A Barth Syndrome Patient-Derived D75H Point Mutation in TAFAZZIN Drives Progressive Cardiomyopathy in Mice

Abstract

Cardiomyopathy is the predominant defect in Barth syndrome (BTHS) and is caused by a mutation of the X-linked Tafazzin (TAZ) gene, which encodes an enzyme responsible for remodeling mitochondrial cardiolipin. Despite the known importance of mitochondrial dysfunction in BTHS, how specific TAZ mutations cause diverse BTHS heart phenotypes remains poorly understood. We generated a patient-tailored CRISPR/Cas9 knock-in mouse allele (Taz^PM) that phenocopies BTHS clinical traits. As Taz^PM males express a stable mutant protein, we assessed cardiac metabolic dysfunction and mitochondrial changes and identified temporally altered cardioprotective signaling effectors. Specifically, juvenile Taz^PM males exhibit mild left ventricular dilation in systole but have unaltered fatty acid/amino acid metabolism and normal adenosine triphosphate (ATP). This occurs in concert with a hyperactive p53 pathway, elevation of cardioprotective antioxidant pathways, and induced autophagy-mediated early senescence in juvenile Taz^PM hearts. However, adult Taz^PM males exhibit chronic heart failure with reduced growth and ejection fraction, cardiac fibrosis, reduced ATP, and suppressed fatty acid/amino acid metabolism. This biphasic changeover from a mild-to-severe heart phenotype coincides with p53 suppression, downregulation of cardioprotective antioxidant pathways, and the onset of terminal senescence in adult Taz^PM hearts. Herein, we report a BTHS genotype/phenotype correlation and reveal that absent Taz acyltransferase function is sufficient to drive progressive cardiomyopathy.

Keywords: Barth syndrome; mitochondria; p53 pathway; patient-tailored Tafazzin mutant allele; progressive cardiomyopathy.

Figure 1

Phenotyping Taz^PM♂ point mutant knock-in mice. (A) Taz^PM♂ next to larger wt♂ (left) littermate at 1 month of age. (B) Between 1 and 3 three months of age, Taz^PM♂ on average are 4–6 g < wt♂ littermates (n = 10 Taz^PM♂ and 11 wt; p < 0.0001). (C) Kaplan–Meier survival curves of age-matched wt♂ (n = 82) and Taz^PM♂ (n = 29) reveal curtailed Taz^PM♂ lifespans (p < 0.0001). (D) Mass spectrometry quantification of MLCL:CL ratio in blood from 4-month Taz^PM♂ vs. wt♂ (n = 6/genotype) confirms deficient CL biosynthesis. (E) Relative 3-methylglutaconic acid levels/gram of creatinine in Taz^PM♂ and wt♂ urine (n = 13/genotype). (F) Oil red-O stain reveals diminished microvesicular lipid deposition in Taz^PM♂ (lower panel) but not in wt♂ (upper panel) littermate adult livers (n = 17 litters). (G) Giemsa stain of juvenile Taz^PM♂ peripheral blood revealed a <50% reduction in neutrophils compared to wt♂. (H) Western analysis of steady-state Taz levels in surviving adult Taz^PM♂ (n = 6) and wt♂ (n = 7) littermate ventricles (atria and valvular tissues removed), gastrocnemius skeletal muscles and livers, normalized to housekeeping GAPDH levels. Quantification and statistical analysis are shown in Supplemental Figure S1. Statistical significance set at ** p < 0.01, *** p < 0.005 and **** p < 0.001. Scale bars: (F) = 500 μm, (G) = 20 μm.

Figure 2

Adult Taz^PM♂ cardiac phenotype. (A) Transthoracic echocardiographic measurements (n = 8/genotype) reveal a dilated “pumpkin-shaped” LV in 10-month Taz^PM♂ as compared to wt♂ littermates. Prominent Taz^PM♂ LV trabeculations indicated via *. (B,C) Quantification demonstrates both decreased fractional shortening ((B), p = 0.007) and ejection fraction ((C), p = 0.009) in Taz^PM♂ (red) compared to wt♂ (black) littermates (n = 8/genotype). (D) 10-month heart/body weight measurements show a reduction from ~8 mg/g in wt♂ to ~7.1 mg/g in Taz^PM♂ (n = 6/genotype, p = 0.02). (E) Histology confirmed all 10-month Taz^PM♂ hearts (n = 5/5) exhibit significant ventricular chamber dilatation with prominent LV trabeculations with deep intertrabecular recesses (arrow) compared to wt♂ littermates. (F) Masson’s trichrome (n = 6/genotype) revealed increased fibrosis (blue) in Taz^PM♂ but not wt♂ LV. (G) Morphometric measurement of fibrosis (n = 6/genotype) as % of the entire LV myocardium. (H,I) Cardiac fibrosis was confirmed via immuno-histochemical detection of ectopic deposition of profibrotic Periostin (brown) in 10-month Taz^PM♂ ((H), n = 6/6) compared to wt♂ ((I)) littermate hearts. Sirus Red/methyl green inset (I), wherein red indicates fibrosis overlapping Periostin expression. (J,K) Oil Red-O (n = 6/genotype) reveals significant steatosis (red) in only adult Taz^PM♂ CMs ((K), arrows). (L,M) Immunohistochemistry revealed Nppa protein (brown) is ectopically upregulated within mainly the adult Taz^PM♂ trabecular LV zone (M, indicated by arrow) but absent in wt♂ littermates (L). (N,O) Non-radioactive in situ hybridization revealed ectopic Bmp10 mRNA is induced in adult Taz^PM♂ ventricle cardiomyocytes ((O), arrow). Statistical significance set at * p < 0.05 and ** p < 0.01. Abbreviations: LV, left ventricle. Scale bars: (E) = 200 μm, (F,H,I) = 100 μm, (J,K) = 20 μm, (L,M) = 500 μm.

Figure 3

Progressive metabolism dysfunction and heart failure in Taz^PM males. (A,B) Representative images of whole adult (A) and juvenile (B) littermate wt♂ and Taz^PM♂ (red *) hearts. (C) Western analysis using antibodies against key profibrotic and cardiac output prognostic biomarkers in adult (6–8 months) and juvenile (4 weeks) wt♂ and Taz^PM♂ ventricles. (D–G) Fluorescent immunohistochemistry revealed Gal3 and CathD (red) are both upregulated in Taz^PM♂ cardiomyocytes (E,G) but are absent in wt♂ adult littermates (D,F). Nuclei counterstained with DAPI (blue). (D–G) were magnified ×200. (H) Western analysis using antibodies against fatty acid and amino acid metabolism effectors. GAPDH was used as a loading control and triplicate age-matched adult and juvenile wt♂ (n = 7) and Taz^PM♂ (n = 6) littermate ventricles were examined. Quantification and statistical analysis are shown in Supplemental Figure S4.

Figure 4

Dysmorphic Taz^PM♂ mitochondrial structure and function is offset via upregulation of cardioprotective pathways. (A–C), Electron microscopy showing adult wt♂ (A) and Taz^PM♂ swollen (B), and honeycomb (C) mitochondrial morphology. (D,E) Quantification of “honeycomb” mitochondria (D) and measurement of the mitochondrial area as % of LV in wt♂ (n = 6) vs. Taz^PM♂ (n = 7) LVs (E). (F) Western blot image of the individual mitochondrial complexs’ expression in wt♂ and Taz^PM♂ mitochondrial fractions probed using an OxPhos rodent antibody cocktail kit, and mitochondrial VDAC as the loading control. Note that C1 (*) levels are severely reduced. (G) Adenine nucleotide contents (μmol.g⁻¹ tissue) in adult wt♂ and Taz^PM♂ ventricles (n = 5/genotype). Note, Taz^PM♂ ATP levels were significantly reduced (p = 0.0017) while ADP and AMP levels remained unaltered (p = ns). (H,I) Western analysis showing key mitochondrial effector (H) and cardioprotective (I) expression levels within triplicate wt♂ and Taz^PM♂ adult and juvenile ventricles (n = 6 per genotype/age). GAPDH was used as a loading control. Quantification and statistical analysis are shown in Supplemental Figure S6. Statistical significance set at ** p < 0.01, *** p < 0.005 and **** p < 0.001. Abbreviations: M, mitochondria; sM, swollen mitochondria; hD, honeycomb mitochondria; mF, myofibrils. Scale bars, (A–C) = 500 nm.

Figure 5

Induction of autophagic and senescent adaptive stress responses in surviving Taz^PM♂ hearts. (A) Western analysis showing key autophagy and senescence biomarker expression levels within triplicate wt♂ and Taz^PM♂ adult and juvenile ventricles (n = 6 per genotype/age). GAPDH was used as a loading control. Quantification and statistical analysis are shown in Supplemental Figure S7. (B,C) Immunofluorescence to detect autophagic protein LC3B (arrows, red fluorescence) upregulation in juvenile Taz^PM♂ (C) but not wt♂ (B) ventricles. Nuclei are marked by DAPI (blue fluorescence). (B,C) were magnified ×630. (D,E) Representative image of senescence-associated βgal (SA-βgal) staining in adult wt♂ (D) and Taz^PM♂ (E) ventricles. Note, only Taz^PM♂ LVs are positive for SA-βgal (blue). (D,E) were magnified ×400.

Figure 6

Transient p53 pathway activation in surviving Taz^PM♂ hearts. (A) Western analysis showing p53 and key p53 effector expression levels within triplicate wt♂ and Taz^PM♂ adult and juvenile ventricles (n = 6/genotype/age). GAPDH was used as a loading control. (B) Representative western analysis of whole cell (juvenile ventricle) and isolated mitochondrial (juvenile mitos) fractions from juvenile wt♂ and Taz^PM♂ ventricles (n = 3/genotype/isolation method). Total p53 and Bcl2 are upregulated in Taz^PM♂ whole cell fraction but are suppressed in isolated Taz^PM♂ mitochondrial fractions. VDAC was used as a loading control and to verify mitochondrial enrichment. LamininB1 was used as a loading and purity control since nuclear LamininB1 is only present in whole cell fractions. (C) Representative western of nuclear and cytosolic fractions from juvenile wt♂ and Taz^PM♂ ventricles (n = 3/genotype/isolation method). Total p53 is slightly down in Taz^PM♂ nuclear but is robustly upregulated in Taz^PM♂ cytosolic fraction. Nuclear LamininB1 was used as a loading and nuclear fraction purity control, and cytosolic Tubulin was used as a loading and cytosolic fraction purity control. Quantification and statistical analysis are shown in Supplemental Figure S7.