Troponin I phosphorylation enhances crossbridge kinetics during β‐adrenergic stimulation in rat cardiac tissue

Abstract

Inotropic agents that increase the intracellular levels of cAMP have been shown to increase crossbridge turnover kinetics in intact rat ventricular muscle, as measured by the parameter f min (the frequency at which dynamic stiffness is minimum). These agents are also known to increase the level of phosphorylation of two candidate myofibrillar proteins: myosin binding protein C (MyBPC) and Troponin I (TnI), but have no effect on myosin light chain 2 phosphorylation (MyLC2). The aim of this study was to investigate whether the phosphorylation of TnI and/or MyBPC was responsible for the increase in crossbridge cycling kinetics (as captured by f min ) seen with the elevation of cAMP within cardiac tissue. Using barium-activated intact rat papillary muscle, we investigated the actions of isobutylmethylxanthine (IBMX), an inhibitor of cAMP-dependent phosphatase, which simulates the action of b-adrenergic agents, and the chemical phosphatase 2,3-butanedione monoxime (BDM), which has been shown to dephosphorylate a number of contractile proteins. The presence of 0.6 mM IBMX approximately doubled the f min value of intact rat papillary muscle. This action was unaffected by the addition of BDM. In the presence of IBMX and BDM, the level of phosphorylation of MyBPC was unchanged, that of MyLC2 was reduced to 60 % of control, yet that of TnI was markedly increased (to 30 % above control levels). We conclude that TnI phosphorylation, mediated by cAMP-dependent protein kinase A, is the molecular basis for the enhanced crossbridge cycling seen during b-adrenergic stimulation of the heart.

Impact of site-specific phosphorylation of protein kinase A sites Ser23and Ser24of cardiac troponin I in human cardiomyocytes

“…In accordance with our findings, it has been demonstrated that PKA treatment did not affect crossbridge cycling in skinned trabeculae (8,17) and cardiomyocytes (15) from rat hearts and in human myofibrils (44). Other studies (11,20,36) in rodents showed that PKA-mediated phosphorylation of cTnI did increase cross-bridge kinetics. More recently, it has been shown that PKA-mediated phosphorylation of cMyBP-C increases cross-bridge kinetics in transgenic mice, independent of cTnI phosphorylation (7,33).…”

“…The role of phosphorylation of cTnI at the PKA sites as a regulator of cross-bridge cycling is less clear. Some studies (11,20,36) have reported an increase in cross-bridge kinetics via phosphorylation of cTnI. However, others (7,33) have attributed an increase in cross-bridge kinetics to phosphorylation of cMyBP-C independent of cTnI phosphorylation, whereas several studies (8,15,17,44) did not find an effect of PKA on cross-bridge kinetics at all.…”

Hypertrophic cardiomyopathy-related β-myosin mutations cause highly variable calcium sensitivity with functional imbalances among individual muscle cells

“…10). On the other hand, treatment of the muscle fibers with the chemical phosphatase BDM during the skinning procedure, at least in heart muscle, has been reported to dephosphorylate myofibrillar proteins to a large extent (32). Thus an effect of variable phosphorylation of myofibrillar proteins on the force-pCa relations cannot be ruled out completely but appears unlikely.…”

“…1A. The effect of PP1␣ treatment was small, indicating that the skinning procedure of the fibers in the presence of BDM [which acts as a chemical phosphatase (32)] may already have dephosphorylated the fibers to a large extent. Figure 1B shows the individual force-pCa relationships of fibers after treatment with PP1␣ from three control individuals and illustrates the distribution of the pCa 50 values and the steepness of the curves.…”

Interpreting cardiac muscle force-length dynamics using a novel functional model

“…For example, results from steady-state sinusoidal forcings or from approximation of the steady state with continuous changing frequency (13,26) or from frequency analysis of responses to pseudorandom binary sequence perturbations (13,25,30) can be presented in model-independent format as complex stiffness frequency spectra. However, interpretations of such results must eventually rely on some understanding of the system, i.e., a model.…”