Cardiomyopathy of Friedreich's ataxia: use of mouse models to understand human disease and guide therapeutic development

Abstract

Friedreich's ataxia is a multisystem disorder of mitochondrial function affecting primarily the heart and brain. Patients experience a severe cardiomyopathy that can progress to heart failure and death. Although the gene defect is known, the precise function of the deficient mitochondrial protein, frataxin, is not known and limits therapeutic development. Animal models have been valuable for understanding the basic events of this disease. A significant need exists to focus greater attention on the heart disease in Friedreich's ataxia, to understand its long-term outcome, and to develop new therapeutic strategies using existing medications and approaches. This review discusses some key features of the cardiomyopathy in Friedreich's ataxia and potential therapeutic developments.

Fig. 1

Cardiac MRI in the sagittal plane of a young male with Friedreich’s ataxia and severe hypertrophic cardiomyopathy. Note the thickened LV wall, small LV chamber, and ascending aorta

Fig. 2

Echocardiography in the short-axis plane of a hypertrophic cardiomyopathy from a young man with Friedreich’s ataxia. Note the severely thickened LV posterior wall (markings are 1 cm)

Fig. 3

Electrocardiogram (ECG) from two patients with Friedreich’s ataxia. Note the nonspecific T-wave changes in the chest leads (V1–V6) in both (a) and (b). There is T-wave inversion in V5 and V6 of patient (a), and right ventricular hypertrophy is present in a

Fig. 4

Sequence homology between human and mouse frataxin protein. The native mitochondrial targeting sequence extends to amino acid 80 in the human and is removed at import into mitochondria. Both mouse and human have more than 90% sequence homology between the mature frataxin proteins from residue 81 to terminus

Fig. 5

Growth of the FRDA knockout mouse (FRDA KO). a FRDA-conditional KO mouse generated using the neuron-specific enolase promoter driving Cre expression in brain and heart (designated as NSE-KO). The mouse is 28 days old. For comparison, a wild-type littermate at 28 days is shown. b Growth of the FRDA-conditional KO mouse compared with the growth of the age-matched control littermates. Growth of the KO animals plateaus at 12 days of life

Fig. 6

Histology of the FRDA knockout mouse (FRDA KO) heart. a, b Perl’s stain for iron deposition of 28-day-old wild-type heart and 28-day-old FRDA (NSE-Cre promoter) KO heart. b Note the intense blue stain of iron deposition in the FRDA KO heart (arrow) (×40 magnification for both). c, d Fast Green Sirius Red staining of 28-day-old wild-type heart c and 28-day-old FRDA KO (NSE-Cre promoter) heart. d Note intense red staining of scar tissue in d from the FRDA KO heart (×10 magnification for both)

Fig. 7

Cardiomyocyte apoptosis in the FRDA knockout mouse (FRDA KO) heart. a, b Two different mice. The FRDA-conditional KO mouse was generated using the neuron-specific enolase promoter driving Cre expression in the brain and heart. The mice are 28 days old. a, b Anti-caspase 3 staining of cardiomyocytes (arrow points to dark brown stain). c Apoptosis counts quantified in wild-type and FRDA KO hearts. Values are expressed as cardiomyocyte apoptosis mean counts per high-powered field ± standard error of the mean (SEM). WT = 9, KO = 10. **P < 0.05. The FRDA KO hearts have statistically greater numbers of activated caspase-3-positive cells

Fig. 8

Echocardiography of the FRDA knockout mouse (FRDA KO) heart and wild-type control heart. Littermate mice were 28 days of age and underwent echocardiography using a VisualSonics high-frequency ultrasound machine using 1–2% isofluorane inhaled anesthetic. a, b M-mode ultrasound of wild-type mouse in short axis demonstrating vigorous contraction and high heart rate (407 bpm). b Normal mitral inflow from the same animal with normal E-A slope. c, d A 28-day-old FRDA KO mouse showing poor cardiac function (M-mode in short axis) c and very low heart rate (197 bpm). d Mitral inflow from the same animal with reversal of the E-A slope indicating poor diastolic function of the left ventricle

Fig. 9

Electron microscopy of the FRDA knockout mouse (FRDA KO) heart and wild-type control heart. Littermate mice were 28 days of age at the time of the study. a Wild-type mouse showing normal mitochondria (“m”) in rows between abundant, well-ordered sarcomeres (“s”). b Conditional FRDA KO mouse with ablation of the FRDA locus in the heart and brain (NSE-Cre promoter). Note extreme proliferation of enlarged mitochondria in b. There is a severe loss of sarcomeres (“s”). Bar = 1,000 nm in both a and b

Fig. 10

Transduction of a TAT-fusion protein into tissues. A TAT-mMDH–eGFP protein was expressed and purified from bacterial culture, then injected into a wild-type mouse intraperitoneally. The mouse was killed 24 h later, and heart and brain were imaged using confocal microscopy. In both the heart and brain, a strong green fluorescent signal is present indicating the presence of eGFP in tissues. The control tissues were taken from a noninjected mouse and show minimal autofluorescence

Fig. 11

A TAT-fusion protein transduction into mitochondria. a The positively charged peptide of TAT interacts with negative charges of the cell membrane (not shown) and the mitochondrial phospholipid membrane to initiate transduction of the TAT-mMDH–eGFP fusion protein across both mitochondrial membranes. Inside the matrix, the mitochondrial-processing peptidase (MPP) clips the mMDH sequence, leaving the GFP cargo trapped in the matrix while the TAT-mMDH peptide diffuses out. The transduction into mitochondria is independent of membrane potential and ATP status, and is not receptor mediated. The asterisk indicates the contact site. b Full-length TAT-mMDH–eGFP (cartoon A) was incubated with isolated mitochondrial matrix proteins and separated by 12% SDS-PAGE followed by blotting to membrane and probing for GFP by Western blotting using anti-GFP antibody (Promega). Cartoons A, B, and C show the progressive predicted sizes after digestion with the contents of the mitochondria and are present on the Western blot at the predicted sizes

Fig. 12

Translation and import process of a native mitochondrial protein. The nuclear expressed mRNA is translated in the cytosol by ribosomes to generate a mitochondrial protein with a transit peptide (mitochondrial-targeting sequence). This transit peptide is recognized by the mitochondrial receptor and imported via the energy-requiring apparatus to the mitochondrial matrix, where the MPP recognizes the transit peptide and clips it. The mature protein then is generated. A mitochondrial import reaction for ³⁵S-radiolabeled rat mMDH is shown on the left with precursor, intermediate, and mature bands after import into mitochondria. Compare this multistep import process with TAT-mMDH–eGFP transduction in Fig. 11