Post-translational modifications of myofilament proteins involved in length-dependent prolongation of relaxation in rabbit right ventricular myocardium

Abstract

The phosphorylation state of several cardiac myofilament proteins changes with the level of stretch in intact, twitch-contracting cardiac muscles. It remains unclear which kinases are involved in the length-dependent phosphorylation of these proteins. We set out to investigate which kinases are involved after a step-wise change in cardiac muscle length. We hypothesize that myofilament protein phosphorylation by PKCβII and PKA alters contractile kinetics during length-dependent activation. Right ventricular intact trabeculae were isolated from New Zealand White rabbit hearts and stimulated to contract at 1Hz. Twitch force recordings where taken at taut and optimal muscle lengths before and after administration of kinase inhibitors at 37°C. PKCβII inhibition significantly decreased time from stimulation to peak force (TTP), time from peak force to 50% relaxation (RT50), and 90% relaxation (RT90) at optimal muscle length. This led to a loss in the length-dependent increase of RT50 and RT90 in the presence of the PKCβII inhibitor, whereas the length-dependent increase in RT50 and RT90 was seen in the controls. PKA inhibition using H-89 significantly decreased TTP at both taut and optimal muscle lengths. Detection of Ser/Thr phosphorylation with ProQ-diamond staining indicates a role for PKCβII in the phosphorylation of tropomyosin and myosin light chain-2 (MLC2) and PKA for tropomyosin, troponin-I, MLC2, myosin binding protein-C, troponin-T (TnT) 3 and TnT4. Our data provide evidence for two signaling kinases acting upon myofilament proteins during length-dependent activation, and provide further insight for length-dependent myofilament function.

Fig. 1

Staurosporine decreases developed tension. Developed tension (A), time to peak tension (B), time from peak tension to 50% relaxation (C), and time from peak tension to 90% relaxation (D) before and after application of Staurosporine, a non-specific serine/threonine kinase inhibitor, at taut, intermediate, and optimal muscle lengths. Data collected at 1 Hz, 37 °C, n = 9.

Fig. 2

Non-specific PKC inhibition decreases RT₅₀ and, to an extent, RT₉₀. Developed tension (A), time to peak tension (B), time from peak tension to 50% relaxation (C), and time from peak tension to 90% relaxation (D) before and after application of bisindolylmaleimide VIII acetate salt (Bis), a non-specific PKC inhibitor, at taut and optimal muscle lengths. Data collected at 1 Hz, 37 °C, n = 8.

Fig. 3

PKCβII inhibition decreases contractile and relaxation kinetics. Developed tension (A), time to peak tension (B), time from peak tension to 50% relaxation (C), and time from peak tension to 90% relaxation (D) before and after application of PKCβII peptide inhibitor I trifluoroacetate salt, at taut and optimal muscle lengths. Data collected at 1 Hz, 37 °C, n = 24.

Fig. 4

H89, a non-specific PKA inhibitor, decreases contractile but not relaxation kinetics. Developed tension (A), time to peak tension (B), time from peak tension to 50% relaxation (C), and time from peak tension to 90% relaxation (D) before and after application of H-89, a PKA inhibitor, at taut and optimal muscle lengths. Data collected at 1 Hz, 37 °C, n = 23.

Fig. 5

Representative assessment of myofilament phosphorylation status. Pro-Q and Coomassie Gels demonstrating changes in phosphorylation of various myofilament proteins in intact cardiac trabeculae after or without administration of PKCβII peptide inhibitor I trifluoroacetate salt (Trifluor.) when the muscle is held slack or at optimal muscle length.

Fig. 6

PKCβII inhibition reduces TnI and MLC2 phosphorylation. ProQ results showing phosphorylation status of myofilament proteins troponin-I (TnI) and myosin light chain-2 (MLC2) with or without PKCβII peptide inhibitor I trifluoroacetate salt administration to induce inhibition of PKCβII at a short (muscle slacked) and long (optimal) muscle length.

Fig. 7

PKA inhibition reduces phosphorylation of several myofilament proteins. ProQ results showing phosphorylation status of myofilament proteins myosin binding protein-C (MyBP-C), troponin T-3 (TnT3), troponin T-4 (TnT4), tropomyosin (Tm), troponin-I (TnI), and myosin light chain-2 (MLC2), with or without H89 administration to induce inhibition of PKA at a short (muscle slacked) and long (optimal) muscle length.