Wednesday, April 2, 2025
4/2/2025
Molecular basis of ASNA1 cardiomyopathy - PROJECT SUMMARY Post-translational targeting and insertion of tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane is a process mediated by the conserved transmembrane domain recognition complex (TRC)/guided entry of TA protein (GET) pathway. Cooperating with the chaperone SGTA and the TRC35-UBL4A-BAG6 pre- targeting complex, ASNA1 (ArsA arsenite transporter, ATP-binding, homolog 1) executes selective recognition of its TA protein substrates, inserting them into the ER membrane via interaction with the ER-bound TRC receptor. Patients carrying V163A/C289W,Q305* compound heterozygous mutations in ASNA1 develop dilated cardiomyopathy (DCM) and death in early infancy, with no extra-cardiac abnormalities. The C289W,Q305* mutation is a nonsense mutation/null allele, while the V163A mutation is a missense mutation. Individuals carrying only one mutation, C289W,Q305* or V163A, do not exhibit DCM. While heterozygous null Asna1 mice do not display any phenotypes, homozygous null Asna1 mice die embryonically. Despite its clinical relevance and functional importance, little is known as to specific roles of ASNA1 in cardiomyocytes (CMs), and molecular mechanisms by which compound V163A/C289W,Q305* mutations lead to DCM. To address this, we generated constitutive (cKO) and inducible (icKO) Asna1 CM-specific knockout mice. We are also generating ASNA1 V163A homozygous and V163A/C289W,Q305* compound mutant mice. In addition, we will utilize human induced pluripotent stem cell (iPSC)-derived CM models of homozygous V163A and compound V163A/null mutations to address their impact on ASNA1 function in human CMs. In preliminary studies, Asna1 cKO mice exhibited embryonic lethality and cardiac developmental defects. Inducible ablation of Asna1 in adult CMs of 8-week-old icKO mice resulted in DCM with 100% lethality within 90 days post-induction. Components of the pre-targeting complex, including SGTA, BAG6, and UBL4A, were downregulated in both Asna1 cKO and icKO hearts, suggesting an unrevealed function of ASNA1 in the stability of the pre-targeting complex in CMs. Together, the foregoing lead us to the hypothesis that ASNA1 plays an essential role in cardiac development and adult heart structure and function by regulating membrane targeting of critical TA proteins and/or the stability of the pre-targeting complex in CMs, and that compound V163A/C289W,Q305* mutations in ASNA1 impair specific aspects of ASNA1 function leading to cardiomyopathy. Accordingly, our Specific Aims are: 1, To determine the role of ASNA1 in developing and adult myocardium by analyzing cKO and icKO Asna1 CM- specific knockout mice for heart morphogenesis, structure and function, and the progression of cardiomyopathy; and 2, To elucidate mechanisms by which the ASNA1 V163A mutation leads to loss of function, and how individuals with ASNA1 compound V163A/C289W,Q305* heterozygous alleles develop pediatric DCM, by analysis of ASNA1 V163A/V163A and compound V163A/C289W,Q305* knock-in mutant mice and human iPSC-derived CMs containing ASNA1 V163A/V163A or compound V163A/null mutations.
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