In Sickness and In Health—and Normal Development
Troponin is the key component of the calcium-dependent switch of the contractile apparatus in striated muscle. There are three subunits of troponin: troponin C, a Ca2+-binding calmodulin-like protein; troponin T, which attaches the complex to tropomyosin, anchoring it to the thin filament as well as having a regulatory role; and troponin I (TnI), named for its ability to inhibit actin-myosin interactions at diastolic levels of Ca2+. As cytosolic Ca2+ increases in systole, it binds to a regulatory Ca2+-binding site on TnC, leading to increasing affinity of TnC for TnI and weakening the interactions of TnI and actin. This permits movement of tropomyosin-troponin on the thin filament such that the inhibition of actin-myosin interaction is diminished, increasing the probability of crossbridge cycling and muscle shortening. The ability to inhibit the actin-myosin interaction resides within a 12-amino acid region of the TnI molecule, although its inhibitory function is modulated by other regions of TnI.1
In mature mammals, a different TnI gene is expressed specifically in each of the three types of striated muscle: fast twitch (fsTnI), slow twitch (ssTnI), and cardiac (cTnI). The fetal and neonatal cardiac atria and ventricle also express ssTnI, and there is a gradual downregulation of the mRNA for ssTnI and increased expression of cTnI with maturation.2 This gene switch occurs in all mammalian species studied to date, including human. The tight regulation of this process was made apparent when the cTnI gene was subjected to targeted deletion in mice by Huang et al.3 Rather than persistently maintaining the ssTnI gene expression after its normal developmental stage in the heart, the mice died as this gene was turned off postnatally.
What are the advantages of the expression of ssTnI in the fetal heart? One functional measure of the calcium switch in muscle is the rate of ATP hydrolysis by myofilaments (actin-activated myosin or myofibrillar MgATPase activity), which increases as the available Ca2+ increases up to a maximal saturating level. A similar, although not completely analogous, relationship of steady-state isometric tension development can be measured in skinned muscle fibers exposed to varying Ca2+ concentrations. The presence of ssTnI in the myofilament results in a baseline left shift in this relationship, referred to as Ca2+ sensitization.4,5⇓ This is conceivably advantageous in the fetal heart, in which the sarcoplasmic reticulum is poorly developed, and the contractile force is much more dependent on Ca2+ entry through the cell membrane. Another advantage, first noted by Solaro et al6 before the basis of the TnI isoform shift was known, is that myofibrils from immature heart are resistant to the effect of acidosis on the myofibrillar MgATPase-Ca2+ relation. In contrast, the myofibrillar MgATPase activity of myofibrils isolated from mature heart is significantly desensitized to Ca2+ (ie, right-shifted) when exposed to lower pH, which limits contractile force. Finally, it is notable that the ssTnI isoform lacks the 32-residue amino terminal extension present in cTnI. This region of the molecule contains two adjacent serine residues that are targets of protein kinase A (PKA) phosphorylation. Functionally, PKA phosphorylation desensitizes the myofilament to Ca2+, although it may also affect crossbridge kinetics allowing more rapid cycling of crossbridges.7 The lack of these PKA targets on TnI in the fetus coincides temporally with the lack of sympathetic innervation in immature heart. There are also sites for PKC phosphorylation on TnI, some of which differ between the ssTnI and cTnI isoforms. Alterations in phosphorylation of TnI have been implicated in disease states such as dilated cardiomyopathy in mouse models and end-stage heart failure in humans.8–10⇓⇓
In this issue of Circulation Research, Westfall and colleagues11 use a myocyte gene transfer model to examine an apparent paradox that arose from prior studies reporting the expression of TnI variants in the hearts of transgenic mice. Previously, Fentzke et al5 completely replaced cTnI with ssTnI in adult mouse hearts and found that the mice exhibited some evidence of diastolic dysfunction as well as Ca2+ sensitization of the myofilaments. However, these mice did not have the typical phenotype of hypertrophic cardiomyopathy at the organ level: myocyte disarray and fibrosis were not noted, nor were these hearts hypertrophied. James et al12 then created mice expressing a TnI mutation that had been noted in a pedigree with familial hypertrophic cardiomyopathy (FHC), cTnI R146G, a mutation in the critical inhibitory region of TnI. Although it could not be measured directly, these mice likely had less than full replacement of the native TnI with the mutant, but despite this, two lines with this mutant had early lethality with grossly abnormal hearts, suggesting dose-dependent lethality. The remaining lines demonstrated varying degrees of cardiac fibrosis, myocyte disarray, diastolic dysfunction, and, in the one line in which it was measured, increased Ca2+ sensitization of tension.
Although there is a spectrum of functional changes produced by individual FHC mutations,13 several studies have noted increased Ca2+ sensitivity with disease-causing mutations of α-tropomyosin, TnT, TnI, and essential myosin light chain.14–18⇓⇓⇓⇓ Ca2+ sensitization may result in elevated resting tension and/or impaired relaxation, thus causing diastolic dysfunction. However, the work of Westfall et al11 makes it clear that, although the disease-related TnI mutant and the fetal cardiac ssTnI isoform both caused increased Ca2+ sensitization of steady-state tension to a similar extent, they result in very different organ-level phenotypes. In general, how a perturbation of myofilament protein function relates to the cellular and clinical phenotype of FHC is a key question that remains largely unanswered. One plausible hypothesis advanced by Westfall et al is that the Ca2+ sensitization associated with ssTnI expression also results in a resilience to the detrimental effect of acidosis, whereas the FHC cTnI R146G mutant myofilaments still become Ca2+ desensitized under acidic conditions. This may be relevant in FHC because of the association of cardiac ischemia in this condition with abnormal intramuscular coronary arteries and myocardial bridging.19
However, it is important to note that other factors could also mediate the difference in whole-heart phenotype between mice expressing ssTnI and cTnI R146G. Previous in vivo studies of abnormal phosphorylation mutants of TnI uncovered altered twitch kinetics,20 potentially as a result of altered crossbridge kinetics or feedback of the altered myofilament properties to produce secondary changes in calcium cycling. These types of alterations would not be evident in the steady-state measurements of tension-Ca2+ relationships reported here. Similar perturbations could contribute to the organ-level abnormalities with the cTnI R146G mutation and should ultimately be investigated.
Another interesting finding in the study of Westfall et al11 is the apparent lower affinity of the cTnI R146G mutant for the myofibrils, as demonstrated by a form of competition assay by viral transduction in the cardiac myocytes. The precise level of protein incorporation of site-specific myofilament protein mutants is often difficult to assess in transgenic overexpression models, indicating the value of the approach used in the present study.11 Should in vivo gene therapy ultimately be available in clinical care, this work also suggests the concept of “competing” the abnormal protein off the myofilament with virally transduced native cTnI.
In summary, the study of Westfall et al11 offers some new insight into the relationship between the myofilament function and phenotype in the whole heart. Many additional detailed mechanistic studies are necessary to understand the link between alterations in myofilament proteins and clinical disease in FHC.
I thank Jennifer Van Eyk for critical reading of this manuscript.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- ↵Van Eyk JE, Thomas LT, Tripet B, Wiesner RJ, Pearlstone JR, Farah CS, Reinach FC, Hodges RS. Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament. J Biol Chem. 1997; 272: 10529–10537.
- ↵Huang X, Pi Y, Lee KJ, Henkel AS, Gregg RG, Powers PA, Walker JW. Cardiac troponin I gene knockout: a mouse model of myocardial troponin I deficiency. Circ Res. 1999; 84: 1–8.
- ↵Westfall MV, Rust EM, Metzger JM. Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function. Proc Natl Acad Sci U S A. 1997; 94: 5444–5449.
- ↵Solaro RJ, Kumar P, Blanchard EM, Martin AF. Differential effects of pH on calcium activation of myofilaments of adult and perinatal dog hearts: evidence for developmental differences in thin filament regulation. Circ Res. 1986; 58: 721–729.
- ↵Holroyde MJ, Howe E, Solaro RJ. Modification of calcium requirements for activation of cardiac myofibrillar ATPase by cyclic AMP dependent phosphorylation. Biochim Biophys Acta. 1979; 586: 63–69.
- ↵Zakhary DR, Moravec CS, Stewart RW, Bond M. Protein kinase A (PKA)-dependent troponin-I phosphorylation and PKA regulatory subunits are decreased in human dilated cardiomyopathy. Circulation. 1999; 99: 505–510.
- ↵Westfall MV, Borton AR, Albayya FP, Metzger JM. Myofilament calcium sensitivity and cardiac disease: insights from troponin I isoforms and mutants. Circ Res. 2002; 91: 525–531.
- ↵James J, Zhang Y, Osinska H, Sanbe A, Klevitsky R, Hewett TE, Robbins J. Transgenic modeling of a cardiac troponin I mutation linked to familial hypertrophic cardiomyopathy. Circ Res. 2000; 87: 805–811.
- ↵Yanaga F, Morimoto S, Ohtsuki I. Ca2+ sensitization and potentiation of the maximum level of myofibrillar ATPase activity caused by mutations of troponin T found in familial hypertrophic cardiomyopathy. J Biol Chem. 1999; 274: 8806–8812.
- ↵Muthuchamy M, Pieples K, Rethinasamy P, Hoit B, Grupp IL, Boivin GP, Wolska B, Evans C, Solaro RJ, Wieczorek DF. Mouse model of a familial hypertrophic cardiomyopathy mutation in α-tropomyosin manifests cardiac dysfunction. Circ Res. 1999; 85: 47–56.
- ↵Szczesna D, Zhang R, Zhao J, Jones M, Guzman G, Potter JD. Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy. J Biol Chem. 2000; 275: 624–630.
- ↵Sanbe A, Nelson D, Gulick J, Setser E, Osinska H, Wang X, Hewett TE, Klevitsky R, Hayes E, Warshaw DM, Robbins J. In vivo analysis of an essential myosin light chain mutation linked to familial hypertrophic cardiomyopathy. Circ Res. 2000; 87: 296–302.
- ↵Pi Y, Kemnitz KR, Zhang D, Kranias EG, Walker JW. Phosphorylation of troponin I controls cardiac twitch dynamics: evidence from phosphorylation site mutants expressed on a troponin I-null background in mice. Circ Res. 2002; 90: 649–656.