OBJECTIVETo explore the possible mechanism of lipopolysaccharide (LPS)-induced cardiomyocyte hypertrophy in rats.

METHODSNeonatal rat cardiomyocytes cultured in vitro were stimulated with 100 µg/L LPS for 1, 4 or 8 h and scanned by atomic force microscopy (AFM) for measurement of the two-dimensional area, three-dimensional surface area and volume of each cell. The total proteins and Na(+)-K(+)-ATPase activity in the cardiomyocytes were determined. The same measurements were also carried out in neonatal rat cardiomyocyte cultures stimulated by 0.5 µmol/L ouabain for 8 h and the total protein levels were measured.

RESULTSFollowing a 8-hour stimulation with LPS, the two-dimensional area, three-dimensional surface area and volume of the single cardiomyocyte became enlarged and the total cellular proteins increased significantly as compared with those in the normal control cells (P < 0.05). LPS treatment for 4 and 8 h resulted in significantly decreased activity of Na(+)-K(+)-ATPase in the cardiomyocytes (P < 0.05). In the cells treated with ouabain for 8 h, the two-dimensional area, three-dimensional surface area, volume of the single cardiomyocyte and the total cellular proteins increased significantly in comparison with the normal control group (P < 0.05).

CONCLUSIONLPS can result in cardiomyocyte hypertrophy in rats possibly in relation to lowered Na(+)-K(+)-ATPase activity in the cardiomyocytes after LPS exposure.

Animals ; Animals, Newborn ; Cell Enlargement ; drug effects ; Cells, Cultured ; Hypertrophy ; chemically induced ; Lipopolysaccharides ; Myocytes, Cardiac ; enzymology ; pathology ; Rats ; Rats, Wistar ; Sodium-Potassium-Exchanging ATPase ; metabolism

Cardiac hypertrophy is an adaptive process to an increased hemodynamic overload. However, the adaption may lead to the fragility of myocardium facing pathological stimuli. In the present study, experiments were designed to explore the susceptibility of hypertrophic myocardiocytes to apoptotic stimuli and the role of protein kinase Cdelta (PKCdelta) during the transition from hypertrophy to apoptosis. Endothelin-1 (ET-1)-treated cardiomyocytes were used as model of cardiac hypertrophy. Angiotensin II (Ang II) was used as an apoptotic stimulus. Cell surface area was measured to determine the extent of hypertrophy. The apoptotic rate in cardiomyocytes was detected by Hoechst 33258. (1) Cell surface area was increased by 42.5% and 67.3% following 1 nmol/L and 10 nmol/L ET-1 treatment, respectively, as compared with serum-free cultured myocytes. So the mildly and moderately hypertrophic myocyte models were set up. (2) Apoptotic rates in serum-free cultured, mildly and moderately hypertrophic myocytes after Ang II treatment were (15.54+/-1.32) %, (20.65+/-1.40) % and (29.33+/-3.52) %, respectively. It is suggested that hypertrophic myocytes are more susceptive to apoptotic stimulus. (3) Rottlerin, a specific inhibitor of PKCdelta depressed apoptotic rates induced by Ang II to (15.88+/-2.25) % in mildly hypertrophic myocytes and to (15.01+/-1.37) % in moderately hypertrophic myocytes; but rottlerin did not affect apoptotic rate induced by Ang II in serum-free cultured myocytes. These results suggest that inhibition of PKCdelta can reduce Ang II-induced apoptosis of hypertrophic cardiomyocytes and that PKCdelta is possibly involved in the apoptotic process of hypertrophic cardiomyocytes.
Angiotensin II ; pharmacology ; Animals ; Animals, Newborn ; Apoptosis ; drug effects ; physiology ; Cardiomegaly ; pathology ; physiopathology ; Cell Enlargement ; drug effects ; Endothelin-1 ; pharmacology ; Heart Failure ; physiopathology ; Myocytes, Cardiac ; cytology ; pathology ; Primary Cell Culture ; Protein Kinase C-delta ; physiology ; Rats ; Rats, Sprague-Dawley

To investigate the anti-cardiac hypertrophic mechanism of statins, thirty-eight male Wistar rats were randomly allocated to four groups. Rats in model group received nitric oxide synthase inhibitor, N-nitro-L-arginine (L-NNA) 15 mg/(kg.d) by peritoneal injection. Rats in simvastatin treatment groups were given simultaneously L-NNA as those in model group and simvastatin 5 or 30 mg/(kg.d) intragastrically respectively. Rats in control group received the same volume of normal sodium. Left ventricular function, left ventricular mass index (LVMI), the content of brain natriuretic peptide (BNP) in plasma and myocardium, myocardial hydroxyproline and heme oxygenase activity were determined after 6 weeks. The results showed that rats in model group developed significant cardiac hypertrophy associated with reduced left ventricular function compared with the control group. However, compared with the model group, L-NNA-induced cardiac hypertrophy of rats was significantly relieved in simvastatin treatment groups, associated with improved left ventricular function, decreased LVMI, lower BNP levels in plasma and myocardium, lower content of myocardial hydroxyproline, and increased myocardial heme oxygenase (HO) activity. In cultured rat neonatal cardiomyocytes, simvastatin (30 or 100 mumol/L) significantly increased heme oxygenase-1 (HO-1) mRNA expression, HO activity as well as the production of CO in cardiomyocytes. Cultured with zinc protoporphyrin, a HO inhibitor, or simvastatin alone did not change [(3)H]leucine uptake of cardiomyocytes. However, cocultured with simvastatin significantly inhibited the cardiomyocyte [(3)H]leucine uptake induced by angiotensin II in a concentration-dependent manner. Cotreatment with zinc protoporphyrin significantly abolished the suppressive effect of simvastatin on cardiomyocyte [(3)H]leucine uptake. These data suggest that the activation of HO-1/CO pathway may be one of the important mechanisms by which statins inhibit cardiac hypertrophy caused by hypertension.
Angiotensins ; antagonists & inhibitors ; pharmacology ; Animals ; Carbon Monoxide ; metabolism ; Cardiomegaly ; etiology ; prevention & control ; Cell Enlargement ; drug effects ; Heme Oxygenase-1 ; metabolism ; Hypertension ; complications ; drug therapy ; Male ; Myocytes, Cardiac ; cytology ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Simvastatin ; pharmacology ; therapeutic use

The apoptosis of cardiomyocytes plays a pivotal role in the pathogenesis of cardiac failure transformed from cardiac hypertrophy, so that suppression of cardiomyocytes apoptosis is an effective pharmacotherapeutic target to prevent cardiac failure. This study focused on the relationship between apoptosis and alteration of the energetic metabolism pathways of hypertrophic cardiomyocytes induced by hypoxia-reoxygenation. Cardiomyocyte hypertrophy was induced by angiotensin II (0.1 mumol/L ) and norepinephrine (1 mumol/L), and the cells were cultured under the condition of hypoxia ( 95% N2 and 5% CO2, the O2 partial pressure was regulated at least lower than 5 mmHg ) for 8 h, then were recovered to normal culture environment. Apoptosis was detected with TUNEL. The activity of pyruvate dehydrogenase (PDH) and carnitine palmitoyltransferase 1 (CPT-1), the rate of glycose oxidation and glycolysis, and fatty acid metabolism were detected by liquid scintillation counting. The results are as follows: (1) The activity of active PDH (PDHa) was slightly higher in hypertrophic cardiomyocytes than that in normal cardiomyocytes, but the activity of CPT-1 was significantly lower in hypertrophic cardiomyoctes than that in normal cardiomyocytes.Compared with the hypertrophic cardiomyocytes cultured with normal oxygen concentration, the activities of PDHa and CPT-1 were decreased significantly after hypoxia for 8 h, and the activity of PDHa were decreased further after reoxygenation for 4 h, but the activity of CPT-1 recovered quickly after reoxygenation. (2) The rate of glucose oxidation in hypertrophic cardiomyocytes increased slightly when cultured under normal O2 partial pressure than that in normal cardiac cells. The rate of glucose oxidation reduced (16 +/- 0.9)% and (48 +/- 1.1)% in normal and hypertrophic cardiomyocytes, respectively, after hypoxia. It reduced further in hypertrophic cardiac cells at 4 h of reoxygenation, then recovered gradually. In normal cardiocytes, it recovered quickly after reoxygenation. (3) The rate of glycolysis of hypertrophic cardiocytes increased slightly than that of the normal cardiocytes when cultured in the general O(2) environment. Compared with the normal cardiomyocytes, the rate of glycolysis of hypertrophic cardiac cells was the same during hypoxia-reoxygenation culture, i.e., the rate of glycolysis decreased slightly after hypoxia for 8 h, but increased rapidly and significantly after reoxygenation. (4) The rate of fatty acid oxidation was slightly lower in hypertrophic cardiocytes than that in normal cardiomyocytes. After hypoxia for 8 h, the rate of fatty acid oxidation decreased significantly in normal and hypertrophic cardiomyocytes, there was no difference between normal and hypertrophic cardiomyocytes. But the alterations of fatty acid oxidation after reoxygenation were different between normal and hypertrophic cardiac cells, namely, the fatty acid oxidation of normal cardiomyocytes were activated slowly and slightly, while the rate of fatty acid oxidation of hypertrophic cardiomyocytes increased markedly at the early stage of reoxygenation, and increased further at 8 h of reoxygenation. (5) The rate of apoptosis in hypertrophic cardiocytes increased obviously after hypoxia for 8 h, and increased further and markedly at the early stage of reoxygenation, then gradually decreased to normal level. (6) Dicholoroacetate could inhibit apoptosis of hypertrophic cardiocytes through increasing glucose oxidation and inhibiting the activation of glycolysis and fatty acid oxidation of hypertrophic cardiomyocytes induced by hypoxia-reoxygenation. These data demonstrate that apoptosis in hypertrophic cardiomyocytes after hypoxia-reoxygenation is mainly due to the inhibition of glucose oxidation and the activation of glucolysis and fatty acid oxidation. Furthermore, increasing glucose oxidation may be a new pharmacotherapeutic target to inhibit apoptosis of hypertrophic cardiac cells.
Angiotensin II ; pharmacology ; Animals ; Animals, Newborn ; Apoptosis ; physiology ; Cardiomegaly ; pathology ; Cell Enlargement ; drug effects ; Cell Hypoxia ; Energy Metabolism ; Myocardial Reperfusion Injury ; metabolism ; pathology ; Myocytes, Cardiac ; metabolism ; pathology ; Norepinephrine ; pharmacology ; Oxygen ; metabolism ; Rats

OBJECTIVETo observe the effect of transfection with small interfering RNA (siRNA) targeting connective tissue growth factor (CTGF) on human tubular epithelial hypertrophy induced by high glucose.

METHODSHK-2 cells were cultured in DMEM/F12 medium containing 1 g/L glucose (normal control group), 4.5 g/L glucose (high glucose group), or 1 g/L glucose+3.5 g/L mannitol (iso-osmolar control group). The cells were transfected with pGenesil-1, pGenesil/neg, or pGenesil/siRNA-CTGF and then cultured in DMEM/F12 medium containing 4.5 g/L glucose as the high glucose+blank control group, high glucose+negative control group and high glucose+interference group, respectively. After cell culture for 24, 48 and 96 h, the cells were collected to detect the mRNA and protein levels of CTGF by real-time PCR and Western blotting, respectively. The proliferative activities of the cells were evaluated with MTT assay, and the total cellular protein contents were determined with Bradford method. Flow cytometry was employed to analyzed the cell cycle changes.

RESULTSHigh-glucose significantly up-regulated the CTGF mRNA and protein levels in HK-2 cells. The cell proliferation was inhibited after high-glucose exposure with increased cell percentage in G1 phase and total cellular protein content suggesting cellular hypertrophy. Transfection with siRNA targeting CTGF significantly inhibited high glucose-induced up-regulation of CTGF mRNA and protein and promoted the cell proliferation, resulting also increased cells in S phase and lowered total cellular protein contents.

CONCLUSIONCTGF is an important mediator of high glucose-induced tubular epithelial hypertrophy, and transfection with siRNA targeting CTGF can alleviate the hypertrophy, suggesting the potential value of CTGF-targeted treatment in the management of diabetic nephropathy.

Cell Enlargement ; drug effects ; Cell Line ; Connective Tissue Growth Factor ; genetics ; metabolism ; Epithelial Cells ; pathology ; Glucose ; pharmacology ; Humans ; Hypertrophy ; Kidney Tubules ; pathology ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate the effect of Shenkang Injection (SI) on hypertrophy and expressions of p21 and p27 in glomerular mesangial cells of rats cultured in high glucose fluid.

METHODSCultured rat glomerular mesangial cells were divided into 6 groups: the normal control group, mannitol-treated group, high glucose-treated group, high, middle and low dose SI treated groups. The synthesis of cell protein and DNA were determined with incorporation of 3H-TdR and 3H-Leu. The changes of p21 mRNA and expressions of p21 and p27 protein were determined by reverse transcriptase polymerase chain reaction and Western blot.

RESULTSHigh glucose caused incorporation of 3H-TdR reduced, incorporation of 3H-Leu increased. SI could decrease incorporation of 3H-Leu, increase incorporation of 3H-TdR. Compared with the high glucose-treated group, SI could inhibit the overexpression of p2l mRNA and protein,p27 protein induced by high glucose.

CONCLUSIONSI could inhibit hypertrophy of glomerular mesangial cells cultured in high glucose partly through inhibiting overexpressions of p2l mRNA, p21 and p27 protein.

Animals ; Cell Enlargement ; Cells, Cultured ; Culture Media ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Glucose ; pharmacology ; Mesangial Cells ; cytology ; drug effects ; metabolism ; Rats

OBJECTIVETo investigate the mechanism of rosiglitazone (RSG, the activator of peroxisome proliferators activated receptor lambda) for inhibiting endothelin-1 (ET-1)-induced neonatal rat cardiac myocyte hypertrophy and the role of protein kinase C (PKC) and c-fos.

METHODSIn vitro cultured neonatal rat cardiac myocytes were treated with ET-1, phorbol ester (PMA, the PKC activator), ET-1+RSG, ET-1+chelerythrine (che, the PKC inhibitor), PMA+RSG, or without treatment (control), respectively. The effects of RSG on the protein content, (3)H-leucine incorporation, PKC activity and C-fos protein expression were observed in the cardiac myocytes stimulated with ET-1 or PMA.

RESULTSAfter two days of culture, the intracellular protein content in ET-1 group and PMA group were increased by 15% (339-/+15 microg/ml) and 13% (329-/+14 microg/ml) as compared with the control cells (290-/+13 microg/ml), respectively (P<0.01). Compared with the ET-1 group, cells treated with ET-1+10(-8) mol/L RSG, ET-1+10(-7) mol/L RSG, and ET-1+che showed decreased intracellular protein content by 10% (303-/+14 microg/ml, P<0.05), 12% (292-/+11 microg/ml, P<0.05), and 13% (291-/+12 microg/ml, P<0.01), respectively. The intracellular protein content in PMA+10(-7) mol/LRSG group was decreased by 10% (P<0.05) in comparison with the PMA group. RSG inhibited protein synthesis enhancement and increased (3)H-leucine incorporation induced by ET-1 and PMA, and antagonized the effects of ET-1 and PMA in promoting PKC activity and c-fos protein expression in the myocytes.

CONCLUSIONThe inhibitory effect of RSG on ET-1- or PMA-induced myocyte hypertrophy is associated with PKC-c-fos pathway.

Animals ; Animals, Newborn ; Blotting, Western ; Cell Enlargement ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelin-1 ; pharmacology ; Hypoglycemic Agents ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Protein Kinase C ; metabolism ; Proto-Oncogene Proteins c-fos ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Tetradecanoylphorbol Acetate ; pharmacology ; Thiazolidinediones ; pharmacology

Peroxisome proliferator-activated receptor alpha (PPARalpha) activation in rodents is thought to improve insulin sensitivity by decreasing ectopic lipids in non-adipose tissues. Fenofibrate, a lipid-modifying agent that acts as a PPARalpha agonist, may prevent adipocyte hypertrophy and insulin resistance by increasing intracellular lipolysis from adipose tissue. Consistent with this hypothesis, fenofibrate decreased visceral fat mass and adipocyte size in high fat diet-fed obese mice, and concomitantly increased the expression of PPARalpha target genes involved in fatty acid beta-oxidation in both epididymal adipose tissue and differentiated 3T3-L1 adipocytes. However, mRNA levels of adipose marker genes, such as leptin and TNFalpha, were decreased in epididymal adipose tissue by fenofibrate treatment. Fenofibrate not only reduced circulating levels of free fatty acids and triglycerides, but also normalized hyperinsulinemia and hyperglycemia in obese mice. Blood glucose levels of fenofibrate-treated mice were significantly reduced during intraperitoneal glucose tolerance test compared with obese controls. These results suggest that fenofibrate-induced fatty acid beta-oxidation in visceral adipose tissue may be one of the major factors leading to decreased adipocyte size and improved insulin sensitivity.
3T3 Cells ; Adipocytes/cytology/*drug effects ; Animals ; Antilipemic Agents/*pharmacology ; Blood Glucose ; Body Weight ; Cell Enlargement/*drug effects ; Dietary Fats ; Gene Expression Regulation/drug effects ; Glucose Tolerance Test ; Insulin/blood ; *Insulin Resistance ; Leptin/genetics ; Lipids/blood ; Mice ; Mice, Inbred C57BL ; Mice, Obese ; PPAR alpha/*metabolism ; Procetofen/*pharmacology ; Tumor Necrosis Factor-alpha/genetics

TGF-beta1-induced glomerular mesangial cell (GMC) injury is a prominent characteristic of renal pathology in several kidney diseases, and a ternary protein complex consisting of PINCH-1, integrin-linked kinase (ILK) and alpha-parvin plays a pivotal role in the regulation of cell behavior such as cell proliferation and hypertrophy. We report here that PINCH-1-ILK-alpha-parvin (PIP) complex regulates the TGF-beta1-induced cell proliferation and hypertrophy in cultured rat GMCs. When GMCs were treated with TGF-beta1 for 1, 2 and 3 days, the PIP complex formation was up-regulated after 1 day, but it was down-regulated on day 2. Cell numbers were significantly elevated on day 2, but dramatically decreased on day 3. In contrast, a significant increase in cellular protein contents was observed 3 days after TGF-beta1-treatment. TGF-beta1 induced early increase of caspase-3 activity. In GMCs incubated with TGF-beta1 for 2 days, cytosolic expression of p27(Kip1) was dramatically reduced, but its nuclear expression was remarkably elevated. A significantly decreased expression of phospho-Akt (Ser 473) was observed in the cells treated with TGF-beta1 for 1 day. TGF-beta1 induced early increase of phospho-p27(Kip1) (Thr 157) expression with subsequent decrease, and similar responses to TGF-beta1 were observed in the p38 phosphorylation (Thr 180/Thr 182). Taken together, TGF-beta1 differently regulates the PIP complex formation of GMCs in an incubation period-dependant fashion. The TGF-beta1-induced up- and down-regulation of the PIP complex formation likely contributes to the pleiotropic effects of TGF-beta1 on mesangial cell proliferation and hypertrophy through cellular localization of p27(Kip1) and alteration of Akt and p38 phosphorylation. TGF-beta1-induced alteration of the PIP complex formation may be importantly implicated in the development and progression of glomerular failure shown in several kidney diseases.
Animals ; *Cell Enlargement ; *Cell Proliferation ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p27/metabolism ; Cytoskeletal Proteins/*metabolism ; DNA-Binding Proteins/*metabolism ; Male ; Mesangial Cells/drug effects/*physiology ; Microfilament Proteins/*metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases/*metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Transforming Growth Factor beta1/*pharmacology ; p38 Mitogen-Activated Protein Kinases/metabolism

OBJECTIVETo demonstrate the inhibitory effect of kappa-opioid receptor activation by U50488H on hypertrophy induced by NE in cultured neonatal rat cardiac myocytes and compare its effect with that of prazosin and propranolol.

METHODSThe cellular proliferation was determined with crystal violet staining. The protein content was assayed with Lowry's method. The cardiomyocytes volumes were measured by computer photograph analysis system. The protein synthesis was assayed with [3H]-lencine incorporation method.

RESULTS(1) NE significantly induced the increase of protein content, [3H]-leucine incorporation and cell size without a concomitant increase in cell number in low serum medium. OThese responses were partially suppressed by prazosin or propranolol alone and completely abolished by both in combination. U50488H significantly inhibited the NE-induced increase of protein content, [3H]-leucine incorporation and cell size. The inhibitory effects of U50488H on NE-induced cardiac hypertrophy were greater than either prazosin or propranolol, but comparable to combination of both.

CONCLUSIONNE, acting via both alpha1- and beta-adrenergic pathway, stimulates myocyte hypertrophy. Stimulating kappa-opioid receptor significantly inhibits NE-induced cardiac hypertrophy, which may be related with alpha1- and beta1-adrenergic pathway.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer ; pharmacology ; Adrenergic alpha-1 Receptor Antagonists ; pharmacology ; Adrenergic beta-Antagonists ; pharmacology ; Animals ; Animals, Newborn ; Cardiomegaly ; chemically induced ; pathology ; prevention & control ; Cell Enlargement ; drug effects ; Cells, Cultured ; Female ; Male ; Myocytes, Cardiac ; cytology ; Norepinephrine ; Prazosin ; pharmacology ; Propranolol ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid, kappa ; agonists