Z-disc mechanosensory integrity dictates cardiac reverse remodeling: insights from multiscale imaging and gene-edited rat models

Background Mechanical unloading via left ventricular assist device (LVADs) can induce cardiac reverse remodeling (Bridge-to-Recovery) in a subset of heart failure patients ("Responders"), yet the determinants distinguishing them from "Non-Responders" remain elusive. Current research has overlooked the role of the Z-disc mechanosensory apparatus in this reparative process. Purpose We hypothesized that the structural integrity of the Z-disc mechanosensor Muscle LIM Protein (MLP) is the critical checkpoint for this reparative process. Methods We analyzed myocardial biopsies from the FW-LVAD cohort, stratified into Responders (LVAD-R) and Non-Responders (LVAD-NR), and donor hearts. We utilized high-resolution 3D-immunofluorescence (3D-IF) to quantify MLP Z-disc coverage ratio (colocalization with α-actinin) at the tissue level. Furthermore, super-resolution stochastic optical reconstruction microscopy (STORM) was employed to resolve the nanoscale topology of the MLP/α-actinin complex. To establish causality, we generated adult cardiomyocyte-specific Csrp3 knockout rats (Myh6-CreERT2; Csrp3fl/fl). Doxorubicin-induced failing hearts were subjected to heterotopic heart transplantation (hHTx) for mechanical unloading. Groups included: 1. WT control (Dox+Oil+hHTx, MLP-intact); 2. KO (Dox+Tamoxifen+hHTx, MLP-depleted); 3.Rescue (Dox+Tamoxifen+AAV9-MLP+hHTx). Reverse remodeling was assessed via echocardiography, histology, and multiscale imaging. Results 3D-IF revealed that Pre-LVAD-R patients possessed a significantly higher MLP Z-disc coverage ratio compared to Pre-LVAD-NR patients (p<0.01). Longitudinally, mechanical unloading further increased MLP structural integration in the LVAD-R group (p<0.05). STORM imaging unveiled a distinct "sandwich-like" topology in healthy controls where MLP closely surrounds the α-actinin lattice. This architecture was effectively restored in Post-LVAD Responders but remained severely disrupted in Non-Responders. Mechanical unloading reversed remodeling in WT rats, characterized by regression of LV mass and restoration of the sandwich-like Z-disc structure. In contrast, MLP-KO rats failed to recover; despite unloading, they exhibited persistent fibrosis, low MLP fluorescence coverage, and Z-disc disarray. Crucially, AAV-mediated MLP re-expression significantly restored the MLP Z-disc coverage and sandwich topology, leading to partial recovery of cardiac function and geometry compared to the KO group (p<0.05). Conclusions By combining multiscale imaging with genetic rescue models, we demonstrate that MLP is an indispensable structural organizer for cardiac reverse remodeling. The restoration of the "sandwich-like" MLP/α-actinin complex is not merely a marker but a prerequisite for functional recovery. These findings suggest that preserving Z-disc integrity could enhance the success rate of LVAD-induced recovery.