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Y V Ladilov

Publications and source records attributed to Y V Ladilov.

12 recordsLinked to original sources
PubMed2000

Importance of bicarbonate transport for protection of cardiomyocytes against reoxygenation injury.

Isolated cardiomyocytes from adult rats were incubated in anoxic bicarbonate-buffered media at extracellular pH (pH(o)) 6.4 until a cytosolic Ca(2+) overload and intracellular pH (pH(i)) of 6.4 were reached. On reoxygenation, the pH of the medium was changed to 7.4 to activate the Na(+)/H(+)exchanger (NHE) and the Na(+)-HCO(-)(3) symporter (NBS). The reoxygenation was performed in the absence or presence of the NHE inhibitor HOE-642 (3 micromol/l) and/or the NBS inhibitor DIDS (0.5 mmol/l), as in bicarbonate-free media. In reoxygenated control cells pH(i) rapidly recovered to the preanoxic level, and a burst of spontaneous oscillations of cytosolic Ca(2+) occurred, accompanied by the development of hypercontracture. When NBS and NHE were simultaneously inhibited during reoxygenation, pH(i) recovery was prevented, Ca(2+) oscillations were attenuated, and hypercontracture was abolished. Sole inhibition of NBS or NHE showed no protection against hypercontracture. In the absence of cytosolic acidosis, HOE-642 or DIDS did not prevent hypercontracture induced by Ca(2+) overload. The results demonstrate that simultaneous inhibition of NHE and NBS is needed to protect myocardial cells against reoxygenation-induced hypercontracture.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Inhibition of NHE protects reoxygenated cardiomyocytes independently of anoxic Ca(2+) overload and acidosis.

We investigated the question of whether inhibition of the Na(+)/H(+) exchanger (NHE) during ischemia is protective due to reduction of cytosolic Ca(2+) accumulation or enhanced acidosis in cardiomyocytes. Additionally, the role of the Na(+)-HCO(3)(-) symporter (NBS) was investigated. Adult rat cardiomyocytes were exposed to simulated ischemia and reoxygenation. Cytosolic pH [2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)], Ca(2+) (fura 2), Na(+) [sodium-binding benzolfuran isophthatlate (SBFI)], and cell length were measured. NHE was inhibited with 3 micromol/l HOE 642 or 1 micromol/l 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and NBS was inhibited with HEPES buffer. During anoxia in bicarbonate buffer, cells developed acidosis and intracellular Na and Ca (Na(i) and Ca(i), respectively) overload. During reoxygenation cells underwent hypercontracture (44.0 +/- 4.1% of the preanoxic length). During anoxia in bicarbonate buffer, inhibition of NHE had no effect on changes in intracellular pH (pH(i)), Na(i), and Ca(i), but it significantly reduced the reoxygenation-induced hypercontracture (HOE: 61.0 +/- 1.4%, EIPA: 68.2 +/- 1.8%). The sole inhibition of NBS during anoxia was not protective. We conclude that inhibition of NHE during anoxia protects cardiomyocytes against reoxygenation injury independently of cytosolic acidification and Ca(i) overload.

Amiloride↗
PubMed1999

Pretreatment with PKC activator protects cardiomyocytes against reoxygenation-induced hypercontracture independently of Ca2+ overload.

OBJECTIVE: Although several studies have shown that activation of protein kinase C (PKC) plays an important role in protection through ischemic preconditioning, little is known about the effects of direct PKC activation on the course of ischemia-reperfusion injury. The aim of this study was to analyse the effects of a pretreatment with the PKC activator 1,2-dioctanoyl-sn-glycerol (1,2DOG). METHODS: Isolated adult Wistar rat cardiomyocytes were exposed to 80 min of simulated ischemia (anoxia, pHo 6.4) and 20 min of reoxygenation (pHo 7.4). Cytosolic Ca2+ (fura-2), cytosolic pH (BCECF), Mg2+ (Mg-fura-2), lactate and cell length were measured and compared between control cells and cells treated with 20 mumol/l 1,2DOG before anoxia (10 min treatment and 10 min wash out). RESULTS: 1,2DOG-pretreatment delayed the time to extreme ATP depletion, but had no effect on lactate production and cytosolic pH. The accumulation of cytosolic Ca2+ was markedly accelerated in pretreated cells that developed rigor shortening, but reoxygenation-induced hypercontracture was significantly reduced. 1,2DOG, therefore, completely abolished Ca(2+)-dependence of hypercontracture. The effects of pretreatment were fully abolished with 1 mumol/l bisindolylmalcimide (PKC inhibitor). We conclude that PKC preactivation leads to (1) reduction of energy demand, (2) acceleration of Ca2+ overload during anoxia and (3) prevention of reoxygenation-induced hypercontracture independent of anoxic changes in cytosolic Ca2+ and pH.

Analysis of Variance↗
PubMed1998

Protection of rat cardiomyocytes against simulated ischemia and reoxygenation by treatment with protein kinase C activator.

The aim of this study was to investigate whether treatment with the protein kinase C (PKC) agonist 1,2-dioctanoyl-sn-glycerol (1,2DOG) can protect isolated adult Wistar rat cardiomyocytes against simulated ischemia and reoxygenation. Cytosolic Ca2+ (assessed by fura 2 fluorescence), pHi (assessed by BCECF fluorescence), and cell length were measured during 80 minutes of simulated ischemia (anoxia, pHo 6.4) and 20 minutes of reoxygenation (pHo 7.4) and compared between control cells and cells treated with 20 micromol/L 1,2DOG before anoxia (10-minute treatment and 10-minute washout), before and during anoxia (two-step treatment), or only during anoxia. Treatment before anoxia attenuated rigor contracture but did not influence anoxic Ca2+ overload. In contrast, two-step treatment before and during anoxia accelerated rigor contracture but reduced the rate of anoxic Ca2+ accumulation. During reoxygenation, control cells developed irreversible hypercontracture (reduction of cell length to 43+/-2% of the initial cell length, n=62), which was accompanied by spontaneous oscillations of cytosolic Ca2+ (19.6+/-1.6 per minute). Two-step treatment with 1,2DOG before and during anoxia significantly reduced hypercontracture (reduction of cell length to 60+/-2%, P<.01 versus control, n=41) and suppressed spontaneous Ca2+ oscillations (2.8+/-0.9 per minute, P<.01 versus control). These effects could not be reproduced by treatment with 1,2DOG before anoxia or during anoxia or by a two-step treatment with the PKC-inactive 1,3-dioctanoyl-sn-glycerol and were fully abolished with 1 micromol/L bisindolylmaleimide (PKC inhibitor). We conclude that a two-step activation of PKC before and during anoxia is required for effective protection of cardiomyocytes against anoxic Ca2+ overload and reoxygenation-induced hypercontracture.

Animals↗
PubMed1998

Protection of isolated cardiomyocytes against reoxygenation-induced hypercontracture by SIN-1C.

Previous studies have shown that SIN-1C (N-morpholinoiminoacetonitrile) can protect ischemic-reperfused myocardium. The aim of the present study was to analyse on the cellular level the mechanism by which SIN-1C may exert this effect. To simulate ischemia-reperfusion, isolated adult rat cardiomyocytes were incubated at pH 6.4 under anoxia and reoxygenated at pH 7.4 in presence or absence of SIN-1C. Reoxygenation was started when intracellular Ca2+ (measured with fura-2) had increased to > or = 10(-5) mol/L and pHi (BCECF) decreased to 6.6. Development of hypercontracture was determined microscopically. In the control group reoxygenation provoked oscillations of cytosolic Ca2+ (60.9 +/- 9.6 min-1 at 5 min of reoxygenation) accompanied by development of hypercontracture (to 77.2 +/- 3.8% of end-ischemic cell length). When SIN-1C was added upon reoxygenation, Ca2+ oscillations were markedly reduced (27.0 +/- 4.5 min-1, p < 0.001) and hypercontracture virtually abolished (90.6 +/- 2.0% of end-ischemic cell length, p < 0.001). SIN-1C did not influence the recovery of pHi during reoxygenation. The results indicate that SIN-1C protects cardiomyocytes against reoxygenation-induced hypercontracture by its ability to suppress oscillations of intracellular Ca2+ during the early phase of reoxygenation.

Acetonitriles↗
PubMed1997

Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture.

BACKGROUND: Resupply of oxygen to the myocardium after extended periods of ischemia or hypoxia can rapidly aggravate the already existing injury by provoking hypercontracture of cardiomyocytes (acute reperfusion injury). Previous studies indicated that halothane can protect ischemic-reperfused myocardium. The aim of the present study was to analyze on the cellular level the mechanism by which halothane may protect against reoxygenation-induced hypercontracture. METHODS AND RESULTS: To simulate ischemia-reperfusion, isolated adult rat cardiomyocytes were incubated at pH 6.4 under anoxia and reoxygenated at pH 7.4 in the presence or absence of 0.4 mmol/L halothane. Reoxygenation was started when intracellular Ca2+ (measured with fura 2) had increased to > or = 10(-5) mol/L and pHi (BCECF) had decreased to 6.5. Development of hypercontracture was determined microscopically. In the control group, reoxygenation provoked oscillations of cytosolic Ca2+ (72+/-9 per minute at fourth minute of reoxygenation) accompanied by development of hypercontracture (to 65+/-3% of end-ischemic cell length). When halothane was added on reoxygenation, Ca2+ oscillations were markedly reduced (4+/-2 per minute, P<.001) and hypercontracture was virtually abolished (90+/-4% of end-ischemic cell length, P<.001). Halothane did not influence the recovery of pHi during reoxygenation. Similar effects on Ca2+ oscillations and hypercontracture were observed when ryanodine (3 micromol/L), an inhibitor of the sarcoplasmic reticulum Ca2+ release, or cyclopiazonic acid (10 micromol/L), an inhibitor of the sarcoplasmic reticulum Ca2+ pump, were applied instead of halothane. CONCLUSIONS: Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture by preventing oscillations of intracellular Ca2+ during the early phase of reoxygenation.

Anesthetics, Inhalation↗
PubMed1997

Simulated ischemia increases the susceptibility of rat cardiomyocytes to hypercontracture.

The hypothesis that rat cardiomyocytes become susceptible to hypercontracture after anoxia/reoxygenation was investigated. The cells were gradually overloaded with Ca2+ after different periods of simulated ischemia (substrate-free anoxia, medium at pH 6.4) followed by 20 minutes of reoxygenation. The cytosolic Ca2+ concentration (measured with fura 2) at which the cells developed maximal hypercontracture (Camax) was used as an index for their susceptibility to hypercontracture (SH). SH was increased in cardiomyocytes after prolonged periods of simulated ischemia; ie, these cells developed hypercontracture at significantly lower cytosolic Ca2+ levels than did normoxic cells (Camax, 0.80 +/- 0.05 mumol/L versus 1.27 +/- 0.05 mumol/L; P < .01). To find the possible cause of increased SH, the influence of Ca2+ overload, acidosis, and protein dephosphorylation were studied. Prevention of cytosolic Ca2+ overload in anoxic cardiomyocytes or imitation of ischemic acidosis in normoxic cells did not influence Camax. In contrast, use of 10 mumol/L cantharidin (inhibitor of protein phosphatases 1 and 2A) during anoxic superfusion prevented the reduction of Camax. Furthermore, treatment of normoxic cardiomyocytes with 20 mmol/L of the chemical phosphatase 2,3-butanedione monoxime reduced Camax. Therefore, prolonged simulated ischemia increases susceptibility of cardio-myocytes to hypercontracture. This seems to be due to protein dephosphorylation.

Acidosis↗
PubMed1996

Myocardial protection during reperfusion.

After prolonged periods of energy depletion, myocardial cells may rapidly deteriorate during the early stage of reperfusion. It has now been clearly demonstrated that this kind of acute lethal reperfusion injury is due to specific processes elicited by cellular re-energization. The most prominent single cause of acute harm to the reoxygenated myocardial cells is myofibrillar hypercontraction. Hypercontraction is caused by a resupply of energy of the myofibrils at excessive cytosolic Ca2+ concentrations. Additionally, the ability of the cytoskeleton to withstand large mechanical forces seems to be weakened after a prolonged period of energy depletion. Intracellular acidosis during the early stage of reperfusion represents a natural mechanism of protection against acute reperfusion injury. The reperfused myocardial cell may also suffer from uncontrolled water uptake and increased sarcolemmal fragility, favoring osmotic damage of cell membranes. As yet therapeutical interventions trying to specifically interfere with these pathomechanisms of reperfusion injury have only been tested experimentally. It seems promising to evaluate their utility for myocardial protection in cardio-surgical operations.

Acidosis↗
PubMed1995

Protection of reoxygenated cardiomyocytes against hypercontracture by inhibition of Na+/H+ exchange.

Effects of Na+/H+ exchange inhibition and cytosolic acidosis on reoxygenated adult rat ventricular cardiomyocytes were investigated. Cells were incubated in anoxic media at pH 6.4 until pCa of < or = 5, intracellular pH (pHi) of 6.5, and cytosolic [Na+] of 50 mM were reached. On reoxygenation, medium pH was changed to 7.4 to activate Na+/H+ exchange. In one group, 20 microM HOE-694, an inhibitor of Na+/H+ exchange, was added. With or without HOE-694, cytosolic Ca2+ and Na+ returned to control levels within 10 min of reoxygenation. In the absence of HOE-694, the pHi renormalized (to 7.2) within 8 min, but irreversible hypercontracture and transient Ca2+ oscillations were observed. In the presence of HOE-694, pHi stayed acidotic (at 6.5), hypercontracture was prevented, and Ca2+ oscillations were attenuated. When the Na+ pump was inhibited with 0.1 mM ouabain, even partial recovery of Ca2+ control became impossible unless HOE-694 was added. Our conclusions are 1) activation of Na+/H+ exchange does not impair recovery of cytosolic Na+ and Ca2+ control unless activity of the sarcolemmal Na+ pump is critically reduced, and 2) due to prolongation of cytosolic acidosis, inhibition of Na+/H+ exchange protects against reoxygenation-induced hypercontracture and cytosolic Ca2+ oscillations.

Animals↗
PubMed1994

Importance of sodium for recovery of calcium control in reoxygenated cardiomyocytes.

The role of Na+ in the recovery from severe anoxic Ca2+ overload was investigated in isolated quiescent ventricular cardiomyocytes from adult rat. Changes of cytosolic Ca2+ and Na+ concentrations were followed by the fura 2 and Na(+)-binding benzofuran isophthalate techniques, respectively. When the fura 2 ratio (340/380 nm) reached saturation in anoxic cells, indicating a severe cytosolic Ca2+ overload, the cells were reoxygenated. This caused a rapid initial drop of cytosolic Ca2+ to a lower but still elevated level (phase I), followed by oscillatory Ca2+ transients at this level (phase II) and, within 10 min, the reestablishment of a stable cytosolic Ca2+ concentration at the normal resting level (phase III). As previously shown [B. Siegmund, R. Zude, and H. M. Piper. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1262-H1269, 1992], Ca2+ shifts in phase I and II are mainly due to uptake and release of Ca2+ by the sarcoplasmic reticulum. Phase I was unchanged, and phase II was much prolonged (> 60 min) in cells reoxygenated under Na+ pump inhibition (0.2 mM ouabain) or Na+ depletion. Phase III could only be reestablished (< 10 min) when ouabain was eluted or external Na+ replenished, respectively. The results show that full recovery of cytosolic Ca2+ control (phase III) requires an active sarcolemmal Na+ pump and the availability of external Na+. This indicates that phase III is determined by the transsarcolemmal extrusion of Ca2+ by a tandem mechanism consisting of 1) the Na+ pump, generating an extracellular-to-intracellular Na+ gradient, and 2) the sarcolemmal Na+/Ca2+ exchange, driven by that gradient to extrude Ca2+.

Animals↗
PubMedDate not supplied

The role of Na+/H+ exchange in ischemia-reperfusion.

In ischemia the cytosol of cardiomyocytes acidifies; this is reversed upon reperfusion. One of the major pH(i)-regulating transport systems involved is the Na+/H+ exchanger. Inhibitors of the Na+/H+ exchanger have been found to more effectively protect ischemic-reperfused myocardium when administered before and during ischemia than during reperfusion alone. It has been hypothesized that the protection provided by pre-ischemic administration is due to a reduction in Na+ and secondary Ca2+ influx. Under reperfusion conditions Na+/H/ exchange inhibition also seems protective since it prolongs intracellular acidosis which can prevent hypercontracture. In detail, however, the mechanisms by which Na+/H+ exchange inhibition provides protection in ischemic-reperfused myocardium are still not fully identified.

Acidosis↗