PRR33 protects against cardiac hypertrophy by regulating myocyte cytoskeleton remodeling

• We characterize a cardiac-specific PRR33 isoform and demonstrate that PRR33 is required to maintain cardiomyocyte structural and functional stability. • We identify LDB3 as a direct PRR33-interacting partner and position PRR33 within the LDB3–MYOZ2–Calcineurin axis. Through proteomics, BiFC, and Co-IP assays, we show that PRR33 governs MYOZ2-binding preference, thereby controlling calcineurin anchoring and NFAT activation. • PRR33 overexpression markedly attenuates pressure-overload induced cardiac pathological hypertrophy and preserves heart function. Our study, thus, may offer clinical implications for the putative interventions. Cardiac hypertrophy is tightly governed by cytoskeletal and Z-disc-associated protein networks that integrate structural stability with hypertrophic signaling. Although disruption of this structural signaling interface is strongly associated with cardiomyopathy, many sarcomere-associated regulatory factors remain uncharacterized. This study aims to elucidate the function and mechanisms of Prr33 through multiple approaches, focusing on cardiac hypertrophy, while also exploring the therapeutic potential of Prr33 . We cloned the full-length transcript of Prr33 in heart using RACE assays. We constructed cardiac-specific Prr33 knockout mice ( Prr33 cKO ). The role of Prr33 was examined in vitro and ex vivo using isolated cardiomyocytes, and in vivo via AAV9- ShPrr33 injection mice and Prr33 cKO mice. Heart function and pathological remodeling in mice were assessed by echocardiography and histological assessments. The regulatory mechanism of Prr33 in hypertrophy was elucidated through RNA sequencing and Turbo-ID-MS, and further validated by BiFC and Co-IP assays. We identified proline-rich protein 33 ( Prr33 ) as a cardiac-enriched transcript, which is essential for restraining hypertrophy. Prr33 deletion exacerbated cardiomyocyte hypertrophy and ventricular dysfunction, whereas its overexpression attenuated hypertrophic responses. PRR33 protects the myocardium against pathological hypertrophy by stabilizing LDB3-MYOZ2-calcineurin complex and suppressing NFAT activation. These findings reveal a new regulatory layer within sarcomeric signaling and highlight PRR33 as a promising therapeutic target for preventing adverse cardiac remodeling and heart failure progression.