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Phosphorylation of titin modulates passive stiffness of cardiac muscle in a titin isoform-dependent manner.

Fukuda N, Wu Y, Nair P, Granzier HL - J. Gen. Physiol. (2005)

Bottom Line: Following PKA treatment, passive force was significantly decreased in all muscle types with the effect greatest in RV, lowest in BLA, and intermediate in BLV.PKA was also found to decrease restoring force in skinned ventricular myocytes of the rat that had been shortened to below the slack length.We found that isoprenaline phosphorylated titin and that it reduced diastolic force to a degree similar to that found in skinned RV preparations.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164, USA.

ABSTRACT
We investigated the effect of protein kinase A (PKA) on passive force in skinned cardiac tissues that express different isoforms of titin, i.e., stiff (N2B) and more compliant (N2BA) titins, at different levels. We used rat ventricular (RV), bovine left ventricular (BLV), and bovine left atrial (BLA) muscles (passive force: RV > BLV > BLA, with the ratio of N2B to N2BA titin, approximately 90:10, approximately 40:60, and approximately 10:90%, respectively) and found that N2B and N2BA isoforms can both be phosphorylated by PKA. Under the relaxed condition, sarcomere length was increased and then held constant for 30 min and the peak passive force, stress-relaxation, and steady-state passive force were determined. Following PKA treatment, passive force was significantly decreased in all muscle types with the effect greatest in RV, lowest in BLA, and intermediate in BLV. Fitting the stress-relaxation data to the sum of three exponential decay functions revealed that PKA blunts the magnitude of stress-relaxation and accelerates its time constants. To investigate whether or not PKA-induced decreases in passive force result from possible alteration of titin-thin filament interaction (e.g., via troponin I phosphorylation), we conducted the same experiments using RV preparations that had been treated with gelsolin to extract thin filaments. PKA decreased passive force in gelsolin-treated RV preparations with a magnitude similar to that observed in control preparations. PKA was also found to decrease restoring force in skinned ventricular myocytes of the rat that had been shortened to below the slack length. Finally, we investigated the effect of the beta-adrenergic receptor agonist isoprenaline on diastolic force in intact rat ventricular trabeculae. We found that isoprenaline phosphorylated titin and that it reduced diastolic force to a degree similar to that found in skinned RV preparations. Taken together, these results suggest that during beta-adrenergic stimulation, PKA increases ventricular compliance in a titin isoform-dependent manner.

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Effect of PKA on passive force in various types of skinned cardiac muscle. (A) Typical chart recording showing the effect of PKA on total and titin-based passive force in RV. SL was increased from 1.90 to 2.25 μm as indicated. Black, before PKA; red, after PKA. Titin-based passive force was obtained by subtracting collagen-based passive force from total passive force. Arrows indicate the onset and end of stress-relaxation. (B) Effect of PKA on titin-based passive force in RV, BLV, and BLA. SL was increased from 1.90 to 2.25 μm. The values of maximal Ca2+-activated force before PKA treatment were 51.52 ± 8.53, 26.08 ± 2.56, and 18.42 ± 1.63 mN/mm2 in RV, BLV, and BLA, respectively (at SL 1.90 μm). Data obtained from each preparation (see A for example) were fitted to three exponential decays as described in text, and curves with mean values of exponential parameters are shown (compare Table I). RV, n = 6; BLV, n = 6; BLA, n = 8. Inset, effect of PKA+PKI on titin-based passive force in RV preparations (SL increased from 1.90 to 2.25 μm; n = 6). PKA+PKI does not significantly affect passive force. (C) Summary of stress-relaxation measurements. Percent changes compared with pre-PKA values (offsets, amplitudes, and time constants) are shown. *, P < 0.05 compared with pre-PKA values.
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fig2: Effect of PKA on passive force in various types of skinned cardiac muscle. (A) Typical chart recording showing the effect of PKA on total and titin-based passive force in RV. SL was increased from 1.90 to 2.25 μm as indicated. Black, before PKA; red, after PKA. Titin-based passive force was obtained by subtracting collagen-based passive force from total passive force. Arrows indicate the onset and end of stress-relaxation. (B) Effect of PKA on titin-based passive force in RV, BLV, and BLA. SL was increased from 1.90 to 2.25 μm. The values of maximal Ca2+-activated force before PKA treatment were 51.52 ± 8.53, 26.08 ± 2.56, and 18.42 ± 1.63 mN/mm2 in RV, BLV, and BLA, respectively (at SL 1.90 μm). Data obtained from each preparation (see A for example) were fitted to three exponential decays as described in text, and curves with mean values of exponential parameters are shown (compare Table I). RV, n = 6; BLV, n = 6; BLA, n = 8. Inset, effect of PKA+PKI on titin-based passive force in RV preparations (SL increased from 1.90 to 2.25 μm; n = 6). PKA+PKI does not significantly affect passive force. (C) Summary of stress-relaxation measurements. Percent changes compared with pre-PKA values (offsets, amplitudes, and time constants) are shown. *, P < 0.05 compared with pre-PKA values.

Mentions: Fig. 2 A is a typical chart trace showing stress-relaxation of a skinned RV preparation before and after PKA treatment (SL increased from 1.9 to 2.25 μm). It is clearly seen that passive force, both total and titin-based, is reduced following incubation with PKA. Fig. 2 B summarizes the effect of PKA on titin-based passive force from six to eight preparations for RV, BLV, and BLA (SL increased from 1.9 to 2.25 μm). As reported previously (Cazorla et al., 2000; Wu et al., 2000; Fukuda et al., 2003), because of differential expressions of titin (compare Fig. 1), passive force was highest in RV, intermediate in BLV, and lowest in BLA. PKA significantly decreased passive force in all types of muscle, and the magnitude of force decrease (both peak and steady-state force) was greatest in RV, smallest in BLA, and intermediate in BLV. The PKA-induced decreases in passive force are relatively small in BLV and especially in BLA, but changes are statistically significant. Therefore, PKA decreases passive force in cardiac muscle regardless of the expression profile of titin, and its effect is more pronounced when N2B expression levels are high. PKI inhibited PKA-induced decreases in passive force in RV (Fig. 2 B, inset) and also in BLV and BLA (not depicted).


Phosphorylation of titin modulates passive stiffness of cardiac muscle in a titin isoform-dependent manner.

Fukuda N, Wu Y, Nair P, Granzier HL - J. Gen. Physiol. (2005)

Effect of PKA on passive force in various types of skinned cardiac muscle. (A) Typical chart recording showing the effect of PKA on total and titin-based passive force in RV. SL was increased from 1.90 to 2.25 μm as indicated. Black, before PKA; red, after PKA. Titin-based passive force was obtained by subtracting collagen-based passive force from total passive force. Arrows indicate the onset and end of stress-relaxation. (B) Effect of PKA on titin-based passive force in RV, BLV, and BLA. SL was increased from 1.90 to 2.25 μm. The values of maximal Ca2+-activated force before PKA treatment were 51.52 ± 8.53, 26.08 ± 2.56, and 18.42 ± 1.63 mN/mm2 in RV, BLV, and BLA, respectively (at SL 1.90 μm). Data obtained from each preparation (see A for example) were fitted to three exponential decays as described in text, and curves with mean values of exponential parameters are shown (compare Table I). RV, n = 6; BLV, n = 6; BLA, n = 8. Inset, effect of PKA+PKI on titin-based passive force in RV preparations (SL increased from 1.90 to 2.25 μm; n = 6). PKA+PKI does not significantly affect passive force. (C) Summary of stress-relaxation measurements. Percent changes compared with pre-PKA values (offsets, amplitudes, and time constants) are shown. *, P < 0.05 compared with pre-PKA values.
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fig2: Effect of PKA on passive force in various types of skinned cardiac muscle. (A) Typical chart recording showing the effect of PKA on total and titin-based passive force in RV. SL was increased from 1.90 to 2.25 μm as indicated. Black, before PKA; red, after PKA. Titin-based passive force was obtained by subtracting collagen-based passive force from total passive force. Arrows indicate the onset and end of stress-relaxation. (B) Effect of PKA on titin-based passive force in RV, BLV, and BLA. SL was increased from 1.90 to 2.25 μm. The values of maximal Ca2+-activated force before PKA treatment were 51.52 ± 8.53, 26.08 ± 2.56, and 18.42 ± 1.63 mN/mm2 in RV, BLV, and BLA, respectively (at SL 1.90 μm). Data obtained from each preparation (see A for example) were fitted to three exponential decays as described in text, and curves with mean values of exponential parameters are shown (compare Table I). RV, n = 6; BLV, n = 6; BLA, n = 8. Inset, effect of PKA+PKI on titin-based passive force in RV preparations (SL increased from 1.90 to 2.25 μm; n = 6). PKA+PKI does not significantly affect passive force. (C) Summary of stress-relaxation measurements. Percent changes compared with pre-PKA values (offsets, amplitudes, and time constants) are shown. *, P < 0.05 compared with pre-PKA values.
Mentions: Fig. 2 A is a typical chart trace showing stress-relaxation of a skinned RV preparation before and after PKA treatment (SL increased from 1.9 to 2.25 μm). It is clearly seen that passive force, both total and titin-based, is reduced following incubation with PKA. Fig. 2 B summarizes the effect of PKA on titin-based passive force from six to eight preparations for RV, BLV, and BLA (SL increased from 1.9 to 2.25 μm). As reported previously (Cazorla et al., 2000; Wu et al., 2000; Fukuda et al., 2003), because of differential expressions of titin (compare Fig. 1), passive force was highest in RV, intermediate in BLV, and lowest in BLA. PKA significantly decreased passive force in all types of muscle, and the magnitude of force decrease (both peak and steady-state force) was greatest in RV, smallest in BLA, and intermediate in BLV. The PKA-induced decreases in passive force are relatively small in BLV and especially in BLA, but changes are statistically significant. Therefore, PKA decreases passive force in cardiac muscle regardless of the expression profile of titin, and its effect is more pronounced when N2B expression levels are high. PKI inhibited PKA-induced decreases in passive force in RV (Fig. 2 B, inset) and also in BLV and BLA (not depicted).

Bottom Line: Following PKA treatment, passive force was significantly decreased in all muscle types with the effect greatest in RV, lowest in BLA, and intermediate in BLV.PKA was also found to decrease restoring force in skinned ventricular myocytes of the rat that had been shortened to below the slack length.We found that isoprenaline phosphorylated titin and that it reduced diastolic force to a degree similar to that found in skinned RV preparations.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164, USA.

ABSTRACT
We investigated the effect of protein kinase A (PKA) on passive force in skinned cardiac tissues that express different isoforms of titin, i.e., stiff (N2B) and more compliant (N2BA) titins, at different levels. We used rat ventricular (RV), bovine left ventricular (BLV), and bovine left atrial (BLA) muscles (passive force: RV > BLV > BLA, with the ratio of N2B to N2BA titin, approximately 90:10, approximately 40:60, and approximately 10:90%, respectively) and found that N2B and N2BA isoforms can both be phosphorylated by PKA. Under the relaxed condition, sarcomere length was increased and then held constant for 30 min and the peak passive force, stress-relaxation, and steady-state passive force were determined. Following PKA treatment, passive force was significantly decreased in all muscle types with the effect greatest in RV, lowest in BLA, and intermediate in BLV. Fitting the stress-relaxation data to the sum of three exponential decay functions revealed that PKA blunts the magnitude of stress-relaxation and accelerates its time constants. To investigate whether or not PKA-induced decreases in passive force result from possible alteration of titin-thin filament interaction (e.g., via troponin I phosphorylation), we conducted the same experiments using RV preparations that had been treated with gelsolin to extract thin filaments. PKA decreased passive force in gelsolin-treated RV preparations with a magnitude similar to that observed in control preparations. PKA was also found to decrease restoring force in skinned ventricular myocytes of the rat that had been shortened to below the slack length. Finally, we investigated the effect of the beta-adrenergic receptor agonist isoprenaline on diastolic force in intact rat ventricular trabeculae. We found that isoprenaline phosphorylated titin and that it reduced diastolic force to a degree similar to that found in skinned RV preparations. Taken together, these results suggest that during beta-adrenergic stimulation, PKA increases ventricular compliance in a titin isoform-dependent manner.

Show MeSH
Related in: MedlinePlus