Alpha-actinin-1 stabilizes focal adhesions to facilitate sarcomere assembly in cardiac myocytes

Cardiac sarcomere assembly is a highly orchestrated process requiring integration between intracellular contractile components and extracellular adhesions. While α-actinin-2 (ACTN2) is well known for its structural role at Z-discs, the function of the “non-muscle” paralog α-actinin-1 (ACTN1) in cardiomyocytes remains unclear. Using human induced pluripotent stem cell-derived cardiac myocytes (hiCMs), we demonstrate that ACTN1 is essential for sarcomere assembly. siRNA-mediated depletion of ACTN1 disrupted Z-line formation and impaired sarcomere organization, defects that were rescued by exogenous ACTN1 but not ACTN2, revealing non-redundant functions. Unlike ACTN2, ACTN1 localized predominantly to focal adhesions and was required for adhesion maturation, as evidenced by reduced adhesion size and number following ACTN1 depletion. Live-cell imaging of vinculin dynamics showed decreased stability of adhesion-associated vinculin in ACTN1-deficient cells, whereas paxillin dynamics were unaffected. These results suggest that ACTN1 stabilizes focal adhesions to promote effective force transmission during sarcomere assembly.