To study the effects of different pH HEPES-KH reperfusate solution on immature myocardial protection, isolated perfused Langendorff model from immature rabbit hearts were developed formed. Control group (C) was perfused only with pH 7.4 HEPES-KH solution for 90 min. Ischemia/reperfusion group (group I/R) was perfused with pH 7.4 HEPES-KH solution before ischemia or after ischemia. Experimental group (group E), after ischemia, was perfused with pH 6.8, pH 7.1 and pH 7.4 HEPES-KH solutions for 5 min, 5 min, and 20 min, respectively. The left ventricular function recovery, MWC, LDH and CK leakage, MDA, ATP content, and SOD activity were determined. Our results showed that the left ventricular function recovery, ATP content and SOD activity in group E were higher than those of group I/R (P < 0.05). MWC, MDA content, LDH and CK leakage in group E were lower than those of group I/R (P < 0.05). These findings suggested that pH paradox might be one of important mechanisms for immature myocardial ischemia-reperfusion injury, and acidic perfusate, at the beginning of reperfusion, might attenuate pH paradox and ameliorate functional recovery in isolated perfused immature rabbit hearts.
Myocardium/metabolism ; Random Allocation ; Superoxide Dismutase/metabolism

Heart Failure/metabolism* ; Humans ; Myocardium/metabolism*

OBJECTIVES: To construct a cell line that can stably express human phospholamban(PLN) and initially explore its application in the study of myocardial toxicity mechanism. METHODS: FastCloning method was used to insert the open reading frame sequence of target gene PLN into eukaryotic expression vector pcDNA5/FRT/TO(hereinafter referred to as pDFT) to construct the pDFT-PLN-Flag plasmid. The Flp-In^TM T-REx^TM 293 cells were generated by cotransfection of the constructed plasmid and pOG44 plasmid to express the target gene. Successfully recombined monoclonal cell lines were screened by hygromycin B resistance. Western blot and indirect immunofluorescence (IFA) were used to examine the expression of the target protein in recombinant cells. After the cell line was exposed to aconitine, it was verified by Western blot to detect changes in PLN protein phosphorylation. RESULTS: After PCR amplification of the recombinant plasmid and DNA electrophoresis, the length of the amplified product is the same as the known PLN gene fragment, which is consistent with the open reading frame (ORF) sequence of the human PLN gene after sequencing. IFA and Western blot showed that the constructed proliferation cell line can stably express high levels of human PLN under induction and regulation. Preliminary results showed that the phosphorylation level of Thr17-PLN decreased after two hours of exposure to 1 μmol/L aconitine. CONCLUSIONS This human cell line can stably express PLN and can be used to study the mechanism of action of aconitine on the cell at molecular level.
Calcium-Binding Proteins/metabolism* ; Cell Line ; Humans ; Myocardium/metabolism* ; Phosphorylation

Heart failure (HF) has become a major challenge in the treatment of global cardiovascular diseases. Great progress has been made in the drug treatment of HF, however, rehospitalization rate and mortality of patients with HF are still high. Hence, there is an urgent need to explore new treatment strategy and new underlying pathogenic mechanisms. In recent years, some researchers have suggested that regulation of ketone body metabolism may become a potentially promising therapeutic approach for HF. Some studies showed that the oxidative utilization of fatty acids and glucose was decreased in the failing heart, accompanied by the increase of ketone body oxidative metabolism. The enhancement of ketone body metabolism in HF is a compensatory change during HF. The failing heart preferentially uses ketone body oxidation to provide energy, which helps to improve the body's cardiac function. This review will discuss the potential significance of ketone body metabolism in the treatment of HF from three aspects: normal myocardial ketone body metabolism, the change of ketone body metabolism in HF, the effect of ketogenic therapy on HF and its treatment.
Humans ; Heart Failure/metabolism* ; Myocardium/metabolism* ; Ketone Bodies/metabolism* ; Cardiovascular Diseases ; Fatty Acids/metabolism* ; Energy Metabolism

OBJECTIVEEndotoxemia is a serious syndrome resulting in multi-organ failure. Once it happens, the penetration of small intestine epithelium increases, body liquid losses, then effective circulating blood decreases and serious metabolic acidosis, serious hypotension, systolic failure, and even shock may occur. In this pathological process, endotoxin, tumor necrosis alpha and systolic dysfunction play important roles. Nowadays, many studies have been done to resolve the systolic dysfunction, but too much attention had been paid to the followings: the depressions of myocardium caused by tumor necrosis alpha, other inflammatory factors, endotoxin and metabolic acidosis; the disturbance of blood vessel-nerve regulations; nitric oxide (NO)/inducible nitric oxide synthase (iNOS) over-synthesis and the decreased density of beta-receptors in the myocardium and/or their activities. Little attention has been paid to the relationship between alpha sarcmeric actin (alpha-SA) and systolic dysfunction during endotoxemia. Glutamine (Gln) can be metabolized into glutathione, an eliminator of free radical. It has been used in preventing myocardial damage from reperfusion. This study aimed to observe the dynamic changes of alpha-SA and mRNA expressions in rats with endotoxemia and examine the effects of Gln on them.

METHODSClassical rat model of endotoxemia was established by intraperitoneal injection of LPS (4 mg/kg, Escherichia coli O55:B5, Sigma). 121 Wistar 18-day-rats were divided into three groups randomly, (1) 0 h control group (normal saline: 1 ml/kg, n = 11). (2) LPS group (LPS: 4 mg/kg, n = 55). (3) Gln group (LPS: 4 mg/kg and immediately 13.64%; Gln: 1 ml/kg, Fresenus, n = 55), Furthermore, LPS and Gln groups were divided into 2, 4, 6, 24 and 72 h time points (n = 11). Each time point of LPS and Gln as well as control rats were anaesthetized at each time point with 1% chloral hydrate injected intraperitoneally at the dosage of 1 ml/kg. Then rats were sacrificed at appoint time, and the hearts were isolated. Eight of them were put in 76 degrees C liquid nitrogen and then frozen in minute 80 degrees C icebox in order to measure the expression of alpha-SA mRNA by RT-PCR. Three of them were fixed in 4% formaldehydum polymerisatum for 12 to 16 h, then the expression of alpha-SA was detected by immunohistochemistry.

RESULTS(1) Compared to 0 h, the expressions of alpha-SA and mRNA in LPS group were significantly depressed (P < 0.01). In LPS group, the lowest was at 6 - 24 h, while in Gln group, it was postponed to 24 h. At 72 h, there was no difference in expressions of alpha-SA between Gln and 0 h group (P > 0.05). (2) Comparing at same time point, the expressions of alpha-SA were significant higher in Gln group than those in LPS group, while the expressions of alpha-SA mRNA in Gln group were high at 4-72 h. There was, however, no significant difference at early phase (P > 0.05).

CONCLUSIONAlpha-SA and its mRNA expression were depressed in LPS-induced endotoxemia, especially from 6 to 24 h. It could damage the systolic function. alpha-SA decrease in endotoxemia was due to the inhibited synthesis other than the promoted degradation. Glutamine could inhibit the effects of LPS on both alpha-SA and its mRNA expressions.

Actins ; metabolism ; Animals ; Endotoxemia ; metabolism ; Glutamine ; pharmacology ; Lipopolysaccharides ; Myocardium ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar

Objective To study the effect of overwork stress response on the expression of connexin 43（Cx43） and connexin 45（Cx45） in cardiomyocytes and on cardiac function. Methods The experimental animals were divided into control group, overworked 1-month group and overworked 2-month group. A overworked rat model was established by forcing swimming of overworked group. The expressions of Cx43 and Cx45 in myocardial tissues of experimental animals were detected by Western blotting, while the corresponding myocardial tissues were stained with hematoxylin-eosin （HE） staining and Masson's staining, then histologically observed. Results Western blotting results showed that, compared with the control group, Cx43 expression in myocardial tissues of overworked rats decreased while Cx45 expression increased. HE staining and Masson's staining results showed that hypertrophy, rupture and interstitial fiber tissue hyperplasia were observed in myocardial fibers of overworked rats. Conclusion Overwork stress response may affect cardiac function as an independent factor and may even cause heart failure or arrhythmias and lead to death.
Animals ; Arrhythmias, Cardiac/metabolism* ; Connexin 43/metabolism* ; Connexins/metabolism* ; Heart Failure ; Myocardium ; Myocytes, Cardiac/metabolism* ; Rats

The effect of drugs on calcium-binding to cardiac muscle membrane fragments and its turnover rate was studied. Ouabian, acetylcholine, isoproterenol and norepinephrine did not have any effect either on calcium-binding to membrane fragments or an washout ahnd release curves of previously bound calcium. Local anesthetics inhibited the calcium-binding. Tetracaine at concentrations of 1 and 10 mM inhibited the calcium-binding by 30% and 54%, respectively, while 10 mM lidocaine inhibited it by 17%. Propranolol, a well-known adrenergic beta-blocker, also inhibited calcium-binding at the external calcium concentration of 10(-3) M. This effect of propranolol may be attributed to its local anesthetic-like action, rather than to the adrenergic blocking effect.
Anesthetics, Local/pharmacology* ; Animal ; Calcium/metabolism* ; Cardiotonic Agents/pharmacology* ; Cell Membrane/metabolism ; In Vitro ; Myocardium/metabolism*

The effect of drugs on calcium-binding to cardiac muscle membrane fragments and its turnover rate was studied. Ouabian, acetylcholine, isoproterenol and norepinephrine did not have any effect either on calcium-binding to membrane fragments or an washout ahnd release curves of previously bound calcium. Local anesthetics inhibited the calcium-binding. Tetracaine at concentrations of 1 and 10 mM inhibited the calcium-binding by 30% and 54%, respectively, while 10 mM lidocaine inhibited it by 17%. Propranolol, a well-known adrenergic beta-blocker, also inhibited calcium-binding at the external calcium concentration of 10(-3) M. This effect of propranolol may be attributed to its local anesthetic-like action, rather than to the adrenergic blocking effect.
Anesthetics, Local/pharmacology* ; Animal ; Calcium/metabolism* ; Cardiotonic Agents/pharmacology* ; Cell Membrane/metabolism ; In Vitro ; Myocardium/metabolism*

OBJECTIVEThe cardiac action potential (AP) and the intracellular Ca(2+) transient (CaT) are closely associated under normal physiological conditions, but not during ventricular fibrillation (VF). The purpose of this study was to determine whether this dissociation is directly related to the higher activation rate during VF.

METHODSWe optically mapped AP and CaT simultaneously in nine isolated rabbit hearts. Pinacidil, a K(ATP) channel opener, was used to shorten the action potential duration (APD) in order to capture tissue at fast pacing rates or to induce ventricular tachycardia (VT) comparable to VF activation rates. Mutual information (MI) was used to calculate the degree of AP and CaT coupling.

RESULTSPinacidil (40 micromol/L) infusion significantly shortened APD. The averaged cycle length (CL) of VF without Pinacidil was (77 +/- 13) ms, whereas the shortest CL achieved during VT under Pinacidil infusion was 76 ms. MIs during fast pacing (1.13 +/- 0.15) bits and fast VT (0.88 +/- 0.18) bits were higher than those during baseline VF (0.39 +/- 0.11) bits, VF with Pinacidil infusion (0.21 +/- 0.07) bits and VF after Pinacidil washout (0.36 +/- 0.15) bits. MIs during fast pacing or fast VT were higher than those of VFs at comparable dominant frequencies.

CONCLUSIONSCaT is closely associated with the AP during fast pacing and fast VT, but not during VF. The reduced MI during VF is not secondary to the fast rate of ventricular activation.

Action Potentials ; Animals ; Calcium ; metabolism ; In Vitro Techniques ; Myocardium ; metabolism ; Rabbits ; Ventricular Fibrillation ; metabolism ; physiopathology

OBJECTIVETo explore the expression and distribution of calcineurin (CaN) in normal and failing human myocardium.

METHODSLeft and right ventricles were obtained from end-staged heart failure patients (n = 12) undergoing heart transplantation and donor hearts (n = 5) taken from victims of vehicle accidents. Immunohistochemistry and SDS-PAGE technique were used to demonstrate expression and distribution of CaN.

RESULTSPositive immunoreactive staining for CaN was detected in human cardiomyocytes, cardiac fibroblasts and epicardial mesothelial cells, but not detected in cardiac vascular endothelial cells and smooth muscle cells. There was no difference in CaN protein levels between failing hearts and donor hearts (Band intensity of right ventricle in failing hearts and donor hearts was 130.20 +/- 8.66 and 139.87 +/- 6.21, P = 0.33. Band intensity of left ventricle in failing hearts and donor hearts was 106.45 and 126.34 +/- 12.09) and between left ventricular and right ventricular myocardium (Band intensity of left and right ventricles in failing hearts was 96.99 +/- 10.67 and 104.58 +/- 13.18, P = 0.63. Band intensity of left and right ventricles in failing hearts was 132.12 and 120.74).

CONCLUSIONSCaN is expressed in human cardiomyocytes, fibroblasts and epicardial mesothelial cells and the protein level and distribution of CaN are similar in failing and donor hearts.

Adult ; Calcineurin ; metabolism ; Female ; Heart Failure ; metabolism ; Humans ; Male ; Middle Aged ; Myocardium ; metabolism