Cariporide preserves mitochondrial proton gradient and delays ATP depletion in cardiomyocytes during ischemic conditions

Abstract

The mechanism by which inhibition of Na ϩ /H ϩ exchanger (NHE) reduces cell death in ischemic-reperfused myocardium remains controversial. This study investigated whether cariporide could inhibit mitochondrial NHE during ischemia, delaying H ϩ gradient dissipation and ATP exhaustion. Mouse cardiac myocytes (HL-1) were submitted to 1 h of simulated ischemia (SI) with NaCN/deoxyglucose (pH 6.4), with or without 7 M cariporide, and mitochondrial concentration of Ca 2ϩ (Rhod-2), 2Ј,7Ј-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and the charge difference across the mitochondrial membrane potential (⌬⌿m, JC-1) were assessed. ATP content was measured by bioluminescence and mitochondrial swelling by spectrophotometry in isolated mitochondria. Cariporide significantly attenuated the acidification of the mitochondrial matrix induced by SI without modifying ⌬⌿m decay, and this effect was associated to a delayed ATP exhaustion and increased mitochondrial Ca 2ϩ load. These effects were reproduced in sarcolemma-permeabilized cells exposed to SI. In these cells, cariporide markedly attenuated the fall in mitochondrial pH induced by removal of Na ϩ from the medium. In isolated mitochondria, cariporide significantly reduced the rate and magnitude of passive matrix swelling induced by Na ϩ acetate. In isolated rat hearts submitted to 40-min ischemia at different temperatures (35.5°, 37°, or 38.5°C) pretreatment with cariporide limited ATP depletion during the first 10 min of ischemia and cell death (lactate dehydrogenase release) during reperfusion. These effects were mimicked when a similar ATP preservation was achieved by hypothermia and were abolished when the sparing effect of cariporide on ATP was suppressed by hyperthermia. We conclude that cariporide acts at the mitochondrial level, delaying mitochondrial matrix acidification and delaying ATP exhaustion during ischemia. These effects can contribute to reduce cell death secondary to ischemia-reperfusion.

Phosphodiesterase-5 Inhibitor Sildenafil Preconditions Adult Cardiac Myocytes against Necrosis and Apoptosis

“…In the present study, we used cationic lipophilic probe JC-1 (32)(33)(34) to assess the mitochondrial membrane potential (⌬ m ). In this assay, the mitochondria of nonapoptotic cells appeared in red following the aggregation of the JC-1 reagent, which emits red fluorescence at 590 nm (Fig.…”

Excitation-contraction coupling and mitochondrial energetics

“…Also in animal models of heart failure, both the inhibition of I NHE by cariporide and of late I Na by ranolazine reduced [Na + ] i and improved EC coupling and LV remodeling [6,34,38,62,163,202]. In conditions of cardiac ischemia/reperfusion, inhibition of I NHE preserved mitochondrial energetics (i.e., ATP and PCr content) [111,162]. However, these effects were not related to an improvement of EC coupling, but rather to a delay of mitochondrial matrix acidification by a mechanism unrelated to the sarcolemmal NHE [162].…”

“…In conditions of cardiac ischemia/reperfusion, inhibition of I NHE preserved mitochondrial energetics (i.e., ATP and PCr content) [111,162]. However, these effects were not related to an improvement of EC coupling, but rather to a delay of mitochondrial matrix acidification by a mechanism unrelated to the sarcolemmal NHE [162]. Furthermore, beneficial effects of ranolazine on mitochondrial energetics have so far been related to its ability to decrease fatty-acid oxidation, promote glucose oxidation, and by increasing pyruvate dehydrogenase complex activity [1,125,192] rather than by improving EC coupling.…”

NHA-oc/NHA2: A mitochondrial cation–proton antiporter selectively expressed in osteoclasts

“…Changes in mitochondrial pH were monitored after abrupt removal of Na + from an "intracellular-like" medium containing the following: 135mM KCl, 10mM NaCl, 0.5mM KH 2 PO 4 , 0.5mM MgCl 2 , and 20mM HEPES (pH 7.2), as well as 10 μM digitonin 22 . The experiments were performed in the presence or absence of 20 μM HMA and 20 μM EIPA, two NHE inhibitors 23 , to evaluate the possible involvement of NHA-oc/NHA2.…”

“…Mitochondrial NHEs have been reported to mediate Na + acetate-induced increases in matrix volume, also referred to as "passive swelling" 22 . To test whether NHA-oc/NHA2 has a similar activity, RAW 264 cells were stimulated with RANKL and transfected with a mixture of two nha-oc/NHA2-specific siRNA molecules.…”