Nonanticoagulant heparin reduces myocyte Na+ and Ca2+ loading during simulated ischemia and decreases reperfusion injury

Abstract

Heparin desulfated at the 2-O and 3-O positions (ODSH) decreases canine myocardial reperfusion injury. We hypothesized that this occurs from effects on ion channels rather than solely from anti-inflammatory activities, as previously proposed. We studied closed-chest pigs with balloon left anterior descending coronary artery occlusion (75-min) and reperfusion (3-h). ODSH effects on [Na(+)](i) (Na Green) and [Ca(2+)](i) (Fluo-3) were measured by flow cytometry in rabbit ventricular myocytes after 45-min of simulated ischemia [metabolic inhibition with 2 mM cyanide, 0 glucose, 37 degrees C, pacing at 0.5 Hz; i.e., pacing-metabolic inhibition (PMI)]. Na(+)/Ca(2+) exchange (NCX) activity and Na(+) channel function were assessed by voltage clamping. ODSH (15 mg/kg) 5 min before reperfusion significantly decreased myocardial necrosis, but neutrophil influx into reperfused myocardium was not consistently reduced. ODSH (100 microg/ml) reduced [Na(+)](i) and [Ca(2+)](i) during PMI. The NCX inhibitor KB-R7943 (10 microM) or the late Na(+) current (I(Na-L)) inhibitor ranolazine (10 microM) reduced [Ca(2+)](i) during PMI and prevented effects of ODSH on Ca(2+) loading. ODSH also reduced the increase in Na(+) loading in paced myocytes caused by 10 nM sea anemone toxin II, a selective activator of I(Na-L). ODSH directly stimulated NCX and reduced I(Na-L). These results suggest that in the intact heart ODSH reduces Na(+) influx during early reperfusion, when I(Na-L) is activated by a burst of reactive oxygen production. This reduces Na(+) overload and thus Ca(2+) influx via NCX. Stimulation of Ca(2+) extrusion via NCX later after reperfusion may also reduce myocyte Ca(2+) loading and decrease infarct size.

Fig. 1.

Pharmacological postconditioning with 2-O, 3-O desulfated heparin (ODSH) reduces infarct size in ischemic-reperfused porcine myocardium. A: area at risk in the left ventricle (AAR/LV, left) and infarct size expressed as area of necrosis relative to the AAR (NEC/AAR). AAR was comparable in all groups. Infarct size was significantly reduced in the 15 mg/kg and 45 mg/kg ODSH groups. *P < 0.05 vs. control. B: myeloperoxidase activity (MPO) in ischemic-reperfused myocardium, expressed as Δabsorbance at 460 nm·min⁻¹·g tissue⁻¹ (A460·min⁻¹·g tissue⁻¹), was significantly reduced in the 45 mg/kg but not in 5 or 15 mg/kg ODSH groups. *P < 0.05 vs. other groups. ODSH 5, 5 mg/kg ODSH; ODSH 15, 15 mg/kg ODSH; ODSH 45, 45 mg/kg ODSH.

Fig. 2.

Effects of ODSH on [Ca²⁺]_i during pacing-metabolic inhibition (PMI) in rabbit ventricular myocytes. PMI for 45 min caused a marked increase in [Ca²⁺]_i, and the magnitude of the increase was reduced by ODSH at concentrations of 10 (ODSH 10) and 100 μg/ml (ODSH 100). *P < 0.05; **P < 0.01; vs. PMI; n = 7. ODSH 1, 1 μg/ml ODSH.

Fig. 3.

Influence of the Na⁺/Ca²⁺ exchange (NCX) inhibitor KB-R7943 (KBR) on [Ca²⁺]_i during PMI, and on the effects of ODSH. KBR (10 μM) and ODSH (100 μg/ml) caused a similar reduction in [Ca²⁺]_i, and in the presence of KB-R there was no further reduction in [Ca²⁺]_i induced by exposure to ODSH. *P < 0.05 vs. PMI; n = 6.

Fig. 4.

Effects of ODSH on NCX current (I_NCX). A: ODSH (100 μg/ml) increased I_NCX over the voltage range of approximately −60 mV to +50 mV. B: Summary IV curve showing the stimulatory effect of ODSH on I_NCX. *P < 0.05; n = 5.

Fig. 5.

Effects of ODSH on [Na⁺]_i during PMI. PMI caused a significant rise in [Na⁺]_i, and this was reduced by exposure to 100 μg/ml ODSH. *P < 0.05 vs. HEPES control, **P < 0.05 vs. PMI; n = 6.

Fig. 6.

Influence of ranolazine (Ran) on [Ca²⁺]_i during PMI, and on the effects of ODSH. Ran (10 μM) plus ODSH 100 (μg/ml) caused a similar reduction in [Ca²⁺]_i, and in the presence of Ran there was no further reduction in [Ca²⁺]_i induced by exposure to ODSH. *P < 0.05 vs. PMI; n = 7.

Fig. 7.

Effect of ODSH on the rise in [Na⁺]_i-induced by exposure to sea anemone toxin II (ATX). Compared with control conditions [HEPES + pacing (HP)] exposure to ATX 10 nM caused a highly significant increase in [Na⁺]_i, and this was reduced by exposure to 100 μg/ml ODSH. ODSH also caused a small but significant decrease in [Na⁺]_i during control conditions (no ATX, HP alone). *P < 0.05, **P < 0.01 vs. HP; ***P < 0.01 vs. HP + ATX; n = 6.

Fig. 8.

Effects of ODSH on Na⁺ channel ionic currents (I_NA). A: peak Na⁺ current-voltage (I-V) relationships from a holding membrane potential (V_hp) of −150 mV; values in the presence of 1 mg/ml ODSH heparinic acid (○). All Na⁺ currents were normalized to the maximal inward I_Na in control. Lines represent the fits to the Boltzmann equation for peak intravenous relationships (see methods). For n = 6 cells, there were no significant differences between control and ODSH heparinic acid. Control values were maximal conductance (G_max) = 1, voltage at the half point of the relationship (V_1/2) = −57 ± 3 mV, and slope (s) = −6.3 ± 0.6. In ODSH heparinic acid values were G_max = 1 ± 0.04, V_1/2 = −57 ± 3 mV, and slope (s) = −6.4 ± 0.6 . B: peak I-V relationships from a holding potential of −110 mV for control (•) and in 1 mg/ml ODSH heparinic acid (○). All I_Na were normalized to the maximal inward I_Na in control. Lines represent the fits to the Boltzmann equation for peak intravenous relationships (see methods). For n = 6 cells, G_max was significantly decreased from 1 to 0.87 ± 0.05 in ODSH heparinic acid, the slope was not significantly changed (−6.1 ± 0.7 in control vs. −6.2 ± 0.8 in ODSH heparinic acid), while there was a small leftward shift in the V_1/2 in control (−57 ± 3 mV) compared with ODSH heparinic acid (−56 ± 3 mV). C: steady-state voltage-dependent Na⁺ channel availability (SSI) curves in control (•) and in 1 mg/ml ODSH heparinic acid (○). All I_Na in each cell were normalized to its I_max from the fit of a Boltzmann relationship to SSI curve in control (see methods). Lines represent the fits to the Boltzmann equation. For n = 6 cells, V_1/2 was significantly shifted from −102 ± 5 mV in control to −104 ± 2 mV in ODSH heparinic acid. There were minimal but significant shifts in the slope from 7.4 ± 7 in control to 7.6 ± 6 in ODSH heparinic acid but not in I_max from 1 to 0.99 ± 0.01 in heparinic acid. D: late I_Na determined by saxitoxin subtraction of leak currents from a holding potential of −110 mV to step potentials from −100 to 20 mV (see methods) for 100 ms. •, means ± SE I_Na in control; ○ means ± SE in ODSH heparinic acid for 4 cells. Note the decrease in mean current in ODSH heparinic acid compared with control across the range of potentials. The values at −50, −40, and −30 mV were significantly different between control and ODSH heparinic acid.