Rong Huo

Reciprocal Repression Between MicroRNA-133 and Calcineurin Regulates Cardiac Hypertrophy A Novel Mechanism for Progressive Cardiac Hypertrophy

Cardiac hypertrophy involves a remodeling process of the heart in response to diverse pathological stimuli. Both calcineurin/nuclear factor of activated T cells pathway and microRNA-133 (miR-133) have been shown to play a critical role in cardiac hypertrophy. It has been recognized that the expression and activity of calcineurin increases and miR-133 expression decreases in the hypertrophic heart, and inhibition of calcineurin or increase of miR-133 expression protects against cardiac hypertrophy. Here we tested the interaction between miR-133 and calcineurin in cardiac hypertrophy. Cardiac hypertrophy in vivo and in vitro was induced by transverse aortic constriction and phenylephrine treatment. mRNA levels were measured by using real-time PCR methods. Luciferase assays showed that transfection of miR-133 in HEK293 cells downregulated calcineurin expression, which was reversed by cotransfection with the miR-133–specific 2′-O-methyl antisense inhibitory oligoribonucleotides. These results were confirmed in cultured primary cardiomyocytes. miR-133 expression was downregulated, and calcineurin activity was enhanced in both in vivo and in vitro cardiac hypertrophy models. Treatment of cells and animals with cyclosporin A, an inhibitor of calcineurin, prevented miR-133 downregulation. Moreover, the antisense oligodeoxynucleotides against the catalytic subunits of calcineurin A&bgr; and the decoy oligodeoxynucleotides targeting nuclear factor of activated T cells transcription factor, a calcineurin downstream effector, increased miR-133 expression in cultured primary cardiomyocytes. Our data show that reciprocal repression between miR-133 and calcineurin regulates cardiac hypertrophy.

Bone Morphogenetic Protein-4 Mediates Cardiac Hypertrophy, Apoptosis, and Fibrosis in Experimentally Pathological Cardiac Hypertrophy

Identifying the key factor mediating pathological cardiac hypertrophy is critically important for developing the strategy to protect against heart failure. Bone morphogenetic protein-4 (BMP4) is a mechanosensitive and proinflammatory gene. In this study, we investigated the role of BMP4 in cardiac hypertrophy, apoptosis, and fibrosis in experimentally pathological cardiac hypertrophy. The in vivo pathological cardiac hypertrophy models were induced by pressure-overload and angiotensin (Ang) II constant infusion in mice, and the in vitro model was induced by Ang II exposure to cultured cardiomyocytes. The expression of BMP4 increased in pressure overload, Ang II constant infusion-induced pathological cardiac hypertrophy, but not in swimming exercise-induced physiological cardiac hypertrophy in mice. BMP4 expression also increased in Ang II–induced cardiomyocyte hypertrophy in vitro. In turn, BMP4 induced cardiomyocyte hypertrophy, apoptosis, and cardiac fibrosis, and these pathological consequences were inhibited by the treatment with BMP4 inhibitors noggin and DMH1. Moreover, Ang II–induced cardiomyocyte hypertrophy was inhibited by BMP4 inhibitors. The underlying mechanism that BMP4-induced cardiomyocyte hypertrophy and apoptosis was through increasing NADPH oxidase 4 expression and reactive oxygen species-dependent pathways. Lentivirus-mediated overexpression of BMP4 recapitulated hypertrophy and apoptosis in cultured cardiomyocytes. BMP4 inhibitor DMH1 inhibited pressure overload–induced cardiac hypertrophy in mice in vivo. The plasma BMP4 level of heart failure patients was increased compared with that of subjects without heart failure. In summary, we conclude that BMP4 is a mediator and novel therapeutic target for pathological cardiac hypertrophy.

Mild hypoxia-induced cardiomyocyte hypertrophy via up-regulation of HIF-1α-mediated TRPC signalling.

Hypoxia‐inducible factor‐1 alpha (HIF‐1α) is a central transcriptional regulator of hypoxic response. The present study was designed to investigate the role of HIF‐1α in mild hypoxia‐induced cardiomyocytes hypertrophy and its underlying mechanism. Mild hypoxia (MH, 10% O2) caused hypertrophy in cultured neonatal rat cardiac myocytes, which was accompanied with increase of HIF‐1α mRNA and accumulation of HIF‐1α protein in nuclei. Transient receptor potential canonical (TRPC) channels including TRPC3 and TRPC6, except for TRPC1, were increased, and Ca2+‐calcineurin signals were also enhanced in a time‐dependent manner under MH condition. MH‐induced cardiomyocytes hypertrophy, TRPC up‐regulation and enhanced Ca2+‐calcineurin signals were inhibited by an HIF‐1α specific blocker, SC205346 (30 μM), whereas promoted by HIF‐1α overexpression. Electrophysiological voltage‐clamp demonstrated that DAG analogue, OAG (30 μM), induced TRPC current by as much as 170% in neonatal rat cardiomyocytes overexpressing HIF‐1α compared to negative control. These results implicate that HIF‐1α plays a key role in development of cardiac hypertrophy in responses to hypoxic stress. Its mechanism is associated with up‐regulating TRPC3, TRPC6 expression, activating TRPC current and subsequently leading to enhanced Ca2+‐calcineurin signals.

Large T-antigen up-regulates Kv4.3 K⁺ channels through Sp1, and Kv4.3 K⁺ channels contribute to cell apoptosis and necrosis through activation of calcium/calmodulin-dependent protein kinase II.

Down-regulation of Kv4.3 K⁺ channels commonly occurs in multiple diseases, but the understanding of the regulation of Kv4.3 K⁺ channels and the role of Kv4.3 K⁺ channels in pathological conditions are limited. HEK (human embryonic kidney)-293T cells are derived from HEK-293 cells which are transformed by expression of the large T-antigen. In the present study, by comparing HEK-293 and HEK-293T cells, we find that HEK-293T cells express more Kv4.3 K⁺ channels and more transcription factor Sp1 (specificity protein 1) than HEK-293 cells. Inhibition of Sp1 with Sp1 decoy oligonucleotide reduces Kv4.3 K⁺ channel expression in HEK-293T cells. Transfection of pN3-Sp1FL vector increases Sp1 protein expression and results in increased Kv4.3 K⁺ expression in HEK-293 cells. Since the ultimate determinant of the phenotype difference between HEK-293 and HEK-293T cells is the large T-antigen, we conclude that the large T-antigen up-regulates Kv4.3 K⁺ channel expression through an increase in Sp1. In both HEK-293 and HEK-293T cells, inhibition of Kv4.3 K⁺ channels with 4-AP (4-aminopyridine) or Kv4.3 small interfering RNA induces cell apoptosis and necrosis, which are completely rescued by the specific CaMKII (calcium/calmodulin-dependent protein kinase II) inhibitor KN-93, suggesting that Kv4.3 K⁺ channels contribute to cell apoptosis and necrosis through CaMKII activation. In summary, we establish: (i) the HEK-293 and HEK-293T cell model for Kv4.3 K⁺ channel study; (ii) that large T-antigen up-regulates Kv4.3 K⁺ channels through increasing Sp1 levels; and (iii) that Kv4.3 K⁺ channels contribute to cell apoptosis and necrosis through activating CaMKII. The present study provides deep insights into the mechanism of the regulation of Kv4.3 K⁺ channels and the role of Kv4.3 K⁺ channels in cell death.

Curcumin inhibits hERG potassium channels in vitro.

Curcumin is reported to exert antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, and anti-tumor activities. The human ether-a-go-go related gene (hERG) encodes the rapid component of the delayed rectifier K⁺ currents. Inhibition of hERG K⁺ channels leads to cardiac repolarization prolongation, which contributes to either the anti-arrhythmic effects of anti-arrhythmic drugs, or the pro-arrhythmic effects (induction of long QT syndrome) of some drugs not used for anti-arrhythmias. Since curcumin shows multiple beneficial effects and clinical significance, the aim of the present study is to investigate the effect of curcumin on hERG K⁺ channels, elucidating its potential cardiac therapeutic or toxic effects. In whole-cell patch-clamp experiments, we found that curcumin inhibited hERG K⁺ currents in HEK293 cells stably expressing hERG channels in a dose-dependent manner, with IC₅₀ value of 5.55 μM. The deactivation, inactivation and the recovery time from inactivation of hERG channels were significantly changed by acute treatment of 10 μM curcumin. Incubation of 20 μM curcumin for 24h reduced the HEK293 cell viability. Intravenous injection of maximal amount of curcumin in rabbits (20 mg/animal) did not affect the cardiac repolarization manifested with QTc value. We conclude that curcumin inhibits hERG K⁺ channels in vitro.

Bone Morphogenetic Protein-2 Antagonizes Bone Morphogenetic Protein-4 Induced Cardiomyocyte Hypertrophy and Apoptosis

Our previous work showed that the expression of bone morphogenetic protein‐4 (BMP4) was up‐regulated in pathological cardiac hypertrophy models and BMP4 induced cardiomyocyte hypertrophy and apoptosis. Bone morphogenetic protein‐2 (BMP2) and BMP4 share greater than 80% amino acid homology and there exists an interaction between BMP2 and BMP4, so the aim of the present study was to elucidate the changes of BMP2 in the cardiac hypertrophy models and the effects of BMP2 on BMP4‐induced cardiomyocyte hypertrophy and apoptosis. The in vivo cardiac hypertrophy models were induced by pressure‐overload and swimming exercise in mice. BMP2 mRNA and protein expressions increased in pressure‐overload and swimming‐exercise induced cardiac hypertrophy. BMP2 itself did not elicit cardiomyocyte hypertrophy and apoptosis, but antagonized BMP4‐induced cardiomyocyte hypertrophy and apoptosis. BMP2 stimulated Akt in cardiomyocytes and Akt inhibitor prevented the antagonism of BMP2 on BMP4‐induced cardiomyocyte apoptosis. Furthermore, BMP2 inhibited BMP4‐induced JNK activation in cardiomyocytes. In conclusion, BMP2 antagonizes BMP4‐induced cardiomyocyte hypertrophy and apoptosis. The anti‐apoptotic effects of BMP2 on BMP4‐induced cardiomyocyte apoptosis might be through activating Akt and inhibiting JNK activation. J. Cell. Physiol. 229: 1503–1510, 2014.

Systemic Heme Oxygenase-1 Transgenic Overexpression Aggravates Pressure Overload- induced Cardiac Hypertrophy in Mice

Background/Aims: Heme oxygenase-1(HO-1) has been reported to protect against cardiac hypertrophy in cultured neonatal cardiomyocytes treated with HO-1 inducer, cardiac specific HO-1 transgenic mice, or animals treated with HO-1 inducer. The aim of the present study is to examine the effects of systemic HO-1 transgenic overexpression on pressure overload-induced cardiac hypertrophy in mice. Methods: Pressure-overload cardiac hypertrophy was induced by transverse aortic constriction (TAC) in WT (wild type) and systemic HO-1 transgenic overexpression (TG) mice. Results: We found that systemic HO-1 transgenic overexpression aggravated pressure overload-induced cardiac hypertrophy. Pressure-overload induced the more increases of heart weight/ body weigh index, left ventricular weight/ body weight index, β-MHC protein expression, cardiac interstitial fibrosis in TG mice than in WT mice. Pressure-overload increased cardiac HO-1 protein expression in WT but not TG mice, but the cardiac HO-1 protein level was still higher in TAC-treated TG mice than in TAC-treated WT mice. The basal cardiac calcineurin protein level in TG mice was lower than that in WT mice. Pressure-overload increased calcineurin protein expression in both WT and TG mice; however, pressure-overload induced more calcineurin protein expression in TG mice than in WT mice. Conclusion: This study shows for the first time that systemic HO-1 transgenic overexpression aggravates pressure overload-induced cardiac hypertrophy.

Bone morphogenetic protein-4 contributes to the down-regulation of Kv4.3 K+ channels in pathological cardiac hypertrophy.

Kv4.3 K(+) channels contributing to Ito are involved in the repolarization of cardiac action potential. Kv4.3 K(+) channels decrease in pathological cardiac hypertrophy, but the mechanism remains unclear. Our previous study found that the expression of bone morphogenetic protein 4 (BMP4) increased in pressure-overload and Ang II constant infusion induced cardiac hypertrophy. Since the downregulation of Kv4.3 K(+) channels and the upregulation of BMP4 simultaneously occur in pathological cardiac hypertrophy, we hypothesize that the up-regulated BMP4 would contribute to the downregulation of Kv4.3 K(+) channels in cardiac hypertrophy. We found that BMP4 treatment reduced Kv4.3 but not Kv4.2 and Kv1.4 K(+) channel protein expression, and BMP4-induced decrease of Kv4.3 K(+) channel protein expression was reversed by BMP4 inhibitor noggin and DMH1 in cultured cardiomyocytes in vitro. BMP4-induced decrease of Kv4.3 K(+) channel protein expression was also reversed by the NADPH oxidase inhibitor apocynin and the radical scavenger tempol. In in vivo transverse aortic constriction (TAC)-induced cardiac hypertrophy, constant infusion of DMH1 completely rescued TAC-induced down-regulation of Kv4.3 K(+) channel protein expression. We conclude that BMP4 contributes to the downregulation of Kv4.3 K(+) channels in pathological cardiac hypertrophy and the underlying mechanism might be through increasing ROS production.

Oestrogen inhibits BMP4‐induced BMP4 expression in cardiomyocytes: a potential mechanism of oestrogen‐mediated protection against cardiac hypertrophy

Oestrogen inhibits cardiac hypertrophy and bone morphogenetic protein‐4 (BMP4) induces cardiac hypertrophy. Here we have studied the inhibition by oestrogen of BMP4 expression in cardiomyocytes.

The potential role of Kv4.3 K+ channel in heart hypertrophy

Transient outward K+ current (Ito) plays a crucial role in the early phase of cardiac action potential repolarization. Kv4.3 K+ channel is an important component of Ito. The function and expression of Kv4.3 K+ channel decrease in variety of heart diseases, especially in heart hypertrophy/heart failure. In this review, we summarized the changes of cardiac Kv4.3 K+ channel in heart diseases and discussed the potential role of Kv4.3 K+ channel in heart hypertrophy/heart failure. In heart hypertrophy/heart failure of mice and rats, downregulation of Kv4.3 K+ channel leads to prolongation of action potential duration (APD), which is associated with increased [Ca2+]i, activation of calcineurin and heart hypertrophy/heart failure. However, in canine and human, Kv4.3 K+ channel does not play a major role in setting cardiac APD. So, in addition to Kv4.3 K+ channel/APD/[Ca2+]i pathway, there exits another mechanism of Kv4.3 K+ channel in heart hypertrophy and heart failure: downregulation of Kv4.3 K+ channels leads to CaMKII dissociation from Kv4.3–CaMKII complex and subsequent activation of the dissociated CaMKII, which induces heart hypertrophy/heart failure. Upregulation of Kv4.3 K+ channel inhibits CaMKII activation and its related harmful consequences. We put forward a new point-of-view that Kv4.3 K+ channel is involved in heart hypertrophy/heart failure independently of its electric function, and drugs inhibiting or upregulating Kv4.3 K+ channel might be potentially harmful or beneficial to hearts through CaMKII.