ca 2+ sensitivity or LDA. The present study reveals an increased maximum force (F max ) of myofibrils from knockout compared with wild-type mice, which had not been detected in studies using skinned myocytes (Radke et al, 2007) or skinned papillary muscle (Lee et al, 2010). The reason for this divergent finding in myofibrils compared with the other preparations of higher structural complexity is not clear.…”

“…The idea of improved cross-bridge attachment at reduced radial spacing is related to the inter-filament spacing hypothesis that has been a widely held hypothesis for explaining LDA (McDonald and Moss, 1995), but which has become controversial (Konhilas et al, 2003). In our study, the activation of myofibrils was initiated at a sarcomere length of 2.3 mm, at which myocardium from knockout relative to wild-type animals exhibits significantly increased LDA (+0.07 pCa) and modestly but significantly reduced myofilament lattice spacing (21.9 nm) (Lee et al, 2013;Lee et al, 2010). However, whereas there is consensus among studies that lattice compression using up to 4% dextran usually slightly increases the maximum force, which might explain the increased F max in our study (Kawai and Schulman, 1985;Konhilas et al, 2002;Lee et al, 2013;McDonald and Moss, 1995;Millman, 1998), there is divergence on whether lattice compression slightly decelerates (Kawai and Schulman, 1985) or slightly accelerates cross-bridge turnover (McDonald and Moss, 1995).…”

“…The knockout mice have been used to test the impact of titin stiffness and titin-based passive tension on length-dependent activation (LDA), i.e. the increase in the Ca 2+ sensitivity of force development due to sarcomere stretch that provides the basis of the Frank-Starling mechanism of the heart (Lee et al, 2013;Lee et al, 2010). These studies revealed that LDA is enhanced in the knockout mice compared with LDA of wild-type mice and that LDA correlates under various conditions with titin-based passive tension.…”

Skeletal muscle tissue in movement and health: positives and negatives

“…Titin's elastic properties have been implicated as a key to this phenomenon (Fukuda and Granzier, 2004). Among the purported roles of titin in cardiac muscle are the following: (1) titin has a key function in stress-sensing signaling (Anderson and Granzier, 2012); (2) titin passive tension triggers an increase in Ca 2+ sensitivity and thus increased contractility (Lee et al, 2010); (3) titin is involved in length-dependent thin filament regulation (Kobirumaki-Shimozawa et al, 2014); and finally (4) titin functions "as a 'bidirectional spring' that … determines not only ventricular rigidity and diastolic function, but also systolic cardiac function" (Castro-Ferreira et al, 2011). In other words, once this 'undiscovered' filament found its place in cardiac muscle, a number of elusive features of cardiac muscle gained clarity.…”