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To explore the dose-dependent diastolic dysfunction and the mechanisms of heart failure and early death in transgenic (TG) mice modeling human restrictive cardiomyopathy (RCM).
The first RCM mouse model (cTnI193His mice) carrying cardiac troponin I (cTnI) R193H mutation (mouse cTnI R193H equals to human cTnI R192H) was generated several years ago in our laboratory. The RCM mice manifested a phenotype similar to that observed in RCM patients carrying the same cTnI mutation, i.e. enlarged atria and restricted ventricles. However, the causes of heart failure and early death observed in RCM mice remain unclear.
In this study, we have produced RCM TG mice (cTnI193His-L, cTnI193His-M and cTnI193His-H) that express various levels of mutant cTnI in the heart. Histological examination and echocardiography were performed in these mice to monitor the time course of the disease development and heart failure.
Our data demonstrate that cTnI mutation-caused diastolic dysfunction is dose-dependent. The key mechanism is myofibril hypersensitivity to Ca2+ resulting in an impaired relaxation in the mutant cardiac myocytes. Prolonged relaxation time and delay of Ca2+ decay observed in the mutant cardiac myocytes are correlated with the level of the mutant protein in the heart. Markedly enlarged atria due to the elevated end-diastolic pressure and myocardial ischemia are observed in the heart of the transgenic mice. In the mice with the highest level of the mutant protein, restricted ventricles and systolic dysfunction occurs followed immediately by heart failure and early death.
Diastolic dysfunction caused by R193H troponin I mutation is specific, showing a dose-dependent pattern. These mouse models are useful tools for study of diastolic dysfunction. Impaired diastole can cause myocardial ischemia and fibrosis formation, resulting in the development of systolic dysfunction and heart failure with early death in the RCM mice with a high level of the mutant protein in the heart.
Cardiomyopathy is a disorder that primarily affects cardiac muscles resulting in cardiac dysfunctions (1-3). Among various types of cardiomyopathies, hypertrophic cardiomyopathy (HCM) and restrictive cardiomyopathy (RCM) share a similar key feature characterized by a diastolic dysfunction (4-6). Unlike HCM, RCM is rare accounting roughly for 2-5% of all inherited cardiomyopathies. However, the prognosis of the disease, especially in young patients with RCM, is poor, as this condition often leads to heart failure and early death (7). Treatment of RCM is difficult and often ineffective (8, 9). A linkage study had, for the first time, demonstrated that idiopathic RCM can be a part of clinical expression of six cardiac troponin I (cTnI) mutations (5). Among those mutations, two of the mutations (K178E and R192H) identified in young individuals were de novo mutations with the worst clinical phenotype (5). The first transgenic (TG) mouse model (cTnI193His mice) carrying the RCM cTnI mutation (Arg193His in mouse cTnI protein that equals to human cTnI R192H) was generated several years ago in our laboratory (10). The RCM TG mice manifested a phenotype similar to that observed in RCM patients carrying the same cTnI mutation, i.e. markedly enlarged atria and restricted ventricles due to the increased ventricular pressure and stiffness (11). The cell-based studies further revealed that the key defect in RCM cardiac myocytes was impaired relaxation that resulted from the myofibril hypersensitivity to Ca2+ (12-13). Although the RCM mouse model is well characterized, several questions still remain. The questions we aim to address in this study are: 1) What is the cause for the varied severity of the disease observed in patients suffering from cardiomyopathies caused by myofibril protein mutations? Is the cardiac dysfunction caused by cTnI mutation dose-dependent, i.e. does the impaired relaxation depend on the level of the mutant protein in the heart? 2) We observed that most RCM cTnI TG mice had early onset of impaired relaxation and followed by heart failure at later stage. What is the mechanism that correlates the early diastolic dysfunction to late stage heart failure? 3) What are the causes of death in RCM cTnI TG mice? Is it congestive heart failure, arrhythmia, or both?
Clinically, these questions have not been well answered to date. This is because of the lack of the quantitative data of the mutant protein in the diseased hearts due to the limited cardiac samples from the patients, and the lack of specific antibodies that differentiate mutant proteins from wild type proteins. In the present study, we have produced RCM cTnI TG mice that express various levels of mutant cTnI in the heart by crossing our cTnI knockout mice with RCM TG cTnI193His mice. Histological examination and echocardiography were performed in these TG mice at different time points to monitor the progress of the disease and to determine simultaneously the myocardial ischemia, fibrosis and these damage-caused cardiac dysfunction and heart failure. Our data demonstrate, for the first time, that cTnI mutation-caused diastolic dysfunction is dose-dependent and myocardial ischemia caused by impaired diastole is probably associated with the late development of systolic dysfunction resulting eventually in heart failure and early death in RCM.
This investigation conforms to the Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, revised 1996) and was in accordance with the protocols approved by the Institutional Animal Care and Use Committees at Florida Atlantic University.
The cTnI gene knockout mice (C57BL/6) were generated previously and well characterized (14). The heterozygous cTnI knockout (cTnI+/-) mice are maintained in our laboratory for more than a decade. These mice were paired with transgenic cTnI193His mice (C57BL/6) that exhibit a RCM phenotype (10). By crossing cTnI+/- mice with cTnI193His mice, we produced cTnI193His/cTnI+/- double transgenic mice. By crossing these double transgenic mice, we obtained the transgenic mice expressing various levels of cTnI R193H in the heart: cTnI193His-H mice that express only mutant cTnI R193H at a wild type cTnI null background and cTnI193His-M mice that express about 40% of mutant cTnI with 60% of wild type cTnI. The cTnI193His-L is our original transgenic mouse line that expresses about 20% of mutant cTnI in the heart. The wild type (WT, C57BL/6) mice were used as controls in the study and they have the same genomic background as the transgenic mice. A PCR-based assay was performed to determine the genotypes and the mutant protein replacement rate in most of the experimental animals was confirmed by Western blotting assays after the experiments.
Cardiac myofilament proteins were extracted and the incorporation rate of wild type and mutant cTnI in cardiac myofibrils were determined by Western blotting as described previously (11, 12). Briefly, cardiac myofibril proteins were examined on SDS-PAGE and Western blotting using two mouse monoclonal antibodies (mAb). TnI-1 antibody is specific for the C-terminal epitope of TnI involving the residue of R193 and 4H6 antibody recognizes an epitope in the middle region of cTnI polypeptide chain. The cTnI R193H mutation destroyed the TnI-1 epitope but not the 4H6 epitope, allowing the distinction between cTnI WT and R193H. For Western blotting, cardiac ventricles were homogenized in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer containing 2% SDS to extract myofilament proteins. The protein bands were transferred to a nitrocellulose membrane using a Bio-Rad Lab semidry electrotransfer apparatus. The nitrocellulose membrane was blocked with 1% bovine serum albumin in Tris-buffered saline and incubated with mAbs diluted in TBS-T containing 0.1% bovine serum albumin. Antibodies on immunoblots were visualized by enhanced chemiluminescence (ECL detection kit from GE Healthcare). Cardiac TnT (cTnT) was used as an internal control to normalize the protein sample loading. For quantifications, the protein bands were scanned by densitometry and compared among the samples on the same blot.
Mouse cardiac myocytes were isolated using a Langendorff Perfusion Cell Isolation System (Cellutron Life Technology, Baltimore, MD) and cultured in the medium provided by the same company. Only the rod-shape cells with clear edges and well-defined sarcomere structure and without sarcolemmal blebs or spontaneous contractions were used for experiments. Mechanical properties of cardiac myocytes were determined using an IonOptix Myocam system (IonOptix Inc., Milton, MA). Cardiac myocytes were loaded into the perfusion chamber mounted on the microscope stage and superfused (1 ml/min, 37 °C) with Tyrode solution containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose and 10 HEPES, at pH 7.4. The cells were field stimulated at 8 V at a frequency of 0.5 Hz. Sarcomere shortening was measured and at least 8 contraction cycles were used for analysis as described previously (12).
The intracellular calcium transients were also measured by IonOptix Myocam system. Freshly isolated mouse cardiac myocytes were loaded with 2 μM of Fura-2 AM (Molecular Probes) at room temperature for 20 min in loading buffer as described previously (12). After 20 min of de-esterification of Fura-2 AM, the cells were superfused in Tyrode solution and electrically stimulated at 8 volts at a frequency of 0.5 Hz. For measurement of Ca2+ transients, the cells were excited by UV light (360 and 380 nm, alternately). The Fura-2 emission at 510 nm was collected with a photomultiplier tube. The kinetics of Ca2+ transients was analyzed in conjunction with myocyte mechanical measurements using the software IonWizard 6.0.
Myofibril Ca2+ sensitivity was determined by measuring pCa-force curves in skinned myofibrils isolated from WT or TG mouse hearts as described previously (12).
Echocardiography studies were performed by using a Vevo 770 High-Resolution In Vivo Imaging System (VisualSonics, Toronto, ON, Canada) in our laboratory as described previously (15). To decrease experimental bias, all of the echocardiography measurements were performed by an examiner blinded to the genotype. Experimental mice were anesthetized with isoflurane at a concentration of 3% and then maintained at 1% isoflurane by a facemask during the whole procedure. Hair on the precordial region was cleanly removed with a Nair lotion hair remover (Church & Dwight Canada, Mississauga, ON, Canada), and the region was covered with ultrasound transmission gel (Aquasonic, Parker Laboratory, Fairfield, NJ). The short-axis imaging was taken to view the LV and RV movement during diastole and systole, allowing us to measure the ventricular structure and dimension. Transmitral blood flow was observed with Pulse Doppler and diastolic mitral annulus velocity was observed in Tissue Doppler. Coronary blood flow was measured by applying Pulse Doppler with a modified long-axis view, which enabled us to see the left main coronary artery generating from aorta and going between the left ventricle anterior wall and right ventricular outflow tract. All data and images were saved and analyzed with an Advanced Cardiovascular Package Software (VisualSonics, Toronto, ON, Canada) using an automated analysis or semi-automated analysis to evaluate the cardiac function.
ECG data were collected in the conscious, free-moving mice by implanting a wireless telemetry device (TA10ETA-F20, Data Science International (DSI), St Paul, MN). Mice were anesthetized by isoflurane inhalation and the implantable transmitter units were secured in mice peritoneal cavities. The two electrical leads were lying flat and straight subcutaneously and positioned in a lead II orientation, in which the negative lead (clear) was placed in the area of the right shoulder and the positive lead (red) immediately to the left of the xyphoid space and caudal to the rib cage. The implanted mice were housed separately in cages and biopotentials were transmitted from within the animals via radio-frequency signals to the underneath antenna receivers connected to a computer system for 24-hour data recording. ECG data of first 7 days after surgery were neglected considering possible effects of anesthesia and surgical procedures. Unfiltered ECG data was gained at a 10 s intervals and ECG waveforms and parameters were analyzed with a DSI analysis package (Dataquest ART 4.2, DSI, St. Paul, MN). Raw ECG waveforms were inspected for ischemia and arrhythmia by at least two independent observers.
For light microscopy, mouse hearts were quickly excised after euthanasia, perfused through aorta with PBS 1 min and fixed in 10% neutral buffered formalin solution (Sigma). Fresh lung and liver tissues were washed by PBS and directly immersed in the same fixation solution. The fixed tissues went through a series of graded ethanol baths to be dehydrated then into xylene. After embedded with paraffin, tissues were sectioned into 5 μm thick slice, stained with hematoxylin and eosin, and then viewed under an Olympus IX71 microscope. Gomori's trichrome staining was also performed to detect fibrosis in the heart sections as described previously (14).
Heterozygous cTnI knockout mice and the original transgenic cTnI193His mice (cTnI193His-L) were previously generated and characterized in our laboratory (10-12, 15). By crossing cTnI+/- mice with cTnI193His mice, we generated cTnI+/-/cTnI193His double transgenic mice. Then by crossing these double transgenic mice, we obtained cTnI193His-M mice that expresses about 38% mutant cTnI R193H and has a heterozygous cTnI gene knockout background. By further crossing the cTnI193His-M mice, we obtained cTnI193His-H mice that express about 80% mutant cTnI R193H in a cTnI null background (Fig. 1). Western blotting data confirmed the incorporation rates of wild type cTnI and the mutant cTnI R193H in cardiac myofilaments from these different transgenic mouse lines (Fig. 1). At the age of 3 weeks, no ssTnI could be detected in the ventricles from the WT, cTnI193His-L or cTnI193His-M mice, however, a small portion of ssTnI was detected in cardiac myofilaments from the cTnI193His-H mice (Fig. 1). This is consistent with what we observed in heterozygous cTnI knockout mice in which the expression of ssTnI is extended as a compensation for the deficiency of the wild type cTnI in young mice (14). No significant effect on cell contraction and relaxation has been observed in cardiac myocytes containing the small portion of ssTnI (14). Kaplan-Meier survival curves demonstrated dramatic differences among the mice expressing the different levels of the mutant protein. Based on observations for the first 3 months, no mice in cTnI193His-H group survived over 30 days after birth, while the survival rates were 78% in cTnI193His-M group and 97% in cTnI193His-L group. The earliest death in cTnI193His-H mice occurred at day 21 after birth, however most of them died at day 27-30 (Fig. 1C). Bi-atrial enlargement was a significant cardiac morphological change observed in these mice as reported previously (12).
Our previous studies indicate that prolonged relaxation caused by the increased myofibril sensitivity for Ca2+ is the major mechanism in cTnI R193H caused diastolic dysfunction (11, 12). The cell-based assays further confirmed that the impaired relaxation was specifically caused by cTnI R193H mutation, showing a dose-dependent manner (Fig. 2). The sarcomere contraction (shortening) time did not have significant change among the cardiac myocytes from the different TG mice expressing different levels of the mutant protein. However, the sarcomere relaxation time was significantly prolonged in TG myocytes in a dose-dependent manner (Fig. 2). The end-diastolic sarcomere length was significantly shortened in TG myocytes compared to the WT (Fig. 2). Correspondingly, the intracellular Ca2+ decay time was also prolonged significantly with the increasing levels of mutant protein in the myocardial cells (Fig. 3). The myofibril sensitivity to Ca2+ was significantly enhanced in the mutant myofibrils compared to the WT myofibrils showing a dose-dependent manner (Fig. 4).
Echocardiography studies also showed a dose-dependent cardiac dysfunction in the TG mice expressing various levels of the mutant protein (Table 1). More obviously, the cTnI193His-H mice at age of 4 weeks showed a significant prolongation of left ventricular isovolumetric relaxation time (IVRT) and a dramatic reduction of left ventricular end diastolic dimension (LVEDD) and volume (LVEDV)(Table 1). Both ejection fraction and fractional shortening were significantly reduced in these mice at age of 4 weeks (Fig. 5 and Table 1). However, the TG mice from cTnI193His-L and cTnI193His-M groups did not show any significant change at this age compared to the WT (Fig. 5) except for a significant prolonged IVRT in cTnI193His-M mice (Table 1). The significantly reduced LVEDD and LVEDV were observed in cTnI193His-L and cTnI193His-M mice at age of 3 months (Fig. 5),
Impaired diastolic function with a normal or near normal systolic function is the hallmark of RCM. The prolonged IVRT was first observed with Doppler on cTnI193His-H mice at age of 21 days and on cTnI193His-M mice at age of 4-weeks (Fig. 6A and Table 1). This is consistent with the reports that IVRT is the most sensitive Doppler index to detect impaired relaxation because it is first to become abnormal (16, 17). In addition, the occurrence time of the altered IVRT is dependent on the levels of the mutant protein in the heart. Besides, a damaged systolic function characterized by a significant reduction of ejection fraction in these mice did not occur until at day 27 after birth (Fig. 6B). These data suggest that impaired relaxation is the original defect caused by cTnI mutation in these TG mice. Nevertheless, the systolic dysfunction occurred in these mice at the end-stage of the disease.
To further explore how and why a diastolic dysfunction eventually develops into a systolic dysfunction in these TG mice, we performed histological examination on various organ samples from the TG mice at different times. The data showed that on day 23 after birth the cTnI193His-H mice were still active. Although bi-atrial enlargement was evident at this time as reported previously due to the increased ventricular pressure, the histological examination of cardiac samples showed a little myocardial atrophy and limited fibrosis (Fig. 7). Histological examinations of the lungs and liver did not show any significant damage (Fig. 7). These data suggest that limited cell death might occur in these hearts with diastolic dysfunction, since systolic function was still preserved at this time, which coincided with a normal ejection fraction observed by echocardiography (Fig. 6B). On day 27 after birth when the mice presented significant mortality, histological examination of cardiac tissues from the cTnI193His-H mice showed a dramatic myofibril disarray and massive fibrosis. Significant pulmonary and hepatic edema was observed in these mice at age of 27 days (Fig. 7), indicating a congestive heart failure in these TG mice. The development of systolic dysfunction in cTnI193His-M mice was much delayed since no significant decrease of EF and FS was observed in these mice before the age of 8 months (Table 2).
To further confirm that myocardium ischemia occurs in the TG mouse hearts, we directly measured the coronary blood flow on 23-day-old cTnI193His-H and WT mice. The TG mice showed a significant decrease of blood flow in left main coronary artery, as indicated by a disappeared systolic peak and a significantly lowered diastolic peak in Doppler observations (Fig. 8 and Table 3). The data indicate that although no significant changes are observed in ejection fraction and fractional shortening, the coronary blood supply is significantly reduced due to the impaired diastole in the TG hearts.
ECG recording using a telemetric system was performed on conscious, free-moving cTnI193His-H mice starting at day 23 after birth and continuing till their death (most on days 28-30). The significant abnormality observed in 6 cTnI193His-H mice was a depressed S-T segment (Fig. 9), suggesting myocardial ischemia. No significant arrhythmia or vibration was observed in these mice, except for a few times, arrhythmia was observed just a couple of hours before death. A gradually developed bradycardia was also noticed during the telemetric ECG recording (Fig. 9). The mechanism of the bradycardia observed on these mice is not clear. It is probably a compensatory mechanism against the impaired cardiac relaxation.
Heart failure is a leading cause of morbidity and mortality in the U.S. Single cardiac myofibril gene mutations are associated with cardiomyopathies leading to various heart failures. Both hypertrophic cardiomyopathy (HCM) and RCM share a same mechanism of diastolic dysfunction (18). Diastolic dysfunction is an important clinical problem since up to 40% of heart failure cases are due to diastolic dysfunction. We have generated TG mice expressing RCM mutant cTnI in the heart to investigate the mechanisms connecting the profound deficits in myofilament function to the development of cardiac dysfunction associated with heart failure. The significant signs of diastolic dysfunction in RCM cTnI193His mice are markedly enlarged atria caused by the increased ventricular pressure and an increased IVRT, which is the earliest sign detected by echocardiography in RCM (11, 12, 16). The data from analyzing in vitro reconstituted thin filaments showed that the RCM cTnI mutations had high Ca2+-sensitizing effects on force generation (19, 20). Davis et al. used an acute genetic engineering method to transfer the RCM mutant cTnI genes into isolated adult rat cardiac myocytes and found that permeabilized myocyte Ca2+ sensitivity was increased (21). In our study, we have shown a dose-dependent myofibril hypersensitivity to Ca2+ accompanied with a prolongation of the relaxation time and a delay of Ca2+ decay in TG cardiac myocytes expressing various levels of cTnI R193H. Our data are in consistent with our previous reports (11,12) and the reports from other studies (19-21).
In the present study, we demonstrate that RCM cTnI R193H mutation causes a dose-dependent diastolic dysfunction in experimental mice. The severity and the onset time of the impaired relaxation (prolonged relaxation time and increased IVRT due to prolonged Ca2+ decay in myofibrils) are dependent on the amount of mutant cTnI in the heart. In cTnI193His-H group, the TG mice cannot tolerate the cardiac dysfunction since cTnI193His-H mice contain 80% mutant cTnI R193H in the heart. The impaired relaxation and the change of IVRT occur the earliest in these mice. The disease progresses quickly in these animals leading to 100% early death by the age of 30 days. The altered IVRT is observed as well in cTnI193His-M mice at age of 4 weeks and in cTnI193His-L mice at age of about 2 months (10). The dose-dependent phenotype in transgenic mice suggests that cTnI R193H mutation caused diastolic dysfunction is specific and the cTnI mutation has a direct effect in the development of impaired relaxation and RCM. The severity of the disease and the onset of the overt signs of diastolic dysfunction or heart failure might be associated with the levels of mutant protein in the heart.
RCM has been associated with a high rate of mortality especially in the young. Ischemia is suspected to be a cause of sudden cardiac death (SCD) in RCM patients. Rivenes et al. reported that five RCM patients presented with syncope, chest pain and ischemic ECG, which were associated with their sudden death. Four hearts available for autopsy demonstrated evidences of acute and/or chronic ischemia in the subendocardium and papillary muscle (7). Another clinical study performed by Palka et al. also reported the presence of ischemic signs and the absence of chronic heart failure in RCM patients (22). Hence, the question whether ischemia is the cause of heart failure and SCD in RCM is still open due to insufficient histological evidence. The histological examinations performed in this study on transgenic RCM mice at different time points demonstrate the progressive formation of fibrosis in TG cardiac muscles, from the scattered fibrosis in cardiac muscles in TG hearts at day 23 to a massive fibrosis formation in the TG hearts at day 27 (Fig. 7). ECG recordings also indicate the ischemic signs such as the alteration of S-T segment in RCM TG mice. More direct evidence is that reduced coronary blood circulation is observed by Doppler echocardiography on these mice. It is also observed that the ischemic signs occur after the impaired relaxation in the TG hearts, but before the overt systolic dysfunction (significantly reduced ejection fraction and fractional shortening) and heart failure (Table 3). It is well known that coronary blood flow occurs mostly during diastole and an increase of end-diastolic pressure will decrease coronary artery blood flow (23). So it is not without reasons to expect an occurrence of myocardial ischemia in a restricted heart since the elevated left ventricular pressure and wall stress in RCM hearts can cause an increased extravascular compressive force resulting in a reduced subendomyocardial perfusion.
The data from ECG recordings on RCM TG mice do not show significant arrhythmia. However, we did see arrhythmic signs on ECG from RCM TG mice a couple of hours before death, which is probably due to the disturbance of electrical activities in a late-stage failing heart. In our study, we do not have sufficient evidences to attribute arrhythmia to be a major cause for the early death observed in RCM TG mice. However, we cannot exclude the possible correlation between arrhythmia and SCD in cardiomyopathy cases since arrhythmia is reported to be a cause of SCD in HCM transgenic mice with Ca2+ hypersensitivity caused by ACTC gene mutation (29).
Heart failure related death is the most common outcome of cardiomyopathies. RCM associated with sarcomere protein mutations seems to cause much severe heart failure compared to the RCM resulting from other factors (24-28). However, due to the rare incidence of RCM and the lack of early screening indicators for the disease, there are no completely tracked, large-scale clinical cases that are available for an intensive survey on the clinical course of RCM. The RCM TG mice provide us with a useful tool to investigate the mechanisms and the disease development time-course by performing morphological and functional measurements at cellular, organ and whole animal levels. It is interesting to note that the preserved systolic function sometimes becomes impaired at the end stage of RCM. Ammash et al. observed systolic dysfunction in 16% of 94 RCM patients (30). Weller et al. reported a low cardiac output in 18 RCM children (31). Our data are consistent with these reports, suggesting that systolic heart failure occurs in the late stage of RCM cases and is probably a major cause for early death in RCM.
We produced transgenic mice expressing various levels of mutant cardiac troponin I (cTnI) in the heart.
We find that myofibril Ca2+ hypersensitivity is mutant protein dose-dependent.
Diastolic dysfunction can cause ischemia resulting in systolic dysfunction at late stage.
Dose of the mutant protein is important for the severity of the disease.
The authors would like to thank Dr. Jose Pinto from Florida State University for assisting in myofibril calcium sensitivity measurements. Part of this work was presented in Biophysical Annual meeting in March of 2011 at Baltimore, MD and the American Heart Association annual meeting in November of 2011 at Orlando, FL. This work was supported by grants from the NIH (GM-073621; HL112130-01), NSFC (31271218) and the American Heart Association (11GRNT7860030) to X.P. Huang. P.Y. Jean-Charles is a recipient of William Townsend Porter Pre-doctoral Fellowship from the American Physiological Society.
Disclosure: none declared.
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