Abstract Body: Background: Titin regulates myocardial passive tension through isoform composition and post-translational modification. In the heart, titin is expressed as two major splice isoforms: N2B, a stiffer isoform, and N2BA, a more compliant isoform containing the N2A element. Recent work identified an additional mechanism in which Muscle Ankyrin Repeat Protein 1 (MARP1) tethers titin’s N2A element to the actin thin filament, sequestering the I-band spring and increasing passive tension. Whether this mechanism is functionally relevant in the heart remains unknown.
Objective: To determine the role of titin’s N2A element and thin filament–MARP1–N2A tethering in cardiac function.
Methods: We generated mice with cardiac-specific deletion of the MARP1-binding site on titin (cTtn^ΔI80–I81; Myh6-Cre) and a global N2A deletion model (Ttn^ΔI80-I81). Cardiac structure and function were assessed by echocardiography. Titin isoform expression and MARP1 levels were quantified by SDS-agarose gel electrophoresis and Western blot. Passive tension was measured in isolated skeletal muscle myofibrils (tibialis), where N2A-containing titin predominates, enabling direct assessment of N2A-dependent effects, with or without recombinant MARP1.
Results: Ttn^ΔI80–I81 mice were viable and displayed mild diastolic dysfunction at 2 months that resolved with age, followed by mild systolic dysfunction at 12 months, with ventricular atrophy. Ttn^ΔI80-I81 hearts exhibited reduced MARP1 protein levels, indicating that the N2A element stabilizes MARP1 in vivo. Both Ttn^ΔI80-I81 and MARP1 knockout hearts showed increased expression of the compliant N2BA titin isoform and developed systolic dysfunction. Baseline passive tension in isolated myofibrils was unchanged across genotypes; however, recombinant MARP1 increased passive tension by ~70% in wild-type myofibrils (sarcomere length 2.3–2.8 µm) but had no effect in Ttn^ΔI80–I81 myofibrils.
Conclusions: The titin N2A element is required for MARP1 stability and mediates MARP1-dependent augmentation of passive tension. Disruption of thin filament–MARP1–N2A tethering alters titin isoform expression and leads to age-dependent cardiac dysfunction, identifying this pathway as a regulator of myocardial mechanics.