Troponin I in the murine myocardium: influence on length-dependent activation and interfilament spacing

Abstract

Cyclic AMP-dependent protein kinase (PKA) targets contractile proteins, troponin-I (TnI) and myosin binding protein C (MyBP-C) in the heart and induces a decrease in myofilament Ca2+ sensitivity. Yet, the effect of sarcomere length (SL) change on Ca2+ sensitivity (length-dependent activation: LDA) following PKA-dependent phosphorylation is not clear. To clarify the role of PKA-dependent phosphorylation of TnI and MyBP-C on LDA in the heart, we examined LDA in skinned myocytes from a non-transgenic (NTG) and a transgenic murine model in which the native cardiac isoform (cTnI) was completely replaced by the slow skeletal isoform of TnI (ssTnI-TG) lacking the phosphorylation sites for PKA, while retaining PKA sites on MyBP-C. In NTG myocytes, PKA treatment decreased Ca2+ sensitivity at each SL, but enhanced the impact of SL change on Ca2+ sensitivity. Despite a greater sensitivity to Ca2+ and a reduction in LDA, neither Ca2+ responsiveness nor LDA was affected by PKA treatment in ssTnI-TG myocytes. To determine whether the above observations could be explained by the lateral separation between thick and thin filaments, as suggested by others, we measured interfilament spacing by X-ray diffraction as a function of SL in skinned cardiac trabeculae in the passive state from both NTG and ssTnI-TG models before and following treatment with PKA. Phosphorylation by PKA increased lattice spacing at every SL in NTG trabeculae. However, the relationship between SL and myofilament lattice spacing in ssTnI-TG was markedly shifted downward to an overall decreased myofilament lattice spacing following PKA treatment. We conclude: (1) PKA-dependent phosphorylation enhances length-dependent activation in NTG hearts; (2) replacement of native TnI with ssTnI increases Ca2+ sensitivity of tension but reduces length-dependent activation; (3) MyBP-C phosphorylation by PKA does not alter calcium responsiveness and induces a decrease in myofilament lattice spacing at all sarcomere lengths and (4) length-dependent activation in the heart cannot be entirely explained by alterations in myofilament lattice spacing.

The continuing evolution of cardiac troponin I biomarker analysis: from protein to proteoform

“…This important cellular mechanism can be modulated by fine tuning myofilament Ca 2+ sensitivity at the molecular level, in part through dynamic post-translational modifications (PTMs) to cTnI. Importantly, these modifications to cTnI and other thin and thick filament proteins collectively serve to regulate myofilament contraction and relaxation [11,12]. The counterweights to physiological cTnI modifications are maladaptive and disease-induced modifications that can adversely affect contractility.…”

Desensitization of Myofilaments to Ca 2+ as a Therapeutic Target for Hypertrophic Cardiomyopathy With Mutations in Thin Filament Proteins

“…It is known that the myofilament Ca 2+ sensitivity changes as a result of phosphorylation of the myofilament proteins, not only in pathological conditions as a result of adaptation or maladaptation, but also during physiological conditions such as exercise 50 . Several in vitro studies have shown that phosphorylation of TnI by PKA can shift the myofilament Ca 2+ sensitivity as much as 0.2 pCa units 46,51 , suggesting that similar changes occur in vivo . Also, we have previously shown that another TG mouse model of HCM with the mutation D175N in Tm, which shows a small relative increase of myofilament sensitivity to Ca 2+ , also demonstrates only a small relative degree of diastolic dysfunction and almost no hypertrophy 52 .…”

“…Moreover, our goal was to desensitize the myofilaments without altering the expression of mutated Tm to make the studies more clinically relevant and as a proof of concept that treatment of HCM with desensitizers should be considered as a potential new treatment. TnI has been recognized as an important regulatory protein for Ca 2+ -mediated thin filament activation 44,45 and furthermore, phosphorylation of TnI at Ser-23/24 reduces the myofilament Ca 2+ sensitivity 46,47 . This makes TnI an excellent potential target for the development of Ca 2+ desensitizers.…”

Cardiac thin filament regulation and the Frank–Starling mechanism

“…Therefore, the series of experimental findings by the de Tombe group [55–57] suggests that the lattice spacing does not solely determine the magnitude of length-dependent activation, and it does not rule out the involvement of the lattice spacing in the regulation of length-dependent activation.…”