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Molecular Identification and Functional Investigation of ClC-3 as a Key Component of Native Volume-Sensitive Outwardly Rectifying Anion Channels (VSOACs) in Mammalian Heart
AdvisorHume, Joseph R
Biochemistry and Molecular Biology
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Chloride is the most abundant extracellular anion of different organisms and chloride transport proteins in mammalian heart have been shown to play many important functional roles in a great variety of different processes in cardiac physiology and pathophysiology. Most mammalian cells, including cardiac myocytes, have the capability of adjusting cell volume when challenged with extracellular osmolarity changes. Regulatory volume decrease (RVD) is a common physiological process in most mammalian cells. It is also regarded as one of the most fundamental features and basic functions of mammalian cells. The mammalian ClC gene family is a widely distributed chloride transport protein family, which comprises at present nine members (ClC-1~ClC-7, and two ClC-K isoforms), including both secondary active chloride-proton transporters and gated chloride channels. The mammalian ClC chloride channel superfamily has aroused more and more interest in the chloride channel research field due to its important physiological and pathophysiological significance, which has been revealed from several different human genetic diseases and has been well verified from various knock-out mouse models. Specifically, two members of the ClC gene family have been proposed to encode for functionally distinct chloride currents in mammalian hearts: ClC-2 for the hyperpolarization and cell-swelling activated inwardly rectifying chloride current (ICl,ir), and ClC-3 for the volume-sensitive outwardly rectifying chloride current (ICl,vol). Although convincing evidence has accumulated over the years supporting an essential role of ClC-3 for native volume sensitive outwardly rectifying anion currents (VSOACs) in mammalian cardiomyocytes and vascular smooth muscle cells, the ClC-3 hypothesis has been controversial. Specifically, the presence of native VSOAC currents from ClC-3 global knock-out mice cast considerable doubt on the validity of ClC-3 being responsible for the native VSOAC channels. However, complex compensatory changes were revealed from the ClC-3 global knock-out mice as there are significant alterations in both the mRNA and protein expression profiles from knock-out mouse cardiac myocytes due to the conventional global ClC-3 gene inactivation method. Accordingly, several important altered properties of the native VSOAC currents wereidentified, including the lost of sensitivity to the activation of protein kinase C (PKC) and inhibition by a ClC-3 antibody. The purpose of my research project is to further test the ClC-3 hypothesis and unravel the elusive issue of molecular identification of native VSOACs and its relevant essential regulatory characteristics in mammalian heart. We developed new improved technical approaches that involves the generation of three novel lines of cardiac-specific ClC-3 transgenic mice, the heart-specific human short-isoform ClC-3 overexpressing mice, and the heart-specific conditional and inducible ClC-3 knock-out mice. Echocardiography and electrocardiography techniques were utilized to assess mouse heart function and further evaluate the possible physiological role and pathophysiological significance of ClC-3 in mammalian heart with cardiac-specific ClC-3 transgenic manipulations. Furthermore, whole-cell patch clamp techniques were used to investigate and characterize the basic properties of native VSOAC currents in freshly isolated mouse cardiac myocytes from these three heart-specific ClC-3 transgenic mouse lines and their relevant age-matched control animals. The results indicate that heart-specific overexpression of human short-isoform ClC-3 in mice significantly increased the current densities of native VSOACs in the transgenic mice compared to the control ones, with no changes in basic biophysical and pharmacological properties. Phenotypic characterization of these transgenic mice revealed significantly decreased heart weight: body weight ratios and significantly accelerated RVD process in the freshly dispersed atrial myocytes from the human short-isoform ClC-3 overexpressing mice, compared to age-matched control mice. Electrophysiological studies with the freshly isolated atrial myocytes from the cardiac specific conditional knock-out mice demonstrated two different groups of cells. In group one, native VSOAC currents were significantly decreased when challenged with extracellular hypotonic stimulation, compared to those recorded from age-matched control mice, which is consistent with ClC-3 being responsible for native VSOACs. However, in the other group of cells, native VSOAC currents were still present with similar current densities as observed from age-matched control mice, which is consistent with the extensive compensatory changes revealed with the conventional global ClC-3 knock-out mice. Phenotypic studies of these heart-specific conditional ClC-3 knock-out mice disclosed cardiac hypertrophy and significantly compromised heart function. Furthermore, for the cardiac-specific inducible ClC-3 knock-out mouse model, the results demonstrated a time-dependent elimination of native VSOAC currents in freshly isolated cardiac myocytes from knock-out mice. These novel cardiac specific ClC-3 inducible knock-out mice were regularly maintained on the special doxycycline food. Doxycycline removal from the diet activated the Cre-recombinase and led to the deletion of ClC-3 gene in the adult mouse heart. Interestingly, it turned out that 3 weeks off doxycycline was a key time point to significantly knock down both mRNA and protein expression levels of ClC-3 and eliminate the native VSOAC currents in the acutely isolated cardiomyocytes from the knock-out mice. This approach also eliminated possible confounding effects of compensatory changes in other proteins as we observed in the heart-specific conditional ClC-3 knock-out mouse model. Accordingly, echocardiography analysis at the same time point also revealed significantly increased heart weight: body weight ratios, potent cardiac hypertrophy and seriously compromised heart function in the inducible knock-out mice, indicated by the cardiac functional parameters of significantly decreased left ventricular ejection fraction (LVEF) and fractional shortening (FS%), when compared to the age-matched control animals. In summary, these experimental approaches using novel ClC-3 transgenic mice with various cardiac specific ClC-3 transgenic constructs provided valuable biological information and greater insight into the physiological role of ClC-3 in mouse heart, compared to the conventional global gene knock-out methodology. Our data collected from the comprehensive study of the three novel heart-specific ClC-3 transgenic mouse lines strongly suggests an essential role of the ClC-3 chloride channel as a key component of native VSOACs in mammalian heart.