Influence of microtubule depolymerization of myocardial cells on mitochondria distribution and energy metabolism in adult rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of microtubule depolymerization of myocardial cells on distribution and activity of mitochondria, and energy metabolism of cells in adult rats.</p><p><b>METHODS</b>Myocardial cells of SD adult rats and SD suckling rats were isolated and cultured. They were divided into adult and suckling rats control groups (AC and SC, normally cultured without any stimulating factor), adult and suckling rats microtubule depolymerization agent groups (AMDA and SMDA, cultured with 8 micromol/L colchicine containing nutrient solution for 30 minutes) according to the random number table. (1) The expression of polymerized beta tubulin in myocardial cells of adult and suckling rats was detected with Western blot. (2) Myocardial cells of rats in AC and AMDA groups were collected. The expression of cytochrome c was detected with Western blot. Distribution of voltage-dependent anion channels (VDAC) and polymerized beta tubulin in myocardial cells were observed with immunofluorescent staining. Mitochondrial inner membrane potential was determined with immunocytochemical method. Activity of myocardial cells was detected with MTT method. Contents of ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) and energy charge of cells were determined with high performance liquid chromatography.</p><p><b>RESULTS</b>(1) The expression of polymerized beta tubulin:in AMDA group it was 0.52 + or - 0.07, which was obviously lower than that (1.25 + or - 0.12) in AC group (F = 31.002, P = 0.000); in SMDA group it was 0.76 + or - 0.12, which was significantly lower than that (1.11 + or - 0.24) in SC group (F = 31.002, P = 0.000), but was obviously higher than that in AMDA group (F = 31.002, P = 0.009). (2) The expression of cytochrome c in AC group was 0.26 + or - 0.03, which was obviously lower than that (1.55 + or - 0.13) in AMDA group (t = -24.056, P = 0.000). (3) Immunofluorescent staining result: in AC group, microtubules of myocardial cells were in linear tubiform, distributed in parallel with myocardial fiber; VDAC staining result showed that mitochondria were in granular form, distributed in the same direction as microtubules. In AMDA group, the normal distribution regularity of microtubules was destroyed, with weakened immune fluorescence intensity, microtubules structure indistinct, continuity lost, rough in appearance, and the distribution of mitochondria became disrupted. (4) Mitochondrial inner membrane potential in AC group fluorescent intensity was 1288 + or - 84, which was obviously higher than that (331 + or - 27) in AMDA group (t = 26.508, P = 0.000). (5) Cellular activity: in AC group absorbance value was 1.75 + or - 0.11, which was obviously lower than that (0.81 + or - 0.07) in AMDA group (t = 17.348, P = 0.000). (6) Energy metabolism: compared with those in AC group, content of ATP decreased, contents of ADP and AMP increased, and ATP/ADP value and energy charge decreased in AMDA group.</p><p><b>CONCLUSIONS</b>Microtubules and mitochondria distribute in the same direction in normal myocardial cells in adult rats. After microtubule depolymerization, mitochondria are arranged in disorder fashion; cytochrome c leaks from mitochondria; mitochondrial membrane potential, energy supply, and cellular activity decrease in the myocardial cells.</p>

The influence of microtubule intervention drugs on glycolytic key enzymes in myocardial cells after hypoxia / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of microtubule intervention drugs on glycolytic key enzymes in myocardial cells after hypoxia.</p><p><b>METHODS</b>The primary passage of cultured myocardial cells from neonatal rats were divided into A group (with hypoxia), B group (with hypoxia and administration of l0 micromol/L colchicine), C group (with hypoxia and administration of 5 micromol/L taxol), D group (with hypoxia and administration of 10 micromol/L taxol), E group (with hypoxia and administration of 15 micromol/L taxol). The morphology of microtubule was observed with laser scanning microscope (LSM). The cell vitality was assayed by cell counting kit (CCK). The activities of hexokinase (HK), pyruvate kinase (PK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) were assayed with colorimetry.</p><p><b>RESULTS</b>In group B and E, the microtubule structure was damaged heavily, and the cell vitality was decreased significantly [The cell vitality was (89.99 +/- 3.47)% in B group and (84.56 +/- 6.61)% in E group, respectively, at 1.0 post hypoxia hour (PHH), and hoth values were obviously lower than that in A group (97.44 +/- 1.76)%, P < 0.01]. The HK, PK and PFK activities decreased obviously. The activities of HK, PK and PFK in group C were similar to those of the A group. Compared with that in other groups, the degree of damage of microtubule structure in D group was milden. The activities of HK, PK and PFK in D group during 0.5 - 6.0 PHH were significantly higher than those in A group. The activity of LDH in each group was increased after hypoxia.</p><p><b>CONCLUSION</b>Proper concentration of microtubule-stabilizing drugs can alleviate the damages to microtubule structure, and enhance the activity of glycolytic key enzymes of myocardial cells at early stage of hypoxia.</p>

Study on the influence of hypoxia induced microtubule damage on the opening of mitochondrial permeable transition pore of cardiac myocytes in rat / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of hypoxia induced microtubule damage on the opening of mitochondrial permeable transition pore (MPTP)of cardiac myocytes and on the decrease of respiratory function in rat.</p><p><b>METHODS</b>Primary cultured myocardial cells from 30 neonatal rats were randomized as normoxic group (A), hypoxia group (B), normoxia with microtubule destabilizing agent group (C, with treatment of 8 micromol/L colchicines for 30 minutes before normoxia), and hypoxia with microtubule stabilizing agent group (D, with treatment of 10 micromol/L taxol for 30 minutes before hypoxia). beta-tubulin immunofluorescence ,the opening of mitochondria permeability transition pore, and the mitochondrial inner membrane potential were detected at 0.5, 1, 3, 6 and 12 post-treatment hours (PTH), and the mitochondrial respiratory function was determined by MTT method. The changes in these indices were also determined in A group at the corresponding time-points.</p><p><b>RESULTS</b>Obvious damage of polymerized microtubule, opening of MPTP, mitochondrial inner membrane potential loss and decrease of myocardial respiratory activity were observed in both group B and C at 0.5 PTH, and they became more and more serious afterwards. However, the changes in the above indices in D group were much better than those in B group (P < 0.05 or 0.01), and no difference was found between D (92.8 +/- 4.0)% and C [(100.0 +/- 0.0) %, P > 0.05] groups.</p><p><b>CONCLUSION</b>Hypoxia played a role in the myocardial microtubule damage as well as in the opening of MPTP. Moreover, hypoxia could also impair the mitochondrial respiratory function. Microtubule destabilizing agent could reproduce well the process of hypoxia induced microtubule damage, while the stabilizing agent exerted protective effect by improving the transition of mitochondrial permeability and the mitochondria respiratory function.</p>