Transgenic Rabbit Model for Human Troponin I–Based Hypertrophic Cardiomyopathy
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《循环学杂志》
the Divisions of Molecular Cardiovascular Biology (A.S., J.J., L.M., J.G., H.O., S.S., R.K., J.R.) and Pediatric Cardiology (V.T., S.N.), Cincinnati Children’s Hospital Research Foundation, Cincinnati, Ohio
Loyola University Chicago, Department of Physiology and Cardiovascular Institute, Maywood, Ill (K.S.G., D.M.B.)
National Institute on Aging, Intramural Research Program, Gerontology Research Center, National Institutes of Health, Baltimore, Md (B.Z., E.G.L.).
Abstract
Background— Transgenic and gene-targeted models have focused on the mouse. Fundamental differences between the mouse and human exist in Ca2+ handling during contraction/relaxation and in alterations in Ca2+ flux during heart failure, with the rabbit more accurately reflecting the human system.
Methods and Results— Cardiac troponin I (cTnI) mutations can cause familial hypertrophic cardiomyopathy. An inhibitory domain mutation, arginine146glycine (cTnI146Gly), was modeled with the use of transgenic expression in the rabbit ventricle. cTnI146Gly levels >40% of total cTnI were perinatally lethal, whereas replacement levels of 15% to 25% were well tolerated. cTnI146Gly expression led to a leftward shift in the force-pCa2+ curves with cardiomyocyte disarray, fibrosis, and altered connexin43 organization. In isolated cTnI146Gly myocytes, twitch relaxation amplitudes were smaller than in normal cells, but [Ca]i transients and sarcoplasmic reticulum Ca2+ load were not different. Detrended fluctuation analysis of the QTmax intervals was used to evaluate the cardiac repolarization phase and showed a significantly higher scaling exponent in the transgenic animals.
Conclusions— Expression of modest amounts of cTnI146Gly led to subtle defects without severely affecting cardiac function. Aberrant connexin organization, subtle morphological deficits, and an altered fractal pattern of the repolarization phase of transgenic rabbits, in the absence of entropy or other ECG abnormalities, may indicate an early developing pathology before the onset of more obvious repolarization abnormalities or major alterations in cardiac mechanics.
Key Words: cardiovascular diseases ; heart diseases ; hypertrophy
Introduction
Familial hypertrophic cardiomyopathy (FHC) displays an autosomal dominant mode of inheritance and a diverse genetic etiology: FHC or a phenocopy can be caused by multiple mutations in genes encoding various contractile, structural, channel, and kinase proteins.1,2 Commonly, arrhythmias, particularly ventricular tachycardia and fibrillation, are associated with sudden death.3,4 The absence of multiple FHC models that faithfully recapitulate important aspects of human disease limits our ability to explore the underlying disease mechanism(s) and develop therapeutic strategies.
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Multiple mutations in the inhibitory subunit of cardiac troponin (cTnI) can cause hypertrophic cardiomyopathy.5,6 Previously, we modeled the cTnIR145G mutation (residue 146 in the human) in transgenic mice.7 Cardiac functional analysis revealed impaired relaxation, and permeabilized cardiac muscle fiber studies showed an increase in Ca2+ sensitivity. These and other data indicate that increased myofibril Ca2+ sensitivity in the cTnI FHC mutations may play a major pathological role.8
The rabbit has advantages, compared with the mouse, for studying cardiovascular disease.9 Substantial differences exist between the mouse and human in the manner in which Ca2+ is handled during contraction/relaxation and in alterations in Ca2+ flux during heart failure, with the rabbit more accurately reflecting the human system.10,11 Human atria express -myosin heavy chain (MHC), and the ventricles express predominantly ;-MHC. The adult rabbit heart reflects this, whereas the mouse expresses -MHC in both the postnatal atria and ventricles.12 That the mouse heart beats 10 times faster than the human heart is another limitation because the faster heart rate influences the refractory period associated with the occurrence of arrhythmia.13 To study the pathogenicity and presentation of an FHC cTnI mutation in the rabbit heart, we generated rabbits with high and low levels of ventricle-specific expression of cTnIR146G. Although high-expressing lines died perinatally, a low-expressing line survived and appeared outwardly normal. However, the cTnIR146G transgenic ventricles showed apical myocyte disarray, interstitial fibrosis, and mild ventricular hypertrophy at 1.5 to 2 years. Increased Ca2+ sensitivity was accompanied by altered patterns of connexin deposition at the gap junctions, whereas detrended fluctuation analysis (DFA) of the QTmax intervals in these rabbits showed a higher scaling exponent.
Methods
Isolation and Construction of the Rabbit ;-MHC Promoter and Transgenic Constructs
Rabbit ;-MHC sequences were obtained from a EMBL3 genomic library. An EcoRI site was introduced into the ;-MHC promoter, and a polyadenylation signal was placed downstream of the site (Figure 1). After insertion of the transgenes, DNA was digested free of vector sequence with NotI, purified, and used to generate transgenic rabbits. The rabbit cTnI cDNA was isolated with the use of reverse transcription–polymerase chain reaction (RT-PCR), and an arginineglycine missense mutation at position 146 was made with the use of PCR mutagenesis. A FLAG-epitope tag was introduced at the N-terminus by PCR. A wild-type cTnI construct (cTnIWT) was also made so that transgenic rabbits expressing FLAG-tagged cTnI at the same levels as the mutant cTnI could serve as controls.
Isolated Cardiomyocytes
Isolated myocytes were loaded with Indo-1 or Fluo-3, bathed in normal Tyrode’s solution with 2 to 2.5 mmol/L Ca2+, and stimulated at 0.5 Hz. Twitch fluorescence transients were recorded with a microscope-based photometer and converted to [Ca]i by standard methods.14 Contraction was recorded by video edge detection and expressed as the fractional change in resting cell length. Caffeine (10 mmol/L) was applied to some cells to measure sarcoplasmic reticulum Ca2+ loading and allow analysis of Ca2+ fluxes contributing to Ca2+ transient decay.15 Comparisons were based on data from 8 to 33 cells from 5 rabbits of either genotype. The significance level for differences of means was P0.05.
DFA of the QTmax Intervals
Four transgenic and 4 nontransgenic rabbits aged 21 months were used. The digital ECGs were obtained over a duration of 5 minutes, and the QTmax intervals (onset of QRS to T wave peak) and R-R intervals were measured. Uncorrected heart rate values of QTmax intervals were used for the analysis of repolarization variability assessment.16 DFA and the entropy of the QTmax intervals with the use of approximate and sample entropy methods were also evaluated. The data were analyzed by these nonlinear methods to assess possible abnormal changes of the heart rate dynamics. The standard deviation (SD) of QTmax and the square root of the mean squared differences of successive repolarization intervals were calculated by standard methods of variability analysis.
Miscellaneous Methods
Myofibrillar sample preparation, gel preparation, electrophoretic conditions, and gel staining have been described.17 Western analyses were performed with the use of anti-TnI, anti-GAPDH (Chemicon International), anti-FLAG (Sigma), anti-connexin43 (Zymed Laboratories Inc), and [Ser 368] anti–phospho-connexin43 (Cell Signaling Technologies). Immunohistochemistry and fiber isolation and analyses have been described.18 Quantitative analysis of interstitial fibrosis was measured in formalin-fixed and paraffin-embedded tissue sections with the use of Sirius red F3BA and fast green FCF.19 After the dye was eluted from the tissue sections, the absorbance at 540 and 605 nm was determined for Sirius red F3BA and fast green FCF binding protein, respectively.
Results
Ventricle-Restricted Transgenic Expression in the Rabbit
Previous transgenic studies affecting the protein complement of the rabbit heart used the mouse ;-MHC promoter.9,20 Although the mouse promoter shows striated muscle specific expression, it does not completely mimic endogenous ;-MHC expression in the rabbit.20 Therefore, we isolated and tested the rabbit ;-MHC promoter in an attempt to drive high levels of transgene expression in the rabbit ventricle. The basic promoter construct consists of 6000 bp of sequence upstream of the transcriptional start site, as well as the exons that encode the 5 untranslated region. A polyadenylation signaling sequence, derived from the human growth hormone gene, is inserted downstream of the unique EcoRI restriction enzyme site, into which the various transgenes can be inserted (Figure 1A). In preliminary experiments, the gene encoding chloramphenicol acetyltransferase was used to titrate promoter activity. Multiple lines were generated, and chloramphenicol acetyltransferase (CAT) expression was determined. The promoter drove high levels of expression in all regions of the ventricle in a copy number–dependent manner as well as in muscles containing slow-type fibers (Figure 1B to 1D). In the atria, no expression was detected in 3 lines and only very low expression in 1 line showing high copy numbers (428H, 39 copies), suggesting that the rabbit ;-MHC promoter drives predominantly ventricular-specific transcription, mimicking endogenous expression. Expression was stable throughout the juvenile and adult periods, and the expression patterns were conserved across multiple lines (data not shown). CAT expression was also determined during embryogenesis. At embryonic day 10, robust expression occurred in the ventricle and outflow tract, with relatively low levels of expression in the atria (Figure 1E), a pattern consistent with endogenous promoter activity in the mouse21 and rabbit (J. James, MD, and J. Robbins, PhD, unpublished data, 2003). To determine homogeneity of CAT expression in the postbirth ventricle, in situ immunohistochemistry was performed on different regions. Transgene expression was homogeneous throughout (Figure 1F, 1G), and the data show that the rabbit ;-MHC promoter efficiently drives ventricular-restricted and slow-type skeletal muscle transgene expression.
Cardiac TnIR146G Transgenic Expression
We used the rabbit ;-MHC promoter to create rabbits in which cTnI was partially replaced by either cTnIR146G or cTnIWT in the ventricle (Figure 2A). Three cTnIR146G transgenic founders and 1 cTnIWT transgenic line were generated. In lines 52 and 71, with copy numbers of 5 and 4, respectively, the mutant protein accounted for >50% of total cTnI by the neonatal stage (Figure 2C). As described by us and others for both cTnI and other contractile proteins, increases observed at the RNA level are not translated into absolute increases of protein.7,22 Rather, endogenous protein levels are reduced such that contractile protein stoichiometry is conserved, resulting in replacement of the endogenous protein with the transgenically encoded species (Figure 2B).23 In both lines, death occurred within 1 week after birth. No obvious phenotype presented in the cTnIWT line, in which the transgenic protein replaced 64% of the endogenous cTnI (Figure 2C), indicating that transgenic expression of cTnI, by itself, was not lethal. At the time of death, the pups from both lines 52 and 71 showed symptoms consistent with heart failure, and electron microscopy confirmed major sarcomere disruption and contractures (data not shown). Premature death was also observed in the severely affected cTnIR146G transgenic mice,7 confirming that >40% to 50% replacement with cTnIR146G is lethal.
Line 51, with a copy number of 2 and protein replacement of 28% (Figure 2C, 2D), was asymptomatic. Mutant protein levels were maintained equally in both adult ventricles (Figure 2E). In the young adults, no obvious cardiac remodeling occurred (Table 1), nor were there any histological abnormalities (data not shown). However, by 18 months there was significant fibrosis in the septum and apex and isolated areas of myocyte disarray (Figure 3), whereas no abnormalities could be detected in either the nontransgenic or cTnIWT transgenic hearts. Interestingly, histological abnormalities were only detected in focal areas of the ventricle despite a uniform mutant protein distribution. Echocardiography detected a slight but significant increase in left ventricular wall thickness, as well as increased septal thickness and improved fractional shortening (Table 2).
Fiber and Cardiomyocyte Studies
In the cTnIR146G transgenic mice, functional analyses revealed impaired relaxation, and fiber studies showed an increase in Ca2+ sensitivity.7 In vitro reconstitution experiments have shown that FHC cTnI mutations (cTnIR146G and cTnIR162W) resulted in reduced inhibition of myosin ATPase by the troponin complex and increased Ca2+ sensitivity of actin-tropomyosin–activated myosin ATPase activity.8 To determine the effect of cTnIR146G on the development of force, we generated force-pCa2+ curves using permeabilized muscle fibers. To obtain a dose response, neonatal fibers were used because line 52, in which 55% replacement was obtained, died between 5 to 9 days after birth. Before the development of overt heart failure in this line, fibers were isolated from the left ventricular papillary muscles and compared with fibers derived from line 51 (28% replacement) and nontransgenic animals. A marked leftward shift of the force-pCa2+ curve was observed in line 52, and a slight leftward shift was seen in line 51 (Figure 4), indicating that the mutant protein affected myofibrillar Ca2+ sensitivity in a dose-dependent but nonlinear manner.
Despite the changes in Ca2+ sensitivity, functional parameters determined by cardiac catheterization were conserved at 6 months (Table 1), with modestly enhanced cardiac function apparent by 2 years (Table 2). These increases are consistent with the data obtained in the cTnIR146G transgenic mice as well as in vitro reconstitution studies.7,8 At the whole-organ level, it appears that increased Ca2+ sensitivity results in modest increases in cardiac function, mild hypertrophy, and focal cardiomyocyte abnormalities.
Data that bear directly on the effect(s) of changes in cTnI on isolated cardiomyocytes are limited, although recent data suggest that, in principle, one should be able to detect alterations in myofilament sensitivity in isolated myocyte experiments.24 Isolated cardiomyocytes were therefore derived from nontransgenic and transgenic cTnIR146G hearts. Figure 5A shows [Ca]i transients followed by a caffeine-induced Ca2+ transient in a transgenic myocyte. Peak fractional shortening was not different, but there was a trend toward larger [Ca]i transient amplitude in the transgenic cells (Figure 5B). These relative amplitudes did not give any indication of increased myofilament Ca2+ sensitivity despite the results in skinned fibers. Twitch relaxation kinetics were unaltered, whereas [Ca]i decline was slightly faster in the transgenic myocytes (Figure 5C). From caffeine-induced Ca2+ transients, we inferred that sarcoplasmic reticulum Ca2+ loading was not different between transgenic and nontransgenic cells (Figure 5D). However, [Ca]i decline was faster in transgenic cells, indicative of greater Na-Ca2+ exchange function in the transgenic rabbits (Figure 5D). The apparently faster Na-Ca2+ exchange rate may partly explain the faster twitch [Ca]i decline (Figure 5C). [Ca]i transients and contraction were also measured with [Ca]o=1 mmol/L or 5 mmol/L and at different frequencies (0.2, 0.5, and 1 Hz) with similar trends. The expected Ca2+ and frequency effects on amplitude and kinetics of the Ca2+ transient and contraction did not differ between the 2 groups (data not shown).
We reasoned that despite no increase in mortality or overt presentation of disease, the visible areas of myocyte disarray might lead to subtle alterations in the gap junctions and electrophysiology of the hearts. Connexin43 (Cx43) is the major component of the ventricular gap junction.25 Changes in subcellular distribution or structural remodeling have been correlated with cardiomyocytes in distress, disarray, border infarct zones, acute responses to injury, or the initial stages of compensated hypertrophy.26,27 Absolute levels of Cx43 were significantly increased at 21 months, but their organization at the gap junction was compromised in the cTnIR146G ventricles (Figure 6A, 6B). Cx43 phosphorylation results in a reduction in gap-junctional intercellular communication,28 and we noted that phospho-Cx43 levels were also significantly elevated (Figure 6E). Elevated levels of Cx43 and phospho-Cx43 were found throughout the heart despite the focal pattern of interstitial fibrosis and myocyte disarray, which were largely restricted to the apical and septal regions. Although Cx43 heterogeneity of expression as well as the absolute levels may predispose the heart to arrhythmias,26 and increases observed in Cx43 phosphorylation can lead to impaired intracellular conduction,29 ECG studies were inconclusive, with no abnormalities presenting (Figure 6C, 6D). To increase the resolution of the study, the cardiac repolarization phase of the digitized ECGs was subjected to DFA (Figure 6F). The QTmax interval was significantly higher in the cTnIR146G rabbits than in the nontransgenic group.
Discussion
The extent of expression and incorporation of mutant cTnI in human cardiomyocytes is not known, although presumably most patients have 1 mutant allele.5 A recent study in which isolated adult rat ventricular myocytes were used suggested that incorporation of cTnIR146G differed significantly from that of the normal protein. Maximum protein replacement of endogenous cTnI with normal cTnI reached 85%, with significantly less replacement (43%) achieved when cTnIR146G was expressed.32 These results suggest that the capacity of myocytes to express and incorporate cTnIR146G into the sarcomere is significantly impaired compared with normal cTnI. This may be due either to mutant protein instability or to a reduced ability of cTnIR146G, relative to cTnI, to incorporate into myofilaments. Consistent with this hypothesis, an FHC-mutant ;-MHC in the soleus represented only 12% to 23% of the ;-myosin.33 The rabbit data show nonviability in lines 52 and 71, in which replacement reached 50%, and it appears that, for this isoform in the rabbit, 50% replacement is not compatible with life.
The lethality of the mutation in the transgenic rabbits is clearly dependent on the degree of replacement by the mutant protein. However, the relationship is not linear but rather exhibits a threshold effect. If the mutant protein is incorporated at >50% of the total cTnI, the muscle fibers exhibit a marked increase in Ca2+ sensitivity, and all of the F1 rabbits die between 5 and 7 days after birth. In contrast, line 51, with 28% replacement, shows only a slight increase in muscle fiber Ca2+ sensitivity and no lethality. In vitro reconstitution studies with mixtures of wild-type and mutant cTnIs show that the effects on Ca2+ sensitivity and in vitro motility are complex and nonlinear.34,35 The data have been interpreted to mean that wild-type cTnI can compensate up to a point for the compromised ATPase inhibitory ability of the mutant.35 This consideration and the biphasic nature of the transition observed with mixtures of different cTnIs emphasize the nonlinearity of the response. This is consistent with the rabbit data, in which a relatively mild phenotype is observed in line 51 compared with the other transgenic lines (52 and 71), as well as with our previous data from the transgenic mice.7 Although the exact degree of replacement by the mutant protein in FHC patients is unknown, our results may help to explain the mechanism underlying why patients with hypertrophic cardiomyopathy linked to this specific cTnI mutation have a relatively mild phenotype with elderly onset penetration35,36 and show that the limits of substitution tolerated lie below 50%.
The histological and morphological alterations observed in the mutant transgenic rabbits were asymmetrical and progressive. Because rabbit ;-MHC promoter activity is relatively uniform in the heart (Figure 1), heterogeneous localization of the mutant protein is an unlikely explanation for the focal defects. The mechanism responsible for the heterogeneity of pathology is unknown. One possible explanation is an increase in contractility, as reflected by a slight enhancement of fractional shortening. Slight but sustained alterations in regional contractility may compromise myocardial blood flow, triggering regional ischemia and/or hypoxia, which can cause histological and morphological alterations.3,4 As an example, the cellular localization of Cx43 as well as its phosphorylation status is responsive to environmental factors; thus, abnormal cardiac function may trigger a focally restricted abnormal pattern of Cx43 expression or posttranslational modification.37
The expected increase in myofibril Ca2+ sensitivity as a result of mutant cTnI expression was confirmed in the intact fibers and could play a major role in the induction of the cardiac FHC phenotype. However, the myocyte data (Figure 5) indicate no enhancement of myofilament Ca2+ responsiveness, contrary to our expectation based on the steady state skinned fiber experiments. Indeed, there was a slight tendency in the reverse direction (ie, reduced sensitivity). This may be a result of the dynamic interplay between Ca2+ binding to the myofilaments and the influence of other cytosolic Ca2+ buffers. Additionally, the sarcomeres are loaded in the fibers, whereas the isolated cardiomyocytes constitute an unloaded system, and thus the 2 preparations are at different points on the length-dependent activation curve. We were not able to detect alterations in either the amounts or phosphorylation patterns of the proteins responsible for Ca2+ handling such as phospholamban or SERCA2a, and Na-Ca2+ exchange levels were also unaffected (data not shown).
Histological and morphological alterations such as interstitial fibrosis, myocyte disarray, and cardiac hypertrophy can also be arrhythmogenic.3 However, although significant fibrosis could be detected by 18 months (Figure 3), areas of disarray, although apparent, were not widespread. Similarly, although there were spatial changes of Cx43 expression and alterations in the absolute levels of phosphorylated Cx43 in the cTnIR146G rabbits, it is doubtful that these are directly arrhythmogenic, and we were unable to detect the presence of malignant ventricular tachyarrhythmia in conscious 2-year-old cTnIR146G transgenic rabbits via continuous ECG monitoring. However, DFA, a more sensitive analytical modality, revealed statistically significant alterations in the QTmax intervals (Figure 6F). In cTnIR146G rabbits, the higher 1 parameter compared with the control group may be an early pathological sign before more obvious ECG changes in repolarization occur. The higher 1 value reflects an altered temporal fractal organization pattern of the QTmax interval. Fractal methods analyze the self-similar fluctuations on multiple different orders of temporal magnitude. Therefore, the transgenic rabbits demonstrated a more pronounced self-similarity of their repolarization phase, showing an altered pattern compared with nontransgenic rabbits. The clear separation of the study group from the control group with DFA was a reinforcing finding of altered fractal organization pattern. The altered fractal organization pattern with an increased value, in the absence of entropy or other ECG abnormalities, has been interpreted as a significant sign for a pathological change in the repolarization phase before the onset of more obvious abnormalities.38 As the transgenic rabbits age, it will be of interest to monitor them for ventricular tachyarrhythmias.
Acknowledgments
This study was supported by grants from the National Institutes of Health (Dr Robbins) and by a Beginning Grant-in-Aid from the American Heart Association (Dr Sanbe).
References
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Loyola University Chicago, Department of Physiology and Cardiovascular Institute, Maywood, Ill (K.S.G., D.M.B.)
National Institute on Aging, Intramural Research Program, Gerontology Research Center, National Institutes of Health, Baltimore, Md (B.Z., E.G.L.).
Abstract
Background— Transgenic and gene-targeted models have focused on the mouse. Fundamental differences between the mouse and human exist in Ca2+ handling during contraction/relaxation and in alterations in Ca2+ flux during heart failure, with the rabbit more accurately reflecting the human system.
Methods and Results— Cardiac troponin I (cTnI) mutations can cause familial hypertrophic cardiomyopathy. An inhibitory domain mutation, arginine146glycine (cTnI146Gly), was modeled with the use of transgenic expression in the rabbit ventricle. cTnI146Gly levels >40% of total cTnI were perinatally lethal, whereas replacement levels of 15% to 25% were well tolerated. cTnI146Gly expression led to a leftward shift in the force-pCa2+ curves with cardiomyocyte disarray, fibrosis, and altered connexin43 organization. In isolated cTnI146Gly myocytes, twitch relaxation amplitudes were smaller than in normal cells, but [Ca]i transients and sarcoplasmic reticulum Ca2+ load were not different. Detrended fluctuation analysis of the QTmax intervals was used to evaluate the cardiac repolarization phase and showed a significantly higher scaling exponent in the transgenic animals.
Conclusions— Expression of modest amounts of cTnI146Gly led to subtle defects without severely affecting cardiac function. Aberrant connexin organization, subtle morphological deficits, and an altered fractal pattern of the repolarization phase of transgenic rabbits, in the absence of entropy or other ECG abnormalities, may indicate an early developing pathology before the onset of more obvious repolarization abnormalities or major alterations in cardiac mechanics.
Key Words: cardiovascular diseases ; heart diseases ; hypertrophy
Introduction
Familial hypertrophic cardiomyopathy (FHC) displays an autosomal dominant mode of inheritance and a diverse genetic etiology: FHC or a phenocopy can be caused by multiple mutations in genes encoding various contractile, structural, channel, and kinase proteins.1,2 Commonly, arrhythmias, particularly ventricular tachycardia and fibrillation, are associated with sudden death.3,4 The absence of multiple FHC models that faithfully recapitulate important aspects of human disease limits our ability to explore the underlying disease mechanism(s) and develop therapeutic strategies.
See p 2276
Multiple mutations in the inhibitory subunit of cardiac troponin (cTnI) can cause hypertrophic cardiomyopathy.5,6 Previously, we modeled the cTnIR145G mutation (residue 146 in the human) in transgenic mice.7 Cardiac functional analysis revealed impaired relaxation, and permeabilized cardiac muscle fiber studies showed an increase in Ca2+ sensitivity. These and other data indicate that increased myofibril Ca2+ sensitivity in the cTnI FHC mutations may play a major pathological role.8
The rabbit has advantages, compared with the mouse, for studying cardiovascular disease.9 Substantial differences exist between the mouse and human in the manner in which Ca2+ is handled during contraction/relaxation and in alterations in Ca2+ flux during heart failure, with the rabbit more accurately reflecting the human system.10,11 Human atria express -myosin heavy chain (MHC), and the ventricles express predominantly ;-MHC. The adult rabbit heart reflects this, whereas the mouse expresses -MHC in both the postnatal atria and ventricles.12 That the mouse heart beats 10 times faster than the human heart is another limitation because the faster heart rate influences the refractory period associated with the occurrence of arrhythmia.13 To study the pathogenicity and presentation of an FHC cTnI mutation in the rabbit heart, we generated rabbits with high and low levels of ventricle-specific expression of cTnIR146G. Although high-expressing lines died perinatally, a low-expressing line survived and appeared outwardly normal. However, the cTnIR146G transgenic ventricles showed apical myocyte disarray, interstitial fibrosis, and mild ventricular hypertrophy at 1.5 to 2 years. Increased Ca2+ sensitivity was accompanied by altered patterns of connexin deposition at the gap junctions, whereas detrended fluctuation analysis (DFA) of the QTmax intervals in these rabbits showed a higher scaling exponent.
Methods
Isolation and Construction of the Rabbit ;-MHC Promoter and Transgenic Constructs
Rabbit ;-MHC sequences were obtained from a EMBL3 genomic library. An EcoRI site was introduced into the ;-MHC promoter, and a polyadenylation signal was placed downstream of the site (Figure 1). After insertion of the transgenes, DNA was digested free of vector sequence with NotI, purified, and used to generate transgenic rabbits. The rabbit cTnI cDNA was isolated with the use of reverse transcription–polymerase chain reaction (RT-PCR), and an arginineglycine missense mutation at position 146 was made with the use of PCR mutagenesis. A FLAG-epitope tag was introduced at the N-terminus by PCR. A wild-type cTnI construct (cTnIWT) was also made so that transgenic rabbits expressing FLAG-tagged cTnI at the same levels as the mutant cTnI could serve as controls.
Isolated Cardiomyocytes
Isolated myocytes were loaded with Indo-1 or Fluo-3, bathed in normal Tyrode’s solution with 2 to 2.5 mmol/L Ca2+, and stimulated at 0.5 Hz. Twitch fluorescence transients were recorded with a microscope-based photometer and converted to [Ca]i by standard methods.14 Contraction was recorded by video edge detection and expressed as the fractional change in resting cell length. Caffeine (10 mmol/L) was applied to some cells to measure sarcoplasmic reticulum Ca2+ loading and allow analysis of Ca2+ fluxes contributing to Ca2+ transient decay.15 Comparisons were based on data from 8 to 33 cells from 5 rabbits of either genotype. The significance level for differences of means was P0.05.
DFA of the QTmax Intervals
Four transgenic and 4 nontransgenic rabbits aged 21 months were used. The digital ECGs were obtained over a duration of 5 minutes, and the QTmax intervals (onset of QRS to T wave peak) and R-R intervals were measured. Uncorrected heart rate values of QTmax intervals were used for the analysis of repolarization variability assessment.16 DFA and the entropy of the QTmax intervals with the use of approximate and sample entropy methods were also evaluated. The data were analyzed by these nonlinear methods to assess possible abnormal changes of the heart rate dynamics. The standard deviation (SD) of QTmax and the square root of the mean squared differences of successive repolarization intervals were calculated by standard methods of variability analysis.
Miscellaneous Methods
Myofibrillar sample preparation, gel preparation, electrophoretic conditions, and gel staining have been described.17 Western analyses were performed with the use of anti-TnI, anti-GAPDH (Chemicon International), anti-FLAG (Sigma), anti-connexin43 (Zymed Laboratories Inc), and [Ser 368] anti–phospho-connexin43 (Cell Signaling Technologies). Immunohistochemistry and fiber isolation and analyses have been described.18 Quantitative analysis of interstitial fibrosis was measured in formalin-fixed and paraffin-embedded tissue sections with the use of Sirius red F3BA and fast green FCF.19 After the dye was eluted from the tissue sections, the absorbance at 540 and 605 nm was determined for Sirius red F3BA and fast green FCF binding protein, respectively.
Results
Ventricle-Restricted Transgenic Expression in the Rabbit
Previous transgenic studies affecting the protein complement of the rabbit heart used the mouse ;-MHC promoter.9,20 Although the mouse promoter shows striated muscle specific expression, it does not completely mimic endogenous ;-MHC expression in the rabbit.20 Therefore, we isolated and tested the rabbit ;-MHC promoter in an attempt to drive high levels of transgene expression in the rabbit ventricle. The basic promoter construct consists of 6000 bp of sequence upstream of the transcriptional start site, as well as the exons that encode the 5 untranslated region. A polyadenylation signaling sequence, derived from the human growth hormone gene, is inserted downstream of the unique EcoRI restriction enzyme site, into which the various transgenes can be inserted (Figure 1A). In preliminary experiments, the gene encoding chloramphenicol acetyltransferase was used to titrate promoter activity. Multiple lines were generated, and chloramphenicol acetyltransferase (CAT) expression was determined. The promoter drove high levels of expression in all regions of the ventricle in a copy number–dependent manner as well as in muscles containing slow-type fibers (Figure 1B to 1D). In the atria, no expression was detected in 3 lines and only very low expression in 1 line showing high copy numbers (428H, 39 copies), suggesting that the rabbit ;-MHC promoter drives predominantly ventricular-specific transcription, mimicking endogenous expression. Expression was stable throughout the juvenile and adult periods, and the expression patterns were conserved across multiple lines (data not shown). CAT expression was also determined during embryogenesis. At embryonic day 10, robust expression occurred in the ventricle and outflow tract, with relatively low levels of expression in the atria (Figure 1E), a pattern consistent with endogenous promoter activity in the mouse21 and rabbit (J. James, MD, and J. Robbins, PhD, unpublished data, 2003). To determine homogeneity of CAT expression in the postbirth ventricle, in situ immunohistochemistry was performed on different regions. Transgene expression was homogeneous throughout (Figure 1F, 1G), and the data show that the rabbit ;-MHC promoter efficiently drives ventricular-restricted and slow-type skeletal muscle transgene expression.
Cardiac TnIR146G Transgenic Expression
We used the rabbit ;-MHC promoter to create rabbits in which cTnI was partially replaced by either cTnIR146G or cTnIWT in the ventricle (Figure 2A). Three cTnIR146G transgenic founders and 1 cTnIWT transgenic line were generated. In lines 52 and 71, with copy numbers of 5 and 4, respectively, the mutant protein accounted for >50% of total cTnI by the neonatal stage (Figure 2C). As described by us and others for both cTnI and other contractile proteins, increases observed at the RNA level are not translated into absolute increases of protein.7,22 Rather, endogenous protein levels are reduced such that contractile protein stoichiometry is conserved, resulting in replacement of the endogenous protein with the transgenically encoded species (Figure 2B).23 In both lines, death occurred within 1 week after birth. No obvious phenotype presented in the cTnIWT line, in which the transgenic protein replaced 64% of the endogenous cTnI (Figure 2C), indicating that transgenic expression of cTnI, by itself, was not lethal. At the time of death, the pups from both lines 52 and 71 showed symptoms consistent with heart failure, and electron microscopy confirmed major sarcomere disruption and contractures (data not shown). Premature death was also observed in the severely affected cTnIR146G transgenic mice,7 confirming that >40% to 50% replacement with cTnIR146G is lethal.
Line 51, with a copy number of 2 and protein replacement of 28% (Figure 2C, 2D), was asymptomatic. Mutant protein levels were maintained equally in both adult ventricles (Figure 2E). In the young adults, no obvious cardiac remodeling occurred (Table 1), nor were there any histological abnormalities (data not shown). However, by 18 months there was significant fibrosis in the septum and apex and isolated areas of myocyte disarray (Figure 3), whereas no abnormalities could be detected in either the nontransgenic or cTnIWT transgenic hearts. Interestingly, histological abnormalities were only detected in focal areas of the ventricle despite a uniform mutant protein distribution. Echocardiography detected a slight but significant increase in left ventricular wall thickness, as well as increased septal thickness and improved fractional shortening (Table 2).
Fiber and Cardiomyocyte Studies
In the cTnIR146G transgenic mice, functional analyses revealed impaired relaxation, and fiber studies showed an increase in Ca2+ sensitivity.7 In vitro reconstitution experiments have shown that FHC cTnI mutations (cTnIR146G and cTnIR162W) resulted in reduced inhibition of myosin ATPase by the troponin complex and increased Ca2+ sensitivity of actin-tropomyosin–activated myosin ATPase activity.8 To determine the effect of cTnIR146G on the development of force, we generated force-pCa2+ curves using permeabilized muscle fibers. To obtain a dose response, neonatal fibers were used because line 52, in which 55% replacement was obtained, died between 5 to 9 days after birth. Before the development of overt heart failure in this line, fibers were isolated from the left ventricular papillary muscles and compared with fibers derived from line 51 (28% replacement) and nontransgenic animals. A marked leftward shift of the force-pCa2+ curve was observed in line 52, and a slight leftward shift was seen in line 51 (Figure 4), indicating that the mutant protein affected myofibrillar Ca2+ sensitivity in a dose-dependent but nonlinear manner.
Despite the changes in Ca2+ sensitivity, functional parameters determined by cardiac catheterization were conserved at 6 months (Table 1), with modestly enhanced cardiac function apparent by 2 years (Table 2). These increases are consistent with the data obtained in the cTnIR146G transgenic mice as well as in vitro reconstitution studies.7,8 At the whole-organ level, it appears that increased Ca2+ sensitivity results in modest increases in cardiac function, mild hypertrophy, and focal cardiomyocyte abnormalities.
Data that bear directly on the effect(s) of changes in cTnI on isolated cardiomyocytes are limited, although recent data suggest that, in principle, one should be able to detect alterations in myofilament sensitivity in isolated myocyte experiments.24 Isolated cardiomyocytes were therefore derived from nontransgenic and transgenic cTnIR146G hearts. Figure 5A shows [Ca]i transients followed by a caffeine-induced Ca2+ transient in a transgenic myocyte. Peak fractional shortening was not different, but there was a trend toward larger [Ca]i transient amplitude in the transgenic cells (Figure 5B). These relative amplitudes did not give any indication of increased myofilament Ca2+ sensitivity despite the results in skinned fibers. Twitch relaxation kinetics were unaltered, whereas [Ca]i decline was slightly faster in the transgenic myocytes (Figure 5C). From caffeine-induced Ca2+ transients, we inferred that sarcoplasmic reticulum Ca2+ loading was not different between transgenic and nontransgenic cells (Figure 5D). However, [Ca]i decline was faster in transgenic cells, indicative of greater Na-Ca2+ exchange function in the transgenic rabbits (Figure 5D). The apparently faster Na-Ca2+ exchange rate may partly explain the faster twitch [Ca]i decline (Figure 5C). [Ca]i transients and contraction were also measured with [Ca]o=1 mmol/L or 5 mmol/L and at different frequencies (0.2, 0.5, and 1 Hz) with similar trends. The expected Ca2+ and frequency effects on amplitude and kinetics of the Ca2+ transient and contraction did not differ between the 2 groups (data not shown).
We reasoned that despite no increase in mortality or overt presentation of disease, the visible areas of myocyte disarray might lead to subtle alterations in the gap junctions and electrophysiology of the hearts. Connexin43 (Cx43) is the major component of the ventricular gap junction.25 Changes in subcellular distribution or structural remodeling have been correlated with cardiomyocytes in distress, disarray, border infarct zones, acute responses to injury, or the initial stages of compensated hypertrophy.26,27 Absolute levels of Cx43 were significantly increased at 21 months, but their organization at the gap junction was compromised in the cTnIR146G ventricles (Figure 6A, 6B). Cx43 phosphorylation results in a reduction in gap-junctional intercellular communication,28 and we noted that phospho-Cx43 levels were also significantly elevated (Figure 6E). Elevated levels of Cx43 and phospho-Cx43 were found throughout the heart despite the focal pattern of interstitial fibrosis and myocyte disarray, which were largely restricted to the apical and septal regions. Although Cx43 heterogeneity of expression as well as the absolute levels may predispose the heart to arrhythmias,26 and increases observed in Cx43 phosphorylation can lead to impaired intracellular conduction,29 ECG studies were inconclusive, with no abnormalities presenting (Figure 6C, 6D). To increase the resolution of the study, the cardiac repolarization phase of the digitized ECGs was subjected to DFA (Figure 6F). The QTmax interval was significantly higher in the cTnIR146G rabbits than in the nontransgenic group.
Discussion
The extent of expression and incorporation of mutant cTnI in human cardiomyocytes is not known, although presumably most patients have 1 mutant allele.5 A recent study in which isolated adult rat ventricular myocytes were used suggested that incorporation of cTnIR146G differed significantly from that of the normal protein. Maximum protein replacement of endogenous cTnI with normal cTnI reached 85%, with significantly less replacement (43%) achieved when cTnIR146G was expressed.32 These results suggest that the capacity of myocytes to express and incorporate cTnIR146G into the sarcomere is significantly impaired compared with normal cTnI. This may be due either to mutant protein instability or to a reduced ability of cTnIR146G, relative to cTnI, to incorporate into myofilaments. Consistent with this hypothesis, an FHC-mutant ;-MHC in the soleus represented only 12% to 23% of the ;-myosin.33 The rabbit data show nonviability in lines 52 and 71, in which replacement reached 50%, and it appears that, for this isoform in the rabbit, 50% replacement is not compatible with life.
The lethality of the mutation in the transgenic rabbits is clearly dependent on the degree of replacement by the mutant protein. However, the relationship is not linear but rather exhibits a threshold effect. If the mutant protein is incorporated at >50% of the total cTnI, the muscle fibers exhibit a marked increase in Ca2+ sensitivity, and all of the F1 rabbits die between 5 and 7 days after birth. In contrast, line 51, with 28% replacement, shows only a slight increase in muscle fiber Ca2+ sensitivity and no lethality. In vitro reconstitution studies with mixtures of wild-type and mutant cTnIs show that the effects on Ca2+ sensitivity and in vitro motility are complex and nonlinear.34,35 The data have been interpreted to mean that wild-type cTnI can compensate up to a point for the compromised ATPase inhibitory ability of the mutant.35 This consideration and the biphasic nature of the transition observed with mixtures of different cTnIs emphasize the nonlinearity of the response. This is consistent with the rabbit data, in which a relatively mild phenotype is observed in line 51 compared with the other transgenic lines (52 and 71), as well as with our previous data from the transgenic mice.7 Although the exact degree of replacement by the mutant protein in FHC patients is unknown, our results may help to explain the mechanism underlying why patients with hypertrophic cardiomyopathy linked to this specific cTnI mutation have a relatively mild phenotype with elderly onset penetration35,36 and show that the limits of substitution tolerated lie below 50%.
The histological and morphological alterations observed in the mutant transgenic rabbits were asymmetrical and progressive. Because rabbit ;-MHC promoter activity is relatively uniform in the heart (Figure 1), heterogeneous localization of the mutant protein is an unlikely explanation for the focal defects. The mechanism responsible for the heterogeneity of pathology is unknown. One possible explanation is an increase in contractility, as reflected by a slight enhancement of fractional shortening. Slight but sustained alterations in regional contractility may compromise myocardial blood flow, triggering regional ischemia and/or hypoxia, which can cause histological and morphological alterations.3,4 As an example, the cellular localization of Cx43 as well as its phosphorylation status is responsive to environmental factors; thus, abnormal cardiac function may trigger a focally restricted abnormal pattern of Cx43 expression or posttranslational modification.37
The expected increase in myofibril Ca2+ sensitivity as a result of mutant cTnI expression was confirmed in the intact fibers and could play a major role in the induction of the cardiac FHC phenotype. However, the myocyte data (Figure 5) indicate no enhancement of myofilament Ca2+ responsiveness, contrary to our expectation based on the steady state skinned fiber experiments. Indeed, there was a slight tendency in the reverse direction (ie, reduced sensitivity). This may be a result of the dynamic interplay between Ca2+ binding to the myofilaments and the influence of other cytosolic Ca2+ buffers. Additionally, the sarcomeres are loaded in the fibers, whereas the isolated cardiomyocytes constitute an unloaded system, and thus the 2 preparations are at different points on the length-dependent activation curve. We were not able to detect alterations in either the amounts or phosphorylation patterns of the proteins responsible for Ca2+ handling such as phospholamban or SERCA2a, and Na-Ca2+ exchange levels were also unaffected (data not shown).
Histological and morphological alterations such as interstitial fibrosis, myocyte disarray, and cardiac hypertrophy can also be arrhythmogenic.3 However, although significant fibrosis could be detected by 18 months (Figure 3), areas of disarray, although apparent, were not widespread. Similarly, although there were spatial changes of Cx43 expression and alterations in the absolute levels of phosphorylated Cx43 in the cTnIR146G rabbits, it is doubtful that these are directly arrhythmogenic, and we were unable to detect the presence of malignant ventricular tachyarrhythmia in conscious 2-year-old cTnIR146G transgenic rabbits via continuous ECG monitoring. However, DFA, a more sensitive analytical modality, revealed statistically significant alterations in the QTmax intervals (Figure 6F). In cTnIR146G rabbits, the higher 1 parameter compared with the control group may be an early pathological sign before more obvious ECG changes in repolarization occur. The higher 1 value reflects an altered temporal fractal organization pattern of the QTmax interval. Fractal methods analyze the self-similar fluctuations on multiple different orders of temporal magnitude. Therefore, the transgenic rabbits demonstrated a more pronounced self-similarity of their repolarization phase, showing an altered pattern compared with nontransgenic rabbits. The clear separation of the study group from the control group with DFA was a reinforcing finding of altered fractal organization pattern. The altered fractal organization pattern with an increased value, in the absence of entropy or other ECG abnormalities, has been interpreted as a significant sign for a pathological change in the repolarization phase before the onset of more obvious abnormalities.38 As the transgenic rabbits age, it will be of interest to monitor them for ventricular tachyarrhythmias.
Acknowledgments
This study was supported by grants from the National Institutes of Health (Dr Robbins) and by a Beginning Grant-in-Aid from the American Heart Association (Dr Sanbe).
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