The role of the Ca²⁺ regulatory sites of skeletal troponin C in modulating muscle fibre reactivity to the Ca²⁺ sensitizer bepridil

Abstract

1 The Ca 2+ -sensor protein troponin C (TnC) exerts a key role in the regulation of muscle contraction, and constitutes a target for Ca 2+ sensitizer compounds, such as bepridil, known to increase its apparent Ca 2+ a nity. Moreover, bepridil has been reported to exert a di erential e ect in slow and fast skeletal muscle ®bres, which express the slow/cardiac and fast TnC isoform, respectively. 2 The role of the TnC isoform in establishing the di erential e ect of bepridil was assessed in slow soleus and fast tibialis rat skinned ®bres, by extraction of endogenous TnC and consecutive reconstitution with either slow or fast recombinant TnC. A mutant (VG2), lacking the regulatory site II, was also used to distinguish the role of each regulatory site. 3 Fast tibialis ®bres reconstituted with cardiac TnC exhibited a typical slow bepridil reactivity, while slow soleus ®bres reincorporated with fast TnC displayed a typically fast reactivity to bepridil. These results indicated that the di erential e ect of bepridil in slow and fast ®bres is related to the TnC isoform predominantly expressed in a ®bre. 4 Experiments with the VG2 mutant demonstrated that BPD can achieve an increase in the Ca 2+ a nity in the absence of a functional site II. Thus, site I was necessary for the BPD e ect to be independent of the Ca 2+ concentration. Moreover, the amplitude of the reinforcement in the Ca 2+ a nity, induced by the binding of bepridil to the TnC molecule, is dependent on the number of functional regulatory sites, the larger a nity reinforcement being detected when only one regulatory site (either site I or II) is functional.

O-Linked N-Acetylglucosaminylation Is Involved in the Ca2+ Activation Properties of Rat Skeletal Muscle

“…The level of endogenous RLC phosphorylation was shown to be a crucial determinant of the Ca 2ϩ sensitivity of force development. Moreover, increased phosphorylation of myosin light chains was associated with slow-to-fast transition in rat atrophied soleus (46). These data showed that posttranslational modifications can play an important role even if a large part of the protein is not constitutively modified.…”

“…Some of these proteins, MLC2 and MLC1, corresponded also to phosphoproteins. Indeed, 30% of MLC2 slow and fast isoforms are phosphorylated in soleus and EDL, respectively (46). The level of endogenous RLC phosphorylation was shown to be a crucial determinant of the Ca 2ϩ sensitivity of force development.…”

Structure of the Regulatory N-domain of Human Cardiac Troponin C in Complex with Human Cardiac Troponin I147–163 and Bepridil

“…This observation is reminiscent of our previous work on EMD 57033 and cTnI 128 -147 [147][148][149][150][151][152][153][154][155][156][157][158][159][160][161][162][163] . This is somewhat surprising, because bepridil has been shown to increase both the activity of actomyosin ATPase and force generation in vitro (19,20), and this increase was observed in both slow/cardiac and fast skeletal muscle fibers, with the effect being greater in slow/cardiac fibers (37). However, this is in agreement with a recent study (21) that shows an apparent weaker binding of bepridil to the cTnC regulatory domain in the cTnC⅐cTnI complex (ϳ140 M) than to the isolated cTnC (ϳ10 M), and bepridil is also shown to inhibit calmodulin from binding to its target proteins (38 Table I.…”

Expression and functional properties of four slow skeletal troponin T isoforms in rat muscles

“…The tension (T)-pCa curves were obtained as previously described (16). The steepness of the T-pCa curve was determined by the Hill coefficients n H, either n1 or n2, according to the following equation (4)…”

“…All solutions were described previously (16), and pCa values were calculated with the Fabiato (8) computer program. The calculation was performed with the stability constants listed for Ca 2ϩ (24).…”