Changlong Nan

Correcting diastolic dysfunction by Ca2+ desensitizing troponin in a transgenic mouse model of restrictive cardiomyopathy.

Several cardiac troponin I (cTnI) mutations are associated with restrictive cardiomyopathy (RCM) in humans. We have created transgenic mice (cTnI(193His) mice) that express the corresponding human RCM R192H mutation. Phenotype of this RCM animal model includes restrictive ventricles, biatrial enlargement and sudden cardiac death, which are similar to those observed in RCM patients carrying the same cTnI mutation. In the present study, we modified the overall cTnI in cardiac muscle by crossing cTnI(193His) mice with transgenic mice expressing an N-terminal truncated cTnI (cTnI-ND) that enhances relaxation. Protein analyses determined that wild type cTnI was replaced by cTnI-ND in the heart of double transgenic mice (Double TG), which express only cTnI-ND and cTnI R193H in cardiac myocytes. The presence of cTnI-ND effectively rescued the lethal phenotype of RCM mice by reducing the mortality rate. Cardiac function was significantly improved in Double TG mice when measured by echocardiography. The hypersensitivity to Ca(2+) and the prolonged relaxation of RCM cTnI(193His) cardiac myocytes were completely reversed by the presence of cTnI-ND in RCM hearts. The results demonstrate that myofibril hypersensitivity to Ca(2+) is a key mechanism that causes impaired relaxation in RCM cTnI mutant hearts and Ca(2+) desensitization by cTnI-ND can correct diastolic dysfunction and rescue the RCM phenotypes, suggesting that Ca(2+) desensitization in myofibrils is a therapeutic option for treatment of diastolic dysfunction without interventions directed at the systemic beta-adrenergic-PKA pathways.

Sodium valproate-induced congenital cardiac abnormalities in mice are associated with the inhibition of histone deacetylase

BackgroundValproic acid, a widely used anticonvulsant drug, is a potent teratogen resulting in various congenital abnormalities. However, the mechanisms underlying valproic acid induced teratogenesis are nor clear. Recent studies indicate that histone deacetylase is a direct target of valproic acid.MethodsIn the present study, we have used histological analysis and RT-PCR assays to examine the cardiac abnormalities in mice treated with sodium valproate (NaVP) and determined the effects of NaVP on histone deacetylase activity and the expression of heart development-related genes in mouse myocardial cells.ResultsThe experimental data show that NaVP can induce cardiac abnormalities in fetal mice in a dose-dependent manner. NaVP causes a dose-dependent inhibition of hitone deacetylase (HDAC) activity in mouse myocardial cells. However, the expression levels of HDAC (both HDAC1 and HDAC2) are not significantly changed in fetal mouse hearts after administration of NaVP in pregnant mice. The transcriptional levels of other heart development-related genes, such as CHF1, Tbx5 and MEF2, are significantly increased in fetal mouse hearts treated with NaVP.ConclusionsThe study indicates that administration of NaVP in pregnant mice can result in various cardiac abnormalities in fetal hearts, which is associated with an inhibition of histone deacetylase without altering the transcription of this enzyme.

Deficiency of methionine sulfoxide reductase A causes cellular dysfunction and mitochondrial damage in cardiac myocytes under physical and oxidative stresses

Methionine sulfoxide reductase A (MsrA) is an enzyme that reverses oxidation of methionine in proteins. Using a MsrA gene knockout (MsrA(-/-)) mouse model, we have investigated the role of MsrA in the heart. Our data indicate that cellular contractility and cardiac function are not significantly changed in MsrA(-/-) mice if the hearts are not stressed. However, the cellular contractility, when stressed using a higher stimulation frequency (2Hz), is significantly reduced in MsrA(-/-) cardiac myocytes. MsrA(-/-) cardiac myocytes also show a significant decrease in contractility after oxidative stress using H(2)O(2). Corresponding changes in Ca(2+) transients are observed in MsrA(-/-) cardiomyocytes treated with 2Hz stimulation or with H(2)O(2). Electron microscope analyses reveal a dramatic morphological change of mitochondria in MsrA(-/-) mouse hearts. Further biochemical measurements indicate that protein oxidation levels in MsrA(-/-) mouse hearts are significantly higher than those in wild type controls. Our study demonstrates that the lack of MsrA in cardiac myocytes reduces myocardial cells capability against stress stimulations resulting in a cellular dysfunction in the heart.

Dose-dependent diastolic dysfunction and early death in a mouse model with cardiac troponin mutations

Our aim was 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 (cTnI(193His) 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 (cTnI(193His)-L, cTnI(193His)-M and cTnI(193His)-H) that express various levels of mutant cTnI in the heart. Histological examination and echocardiography were performed on 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 Ca(2+) resulting in an impaired relaxation in the mutant cardiac myocytes. Prolonged relaxation time and delay of Ca(2+) 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 occur 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 the 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.

Insights into restrictive cardiomyopathy from clinical and animal studies

Cardiomyopathies are diseases that primarily affect the myocardium, leading to serious cardiac dysfunction and heart failure. Out of the three major categories of cardiomyopathies (hypertrophic, dilated and restrictive), restrictive cardiomyopathy (RCM) is less common and also the least studied. However, the prognosis for RCM is poor as some patients dying in their childhood. The molecular mechanisms behind the disease development and progression are not very clear and the treatment of RCM is very difficult and often ineffective. In this article, we reviewed the recent progress in RCM research from the clinical studies and the translational studies done on diseased transgenic animal models. This will help for a better understanding of the mechanisms underlying the etiology and development of RCM and for the design of better treatments for the disease.

Transcription factor Yin Yang 1 represses fetal troponin I gene expression in neonatal myocardial cells.

Yin Yang 1 (YY1) is a transcription factor that can activate or repress expression of a variety of genes and is involved in several developmental processes. While some transcription factors are known to modulate skeletal myogenesis, the regulation of fetal troponin I (ssTnI) expression by YY1 in cardiac development has not been studied. The present study shows that the fetal troponin I gene expression in neonatal myocardium was reduced by overexpression of YY1, while cardiac troponin I (cTnI) did not show any significant decrease. And a dose-response inhibition by YY1 was observed in fetal troponin I promoter induced transcriptional activities. Mutation of YY1-binding site can abolish the inhibitory effect and YY1 silencing in neonatal myocardium resulted in an increase of ssTnI protein expression. Our results indicate that YY1 is a novel regulator of fetal TnI transcription in the heart.

Functional characterization of mouse fetal TnI gene promoters in myocardial cells

Two major troponin I (TnI) genes, fetal TnI (ssTnI) and adult TnI (cTnI), are expressed in the mammalian heart under the control of a developmentally regulated program. In this study, the up-stream domain ( approximately 1,800 bp) of mouse fetal TnI gene has been cloned and characterized. There is a high homology of this region among mouse, rat and human. Analysis of the sequence revealed several putative regulatory domains and binding sites (Sp1 binding sites, GATA binding site, MyoD, CREB, MEF2, AP1, NFkappaB, etc). Transfection assays indicated that conserved GA-rich sequences, CREB and a CCAAT box within the first 300 bp upstream of the transcription start site were critical for the gene expression. Electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays revealed binding proteins to CREB site in nuclear extracts from myocardial cells. An inhibitory domain was revealed within the sequence between -1,700 to -1,780. Thyroid hormone (T(3)) caused a significant inhibitory effect on ssTnI expression in myocardial cells whereas this effect was not evident in CHO cells.

Application of Echocardiography on Transgenic Mice with Cardiomyopathies

Cardiomyopathies are common cardiac disorders that primarily affect cardiac muscle resulting in cardiac dysfunction and heart failure. Transgenic mouse disease models have been developed to investigate the cellular mechanisms underlying heart failure and sudden cardiac death observed in cardiomyopathy cases and to explore the therapeutic outcomes in experimental animals in vivo. Echocardiography is an essential diagnostic tool for accurate and noninvasive assessment of cardiac structure and function in experimental animals. Our laboratory has been among the first to apply high-frequency research echocardiography on transgenic mice with cardiomyopathies. In this work, we have summarized our and other studies on assessment of systolic and diastolic dysfunction using conventional echocardiography, pulsed Doppler, and tissue Doppler imaging in transgenic mice with various cardiomyopathies. Estimation of embryonic mouse hearts has been performed as well using this high-resolution echocardiography. Some technical considerations in mouse echocardiography have also been discussed.

Calcium desensitizer catechin reverses diastolic dysfunction in mice with restrictive cardiomyopathy

Diastolic dysfunction refers to an impaired relaxation and an abnormality in ventricular blood filling during diastole while systolic function is preserved. Cardiac myofibril hypersensitivity to Ca(2+) is a major factor that causes impaired relaxation of myocardial cells. The present study investigates the effect of the green tea extract catechins on myofibril calcium desensitization and restoration of diastolic function in a restrictive cardiomyopathy (RCM) mouse model with cardiac troponin mutations. Wild type (WT) and RCM mice were treated daily with catechin (epigallocatechin-3-gallate, EGCg, 50 mg/kg body weight) for 3 months. Echocardiography and cell based assays were performed to measure cardiac structure and flow-related variables including chamber dimensions, fraction shortening, trans-mitral flow patterns in the experimental mice. In addition, myocyte contractility and calcium dynamics were measured in WT and RCM cardiomyocytes treated in vitro with 5 μM EGCg. Our data indicated that RCM mice treated with EGCg showed an improved diastolic function while systolic function remained unchanged. At the cellular level, sarcomere relaxation and calcium decay were accelerated in RCM myocardial cells treated with EGCg. These results suggest that catechin is effective in reversing the impaired relaxation in RCM myocardial cells and rescuing the RCM mice with diastolic dysfunction.

Diastolic dysfunction and cardiac troponin I decrease in aging hearts.

Cardiac tropnoin I (cTnI) plays a critical role in the regulation of diastolic function, and its low expression may result in cardiac diastolic dysfunction, which is the most common form of cardiovascular disorders in older adults. In this study, cTnI expression levels were determined in mice at various ages and cardiac function was measured and compared between young adult mice (3 and 10 months) and older mice (18 months). The data indicated that the cTnI levels reached a peak high in young adult hearts (3 months), but decreased in older hearts (18 months). Furthermore, the older hearts showed a significant diastolic dysfunction observed by P-V loop and echocardiography measurements. To further define the mechanism underlying the cTnI decrease in aging hearts, we tested DNA methylation and histone acetylation modifications of cTnI gene. We found that acetylation of histone near the promoter region of cTnI gene played an important role in regulation of cTnI expression in the heart at different ages. Our study indicates that epigenetic modification caused cTnI expression decrease is one of the possible causes that result in a reduced cTnI level and diastolic dysfunction in the older hearts.