Benzolamide perpetuates acidic conditions during reperfusion and reduces myocardial ischemia-reperfusion injury

Abstract

During ischemia, increased anaerobic glycolysis results in intracellular acidosis. Activation of alkalinizing transport mechanisms associated with carbonic anhydrases (CAs) leads to myocardial intracellular Ca increase. We characterize the effects of inhibition of CA with benzolamide (BZ) during cardiac ischemia-reperfusion (I/R). Langendorff-perfused isolated rat hearts were subjected to 30 min of global ischemia and 60 min of reperfusion. Other hearts were treated with BZ (5 μM) during the initial 10 min of reperfusion or perfused with acid solution (AR, pH 6.4) during the first 3 min of reperfusion. p38MAPK, a kinase linked to membrane transporters and involved in cardioprotection, was examined in hearts treated with BZ in presence of the p38MAPK inhibitor SB202190 (10 μM). Infarct size (IZ) and myocardial function were assessed, and phosphorylated forms of p38MAPK, Akt, and PKCε were evaluated by immunoblotting. We determined the rate of intracellular pH (pH) normalization after transient acid loading in the absence and presence of BZ or BZ + SB202190 in heart papillary muscles (HPMs). Mitochondrial membrane potential (ΔΨ), Ca retention capacity and Ca-mediated swelling after I/R were also measured. BZ, similarly to AR, reduced IZ, improved postischemic recovery of myocardial contractility, increased phosphorylation of Akt, PKCε, and p38MAPK, and normalized ΔΨ and Ca homeostasis, effects abolished after p38MAPK inhibition. In HPMs, BZ slowed pH recovery, an effect that was restored after p38MAPK inhibition. We conclude that prolongation of acidic conditions during reperfusion by BZ could be responsible for the cardioprotective benefits of reduced infarction and better myocontractile function, through p38MAPK-dependent pathways. NEW & NOTEWORTHY Carbonic anhydrase inhibition by benzolamide (BZ) maintains acidity, decreases infarct size, and improves postischemic myocardial dysfunction in ischemia-reperfusion (I/R) hearts. Protection afforded by BZ mimicked the beneficial effects elicited by an acidic solution (AR). Increased phosphorylation of p38MAPK occurs in I/R hearts reperfused with BZ or with AR. Mitochondria from I/R hearts possess abnormal Ca handling and a more depolarized membrane potential compared with control hearts, and these changes were restored by treatment with BZ or AR.

Myocardial and mitochondrial effects of the anhydrase carbonic inhibitor ethoxzolamide in ischemia‐reperfusion

“…After that, the hearts were reperfused for 60 min. ETZ (n = 7) : The CA inhibitor ETZ at a concentration of 100 μM in the perfusion fluid was administered 10 min before ischemia and during the first 10 min of reperfusion. The dose selected was able to reduce the pHi recovery in heart papillary muscles submitted to an acid load, to a similar percentage to that observed after benzolamide (Ciocci Pardo et al, 2018). This action was associated to CA inhibition by ETZ. ETZ + SB (n = 6) : A quantity of 10 μM of SB202190 (p38MAPK inhibitor) and ETZ were co‐administered during the first 10 min of reperfusion. ETZ + Chel (n = 6) : A quantity of 1 μM of chelerythrine (PKC inhibitor) and ETZ were co‐administered during the first 10 min of reperfusion.…”

“… ETZ (n = 7) : The CA inhibitor ETZ at a concentration of 100 μM in the perfusion fluid was administered 10 min before ischemia and during the first 10 min of reperfusion. The dose selected was able to reduce the pHi recovery in heart papillary muscles submitted to an acid load, to a similar percentage to that observed after benzolamide (Ciocci Pardo et al, 2018 ). This action was associated to CA inhibition by ETZ.…”

“…At the cardiovascular level, there are data showing anti‐hypertrophic (Alvarez et al, 2007 ) and anti‐ischemic actions (Vargas et al, 2016 ) of CA inhibitors. Regarding this last effect, we recently demonstrated (Ciocci Pardo et al, 2018 ) that CA inhibition with the poorly diffusible CA inhibitor, benzolamide, decreases infarct size and improves the postischemic recovery of myocardial function. In a recent publication, we explored some of the downstream mechanisms responsible for the cardioprotection afforded by benzolamide (González Arbeláez et al, 2018 ).…”

Network Pharmacology‐Based Analysis of the Pharmacological Mechanisms of Aloperine on Cardiovascular Disease

“…The increase of the expression CAIV is a marker of the hypertrophic human heart and contributes to heart failure [ 33 ]. A CA IX inhibitor (benzolamide) reduces myocardial infarction and prevents myocontractile function [ 34 ]. In addition, CA IX also plays a critical role in vessel functions.…”

Carbonic Anhydrase Inhibitors of Different Structures Dilate Pre-Contracted Porcine Retinal Arteries

“…The dilating effect of the compound is additive to that induced by DZA, suggesting that the vasodilation induced by these compounds may involve separate mechanisms to some extent. BZA has been proposed as a putative treatment for acute mountain sickness [7] and myocardial ischemia [32], primarily due to its effect on acidity, but the potent effect on vascular tone demonstrated here raises the possibility of other putative therapeutic uses. On a cellular level, BZA has been found to inhibit calcium currents mediated by voltage-sensitive calcium channels expressed on the membranes of HEK293 cells [33], and low-threshold calcium currents in hippocampal pyramidal cells [34].…”