This Article Abstract Full Text (PDF) All Versions of this Article: 111/18/2330    most recent 01.CIR.0000164234.24957.75v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sanbe, A. Articles by Robbins, J. Search for Related Content PubMed PubMed Citation Articles by Sanbe, A. Articles by Robbins, J. Related Collections Contractile function Animal models of human disease Related Article

(Circulation. 2005;111:2330-2338.)
© 2005 American Heart Association, Inc.

Molecular Cardiology

Transgenic Rabbit Model for Human Troponin I–Based Hypertrophic Cardiomyopathy

Atsushi Sanbe, PhD; Jeanne James, MD; Volkan Tuzcu, MD; Selman Nas, PhD; Lisa Martin, AS; James Gulick, MS; Hanna Osinska, PhD; Sadayappan Sakthivel, PhD; Raisa Klevitsky, PhD; Kenneth S. Ginsburg, PhD; Donald M. Bers, PhD; Bruce Zinman, MS; Edward G. Lakatta, MD; Jeffrey Robbins, PhD

From 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.); and National Institute on Aging, Intramural Research Program, Gerontology Research Center, National Institutes of Health, Baltimore, Md (B.Z., E.G.L.).

Correspondence to Jeffrey Robbins, PhD, Division of Molecular Cardiovascular Biology, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail jeff.robbins{at}cchmc.org

Received August 2, 2004; revision received December 29, 2004; accepted January 4, 2005.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
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 (cTnI^146Gly), was modeled with the use of transgenic expression in the rabbit ventricle. cTnI^146Gly levels >40% of total cTnI were perinatally lethal, whereas replacement levels of 15% to 25% were well tolerated. cTnI^146Gly expression led to a leftward shift in the force-pCa²⁺ curves with cardiomyocyte disarray, fibrosis, and altered connexin43 organization. In isolated cTnI^146Gly 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 QT_max 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 cTnI^146Gly 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

Top Abstract Introduction Methods Results Discussion References

 
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 cTnI^R145G 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 ß-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 cTnI^R146G. Although high-expressing lines died perinatally, a low-expressing line survived and appeared outwardly normal. However, the cTnI^R146G 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 QT_max intervals in these rabbits showed a higher scaling exponent.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Isolation and Construction of the Rabbit ß-MHC Promoter and Transgenic Constructs
Rabbit ß-MHC sequences were obtained from a EMBL3 genomic library. An EcoRI site was introduced into the ß-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 (cTnI^WT) was also made so that transgenic rabbits expressing FLAG-tagged cTnI at the same levels as the mutant cTnI could serve as controls.

View larger version (55K): [in this window] [in a new window]   Figure 1. Transgenic modulation in the rabbit heart. A, Construct organization. The promoter region was isolated, and an EcoRI site was introduced as a unique cloning site in the noncoding region of exon 3. A fragment containing the human growth hormone polyadenylation signal (hGH polyA) was inserted, and the reporter gene cat was used to measure transcriptional activity. B to D, Rabbit ß-MHC promoter activity. Cardiac muscle (B), the slow skeletal diaphragm and soleus muscles (C), and other muscle and nonmuscle tissues were examined (D). E, Embryonic day 10. Staining is shown in the ventricle and outflow tract as well as minor staining in the atrium. A indicates atrium, V; ventricle, OT; outflow tract. F and G, Immunohistochemistry showed no signal in the nontransgenic ventricle (F) and uniform CAT staining in the transgenic apex (G). Tissues were derived from 5-week-old animals (n=3 to 4). LV indicates left ventricle; RV, right ventricle; LAtr, left atria; RAtr, right atria; Dia, diaphragm; Sol, soleus muscle; Bic, biceps muscle; Tibi, tibial muscle; Mas, masseter muscle; Ton, tongue; Sto, stomach; S.In, small intestine; Lun, lung; Liv, liver; and Sple, spleen.

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 QT_max 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 QT_max intervals (onset of QRS to T wave peak) and R-R intervals were measured. Uncorrected heart rate values of QT_max intervals were used for the analysis of repolarization variability assessment.¹⁶ DFA and the entropy of the QT_max 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 QT_max 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

Top Abstract Introduction Methods Results Discussion References

 
Ventricle-Restricted Transgenic Expression in the Rabbit
Previous transgenic studies affecting the protein complement of the rabbit heart used the mouse ß-MHC promoter.^9,20 Although the mouse promoter shows striated muscle specific expression, it does not completely mimic endogenous ß-MHC expression in the rabbit.²⁰ Therefore, we isolated and tested the rabbit ß-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 ß-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 ß-MHC promoter efficiently drives ventricular-restricted and slow-type skeletal muscle transgene expression.

Cardiac TnI^R146G Transgenic Expression
We used the rabbit ß-MHC promoter to create rabbits in which cTnI was partially replaced by either cTnI^R146G or cTnI^WT in the ventricle (Figure 2A). Three cTnI^R146G transgenic founders and 1 cTnI^WT 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 cTnI^WT 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 cTnI^R146G transgenic mice,⁷ confirming that >40% to 50% replacement with cTnI^R146G is lethal.

View larger version (31K): [in this window] [in a new window]   Figure 2. cTnIR146G protein levels in transgenic rabbit hearts at 6 months. A, Transgenic constructs for expressing either wild-type (WT) cTnI or cTnIR146G. B, Conservation of overall cTnI and sarcomeric protein levels in transgenic (TG) animals. Shown is a PAGE analysis that compares nontransgenic (NTG) and line 51 sarcomeric proteins. As expected from previous experiments with cardiac-specific expression of the sarcomeric proteins,7,23 the absolute levels are conserved. Line 15 (cTnIWT) proteins were indistinguishable as well (data not shown). C, Three cTnIR146G lines (lines 51, 52, and 71) and 1 WT line (line 15) expressed high levels of transgenic protein. The degree of replacement at the neonatal stage is shown. D and E, At 6 months in line 51, cTnIR146G made up 28% of the total cTnI complement. E, Equal amounts of cTnIR146G were detected in both the left ventricle (LV) and right ventricle (RV). Atr indicates atria.

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 cTnI^WT 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).

View this table: [in this window] [in a new window]   TABLE 1. Tissue Weights and Hemodynamic Parameters

View larger version (72K): [in this window] [in a new window]   Figure 3. A, Apical and septal sections from 18-month nontransgenic (NTG), cTnIWT (WT), and cTnIR146G (R146G) hearts. Sections were stained with Masson’s trichrome. Bar=125 µm. B, Interstitial fibrosis was quantified with the use of Sirius red F3BA and fast green FCF as described in Methods. LV indicates left ventricle; Sep, septum; and RV, right ventricle. *P<0.05 vs nontransgenic.

View this table: [in this window] [in a new window]   TABLE 2. Echocardiographic Parameters

Fiber and Cardiomyocyte Studies
In the cTnI^R146G 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 (cTnI^R146G and cTnI^R162W) 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 cTnI^R146G 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.

View larger version (22K): [in this window] [in a new window]   Figure 4. Force-pCa2+ curves of skinned fibers from ventricular papillary muscle. Force (A) and normalized force (B) were graphed. The ventricular skinned fibers were obtained from neonatal left ventricular trabeculae. Data were analyzed by 1-way ANOVA. No alterations in maximum shortening velocity were observed (data not shown). NTG indicates nontransgenic. *P<0.05 vs nontransgenic; **P<0.01 vs nontransgenic; ***P<0.001, #P<0.05 vs line 51; ##P<0.01 vs line 51.

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 cTnI^R146G 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 cTnI^R146G 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).

View larger version (25K): [in this window] [in a new window]   Figure 5. Isolated myocyte studies. A, Twitch Ca transients and a caffeine-induced Ca2+ transient in a transgenic myocyte. B, Mean data for twitch parameters at 0.5 Hz steady state are shown for amplitudes of contraction (as percentage of resting cell length [rcl]) and Ca2+ transient (as [Ca]i). C, Kinetics of relaxation half-time (t1/2) and time constant () of [Ca]i decline. D, Caffeine-induced Ca2+ transients were analyzed to infer sarcoplasmic reticulum Ca2+ load and Na-Ca2+ exchange (NCX) function, which is the primary mechanism responsible for the rate of [Ca]i decline in the presence of caffeine.14 NTG indicates nontransgenic; TG, transgenic; and SR, sarcoplasmic reticulum. *P<0.05 based on Student t test; #P<0.05 based on Mann-Whitney test.

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 cTnI^R146G 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 QT_max interval was significantly higher in the cTnI^R146G rabbits than in the nontransgenic group.

View larger version (57K): [in this window] [in a new window]   Figure 6. Cx43 expression and phosphorylation. A and B, Patterns of Cx43 localization in sections derived from the nontransgenic (NTG) and 6-month cTnIR146G hearts. Arrowheads show regions of aberrant accumulation of Cx43. C and D, Representative ECGs. Lead I tracing is from a nontransgenic and line 51 rabbit, respectively. The time and voltage scales are indicated. E, Western blots of cardiac samples derived from 21-month cTnIR146G and nontransgenic rabbits. Both total and phospho-Cx43 were elevated in the cTnIR146G hearts. The broadening of the transgenic (TG) bands in the top panel is due to the retarded migration of the phosphorylated species, which, if small amounts of protein are electrophoresed, can be resolved from the nonphosphorylated species. LV indicates left ventricle; RV, right ventricle. F, DFA in nontransgenic and transgenic rabbits. DFA of nontransgenic and transgenic groups was used to quantify the fractal correlation properties of the R-R interval data. This method is a modified root mean square analysis of a random walk (=0.85±0.13, =0.62±0.04, respectively; P=0.015).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Patients suffering from FHC as a result of mutations in cTnI are often asymptomatic, with the disease being diagnosed only after sudden death occurs. Previously, we constructed cTnI^R146G transgenic mice that, although displaying increased Ca²⁺ sensitivity, never showed hypertrophy or cellular pathology, except in parous females.⁷ This lack of pathology, even at high replacement levels, is significantly different from the rabbit model because the 2-year-old transgenic rabbit population that had only 25% replacement displayed significant levels of interstitial fibrosis and focal areas of myocyte disarray as well as prolongation of the QT_max interval and alterations in Cx43 distribution and phosphorylation. Indeed, rabbits that exhibited replacement levels of 40%, which approached those obtained in the mouse, did not survive past the perinatal period. The relative strengths of the rabbit versus the mouse for studying cardiovascular disease have been discussed above and in other studies.^9,20,30,31 Major weaknesses of the rabbit revolve around the time it takes to raise the animals and the innate costs of dealing with a larger animal model. The mouse and rabbit models are compared in Table 3. The data suggest that the rabbit model more closely resembles human disease relative to the mouse and, when the cellular pathology and early mortality that distinguishes the 2 models are considered, may provide a unique tool to investigate the pathogenic mechanisms and evaluate potential therapies.

View this table: [in this window] [in a new window]   TABLE 3. Comparison Between the Mouse and Rabbit cTnI Models

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 cTnI^R146G 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 cTnI^R146G was expressed.³² These results suggest that the capacity of myocytes to express and incorporate cTnI^R146G 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 cTnI^R146G, relative to cTnI, to incorporate into myofilaments. Consistent with this hypothesis, an FHC-mutant ß-MHC in the soleus represented only 12% to 23% of the ß-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 penetration^35,36 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 ß-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 cTnI^R146G 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 cTnI^R146G transgenic rabbits via continuous ECG monitoring. However, DFA, a more sensitive analytical modality, revealed statistically significant alterations in the QT_max intervals (Figure 6F). In cTnI^R146G 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 QT_max 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

Top Abstract Introduction Methods Results Discussion References

 
1. Marian AJ, Roberts R. The molecular genetic basis for hypertrophic cardiomyopathy. J Mol Cell Cardiol. 2001; 33: 655–670.[CrossRef][Medline] [Order article via Infotrieve]

2. Hernandez OM, Housmans PR, Potter JD. Invited review: pathophysiology of cardiac muscle contraction and relaxation as a result of alterations in thin filament regulation. J Appl Physiol. 2001; 90: 1125–1136.[Abstract/Free Full Text]

3. Maron BJ, Bonow RO, Cannon RO III, Leon MB, Epstein SE. Hypertrophic cardiomyopathy: interrelations of clinical manifestations, pathophysiology, and therapy (1). N Engl J Med. 1987; 316: 780–789.[Medline] [Order article via Infotrieve]

4. Maron BJ, Bonow RO, Cannon RO III, Leon MB, Epstein SE. Hypertrophic cardiomyopathy. Interrelations of clinical manifestations, pathophysiology, and therapy (2). N Engl J Med. 1987; 316: 844–852.[Medline] [Order article via Infotrieve]

5. Kimura A, Harada H, Park JE, Nishi H, Satoh M, Takahashi M, Hiroi S, Sasaoka T, Ohbuchi N, Nakamura T, Koyanagi T, Hwang TH, Choo JA, Chung KS, Hasegawa A, Nagai R, Okazaki O, Nakamura H, Matsuzaki M, Sakamoto T, Toshima H, Koga Y, Imaizumi T, Sasazuki T. Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat Genet. 1997; 16: 379–382.[CrossRef][Medline] [Order article via Infotrieve]

6. Morner S, Richard P, Kazzam E, Hainque B, Schwartz K, Waldenstrom A. Deletion in the cardiac troponin I gene in a family from northern Sweden with hypertrophic cardiomyopathy. J Mol Cell Cardiol. 2000; 32: 521–525.[CrossRef][Medline] [Order article via Infotrieve]

7. 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.[Abstract/Free Full Text]

8. Takahashi-Yanaga F, Morimoto S, Harada K, Minakami R, Shiraishi F, Ohta M, Lu QW, Sasaguri T, Ohtsuki I. Functional consequences of the mutations in human cardiac troponin I gene found in familial hypertrophic cardiomyopathy. J Mol Cell Cardiol. 2001; 33: 2095–2107.[CrossRef][Medline] [Order article via Infotrieve]

9. Marian AJ, Wu Y, Lim DS, McCluggage M, Youker K, Yu QT, Brugada R, DeMayo F, Quinones M, Roberts R. A transgenic rabbit model for human hypertrophic cardiomyopathy. J Clin Invest. 1999; 104: 1683–1692.[Medline] [Order article via Infotrieve]

10. Bers DM. Cardiac Na/Ca exchange function in rabbit, mouse and man: what’s the difference? J Mol Cell Cardiol. 2002; 34: 369–373.[CrossRef][Medline] [Order article via Infotrieve]

11. Bers DM. Cardiac excitation-contraction coupling. Nature. 2002; 415: 198–205.[CrossRef][Medline] [Order article via Infotrieve]

12. Everett AW, Clark WA, Chizzonite RA, Zak R. Change in synthesis rates of alpha- and beta-myosin heavy chains in rabbit heart after treatment with thyroid hormone. J Biol Chem. 1983; 258: 2421–2425.[Abstract/Free Full Text]

13. Boyett MR, Jewell BR. A study of the factors responsible for rate-dependent shortening of the action potential in mammalian ventricular muscle. J Physiol. 1978; 285: 359–380.[Abstract/Free Full Text]

14. Ginsburg KS, Bers DM. Modulation of excitation-contraction coupling by isoproterenol in cardiomyocytes with controlled SR Ca2+ load and Ca2+ current trigger. J Physiol. 2004; 556: 463–480.[Abstract/Free Full Text]

15. Bassani JW, Bassani RA, Bers DM. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol. 1994; 476: 279–293.[Abstract/Free Full Text]

16. Perkiomaki JS, Couderc JP, Daubert JP, Zareba W. Temporal complexity of repolarization and mortality in patients with implantable cardioverter defibrillators. Pacing Clin Electrophysiol. 2003; 26: 1931–1936.[CrossRef][Medline] [Order article via Infotrieve]

17. Sanbe A, Fewell JG, Gulick J, Osinska H, Lorenz J, Hall DG, Murray LA, Kimball TR, Witt SA, Robbins J. Abnormal cardiac structure and function in mice expressing nonphosphorylatable cardiac regulatory myosin light chain 2. J Biol Chem. 1999; 274: 21085–21094.[Abstract/Free Full Text]

18. Sanbe A, Gulick J, Hanks MC, Liang Q, Osinska H, Robbins J. Reengineering inducible cardiac-specific transgenesis with an attenuated myosin heavy chain promoter. Circ Res. 2003; 92: 609–616.[Abstract/Free Full Text]

19. Chiu YT, Liu SK, Liu M, Chen SP, Lin YH, Mao SJ, Chu R. Characterization and quantitation of extracellular collagen matrix in myocardium of pigs with spontaneously occurring hypertrophic cardiomyopathy. Cardiovasc Pathol. 1999; 8: 169–175.[CrossRef][Medline] [Order article via Infotrieve]

20. James J, Sanbe A, Yager K, Martin L, Klevitsky R, Robbins J. Genetic manipulation of the rabbit heart via transgenesis. Circulation. 2000; 101: 1715–1721.[Abstract/Free Full Text]

21. Knotts S, Sanchez A, Rindt H, Robbins J. Developmental modulation of a ß myosin heavy chain promoter–driven transgene. Dev Dyn. 1996; 206: 182–192.[CrossRef][Medline] [Order article via Infotrieve]

22. Fewell JG, Hewett TE, Sanbe A, Klevitsky R, Hayes E, Warshaw D, Maughan D, Robbins J. Functional significance of cardiac myosin essential light chain isoform switching in transgenic mice. J Clin Invest. 1998; 101: 2630–2639.[Medline] [Order article via Infotrieve]

23. Sanbe A, Gulick J, Hayes E, Warshaw D, Osinska H, Chan CB, Klevitsky R, Robbins J. Myosin light chain replacement in the heart. Am J Physiol. 2000; 279: H1355–H1364.

24. Layland J, Grieve DJ, Cave AC, Sparks E, Solaro RJ, Shah AM. Essential role of troponin I in the positive inotropic response to isoprenaline in mouse hearts contracting auxotonically. J Physiol. 2004; 556: 835–847.[Abstract/Free Full Text]

25. Severs NJ. Cardiac muscle cell interaction: from microanatomy to the molecular make-up of the gap junction. Histol Histopathol. 1995; 10: 481–501.[Medline] [Order article via Infotrieve]

26. Severs NJ, Coppen SR, Dupont E, Yeh HI, Ko YS, Matsushita T. Gap junction alterations in human cardiac disease. Cardiovasc Res. 2004; 62: 368–377.[Abstract/Free Full Text]

27. Peters NS. New insights into myocardial arrhythmogenesis: distribution of gap-junctional coupling in normal, ischaemic and hypertrophied human hearts. Clin Sci (Lond). 1996; 90: 447–452.[Medline] [Order article via Infotrieve]

28. Ogawa T, Hayashi T, Kyoizumi S, Kusunoki Y, Nakachi K, MacPhee DG, Trosko JE, Kataoka K, Yorioka N. Anisomycin downregulates gap-junctional intercellular communication via the p38 MAP-kinase pathway. J Cell Sci. 2004; 117: 2087–2096.[Abstract/Free Full Text]

29. Moreno AP, Saez JC, Fishman GI, Spray DC. Human connexin43 gap junction channels: regulation of unitary conductances by phosphorylation. Circ Res. 1994; 74: 1050–1057.[Abstract/Free Full Text]

30. Patel R, Nagueh SF, Tsybouleva N, Abdellatif M, Lutucuta S, Kopelen HA, Quinones MA, Zoghbi WA, Entman ML, Roberts R, Marian AJ. Simvastatin induces regression of cardiac hypertrophy and fibrosis and improves cardiac function in a transgenic rabbit model of human hypertrophic cardiomyopathy. Circulation. 2001; 104: 317–324.[Abstract/Free Full Text]

31. Sanbe A, Gulick JD, Martin L, Yager K, Robbins J. Construction and characterization of cardiac specific promoters for rabbit transgenesis. Circulation. 2000; 102: 212–218.

32. 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.[Abstract/Free Full Text]

33. Nier V, Schultz I, Brenner B, Forssmann W, Raida M. Variability in the ratio of mutant to wildtype myosin heavy chain present in the soleus muscle of patients with familial hypertrophic cardiomyopathy: a new approach for the quantification of mutant to wildtype protein. FEBS Lett. 1999; 461: 246–252.[CrossRef][Medline] [Order article via Infotrieve]

34. Redwood C, Lohmann K, Bing W, Esposito GM, Elliott K, Abdulrazzak H, Knott A, Purcell I, Marston S, Watkins H. Investigation of a truncated cardiac troponin T that causes familial hypertrophic cardiomyopathy: Ca(2+) regulatory properties of reconstituted thin filaments depend on the ratio of mutant to wild-type protein. Circ Res. 2000; 86: 1146–1152.[Abstract/Free Full Text]

35. Elliott K, Watkins H, Redwood CS. Altered regulatory properties of human cardiac troponin I mutants that cause hypertrophic cardiomyopathy. J Biol Chem. 2000; 275: 22069–22074.[Abstract/Free Full Text]

36. Arad M, Seidman JG, Seidman CE. Phenotypic diversity in hypertrophic cardiomyopathy. Hum Mol Genet. 2002; 11: 2499–2506.[Abstract/Free Full Text]

37. Beardslee MA, Lerner DL, Tadros PN, Laing JG, Beyer EC, Yamada KA, Kleber AG, Schuessler RB, Saffitz JE. Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia. Circ Res. 2000; 87: 656–662.[Abstract/Free Full Text]

38. Miorelli M, Buja G, Melacini P, Fasoli G, Nava A. QT-interval variability in hypertrophic cardiomyopathy patients with cardiac arrest. Int J Cardiol. 1994; 45: 121–127.[CrossRef][Medline] [Order article via Infotrieve]

Related Article:

On Mice, Rabbits, and Human Heart Failure

A.J. Marian
Circulation 2005 111: 2276-2279. [Extract] [Full Text]

This article has been cited by other articles:

				Y. Wen, J. R. Pinto, A. V. Gomes, Y. Xu, Y. Wang, Y. Wang, J. D. Potter, and W. G. L. Kerrick Functional Consequences of the Human Cardiac Troponin I Hypertrophic Cardiomyopathy Mutation R145G in Transgenic Mice J. Biol. Chem., July 18, 2008; 283(29): 20484 - 20494. [Abstract] [Full Text] [PDF]

				J. Du, J. Liu, H.-Z. Feng, M. M. Hossain, N. Gobara, C. Zhang, Y. Li, P.-Y. Jean-Charles, J.-P. Jin, and X.-P. Huang Impaired relaxation is the main manifestation in transgenic mice expressing a restrictive cardiomyopathy mutation, R193H, in cardiac TnI Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2604 - H2613. [Abstract] [Full Text] [PDF]

				T. Tsoutsman, M. Kelly, D. C.H. Ng, J.-E. Tan, E. Tu, L. Lam, M. A. Bogoyevitch, C. E. Seidman, J.G. Seidman, and C. Semsarian Severe Heart Failure and Early Mortality in a Double-Mutation Mouse Model of Familial Hypertrophic Cardiomyopathy Circulation, April 8, 2008; 117(14): 1820 - 1831. [Abstract] [Full Text] [PDF]

				S. Sadayappan, N. Finley, J. W. Howarth, H. Osinska, R. Klevitsky, J. N. Lorenz, P. R. Rosevear, and J. Robbins Role of the acidic N' region of cardiac troponin I in regulating myocardial function FASEB J, April 1, 2008; 22(4): 1246 - 1257. [Abstract] [Full Text] [PDF]

				C. M. Ripplinger, W. Li, J. Hadley, J. Chen, F. Rothenberg, R. Lombardi, S. A. Wickline, A. J. Marian, and I. R. Efimov Enhanced Transmural Fiber Rotation and Connexin 43 Heterogeneity Are Associated With an Increased Upper Limit of Vulnerability in a Transgenic Rabbit Model of Human Hypertrophic Cardiomyopathy Circ. Res., November 9, 2007; 101(10): 1049 - 1057. [Abstract] [Full Text] [PDF]

				J. Liu, J. Du, C. Zhang, J. W. Walker, and X. Huang Progressive troponin I loss impairs cardiac relaxation and causes heart failure in mice Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H1273 - H1281. [Abstract] [Full Text] [PDF]

				W. Liu, M. W. Ashford, J. Chen, M. P. Watkins, T. A. Williams, S. A. Wickline, and X. Yu MR tagging demonstrates quantitative differences in regional ventricular wall motion in mice, rats, and men Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2515 - H2521. [Abstract] [Full Text] [PDF]

				A.J. Marian On Mice, Rabbits, and Human Heart Failure Circulation, May 10, 2005; 111(18): 2276 - 2279. [Full Text] [PDF]

				J. James, L. Martin, M. Krenz, C. Quatman, F. Jones, R. Klevitsky, J. Gulick, and J. Robbins Forced Expression of {alpha}-Myosin Heavy Chain in the Rabbit Ventricle Results in Cardioprotection Under Cardiomyopathic Conditions Circulation, May 10, 2005; 111(18): 2339 - 2346. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 111/18/2330    most recent 01.CIR.0000164234.24957.75v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sanbe, A. Articles by Robbins, J. Search for Related Content PubMed PubMed Citation Articles by Sanbe, A. Articles by Robbins, J. Related Collections Contractile function Animal models of human disease Related Article