Thin Filament Disinhibition by Restrictive Cardiomyopathy Mutant R193H Troponin I Induces Ca2+-Independent Mechanical Tone and Acute Myocyte Remodeling
Inherited restrictive cardiomyopathy (RCM) is a debilitating disease characterized by a stiff heart with impaired ventricular relaxation. Mutations in cardiac troponin I (cTnI) were identified as causal for RCM. Acute genetic engineering of adult cardiac myocytes was used to identify primary structure/function effects of mutant cTnI. Studies focused on R193H cTnI owing to the poor prognosis of this allele. Compared with wild-type cTnI, R193H mutant cTnI more effectively incorporated into the sarcomere, where it exerted dose-dependent effects on basal and dynamic contractile function. Under loaded conditions, permeabilized myocyte Ca2+ sensitivity of tension was increased, whereas the passive tension–extension relationship was not altered by R193H cTnI. Normal rod-shaped myocyte morphology acutely transitioned to a “short-squat” phenotype in concert with progressive stoichiometric incorporation of R193H in the absence of altered diastolic Ca2+. The specific myosin inhibitor blebbistatin fully blocked this transition. Heightened Ca2+ buffering by the R193H myofilaments, and not alterations in Ca2+ handling by the sarcoplasmic reticulum, slowed the decay rate of the Ca2+ transient. Incomplete mechanical relaxation conferred by R193H was exacerbated at increasing pacing frequencies independent of elevated diastolic Ca2+. R193H cTnI–dependent mechanical tone caused acute remodeling to a quasicontracted state not elicited by other Ca2+-sensitizing proteins and is a direct correlate of the stiff heart characteristic of RCM in vivo. These results point toward targets downstream of Ca2+ handling, notably thin filament regulation and actin–myosin interaction, in designing therapeutic strategies to redress the primary cell morphological and mechanical underpinnings of RCM.
Inherited cardiomyopathies represent a clinically diverse group of progressive heart muscle diseases that can be caused by mutations in specific sarcomeric genes.1 Cardiomyopathies are classified into several distinct clinical subtypes based on a range of morphological and functional criteria.1 The most malignant and least studied of the subtypes is restrictive cardiomyopathy (RCM).2 The distinguishing clinical features of RCM patients include markedly impaired ventricular filling, pronounced diastolic dysfunction from an extremely stiff heart,1,2 and the potential for rapid progression to overt heart failure.2,3
Recently, mutations were identified in the gene encoding cardiac troponin I (cTnI), TNNI3, in human patients with autosomal dominant RCM.3 RCM-linked mutant cTnIs result in amino acid substitutions in the most highly conserved regions of cTnI.4 cTnI is known to function as a molecular switch within the sarcomere by regulating the Ca2+ dependent cardiac muscle contraction.4 During diastole, myocyte intracellular Ca2+ is low and cTnI binds tightly to actin inhibiting strong actin–myosin interactions. Elevation of intracellular Ca2+ initiates systole by weakening cTnI–actin interactions, promoting a strong TnI–TnC interaction, permissive of cross-bridge cycling.4
Recent biochemical reconstitution studies provide evidence that RCM mutant cTnIs hypersensitize the myofilaments to Ca2+ relative to hypertrophic cardiomyopathy (HCM) mutant TnIs.5–7 These data in reconstituted preparations are important, but the primary effects of RCM mutants on membrane-intact cardiac myocytes studied under physiologic conditions remain unknown. This is important as recent evidence suggests a vital interplay between myocyte Ca2+ handling and the sarcomere in diseased myocardial tissue.8 Accordingly, we used an acute genetic engineering strategy to study myocyte structure and function. This study focused on the RCM-linked mutant cTnI R193H owing to its severe phenotype in humans.3 Simultaneous dual gene transfer of wild-type (WT) and cTnI mutant R193H showed a selective advantage of RCM cTnI over WT cTnI for stoichiometric incorporation into the sarcomere. This outcome has implications for gene dose–response relationships given the autosomal dominant genetic underpinnings of inherited RCM. Functional studies demonstrated that R193H cTnI blunted the response to positive inotropes and resulted in a myofilament-based remodeling of the Ca2+ transient in the absence of changes in Ca2+ cycling protein expression and sarcoplasmic reticulum (SR) Ca2+ load. A novel Ca2+-independent precontracted state was observed in intact mutant myocytes coinciding with the progressive incorporation of R193H cTnI into the cardiac sarcomere. This R193H mediated cellular remodeling cannot be explained by Ca2+ sensitivity alterations alone as other Ca2+-sensitizing TnI molecules, including slow skeletal TnI and HCM R146G TnI, do not cause a “quasicontracted” basal state.4,9,10 These findings suggest that events downstream of Ca2+ handling are causal for the RCM R193H defects and points to the thin filament as a unique target for possible therapeutic intervention.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Mutagenesis and Generation of Recombinant Adenovirus
Stratagene Quick Change site directed mutagenesis kits were used to engineer the RCM-linked R193H mutation into the full-length rat cTnI cDNA. The Admax vector system was used to generate recombinant adenoviral vectors, yielding a titer of 1010 to 1012 pfu/mL.
Isolated Cardiac Myocyte Preparation and Adenoviral Gene Transfer
Hearts were removed from heparinized and anesthetized adult rats, and ventricular cardiac myocytes were isolated by collagenase-hyaluronidase digestion.10
Unloaded Dynamic Sarcomere Contractility, Calcium Transients, and Isometric Tension
Methods are detailed in the online data supplement.
Isolated adult rat cardiac myocytes from control (WT) and flag- and R193H-transduced experimental groups were cultured in DMEM with or without 10 μmol/L blebbistatin (Sigma), with or without 10 mmol/L 2,3-butanedione monoxime (BDM) (Sigma), or with or without acidic DMEM (pH 6.5, without NaHCO3 and HEPES modified) for 96 hours and microscopically examined using Image-Pro Express software (Media Cybernetics, Silver Spring, Md).
Targeted Stoichiometric Replacement and Localization of RCM cTnI R193H
All vector-constructed cTnIs (mutant and WT) were engineered with or without C-terminal flag epitope tag to directly assess expression relative to native cTnI. The flag epitope had no detectable effect on myocyte structure or contractile performance as shown previously.10–12 Myofilament stoichiometry after TnI gene transfer was unchanged across time in all groups as determined by cTnI/αTm ratio.10 The rate of replacement was significantly greater for R193H than cTnI flag (Figure 1A), such that R193H mutant cTnI almost fully replaced the native cTnI, whereas cTnI flag had reached ≈45% replacement at 4 days after gene transfer. By day 6, cTnI flag can achieve complete replacement without affecting Ca2+-activated tension.10 There was no significant difference in the relative expression of the mutant cTnIs between permeabilized and nonpermeabilized myocytes (Figure 1A, column P; t test, P>0.05), evidence that the mutant cTnIs were incorporating into the sarcomeric lattice.
The increased magnitude of replacement by vector-derived R193H relative to cTnI flag suggested that the R193H mutant has an advantage over WT cTnI when incorporating into the sarcomere (Figure 1A). To directly test this hypothesis, a dual gene transfer approach was used, whereby myocytes were simultaneously transduced with 2 recombinant vectors and then assessed for incorporation efficiency (Figure 1B and supplemental Figure I). As cTnI WT titer was increased, cTnI flag could be effectively out-competed by the increase in cTnI WT titer (Figure 1B, right). By contrast, cTnI R193H flag replacement was still markedly evident even in the presence of 500 multiplicities of infection of cTnI WT vector (Figure 1B, left). These findings are evidence for the preferential incorporation of the cTnI R193H over cTnI WT into the intact sarcomere.
Confocal imaging of myocytes dual labeled with α-actinin and flag antibodies provided evidence of the correct sarcomeric localization and striated incorporation of cTnI R193H (Figure 1C). More than 95% of the myocytes incorporated mutant cTnIs, and only R193H mutant myocytes had a distinct alteration in cellular morphology, whereby >87% of R193H myocytes had a short-squat phenotype after gene transfer (Figure 1C).
R193H cTnI Increases Myofilament Ca2+-Activated Tension
The steady-state isometric tension–Ca2+ relationship was determined at a preset sarcomere length (SL) (2.1 μm) in single permeabilized cardiac myocytes after gene transfer. There was no significant difference in maximal isometric force generation or inhibition at nominal [Ca2+] between R193H and control myocytes (supplemental Table II). R193H mutants demonstrated higher isometric tension than control myocytes at submaximal [Ca2+]s as represented by a marked leftward shift in the tension–Ca2+ relationship (Figure 2A and 2B). Under acidic conditions, R193H mutants showed heightened myofilament Ca2+ sensitivity relative to controls (Figure 2C). The passive tension–SL extension relationships for R193H and control myocytes were not significantly different at nominal [Ca2+], providing functional evidence that titin-dependent myocyte elasticity13 is not altered by R193H mutant cTnI (Figure 2D).
RCM cTnI R193H Causes a Ca2+-Independent Shortening of Resting SL and Slows Relaxation and Ca2+ Transient Decay
Basal contractile function and Ca2+ transients were simultaneously measured in unloaded intact adult rat cardiac myocytes at 37°C (Figure 3). A summary of contractility, defined as the amplitude of SL shortening, and Ca2+ transient parameters from myocytes 4 days after gene transfer is presented in supplemental Table I. Efficiency of gene transfer was >95%, with 90±3% replacement of native cTnI with vector-derived R193H mutant cTnI (Figure 1A).
RCM R193H gene transfer and sarcomeric incorporation significantly altered the normal rod-shaped cell morphology and decreased resting SLs in the absence of altered baseline [Ca2+] (P<0.001; Figure 3B and supplemental Table I). The R193H myocytes had baseline SLs that were on average 110 nm per SL shorter than control myocytes (P<0.001; Figure 3A and supplemental Table I). The significant Ca2+-independent shortening of SLs in R193H mutant myocytes could be attributed to the heightened magnitude of replacement by RCM R193H mutant relative to cTnI flag at this time point; therefore, the relationship between gene dosage and resting SL was determined by linear regression. With progressive cTnI flag incorporation, there was no change in resting SL (Figure 3A; y=−4.1e−6+1.801). By contrast SLs were significantly shorter even at 20% R193H replacement and demonstrated a significant dose-dependent response (Figure 3A; R2=0.88, y=−0.0011x+1.7815).
Representative sarcomere shortening traces and the corresponding Ca2+ transients of nontransduced (control), cTnI flag, and RCM-linked R193H mutant myocytes are shown in Figure 3B. RCM R193H cTnI caused a marked slowing of relaxation with a concomitant slowing of the Ca2+ transient decay, whereas cTnI flag had no effect. There was no significant difference in peak shortening/fluorescence amplitudes between groups (supplemental Table I). The length of time from peak shortening to 50%, 75%, and 90% relaxation in R193H mutant myocytes was more than tripled relative to controls; likewise, the corresponding decay time of the R193H mutant myocyte Ca2+ transient was more than doubled (Figure 3C and supplemental Table I). Progressive incorporation of R193H cTnI into the sarcomere produced dose-dependent slowing of the time to 90% relaxation even at 20% replacement in comparison to control and cTnI flag myocytes (Figure 3C; R2=0.79, y=0.003x+0.179). The cTnI flag myocytes had no change in relaxation time with increasing incorporation of cTnI flag (Figure 3C; R2=0.69, y=4e−05x+0.128). The shortened resting SLs of the R193H myocyte are not predicted to affect relaxation times, as the dynamics of tension decay are independent of preloaded SL.14,15
Pair-wise comparisons of myocyte contractile function was measured with 10 nmol/L isoproterenol (Figure 3D) or with or without 5.0 mmol/L extracellular Ca2+ (supplemental Figure IIA and IIB). Relative to controls, the R193H myocytes did not significantly increase contractility in the presence of either isoproterenol or 5.0 mmol/L extracellular Ca2+ (Figure 3D and supplemental Figure IIA) but did have a significant lusitropic response to isoproterenol, which almost completely corrected their slow relaxation kinetics (Figure 3D). Steady-state Ca2+-activated tension experiments revealed that the mutant myofilaments responded to the protein kinase A (PKA) catalytic subunit with the same magnitude of desensitization of the myofilaments to Ca2+ as seen with WT myocytes (Figure 3D). These data suggest that R193H cTnI does not abrogate the intramolecular cTnI switch function on serine phosphorylation by PKA to facilitate fast relaxation.
The marked slowing of the late Ca2+ transient decay phase in R193H mutant myocytes suggests that the R193H cTnI may alter Ca2+ cycling. To test this hypothesis, transduced myocytes were rapidly exposed to caffeine (10 mmol/L) to induce Ca2+ release from the SR. There was no significant difference (P=0.1) in Ca2+ transient amplitude between control (0.19±0.02) and R193H (0.24±0.02) myocytes, as summarized in Figure 3E and 3F, suggesting that the R193H cTnI does not directly affect functional SR Ca2+ load. Expression of critical Ca2+ handling proteins including Na+–Ca2+ exchanger, SR Ca2+-ATPase pump, phospholamban, and the serine 16 phosphorylated form of phospholamban were assessed by Western blot (Figure 3G). There were no significant differences in Ca2+ handling protein expression between groups, suggesting that Ca2+ reuptake and extrusion processes are functioning appropriately in R193H mutant myocytes.
Increased Electrical Pacing Exacerbates Ca2+-Independent Diastolic Tone in RCM Mutant Myocytes
Frequency-response experiments highlighted a significant frequency-dependent diastolic dysfunction in R193H mutant myocytes (Figure 4A and 4B), as demonstrated by R193H SLs remaining significantly shorter during relaxation throughout a train of pulses relative to controls. This effect, which is summarized in Figure 4C, becomes more pronounced at higher frequencies, indicating that unlike controls the mutant myofilaments only partially relax with increasing demands. To quantify this effect the average resting SLs and fluorescence ratios were measured at each frequency >0.2 Hz, and these values were subtracted from the diastolic SL/fluorescence ratio measured at 0.2 Hz, yielding the following values (Figure 4C): ΔA (0.5 Hz), ΔB (1.0 Hz), and ΔC (2.0 Hz). Control mechanical transients remained tightly coupled to the Ca2+ transient, but the R193H mutant myocytes developed progressively shorter diastolic SLs that were 18±3 nm shorter at 2.0 Hz compared with baseline, despite the Ca2+ transient returning to baseline values (Figure 4C). Five seconds after the pacing protocol (“off” bar in Figure 4A and 4B), resting SLs in R193H mutant myocytes were 10% (17±5 nm) shorter than their prefrequency response values in the absence of elevated diastolic Ca2+ (Figure 4C, ΔD). In control myocytes, however, there was no significant difference in diastolic SLs and Ca2+ ratios before and after frequency response. Thus, pacing exacerbated the already increased basal mechanical tone in R193H myocytes independent of the Ca2+ transient, providing further evidence that RCM R193H mutation causes myofilament disinhibition at rest.
These experiments also revealed that R193H mutant myocytes have an abnormal contractile response to increasing stimulation frequency. Classically, rodent myocytes respond to increased pacing with decreased shortening and Ca2+ transient amplitudes (negative staircase), as seen in control myocytes (Figure 4D). The sarcomere shortening and Ca2+ transient amplitudes of the R193H mutant remained unchanged with escalating frequency (Figure 4D). In all experimental groups, relaxation and Ca2+ transient decay increased as a function of increasing stimulation frequency (data not shown). Despite the global increase in sarcomere relaxation rates, R193H mutant myocytes were still 50% slower than control myocytes at each frequency. The Ca2+ transient decay rate, however, was only significantly slower than controls at the lower frequencies (0.2 and 0.5 Hz), providing further evidence of a frequency-dependent uncoupling between mechanical and Ca2+-handling events. The frequency-dependent hastening of R193H mutant Ca2+ transients also suggests that SR Ca2+ handling is not directly impaired.
RCM cTnI Causes a Ca2+-Independent Cellular Mechanical Tone
Whole cell morphology of R193H myocytes was 18% shorter and 32% wider (P<0.05) than WT myocytes, which maintained a classic rod-shaped morphology in primary culture (P<0.05, Figure 5A and 5B). To gain insight into the mechanism of this acute cellular remodeling, myocytes were treated with BDM (10 mmol/L), a compound that has pleiotropic inhibitory effects on myocyte E-C coupling. Chronic application of BDM in cell culture blocked the morphological transition of R193H mutant myocytes (Figure 5C), suggesting that acute remodeling is mediated either through Ca2+ handling or downstream at the level of actin-myosin interaction. Application of the specific myosin II inhibitor, blebbistatin, also sustained the R193H mutant myocyte with normal cellular morphology with a relative 30% increase in length and 50% decrease in width (Figure 5C), an effect not seen with the Ca2+ channel blocker, diltiazem (data shown in supplemental Figure IV). These data suggest that the R193H mutant myocyte remodeling that we refer to as basal mechanical tone is caused by insufficient actin–myosin inhibition at physiologic diastolic [Ca2+].
The loss of the inhibitory function of R193H mutant cTnI that results in an elevated mechanical tone may be a function of the loss of positive charge in the highly basic C-terminal domain of cTnI that is associated with the arginine-to-histidine substitution at codon 193. Because histidine becomes protonated at a pH of <7.0, mimicking the positive charge of arginine, we tested the hypothesis that an acidic media (pH 6.5) would block R193H mutant myocyte from developing diastolic tone. To test this hypothesis, transduced myocytes were chronically cultured in an acidic media (pH 6.5) without bicarbonate. Figure 5C demonstrates that acidic media blocked R193H cTnI-mediated alterations in myocyte length and width (DMEM, pH 7.4) and were similar to control myocyte dimensions (supplemental Figure III). Acidosis (pH 6.2) in steady-state Ca2+-activated tension assays showed that the R193H myocytes had a blunted pH-dependent desensitization of the myofilaments to Ca2+, unlike WT myocytes, which showed more significant desensitization of the myofilaments to Ca2+ (Figure 2C).
We report the new finding that the RCM mutant cTnI R193H produces a direct cellular defect of a Ca2+-independent heightened mechanical tone at baseline (Figure 6). Myofilament Ca2+ sensitization cannot account for these findings as other potent TnI Ca2+ sensitizers, shown previously to confer equal or greater increases in Ca2+ sensitivity, do not produce these effects when tested under identical experimental conditions.4,9–12 The increased basal mechanical tone occurred without associated alterations in diastolic [Ca2+], thus uncoupling cellular mechanical function from Ca2+ signaling. Blebbistatin, the specific inhibitor of myosin II function, fully blocked this effect, providing evidence that active myosin–actin interaction is required and is secondary to effects initiated by RCM cTnI sarcomeric incorporation.
The primary and novel defect of increased basal mechanical tone manifests as progressively shortened SLs commensurate with the replacement-dependent increase in incorporation of RCM TnI R193H into the sarcomere. Recent echocardiography results on a R193H transgenic mouse model reported significantly reduced left ventricular end-diastolic volumes.16 This organ-level phenotype under physiologic loaded conditions is interesting and may relate to our finding of reduced SLs after R193H gene transfer in vitro. Based on these findings, we hypothesize that R193H myocytes operate at reduced SLs under load in vivo. Passive tension–extension measurements at nominal [Ca2+] were not affected by cTnI R193H, providing mechanical evidence that passive elements (titin) are not responsible for the heightened basal mechanical tone. We interpret this effect as a partial disinhibition of the thin filament causing a quasicontracted state of the intact myocyte at physiologic diastolic [Ca2+]i.
Another primary effect of cTnI R193H relates to its heightened incorporation efficiency into the adult cardiac sarcomere. Simultaneous dual gene transfer of cTnI R193H and cTnI WT to cardiac myocytes showed that R193H could not be readily “competed off” by increasing cTnI WT viral titer in contrast to nonmutated cTnI (Figure 1B). This is interpreted as an advantage of R193H over cTnI WT to stoichiometrically replace endogenous cTnI and incorporate into the sarcomere. Because RCM is inherited in an autosomal-dominant manner, affected individuals have 1 normal cTnI allele and 1 R193H allele. Our results suggest, in contrast to the estimated 50:50 ratio of WT to diseased protein content reported in hearts for other inherited cardiomyopathy alleles,8,17 R193H individuals may be predicted to have greater than 50% R193H protein content in the sarcomere. This may prove physiologically important as genetic titration of R193H in this study demonstrated significant dose-dependent effects on myocyte structure/function (Figure 3A and 3C). In addition, novel gene suppression strategies for treating inherited cardiomyopathies18 may be more challenging for cTnI R193H or similar functioning alleles given its preferential incorporation into the sarcomere.
Chronic exposure of the R193H mutant myocytes with the myosin II–specific inhibitor blebbistatin fully blocked cellular remodeling and is evidence that the R193H weakens the inhibitory function of cTnI, resulting in a low level of actin–myosin interaction under normal diastolic [Ca2+]. This partial loss of inhibitory function of cTnI attributable to R193H may be caused by the charge change that results from an arginine-to-histidine substitution in the highly basic C-terminal region of TnI. Exon 8, which encodes cTnI amino acids 185 to 211, is highly conserved across all TnI isoforms and species, ranging from human to fish (>72% similarity),4 indicating that there is high selection pressure in this region of TnI. Of the more than 10 known RCM and HCM mutations located within the cTnI C-terminal region, 40% result in a loss of positive charge. The elucidation of the crystal structure of TnI in conjunction with recent NMR studies show that the C-terminal region in the Ca2+ bound state is highly dynamic and lacking secondary structure.19,20 In the “fly casting” model,19,21 the cTnI C terminus is unstructured in the presence of Ca2+ and tethered through upstream TnI interactions with TnC.19 After Ca2+ removal, the cTnI switch domain interacts with actin and the C-terminal domain “reels in” the rest of the troponin complex to its new position on the thin filament.19,22 The fly casting model necessitates electrostatic interactions to facilitate rapid protein–protein interface transitions. We speculate, for cTnI R193H in particular, electrostatic tethering is critical and also pH-dependent. At physiologic pH, the histidine mutation is postulated to adversely affect cTnI–actin electrostatic interactions through loss of positive charge in this region of cTnI. We further hypothesize that at acidic pH, at which the imidazole group of histidine becomes positively charged, the cTnI–actin interaction would stabilize cTnI–actin interactions similar to control. In support of this idea, when R193H transduced cardiac myocytes were maintained in acidic media (pH 6.5) throughout the entire culture period, the increased basal mechanical tone was blocked. The caveat is that acidosis is a global inhibitor of contractile function affecting both Ca2+ handling and actin–myosin interactions; however, this experiment highlights the importance of actin–myosin interactions as a precursor to increased mechanical tone.
Our results of a primary effect of an increased cellular mechanical tone contrasts with an apparent secondary outcome of myocyte remodeling that accompanies HCM mutant troponin in the context of the working myocardium in vivo.23,24 Isolated myocytes from the α-myosin heavy chain R403Q mice exhibited shortened morphology, albeit in the absence of changes in resting SL,23 suggesting that different mechanisms, such as compensatory changes rather than the direct effect of heightening resting cross-bridge activation, govern the altered morphology. In addition, our results are distinct from previously reported shortened myocytes isolated from adult transgenic mice harboring HCM mutant cTnT, which could be readily reversed by acute application of BDM.24 In our study, acute application of BDM had no effect on the short-squat myocyte morphology, but after membrane permeabilization, the R193H myocytes could be readily mechanically extended to normal rod-shaped morphology and SLs via micropipette attachment to each end of the cell.25 Under these conditions, cell widths and resting passive tension were not different between RCM R193H and WT myocytes. Gomes et al did find that R193H mutants had a decreased ability to inhibit ATPase activity, but they did not see this result in isometric tension assays.5 Together, these results underscore the role of the intact myocyte and myofilament response to diastolic [Ca2+] as being required to induce cellular morphological alterations seen for R193H mutant cTnI. As such, our findings in intact myocytes would not be detected in protocols in which TnI is exchanged in membrane-permeabilized preparations at nominal [Ca2+].5–7
The slowing in SL relaxation and Ca2+ transient decay kinetics may be explained by cTnI-mediated thin filament disinhibition, as there was no change in functional SR Ca2+ load or Ca2+-handling protein expression (Figure 3E and 3F) in R193H myocytes. Collectively, these data suggest a role of the mutant myofilaments in altering the Ca2+ transient and lend support to our proposed mechanism that the heightened Ca2+-buffering capacity of R193H myofilaments altered Ca2+-cycling dynamics to directly remodel the Ca2+ transient as previously suggested in mouse models of HCM.8
This study shows that the RCM-linked mutation R193H in TNNI3 directly induces a basal mechanical tone that cannot be explained simply by a gain in myofilament Ca2+ sensitivity. This basal quasicontracted state arises without detected alterations in resting [Ca2+]. We infer that the primary cellular defect of cTnI R193H results from TnI-based disinhibition in actin–myosin interaction at normal diastolic [Ca2+]. This Ca2+-independent mechanical tone is blocked by direct chronic inhibition of actin–myosin interaction but not by diltiazem. Therapeutic strategies for inherited and acquired cardiomyopathies have primarily focused on myocyte Ca2+ handling.8 The present results suggest that targets downstream of Ca2+ handling, specifically thin filament regulation and actin–myosin interaction, may represent new targets to redress the primary cell morphological and mechanical underpinnings of RCM cTnI.
We thank Drs Margaret Westfall, Sharlene Day, and Dan Michele for helpful discussions.
Sources of Funding
Supported by the NIH and an American Heart Association Greater Midwest Predoctoral Fellowship.
Original received October 6, 2006; first resubmission received January 16, 2007; second resubmission received March 21, 2007; revised second resubmission received April 12, 2007;
Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of Cardiomyopathies. Circulation. 1996; 93: 841–842.
Rivenes SM, Kearney DL, Smith EO, Towbin JA, Denfield SW. Sudden death and cardiovascular collapse in children with restrictive cardiomyopathy. Circulation. 2000; 102: 876–882.
Metzger JM, Westfall MV. Covalent and noncovalent modification of thin filament action: the essential role of troponin in cardiac muscle regulation. Circ Res. 2004; 94: 146–158.
Gomes AV, Liang JS, Potter JD. Mutations in human cardiac troponin I that are associated with restrictive cardiomyopathy affect basal ATPase activity and the calcium sensitivity of force development. J Biol Chem. 2005; 280: 30909–30915.
Yumoto F, Lu QW, Morimoto S, Tanaka H, Kono N, Nagata K, Ojima T, Takahashi-Yanaga F, Miwa Y, Sasaguri T, Nishita K, Tanokura M, Ohtsuki I. Drastic Ca2+ sensitization of myofilament associated with a small structural change in troponin I in inherited restrictive cardiomyopathy. Biochem Biophys Res Commun. 2005; 338: 1519–1526.
Kobayashi T, Solaro RJ. Increased Ca2+ affinity of cardiac thin filaments reconstituted with cardiomyopathy-related mutant cardiac troponin I. J Biol Chem. 2006; 281: 13471–13477.
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.
Buvoli M, Buvoli A, Leinwand LA. Suppression of nonsense mutations in cell culture and mice by multimerized suppressor tRNA genes. Mol Cell Biol. 2000; 20: 3116–3124.
Shoemaker BA, Portman JJ, Wolynes PG. Speeding molecular recognition by using the folding funnel: the fly-casting mechanism. Proc Natl Acad Sci U S A. 2000; 97: 8868–8873.