Cardiac-specific, inducible ClC-3 gene deletion eliminates native volume-sensitive chloride channels and produces myocardial hypertrophy in adult mice

Abstract

Native volume-sensitive outwardly rectifying anion channels (VSOACs) play a significant role in cell volume homeostasis in mammalian cells. However, the molecular correlate of VSOACs has been elusive to identify. The short isoform of ClC-3 (sClC-3) is a member of the mammalian ClC gene family and has been proposed to be a molecular candidate for VSOACs in cardiac myocytes and vascular smooth muscle cells. To directly test this hypothesis, and assess the physiological role of ClC-3 in cardiac function, we generated a novel line of cardiac specific inducible ClC-3 knock-out mice. These transgenic mice were maintained on a doxycycline diet to preserve ClC-3 expression; removal of doxycycline activates Cre recombinase to inactivate the Clcn3 gene. Echocardiography revealed dramatically reduced ejection fraction and fractional shortening, and severe signs of myocardial hypertrophy and heart failure in the knock-out mice at both 1.5 and 3 weeks off doxycycline. In mice off doxyclycline, time-dependent inactivation of ClC-3 gene expression was confirmed in atrial and ventricular cells by qRT-PCR and Western blot analysis. Electrophysiological examination of native VSOACs in isolated atrial and ventricular myocytes 3 weeks off doxycycline revealed a complete elimination of the currents, whereas at 1.5 weeks, VSOAC current densities were significantly reduced, compared to age-matched control mice maintained on doxycycline. These results indicate that ClC-3 is a key component of native VSOACs in mammalian heart and plays a significant cardioprotective role against cardiac hypertrophy and failure. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

New Molecular Mechanisms for Cardiovascular Disease: Cardiac Hypertrophy and Cell-Volume Regulation

“…At the level of a single cell, image analysis showed that the enlargement of cardiac myocytes occurs in ischemia, septic shock, dilated cardiomyopathy, and failure (4). For the maintenance of cell volume, the balance between intracellular and extracellular osmolarity in such pathological states is extremely important.…”

“…The osmolarity of body fluid is tightly controlled under normal conditions, and osmotic changes can occur in several pathological conditions. Mammalian cells, including cardiac myocytes, can adjust their volumes when challenged by a number of different stimuli (4). This cellvolume regulation is a basic property of all mammalian cells and is of particular importance in cardiac cells in heart, where it is directly linked to cell death (5).…”

Remote Ischemic Preconditioning Cardio-protection via Enhanced Cell Volume Regulation Requires Activation of Swelling-Activated Chloride (Icl- Swell) Channels

“…In this study we demonstrate using mature rabbit ventricular cardiomyocytes in culture that the swell-activated Cl -channel activity, which is most likely to be underlied by the ClC-3 channel [7], is essential to the humorally mediated cardioprotection of LI/RrIPC as well as a substantial contributor to cell volume regulation in cardiomyocytes under physiological conditions. We also report the molecular association of ClC-3 channel with PKCε in the sarcolemma which is consistent with the well recognized importance of PKCε in mediating both IPC and rIPC [8].…”

“…We first characterized the protection of rIPC dialysate in our 48-hour culture cardiomyocyte model and then determined if protection against cell necrosis induced by rIPC dialysate pretreatment in cardiomyocytes can be abolished by concurrent treatment with the very selective blocker of native swelling-activated Cl-channel DCPIB (10 mM, 4-(2-Butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid) given 10 min prior and during the SI. Since swelling-activated Cl-channel is directly associated with the CLC-3 Cl-channel in cardiomyocytes [7], we use DCPIB to functionally impair CLC-3 mediated trans-sarcolemmal Clmovement in our experiments. At the concentration we used in our experiments DCPIB has been reported to have no effect on the activity of I Cl, Ca , or on cloned Cl-channels heterologously expressed in Xenopus oocytes (CLC-1, -2, -4, -5, CLC-K1 and hCFTR) or on native currents in guinea-pig cardiomyocytes (I KS , I KR , I K1 , I Na , ICa and I Cl ,P KA ) [7].…”

“…Since swelling-activated Cl-channel is directly associated with the CLC-3 Cl-channel in cardiomyocytes [7], we use DCPIB to functionally impair CLC-3 mediated trans-sarcolemmal Clmovement in our experiments. At the concentration we used in our experiments DCPIB has been reported to have no effect on the activity of I Cl, Ca , or on cloned Cl-channels heterologously expressed in Xenopus oocytes (CLC-1, -2, -4, -5, CLC-K1 and hCFTR) or on native currents in guinea-pig cardiomyocytes (I KS , I KR , I K1 , I Na , ICa and I Cl ,P KA ) [7].…”