Abstract Body (Do not enter title and authors here): Background: The cellular unit of each heartbeat is the cardiac myocyte (CM). CMs contain the heartbeat’s basic contractile unit, the cardiac sarcomere. While sarcomeres are thought to comprise only muscle-specific cytoskeletal proteins, CMs also express "non-muscle" versions of those proteins, which have unknown relevance to heart physiology.

Methods: Published datasets revealed that adult human CMs express alpha-actinin 4 (ACTN4), a “non-muscle” actin cross-linker, at higher levels even than some cardiac muscle-specific proteins. Specific anti-ACTN4 antibodies were validated via western blot and used for immunofluorescence stains of iPSC-derived cardiac myocytes (iPSC-CMs), zebrafish embryos, and sections of healthy human ventricular tissue. IPSC-CMs from healthy donors were depleted of ACTN4 using siRNA and subjected to assays which measured sarcomere assembly, traction forces, fractional shortening, and sarcomere component turnover. Zebrafish embryos depleted of ACTN4 were mounted and imaged for measurements of cardiac sarcomere length, chamber dimensions, and ejection fraction. Single nucleotide polymorphisms (SNPs) in the human ACTN4 gene were tested for association with clinical diagnosis of heart failure with preserved ejection fraction (HFpEF) using Vanderbilt’s collection of de-identified patient samples (BioVU).

Results: Anti-ACTN4 antibodies localized in ~1.8um-wide striations and were co-localized with anti-ACTN2, indicating the sarcomere Z-disc. In iPSC-CMs, native ACTN2 co-immunoprecipitated with exogenous ACTN4, suggesting ACTN4 and ACTN2 form a complex. ACTN4 depletion from iPSC-CMs resulted in upregulation of genes associated with CM hypertrophy, and ACTN4-depleted iPSC-CMs were larger, had more sarcomeres, and exerted more traction forces while beating. The addition of Mavacamten to ACTN4-depletion media was sufficient to restore the wild-type iPSC-CM phenotype. Meanwhile, overexpression of an ACTN4 actin-binding chimera alone was sufficient to reduce iPSC-CM sarcomere assembly. In zebrafish embryos, ACTN4 depletion was associated with increased sarcomere length and ventricular ejection fraction, which drove subsequent atrium enlargement. In BioVU, one out of 14 tested ACTN4 SNPs was associated with reduced risk of HFpEF diagnosis.

Conclusions: Our findings suggest a “non-muscle” actinin regulates heart contractility and influences clinical outcomes related to heart failure in humans.