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 microm 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 microm, respectively. There was no difference in protein expression or total 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.

Figure 1. Characterization of the N2B KO mouse model

(A) Titin expression in LV myocardium of WT and N2B KO mice (1% agarose gels). WT myocardium of the mouse expresses predominately N2B titin with a small level of N2BA titin. In the KO both N2B titin and N2BA titin have a slightly higher mobility than in the WT, consistent with the excision of the N2B element. (B) Titin-based passive tension in WT and N2B KO skinned myocardium (results from 8 WT and 8 KO mice). (Tension is steady-state tension and was measured after 5 min stress relaxation.) Asterisks: comparison between KO and WT myocardium.

Figure 2. Expression level and phosphorylation status of thin- and thick- filament proteins

A) Left top: Representative Western blots (WBs) of cMyBP-C, cTnT, cTnI, α-Tm, and MLC-2 in WT and KO left ventricular (LV) myocardium. Left bottom: MHC gels loaded with WT and N2B KO LV myocardial proteins and bovine left ventricular (BLV) proteins. Right: Pro-Q diamond (Pro-Q) stained 4–20% gradient gels. B) Left: expression analysis; right: phosphorylation analysis. Protein expression levels (n=6 per genotype) and phosphorylation levels (n=8 per genotype) in KO are not significantly different from WT.

Figure 3. Force–pCa relations in WT and N2B KO skinned myocardium

(A) Explanation of experimental protocol. The preparation was stretched, held for 9 min and then released. During the hold phase, the muscle was first in relaxing solution (pCa ~9.0), followed by pre-activating solution (Pre-A) and pCa 6.05, 5.85, 5.75, 5.6, 4.5 activating solutions, and finally relaxing solution again. Passive tension was measured just prior to activation and active tension in each activating solution was measured from the steady-state tension (arrows) minus passive tension. Active tensions were normalized by the maximal active tension at pCa 4.5. (B and C) Average tension-pCa curves and pCa₅₀ (inset) of WT (B) and KO (C) at SL 1.95, 2.1, and 2.3 µm. In both genotypes, increasing sarcomere length left-shifts the tension-pCa curves and increases pCa₅₀ values. Results from 8 WT and 8 KO mice. Asterisks: comparison between different sarcomere lengths in each genotype.

Figure 4. Tension increase in KO at submaximal calcium levels

Active tensions of KO myocardium are expressed relative to those of WT. At all pCa’s (except pCa 5.5) tensions are significantly higher in KO than in WT muscles. Results from 8 WT and 8 KO mice. KO/WT ratio is significantly greater than 1 at SL2.1um (asterisks) and SL2.3um (number sign).

Figure 5. Length dependence of activation in WT and N2B KO skinned myocardium

(A) Average tension-pCa curves of WT (open symbols) and KO (closed symbols) at SL 1.95 and 2.3µm. Inset, ΔpCa₅₀ values (asterisks: comparison between WT and KO). (B) ΔTitin-based passive tension is significantly correlated with LDA (ΔpCa₅₀ from SL1.95 to 2.3µm) in WT (open symbols) and KO (closed symbols) myocardium. Dashed line is the linear regression fit (p<0.002). Results from 8 WT and 8 KO mice

Figure 6. Length dependence of activation in WT and N2B KO skinned myocardium before and after PKA treatment

ΔTitin-based passive tension is significantly correlated with LDA (ΔpCa₅₀ from SL1.95 to 2.3µm) in WT (open symbols) and KO (closed symbols) myocardium. Dashed line is the linear regression fit (p<0.002).

Figure 7. Assessment of FSM in isolated hearts

(A) Left: Schematic of isolated heart setup. The heart is perfused, twitch activated, and a small fluid-filled balloon, introduced into the LV and connected to a fast servomotor, rapidly changes LV volume; a pressure sensor inserted in the balloon measure LV pressure. (B) Superimposed family of set of contractions before and after (test contraction) volume change. Pressure was converted to wall stress (σ) and developed stress (σ_dev) and diastolic stress (σ_d) were determined. (C) Example of results of control (open symbols) and KO heart (closed symbols) obtained in the presence of dobutamine. Note that the developed stress (σ_dev) – volume relation is steeper in the KO than in the WT heart. (D) Developed stiffness (slope of σ - volume relation) is positively correlated with diastolic stiffness (p<0.01). Individual results from WT and KO hearts are shown. Inset: comparison of mean values (asterisks: comparison between WT and KO). Results from 6 WT and 6 KO mice