Titin isoform variance and length dependence of activation in skinned bovine cardiac muscle

Abstract

We have explored the role of the giant elastic protein titin in the Frank-Starling mechanism of the heart by measuring the sarcomere length (SL) dependence of activation in skinned cardiac muscles with different titin-based passive stiffness characteristics. We studied muscle from the bovine left ventricle (BLV), which expresses a high level of a stiff titin isoform, and muscle from the bovine left atrium (BLA), which expresses more compliant titin isoforms. Passive tension was also varied in each muscle type by manipulating the pre-history of stretch prior to activation. We found that the SL-dependent increases in Ca2+ sensitivity and maximal Ca2+-activated tension were markedly more pronounced when titin-based passive tension was high. Small-angle X-ray diffraction experiments revealed that the SL dependence of reduction of interfilament lattice spacing is greater in BLV than in BLA and that the lattice spacing is coupled with titin-based passive tension. These results support the notion that titin-based passive tension promotes actomyosin interaction by reducing the lattice spacing. This work indicates that titin may be a factor involved in the Frank-Starling mechanism of the heart by promoting actomyosin interaction in response to stretch.

Figure 1. Changes in passive tension with time following stretch

SL was increased from 1.9 to 2.4 µm and then held constant. Titin-based passive tension is shown in upper-right inset. * P < 0.05 compared with corresponding values for BLA. n = 5 for BLV and BLA. Expression profile of N2B and N2BA titins is also shown in upper left inset (T2, degradation product of titin).

Figure 6. Interfilament lattice spacing in BLV and BLA

A, changes in d_1,0 with SL in BLV and BLA. Lines are linear regression fits for BLV (coninuous line) (y = −9.06x + 62.36 (r² = 0.865, P < 0.0001)) and BLA (dashed line) (y = −4.66x + 51.35 (r² = 0.783, P < 0.0001)). The slope of the linear regression line was significantly (P < 0.001) greater in BLV than in BLA. Eleven preparations were used (BLV and BLA). Inset, X-ray patterns from BLA at SL 2.4 µm showing sharp equatorial reflections. B, linear regression analysis of the relation between titin-based passive tension and d_1,0 when SL was increased from the slack length (1.9-2.0 µm) to ∼2.4 µm. ▵d_1,0, difference in d_1,0 at the slack length and SL ∼2.4 µm. A significant correlation is present (y = 0.33x + 0.19 (r² = 0.572, P < 0.0001)). C, ▵Titin-based passive tension, ▵d_1,0 and ▵pCa₅₀ (same as in Fig. 2A inset) in BLV and BLA. All measurements were made at quasi steady-state passive tension.

Figure 2. SL dependence of Ca²⁺ sensitivity and passive tension

A, coninuous and dashed lines indicate BLV and BLA, respectively. Top, pCa-tension relationships in BLV and BLA at SL 1.9 and 2.4 µm. Inset, ▵pCa₅₀. Bottom, passive tension (total) just prior to activating muscle at each pCa. Note that changes in passive tension throughout the protocol are small. * P < 0.05 compared with corresponding values for BLA at SL 2.4 µm. B, top, SL dependence of Ca²⁺ sensitivity at high passive tension in BLV (shown in blue; black symbols and lines are taken from A (BLV) for comparison). Inset, ▵pCa₅₀ (low passive tension, taken from A (BLV)). Bottom, passive tension (total) just prior to activating muscle at each pCa. ‡ P < 0.05 compared with corresponding values obtained in A for BLV at SL 2.4 µm. C, same as in B for BLA. pCa-tension relationships and passive tension changes are shown in red (black symbols and lines are taken from A (BLA) for comparison). ‡ P < 0.05 compared with corresponding values obtained in A for BLA at SL 2.4 µm.

Figure 3. Effect of titin-based passive tension on SL-dependent activation

Data taken from Fig. 2 and Table 1. Lines are linear regression lines. Titin-based passive tension was obtained as described in Methods. ▵Titin-based passive tension, difference in titin-based passive tension at SL 1.9 and 2.4 µm. Passive tension data in Table 1 were used for analysis. Passive tension measured immediately after stretch is indicated as ‘high’. A, effect of titin-based passive tension on Ca²⁺ sensitivity (y = 0.037x + 0.003 (r² = 0.979, P < 0.01)). B, effect of titin-based passive tension on maximal Ca²⁺-activated tension (y = 2.857x − 0.294 (r² = 0.989, P < 0.005)).

Figure 4. SDS-PAGE analysis showing isoforms of thick and thin filament-based proteins in BLV and BLA

A, 5 % acrylamide gel showing separation of MHC in BLV and BLA. B, 15 % acrylamide gel showing regulatory proteins as well as MLCs in BLV and BLA.

Figure 5. Expression ratio of atrial MLC to ventricular MLC plotted against ▵pCa₅₀ (A and B) and ▵maximal tension (C and D) in BLV preparations

Preparations were obtained from seven different hearts. No correlation exists between parameters.