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

The normal adult heart is a well maintained machine that has a mechanism for growth replacement of the sarcomere that is lost by natural degeneration. This process ensures the heart has the strength of contraction to function correctly giving blood supply to the whole body. Some of the force of contraction of the sarcomere is transmitted to its major protein titin where its strength results in unfolding of a lexible section and release of a growth stimulant. The origin of all the cardiomyopathies can be traced to errors in this system resulting from mutations in a wide variety of the sarcomeric proteins. Too much or chronic tension transfer to titin giving increased growth resulting in hypertrophic cardiomyopathy (HCM) and too little leading to muscle wastage, dilated cardiomyopathy (DCM). HCM can ultimately lead to sudden cardiac death and DCM to heart failure. In this paper I show (1) a collection of the tension/ATPase calcium dependencies of cardiac myo ibrils that de ine the mechanism of Ca2+ cooperativity. (2) I then reintroduce the stress/ strain relationship to cardiomyopathies. (3) I then review the cardiomyopathy literature that contains similar Ca2+ dependency data to throw light on the mechanisms involved in generation of the types of myopathies from the mutations involved. In the review of cardiomyopathy there are two sections on mutations, the irst dealing with those disrupting the Ca2+ cooperativity, i.e. the Hill coef icient of activation, leading to incomplete relaxation in diastole, chronic tension, and increased growth. Secondly dealing with those where the Ca2+ cooperativity is not affected giving either increased or decreased tension transfer to titin and changes in sarcomere growth.


Introduction
The normal adult heart is a well maintained machine that has a mechanism for growth replacement of the sarcomere that is lost by natural degeneration. This process ensures the heart has the strength of contraction to function correctly giving blood supply to the whole body. Some of the force of contraction of the sarcomere is transmitted to its major protein titin where its strength results in unfolding of a lexible section and release of a growth stimulant. The origin of all the cardiomyopathies can be traced to errors in this system resulting from mutations in a wide variety of the sarcomeric proteins. Too much or chronic tension transfer to titin giving increased growth resulting in hypertrophic cardiomyopathy (HCM) and too little leading to muscle wastage, dilated cardiomyopathy (DCM). HCM can ultimately lead to sudden cardiac death and DCM to heart failure. In this paper I show (1) a collection of the tension/ATPase calcium dependencies of cardiac myo ibrils that de ine the mechanism of Ca 2+ cooperativity. (2) I then reintroduce the stress/ strain relationship to cardiomyopathies. (3) I then review the cardiomyopathy literature that contains similar Ca 2+ dependency data to throw light on the mechanisms involved in generation of the types of myopathies from the mutations involved. In the review of cardiomyopathy there are two sections on mutations, the irst dealing with those disrupting the Ca 2+ cooperativity, i.e. the Hill coef icient of activation, leading to incomplete relaxation in diastole, chronic tension, and increased growth. Secondly dealing with those where the Ca 2+ cooperativity is not affected giving either increased or decreased tension transfer to titin and changes in sarcomere growth.
These considerations have con irmed that the inhibitory function of cardiac troponin-I (cTnI) is part of a concerted process with the regulatory cardiac myosin binding protein-C (cMyBP-C) to block the use of myosin light chain (MLC) bound magnesium adenosine triphosphate (MgATP) from being used as cross-bridge substrate until the magnesium bound is exchanged for calcium. This Ca 2+ dependency added to the trigger troponin-C (cTnC) Ca 2+ binding is the origin of the Ca 2+ cooperativity ( Figure 1).

The Ca 2+ activation of the myofi brils is cooperative
The conclusion is that two Ca 2+ binding sites are occupied cooperatively for activation of the cross bridge ATPase. The ATP usage is unimolecular.
The measurable binding of Ca 2+ to the myo ibrils is unimolecular, not cooperative ( Figure 2).
On cardiac cTnC there are two high af inity and two low af inity Ca 2+ binding sites but only a total of three bind, i.e. only one for the weaker, activating sites. The strong binding sites are fully occupied under all physiological conditions. Mg 2+ binding to any cTnC site is too weak to have the effect seen later under Magnesium inhibition. The conclusion is under physiological conditions the measurable binding of Ca 2+ to the myo ibrils is unimolecular, not cooperative. This is fully con irmed by Morimoto and Ohsuki [3]. Only one of the low active sites is used on activation.
The conclusion is that activation is by Ca 2+ bound to cTnC cooperatively with Ca 2+ bound to the ATP on the myosin light chain in equilibrium with inactive myosin bound MgATP. Mg 2+ does not competitively bind to cTnC. This ensures that the initial binding of MgATP in diastole gives the completely relaxed myo ibril maintaining the status quo of the stressgrowth equilibrium. Ca 2+ is replaced by Mg 2+ on cleavage of the pyrophosphate bond in ATP. MgATP bound to myosin light chain is derived from creatine kinase rephosphorylation of the myosin bound MgADP product of the cross-bridge ATPase. Creatine kinase is piggy-back bound to cardiac myosin binding protein-C (cMyBP-C) on the thick ilament.
(a) The reversible extraction of cMyBP-C.
The displacement is to the left, lower [Ca 2+ ] approaching the K m for cTnC Ca 2+ binding ( Table 2). The Ca 2+ cooperativity is lost, lower Hill coe icient (n H ), CaATP bound is not required. This demonstrates that MgATP can act as substrate when cMyBP-C is not bound to myosin-actin.
The process of removal is reversible (Figure 4).
The conclusion is that cMyBP-C (along with cTnI see later) has the biochemical function of ensuring myosin bound MgATP is not the functional substrate of the cross-bridge ATPase in the normal heart. This ensures that the system is fully relaxed in diastole on completion of the cycle with binding of MgATP. cMyBP-C clearly has little structural function from the reversibility of its removal, save for its localisation of the creatine kinase.
(b) The addition of an N-terminal fragment of cMyBP-C that binds the myosin S2 site and actin.  : Tension-pCa data obtained before and after partial extraction of cMyBP-C (C-protein) from rat ventricular myocytes, from Hofmann, et al. [6], see cMyBP-C-nul later. Kampourakis, et al. [7] demonstrated the result of occupation of the free cMyBP-C binding sites (S2) on myosin by cMyBP-C myosin binding fragment C1mC2, see later. The free S2 binding sites outnumber those bound by a factor of at least 5. This N-terminal fragment of cMyBP-C contains the binding sites for myosin and actin binding. On addition this fragment binds to the free myosin and actin sites overriding the effect of bound cMyBP-C [7]. At the concentration of C1mC2 used (2 μM) it almost certainly does not displace the bound cMyBP-C, as its K m for maximal Ca 2+ free activation is more than 20 μM. This ability to fully activate in the absence of Ca 2+ is the irst indication that the action of cMyBP-C also involves the cTnI, i.e. is concerted ( Figure 5).
The displacement is to the left, lower [Ca 2+ ], and the cooperativity is lost. Only Ca 2+ bound to cTnC is required for activation, therefore MgATP can act as substrate when C1mC2 is bound to myosin-actin. cMyBP-C bound to Myosin and/or the presence of free Myosin S2 sites are required to maintain Ca 2+ cooperativity.

The stress/strain relationship to cardiomyopathies
I have previously suggested [8] that any mutation giving rise to the above loss of Ca 2+ cooperativity would mean that true diastole would not be reached and the resulting chronic strain would be transmitted to the sensor in titin, releasing LIM protein and hence promoting growth. The result of increased growth is hypertrophic cardiomyopathy (HCM) [8]. Any reduction in transmission of tension to the titin would result in not maintaining the status quo of growth v. tissue loss, less Lim activity reduced maintenance growth and dilated cardiomyopathy (DCM). When mediated through change in the cMyBP-C resulting in HCM the shift in sensitivity is to the left (lower Ca 2+ ) and the opposite for DCM.
Sarcomeric mutations giving rise to a reduction in the Ca 2+ cooperativity of activation giving rise to HCM.
Disruption of either the cTnI or the cMyBP-C functions results in the myosin bound MgATP being used as substrate, with loss of calcium cooperativity and a shift to increased Ca 2+ sensitivity. In general the result of this is incomplete relaxation at the end of the cross-bridge cycle when MgATP is rebound and immediately used when some cTnC at diastolic calcium level ([Ca 2+ ] D ) is still Ca 2+ bound, causing chronic tension in the myo ibril. This tension is transmitted through the troponinactin-titin system, with release of growth activators (LIM protein) and resulting hypertrophic growth of the myocytes.
HCM has been recently reviewed by Teekakirikul, [9]. Along with DCM it is one of the most common heritable cardiovascular disorders. HCM is characterized by left ventricular hypertrophy (LVH) that is unexplained by abnormal loading conditions, with myocyte hypertrophy and disarray, and increased myocardial ibrosis as key histopathological hallmarks. Genetic studies reveal the multiple variants in sarcomere protein genes in approximately 40% -60% of patients with HCM, establishing HCM as a disease of sarcomere proteins, similarly for DCM. Most HCM disease-causing variants occur in the myosin, cMyBP-C and cTnI. HCM variants do also occur, along with other myopathy causing mutations, in the thin ilament proteins.
I now give examples of instances of HCM where the kinetics have been graphically reported. Most of these show a small consistent loss of Ca 2+ cooperativity re lected in reduced Hill coef icient (n H ) for Ca 2+ activation, accompanied by increased Ca 2+ sensitivity.
Along with Myosin mutations these are the main sources of HCM and here the effect of cMyBP-C knockout is included.
The mutant cMyBP-C showed a shift to lower [Ca 2+ ] and reduced cooperativity. Expression of cMyBP-C was also reduced by 30% (Figure 7).
Here they have also shown that for this mutation of cMyBP-C  there is almost complete absence of cTnI phosphorylation compared to normal. This appears to be accompanied with a decrease in phosphoryl cMyBP-C, more than the protein level decrease, and an increase in that of desmin [10]. This is the second strong link in the activities of cMyBP-C and cTnI.
The KO Y235S myocardium shows a left-ward shift in the pCa curve, indicating increased calcium sensitivity and reduced Ca 2+ cooperativity. Unlike the frameshift mutants above there is no accompanying reduction in cMyBP-C expression or phosphorylation of it or the cTnI.
Parbhudayal, et al. [12] have also made the irst study to demonstrate an intercellular variation of myo ilament cMyBP-C protein expression within the myocardium from HCM patients with heterozygous cMyBP-C mutations.
Some of the observations on the genetic elimination of the cMyBP-C are contrary to those on the extraction of cMyBP-C [6] or binding of C1C2 [7] fragment. These studies used gene targeting to produce knockout mice that lack cMyBP-C in heart. The results show that cMyBP-C is not essential for sarcomere assembly or cardiac development but that the absence of cMyBP-C is suf icient to trigger profound cardiac hypertrophy and depressed myocyte contractile properties. Clearly in the absence of the substrate control by cMyBP-C one would expect both an increase in Ca 2+ sensitivity and loss of cooperativity. However, the inding that Ca 2+ sensitivity of tension was reduced in cMyBP-C-nul mice contrasts with enhanced Ca 2+ sensitivity reported after extraction of ∼60% of cMyBP-C from rat myocytes using biochemical techniques. Potential explanation to account for the different results have been suggested. These are that the biochemical extractions occurred in vitro over short time periods, thus precluding adaptive responses, whereas compensatory effects (eg, protein phosphorylations or structural changes during hypertrophy) after genetic elimination of cMyBP-C may be additional factors in the present study. However a better explanation is in binding of the cMyBP-C to the actin-myosin, see effect of N-terminal fragments later.
The irst study of cMyBP-C-nul By Harris, et al ( Figure 9).
Here the anomalies found were a less than expected reduction in the Ca 2+ cooperativity and much less than expected Ca 2+ sensitivity, cf ref [6,7].
In a follow up Harris, et al. found [14] a more expected effect of the cMyBP-C knockout on the Ca 2+ cooperativity, cf Hofmann [6]. But retained the lower sensitivity. In this study they surprisingly looked at the effects of adding binding fragments a la Kampourakis, et al. [7] (Figures 10-12).
Of note is the change of pCa 50 with lack of reduction in Hill coef icient on addition of C1C2 fragment to the wild-type in this later study [14] in contrast to that of others, Kampourakis, [7]. Figure 5 and Razumova [15], Figure 13. The shift to greater   Ca 2+ sensitivity on addition of C1C2 in the absence of cMyBP-C con irms the effects of C1C2 fragment are by direct binding to unoccupied sites on the myosin (S2) and/or the actin, as suspected from the concentration used by Kampourakis. et al. [7] and the concentration they required for Ca 2+ free activation. The dose dependency of C1C2 compared to that of C0C2 or C0C1m on the cMyBP-C KO would be interesting in this regard. The lack of shift to lower Ca 2+ by knockout indicates a weaker af inity of the cTnC for Ca 2+ in the absence of cMyBP-C, i.e. cMyBP-C binding to the actin-myosin increases the af inity of cTnC for Ca 2+ . The large leftward shift to greater Ca 2+ sensitivity on addition of C1C2 fragment in the KO case does con irm this. Only partial removal of cMyBP-C, Hofmann, [6], leaves some cTnC with the higher af inity.

Limitations on use of cMyBP-C fragments.
For convenience I include here the mapping of the N-terminal fragments of cMyBP-C (Figures 11,12).
Incubation of trabeculae with 10 μM C1C2 resulted in a leftward shift of the tension-pCa relationship relative to control, indicating an increase in Ca 2+ sensitivity of tension (ΔpCa 50 = 0.30 ± 0.05). This con irms the incorrectness of the observation of Harris, et al. [14] ( Table 3).
The addition of 20 μM C1C2(-m) that lacks the cMyBP-C motif was without effect on the tension-pCa relationship.
Note the use of only 5 μM of the N-terminal domain peptides. K m for C1C2 Ca 2+ free activation is 20 μM [7], when m is phosphorylated C1C2 becomes inactive. Kampourakis, et al. [7] only used 2 μM so minimising Ca 2+ free stimulation. In experiments with cMyBP-C nulls Raumova, et al. [16] use 10 μM. The 20 μM K m for maximum activation without Ca 2+ probably re lects displacement of bound cMyBP-C, 2 μM only binds at free Myosin S2 and or actin sites. Hence the large reduction of Hill-coe icient on addition.
The much later observations of Harris, et al. [17] have been ignored as.
They used 100 μM of the fragments and in doing so even C1-m was found to act alone. (Figure 14).

Mutation in myosin light chain resulting in HCM
The shift to lower [Ca 2+ ] and loss of cooperativity was accompanied by considerable loss of contraction strength, however this does bypass the cMyBP-C substrate control and the linkage is not loppy as with DCM mutations in light chain, see later.
The observed results of this mutation were; 1) the complex containing HCTnIR145G only inhibited    PKA treatment shifts both curves to the right, less Ca 2+ sensitive without change in Hill coef icients.
RCM is when the walls of the lower chambers of the ventricles are too rigid to expand as they ill with blood. The pumping ability of the ventricles may be normal, but it's harder for the ventricles to get enough blood. With time, the heart can't pump properly. This leads to heart failure. With RCM mutants the loss of cooperativity and shift to greater Ca 2+ sensitivity are the largest of those reported for HCM and more re lect those found when cMyBP-C is nulli ied by addition of its S2 binding moiety or partially removed. There is considerable activation and lack of relaxation at low [Ca 2+ ] D [20,21]. There is no evidence of amyloidosis giving rigidity in this case, as shown by Du, et al. [22], however increases in desmin and α-actinin have been demonstrated in diastolic failure by Zhang, [23] and Sheng, et al. [24]. The latter may very well be the basis of the increase in rigidity found in RCM.
With the above large shift to increased Ca 2+ sensitivity the suggestion is that cTnI is involved with the binding of cMyBP-C to the actin-myosin, the third strong indication of this.

A mutation cTnC giving unexpectedly DCM although loss of Ca 2+ cooperativity.
There is one instance of mutated cTnC where the use of MgATP as substrate will occur and should lead to hypertrophy but this is over-ridden by breakdown in stress transfer to the thin ilament causing dilated myopathy ( Figure 18). I conclude here that the Ca 2+ sensitivity increase is entirely down to a structural change i.e. cTnI not being G159C-cTnC   bound [27] and thus not cooperating with the cMyBP-C in blocking the use of MgATP as substrate. It is also likely that G159C-cTnC does not properly bind other thin ilament proteins, actin, cTnT or tropomyosin. Similar results were found by Swindle, et al. [30].

Sarcomeric mutations that do not show loss of Hill coeffi cient, Ca 2+ cooperativity, giving rise to either HCM or DCM
These mutations are mostly in the Troponin-Ttropomyosin-titin genes although those in myosin are reported. The mutations either increase or decrease the transfer of stress to the titin and its bound Lim protein, giving rise to HCM or DCM. The Ca 2+ cooperativity is maintained, i.e. both the cTnC and myosin bound ATP are Ca 2+ bound with these mutations. The central roles of Troponin-T (cTnT) and tropomyosin (∝-Tm) in the thin ilament transfer of stress is well demonstrated by the presence of assorted variants with either hypertrophic or dilated outcome. The HCM mutants of ∝-tropomyosin (∝-Tm) all display elevated Ca 2+ free ATPase (or tension), a form of inhibited diastolic relaxation, different to loss of Ca 2+ cooperativity, again giving chronic stress in the titin. In general the various mutants giving DCM show reduced maximum ATPase/tension, little or no shift in either the pCa 50 or Hill coef icient.

Mutations in the Myosin Heavy Chain
For the myosin heavy chain variants there are very little data on the variation of ATPase or contractility with [Ca 2+ ] although there are a very large number of mutations known. All available are HCM and show increased maximum contractility at high [Ca 2+ ]. The earliest found was Keller, et al. [31] who showed that mutant myosin R403W exhibited a large increase in maximal actin-activated ATPase activity (+114%; p < 0.05) and Km for actin (+87%; p < 0.05) when compared to WT. Kraft, et al. [32] found increase in contractile strength for R723G in cardiomyocytes. Spudich, et al. [33] studied the mutations R21C, S166F (small n H increase) and DK177 all showed increase maximal ATPase. Nag, et al. [34] for HCM R403Q Show a lowering of contractile strength. It lacks the S2 fragment (cMyBP-C binding domain) and thus must surely behave like cMyBP-C-nul. Sarkar, et al. [35] con irm the R403Q conclusions. These all increase tension transfer to titin.

Mutation in myosin light chain giving DCM (Figure 19)
This light-chain mutation was seen to impair the binding of the regulatory light chain (RLC) to the myosin heavy chain (MHC). Less binding to MHC, more loppy less strain is transferred to the thin ilament.

Tropomyosin -Tm) mutations giving HCM
Michele, et al. [37] report the functional effects of cardiacspeci ic expression of human E180G mutant ∝-Tm in transgenic mice (Figure 20). E180G ∝-Tm mice had signi icantly slowed relaxation under physiological conditions. This dysfunction was eliminated by propranolol. In a follow up by Michele, et al. [38] they use adenoviral-mediated gene transfer of four point mutations in α-tropomyosin (∝-Tm) into adult cardiac myocytes in vitro to show that all four HCM α-Tm proteins can be expressed and incorporated into normal sarcomeric structures in cardiac myocytes at similar levels as normal α-Tm proteins.
Muthhuchamy, [39] report the functional effects of cardiacspeci ic expression of HCM human α-TM 175 TG in transgenic mice ( Figure 21).   Increased expression of the α-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous α-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of relaxation in hearts of the HCM mice. In this case the stress was elevated in diastole. Chang, et al. [40] report similar results with other HCM-associated ∝-Tm mutations ( Figure 22).
All three HCM-associated ∝-Tm mutations increased the Ca 2+ sensitivity of ATPase activity. All three had signi icantly decreased abilities to inhibit ATPase activity at effectively zero [Ca 2+ ]. This is clearly the same as with the α-TM 175 transgenic above [39]. In all these the [Ca 2+ ] D chronic stress is transferred to the titin.
Statistical analysis of activities at pCa 9.1 and 5.0 shows no signi icant differences; Both mutations caused a small but signi icant decrease in the pCa 50 values, and had no effect on the inhibition of ATPase activity. from Chang, et al. [40]. Somehow less tension in titin, need to look for altered interaction with cTnT etc.

Various reports of other HCM and DCM Tm mutants
Gupte, et al. [41] showed DCM mutants D84N and D230N shift the pCa 50 stimulated ATPase activity to the right relative to WT and HCM mutants shift the pCa 50 to the left. The changes in pCa, n H and ATPase max for the DCM cases are small. The changes in the curves for the HCM mutants are again similar to above ∝-Tm reports and there is residual activity at diastolic [Ca 2+ ] D . Similar DCM results are reported by Rajan, et al. [42] for a-TM54 TG hearts with more signi icant loss in contraction force (Table 4) (Figure 24).

Actin mutant giving DCM
There is no cooperativity change and a small movement to lower [Ca 2+ ]. There was no signi icant difference in sliding speed or fraction of ilaments motile. When troponin was dephosphorylated the Ca 2+ -sensitivity of E361G-containing thin ilaments was now much lower than NTG, this was due to uncoupling of Ca 2+ -sensitivity from cTnI phosphorylation.

cTnT mutations giving HCM
Hernandez, et al. [44] report both ATPase and force measurements for skinned papillary muscle ibers of F110I-cTnT and R278C-TnT transgenic mice. In both F110I-TnT and R278C-TnT the maximum force was greatly reduced from WT but the ATPase not affected. In this case it is clear that the force instead of transmitting to the Z disc was absorbed by the titin spring, and thus releasing increased LIM protein.  Venkatraman, et al. [47] report similar results for other DCM mutants.
It appears that the transfer of stress to the titin is opposite in the two cases and when both are included together they nullify each other. In this case there are small changes in Hill coef icient, origin of which is not clear.
Total disagreement with Cooperativity of WT reported by Holroyde [1] and Smith [2] and very little Hill coef icient (n H ) change with mutations. This is expected as only the subfragment S1 is used. It is S2 that binds the cMyBP-C. HCM, DCM mechanisms unclear.

DCM From several patients no protein mutation known
In samples from patients Wolff, et al. [50] report that the calcium concentration producing half-maximal tension Figure 25: Force-pCa relationships in the rabbit skinned cardiac muscle fi bers into which human wild-type, ΔK210 (DCM), or ΔE160 (HCM) cTnT was incorporated. Maximum force levels were both ca. 30% down compared with wild-type, from Morimoto, et al. [45].

Figure 26:
Diff erential phosphorylation of sarcomeric proteins in isolated myofi brils from ΔK210 homozygous hearts (DCM). A, cardiac myofi brillar proteins stained with ProQ Diamond for phospho-proteins detection and Sypro Ruby for total protein detection. They show equal protein content (Sypro) and disparity in phosphorylation levels (ProQ), from Sfi chi-Duke, et al. [46]. 50 ) was less in cardiomyopathic preparations than in donor preparations demonstrating an increase in myo ibrillar calcium sensitivity of isometric tension which is lost on pKA phosphorylation. These results are contradictory to all other reports and probably re lect/suggest that the increased calcium sensitivity in this DCM report may be due at least in part to an in vivo reduction of the beta-adrenergically mediated phosphorylation of myo ibrillar regulatory proteins such as cTnI and/or cMyBPC or their degradation.

Conclusion
The effects of HCM mutants of cTnI, in particular the large shifts in pCa 50 for activation of the RCM mutants along with the two earlier indications of a concerted effect of cMyBP-C  in conjunction with cTnI lead to a de inite observation. The conclusion is together in the normal heart the cMyBP-C in conjunction with cTnI have the biochemical function of ensuring myosin bound MgATP is not the functional substrate of the cross-bridge ATPase. Disruption of either cTnI or cMyBP-C is suf icient to break this and allow MgATP to be used as substrate of the cross-bridge ATPase. The result of this is clear with a reduction in the Ca 2+ cooperativity of activation and HCM. When unimpaired the joint action ensures that the system is fully relaxed in diastole on completion of the cycle with binding of MgATP. The cMyBP-C knock out myo ibril does not display the commonly accepted cTnC af inity for Ca 2+ it shows activation at higher [Ca 2+ ]. Clearly one of the functions of cMyBP-C is to increase the calcium af inity of cTnC to that we are familiar with. The interaction with actinmyosin gives a change in Ca 2+ sensitivity when an N-terminal fragment of cMyBP-C is added to the knock out myo ibril. The phosphorylation state of the cTnI or others may have a bearing on this as well as the degree of pCa 50 change with the mutants. The subtle interaction between cTnI and cMyBP-C is in need of much careful study. The overlap of the actin and myosin binding regions of cMyBP-C are certain candidates for the concerted action in substrate control. The possible binding of C1mC2 fragment of cMyBP-C to the actin along with the myosin is not clear in the literature reports. The effect of binding assorted segments of cMyBP-C in its absence may illuminate this.
The mechanisms of the effects, not arising from the cMyBP-C/cTnI system, of a range of mutations giving HCM and DCM are not clear. However when studying the response to calcium with an array of mutant sarcomeric proteins one may rationalise the effect of a mutation from the accepted function and interactions of the particular protein. Generally stronger interaction, more stress in ilaments, more LIM release giving HCM and the reverse for DCM. Examples include loppy binding of MLC to the myosin giving DCM with retained ATPase function. Tropomyosin (α-TM) is involved in the interaction of the MLC with the actin ilament, the crossbridge. All the HCM mutants of α-TM show a hindered dissociation of the crossbridge, slowed relaxation, resulting in transfer of chronic tension to the titin. Other α-TM mutants show reduced tension, at both or either [Ca 2+ ] D and [Ca 2+ ] sys resulting in DCM. TnT is directly involved with the stress transfer to the thin ilament titin and hence mutations result in either HCM or DCM. Only one actin mutant is quoted giving weaker thin ilament stress. Clues to the effect of mutations may be found in structural studies such as given by Lu, Wu and Morimoto, [51] and a review by de A. Marques M and de Oliveira [52].
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]