Deuterium oxide protects against myocardial injury induced by ischemia and reperfusion in rats.

Objectives. Although deuterium oxide (D2O) has preservative property on the extracted organ, whether D2O also protects the in situ myocardial injury remains unknown. Using cardiac microdialysis, local administration of D2O through dialysis probe was applied in situ rat heart. We examined the effect of the D2O on the myocardial injury induced ischemia, reperfusion, and chemical hypoxia. Methodology. We measured dialysate myoglobin levels during 30 min of coronary occlusion and reperfusion in the absence and presence of D2O. Furthermore, to confirm the effect of D2O on NaCN induced myocardial injury, we measured the dialysate myoglobin levels with local perfusion of NaCN in the absence and presence of D2O. Results. The dialysate myoglobin levels increased from 177 ± 45 ng/mL at baseline to 3030 ± 1523 ng/mL during 15-30 min of coronary occlusion and further increased to 8588 ± 1684ng/mL at 0-15 min of reperfusion. The dialysate myoglobin levels with 60 min local perfusion of NaCN increased to 1214 ± 279 ng/mL. D2O attenuated myocardial myoglobin release during 15-30 min of coronary occlusion and 0-30 min of reperfusion and 15-60 min of local perfusion of NaCN. Conclusions. D2O might have a beneficial effect of myocardium against ischemia, reperfusion and chemical hypoxia.

Mild Hypothermia Is Ineffective to Protect Against Myocardial Injury Induced by Chemical Anoxia or Forced Calcium Overload.

Although hypothermia suppresses myocardial ischemia/reperfusion injury, whether it also protects the myocardium against cellular stresses such as chemical anoxia and calcium overload remains unknown. We examined the effect of mild hypothermia (33°C) on myocardial injury during ischemia/reperfusion, local administration of sodium cyanide (chemical anoxia), or local administration of maitotoxin (forced Ca overload) using cardiac microdialysis applied to the feline left ventricle. Baseline myoglobin levels (in ng/mL) were 237 ± 57 and 150 ± 46 under normothermia and hypothermia, respectively (mean ± SE, n = 6 probes each). Coronary artery occlusion increased the myoglobin level to 2600 ± 424 under normothermia, which was suppressed to 1160 ± 149 under hypothermia (P < 0.05). Reperfusion further increased the myoglobin level to 6790 ± 1550 under normothermia, which was also suppressed to 2060 ± 343 under hypothermia (P < 0.05). By contrast, hypothermia did not affect the cyanide-induced myoglobin release (930 ± 130 vs. 912 ± 62, n = 6 probes each) or the maitotoxin-induced myoglobin release (2070 ± 511 vs. 2110 ± 567, n = 6 probes each). In conclusion, mild hypothermia does not make the myocardium resistant to cellular stresses such as chemical anoxia and forced Ca overload.

Effects of intravenous cariporide on release of norepinephrine and myoglobin during myocardial ischemia/reperfusion in rabbits.

AIMS: To examine the effects of cariporide, a Na(+)/H(+) exchanger-1 inhibitor, on cardiac norepinephrine (NE) and myoglobin release during myocardial ischemia/reperfusion by applying a microdialysis technique to the rabbit heart. MAIN METHODS: In anesthetized rabbits, two dialysis probes were implanted into the left ventricular myocardium and were perfused with Ringer's solution. Cariporide (0.3mg/kg) was injected intravenously, followed by occlusion of the left circumflex coronary artery. During 30-min coronary occlusion followed by 30-min reperfusion, four consecutive 15-min dialysate samples (two during ischemia and two during reperfusion) were collected in vehicle and cariporide-treated groups. Dialysate myoglobin and NE concentrations were measured by immunochemistry and high-performance liquid chromatography, respectively. KEY FINDINGS: Dialysate myoglobin and NE concentrations increased significantly during myocardial ischemia/reperfusion in both vehicle and cariporide-treated groups (P<0.01 vs. baseline). In cariporide-treated group, dialysate myoglobin concentrations were significantly lower than those in vehicle group throughout ischemia/reperfusion (P<0.01 at 0-15 min of ischemia, P<0.05 at 15-30 min of ischemia, P<0.01 at 0-15 min of reperfusion, and P<0.01 at 15-30 min of reperfusion). However, dialysate NE concentrations in cariporide-treated group were lower than those in vehicle group only during ischemia (P<0.01 at 0-15 min of ischemia, and P<0.05 at 15-30 min of ischemia). SIGNIFICANCE: When administered before ischemia, cariporide reduces myoglobin release during ischemia/reperfusion and decreases NE release during ischemia.

Effects of intravenous magnesium infusion on in vivo release of acetylcholine and catecholamine in rat adrenal medulla.

We applied microdialysis technique to the left adrenal medulla of anesthetized rats and examined the effects of intravenous Mg(2+) infusion on presynaptic acetylcholine (ACh) release and postsynaptic catecholamine release induced by electrical stimulation of splanchnic nerves. The dialysis probes were perfused with Ringer's solution containing neostigmine. Low-dose MgSO4 (25 µmol/kg/min for 30 min i.v.) increased mean plasma Mg(2+) concentration to 2.5mM; the administration suppressed norepinephrine (NE) release by approximately 30% and epinephrine (Epi) release by approximately 20%, but did not affect ACh release. High-dose MgSO4 (50 µmol/kg/min for 30 min i.v.) increased mean plasma Mg(2+) concentration to 3.8mM; the administration suppressed ACh release by approximately 25%, NE release by approximately 60% and Epi release by approximately 45%. Administration of Na2SO4 (50 µmol/kg/min for 30 min i.v.) did not change the release of ACh, NE or Epi. Local administration of nifedipine (200 µM) suppressed NE release by approximately 40% and Epi release by approximately 30%, but did not affect ACh release. In the presence of nifedipine, low-dose MgSO4 did not suppress the release of ACh, or further suppress NE or Epi compared to nifedipine alone, but high-dose MgSO4 suppressed ACh release by approximately 25% and further suppressed NE release by approximately 60% and Epi release by approximately 50% compared to nifedipine alone. In conclusion, intravenous administration of Mg(2+) inhibits both presynaptic ACh release and postsynaptic catecholamine release in the adrenal medulla, but L-type Ca(2+) channel-controlled catecholamine release may be more sensitive to Mg(2+) than non-L-type Ca(2+) channel-controlled ACh release.

In vivo direct monitoring of interstitial norepinephrine levels at the sinoatrial node.

We assessed in vivo interstitial norepinephrine (NE) levels at the sinoatrial node in rabbits, using microdialysis technique. A dialysis probe was implanted adjacent to the sinoatrial node of an anesthetized rabbit and dialysate was sampled during sympathetic nerve stimulation. Atrial dialysate NE concentration correlated well with heart rate. Desipramine significantly increased dialysate NE concentrations both before and during sympathetic nerve stimulation compared with the absence of desipramine. However, desipramine did not affect the relation between heart rate and dialysate NE concentration. These results suggest that atrial dialysate NE level reflects the relative change of NE concentration in the synaptic cleft. Microdialysis is a powerful tool to assess in vivo interstitial NE levels at the sinoatrial node.

Role of Ca2+-activated K+ channels in catecholamine release from in vivo rat adrenal medulla.

To elucidate the role of Ca(2+)-activated K(+) (K(Ca)) channels in the presynaptic acetylcholine (ACh) release from splanchnic nerve endings and the postsynaptic catecholamine release from chromaffin cells, we applied microdialysis technique to the left adrenal medulla of anesthetized rats and investigated the effects of local administration of K(Ca) channel antagonists through dialysis probes on the release of ACh and/or catecholamine, induced by electrical stimulation of splanchnic nerves or local administration of ACh through the dialysis probes. Nerve stimulation-induced release: in the presence of a cholinesterase inhibitor, neostigmine, large-conductance K(Ca) (BK) channel antagonists, iberiotoxin and paxilline enhanced the presynaptic ACh release and postsynaptic norepinephrine (NE) and epinephrine (Epi) release. Small-conductance K(Ca) (SK) channel antagonists, apamin and scyllatoxin enhanced the Epi release without any changes in ACh or NE release. In the absence of neostigmine, ACh release was not detected. Iberiotoxin and paxilline enhanced NE and Epi release. Apamin and scyllatoxin had no effect on NE or Epi release. Exogenous ACh-induced release: iberiotoxin and paxilline enhanced the Epi release, but had no effect on the NE release. Apamin and scyllatoxin enhanced both NE and Epi release. In conclusion, BK channels on splanchnic nerve endings play an inhibitory role in the physiological catecholamine release from adrenal medulla by limiting presynaptic ACh release while SK channels do not. BK channels on Epi-storing cells may play an inhibitory role in nerve stimulation-induced Epi release. SK channels on NE- and Epi-storing cells play a minor role in nerve stimulation-induced catecholamine release.

In vivo direct monitoring of vagal acetylcholine release to the sinoatrial node.

To directly monitor vagal acetylcholine (ACh) release into the sinoatrial node, which regulates heart rate, we implanted a microdialysis probe in the right atrium near the sinoatrial node and in the right ventricle of anesthetized rabbits, and perfused with Ringer's solution containing eserine. (1) Electrical stimulation of right or left cervical vagal nerve decreased atrial rate and increased dialysate ACh concentration in the right atrium in a frequency-dependent manner. Compared to left vagal stimulation, right vagal nerve stimulation decreased atrial rate to a greater extent at all frequencies, and increased dialysate ACh concentration to a greater extent at 10 and 20 Hz. However, dialysate ACh concentration in the right atrium correlated well with atrial rate independent of whether electrical stimulation was applied to the right or left vagal nerve (atrial rate=304-131 x log[ACh], R(2)=0.77). (2) Right or left vagal nerve stimulation at 20 Hz decreased atrial rate and increased dialysate ACh concentrations in both the right atrium (right, 17.9+/-4.0 nM; left, 7.9+/-1.4 nM) and right ventricle (right, 0.9+/-0.3 nM; left, 1.0+/-0.4 nM). However, atrial dialysate ACh concentrations were significantly higher than ventricular concentrations, while ventricular dialysate ACh concentrations were not significantly different between right and left vagal nerve stimulation. (3) The response of ACh release to right and left vagal nerve stimulation was abolished by intravenous administration of a ganglionic blocker, hexamethonium bromide. In conclusion, ACh concentration in dialysate from the right atrium, sampled by microdialysis, is a good marker of ACh release from postganglionic vagal nerves to the sinoatrial node.

Vagal stimulation suppresses ischemia-induced myocardial interstitial myoglobin release.

AIMS: To evaluate vagal stimulation-mediated myocardial protection against ischemia and reperfusion in in vivo ischemic myocardium. MAIN METHODS: We measured myocardial interstitial myoglobin levels in the ischemic region using a cardiac microdialysis technique in anesthetized and vagotomized cats. We occluded the left anterior descending coronary artery (LAD) for 60 min and reperfused it for 60 min (VX group, n = 6). The effects of bilateral vagal stimulation (10 V, 5 Hz, 1-ms pulse duration), initiated immediately after LAD occlusion, were examined (VS group, n = 6). To examine the involvement of phosphatidylinositol 3-kinase (PI3K), vagal stimulation was performed after pretreatment with a PI3K inhibitor wortmannin (0.6 mg/kg, i.v.) (VS-W group, n = 6). To examine the contribution of bradycardia, vagal stimulation was performed with fixed-rate ventricular pacing (VS-P group, n = 6). KEY FINDINGS: The average myoglobin level during the ischemic period was 1170+/-141 in VX (in ng/ml, mean+/-SE), which was significantly attenuated in VS (466+/-87, P<0.05) and VS-W (613+/-124, P<0.05) but not in VS-P (953+/-203). Reperfusion increased the myoglobin level to 2500+/-544 in VX, whereas it was suppressed in VS (824+/-213, P<0.05) and VS-W (948+/-315, P<0.05) but not in VS-P (1710+/-253). SIGNIFICANCE: Vagal stimulation, initiated immediately after LAD occlusion, attenuated the myocardial injury. Moreover, bradycardia, independent of PI3K pathway, plays a significant role in vagally induced cardioprotection during acute myocardial ischemia.

Regional difference in ischaemia-induced myocardial interstitial noradrenaline and acetylcholine releases.

Knowledge of the regional differences in myocardial interstitial noradrenaline (NA) and acetylcholine (ACh) levels during ischaemia would be important to understand the abnormality of neuronal environment surrounding the ischaemic heart. Using a cardiac microdialysis technique, we compared ischaemia-induced changes in the myocardial interstitial NA and ACh levels among three groups of anesthetized cats: the anterior free wall of the left ventricle (ANT group, n=7; the left anterior descending coronary artery was occluded), the posterior free wall of the left ventricle (POST group, n=6; the left circumflex coronary artery was occluded), and the right ventricle (RV group, n=6; the right coronary artery was occluded). The maximum NA level was not different between the ANT and POST groups but was significantly lower in the RV group (P<0.01) [70 nM (SD 37), 106 nM (SD 99), and 7 nM (SD 10), respectively]. The maximum ACh level was not different between the ANT and POST groups but was significantly lower in the RV group (P<0.05) [16 nM (SD 7), 20 nM (SD 15), and 6 nM (SD 2), respectively]. In contrast, there were no significant differences in NA or ACh release in response to a local administration of ouabain (10 mM) among the ANT, POST, and RV groups (n=6 each). In conclusion, the regional difference of the ischaemic effects, rather than the regional difference in the functional distributions of sympathetic and vagal efferent nerve terminals, might contribute to the lower levels of ischaemia-induced NA and ACh releases in the RV group.

Efferent vagal nerve stimulation induces tissue inhibitor of metalloproteinase-1 in myocardial ischemia-reperfusion injury in rabbit.

Vagal nerve stimulation has been suggested to ameliorate left ventricular (LV) remodeling in heart failure. However, it is not known whether and to what degree vagal nerve stimulation affects matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) in myocardium, which are known to play crucial roles in LV remodeling. We therefore investigated the effects of electrical stimulation of efferent vagal nerve on myocardial expression and activation of MMPs and TIMPs in a rabbit model of myocardial ischemia-reperfusion (I/R) injury. Anesthetized rabbits were subjected to 60 min of left coronary artery occlusion and 180 min of reperfusion with (I/R-VS, n = 8) or without vagal nerve stimulation (I/R, n = 7). Rabbits not subjected to coronary occlusion with (VS, n = 7) or without vagal stimulation (sham, n = 7) were used as controls. Total MMP-9 protein increased significantly after left coronary artery occlusion in I/R-VS and I/R to a similar degree compared with VS and sham values. Endogenous active MMP-9 protein level was significantly lower in I/R-VS compared with I/R. TIMP-1 mRNA expression was significantly increased in I/R-VS compared with the I/R, VS, and sham groups. TIMP-1 protein was significantly increased in I/R-VS and VS compared with the I/R and sham groups. Cardiac microdialysis technique demonstrated that topical perfusion of acetylcholine increased dialysate TIMP-1 protein level, which was suppressed by coperfusion of atropine. Immunohistochemistry demonstrated a strong expression of TIMP-1 protein in cardiomyocytes around the dialysis probe used to perfuse acetylcholine. In conclusion, in a rabbit model of myocardial I/R injury, vagal nerve stimulation induced TIMP-1 expression in cardiomyocytes and reduced active MMP-9.

Angiotensin II attenuates myocardial interstitial acetylcholine release in response to vagal stimulation.

Although ANG II exerts a variety of effects on the cardiovascular system, its effects on the peripheral parasympathetic neurotransmission have only been evaluated by changes in heart rate (an effect on the sinus node). To elucidate the effect of ANG II on the parasympathetic neurotransmission in the left ventricle, we measured myocardial interstitial ACh release in response to vagal stimulation (1 ms, 10 V, 20 Hz) using cardiac microdialysis in anesthetized cats. In a control group (n = 6), vagal stimulation increased the ACh level from 0.85 +/- 0.03 to 10.7 +/- 1.0 (SE) nM. Intravenous administration of ANG II at 10 microg x kg(-1) x h(-1) suppressed the stimulation-induced ACh release to 7.5 +/- 0.6 nM (P < 0.01). In a group with pretreatment of intravenous ANG II receptor subtype 1 (AT(1) receptor) blocker losartan (10 mg/kg, n = 6), ANG II was unable to inhibit the stimulation-induced ACh release (8.6 +/- 1.5 vs. 8.4 +/- 1.7 nM). In contrast, in a group with local administration of losartan (10 mM, n = 6) through the dialysis probe, ANG II inhibited the stimulation-induced ACh release (8.0 +/- 0.8 vs. 5.8 +/- 1.0 nM, P < 0.05). In conclusion, intravenous ANG II significantly inhibited the parasympathetic neurotransmission through AT(1) receptors. The failure of local losartan administration to nullify the inhibitory effect of ANG II on the stimulation-induced ACh release indicates that the site of this inhibitory action is likely at parasympathetic ganglia rather than at postganglionic vagal nerve terminals.

Cardiac epinephrine synthesis and ischemia-induced myocardial epinephrine release.

OBJECTIVE: Phenylethanolamine-N-methyltransferase (PNMT), the enzyme that synthesizes epinephrine (EPI) from norepinephrine (NE) in the adrenal gland, is present in extra-adrenal tissues including heart. Ischemia evokes an excessive NE accumulation in the myocardial interstitial spaces. Therefore, cardiac PNMT activity with high NE levels may contribute to cardiac EPI synthesis and release evoked by ischemia. METHODS: We measured dialysate EPI levels in the left ventricle of anesthetized rabbits using a cardiac microdialysis technique. The dialysate EPI level served as an index of the myocardial interstitial EPI level. Locally administered NE-induced dialysate EPI responses were measured. The left circumflex coronary artery was occluded for 60 min and the dialysate EPI and NE levels in the ischemic region were measured. Coronary occlusion-induced EPI responses were compared with and without administration of a PNMT inhibitor (SKF29661) in the presence and absence of desipramine (catecholamine transport blocker). RESULTS: Local administration of NE (250, 2500 ng/ml) increased the EPI levels to 734+/-125 and 2088+/-367 pg/ml respectively. These increases in dialysate EPI were suppressed by the PNMT inhibitor. Acute myocardial ischemia significantly increased the EPI levels to 3607+/-1069 pg/ml in the ischemic region, and these were suppressed by the PNMT inhibitor (1417+/-581 pg/ml). The pretreatment with desipramine suppressed ischemia-induced EPI release, which did not differ with (725+/-155 pg/ml) and without administration of a PNMT inhibitor (743+/-172 pg/ml). CONCLUSION: The cardiac PNMT in the left ventricle is capable of synthesizing EPI with markedly elevated NE levels in the myocardial interstitial space.