Rowans Scientific Publications
Milton, Cambridge, United Kingdom
|New Concepts in the Control of Muscle Contraction|
This is a collection of papers that presents a novel
interpretation of data from the literature to reason logically for an
overlooked mechanism of stimulus-contraction coupling in muscle.
This mechanism is then used to explain aspects of the puzzles
relating to both an important physiological function of the heart,
The Frank–Starling Law, and the basis of a common inherited
disease state, familial hypertrophic cardiomyopathy (FHCM).
“a cross-bridge model of muscle
contraction activated both
This encompasses the established principles of muscle stimulus-contraction coupling but adds the thick-ﬁlament activation that has been mooted often in the literature but never attributed a mechanistic basis.
Gerry A Smith
This series of papers was initiated by the availability of a range of Ca2+ indicators whose degree of saturation changes the nuclear magnetic resonance of the 19F they contain. Having differing dissociation constants and resultant Ca2+ buffering ranges, they have allowed us to extend the measurement of cytosolic [Ca2+] in the intact beating heart. This methodology has overcome the age old bugbear of science where the method used perturbs the target to be measured.
The ﬁrst Paper (I) in this series [previously reported in the Biochemical Journal (2000)] entitled Estimation of systolic and diastolic free intracellular Ca2+ by titration of Ca2+ buffering in the ferret heart. Loading hearts with indicators resulted in the expected variation of buffering of the Ca2+ transient. The measured end-diastolic- and peak-systolic-[Ca2+]i were both positively correlated with indicator dissociation constant (Kd,). These correlations were used to estimate the unperturbed values by extrapolation to Kd = 0 (diastolic-[Ca2+]i) and to Kd = ∞(systolic- [Ca2+]i) because indicators with these Kd values will be fully bound and fully free under all conditions and hence not act as buffers.
The extrapolated values in the intact beating heart are the ﬁrst reported reliable end-diastolic- and peak-systolic [Ca2+]i measurements. With some minor assumptions, knowing these values and those actually observed in the presence of known concentrations of the various indicator-buffers has allowed access to calculation of the relative buffering capacity of the myoﬁbrils in the intact heart. This second Paper (II), Ca2+ buffering in the heart: Ca2+ binding to and activation of cardiac myoﬁbrils, accompanied the previous Paper (I) in the Biochemical Journal (2000). The generally used concentrations of other spectroscopic indicators, e.g. Fura-2, also have a massive effect on the Ca2+ transient. During preparation of this paper it became obvious that the simple model of activation by binding of Ca2+ to troponin-C was not quite sufﬁcient to explain the data. Incorporation of an ATP-dependent additional transient Ca2+-binding, as indicated by the work of Morimoto and Ohtsuki (1994, see Paper (II) reference section) not only improved the modelling ﬁt but also produced Ca2+ and ATP afﬁnities that closely predicted the Ca2+ ATPase activity of myoﬁbrils measured in vitro.
At this stage of the studies the large amount of data (e.g. those from Solaro 1976, see Paper (III) reference section) relating to the inhibitory effects of Mg2+ on the myoﬁbrillar ATPase and related came to the authors’ attention. These data on the effects of the concentration of [Mg2+], along with related effects found in studies of the poorly explained mechanisms of both the Frank–Starling Law and familial cardiac myopathies, have not yet been absorbed into mainstream muscle research. The lack of a known Mg2+ binding of relevant strengths, that is measurable by static methods, most probably underlies the current position.
The third of the published papers in this series (III) is to date the only serious attempt to marry the Mg2+ inhibition with the accepted troponin-C based mechanism of myoﬁbril activation. This work is entitled, The effect of Mg2+ on cardiac muscle function; is CaATP the substrate for priming myoﬁbril cross-bridge formation and Ca2+ re-uptake by the sarcoplasmic reticulum? The earlier work above (II), indicated two Ca2+-binding sites, possibly one for each paired myosin head, to act as a single unit in initial cross-bridge formation. A large positive inotropic (contraction-intensifying) effect of reducing the cytosolic [Mg2+] by introduction of a suitable chelator, citrate, 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 myosin-bound ATP is proposed as the second activating (cross-bridge recruiting) Ca2+-binding site. Published physico-chemical studies on skeletal muscle have shown that CaATP is potentially a more effective substrate than MgATP for cross-bridge formation. Calculations from this model show the Mg2+ effect on Ca2+-dependency and its associated changes of Hill coefﬁcient.
Also explained is the burst ATPase, i.e. the slowing of phosphate release occurring in second and third cycles of ATPase. The speciﬁc requirement for phosphocreatine (PCr) for full ATPase activity in situ and its effect on the apparent afﬁnity of the myoﬁbrils for ATP is simply a result of limited diffusion rates within the myoﬁbrils and the relative Mg2+ -binding afﬁnities of PCr, phosphate and creatine contrasted with those of ATP and phosphoenolpyruvate and their hydrolysis products.
The ﬁrst previously unpublished paper in this series (IV) is entitled, The Frank–Starling Law of the Heart; cross-bridge recruitment and Ca2+ cooperativity. This Paper starts with the insight made by the author and his colleagues into the mechanism of the control of cross-bridge recruitment by the little understood Mg2+ -inhibited Ca2+ binding to the myoﬁbril. This is then used to rationalize the many observations made in the literature relating the effect of changes in the concentrations of reactants and products of the actomyosin ATPase activity on the level of increased contractile strength resulting from stretch of cardiac muscle in diastole. We concluded that stretch in diastole, i.e. where there are no cross-bridges present, results in abrogation of the need for the Mg2+ -inhibited, non-troponin-C related, Ca2+ binding for activation. Direct evidence is cited to demonstrate the reduced Ca2+ binding related to activation and the resultant slowing of the decay of cross-bridges during the relaxation.
The Ca2+ cooperativity observed in contraction is central to the logical arguments used in reaching the conclusion in Paper (IV) and the theoretical base of this is expanded in Paper (V), a purely biochemical kinetics paper; Dual Ca2+ Site Activation of Cardiac Muscle Contraction; Experimental isolation of the Ca2+ controls. Here evidence against long-range cooperativity of both cross-bridges and multiple troponin-C Ca2+ binding is presented. A fundamental biochemical kinetic study is cited to show the most likely origins of the Ca2+ cooperativity in the actomyosin ATPase. Furthermore, studies are cited that demonstrate the independent and reversible removal in turn of both the troponin-C based Ca2+ control (thin-ﬁlament activation) and the Mg2+ -inhibited Ca2+ control (thick-ﬁlament activation). A conclusion is drawn that the Mg2+ -inhibited Ca2+ binding is overriding in limiting cross-bridge recruitment as a result of having a much lower afﬁnity than that of troponin-C. The biochemical relationship of the kinetic model to the Frank–Starling Law (1915, see Paper (IV) reference section) is further strengthened, i.e. the thick-ﬁlament activation is by-passed by diastolic stretch.
The above leads directly to the question posed in the short review Paper (VI); The Frank–Starling Law of the Heart Coupling of stretch to substrate change: A Review. This demonstrates the current state of confusion in the literature regarding the mechanism. Evidence exists both for and against a relationship between the stretch-induced change in separation of the myoﬁlaments and the strength of contraction. However, it is clear that some change in a sarcomeric dimension has to be involved in considerations of the Frank–Starling Law. These questions are resolved in the next Paper, see below.
At this stage the question arises of whether there is a relationship between the new insight into the Ca2+/Mg2+ control of cardiac muscle contraction and the common inherited disorder familial hypertrophic cardiomyopathy (FHCM). This question leads to Paper (VII) in the series; The Frank–Starling Law and Familial Hypertrophic Cardiomyopathy; Some new striking aspects of muscle structures. This disease state (FHCM) is odd in that it results from any one of around 200 mutations spread over a dozen or so genes that encode for the proteins of the sarcomere. The outstanding candidate as the unifying root cause of this syndrome has to be within the dimensions of the sarcomere; nothing else is likely to be commonly affected by so many different changes in so many different proteins. The relationship of both Ca2+ sensitivity and cooperativity changes brought about by FHCM and diastolic stretch add strength to the sarcomeric dimension ideas. Commonly cited electron micrograph data, biochemical composition data, X-ray structural studies and differences between skeletal and cardiac muscle lead to the inevitable conclusion that the previously used model of muscle structure included a major error.
It is concluded that for mammalian muscle there are two types of thin ﬁlament associated with each thick ﬁlament, one attached to the Z-band and C-region, the other being shorter and only strongly associated with the C-region. The differences between muscle types rest, along with isomeric protein forms, e.g. nebulin in skeletal and nebulette in cardiac, largely in the relative lengths of these two types of thin ﬁlament. In cardiac muscle the thin ﬁlaments that are not attached to the Z-band are, along with the nebulette, too short to span the full length of the cross bridge forming C-region through the i-region to the Z-band. Therein lies the ﬁxed resting length peculiar to the cardiac sarcomere and hence the effect of stretch. We suggest that this critical dimension is also at the root of FHCM.
The above studies allow the construction of a biochemical kinetic model to test further possible predictions. This is presented in Paper (VIII) Biochemical Kinetic Calculations of Dual Ca2+ Site Activation of Cardiac Muscle Contraction The mechanisms of effectors. In this paper a biochemical kinetic model is constructed (reﬁned from that previously given by the author). A new approach is used to correlate ATPase activity to rigidity and the resultant force of contraction. Generally accepted literature kinetic constants and afﬁnities are used to establish the control situation. Application of the known [Ca2+] transient predicts a contractile strength that is approximately 20+% of maximum, in agreement with the increases inducible by stretch or drug treatment.
Which kinetic constants are altered by the given perturbations are shown along with expected and found changes in sensitivity to Ca2+ and the cooperativity involved. For example, the phosphorylation of troponin-I by adrenergic stimulation has, as observed, a reverse effect to that expected, the myoﬁbril becomes less sensitive to Ca2+ concentration with reduced cooperativity, but also results in the necessary faster relaxation. A major ﬁnding is that increases in contractile strength induced by Levosimendan are a result of promotion of thick ﬁlament (CaATP) activation of cross-bridge recruitment that is manifest as a small reduction in static 45Ca2+ binding to the myoﬁlaments, contrary to belief that the drug’s action is a result of increased Ca2+-troponin-C binding cooperativity.
The work presented promotes novel approaches to the understanding and treatment of many heart conditions.