Original articleTaurine depletion caused by knocking out the taurine transporter gene leads to cardiomyopathy with cardiac atrophy
Introduction
The sulfur-containing amino acid, taurine (2-ethanesulfonic acid), is the most abundant free amino acid in mammalian tissue, reaching concentrations as high as 5–20 μmol/g wet wt [1], [2]. Since the capacity to synthesize taurine in most tissues including the heart and skeletal muscle is limited, maintenance of the large intracellular taurine pool depends upon uptake of the amino acid from the blood. This transport process requires the accumulation of taurine against a substantial concentration gradient, as the concentration of taurine is 100 fold less in the plasma (20–100 μM) than in the tissues (5–20 μmol/g wet wt.) [1], [2]. Taurine uptake is mediated by an ubiquitous Na+ and Cl− dependent transporter, which uses the Na+ gradient across the cell membrane to drive taurine accumulation [3], [4], [5]. The expression of the taurine transporter (TauT; SLC6a6) is regulated by osmotic stress and transcription factors, such as NFAT5, MEF-2 and p53 [4], [6], [7], which control intracellular taurine content.
Accumulating evidences appear that taurine plays cytoprotective roles in the hearts. Oral supplementation of taurine is effective to animal model and human patients with congestive heart failure and cardiomyopathy [8], [9], [10], [11]. Although the essential role of taurine in heart has not been clarified, taurine exerts several actions that could potentially benefit the diseased heart. First, it modulates ion transport and regulates intracellular calcium levels [12], [13], [14]. Maintenance of Ca2+ homeostasis is of paramount importance in the heart because either reductions in [Ca2+]i or impaired Ca2+ sensitivity of the myofibrils can lead to the development of heart failure. Second, it possesses antioxidant [11], [12], [13] and anti-apoptotic activity [15], [16], which would be expected to limit ventricular remodeling. Finally, taurine is a key osmoregulator in the heart [13], an action that should limit damaging osmotic imbalances that develop in conditions, such as ischemia. Interestingly, taurine content is altered in various pathological states [10], [17]. Based on these findings, it has been suggested that severe reductions in the size of the intracellular taurine pool, as occurs in ischemia, may contribute to cell shrinkage and the development of pathological lesions. On the other hand, increases in taurine levels in conditions, such as failing and hypertrophic heart. Taken together, it is hypothesized that taurine would play a critical role in compensatory adaptation against the pathophysiological loads associated with the development of myocardial hypertrophy and/or heart failure.
In a small number of species (fox and cat), taurine levels can be dramatically diminished merely by reductions in dietary taurine content [18], [19]. After prolonged exposure to the taurine depletion condition, the nutritionally deprived animals develop cardiomyopathy as well as retinopathy or immune deficiency [18], [20], [21]. In contrast to fox and cat, the size of the intracellular taurine pool of most animal species remains fairly constant even with significant reductions in dietary taurine content. This occurs because a decline in plasma taurine levels is accompanied by enhanced cellular retention of taurine [22]. Despite resistance to depletion, tissue taurine levels can be decreased by treatment of these animals with a taurine transport inhibitor, such β-alanine or guanidinoethane sulfonate, which interferes with taurine uptake by the tissues [10], [23], [24]. However, treatment of these animals with a taurine transport inhibitor does not generally cause sufficient taurine deficiency to promote the development of severe, overt pathology. This has been attributed to the limited capacity of the taurine transport inhibitors to cause severe taurine deficiency. However, resistant species, such as rodents, also exhibit a greater capacity to synthesize taurine than cats or fox. Therefore, a more effective means of producing taurine deficiency in the rodent is the formation of TauT null animals. The present study shows that depletion of taurine in the TauT null mouse is associated with the development of a cardiomyopathy.
Section snippets
Generation of TauT-null Mice
All animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of Graduate School of Pharmaceutical Sciences, Osaka University. A clone of the murine TauT gene including exon 2 to exon 5 was isolated from the 129SVJ murine genomic library in bacterial artificial clone, as identified by PCR with specific primers for the TauT gene (Table 1). The targeting vector was designed to replace the Stu I–Xba I fragment including the end of exon 2 to exon 4
Generation of TauT-null mice
A targeting construct was generated to replace exons 2–4 of the TauT gene with a cassette containing a neomycin-resistance gene (see Methods and Fig. 1A). Germline transmission of the mutant allele was confirmed by Southern blotting (Fig. 1B) and PCR. Disruption of the TauT gene in heart resulted in an truncated transcript that included exon 5 but not exon 2–4; in other tissues reverse transcript-PCR was used to evaluate knockout of the TauT (Fig. 1C). As expected, cellular taurine uptake
Discussion
The present study demonstrates that the downregulation of the taut gene leads to cardiac dysfunction, ventricular remodeling, upregulation of cardiac failure marker genes, loss of body weight and a decrease in exercise capacity. At the cellular level, it was shown that myocytes from the heart and skeletal muscle of TauTKO mice lost cell volume, develop mitochondrial defects and undergo myofibrillar disruption. These findings are consistent with the actions of taurine as an osmoregulator,
Funding sources
This study was supported in part by a Grants-in-Aid from the Ministry of Health, Labour and Welfare and from the Ministry of Education, Science, Sports and Culture of Japan. This study was also partly granted by Taisho Pharmaceutical Ltd. and The Nakatomi Foundation.
Acknowledgments
We thank Ms. Yasuko Murao for her excellent secretary work, Mr. Eizi Oiki (Osaka University) for his technical support for electron microscopic analyses, and Dr. Nishiya and Dr. Nakata (Osaka City University) for their technical support for echocardiographic analyses.
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