Cheng-Kun Du

Knock-In Mouse Model of Dilated Cardiomyopathy Caused by Troponin Mutation

We created knock-in mice in which a deletion of 3 base pairs coding for K210 in cardiac troponin (cTn)T found in familial dilated cardiomyopathy patients was introduced into endogenous genes. Membrane-permeabilized cardiac muscle fibers from mutant mice showed significantly lower Ca2+ sensitivity in force generation than those from wild-type mice. Peak amplitude of Ca2+ transient in cardiomyocytes was increased in mutant mice, and maximum isometric force produced by intact cardiac muscle fibers of mutant mice was not significantly different from that of wild-type mice, suggesting that Ca2+ transient was augmented to compensate for decreased myofilament Ca2+ sensitivity. Nevertheless, mutant mice developed marked cardiac enlargement, heart failure, and frequent sudden death recapitulating the phenotypes of dilated cardiomyopathy patients, indicating that global functional defect of the heart attributable to decreased myofilament Ca2+ sensitivity could not be fully compensated by only increasing the intracellular Ca2+ transient. We found that a positive inotropic agent, pimobendan, which directly increases myofilament Ca2+ sensitivity, had profound effects of preventing cardiac enlargement, heart failure, and sudden death. These results verify the hypothesis that Ca2+ desensitization of cardiac myofilament is the absolute cause of the pathogenesis of dilated cardiomyopathy associated with this mutation and strongly suggest that Ca2+ sensitizers are beneficial for the treatment of dilated cardiomyopathy patients affected by sarcomeric regulatory protein mutations.

Biological actions of green tea catechins on cardiac troponin C

BACKGROUND AND PURPOSE Catechins, biologically active polyphenols in green tea, are known to have a protective effect against cardiovascular diseases. In this study, we investigated direct actions of green tea catechins on cardiac muscle function to explore their uses as potential drugs for cardiac muscle disease.

Up-regulation of type 2 iodothyronine deiodinase in dilated cardiomyopathy

AIMS Thyroid hormone (TH) has prominent effects on the heart, and hyperthyroidism is occasionally found to be a cause of dilated cardiomyopathy (DCM). We aim to explore the potential role of TH in the pathogenesis of DCM. METHODS AND RESULTS The pathophysiological role of TH in the heart was investigated using a knock-in mouse model of inherited DCM with a deletion mutation DeltaK210 in the cardiac troponin T gene. Serum tri-iodothyronine (T(3)) levels showed no significant difference between wild-type (WT) and DCM mice, whereas cardiac T(3) levels in DCM mice were significantly higher than those in WT mice. Type 2 iodothyronine deiodinase (Dio2), which produces T(3) from thyroxin, was up-regulated in the DCM mice hearts. The cAMP levels were increased in DCM mice hearts, suggesting that transcriptional up-regulation of Dio2 gene is mediated through the evolutionarily conserved cAMP-response element site in its promoter. Propylthiouracil (PTU), an anti-thyroid drug, prevented the hypertrophic remodelling of the heart in DCM mice and improved their cardiac function and life expectancy. Akt and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation increased in the DCM mice hearts and PTU treatment significantly reduced the phosphorylation levels, strongly suggesting that Dio2 up-regulation is involved in cardiac remodelling in DCM through activating the TH-signalling pathways involving Akt and p38 MAPK. Dio2 gene expression was also markedly up-regulated in the mice hearts developing similar eccentric hypertrophy after myocardial infarction. CONCLUSION Local hyperthyroidism via transcriptional up-regulation of the Dio2 gene may be an important underlying mechanism for the hypertrophic cardiac remodelling in DCM.

Therapeutic effect of β-adrenoceptor blockers using a mouse model of dilated cardiomyopathy with a troponin mutation

AIMS Extensive clinical studies have demonstrated that beta-adrenoceptor blocking agents (beta-blockers) are beneficial in the treatment of chronic heart failure, which is due to various aetiologies, including idiopathic dilated cardiomyopathy (DCM) and ischaemic heart disease. However, little is known about the therapeutic efficacy of beta-blockers in the treatment of the inherited form of DCM, of which causative mutations have recently been identified in various genes, including those encoding cardiac sarcomeric proteins. Using a mouse model of inherited DCM with a troponin mutation, we aim to study the treatment benefits of beta-blockers. METHODS AND RESULTS Three different types of beta-blockers, carvedilol, metoprolol, and atenolol, were orally administered to a knock-in mouse model of inherited DCM with a deletion mutation DeltaK210 in the cardiac troponin T gene (TNNT2). Therapeutic effects were examined on the basis of survival and myocardial remodelling. The lipophilic beta(1)-selective beta-blocker metoprolol was found to prevent cardiac dysfunction and remodelling and extend the survival of knock-in mice. Conversely, both the non-selective beta-blocker carvedilol and the hydrophilic beta(1)-selective beta-blocker atenolol had no beneficial effects on survival and myocardial remodelling in this mouse model of inherited DCM. CONCLUSION The highly lipophilic beta(1)-selective beta-blocker metoprolol, known to prevent ventricular fibrillation via central nervous system-mediated vagal activation, may be especially beneficial to DCM patients showing a family history of frequent sudden cardiac death, such as those with a deletion mutation DeltaK210 in the TNNT2 gene.

Identification and physiological activity of survival factor released from cardiomyocytes during ischaemia and reperfusion

AIMS We carried out a screening of survival factors released from cells exposed to simulated ischaemia and reperfusion (sI/R) using the embryonic rat heart-derived cell line, H9c2 cells, and examined the physiological role of the identified factor. METHOD AND RESULTS The culture medium supernatant of H9c2 cells exposed to sI/R was separated by column chromatography and the fractions examined for survival activity. The protein with survival activity was identified by mass spectrometry, and its physiological role was examined in the models of ischaemia. Cell survival activity was detected in at least three fractions of the cell supernatant collected during sI/R and subjected to a series of column chromatographic steps. Among the proteins measured by mass spectrometry and western blotting, a p36 protein identified as a glycolytic enzyme, lactate dehydrogenase muscle subunit (M-LDH), showed strong survival activity. H(2)O(2)-induced intracellular calcium overload in H9c2 cells and irregular Ca(2+) transients in adult rat cardiomyocytes were both found to be inhibited by pretreatment with M-LDH. M-LDH also lowered the frequency and amplitude of early afterdepolarizations induced by H(2)O(2) in adult rat cardiomyocytes and suppressed the ischaemia-reperfusion-induced reduction of cardiac output from mouse working heart preparations. M-LDH was found to increase the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), which plays a role in H9c2 cell survival. CONCLUSION M-LDH released from cardiomyocytes after hypoxia and reoxygenation has a role in protecting the heart from oxidative stress-induced injury through an intracellular signal transduction pathway involving ERK1/2.

Survival benefit of ghrelin in the heart failure due to dilated cardiomyopathy.

Although ghrelin has been demonstrated to improve cardiac function in heart failure, its therapeutic efficacy on the life expectancy remains unknown. We aim to examine whether ghrelin can improve the life survival in heart failure using a mouse model of inherited dilated cardiomyopathy (DCM) caused by a deletion mutation ΔK210 in cardiac troponin T (cTnT). From 30 days of age, ghrelin (150 μg/kg) was administered subcutaneously to DCM mice once daily, control mice received saline only. The survival rates were compared between the two groups for 30 days. After 30‐day treatment, functional and morphological measurements were conducted. Ghrelin‐treated DCM mice had significantly prolonged life spans compared with saline‐treated control DCM mice. Echocardiography showed that ghrelin reduced left ventricular (LV) end‐diastolic dimensions and increased LV ejection fraction. Moreover, histoanatomical data revealed that ghrelin decreased the heart‐to‐body weight ratio, prevented cardiac remodeling and fibrosis, and markedly decreased the expression of brain natriuretic peptide. Telemetry recording and heart rate variability analysis showed that ghrelin suppressed the excessive cardiac sympathetic nerve activity (CSNA) and recovered the cardiac parasympathetic nerve activity. These results suggest that ghrelin has therapeutic benefits for survival as well as for the cardiac function and remodeling in heart failure probably through suppression of CSNA and recovery of cardiac parasympathetic nerve activity.

In vivo monitoring of acetylcholine release from cardiac vagal nerve endings in anesthetized mice

We applied a microdialysis technique to the left ventricular myocardium of anesthetized mice and tried to monitor acetylcholine (ACh) release from cardiac vagal nerves. Transection of bilateral cervical vagal nerves decreased dialysate ACh concentration. Electrical stimulation of the left cervical vagal nerve increased dialysate ACh concentration in proportion to the frequency of stimulation. Intravenous administration of hexamethonium, prevented the increase in dialysate ACh concentration during vagal nerve stimulation, indicating that ACh in the dialysate primarily reflects ACh released from post-ganglionic cardiac vagal nerves. Microdialysis permits monitoring of ACh release from post-ganglionic cardiac vagal nerves that are most likely to be innervating the left ventricle in mice.

Contribution of serotonin uptake and degradation to myocardial interstitial serotonin levels during ischaemia‐reperfusion in rabbits

Although deleterious effects of serotonin (5‐HT) have been demonstrated during myocardial ischaemia‐reperfusion, little information is available on myocardial interstitial 5‐HT kinetics. This study evaluated the contribution of 5‐HT reuptake and degradation to myocardial interstitial 5‐HT levels during ischaemia‐reperfusion.

Role of brain serotonin dysfunction in the pathophysiology of congestive heart failure

Inherited or non-inherited dilated cardiomyopathy (DCM) patients develop varied disease phenotypes leading to death after developing congestive heart failure (HF) or sudden death with mild or no overt HF symptoms, suggesting that environmental and/or genetic factors may modify the disease phenotype of DCM. In this study, we sought to explore unknown genetic factors affecting the disease phenotype of monogenic inherited human DCM. Knock-in mice bearing a sarcomeric protein mutation that causes DCM were created on different genetic backgrounds; BALB/c and C57Bl/6. DCM mice on the BALB/c background showed cardiac enlargement and systolic dysfunction and developed congestive HF before died. In contrast, DCM mice on the C57Bl/6 background developed no overt HF symptoms and died suddenly, although they showed considerable cardiac enlargement and systolic dysfunction. BALB/c mice have brain serotonin dysfunction due to a single nucleotide polymorphism (SNP) in tryptophan hydroxylase 2 (TPH2). Brain serotonin dysfunction plays a critical role in depression and anxiety and BALB/c mice exhibit depression- and anxiety-related behaviors. Since depression is common and associated with poor prognosis in HF patients, we examined therapeutic effects of anti-depression drug paroxetine and anti-anxiety drug buspirone that could improve the brain serotonin function in mice. Both drugs reduced cardiac enlargement and improved systolic dysfunction and symptoms of severe congestive HF in DCM mice on the BALB/c background. These results strongly suggest that genetic backgrounds involving brain serotonin dysfunction, such as TPH2 gene SNP, may play an important role in the development of congestive HF in DCM.

In vivo effects of propyl gallate, a novel Ca2 + sensitizer, in a mouse model of dilated cardiomyopathy caused by cardiac troponin T mutation

AIMS We have previously demonstrated that propyl gallate has a Ca(2+) sensitizing effect on the force generation in membrane-permeabilized (skinned) cardiac muscle fibers. However, in vivo beneficial effects of propyl gallate as a novel Ca(2+) sensitizer remain uncertain. In the present study, we aim to explore in vivo effects of propyl gallate. MAIN METHODS We compared effects of propyl gallate on ex vivo intact cardiac muscle fibers and in vivo hearts in healthy mice with those of pimobendan, a clinically used Ca(2+) sensitizer. The therapeutic effect of propyl gallate was investigated using a mouse model of dilated cardiomyopathy (DCM) with reduced myofilament Ca(2+) sensitivity due to a deletion mutation ΔK210 in cardiac troponin T. KEY FINDINGS Propyl gallate, as well as pimobendan, showed a positive inotropic effect. Propyl gallate slightly increased the blood pressure without changing the heart rate in healthy mice, whereas pimobendan decreased the blood pressure probably through vasodilation via inhibition of phosphodiesterase and increased the heart rate. Propyl gallate prevented cardiac remodeling and systolic dysfunction and significantly improved the life-expectancy of knock-in mouse model of DCM with reduced myofilament Ca(2+) sensitivity due to a mutation in cardiac troponin T. On the other hand, gallate, a similarly strong antioxidant polyphenol lacking Ca(2+) sensitizing action, had no beneficial effects on the DCM mice. SIGNIFICANCE These results suggest that propyl gallate might be useful for the treatment of inherited DCM caused by a reduction in the myofilament Ca(2+) sensitivity.