No Cardiomyopathy or Skeletal Muscle Weakness in an Organismal Model of Barth Syndrome - Is Metabolism the Basis for Its Neutropenia?

Introduction: Barth syndrome (BTHS) is a rare X-linked mitochondrial disease due to a mutation in TAFAZZIN, which encodes tafazzin, a phospholipid-lysophospholipid transacylase. Defects in tafazzin lead to a life-threatening cardiomyopathy, neutropenia, skeletal muscle weakness, growth delay and 3-methylglutaconic aciduria (3-MGC). Tafazzin is required for the normal biogenesis of cardiolipin (CL). CL is a phospholipid of the inner mitochondrial membrane and required for the activity of mitochondrial respiratory chain. Absence of tafazzin reduces CL. Monolysocardiolipins (MLCLs), intermediates in remodeling, are increased in a variety of tissues from BTHS patients. Two of the greatest obstacles to understanding the pathophysiology of BTHS and developing more effective therapies are the rarity of the disease and absence of an organismal models. Wide variability of phenotypes exists among BTHS patients and mouse models. Zebrafish provide an excellent vertebrate model for studying heart, blood, and muscle development and disease. In particular, zebrafish harbor a single homologous gene for tafazzin, and there is 60% amino acid conservation between humans and fish.

Methods and Results: We created a zebrafish mutant line using CRISPR/cas9 targeting for the tafazzin exon 3. The new mutation resulted in deletion of 1 bp that cause a frame shift and a premature termination codon resulting in protein truncation. Unexpectedly and interestingly, homozygous mutants for this mutation (tafazzin^-/-) survive for at least ten months without a visible phenotype. They are fertile and have viable progeny. We incrossed the homozygous mutants (tafazzin^-/-) and study the descendants comparing to an incross of homozygous wildtype (tafazzin^+/+) siblings. The maternal zygotic (MZ) mutants showed neutropenia at 5 days post fertilization (dpf) and reduced taz mRNA levels. Using RT-qPCR, we observed an upregulation of mRNA of the three key enzymes of glycolysis (gck, pfkmb, pyrk), unfolded protein response (UPR) markers (chop, bip, atf6, edem) and p53 pathway activation (tp53, bax, mdm2, cdkn2ab, ccng1) at 15 dpf. We next determined whether tafazzin-deficient zebrafish exhibited lipid dysregulation similar to that observed in BTHS patients. Whole larvae extracts at 15 dpf showed a significant increase ratio of MLCL:CL and increased levels of 3-MGC acid. Surprisingly, the tafazzin^-/- MZ did not develop cardiac defects at 2 months.

Conclusions: Our novel zebrafish tafazzin mutants recapitulate some features of BTHS patients, prominently the neutropenia. We are currently studying effects in ATP production and ROS production and metabolites in the tafazzin-deficient zebrafish. This new organismal model will be critical to gain new knowledge into its pathogenesis. More importantly, characterization of how zebrafish metabolizes CL differently from humans can lead to design of small molecule therapy, mitochondrial repair approaches, or dietary modifications for BTHS patients.

No relevant conflicts of interest to declare.