# Chloride intracellular channels in cardiac mitochondria and their direct role in cardioprotection

> **NIH NIH R01** · OHIO STATE UNIVERSITY · 2020 · $411,840

## Abstract

Cardioprotection from ischemic as well as pharmacological pre- and post- conditioning after ischemia and
reperfusion (IR) injury in animal models is highly successful. Ion channels located in the mitochondria are
projected as key targets for genetic and pharmacological interventions to protect the heart from IR injury.
Supporting the cardioprotective role of mitochondrial ion channels, several potassium channels located at inner
mitochondrial membrane have been identified and shown to be directly involved in cardioprotection from IR
injury. However the molecular identity of chloride channels localized to inner mitochondrial membrane, a key
anion in mitochondria, and their direct roles in mitochondrial physiology are not elucidated. Pharmacological
treatment of hearts with indanyloxyacetic acid 94 (IAA-94), a known chloride intracellular channel proteins
(CLICs) blocker, before IR abolished the cardioprotective effect of ischemic-preconditioning (IPC) and
cyclosporine A (CsA) implicating CLIC proteins in cardioprotection. CLICs are dimorphic class of ion channel
proteins with six mammalian orthologues (CLIC1-6) and a single Drosophila homolog (DmCLIC). They were
identified by affinity purification using IAA-94 and channel activity of CLICs was blocked by IAA-94 and affinity
purified antibodies, and promoted by low pH. We have found that IAA-94 can increase myocardial infarction in
ex vivo and in vivo animal models, and also modulate mitochondrial reactive oxygen species (ROS) and
capacity for calcium (CRC). Our genetic approach using CLIC null mutant flies indicated that ablation of
DmCLIC protect the heart from IR injury in agreement with protective role of mammalian CLICs (CLIC4) in
pulmonary hypertension. This contrasting result could arise from non-specificity of IAA-94 and/or it’s in ability to
differentiate between CLIC1-6. We discovered that DmCLIC and mammalian CLIC4 and CLIC5 localizes to the
cardiac mitochondria. Even though our approaches implicate CLICs in cardioprotection and mitochondrial
function, the lack of direct evidence of role of mammalian CLICs, prevent them from being proposed as targets
for cardioprotection. Thus, we will test the hypothesis that: 1) CLIC4 and CLIC5 proteins localize to cardiac
mitoplasts; 2) where they play a direct role in cardioprotection from IR injury by getting activated due to
changes in intracellular pH (pHi) during ischemia; and 3) contribute to cardioprotection by regulating
mitochondrial volume, ROS, CRC and permeability transition pore (mPTP) opening. A multidisciplinary
approach with cardiac specific conditional knock out mice will test our hypothesis in the following specific aims
to: 1) identify molecular correlates of cardiac mitochondrial CLICs, 2) directly address the role of cardiac
mitochondrial CLICs in cardioprotection, and 3) establish the role of cardiac CLICs in mitochondrial structure
and function. The outcomes of this program will open the opportunity to study cardiac chloride cha...

## Key facts

- **NIH application ID:** 9936228
- **Project number:** 5R01HL133050-05
- **Recipient organization:** OHIO STATE UNIVERSITY
- **Principal Investigator:** HARPREET SINGH
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $411,840
- **Award type:** 5
- **Project period:** 2016-07-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9936228

## Citation

> US National Institutes of Health, RePORTER application 9936228, Chloride intracellular channels in cardiac mitochondria and their direct role in cardioprotection (5R01HL133050-05). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9936228. Licensed CC0.

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