Proceedings for Annual Meeting of The Japanese Pharmacological Society, 2019
Background: The present study is to estimate the geometrical and constituent heterogeneity effect... more Background: The present study is to estimate the geometrical and constituent heterogeneity effects on electrical conduction on in vitro monolayer consisting of atrial-like cardiomyocytes (ALCMs) derived from human induced pluripotent stem cells (hiPSCs) and human atrial fibroblasts (HAFbs) under HF field stimuli (HFFS). Method: We induced hiPSCs into ALCMs by adding all-trans retinoic acid (ATRA). The ALCMs and HAFbs were transferred in defined ratios on manually fabricated plate with geometrical transition. HFFS were delivered, and the electrical propagation was assessed by optical mapping. Results: ATRA-treated CMs showed atrial specific properties compared to untreated CMs. HFFS preferentially induced impaired conduction on ALCMs with an abrupt geometrical transition, but not on ALCMs with uniform geometry. In addition, the co-culture of HAFbs with the ALCMs deteriorated the stability of electrical conduction than in mono-culture of ALCMs. Conclusion: Geometrical heterogeneity under HFFS jeopardizes electrical conduction on in vitro ALCMs monolayer. Constituent heterogeneity represented by HAFbs contributes to the deterioration of the electrical conduction stability.
A correction to this article has been published and is linked from the HTML and PDF versions of t... more A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
Table 1 shows, Teneligliptin attenuated cardiomyocyte hypertrophy, fibrosis and lung congestion, ... more Table 1 shows, Teneligliptin attenuated cardiomyocyte hypertrophy, fibrosis and lung congestion, however, it induced left ventricular dilatation. Conclusion: Teneligliptin have potential to prevent hypertrophy, fibrosis and development of heart failure, however, it showed not only beneficial but harmful effects. Teneligliptin may induce left ventricular dilation by inhibiting normal compensation for pressure overload.
We reviewed the concept that de-d lialion is a mechvnisn of electrical rcmodeing m d eased hearls... more We reviewed the concept that de-d lialion is a mechvnisn of electrical rcmodeing m d eased hearls,rekrrnngloourdataon 1)d eutialchanges in action potential duration (APD) and transimt outward current (Ito) of cultured ventricular myocylcs of newborn rats and 2) changes in APD, Ito and us gene expression m hypertrophied adult nil venom. The latter changes associated with on of the hypertrophy appeared m the reverse order of those m newborn rats. Gene switching of ionic channels to d liation was oonckuded to be essential for electrical mnodebng of the died hearts.
type Ca 2+ channels are pivotal for cardiac excitation and contraction 1 and they are coded by 4 ... more type Ca 2+ channels are pivotal for cardiac excitation and contraction 1 and they are coded by 4 distinct genes: Cav1.1 (1S, skeletal muscle), Cav1.2 (1C, cardiac and smooth muscle), Cav1.3 (1D, neuron, neurosecretory cells and heart) and Cav1.4 (1F, retina). 2-4 The Cav1.2 Ca 2+ channel is predominant in cardiac muscle. 5,6 Ca 2+ entry into the cytosol through this channel triggers Ca 2+ release from the sarcoplasmic reticulum and underlies the Ca 2+ transient for contraction. 1 The Cav1.3 Ca 2+ channel activates at more negative membrane potentials than the Cav1.2 channel 7 and contributes to diastolic depolarization of sinoatrial (SA) nodal cells. This role of Cav1.3 Ca 2+ channels in SA nodal automaticity is further substantiated by the observation that Cav1.3 ablation leads to SA node dysfunction. 8-10 The murine embryonic heart starts to contract at 8.5 days post coitum (dpc) and the heart beat becomes regular at 9.5 dpc, when the heart shape is still tube-like. 11 Ventricles isolated from embryonic hearts at 9.5 dpc also beat spontaneously and regularly. Cardiac excitation and contraction in this early embryonic stage depend on Ca 2+ influx through
ongestive heart failure (CHF) is associated with an augmentation of sympathetic nerve activity an... more ongestive heart failure (CHF) is associated with an augmentation of sympathetic nerve activity and a reduction in parasympathetic nerve activity. 1,2 This autonomic imbalance may underlie the increased risk of sudden cardiac death in CHF patients. 3 In normal individuals, parasympathetic impulses are transmitted from the solitary nucleus in the brain to the heart through efferent preand post-ganglionic vagal neurons. Upon vagal stimulation, acetylcholine (ACh) released from the nerve terminals binds to muscarinic (M2) receptors of the heart. Emerging evidence shows that the ACh release at both pre-and postganglionic levels is facilitated by nitric oxide (NO) generated by neuronal NO synthase (nNOS) at nerve terminals. 4,5 Stimulation of the M2 receptor in the sinoatrial (SA) node pacemaker cells results in a reduction of heart rate (HR) via increase of ACh-sensitive potassium channel current (IK,ACh) and decrease of hyperpolarization-activated cation channel current (If). 6 The level within the vagal cascade, at which a defect results in parasympathetic withdrawal, is unknown. In a dog model of CHF induced by rapid ventricular
American Journal of Physiology-Heart and Circulatory Physiology, 2004
T-type Ca2+ channels are implicated in cardiac automaticity, cell growth, and cardiovascular remo... more T-type Ca2+ channels are implicated in cardiac automaticity, cell growth, and cardiovascular remodeling. Two voltage-gated Ca2+ subtypes (Cav3.1 and Cav3.2) have been cloned for the pore-forming α1-subunit of the T-type Ca2+ channel in cardiac muscle, but their differential roles remain to be clarified. The aim of this study was to elucidate the relative contribution of the two subtypes in the normal development of mouse hearts. A whole cell patch clamp was used to record ionic currents from ventricular myocytes isolated from mice of early (E9.5) and late embryonic days (E18) and from adult 10-wk-old mice. Large T-type Ca2+ current ( ICa,T) was observed at both E9.5 and E18, displaying similar voltage-dependence and kinetics of activation and inactivation. The current was inhibited by Ni2+ at relatively low concentrations (IC50 26–31 μM). ICa,T was undetectable in adult myocytes. Quantitative PCR analysis revealed that Cav3.2 mRNA is the predominant subtype encoding T-type Ca2+ chan...
Human induced pluripotent stem (hiPS) cells have been used as a cell source for regenerative ther... more Human induced pluripotent stem (hiPS) cells have been used as a cell source for regenerative therapy and disease modeling. The purity of hiPS-cardiomyocytes (hiPS-CMs) has markedly improved with advancements in cell culture and differentiation protocols. However, the morphological features and molecular properties of the relatively immature cells are still unclear, which has hampered their clinical application. The aim of the present study was to investigate the extent to which topographic substrates actively influence hiPS-CMs. hiPS-CMs were seeded on randomized oriented fiber substrate (random), anisotropic aligned fiber substrate (align), and flat non-scaffold substrate (flat). After culturing for one week, the hiPS-CMs on the aligned patterns showed more mature-like properties, including elongated rod shape, shorter duration of action potential, accelerated conduction velocity, and elevated cardiac gene expression. Subsequently, to determine whether this development was irrevers...
Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration accompanied... more Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration accompanied by dilated cardiomyopathy. Recently, abnormality of yes-associated protein (YAP) has been reported as the pathogenesis of muscle degeneration of DMD; however YAP activity remains unclear in dystrophic heart of DMD. Herein, we investigated YAP activity using disease-specific induced pluripotent stem cell (iPSC) derived cardiomyocytes (CMs) in DMD. DMD-iPSCs were generated from DMD patient with exon 48–54 deletion in DMD, and genome-edited (Ed)-DMD-iPSCs with in-frame (Ed-DMD-iPSCs) were created using CRISPR/Cas9. Nuclear translocation of YAP [nuclear (N)/cytoplasmic (C) ratio] was significantly lower in DMD-iPSC-CMs than in Ed-DMD-iPSC-CMs. In addition, Ki67 expression, indicating proliferative ability, was significantly lower in DMD-iPSC-CMs than Ed-DMD-iPSC-CMs. Therefore, immunofluorescent staining showed that actin stress fibers associated with YAP activity by mechanotransduction wer...
Molecular signaling of sympathetic innervation of myocardium is an unresolved issue. The purpose ... more Molecular signaling of sympathetic innervation of myocardium is an unresolved issue. The purpose of this study was to investigate the effect of neurotrophic factors on sympathetic neurite growth towards cardiomyocytes. Cardiomyocytes (CMs) and sympathetic neurons (SNs) were isolated from neonatal rat hearts and superior cervical ganglia, and were co-cultured, either in a random or localized way. Neurite growth from SNs toward CMs was assessed by immunohistochemistry for neurofilament M and α-actinin in response to neurotrophic factors-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and a chemical repellent, semaphorin 3A. As a result, GDNF as well as NGF and BDNF stimulated neurite growth. GDNF enhanced neurite outgrowth even under the NGF-depleted culture condition, excluding an indirect effect of GDNF via NGF. Quantification of mRNA and protein by real-time PCR and immunohis...
American Journal of Physiology-Heart and Circulatory Physiology
The effects of myocardial hypertrophy on mRNA expression levels of voltage-gated K+ channels were... more The effects of myocardial hypertrophy on mRNA expression levels of voltage-gated K+ channels were investigated using monocrotaline (MCT)-induced pulmonary hypertensive rats. The ratio of right ventricle weight to left ventricle plus septum weight on day 28 was increased significantly compared with control rats [control vs. MCT: 0.27 ± 0.01 vs. 0.58 ± 0.03 ms ( n = 8–13); P < 0.05]. Electrocardiograms showed that QRS duration [control vs. MCT: 26.4 ± 2.6 ms vs. 31.5 ± 5.8 ms ( n = 6); P < 0.05], Q-T interval [control vs. MCT: 100.8 ± 8.9 ms vs. 110.0 ± 4.2 ms ( n = 6); P < 0.05] and corrected Q-T interval [Q-Tc; control vs. MCT: 8.4 ± 0.7 ms vs. 10.2 ± 0.4 ms ( n = 6); P < 0.05] were prolonged significantly on day 28. mRNA levels of Kv1.2, 1.5, 2.1, 4.2, and 4.3 for day 28 assessed by ribonuclease protection assays were decreased significantly from control by 60 ± 10, 76 ± 3, 58 ± 5, 81 ± 5, and 45 ± 12%, respectively ( n = 3; P < 0.005), and Kv1.4 mRNA level for day 2...
The DNA damage response (DDR) plays a pivotal role in maintaining genome integrity. DNA damage an... more The DNA damage response (DDR) plays a pivotal role in maintaining genome integrity. DNA damage and DDR activation are observed in the failing heart, however, the type of DNA damage and its role in the pathogenesis of heart failure remain elusive. Here we show the critical role of DNA single-strand break (SSB) in the pathogenesis of pressure overload-induced heart failure. Accumulation of unrepaired SSB is observed in cardiomyocytes of the failing heart. Unrepaired SSB activates DDR and increases the expression of inflammatory cytokines through NF-κB signalling. Pressure overload-induced heart failure is more severe in the mice lacking XRCC1, an essential protein for SSB repair, which is rescued by blocking DDR activation through genetic deletion of ATM, suggesting the causative role of SSB accumulation and DDR activation in the pathogenesis of heart failure. Prevention of SSB accumulation or persistent DDR activation may become a new therapeutic strategy against heart failure.
Disruption of angiotensin II type 1 (AT 1) receptor prolonged life span in mice. Since aging-rela... more Disruption of angiotensin II type 1 (AT 1) receptor prolonged life span in mice. Since aging-related decline in skeletal muscle function was retarded in Atgr1a −/− mice, we examined the role of AT 1 receptor in muscle regeneration after injury. Administration of AT 1 receptor blocker irbesartan increased the size of regenerating myofibers, decreased fibrosis, and enhanced functional muscle recovery after cryoinjury. We recently reported that complement C1q, secreted by macrophages, activated Wnt/β-catenin signaling and promoted aging-related decline in regenerative capacity of skeletal muscle. Notably, irbesartan induced M2 polarization of macrophages, but reduced C1q expression in cryoinjured muscles and in cultured macrophage cells. Irbesartan inhibited upregulation of Axin2, a downstream gene of Wnt/β-catenin pathway, in cryoinjured muscles. In addition, topical administration of C1q reversed beneficial effects of irbesartan on skeletal muscle regeneration after injury. These results suggest that AT 1 receptor blockade improves muscle repair and regeneration through down-regulation of the aging-promoting C1q-Wnt/β-catenin signaling pathway. Angiotensin (Ang) II is the crucial bioactive molecule of the renin-angiotensin system, and exerts most of its pathophysiological actions through binding to Ang II type 1 (AT 1) receptor 1,2. While humans have a single AGTR1 gene that encodes AT 1 receptor, mice have Agtr1a and Agtr1b genes encoding two isoforms (AT 1a and AT 1b) of AT 1 receptor, and the major isoform of mouse AT 1 receptor is AT 1a receptor 1,2. AT 1 receptor is a member of the G protein-coupled receptor family, which shares typical conformation of seven transmembrane-spanning α-helices 3. Upon stimulation by binding to Ang II or mechanical stretch 4,5 , AT 1 receptor activates multiple G protein-dependent and-independent signaling pathways and promote the formation of reactive oxygen species (ROS), leading to a variety of responses such as
Circulation journal : official journal of the Japanese Circulation Society, Jan 28, 2015
Adipose tissue is one of the sources of mesenchymal stem cells, which have the potential to diffe... more Adipose tissue is one of the sources of mesenchymal stem cells, which have the potential to differentiate into various types of cells, including myocytes. Whether brown adipose tissue (BAT)-derived cells might differentiate into the cardiac pacemaking-conducting cells, and have the potential to regenerate the cardiac conduction system (CCS), is investigated in this study.Methods and Results:BAT was isolated from the interscapular area of mice and enzymatically digested before culture. Round or fusiform cells showed spontaneous beating at 4-7 days after culturing of BAT-derived cells. Reverse transcriptase-polymerase chain reaction analysis and immunocytochemical analysis revealed that BAT-derived cells expressed several cardiomyocytes, the CCS and pacemaker (PM) cell marker genes and proteins. Patch-clamp techniques revealed that spontaneous electrical activity and the shape of the action potential showed properties of cardiac PM cells. Next, a complete atrioventricular (AV) block w...
American journal of physiology. Heart and circulatory physiology, Jan 8, 2015
Although cardiac fibrosis causes heart failure, its molecular mechanisms remain elusive. In this ... more Although cardiac fibrosis causes heart failure, its molecular mechanisms remain elusive. In this study, we investigated the mechanisms of cardiac fibrosis and examined the effects of the anti-fibrotic drug pirfenidone (PFD) on chronic heart failure. To understand the responsible mechanisms, we generated an in vivo pressure-overloaded heart failure model via transverse aortic constriction (TAC) and examined the effects of PFD on chronic-phase cardiac fibrosis and function. In the vehicle group, contractile dysfunction and left ventricle fibrosis progressed further from 4 to 8 weeks after TAC but were prevented by PFD treatment beginning 4 weeks after TAC. We isolated cardiac fibroblasts and vascular endothelial cells from the left ventricles of adult male mice and investigated the cell type-specific effects of PFD. TGF-β induced upregulated collagen 1 expression via p38 phosphorylation and downregulated claudin 5 (Cldn5) expression in cardiac fibroblasts and endothelial cells, respec...
E xercise intolerance is a common clinical manifestation associated with chronic heart failure (C... more E xercise intolerance is a common clinical manifestation associated with chronic heart failure (CHF). 1 Various morphological, histological, and biochemical changes are observed in the skeletal muscle of patients with CHF. These changes are collectively called skeletal myopathy and are the major cause of exercise intolerance. 2,3 Fibers of the skeletal muscle are classified into 4 types according to their biochemical characters; slow-twitch type I fatigue-resistant fiber, fast-twitch type IIa fatigue-resistant fiber, fast-twitch type IIx fiber with intermediate fatigue-resistance, and fasttwitch type IIb fatigable fiber. 4 Fiber type shift toward more fatigable type IIb fiber is one of the characteristics observed during the development of skeletal myopathy in patients with CHF. 1,5 Clinical Perspective on p 808 Forkhead box O (FoxO) transcription factors play important roles in the regulation of various cellular processes,
Hypertension induces structural remodelling of arteries, which leads to arteriosclerosis and end-... more Hypertension induces structural remodelling of arteries, which leads to arteriosclerosis and end-organ damage. Hyperplasia of vascular smooth muscle cells (VSMCs) and infiltration of immune cells are the hallmark of hypertensive arterial remodelling. However, the precise molecular mechanisms of arterial remodelling remain elusive. We have recently reported that complement C1q activates β-catenin signalling independent of Wnts. Here, we show a critical role of complement C1-induced activation of β-catenin signalling in hypertensive arterial remodelling. Activation of β-catenin and proliferation of VSMCs were observed after blood-pressure elevation, which were prevented by genetic and chemical inhibition of β-catenin signalling. Macrophage depletion and C1qa gene deletion attenuated the hypertension-induced β-catenin signalling, proliferation of VSMCs and pathological arterial remodelling. Our findings unveil the link between complement C1 and arterial remodelling and suggest that C1-...
Enzymatic proteolysis by calpains, Ca(2+)-dependent intracellular cysteine proteases, has been im... more Enzymatic proteolysis by calpains, Ca(2+)-dependent intracellular cysteine proteases, has been implicated in pathological processes such as cellular degeneration or death. Here, we investigated the role of calpain activation in the hearts subjected to myocardial infarction. We produced myocardial infarction in Cast(-/-) mice deficient for calpastatin, the specific endogenous inhibitory protein for calpains, and Cast(+/+) mice. The activity of cardiac calpains in Cast(+/+) mice was not elevated within 1 day but showed a gradual elevation after 7 days following myocardial infarction, which was further pronounced in Cast(-/-) mice. Although the prevalence of cardiomyocyte death was indistinguishable between Cast(-/-) and Cast(+/+) mice, Cast(-/-) mice exhibited profound contractile dysfunction and chamber dilatation and showed a significant reduction in survival rate after myocardial infarction as compared with Cast(+/+) mice. Notably, immunofluorescence revealed that at 28 days after ...
Proceedings for Annual Meeting of The Japanese Pharmacological Society, 2019
Background: The present study is to estimate the geometrical and constituent heterogeneity effect... more Background: The present study is to estimate the geometrical and constituent heterogeneity effects on electrical conduction on in vitro monolayer consisting of atrial-like cardiomyocytes (ALCMs) derived from human induced pluripotent stem cells (hiPSCs) and human atrial fibroblasts (HAFbs) under HF field stimuli (HFFS). Method: We induced hiPSCs into ALCMs by adding all-trans retinoic acid (ATRA). The ALCMs and HAFbs were transferred in defined ratios on manually fabricated plate with geometrical transition. HFFS were delivered, and the electrical propagation was assessed by optical mapping. Results: ATRA-treated CMs showed atrial specific properties compared to untreated CMs. HFFS preferentially induced impaired conduction on ALCMs with an abrupt geometrical transition, but not on ALCMs with uniform geometry. In addition, the co-culture of HAFbs with the ALCMs deteriorated the stability of electrical conduction than in mono-culture of ALCMs. Conclusion: Geometrical heterogeneity under HFFS jeopardizes electrical conduction on in vitro ALCMs monolayer. Constituent heterogeneity represented by HAFbs contributes to the deterioration of the electrical conduction stability.
A correction to this article has been published and is linked from the HTML and PDF versions of t... more A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
Table 1 shows, Teneligliptin attenuated cardiomyocyte hypertrophy, fibrosis and lung congestion, ... more Table 1 shows, Teneligliptin attenuated cardiomyocyte hypertrophy, fibrosis and lung congestion, however, it induced left ventricular dilatation. Conclusion: Teneligliptin have potential to prevent hypertrophy, fibrosis and development of heart failure, however, it showed not only beneficial but harmful effects. Teneligliptin may induce left ventricular dilation by inhibiting normal compensation for pressure overload.
We reviewed the concept that de-d lialion is a mechvnisn of electrical rcmodeing m d eased hearls... more We reviewed the concept that de-d lialion is a mechvnisn of electrical rcmodeing m d eased hearls,rekrrnngloourdataon 1)d eutialchanges in action potential duration (APD) and transimt outward current (Ito) of cultured ventricular myocylcs of newborn rats and 2) changes in APD, Ito and us gene expression m hypertrophied adult nil venom. The latter changes associated with on of the hypertrophy appeared m the reverse order of those m newborn rats. Gene switching of ionic channels to d liation was oonckuded to be essential for electrical mnodebng of the died hearts.
type Ca 2+ channels are pivotal for cardiac excitation and contraction 1 and they are coded by 4 ... more type Ca 2+ channels are pivotal for cardiac excitation and contraction 1 and they are coded by 4 distinct genes: Cav1.1 (1S, skeletal muscle), Cav1.2 (1C, cardiac and smooth muscle), Cav1.3 (1D, neuron, neurosecretory cells and heart) and Cav1.4 (1F, retina). 2-4 The Cav1.2 Ca 2+ channel is predominant in cardiac muscle. 5,6 Ca 2+ entry into the cytosol through this channel triggers Ca 2+ release from the sarcoplasmic reticulum and underlies the Ca 2+ transient for contraction. 1 The Cav1.3 Ca 2+ channel activates at more negative membrane potentials than the Cav1.2 channel 7 and contributes to diastolic depolarization of sinoatrial (SA) nodal cells. This role of Cav1.3 Ca 2+ channels in SA nodal automaticity is further substantiated by the observation that Cav1.3 ablation leads to SA node dysfunction. 8-10 The murine embryonic heart starts to contract at 8.5 days post coitum (dpc) and the heart beat becomes regular at 9.5 dpc, when the heart shape is still tube-like. 11 Ventricles isolated from embryonic hearts at 9.5 dpc also beat spontaneously and regularly. Cardiac excitation and contraction in this early embryonic stage depend on Ca 2+ influx through
ongestive heart failure (CHF) is associated with an augmentation of sympathetic nerve activity an... more ongestive heart failure (CHF) is associated with an augmentation of sympathetic nerve activity and a reduction in parasympathetic nerve activity. 1,2 This autonomic imbalance may underlie the increased risk of sudden cardiac death in CHF patients. 3 In normal individuals, parasympathetic impulses are transmitted from the solitary nucleus in the brain to the heart through efferent preand post-ganglionic vagal neurons. Upon vagal stimulation, acetylcholine (ACh) released from the nerve terminals binds to muscarinic (M2) receptors of the heart. Emerging evidence shows that the ACh release at both pre-and postganglionic levels is facilitated by nitric oxide (NO) generated by neuronal NO synthase (nNOS) at nerve terminals. 4,5 Stimulation of the M2 receptor in the sinoatrial (SA) node pacemaker cells results in a reduction of heart rate (HR) via increase of ACh-sensitive potassium channel current (IK,ACh) and decrease of hyperpolarization-activated cation channel current (If). 6 The level within the vagal cascade, at which a defect results in parasympathetic withdrawal, is unknown. In a dog model of CHF induced by rapid ventricular
American Journal of Physiology-Heart and Circulatory Physiology, 2004
T-type Ca2+ channels are implicated in cardiac automaticity, cell growth, and cardiovascular remo... more T-type Ca2+ channels are implicated in cardiac automaticity, cell growth, and cardiovascular remodeling. Two voltage-gated Ca2+ subtypes (Cav3.1 and Cav3.2) have been cloned for the pore-forming α1-subunit of the T-type Ca2+ channel in cardiac muscle, but their differential roles remain to be clarified. The aim of this study was to elucidate the relative contribution of the two subtypes in the normal development of mouse hearts. A whole cell patch clamp was used to record ionic currents from ventricular myocytes isolated from mice of early (E9.5) and late embryonic days (E18) and from adult 10-wk-old mice. Large T-type Ca2+ current ( ICa,T) was observed at both E9.5 and E18, displaying similar voltage-dependence and kinetics of activation and inactivation. The current was inhibited by Ni2+ at relatively low concentrations (IC50 26–31 μM). ICa,T was undetectable in adult myocytes. Quantitative PCR analysis revealed that Cav3.2 mRNA is the predominant subtype encoding T-type Ca2+ chan...
Human induced pluripotent stem (hiPS) cells have been used as a cell source for regenerative ther... more Human induced pluripotent stem (hiPS) cells have been used as a cell source for regenerative therapy and disease modeling. The purity of hiPS-cardiomyocytes (hiPS-CMs) has markedly improved with advancements in cell culture and differentiation protocols. However, the morphological features and molecular properties of the relatively immature cells are still unclear, which has hampered their clinical application. The aim of the present study was to investigate the extent to which topographic substrates actively influence hiPS-CMs. hiPS-CMs were seeded on randomized oriented fiber substrate (random), anisotropic aligned fiber substrate (align), and flat non-scaffold substrate (flat). After culturing for one week, the hiPS-CMs on the aligned patterns showed more mature-like properties, including elongated rod shape, shorter duration of action potential, accelerated conduction velocity, and elevated cardiac gene expression. Subsequently, to determine whether this development was irrevers...
Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration accompanied... more Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration accompanied by dilated cardiomyopathy. Recently, abnormality of yes-associated protein (YAP) has been reported as the pathogenesis of muscle degeneration of DMD; however YAP activity remains unclear in dystrophic heart of DMD. Herein, we investigated YAP activity using disease-specific induced pluripotent stem cell (iPSC) derived cardiomyocytes (CMs) in DMD. DMD-iPSCs were generated from DMD patient with exon 48–54 deletion in DMD, and genome-edited (Ed)-DMD-iPSCs with in-frame (Ed-DMD-iPSCs) were created using CRISPR/Cas9. Nuclear translocation of YAP [nuclear (N)/cytoplasmic (C) ratio] was significantly lower in DMD-iPSC-CMs than in Ed-DMD-iPSC-CMs. In addition, Ki67 expression, indicating proliferative ability, was significantly lower in DMD-iPSC-CMs than Ed-DMD-iPSC-CMs. Therefore, immunofluorescent staining showed that actin stress fibers associated with YAP activity by mechanotransduction wer...
Molecular signaling of sympathetic innervation of myocardium is an unresolved issue. The purpose ... more Molecular signaling of sympathetic innervation of myocardium is an unresolved issue. The purpose of this study was to investigate the effect of neurotrophic factors on sympathetic neurite growth towards cardiomyocytes. Cardiomyocytes (CMs) and sympathetic neurons (SNs) were isolated from neonatal rat hearts and superior cervical ganglia, and were co-cultured, either in a random or localized way. Neurite growth from SNs toward CMs was assessed by immunohistochemistry for neurofilament M and α-actinin in response to neurotrophic factors-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and a chemical repellent, semaphorin 3A. As a result, GDNF as well as NGF and BDNF stimulated neurite growth. GDNF enhanced neurite outgrowth even under the NGF-depleted culture condition, excluding an indirect effect of GDNF via NGF. Quantification of mRNA and protein by real-time PCR and immunohis...
American Journal of Physiology-Heart and Circulatory Physiology
The effects of myocardial hypertrophy on mRNA expression levels of voltage-gated K+ channels were... more The effects of myocardial hypertrophy on mRNA expression levels of voltage-gated K+ channels were investigated using monocrotaline (MCT)-induced pulmonary hypertensive rats. The ratio of right ventricle weight to left ventricle plus septum weight on day 28 was increased significantly compared with control rats [control vs. MCT: 0.27 ± 0.01 vs. 0.58 ± 0.03 ms ( n = 8–13); P < 0.05]. Electrocardiograms showed that QRS duration [control vs. MCT: 26.4 ± 2.6 ms vs. 31.5 ± 5.8 ms ( n = 6); P < 0.05], Q-T interval [control vs. MCT: 100.8 ± 8.9 ms vs. 110.0 ± 4.2 ms ( n = 6); P < 0.05] and corrected Q-T interval [Q-Tc; control vs. MCT: 8.4 ± 0.7 ms vs. 10.2 ± 0.4 ms ( n = 6); P < 0.05] were prolonged significantly on day 28. mRNA levels of Kv1.2, 1.5, 2.1, 4.2, and 4.3 for day 28 assessed by ribonuclease protection assays were decreased significantly from control by 60 ± 10, 76 ± 3, 58 ± 5, 81 ± 5, and 45 ± 12%, respectively ( n = 3; P < 0.005), and Kv1.4 mRNA level for day 2...
The DNA damage response (DDR) plays a pivotal role in maintaining genome integrity. DNA damage an... more The DNA damage response (DDR) plays a pivotal role in maintaining genome integrity. DNA damage and DDR activation are observed in the failing heart, however, the type of DNA damage and its role in the pathogenesis of heart failure remain elusive. Here we show the critical role of DNA single-strand break (SSB) in the pathogenesis of pressure overload-induced heart failure. Accumulation of unrepaired SSB is observed in cardiomyocytes of the failing heart. Unrepaired SSB activates DDR and increases the expression of inflammatory cytokines through NF-κB signalling. Pressure overload-induced heart failure is more severe in the mice lacking XRCC1, an essential protein for SSB repair, which is rescued by blocking DDR activation through genetic deletion of ATM, suggesting the causative role of SSB accumulation and DDR activation in the pathogenesis of heart failure. Prevention of SSB accumulation or persistent DDR activation may become a new therapeutic strategy against heart failure.
Disruption of angiotensin II type 1 (AT 1) receptor prolonged life span in mice. Since aging-rela... more Disruption of angiotensin II type 1 (AT 1) receptor prolonged life span in mice. Since aging-related decline in skeletal muscle function was retarded in Atgr1a −/− mice, we examined the role of AT 1 receptor in muscle regeneration after injury. Administration of AT 1 receptor blocker irbesartan increased the size of regenerating myofibers, decreased fibrosis, and enhanced functional muscle recovery after cryoinjury. We recently reported that complement C1q, secreted by macrophages, activated Wnt/β-catenin signaling and promoted aging-related decline in regenerative capacity of skeletal muscle. Notably, irbesartan induced M2 polarization of macrophages, but reduced C1q expression in cryoinjured muscles and in cultured macrophage cells. Irbesartan inhibited upregulation of Axin2, a downstream gene of Wnt/β-catenin pathway, in cryoinjured muscles. In addition, topical administration of C1q reversed beneficial effects of irbesartan on skeletal muscle regeneration after injury. These results suggest that AT 1 receptor blockade improves muscle repair and regeneration through down-regulation of the aging-promoting C1q-Wnt/β-catenin signaling pathway. Angiotensin (Ang) II is the crucial bioactive molecule of the renin-angiotensin system, and exerts most of its pathophysiological actions through binding to Ang II type 1 (AT 1) receptor 1,2. While humans have a single AGTR1 gene that encodes AT 1 receptor, mice have Agtr1a and Agtr1b genes encoding two isoforms (AT 1a and AT 1b) of AT 1 receptor, and the major isoform of mouse AT 1 receptor is AT 1a receptor 1,2. AT 1 receptor is a member of the G protein-coupled receptor family, which shares typical conformation of seven transmembrane-spanning α-helices 3. Upon stimulation by binding to Ang II or mechanical stretch 4,5 , AT 1 receptor activates multiple G protein-dependent and-independent signaling pathways and promote the formation of reactive oxygen species (ROS), leading to a variety of responses such as
Circulation journal : official journal of the Japanese Circulation Society, Jan 28, 2015
Adipose tissue is one of the sources of mesenchymal stem cells, which have the potential to diffe... more Adipose tissue is one of the sources of mesenchymal stem cells, which have the potential to differentiate into various types of cells, including myocytes. Whether brown adipose tissue (BAT)-derived cells might differentiate into the cardiac pacemaking-conducting cells, and have the potential to regenerate the cardiac conduction system (CCS), is investigated in this study.Methods and Results:BAT was isolated from the interscapular area of mice and enzymatically digested before culture. Round or fusiform cells showed spontaneous beating at 4-7 days after culturing of BAT-derived cells. Reverse transcriptase-polymerase chain reaction analysis and immunocytochemical analysis revealed that BAT-derived cells expressed several cardiomyocytes, the CCS and pacemaker (PM) cell marker genes and proteins. Patch-clamp techniques revealed that spontaneous electrical activity and the shape of the action potential showed properties of cardiac PM cells. Next, a complete atrioventricular (AV) block w...
American journal of physiology. Heart and circulatory physiology, Jan 8, 2015
Although cardiac fibrosis causes heart failure, its molecular mechanisms remain elusive. In this ... more Although cardiac fibrosis causes heart failure, its molecular mechanisms remain elusive. In this study, we investigated the mechanisms of cardiac fibrosis and examined the effects of the anti-fibrotic drug pirfenidone (PFD) on chronic heart failure. To understand the responsible mechanisms, we generated an in vivo pressure-overloaded heart failure model via transverse aortic constriction (TAC) and examined the effects of PFD on chronic-phase cardiac fibrosis and function. In the vehicle group, contractile dysfunction and left ventricle fibrosis progressed further from 4 to 8 weeks after TAC but were prevented by PFD treatment beginning 4 weeks after TAC. We isolated cardiac fibroblasts and vascular endothelial cells from the left ventricles of adult male mice and investigated the cell type-specific effects of PFD. TGF-β induced upregulated collagen 1 expression via p38 phosphorylation and downregulated claudin 5 (Cldn5) expression in cardiac fibroblasts and endothelial cells, respec...
E xercise intolerance is a common clinical manifestation associated with chronic heart failure (C... more E xercise intolerance is a common clinical manifestation associated with chronic heart failure (CHF). 1 Various morphological, histological, and biochemical changes are observed in the skeletal muscle of patients with CHF. These changes are collectively called skeletal myopathy and are the major cause of exercise intolerance. 2,3 Fibers of the skeletal muscle are classified into 4 types according to their biochemical characters; slow-twitch type I fatigue-resistant fiber, fast-twitch type IIa fatigue-resistant fiber, fast-twitch type IIx fiber with intermediate fatigue-resistance, and fasttwitch type IIb fatigable fiber. 4 Fiber type shift toward more fatigable type IIb fiber is one of the characteristics observed during the development of skeletal myopathy in patients with CHF. 1,5 Clinical Perspective on p 808 Forkhead box O (FoxO) transcription factors play important roles in the regulation of various cellular processes,
Hypertension induces structural remodelling of arteries, which leads to arteriosclerosis and end-... more Hypertension induces structural remodelling of arteries, which leads to arteriosclerosis and end-organ damage. Hyperplasia of vascular smooth muscle cells (VSMCs) and infiltration of immune cells are the hallmark of hypertensive arterial remodelling. However, the precise molecular mechanisms of arterial remodelling remain elusive. We have recently reported that complement C1q activates β-catenin signalling independent of Wnts. Here, we show a critical role of complement C1-induced activation of β-catenin signalling in hypertensive arterial remodelling. Activation of β-catenin and proliferation of VSMCs were observed after blood-pressure elevation, which were prevented by genetic and chemical inhibition of β-catenin signalling. Macrophage depletion and C1qa gene deletion attenuated the hypertension-induced β-catenin signalling, proliferation of VSMCs and pathological arterial remodelling. Our findings unveil the link between complement C1 and arterial remodelling and suggest that C1-...
Enzymatic proteolysis by calpains, Ca(2+)-dependent intracellular cysteine proteases, has been im... more Enzymatic proteolysis by calpains, Ca(2+)-dependent intracellular cysteine proteases, has been implicated in pathological processes such as cellular degeneration or death. Here, we investigated the role of calpain activation in the hearts subjected to myocardial infarction. We produced myocardial infarction in Cast(-/-) mice deficient for calpastatin, the specific endogenous inhibitory protein for calpains, and Cast(+/+) mice. The activity of cardiac calpains in Cast(+/+) mice was not elevated within 1 day but showed a gradual elevation after 7 days following myocardial infarction, which was further pronounced in Cast(-/-) mice. Although the prevalence of cardiomyocyte death was indistinguishable between Cast(-/-) and Cast(+/+) mice, Cast(-/-) mice exhibited profound contractile dysfunction and chamber dilatation and showed a significant reduction in survival rate after myocardial infarction as compared with Cast(+/+) mice. Notably, immunofluorescence revealed that at 28 days after ...
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Papers by Jong-kook Lee