The effect of Mg2+ on cardiac muscle function: is CaATP the substrate for priming myofibril cross-bridge formation and Ca2+ reuptake by the sarcoplasmic reticulum?

Abstract

Kinetics are established for the activation of the myofibril from the relaxed state [Smith, Dixon, Kirschenlohr, Grace, Metcalfe and Vandenberg (2000) Biochem. J. 346, 393–402]. These require two troponin Ca2+-binding sites, one for each myosin head, to act as a single unit in initial cross-bridge formation. This defines the first, or activating, ATPase reaction, as distinct from the further activity of the enzyme that continues when a cross-bridge to actin is already established. The pairing of myosin heads to act as one unit suggests a possible alternating mechanism for muscle action. A large positive inotropic (contraction-intensifying) effect of loading the Mg2+ chelator citrate, via its acetoxymethyl ester, into the heart has confirmed the competitive inhibition of the Ca2+ activation by Mg2+, previously seen in vitro. In the absence of a recognized second Ca2+-binding site on the myofibril, with appropriate binding properties, the bound ATP is proposed as the second activating Ca2+-binding site. As ATP, free or bound to protein, can bind either Mg2+ or Ca2+, this leads to competitive inhibition by Mg2+. Published physico-chemical studies on skeletal muscle have shown that CaATP is potentially a more effective substrate than MgATP for cross-bridge formation. The above considerations allow calculation of the observed variation of fractional activation by Ca2+ as a function of [Mg2+] and in turn reveal simple Michaelis–Menten kinetics for the activation of the ATPase by sub-millimolar [Mg2+]. Furthermore the ability of bound ATP to bind either cation, and the much better promotion of cross-bridge formation by CaATP binding, give rise to the observed variation of the Hill coefficient for Ca2+ activation with altered [Mg2+]. The inclusion of CaADP within the initiating cross-bridge and replacement by MgADP during the second cycle is consistent with the observed fall in the rate of the myofibril ATPase that occurs after two phosphates are released. The similarity of the kinetics of the cardiac sarcoplasmic reticulum ATPase to those of the myofibril, in particular the positive co-operativity of both Mg2+ inhibition and Ca2+ activation, leads to the conclusion that this ATPase also has an initiation step that utilizes CaATP. The first-order activation by sub-millimolar [Mg2+], similar to that of the myofibril, may be explained by Mg2+ involvement in the phosphate-release step of the ATPase. The inhibition of both the myofibril and sarcoplasmic reticulum Ca2+-transporting ATPases by Mg2+ offers an explanation for the specific requirement for phosphocreatine (PCr) for full activity of both enzymes in situ and its effect on their apparent affinities for ATP. This explanation is based on the slow diffusion of Mg2+ within the myofibril and on the contrast of PCr with both ATP and phosphoenolpyruvate, in that PCr does not bind Mg2+ under physiological conditions, whereas both the other two bind it more tightly than the products of their hydrolysis do. The switch to supply of energy by diffusion of MgATP into the myofibril when depletion of PCr raises [ATP]/[PCr] greatly, e.g. during anoxia, results in a local [Mg2+] increase, which inhibits the ATPase. It is possible that mechanisms similar to those described above occur in skeletal muscle but the Ca2+ co-operativity involved would be masked by the presence of two Ca2+-binding sites on each troponin.

The mechanisms of cardiac myopathies, a kinetics approach: Leading review

“…I stress that all my conclusions regarding Ca 2+ and Mg 2+ in the myo ibril are based entirely on the well respected measurement made on the intact system by Holroyde, Robertson, Johnson, Solaro and Potter, Morimoto and Ohtsuki [3], Donaldson, best and Kerrick [5], and myself et al [2,4] and others in these references. To emphasise the need to only consider the intact system with all the many interactions of its constituents, I quote a more recent source Harris, Rostkova, Gautel and Moss14 who clearly demonstrate that the binding of cMyBP-C to the system has a major effect on the Ca 2+ af inity of the cTnC, This is especially shown by the effect of adding the actin-myosin binding fragment of cMyBP-C (C1mC2) to the cMyBP-C knockout myo ibrils.…”

“…Variation with pMg of the pCa 50 (half activation) and Hill coeffi cient for Ca 2+ activation of isometric force development in skinned cardiac-muscle fi bres, from Smith, et al[4] and original data from Donaldson[5].…”

Myosin heavy chain and cardiac troponin T damage is associated with impaired myofibrillar ATPase activity contributing to sarcomeric dysfunction in Ca2+-paradox rat hearts

“…Chemical energy required for myofilament contraction originates from ATP hydrolysis through myosin ATPase, while ATP binding again to myosin heads is needed for cardiac muscle relaxation cycle-to-cycle [30, 31]. Basal ATP hydrolysis by the Mg 2+ -dependent myosin ATPase activity triggers myosin off to a high-energy form binding P i and ADP.…”

The mechanisms of the Frank-Starling law and familial cardiomyopathy are different. The function of myosin binding protein-C is retained on myocyte length increase and force generated is kinase controlled

“…On the basis of the measured binding affinities [1,2]. I have argued that the inhibition is not via the troponin-C binding of Ca 2+ but via a second transient effector binding site [3,4] dependant on ATP and hence not measurable by 45 Ca 2+ binding and proposed that this is the myosinbound ATP or the myosin bound products MgADP and Pi or transient phosphoprotein. The Myosin binding Protein-C (MyBP-C) makes Ca 2+ -Mg 2+ exchange or a consequent step rate-limiting in cross-bridge cycling and prevents MgATP appearing as the apparent substrate [3].…”

“…It is possible but unlikely that this dimension is the separation of the thick and thin fillaments [10,11]. As there is no change in the Hill coefficient on shift to lower [Ca 2+ ] it is the result of simply raising the effective Ca 2+ affinity of either the troponin-C [1,2,8,[17][18][19] or the myosin bound ATP/ADP [3,4], i.e. increased exchange rate of Ca 2+ for Mg 2+ .…”