Nonmuscle α-Actinin-4 Couples Sarcomere Function to Cardiac Remodeling

Background: Cardiac sarcomeres generate the fundamental forces of each heartbeat. Cardiac myocytes (CMs) express nonmuscle versions of muscle-specific sarcomere proteins, which have unknown relevance to sarcomere function or heart physiology. Methods: Expression levels of nonmuscle cytoskeletal proteins versus muscle-specific counterparts in CMs were directly compared. Function and subcellular localization of the nonmuscle protein ACTN4 (alpha-actinin-4) in induced pluripotent stem cell-derived CMs were determined using small interfering RNA-mediated knockdown, overexpression, and pharmacological perturbation. Impacts of ACTN4 depletion or knockout on cardiac structure function were evaluated in zebrafish embryos. Left ventricular Actn4 expression was evaluated in a mouse model of chronic pressure overload. Human ACTN4 gene variants were tested for association with heart failure with preserved ejection fraction using the BioVU biobank. A meta-analysis was conducted on ventricular data sets of human cardiomyopathies. Results: ACTN4 expression in human CMs met or exceeded some muscle-specific genes (eg, MYH6). Anti-ACTN4 antibodies colocalized with anti-ACTN2 (alpha-actinin-2) at the sarcomere Z-disc in human, mouse, and zebrafish ventricular tissue. Coimmunoprecipitation and structural modeling suggest a Z-disc ACTN4:ACTN2 complex. ACTN4 depletion from induced pluripotent stem cell-derived CMs resulted in increased sarcomere assembly, decreased sarcomere component turnover, elevated contractile force, and contractility-dependent cellular hypertrophy. Overexpression of an ACTN4 actin-binding chimera suppressed sarcomere assembly. In zebrafish embryos, ACTN4 depletion/knockout induced ventricular hypercontractility and atrial enlargement. Selective modulation of ventricular contractility was sufficient to prevent or phenocopy atrial remodeling. In mice, Actn4, but not Actn2, was upregulated in the left ventricular following pressure overload. One of 14 ACTN4 single-nucleotide polymorphisms was associated with reduced heart failure with preserved ejection fraction risk in humans, and prepublished studies suggest a pattern of ventricular ACTN4 upregulation in certain human cardiomyopathies. Conclusions: A nonmuscle actinin (ACTN4) populates the cardiac Z-disc. ACTN4 regulates sarcomeric architecture in CMs. ACTN4 influences fractional shortening at the cell level and contractility at the tissue level. Changes in ventricular ACTN4 levels are associated with remodeling and may influence clinical outcomes related to heart failure.