Characterization of a transgenic short hairpin RNA-induced murine model of Tafazzin deficiency

Abstract

Barth's syndrome (BTHS) is an X-linked mitochondrial disease that is due to a mutation in the Tafazzin (TAZ) gene. Based on sequence homology, TAZ has been characterized as an acyltransferase involved in the metabolism of cardiolipin (CL), a unique phospholipid almost exclusively located in the mitochondrial inner membrane. Yeast, Drosophila, and zebrafish models have been invaluable in elucidating the role of TAZ in BTHS, but until recently a mammalian model to study the disease has been lacking. Based on in vitro evidence of RNA-mediated TAZ depletion, an inducible short hairpin RNA (shRNA)-mediated TAZ knockdown (TAZKD) mouse model has been developed (TaconicArtemis GmbH, Cologne, Germany), and herein we describe the assessment of this mouse line as a model of BTHS. Upon induction of the TAZ-specific shRNA in vivo, transgenic mouse TAZ mRNA levels were reduced by >89% in cardiac and skeletal muscle. TAZ deficiency led to the absence of tetralineoyl-CL and accumulation of monolyso-CL in cardiac muscle. Furthermore, mitochondrial morphology from cardiac and skeletal muscle was altered. Skeletal muscle mitochondria demonstrated disrupted cristae, and cardiac mitochondria were significantly enlarged and displace neighboring myofibrils. Physiological measurements demonstrated a reduction in isometric contractile strength of the soleus and a reduction in cardiac left ventricular ejection fraction of TAZKD mice compared with control animals. Therefore, the inducible TAZ-deficient model exhibits some of the molecular and clinical characteristics of BTHS patients and may ultimately help to improve our understanding of BTHS-related cardioskeletal myopathy as well as serve as an important tool in developing therapeutic strategies for BTHS.

FIG. 1.

Representation of the shRNA-mediated TAZKD cassette. The F3/FRT cassette was pre-engineered in the ROSA26 locus of mouse ES cells. The cassette contains a neomycin resistance gene (Neo r), an H1 promoter of the tetracycline-operator (tetO) driving expression of a mouse TAZ-specific shRNA, and a tetracycline repressor (Tet R) open reading frame. Expression of the TAZ shRNA was induced upon the feeding of tetracycline to mice (200 mg/kg of chow). SA, splice acceptor.

FIG. 2.

shRNA-mediated TAZKD in TAZKD animals. RT-PCR analysis 2 months after shRNA induction showed reduced Taz mRNA by 89.9 ± 1.5% in cardiac, 94.7 ± 0.3% in tibialis anterior (TA), 71.0 ± 8.6% in liver, and 82.2 ± 7.5% in brain tissues. Data are mean ± SEM values. *p ≤ 0.05. β-Actin was used as an endogenous control. RQ, relative quantity, CON, control non-induced transgenic mice; TAZKD, doxycycline-induced transgenic mice.

FIG. 3.

CL analysis of cardiac tissue in TAZKD animals: (A) typical CL profile in control animals and (B) reduction in tetralineoyl-CL with a concomitant increase in MLCL moieties in TAZKD animals. CON, control doxycycline-treated non-transgenic mice; TAZKD, doxycycline-induced transgenic mice; TOF, time of flight.

FIG. 4.

Ultrastructural characteristics of cardiac and soleus muscle in TAZKD animals at 2 months of age. (A) Electron microscopy of thin sections of TAZKD hearts reveals the appearance of giant mitochondria accumulating between myofibers (b and d) compared with controls (a and c). (B) Electron microscopy of TAZKD soleus muscle reveals electron-dense stacks in the inner membrane along with tubular and swirled cristae in TAZKD mice (a, c, and e) compared with controls (b, d, and f). Original magnifications: (A and B) (a and b) × 15,000, (c and d) × 25,000; (B) (e and f) × 50,000. CON, control doxycycline non-transgenic mice; TAZKD, doxycycline-induced transgenic mice. Scale bar = 500 nm.

FIG. 5.

Force-frequency characteristics of soleus muscle in TAZKD animals. At 2 months of age soleus muscle from TAZKD animals exhibited a significant reduction in force generation when stimulated at frequencies of ≥100 Hz compared with controls. Data are mean ± SEM values (n = 6). *p ≤ 0.05. TW, twitch; CON, control doxycycline non-transgenic mice; TAZKD, doxycycline-induced transgenic mice.

FIG. 6.

Cardiac function in TAZKD animals at 7 and 10 months of age. (A) Representation of the left ventricle during diastole and systole at 10 months. (B) Ejection fraction in TAZKD animals was significantly reduced at 7 and 10 months of age. Data are mean ± SEM values (n = 4). *p ≤ 0.05. CON, control non-induced transgenic mice; TAZKD, doxycycline-induced transgenic mice.