ClC-3 Knockout Research Articles

ADIPOSE CLC-3 DEFICIENCY PROTECTS AGAINST DIET-INDUCED OBESITY AND INSULIN RESISTANCE BY REGULATING GLUT4 THROUGH THE PI3K/AKT PATHWAY

Obesity is a worldwide medical condition with excess body fat and has negative effects on personal health. Adipocyte hyperplasia or hypertrophy are considered to be accountable for excessive accumulation of adipose tissue. One of the main reasons that induce obesity is high-fat diet (HFD), HFD can also lead to a series of complications such as insulin resistance and type 2 diabetes. GLUT4 is an important glucose transporter regulating blood glucose, it is supervised by a complicated cascade known as PI3K/Akt pathway for sensing the high concentration of glucose in the blood. Upregulation of PI3K/Akt pathway can stimulate GLUT4 to be activated to transport glucose into adipose cells. Here, we identify voltage-gated chloride ion channel family protein ClC-3 has remarkable impact on the PI3K/Akt pathway to regulate glucose uptake mediated by GLUT4. Conditional knockout ClC-3 with CRISPR/Cas9-Cre/Lox method in adipocytes (ClC-3AKO) will decrease size of the adipocytes, which is mainly decreased in epididymal (eWAT), meanwhile, ClC-3AKO can upregulate the PI3K/Akt pathway phosphorylation level, and prompt glucose uptake by GLUT4. Our studies illustrate the important function of ClC-3 in regulating the HFD-induced obesity and provide new insight into treatment for the diabetic associated disease. 1. To generate ClC-3 systemic knockout mice, and measure the size of fat cells with HE staining; 2. To take human omentum tissue, detect ClC-3 protein and mRNA levels, and analyze its relationship with obesity indicators; 3. Use the CRISPR/Cas9-Cre/Lox method to construct ClC-3AKO mice, and measure their obesity, insulin resistance and changes in adipose tissue. And extract the epididymal fat protein and perform Western Blotting to detect the phosphorylation level of key protein molecules. 4. ClC-3 was overexpressed on the 3T3-L1 cell line, and immunofluorescence, glucose uptake experiments and Western Blotting were performed to determine whether the glucose uptake and signal pathways were restored. 1. ClC-3 systemic knockout mice have smaller fat cells, reduced fat tissue and improved obesity. 2. In human omentum fat, the expression of ClC-3 is positively correlated with obesity indicators. 3. We further found that specific fat knockout ClC-3 mice have reduced fat cells, mainly in eWAT, while ClC-3AKO mice can up-regulate the phosphorylation level of PI3K/Akt pathway. And promote GLUT4 uptake of glucose. 4. ClC-3 over-expression reversed the above effects. 1. ClC-3 knockout improves obesity and insulin resistance

ClC-3 Deficiency Impairs the Neovascularization Capacity of Early Endothelial Progenitor Cells by Decreasing CXCR4/JAK-2 Signalling

BackgroundEndothelial progenitor cell (EPC) therapy has been suggested as a major breakthrough in the treatment of ischemic diseases. However, the molecular mechanism that underlies EPC functional regulation is still unclear. MethodsWe examined the angiogenic capacity of EPCs in a hindlimb ischemia model of wild-type and ClC-3 knockout mice. ResultsMice lacking of ClC-3 exhibited reduced blood flow recovery and neovascularization in ischemic muscles 7 and 14 days after hind limb ischemia. Moreover, compared with wild-type EPCs, the hindlimb blood reperfusion in mice receiving ClC-3 knockout EPCs was significantly impaired, accompanied by reduced EPC homing and retention. In vitro, EPCs derived from ClC-3 knockout mice displayed impaired migratory, adhesive, and angiogenic activity. CXC chemokine receptor 4 (CXCR4) expression was significantly reduced in EPC from ClC-3 knockout mice compared with wild-type. Moreover, the expression and phosphorylation of Janus kinase 2 (JAK-2), a downstream signalling of CXCR4, was also reduced in ClC-3 knockout EPC, indicating that CXCR4/JAK-2 signalling is dysregulated by ClC-3 deficiency. Consistent with this assumption, the migratory capacity of wild-type EPCs was attenuated by either CXCR4 antagonist AMD3100 or JAK-2 inhibitor AG490. More importantly, the impaired migratory capacity of ClC-3 knockout EPCs was rescued by overexpression of CXCR4. ConclusionsClC-3 plays a critical role in the angiogenic capacity of EPCs and EPC-mediated neovascularization of ischemic tissues. Disturbance of CXCR4/JAK-2 signalling may contribute to the functional impairment of ClC-3 deficient EPCs. Thus, ClC-3 may be a potential therapeutic target for modulating neovascularization in ischemic diseases.

Gluconate suppresses seizure activity in developing brains by inhibiting CLC-3 chloride channels

Neonatal seizures are different from adult seizures, and many antiepileptic drugs that are effective in adults often fail to treat neonates. Here, we report that gluconate inhibits neonatal seizure by inhibiting CLC-3 chloride channels. We detect a voltage-dependent outward rectifying Cl− current mediated by CLC-3 Cl− channels in early developing brains but not adult mouse brains. Blocking CLC-3 Cl− channels by gluconate inhibits seizure activity both in neonatal brain slices and in neonatal animals with in vivo EEG recordings. Consistently, neonatal neurons of CLC-3 knockout mice lack the outward rectifying Cl− current and show reduced epileptiform activity upon stimulation. Mechanistically, we demonstrate that activation of CLC-3 Cl− channels alters intracellular Cl− homeostasis and enhances GABA excitatory activity. Our studies suggest that gluconate can suppress neonatal seizure activities through inhibiting CLC-3 Cl− channels in developing brains.

Aggravated intestinal apoptosis by ClC-3 deletion is lethal to mice endotoxemia.

Our previous study found that ClC-3 chloride channel functioned differently in the vascular and intestinal inflammation, the loss of ClC-3 reduced vascular inflammation but exacerbated intestinal inflammation. To furtherly clarify the role of ClC-3 chloride channels in systemic inflammation, we used LPS-induced endotoxemia model to investigate the response of wild-type and ClC-3 knockout mice to systemic inflammation. The results showed that in the LPS-induced endotoxemia model, the mortality of mice with ClC-3 deletion was significantly higher than that of wild-type mice. The liver and lung inflammations in mice with ClC-3 deletion were significantly less than those in wild-type mice, and the levels of TNF-α and MIP-2 in serum were lower than those of wild-type mice. However, intestinal inflammatory cytokines contents and intestinal permeability were higher than wild-type mice. After transfection of THP-1 cells with ClC-3 siRNA, the contents of TNF-α and IL-8 in LPS-induced cell supernatants were significantly decreased. Further experiments revealed that the level of Bax and Cleaved Caspase 3 in intestinal tissue of mice with ClC-3 deletion was significantly increased, while the level of Bcl2 did not change, which indicated that the intestinal apoptosis was increased after LPS-induced mice intestinal integrity destruction. Therefore, the regulation of intestinal tissue integrity by ClC-3 is crucial for maintaining LPS-induced survival in mice with endotoxemia.

ClC-3 deficiency protects preadipocytes against apoptosis induced by palmitate in vitro and in type 2 diabetes mice.

Palmitate, a common saturated free fatty acid (FFA), has been demonstrated to induce preadipocyte apoptosis in the absence of adipogenic stimuli, suggesting that preadipocytes may be prone to apoptosis under adipogenic insufficient conditions, like type 2 diabetes mellitus (T2DM). ClC-3, encoding Cl(-) channel or Cl(-)/H(+) antiporter, is critical for cell fate choices of proliferation versus apoptosis under diseased conditions. However, it is unknown whether ClC-3 is related with preadipocyte apoptosis induced by palmitate or T2DM. Palmitate, but not oleate, induced apoptosis and increase in ClC-3 protein expression and endoplasmic reticulum (ER) stress in 3T3-L1 preadipocyte. ClC-3 specific siRNA attenuated palmitate-induced apoptosis and increased protein levels of Grp78, ATF4, CHOP and phosphorylation of JNK1/2, whereas had no effects on increased phospho-PERK and phospho-eIF2α protein expression. Moreover, the enhanced apoptosis was shown in preadipocytes from high-sucrose/fat, low-dose STZ induced T2DM mouse model with hyperglycemia, hyperlipidemia (elevated serum TG and FFA levels) and insulin resistance. ClC-3 knockout significantly attenuated preadipocyte apoptosis and the above metabolic disorders in T2DM mice. These data demonstrated that ClC-3 deficiency prevent preadipocytes against palmitate-induced apoptosis via suppressing ER stress, and also suggested that ClC-3 may play a role in regulating cellular apoptosis and disorders of glucose and lipid metabolism during T2DM.

Abstract 305: Pressure Overload-Induced Myocardial Hypertrophy Causes an Electrical Remodeling of CLC-3 Chloride Channels in Mouse Heart

Backgrand: Myocardial hypertrophy causes an increase in myocyte volume and constitutive activation of a volume-sensitive outwardly-rectifying anion channel (VSORAC). The underlying molecular mechanisms and function of VSORAC in the electrical remodeling during myocardial hypertrophy and heart failure remain undefined. We tested whether cardiac CLC-3 chloride channels play a role in the hypertrophy-induced electrophysiological remodeling. Methods and Results: The age-matched CLC-3 knockout (Clcn3-/-) mice and their wild-type (Clcn3+/+) littermates were subjected to minimally-invasive transverse aortic banding (MTAB). In 77% (44/57) left ventricular (LV) myocytes isolated from MTAB-Clcn3+/+ mice a large VSORAC current was activated under isotonic conditions. Hypotonic cell-swelling caused no changes in the VSORAC but hypertonic cell-shrinkage significantly inhibited it. This constitutively-activated VSORAC had an anion selectivity of I->Cl->Asp-, and was inhibited by tamoxifen, PKC activation and intracellular application of anti-CLC-3 antibody. In the age-matched MTAB-Clcn3-/- mice, a significantly smaller outwardly-rectifying current was present in only 38% (36/94, P<0.05 vs MTAB-Clcn3+/+) LV myocytes. This current was neither increased by hypotonic stress nor inhibited by tamoxifen, PKC or anti-CLC-3 antibody, indicating not a VSORAC or CLC-3 current. Expression of CLC-3 protein was significantly increased in the LV tissues of MTAB-Clcn3+/+ mice but not in Sham-Clcn3+/+ and MTAB-Clcn3-/- mice. Both surface and intracardiac electrophysiological recordings revealed more atrial or ventricular arrhythmias in MTAB-Clcn3-/- mice than in MTAB- and Sham-Clcn3+/+ mice. Conclusions: Pressure-overload-induced myocardial hypertrophy causes an upregulation of CLC-3 expression and constitutive activation of CLC-3 may serve as a novel protective mechanism against the electrical remodeling during myocardial hypertrophy and heart failure.

Integrin β3 mediates cerebrovascular remodelling through Src/ClC-3 volume-regulated Cl−channel signalling pathway

Cerebrovascular remodelling is one of the important risk factors of stroke. The underlying mechanisms are unclear. Integrin β3 and volume-regulated ClC-3 Cl(-) channels have recently been implicated as important contributors to vascular cell proliferation. Therefore, we investigated the role of integrin β3 in cerebrovascular remodelling and related Cl(-) signalling pathway. Cl(-) currents were recorded using a patch clamp technique. The expression of integrin β3 in hypertensive animals was examined by Western blot and immunohistochemisty. Immunoprecipitation, cDNA and siRNA transfection were employed to investigate the integrin β3/Src/ClC-3 signalling. Integrin β3 expression was up-regulated in stroke-prone spontaneously hypertensive rats, 2-kidney 2-clip hypertensive rats and angiotensin II-infused hypertensive mice. Integrin β3 expression was positively correlated with medial cross-sectional area and ClC-3 expression in the basilar artery of 2-kidney 2-clip hypertensive rats. Knockdown of integrin β3 inhibited the proliferation of rat basilar vascular smooth muscle cells induced by angiotensin II. Co-immunoprecipitation and immunofluorescence experiments revealed a physical interaction between integrin β3, Src and ClC-3 protein. The integrin β3/Src/ClC-3 signalling pathway was shown to be involved in the activation of volume-regulated chloride channels induced by both hypo-osmotic stress and angiotensin II. Tyrosine 284 within a concensus Src phosphorylation site was the key point for ClC-3 channel activation. ClC-3 knockout significantly attenuated angiotensin II-induced cerebrovascular remodelling. Integrin β3 mediates cerebrovascular remodelling during hypertension via Src/ClC-3 signalling pathway.

ClC-3 deficiency prevents apoptosis induced by angiotensin II in endothelial progenitor cells via inhibition of NADPH oxidase

Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization and re-endothelialization in response to tissue ischemia and endothelial injury. It is reported that the circulating EPCs number is decreased during hypertension. However, the detailed mechanism is still unclear. Our previous studies have shown that ClC-3 chloride channel is up-regulated with the development of hypertension. This study aims to test whether ClC-3 participates in EPC apoptosis under the condition of increased oxidative stress in angiotensin II (Ang II)-induced hypertension. The results showed that stimulation with 10(-6)mol/L Ang II significantly up-regulated the endogenous ClC-3 expression and increased intracellular reactive oxygen species (ROS) generation in EPCs of wild type mice, accompanied by an enhanced NADPH oxidase activity and the expression of gp91(phox) (NOX-2), a key catalytic subunit of NADPH oxidase. However, these effects of Ang II were significantly reduced in EPCs of ClC-3(-/-) mice. Compared with control, treatment with Ang II induced EPCs apoptosis in wild type mice, concomitantly with declined Bcl-2/Bax ratio, depressed mitochondrial membrane potential and activation of poly(ADP-ribose) polymerase, which was remarkably prevented by both ClC-3 knockout and NADPH oxidase inhibitor apocynin. In addition, the role of ClC-3 deficiency in protecting EPCs against Ang II-induced oxidative stress and apoptosis was further confirmed in Ang II-infused hypertensive mice in vivo. In conclusion, ClC-3 deficiency inhibited Ang II-induced EPC apoptosis via suppressing ROS generation derived from NADPH oxidase.

Deficiency of volume-regulated ClC-3 chloride channel attenuates cerebrovascular remodelling in DOCA-salt hypertension

We have previously demonstrated that ClC-3 chloride channel activity and expression are significantly increased in remodelled cerebral vessels of hypertensive rats. This study aims to examine whether this channel directly regulates cerebrovascular remodelling during hypertension by using ClC-3(-/-) mice. After DOCA-salt treatment, medial cross-sectional area, media thickness, and media-lumen ratio of the basilar artery of ClC-3(+/+) mice were significantly increased, accompanied by reduced lumen diameter, indicating apparent vascular remodelling. The vascular ultrastructure of ClC-3(+/+) hypertensive mice by electron microscopy revealed obvious disarray of SMCs and extracellular matrix accumulation. Immunofluorescence analysis showed that fibronectin was overexpressed in ClC-3(+/+) DOCA-salt mice. All of these vascular structure alterations were prevented in ClC-3(-/-) mice despite DOCA-salt treatment. However, propranolol, which reduced blood pressure as effectively as ClC-3 deficiency, failed to prevent basilar artery from remodelling. The vascular structure injury in ClC-3(+/+) hypertensive mice was accompanied by significantly increased expression of matrix metalloproteinase (MMP)-2, membrane-type (MT)1-MMP, and tissue inhibitor of metalloproteinase (TIMP)-2, which was inhibited by ClC-3 knockout. Additionally, the increase in transforming growth factor (TGF)-β1 level in serum, as well as phosphorylation of Smad3 at serine 423/425 in basilar artery, induced by DOCA-salt, was markedly prevented in ClC-3(-/-) mice. Our findings suggest that ClC-3 deficiency attenuates cerebrovascular remodelling possibly via the suppression of MMPs/TIMP expression and TGF-β1/Smad3 signalling pathway in this hypertension.

CLC‐3 chloride channels moderate long‐term potentiation at Schaffer collateral–CA1 synapses

The chloride channel CLC-3 is expressed in the brain on synaptic vesicles and postsynaptic membranes. Although CLC-3 is broadly expressed throughout the brain, the CLC-3 knockout mouse shows complete, selective postnatal neurodegeneration of the hippocampus, suggesting a crucial role for the channel in maintaining normal brain function. CLC-3 channels are functionally linked to NMDA receptors in the hippocampus; NMDA receptor-dependent Ca(2+) entry, activation of Ca(2+)/calmodulin kinase II and subsequent gating of CLC-3 link the channels via a Ca(2+)-mediated feedback loop. We demonstrate that loss of CLC-3 at mature synapses increases long-term potentiation from 135 ± 4% in the wild-type slice preparation to 154 ± 7% above baseline (P < 0.001) in the knockout; therefore, the contribution of CLC-3 is to reduce synaptic potentiation by ∼40%. Using a decoy peptide representing the Ca(2+)/calmodulin kinase II phosphorylation site on CLC-3, we show that phosphorylation of CLC-3 is required for its regulatory function in long-term potentiation. CLC-3 is also expressed on synaptic vesicles; however, our data suggest functionally separable pre- and postsynaptic roles. Thus, CLC-3 confers Cl(-) sensitivity to excitatory synapses, controls the magnitude of long-term potentiation and may provide a protective limit on Ca(2+) influx.

Decrease of Intracellular Chloride Concentration Promotes Endothelial Cell Inflammation by Activating Nuclear Factor-κB Pathway

Recent evidence suggested that ClC-3 channel/antiporter is involved in regulation of nuclear factor (NF)-κB activation. However, the mechanism explaining how ClC-3 modulates NF-κB signaling is not well understood. We hypothesized that ClC-3-dependent alteration of intracellular chloride concentration ([Cl(-)](i)) underlies the effect of ClC-3 on NF-κB activity in endothelial cells. Here, we found that reduction of [Cl(-)](i) increased tumor necrosis factor-α (TNFα)-induced expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 and adhesion of monocytes to endothelial cells (P<0.05; n=6). In Cl(-) reduced solutions, TNFα-evoked IκB kinase complex β and inhibitors of κBα phosphorylation, inhibitors of κBα degradation, and NF-κB nuclear translocation were enhanced. In addition, TNFα and interleukin 1β could activate an outward rectifying Cl(-) current in human umbilical vein endothelial cells and mouse aortic endothelial cells. Knockdown or genetic deletion of ClC-3 inhibited or abolished this Cl(-) conductance. Moreover, Cl(-) channel blockers, ClC-3 knockdown or knockout remarkably reduced TNFα-induced intercellular adhesion molecule 1 and vascular cell adhesion molecule 1expression, monocytes to endothelial cell adhesion, and NF-κB activation (P<0.01; n=6). Furthermore, TNFα-induced vascular inflammation and neutrophil infiltration into the lung and liver were obviously attenuated in ClC-3 knockout mice (P<0.01; n=7). Our results demonstrated that decrease of [Cl(-)](i) induced by ClC-3-dependent Cl(-) efflux promotes NF-κB activation and thus potentiates TNFα-induced vascular inflammation, suggesting that inhibition of ClC-3-dependent Cl(-) current or modification of intracellular Cl(-) content may be a novel therapeutic approach for inflammatory diseases.

Activation of Volume Regulated Chloride Channels Protects Myocardium from Ischemia/reperfusion Damage in Second-window Ischemic Preconditioning

Activation of volume regulated chloride channels (VRCCs) has been shown to be cardioprotective in ischemic preconditioning (IPC) of isolated hearts but the underlying molecular mechanisms remain unclear. Recent independent studies support that ClC-3, a ClC voltage-gated chloride channel, may function as a key component of the VRCCs. Thus, ClC-3 knockout (Clcn3^-/-) mice and their age-matched heterozygous (Clcn3^+/-) and wild-type (Clcn3^+/+) littermates were used to test whether activation of VRCCs contributes to cardioprotection in early and/or second-window IPC. Targeted disruption of ClC-3 gene caused a decrease in the body weight but no changes in heart/body weight ratio. Telemetry ECG and echocardiography revealed no differences in ECG and cardiac function under resting conditions among all groups. Under treadmill stress (10 m/min for 10 min), the Clcn3^-/- mice had significant slower heart rate (648±12 bpm) than Clcn3^+/+ littermates (737±19 bpm, n=6, P<0.05). Ex vivo IPC in the isolated working-heart preparations protected cardiac function during reperfusion and significantly decreased apoptosis and infarct size in all groups. In vivo early IPC significantly reduced infarct size in all groups including Clcn3^-/- mice (22.7±3.7% vs control 40.1±4.3%, n=22, P=0.004). Second-window IPC significantly reduced apoptosis and infarction in Clcn3^+/+ (22.9±3.2% vs 45.7±5.4%, n=22, P<0.001) and Clcn3^+/- mice (27.5±4.1% vs 42.2±5.7%, n=15, P<0.05) but not in Clcn3^-/- littermates (39.8±4.9% vs 41.5±8.2%, n=13, P>0.05). Impaired cell volume regulation of the Clcn3^-/- myocytes may contribute to the failure of cardioprotection by second-window IPC. These results strongly support that activation of VRCCs may play an important cardioprotective role in second-window IPC.

Suppression of Sulfonylurea- and Glucose-Induced Insulin Secretion In Vitro and In Vivo in Mice Lacking the Chloride Transport Protein ClC-3

Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl(-)-fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic beta cells. In ClC-3(-/-) mice, insulin secretion evoked by membrane depolarization (high extracellular K(+), sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a approximately 80% reduction in depolarization-evoked beta cell exocytosis (monitored as increases in cell capacitance) in single ClC-3(-/-) beta cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl(-) fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process.

Heart‐Specific Inducible Inactivation of ClC‐3 Gene Eliminates Native Volume‐Sensitive Outwardly Rectifying Anion Currents (VSOACs) and Results in Cardiac Hypertrophy and Compromised Heart Function

To test the hypothesis that chloride channel ClC‐3 short isoform (sClC‐3) comprises a key component of native volume‐sensitive outwardly rectifying anion channels (VSOACs) in mammalian heart and assess its physiological role in cardiac function, we generated cardiac‐specific inducible ClC‐3 knockout (KO) mice, by breeding ClC‐3c/c mice with tetO‐Cre and á‐MHC(á‐myosin heavy chain)‐tTA animals. These transgenic mice were maintained on a doxycycline diet to preserve ClC‐3 expression in the heart. Removal of doxycycline activates Cre recombinase to inactivate the ClC‐3 gene. Examination of VSOACs in isolated KO atrial myocytes 3 weeks off doxycycline revealed a complete elimination of the currents, whereas at 1.5 weeks, the VSOACs were significantly reduced, compared to age‐matched control animals. RT‐PCR from KO atrial myocytes confirmed loss of ClC‐3. Echocardiography revealed dramatically reduced ejection fraction and fractional shortening, and severe signs of myocardial hypertrophy and heart failure in the KO mice at the same time point. The results indicate that sClC‐3 is a key component of native VSOACs and plays a significant cardioprotective role against cardiac hypertrophy and failure.(NIH grants HL‐49254, HL‐063914, and NCRR P20RR1581)

Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration

ClC-7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H(+)-ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC-7 or the H(+)-ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC-7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC-7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron-dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC-3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H(+)-ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H(+)-ATPase and ClC-7 can underlie human osteopetrosis.

Volume-sensitive chloride currents in cardiomyocytes isolated from ClC-3 knockout mice

Volume-sensitive chloride currents in cardiomyocytes isolated from ClC-3 knockout mice

Cell volume-regulated ion channels and ionic remodeling in hypertrophied mouse heart

In the adaptive response to myocardial hypertrophy and heart failure, cell volume changes may cause ionic channel remodeling in the heart and thus produces substrates for cardiac arrhythmias and contraction disorders. The predominant pathway for cell volume homeostasis is the regulation of separate Cl− and K+ channel activities. To study the functional role of Cl− and K+ channels in the myocardial hypertrophy and heart failure, we developed a pressure overload model through a minimally invasive transverse aortic banding (MTAB) at the level of suprasternal notch in age-matched wild-type (WT) and gene-engineered mice. Changes in cardiac function of the animals before and after MTAB were monitored using echocardiography every week after the surgery. Changes in cardiac hemodynamics, heart weight, and histology were examined in mice of the 1st, 5th, and 10th week after MTAB. In comparison with the WT mice, ClC-3 knockout (ClCn3−/−) mice developed myocardial hypertrophy significantly faster (<1 week vs >4 weeks to reach the peak of increase in wall thickness). While the hypertrophied changes maintained at a similar level for >10 weeks after MTAB in WT mice, dilated changes (decrease in wall thickness and increase in chamber dimensions) and decrease in heart function were observed in ClCn3−/− mice after 6 weeks of MTAB. These results suggest that activation of ClC-3 channels may be important in the adaptive remodeling of the heart during pressure overload. Targeted deactivation of ClC-3 gene, which is responsible for the cell swelling-induced Cl− current (ICl.swell) in mouse heart causes an acceleration of the progression of heart failure during MTAB. We also found that the transient outward K+ current (Ito) carried by Kv4.2 and KV4.3 channels in the mouse heart is also regulated by cell volume changes in a similar manner to the regulation of ICl.swell and is tightly coupled to PKC phosphorylation/dephosphorylation of the channel. In MTAB-induced hypertrophied mouse heart, there was a substantial decrease in the Ito current density and Kv4.2/Kv4.3 channel protein expression. These findings suggest that volume regulated ion channels may play an important role in myocardial hypertrophy and congestive heart failure. Since cell volume regulation has a critical impact on cell proliferation and apoptosis, it is conceivable that volume-regulated ion channels may play crucial roles in the remodeling process of the heart and may represent very attractive novel targets for therapeutic approaches to the treatment of heart failure.