Effects of Spironolactone on Hypoxia-Inducible Factor-1α in the Patients Receiving Coronary Artery Bypass Grafting.

ABSTRACT: We explored the protective effect of spironolactone on cardiac function in the patients undergoing coronary artery bypass grafting (CABG) by determining serum hypoxia-inducible factor-1α (HIF-1α) before and after CABG. We used the propensity score matching method retrospectively to select 174 patients undergoing CABG in our hospital from March 2018 to December 2019. Of the 174 patients, 87 patients taking spironolactone for more than 3 months before CABG were used as a test group and other 87 patients who were not taking spironolactone as a control group. In all patients, serum HIF-1α and troponin I levels were determined before as well as 24 hours and 7 days after CABG, serum N-terminal probrain natriuretic peptide (NT-proBNP) level was determined before as well as 12, 24, and 36 hours after CABG, and electrocardiographic monitoring was performed within 36 hours after CABG. The results indicated that there were no significant differences in the HIF-1α level between the test group and the control group before and 7 days after CABG, but the HIF-1α level was significantly lower in the test group than that in the control group 24 hours after CABG (P < 0.01). The 2 groups were not significantly different in the troponin I level at any time point. There was no significant difference in the serum NT-proBNP level between the test group and the control group before CABG, but NT-proBNP (BNP) levels were all significantly lower in the test group than those in the control group at postoperative 12, 24, and 36 hour time points (all P <0.05). The incidence of postoperative atrial fibrillation was also significantly lower in the test group than that in the control group (P = 0.035). Spironolactone protects cardiac function probably by improving myocardial hypoxia and inhibiting myocardial remodeling.

The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells.

EGFP (enhanced green fluorescent protein) tagged to either the N (amino)-terminus [EGFP/hERG (human ether-a-go-go-related gene)] or C (carboxyl)-terminus (hERG/EGFP) of hERG channel is used to study mutant channel protein trafficking for several years. However, it has been reported that the process can alter hERG channel properties. The aim of the study was to determine whether EGFP tagged to N-terminus of hERG channels would alter the cellular localizations and the electrophysiological properties of hERG channels compared with untagged hERG channels. The hERG channels tagged with or without EGFP were transiently expressed in HEK (human embryonic kidney) 293 cells using a lipofectamine method. HEK 293 cells expressing pCDNA3-hERG or pEGFP-hERG were double immunolabelled with anti-hERG and anti-calnexin (an ER marker protein) followed with FITC- and TRITC (tetramethylrhodamine ß-isothiocyanate)-labelled secondary antibodies, respectively. Confocal laser scanning microscope was used to observe the cellular localization of EGFP-tagged hERG channels and untagged hERG channels. Patch-clamp technique was used to record whole cell currents. We found that the EGFP/hERG fusion protein and untagged hERG channels were both expressed not only on the cell surface membrane but also in the cytoplasm of HEK293 cells. The EGFP/hERG appeared to influence the hERG channel gating properties, including reduction of the peak tail current density, more rapid inactivation process, faster recovery from inactivation and faster deactivation kinetics compared with untagged hERG channels. Our results suggest that the EGFP/hERG channel alter the electrophysiological properties of hERG channel, but it does not seem to alter the cellular location of hERG channels. Thus, EGFP tagging to N-terminus might be used for research of subcellular location of hERG channels but not for the channel electrophysiological properties.

[Functional expression of congenital long QT syndrome related HERG mutation A561V in vitro].

OBJECTIVE: To investigate the functional expression of HERG mutation A561V detected in a Chinese congenital long QT syndrome family. METHODS: The mutation gene A561V was cloned into eukaryotic expressive vector pcDNA3 by quick site-directed mutagenesis PCR and restriction enzymes. The wild-type HERG, heterozygous type HERG and HERG mutation A561V were respectively cotransfected with pRK5-GFP into HEK293 cells by Suprefact transfection regent. The protein expression was measured by immunofluorescence method and Western blot. The electrophysiological characteristics of transfected cells were determined by whole cell patch-clamp technique. RESULTS: Direct sequence analyses revealed a C to T transition at position 1682. A561V mutation was correctly combined to eukaryotic expressive vector pcDNA3 and expressed in HEK293 cells. The protein expression of mutation and heterozygosis were located in cytoplasm and cellular membrane. 155 kDa and 135 kDa protein bands were detected in wild type HERG channel while only 135 kDa protein band was shown in heterozygous and mutational channels. Significant HERG tail-current was recorded in wild type HERG channel but not in mutation and heterozygosis channels. CONCLUSION: This study evidenced a functional dominant-negative current suppression in HEK293 cells transfected with HERG mutation A561V.

[Construction of eukaryotic expression vector of congenital long QT syndrome related HERG gene A561V mutation and its expression in HEK293 cells].

OBJECTIVE: To investigate the protocol of the construction of HERG gene mutations, an A561V mutation which was detected in a Chinese congenital long QT syndrome (LQTS) family had been constructed and expressed in vitro. METHODS: The A561V cloning vector PGEM-HERG-A561V was constructed by quick site-directed mutagenesis PCR. The A561V expressive vector pcDNA3-HERG-A561V was constructed by restriction enzymes. pRK5-GFP was cotransfected with pcDNA3-HERG-A561V or wild type pcDNA3-HERG into HEK293 cells by Superfect transfection reagent. The protein was measured by immunofluorescence. RESULTS: Direct sequence analyses revealed a C to T transition at position 1682. The A561V mutation was correctly combined to eukaryotic expressive vector pcDNA3 and expressed in HEK293 cells. The protein of mutation was expressed in cytoplasm and cellular membrane while the wild type gene was expressed only on cellular membrane. CONCLUSION: The protocol can be used successfully to construct and express HERG A561V mutation and it forms the basement of the further study on functions of mutation.

[Single nucleotide polymorphisms of genes associated with high density lipoprotein metabolism in Chinese population].

OBJECTIVE: To study the single nucleotide polymorphisms in genes associated with the high density lipoprotein (HDL) metabolism in Chinese population. METHODS: Two hundred and nine normal Han ethnic subjects, aged 59+/-10 years, were recruited from 5 medical centers in western part of China. DNA was extracted by proteinase K digestion, phenol and chloroform extraction as well as isopropanol precipitation. The polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) in conjunction with sequencing were employed to test the single nucleotide polymorphisms (SNPs) in ATP-binding cassette transporter (ABCA1), cholesteryl ester transfer protein (CETP) and lipoprotein lipase (LPL) genes. RESULTS: The allelic frequencies of A and G of ABCA1 gene are 53.4% and 46.6%; of B2 and B1 allele of CETP, 41.0% and 59.0%; of HindIII (-) and (+) allele of LPL, 18.9% and 81.1%; and of PvuII(+) and (-) allele of LPL, 66.0% and 34.0%, respectively. All genotype frequencies fit well with the Hardy-Weinberg equilibrium; the significant linkage disequilibrium exists between LPL HindIII(+)and PvuII(+) polymorphisms. All of the RFLP in these genes result from the single nucleic substitution in fragment recognized by corresponding restriction enzymes. CONCLUSION: The genetic polymorphisms of ABCA1, LPL-HindIII and LPL-PvuII in Chinese Han ethnic population are significantly different from Caucasians residing in USA or Europe.

[Heterogeneity of Na+/Ca2+ exchanger current across the left ventricular wall of rabbit].

OBJECTIVE: To investigate the distribution characteristics of Na+/Ca2+ exchanger current (I(Na+/Ca2+)) across the left ventricular wall of rabbit and the relationship between I(Na+/Ca2+) and transmural depolarization heterogeneity. METHODS: By using whole-cell patch clamp techniques, action potentials (AP), I(Na+/Ca2+), and both rapid and slow components of delayed rectifier potassium current (I(Kr) and I(Ka)) were recorded in subendocardial (Endo), midmyocardial (M), and subepicardial (Epi) cells of the left ventricular wall of rabbit. RESULTS: AP duration in M cells was longer than that in Epi cells, P<0.01. At the test potential of +40 mV, outward I(Na+/Ca2+) in M cells was larger than that in Epi and Endo cells, P<0.01, P<0.05, respectively. At the test potential of -100 mV, inward I(Na+/Ca2+) in M cells was larger than that in Epi cells, P<0.05. At the test potential of +50 mV, the tail current density of I(Ka) in M cells was smaller than that in Epi cells, P<0.05, and there was no significant difference among the tail current densities of I(Kr) in Endo, M, and Epi cells. CONCLUSION: The distribution of I(Na+/Ca2+) and I(Ka) across the left ventricular wall of rabbit is unequal, which contributes to the transmural depolarization heterogeneity.

[Long QT syndrome gene diagnosis by haplotype analysis].

OBJECTIVE: Three long QT syndrome(LQTS) pedigrees were brought together for genetic diagnosis by using short tandem repeat(STR) markers. METHODS: Genomic DNA was extracted from blood samples. STR markers (D7S1824, D7S2439, D7S483, D3S1298, D3S1767, D3S3521) in or spanning the HERG and SCN5A gene were amplified; the haplotype analysis for LQTS was performed. RESULTS: Clinical diagnosis showed that 15 are LQTS patients (3 died) and 11 are probable patients. Linkage analysis showed that LQTS patients are linked with the SCN5A gene in family 1, HERG is linked with the disease in family 2 and 3. Fourteen gene carriers were identified, 2 patients and 7 probable patients were excluded. CONCLUSION: Linkage analysis using STR markers can serve as useful tool for presymptomatic diagnosis.

[Na+/Ca2+ exchanger current and K+ current remodeling in midmyocardial cells of hypertrophic left ventricle].

OBJECTIVE: To investigate the characteristics of Na+/Ca2+ exchanger current (INa+ /Ca2+) and K+ current remodeling in midmyocardial cells of hypertrophic left ventricle for understanding the ionic basis of arrhythmia of the hypertrophic heart. METHODS: Twenty New Zealand rabbits were divided equally into normal control group and operation group, and in the latter, left ventricular hypertrophy was induced in the rabbits by partial ligation of the abdominal aorta. Action potentials, INa+/Ca2+, slowly activating delayed rectifier K+ current (IKs) and rapidly activating delayed rectifier K+ current (IKr) were recorded in the two groups by using whole-cell patch-clamp technique. RESULTS: At the basic cycle length of 2 s, 90% action potential duration (APD90) in control and operation groups was 522.0+/-19.5 ms (n=6) and 664.7+/-32.7 ms (n=7) respectively; at the testing potential of +40 mV, outward INa+/Ca2+ density in the two groups was 0.94+/-0.11 pA/pF (n=9) and 1.30+/-0.11 pA/pF (n=8) respectively; the testing potential of -100 mV elicited inward INa+/Ca2+ density of 0.40+/-0.05 pA/pF (n=9) and 0.56+/-0.02 pA/pF (n=8) respectively. The testing potential of +50 mV induced IKs tail current density of 0.26+/-0.03 pA/pF (n=8) and 0.17+/-0.01 pA/pF (n=9), and IKr tail current density of 0.34+/-0.02 pA/pF (n=8) and 0.23+/-0.02 pA/pF (n=9) respectively. Statistically significant differences were identified between the control and operation groups in all the above indices measured. CONCLUSION: The characteristics of electrical remodeling changes in midmyocardial cells of hypertrophic left ventricle, exhibited by prolonged action potential, up-regulated INa+/Ca2+ and down-regulated IKs and IKr.

Curcumin ameliorates left ventricular function in rabbits with pressure overload: inhibition of the remodeling of the left ventricular collagen network associated with suppression of myocardial tumor necrosis factor-alpha and matrix metalloproteinase-2 expression.

OBJECTIVE: Curcumin is a wide-spectrum cellular protector with antiinflammatory, antioxidizant, and antifibrotic effects. This study was conducted to investigate its effects on myocardial collagen remodeling in pressure overloaded rabbits. METHODS AND RESULTS: Pressure overloaded rabbits were established by partial abdominal aorta ligation. The rabbits were divided into the sham-operation group, vehicle group and curcumin group. Curcumin was administered orally at a dose of 100 mg/kg.d in 10 ml of 2.5% polyethylene glycol solution and the other 2 groups were given the same dose of polyethylene glycol solution. Compared with the vehicle group, left ventricular function in the curcumin group was significantly ameliorated, as indicated by decreased left ventricular end-diastolic pressure, left ventricle weight to body weight ratio, and the left ventricular posterior wall thickness. The collagen volume fraction in the curcumin group was also reduced. Myocardial tumor necrosis factor (TNF)-alpha and matrix metalloproteinase (MMP)-2 expression were significantly overexpressed in the vehicle group and markedly suppressed in the curcumin group at both the 4th and 8th weeks. At the end of the 8th week, the ejection fraction in the curcumin group was increased compared with that in the vehicle group. CONCLUSION: Curcumin improved left ventricular function in pressure overloaded rabbits. This might be due to inhibition of collagen remodeling associated with suppression of myocardial expression of tumor necrosis factor-alpha, and matrix metalloproteinase-2.