ZBTB20 Regulates SERCA2a Activity and Myocardial Contractility Through Phospholamban.

BACKGROUND: Intracellular Ca2+ cycling determines myocardial contraction and relaxation in response to physiological demands. SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a) is responsible for the sequestration of cytosolic Ca2+ into intracellular stores during cardiac relaxation, and its activity is reversibly inhibited by PLN (phospholamban). However, the regulatory hierarchy of SERCA2a activity remains unclear. METHODS: Cardiomyocyte-specific ZBTB20 knockout mice were generated by crossing ZBTB20flox mice with Myh6-Cre mice. Echocardiography, blood pressure measurements, Langendorff perfusion, histological analysis and immunohistochemistry, quantitative reverse transcription-PCR, Western blot analysis, electrophysiological measurements, and chromatin immunoprecipitation assay were performed to clarify the phenotype and elucidate the molecular mechanisms. RESULTS: Specific ablation of ZBTB20 in cardiomyocyte led to a significant increase in basal myocardial contractile parameters both in vivo and in vitro, accompanied by an impairment in cardiac reserve and exercise capacity. Moreover, the cardiomyocytes lacking ZBTB20 showed an increase in sarcoplasmic reticular Ca2+ content and exhibited a remarkable enhancement in both SERCA2a activity and electrically stimulated contraction. Mechanistically, PLN expression was dramatically reduced in cardiomyocytes at the mRNA and protein levels by ZBTB20 deletion or silencing, and PLN overexpression could largely restore the basal contractility in ZBTB20-deficient cardiomyocytes. CONCLUSIONS: These data point to ZBTB20 as a fine-tuning modulator of PLN expression and SERCA2a activity, thereby offering new perspective on the regulation of basal contractility in the mammalian heart.

The Role of Prohibitin-2 in Diseases.

Prohibitin-2 (PHB2) is a conserved protein in mitochondria that regulates various biological processes, including cell cycle, proliferation, apoptosis, transcription, signal transduction, and mitochondrial ridge morphogenesis. Recently, there has been growing interest in the biological function of PHB2. This article primarily discusses the recent advances in the role of PHB2 in diseases.

Subcellular Expression Patterns of FKBP Prolyl Isomerase 10 (FKBP10) in Colorectal Cancer and Its Clinical Significance.

FKBP10, a member of the FK506-binding protein (FKBP) family, has been implicated in cancer development, although its prognostic function remains controversial. In this study, we analyzed the expression of FKBP10 in tumor tissues using online databases (TCGA) as well as our CRC cohort, and investigated the relationship between its subcellular expression pattern and patient outcomes. Cox regression analysis was used to determine the associations between different subcellular expression patterns of FKBP10 and clinical features of patients. We also discussed the expression level of FKBP10 based on different subcellular expression patterns. Our results showed that FKBP10 was significantly elevated in CRC tissues and exhibited three different subcellular expression patterns which were defined as 'FKBP10-C' (concentrated), 'FKBP10-T' (transitional) and 'FKBP10-D' (dispersive). The FKBP10-D expression pattern was only found in tumor tissues and was associated with unfavorable disease-free survival in CRC patients. High expression levels of FKBP10-C predicted an unfavorable prognosis of recurrence of CRC, while FKBP10-D did not. Our findings suggest that the subcellular expression patterns and expression level of FKBP10 play crucial prognostic roles in CRC, which revealed that FKBP10 may be a viable prognostic and therapeutic target for the diagnosis and treatment of CRC.

Eukaryotic Ribosomal Protein S5 of the 40S Subunit: Structure and Function.

The ribosomal protein RPS5 is one of the prime proteins to combine with RNA and belongs to the conserved ribosomal protein family. It plays a substantial role in the process of translation and also has some non-ribosome functions. Despite the enormous studies on the relationship between the structure and function of prokaryotic RPS7, the structure and molecular details of the mechanism of eukaryotic RPS5 remain largely unexplored. This article focuses on the structure of RPS5 and its role in cells and diseases, especially the binding to 18S rRNA. The role of RPS5 in translation initiation and its potential use as targets for liver disease and cancer are discussed.

ChREBP-regulated lipogenesis is not required for the thermogenesis of brown adipose tissue.

OBJECTIVES: Brown adipose tissue (BAT) plays a critical role in energy expenditure by uncoupling protein 1 (UCP1)-mediated thermogenesis and represents an important therapeutic target for metabolic diseases. Carbohydrate response element-binding protein (ChREBP) is a key transcription factor regulating de novo lipogenesis, and its activity is associated with UCP1 expression and thermogenesis in BAT. However, the exact physiological role of endogenous ChREBP in BAT thermogenesis remains unclear. METHODS: We used the Cre/LoxP system to generate ChREBP BAT-specific knockout mice, and examined their BAT thermogenesis under acute cold exposure and long-term cold acclimation. Gene expression was analyzed at the mRNA and protein levels, and lipogenesis was examined by 3H-H2O incorporation assay. RESULTS: The mice lacking ChREBP specifically in BAT displayed a significant decrease in the expression levels of lipogenic genes and the activity of de novo lipogenesis in BAT after cold exposure, with UCP1 expression decreased under thermoneutral conditions or after acute cold exposure but not chronic cold acclimation. Unexpectedly, BAT-specific ChREBP deletion did not significantly affect body temperature as well as local temperature or morphology of BAT after acute cold exposure or chronic cold acclimation. Of note, ChREBP deletion mildly aggravated glucose intolerance induced by a high-fat diet. CONCLUSIONS: Our work indicates that ChREBP regulates de novo lipogenesis in BAT and glucose tolerance, but is not required for non-shivering thermogenesis by BAT under acute or long-term cold exposure.

The zinc finger and BTB domain containing protein ZBTB20 regulates plasma triglyceride metabolism by repressing lipoprotein lipase gene transcription in hepatocytes.

BACKGROUND AND AIMS: Lipoprotein lipase (LPL) is responsible for the lipolytic processing of triglyceride-rich lipoproteins, the deficiency of which causes severe hypertriglyceridemia. Liver LPL expression is high in suckling rodents but relatively low at adulthood. However, the regulatory mechanism and functional significance of liver LPL expression are incompletely understood. We have established the zinc finger protein ZBTB20 as a critical factor for hepatic lipogenesis. Here, we evaluated the role of ZBTB20 in regulating liver Lpl gene transcription and plasma triglyceride metabolism. APPROACH AND RESULTS: Hepatocyte-specific inactivation of ZBTB20 in mice led to a remarkable increase in LPL expression at the mRNA and protein levels in adult liver, in which LPL protein was mainly localized onto sinusoidal epithelial cells and Kupffer cells. As a result, the LPL activity in postheparin plasma was substantially increased, and postprandial plasma triglyceride clearance was significantly enhanced, whereas plasma triglyceride levels were decreased. The dysregulated liver LPL expression and low plasma triglyceride levels in ZBTB20-deficient mice were normalized by inactivating hepatic LPL expression. ZBTB20 deficiency protected the mice against high-fat diet-induced hyperlipidemia without causing excessive triglyceride accumulation in the liver. Chromatin immunoprecipitation and gel-shift assay studies revealed that ZBTB20 binds to the LPL promoter in the liver. A luciferase reporter assay revealed that ZBTB20 inhibits the transcriptional activity of LPL promoter. The regulation of LPL expression by ZBTB20 is liver-specific under physiological conditions. CONCLUSIONS: Liver ZBTB20 serves as a key regulator of LPL expression and plasma triglyceride metabolism and could be a therapeutic target for hypertriglyceridemia.

ZBTB20 in Nociceptive Neurons of the Trigeminal Ganglia Regulates Pruritus.

Recent studies have shown that ZBTB20, a zinc-finger protein containing transcription factor, is highly expressed in small-diameter primary sensory neurons in mice, and modulates pain through regulating TRP channels. However, whether ZBTB20 regulates itch sensation has not been demonstrated. In this study, small-diameter primary sensory neuron-specific ZBTB20 knockout (PN-ZB20KO) mice were used to investigate the role of ZBTB20 in the regulation of itch sensation. First, both histamine-dependent and non-histamine-dependent itch behaviors induced by injection of histamine and chloroquine (CQ) into the cheek were significantly diminished in PN-ZB20KO mice. Second, double immunohistochemistry showed that ZBTB20 was mainly expressed in CGRP-labeled small peptidergic neurons and was expressed at low levels in IB4-labeled small non-peptidergic and NF200-labeled large neurons in the trigeminal ganglia (TG). ZBTB20 was also expressed in most TRPV1+ and TRPA1+ neurons and to a lesser extent in TRPM8+ neurons in the TG. Furthermore, cheek injection of histamine and CQ enhanced the mRNA expression of TRPV1 and TRPA1 but not TRPM8 in the TG. Moreover, TRPV1 and TRPA1 knockout (KO) mice exhibited attenuation of itch behavior induced by histamine and CQ, respectively. Finally, silencing endogenous ZBTB20 with recombinant lentivirus expressing a short hairpin RNA against ZBTB20 (LV-shZBTB20) in TG neurons attenuated histamine- and non-histamine-induced itch and downregulated TRP channels in the TG. Our study suggests that ZBTB20 plays an important role in mediating itch in small primary sensory neurons.

Zbtb20 deficiency causes cardiac contractile dysfunction in mice.

The zinc-finger protein ZBTB20 regulates development and metabolism in multiple systems, and is essential for postnatal survival in mice. However, its potential role in the cardiovascular system remains undefined. Here, we demonstrate that ZBTB20 is critically involved in the regulation of cardiac contractility and blood pressure in mice. At the age of 16 days, the relatively healthy Zbtb20-null mice exhibited hypotension without obvious change of heart rate or other evidence for heart failure. Moreover, Zbtb20 deletion led to a marked reduction in heart size, left ventricular wall thickness, and cell size of cardiomyocytes, which was largely proportional to the decreased body growth. Notably, echocardiographic and hemodynamic analyses showed that cardiac contractility was greatly impaired in the absence of ZBTB20. Mechanistically, ZBTB20 deficiency decreased cardiac ATP contents, and compromised the enzyme activity of mitochondrial complex I in heart as well as L-type calcium current density in cardiomyocytes. Furthermore, the developmental activation of some mitochondrial function-related genes was significantly attenuated in Zbtb20-null myocardium, which included Hspb8, Ckmt2, Cox7a1, Tfrc, and Ogdhl. Put together, these results suggest that ZBTB20 plays a crucial role in the regulation of heart development, energy metabolism, and contractility.

ZBTB20 regulates EGFR expression and hepatocyte proliferation in mouse liver regeneration.

Liver has a unique regenerative capacity, however, its regulatory mechanism is not fully defined. We have established the zinc-finger protein ZBTB20 as a key transcriptional repressor for alpha-fetoprotein (AFP) gene in liver. As a marker of hepatic differentiation, AFP expression is closely associated with hepatocyte proliferation. Unexpectedly, here we showed that ZBTB20 acts as a positive regulator of hepatic replication and is required for efficient liver regeneration. The mice specifically lacking ZBTB20 in hepatocytes exhibited a remarkable defect in liver regeneration after partial hepatectomy, which was characterized by impaired hepatocyte proliferation along with delayed cyclin D1 induction and diminished AKT activation. Furthermore, we found that epithelial growth factor receptor (EGFR) expression was dramatically reduced in the liver in the absence of ZBTB20, thereby substantially attenuating the activation of EGFR signaling pathway in regenerating liver. Adenovirus-mediated EGFR overexpression in ZBTB20-deficient hepatocytes could largely restore AKT activation in response to EGFR ligands in vitro, as well as hepatocyte replication in liver regeneration. Furthermore, ZBTB20 overexpression could significantly restore hepatic EGFR expression and cell proliferation after hepatectomy in ZBTB20-deficient liver. Taken together, our data point to ZBTB20 as a critical regulator of EGFR expression and hepatocyte proliferation in mouse liver regeneration, and may serve as a potential therapeutic target in clinical settings of liver regeneration.

ZBTB20 regulates nociception and pain sensation by modulating TRP channel expression in nociceptive sensory neurons.

In mammals, pain sensation is initiated by the detection of noxious stimuli through specialized transduction ion channels and receptors in nociceptive sensory neurons. Transient receptor potential (TRP) channels are the key sensory transducers that confer nociceptors distinct sensory modalities. However, the regulatory mechanisms about their expression are poorly defined. Here we show that the zinc-finger protein ZBTB20 regulates TRP channels expression in nociceptors. ZBTB20 is highly expressed in nociceptive sensory neurons of dorsal root ganglia. Disruption of ZBTB20 in nociceptors led to a marked decrease in the expression levels of TRPV1, TRPA1 and TRPM8 and the response of calcium flux and whole-cell currents evoked by their respective specific agonists. Phenotypically, the mice lacking ZBTB20 specifically in nociceptors showed a defect in nociception and pain sensation in response to thermal, mechanical and inflammatory stimulation. Our findings point to ZBTB20 as a critical regulator of nociception and pain sensation by modulating TRP channels expression in nociceptors.

[Salusins and its cardiovascular effects].

Salusins are newly discovered cardiovascular active peptides, including salusin-alpha and salusin-beta, which are peptides containing 28 and 20 amino acids respectively. Salusins are widely distributed in tissuse and organs of human and rat, and have a series of cardiovascular effects, including lowering blood pressure, slowing down the heart rate, inhibiting myocardial contraction, reducing cardiac ischemic injury, and promoting hypertrophy of cardiomyocytes and proliferation of vascular smooth muscle cells. It is noteworthy to mention that salusin-alpha and salusin-beta are polypeptides produced by the same precursor and play opposite roles in the development and progression of atherosclerosis.

Effects of Salusin-ß on action potential and ionic currents in ventricular myocytes of rats.

AIM: Salusin-ß is a regulatory peptide that exerts negative inotropic effect on ventricular muscle, but its electrophysiological effects on ventricular myocytes are still unknown. METHODS: Action potential and channel currents such as sodium current (I(N) (a) ), transient outward potassium current (I(to) ), steady-state potassium current (I(sus) ), sodium-calcium exchange current (I(N) (aCa) ) and inward rectifier potassium current (I(K) (1) ) were measured in ventricular myocytes isolated from 12 to 16 weeks rats by whole-cell voltage-clamp techniques. RESULTS: Salusin-ß dose-dependently shortened the duration of action potential in rat ventricular myocytes. Furthermore, salusin-ß significantly inhibited I(N) (aCa) and increased I(to) , but did not affect I(N) (a) , I(sus) and I(K) (1) . CONCLUSION: These results suggest that the effect of salusin-ß on action potential may be partly attributed to a decrease in inward currents and an increase in outward currents.

The inhibitory effect of miRNA-1 on ET-1 gene expression.

There is a close correlation between endothelin-1 (ET-1) and microRNA-1 (miRNA-1) expression in the heart, but whether ET-1 expression is regulated by miRNA-1 warrants further research. Our results revealed multiple clues suggesting that miRNA-1 may participate in inhibiting ET-1 gene expression. The inhibitory effect of miRNA-1 on recombinant luciferase reporter gene was mediated by the target sequence at the 127th nucleotide on ET-1 3'UTR. We further confirmed that miRNA-1 could inhibit endogenous ET-1 gene expression at the post-transcriptional level. Our study provides a new perspective on the regulatory mechanism of ET-1 gene.

Altered microRNA expression profiles in retinas with diabetic retinopathy.

Rats with streptozotocin (STZ)-induced diabetes were studied in order to identify abnormal microRNA (miRNA) expression profiles in diabetic retinopathy (DR) and to ascertain miRNAs associated with DR. Histopathologically, we observed characteristic features of DR in rats at 10 weeks after STZ injection. Investigation of miRNA expression profiles in the retinas of control and diabetic rats using miRNA microarrays revealed that many miRNAs were abnormally expressed in DR. On the basis of their fold changes and probability values, a total of 37 miRNAs were selected for further validation by real-time PCR analysis. The results showed that 11 miRNAs were significantly upregulated and 6 miRNAs were notably downregulated in DR. Furthermore, these changes in retinal miRNA expression levels paralleled the course of DR. Levels of miR-182, miR-96, miR-183, miR-211, miR-204, and miR-124 were significantly increased during the progress of DR, whereas miR-10b, miR-10a, miR-219-2-3p, miR-144, miR-338, and miR-199a-3p were significantly decreased. Our data indicate that the aberrant miRNA expression profiles in DR are associated with the development of DR. Modulation of retinal miRNA expression levels may provide a potential therapeutic strategy for DRs.

Overexpression of microRNA-378 attenuates ischemia-induced apoptosis by inhibiting caspase-3 expression in cardiac myocytes.

MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. In an intact rat model of myocardial ischemia caused by coronary artery ligation, this study identified 17 miRNAs that changed more than 1.5-fold in the myocardium subjected to 4-h ischemia. Using miRNA microarray analysis, most of these aberrantly expressed miRNAs were confirmed by quantitative RT-PCR. MiR-378, a significantly down-regulated miRNA, was selected for further function study. In serum deprived rat H9c2 cardiomyocytes exposed to hypoxia (1% O(2)), miR-378 expression was down-regulated as well. The overexpression of miR-378 resulting from miR-378 mimic transfection significantly enhanced cell viability, reduced lactate dehydrogenase release, and inhibited apoptosis and necrosis. By contrast, miR-378 deficiency resulting from miR-378 inhibitor transfection aggravated the hypoxia-induced apoptosis and cell injury. In accordance, miR-378 inhibitor caused significant apoptosis and cell injury to cardiomyocytes cultured under normoxia. Using bioinformatic algorithms, caspase-3, a key apoptosis executioner, was predicted as a putative target of miR-378. The quantitative RT-PCR showed no effects of miR-378 mimic or inhibitor on caspase-3 mRNA level. However, the amount of caspase-3 proteins was reduced by miR-378 mimic, whereas increased by miR-378 inhibitor. Furthermore, the luciferase reporter assay confirmed caspase-3 to be a target of miR-378, and the apoptosis and cell injury caused by miR-378 inhibitor in both normoxic and hypoxic cells were abolished by a caspase-3 inhibitor. This study first showed that miR-378 inhibited caspase-3 expression and attenuated ischemic injury in cardiomyocytes. It may represent a potential novel treatment for apoptosis and ischemic heart disease.

Role of miR-1 and miR-133a in myocardial ischemic postconditioning.

BACKGROUND: Ischemic postconditioning (IPost) has aroused much attention since 2003 when it was firstly reported. The role of microRNAs (miRNAs or miRs) in IPost has rarely been reported. The present study was undertaken to investigate whether miRNAs were involved in the protective effect of IPost against myocardial ischemia-reperfusion (IR) injury and the probable mechanisms involved. METHODS: Thirty SD rats weighing 250-300 g were equally randomized to three groups: Control group, where the rats were treated with thoracotomy only; IR group, where the rats were treated with ischemia for 60 min and reperfusion for 180 min; and IPost group, where the rats were treated with 3 cycles of transient IR just before reperfusion. The extent of myocardial infarction, LDH and CK activities were measured immediately after treatment. Myocardial apoptosis was detected by TUNEL assay. The myocardial tissue was collected after IR or IPost stimulation to evaluate the miRNAs expression level by miRNA-microarray and quantitative real-time RT-PCR. Real-time PCR was conducted to identify changes in mRNA expression of apoptosis-related genes such as Bcl-2, Bax and Caspase-9 (CASP9), and Western blot was used to compare the protein expression level of CASP9 in the three groups. The miRNA mimics and anti-miRNA oligonucleotides (AMO) were transferred into the cultured neonatal cardiomyocytes and myocardium before they were treated with IR. The effect of miRNAs on apoptosis was determined by flow cytometry and TUNEL assay. CASP9, as one of the candidate target of miR-133a, was compared during IR after the miR-133a mimic or AMO-133a was transferred into the myocardium. RESULTS: IPost reduced the IR-induced infarct size of the left ventricle, and decreased CK and LDH levels. TUNEL assay showed that myocardial apoptosis was attenuated by IPost compared with IR. MiRNA-microarray and RT-PCR showed that myocardial-specific miR-1 and miR-133a were down-regulated by IR, and up-regulated by IPost compared with IR. Furthermore, IPost up-regulated the mRNA expression of Bcl-2, down-regulated that of Bax and CASP9. Western blot showed that IPost also down-regulated the CASP9 protein expression compared with IR. The results of flow cytometry and TUNEL assay showed that up-regulation of miR-1 and miR-133a decreased apoptosis of cardiomyocytes. MiR-133a mimic down-regulated CASP9 protein expression and attenuated IR-induced apoptosis. CONCLUSION: MiRNAs are associated with the protective effect of IPost against myocardial IR injury. IPost can up-regulate miR-1 and miR-133a, and decrease apoptosis of cardiomyocyte. Myocardial-specific miR-1 and miR-133a may play an important role in IPost protection by regulating apoptosis-related genes. MiR-133a may attenuate apoptosis of myocardiocytes by targeting CASP9.

Erythropoietin receptor antibody inhibits oxidative stress induced retinal neovascularization in mice.

AIM: To observe the effect of erythropoietin receptor antibody (EpoRA) on oxygen-induced retinal neovascularization. METHODS: C57BL / 6J mice, newly born 7 days, were exposed in high oxygen for 5 days and then placed in normal air for another 5 days, thus the animal models of retinal neovascularization were made. Experimental animals were allocated into 3 groups: normal, experimental and therapeutic. The normal group was fed in the normal environment. Into the vitreous cavity of mice in the therapeutic group were injected 2µL of EpoRA for 5 successive days. And the experimental group was injected the same amount of normal saline. Mice were sacrificed 17 days after birth and their eyeballs were removed for detection of malonaldehyde(MDA) content in the retina and by HE staining endothelial cells were counted the breaking through internal limiting membrane. RESULTS: In the experimental group, MDA content in the retina was 25.11±3.46µmol/g , which was obviously less than those in the normal group(5.34±1.79µmol/g, P<0.01) and those in the therapeutic group (12.04±1.91µmol/g). Pathological sections showed the nuclear number of the endothelial cells breaking through internal limiting membrane was 0.7±0.2 in normal group, and 46.2±6.5 in high oxygen induced experimental group. In the therapeutic group injected with EpoRA, it was lowered to 24.0±5.0 (P<0.01). CONCLUSION: EpoRA can effectively inhibit oxygen-induced neovascularization in retina of mouse by reducing oxidative damage.

Leptin protects cardiomyocytes from serum-deprivation-induced apoptosis by increasing anti-oxidant defence.

1. Leptin, an important adipose-derived hormone, can be associated with cardiac pathophysiology; however, the role of leptin in cardiomyocyte apoptosis is poorly understood. The present study examines serum-deprivation-induced apoptosis in primary cultured cardiomyocytes treated with leptin. 2. Cardiomyocytes were subjected to serum deprivation in the presence or absence of leptin (5 or 50 nmol/L) for 48 h. Apoptosis was determined by Hoechst 33258 and Annexin V-FITC/propidium iodide dual staining. Cell viability, malondialdehyde (MDA) content, caspase 3 activation, and the expression and enzyme activity of superoxide dismutase (SOD) were measured. Small interference RNA (siRNA) targeting SOD1 and SOD2 were used to knockdown their expression and measure apoptosis. 3. Serum deprivation caused nearly 30% of apoptosis in cardiomyocytes, and an approximately 60% decrease in cell viability. The mRNA levels and the activated form of caspase 3 were greatly increased. In the presence of leptin, the apoptotic rate was reduced to approximately 15%, cell viability was increased and the activation of caspase 3 was partially inhibited. Additionally, the augmented lipid peroxidation (MDA formation) was abolished, and the impaired activities of SOD1 and SOD2 were restored by leptin. The mRNA expression of SOD2, but not SOD1, was stimulated by leptin. Transfection with siRNA that cause deficiency of either SOD1 or SOD2 attenuated the anti-apoptotic effects of leptin. 4. The results suggest that leptin inhibits serum-deprivation-induced apoptosis in cardiomyocytes by activating SOD. The present study outlines the direct actions of leptin in cardiac disorders that are related to elevated leptin levels.

MicroRNAs are dynamically regulated in hypertrophic hearts, and miR-199a is essential for the maintenance of cell size in cardiomyocytes.

Cardiac hypertrophy, which is characterized by an increase in cell size and reactivation of fetal genes, occurs as an adaptive response to diverse forms of stress and often results in heart failure and sudden death. Growing evidence indicates that microRNAs (miRNAs) are involved in cardiac hypertrophy, but the function of these miRNAs remains elusive. Here, using real time PCR analysis, we showed that several miRNAs were dynamically regulated in the rat hypertrophic hearts and miR-199a was up-regulated by 10-fold in hypertrophic hearts after abdominal aorta constriction for 12 weeks. With tissue profiling analysis, we showed that miR-199a was predominantly expressed in cardiomyocytes, but was also faintly detected in cardiac fibroblasts. To investigate whether miR-199a was involved in cardiac hypertrophy, both over-expression and knockdown of miR-199a were performed in cultured cardiomyocytes. Over-expression of miR-199a in cardiomyocytes increased the cell size as measured by cell surface area, and also reduced the mRNA expression level of alpha-myosin heavy chain. In accordance, knockdown of endogenous miR-199a in cardiomyocytes reduced the cell size. Down-regulation of miR-199a also attenuated the phenylephrine-induced increase of cell size. Furthermore, bioinformatic algorithms were used to predict the potential targets of miR-199a in cardiac hypertrophy, and hypoxia-inducible factor 1 alpha was confirmed by the luciferase reporter assay to be a potential target of miR-199a. Taken together, our results demonstrated that miR-199a, which was predominantly expressed in cardiomyocytes, was essential for the maintenance of cell size of cardiomyocytes and might play a role in the regulation of cardiac hypertrophy.

Inhibition of L-type calcium currents by salusin-beta in rat cardiac ventricular myocytes.

Salusin-beta is a new regulatory peptide relevant to the cardiovascular system and exerts negative inotropic effect on ventricular muscle. The purpose of the present study was to determine whether salusin-beta can inhibit cardiac L-type calcium channel current (I(Ca,L)). Using whole-cell voltage-clamp techniques, I(Ca,L) was measured in ventricular myocytes isolated from 12 to 16 weeks rats. Salusin-beta dose-dependently and reversibly reduced the magnitude of I(Ca,L) in rat ventricular myocytes. Neither threshold potential nor the peak potential of current-voltage relationship was affected. Salusin-beta increased the rate of I(Ca,L) inactivation without altering its gating properties. These results suggest salusin-beta inhibited I(Ca,L) by increasing the rate of I(Ca,L) inactivation and the inhibition of L-type Ca(2+) channels induced by salusin-beta may contribute to its negative inotropic effect on ventricular muscle.