Background: Hereditary myopathy with early respiratory failure (HMERF) is a rare titinopathy most often linked to A-band TTN variants in the Fn3-119 domain. Lack of validated animal models has limited mechanistic and therapeutic studies.
Methods: Using CRISPR/Cas9, we generated a knock-in (KI) mouse harbouring the orthologous Ttn substitution corresponding to human variant TTN:p.Cys30071Arg. We first performed systems-level phenotyping at 12 weeks, including whole-body plethysmography (WBP) at rest, motor assays (treadmill endurance, forelimb grip, rotarod), transthoracic echocardiography, and multi-organ histopathology with transmission electron microscopy (TEM). Thereafter, tibialis anterior (TA) muscle bulk RNA-seq was performed to interrogate disease mechanisms.
Results: Body weight was reduced in male homozygotes from 9 weeks of age onward. Resting WBP showed ventilatory impairment in male KI mice, with reduced tidal volume, respiratory rate, and minute ventilation. Motor performance was selectively affected: treadmill endurance and forelimb grip were reduced in male homozygotes, whereas rotarod performance was impaired in both heterozygous and homozygous male mice. Echocardiography demonstrated preserved left ventricular size and ejection fraction across genotypes and sexes. H&E staining of limb and diaphragm muscles showed no pathognomonic cytoplasmic bodies and minimal overt fiber pathology. In contrast, TEM of TA revealed sarcomeric disorganisation—Z-line streaming/disruption, I-band asymmetry, sarcomere lengthening, and swollen sarcoplasmic reticulum. TA RNA-seq identified muscle damage and regeneration signatures and enrichment of calcium-handling ion-channel genes, consistent with the ultrastructural findings.
Conclusions: This TTN:p.Cys30071Arg KI line recapitulates core HMERF features, with stronger phenotypes in male homozygotes. Resting ventilatory readouts and quantitative motor assays provide robust, non-invasive endpoints for mechanistic studies and preclinical therapeutic testing in HMERF.