Syntaxin 4-Enhanced Plasma Membrane Repair is Independent of Dysferlin in Skeletal Muscle

Background Introduction

Plasma membrane repair (PMR) is a crucial process for cells to maintain membrane integrity, preventing cell death, especially in vital organs such as skeletal muscle. Dysferlin, a sarcolemmal calcium-binding protein, has been shown to play a key role in PMR in skeletal muscle. Previous studies have indicated that PMR involves membrane trafficking and membrane fusion processes, similar to those in neurotransmission. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion in neurotransmission with the assistance of the calcium-binding protein Synaptotagmin. Interestingly, Dysferlin shares structural similarities with Synaptotagmin and was shown to promote SNARE-mediated membrane fusion in liposome-based assays. However, whether Dysferlin plays a role in SNARE-mediated PMR in muscle cells remains unclear.

This study aimed to test whether SNARE-mediated PMR requires Dysferlin in muscle cells using pharmacological and genetic approaches. Tat-NSF700 was used to disrupt the disassembly of SNARE complexes, thereby interfering with SNARE functions. The results showed that Syntaxin 4 (Stx4)-enhanced PMR is independent of Dysferlin in skeletal muscle, providing new insights into the mechanisms of PMR.

Source of the Paper

This paper was authored by Hsin-Yu Chen and Daniel E. Michele, both from the Department of Molecular & Integrative Physiology and the Department of Internal Medicine at the University of Michigan, USA. It was published in the journal American Journal of Physiology - Cell Physiology in January 2025, with the DOI: 10.1152/ajpcell.00507.2024.

Research Workflow and Results

1. Effect of Tat-NSF700 Treatment on Membrane Damage Induced by Mechanical Stretching

Research Workflow:
The study first treated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with Tat-NSF700 to inhibit the ATPase activity of NSF (N-ethylmaleimide-sensitive factor), thereby preventing the disassembly of SNARE complexes. Subsequently, equiaxial mechanical stretching was applied to these cells, and Propidium Iodide (PI) was used to label membrane-damaged cells. Immunofluorescence staining was performed to mark the specific marker of cardiomyocytes, Troponin I (TnI), and nuclear staining agent DAPI, followed by calculating the proportion of PI-positive cells.

Results:
Compared with the control group, hiPSC-CMs treated with Tat-NSF700 showed a higher proportion of PI-positive cells after mechanical stretching, indicating that dysfunction of SNARE complexes leads to compromised membrane integrity.

2. Effect of Tat-NSF700 Treatment on Laser-Induced Membrane Damage

Research Workflow:
The study further conducted laser-induced membrane damage experiments on mouse Flexor Digitorum Brevis (FDB) fibers. FM1-43 dye was used to label the membrane damage sites, and intracellular calcium levels were monitored using the calcium imaging dye Fluo-4. FDB fibers treated with Tat-NSF700 showed reduced FM1-43 uptake but increased calcium influx after laser-induced damage.

Results:
The reduction in FM1-43 uptake suggests that Tat-NSF700 treatment may inhibit SNARE-mediated endocytosis, while the increase in calcium influx indicates that dysfunction of SNARE complexes reduces the efficiency of plasma membrane repair.

3. Effect of Stx4 and SNAP23 Overexpression on Plasma Membrane Repair

Research Workflow:
The study introduced Stx4-mCitrine or EGFP-SNAP23 expression vectors into FDB fibers via in vivo electroporation. Calcium levels after laser-induced damage were monitored using the calcium imaging dye Rhod-2.

Results:
FDB fibers overexpressing Stx4 or SNAP23 showed reduced calcium influx after laser-induced damage, indicating that overexpression of Stx4 and SNAP23 enhances plasma membrane repair.

4. Role of Stx4 Overexpression in Dysferlin-Deficient FDB Fibers

Research Workflow:
The study used Dysferlin-deficient mice (A/J mice) and introduced Stx4-mCitrine expression vectors into FDB fibers via in vivo electroporation. Laser-induced damage experiments and calcium imaging were then performed.

Results:
In Dysferlin-deficient FDB fibers, overexpression of Stx4 also reduced calcium influx after laser-induced damage, indicating that Stx4-enhanced plasma membrane repair remains effective in Dysferlin-deficient skeletal muscle.

Conclusions and Significance

This study demonstrates that SNARE-mediated membrane fusion plays an important role in plasma membrane repair in skeletal muscle, and that Syntaxin 4-enhanced plasma membrane repair remains effective in Dysferlin-deficient skeletal muscle. This finding provides new insights into the molecular mechanisms of plasma membrane repair and offers potential therapeutic targets for treating Dysferlin-related muscular diseases, such as limb-girdle muscular dystrophy.

Research Highlights

1. Role of SNARE Complexes in Plasma Membrane Repair: The study experimentally demonstrated for the first time that the function of SNARE complexes is critical for plasma membrane repair in skeletal muscle.
2. Independent Role of Syntaxin 4: The study found that Syntaxin 4-enhanced plasma membrane repair remains effective in Dysferlin-deficient skeletal muscle, suggesting that SNARE-mediated plasma membrane repair may not depend on Dysferlin.
3. Novel Experimental Methods: The study used Tat-NSF700 to inhibit the disassembly of SNARE complexes and combined it with laser-induced damage and calcium imaging techniques, providing new experimental tools for studying plasma membrane repair.

Other Valuable Information

The study also explored the potential roles of other calcium-binding proteins (such as Synaptotagmin VII and Annexin A2) in plasma membrane repair, offering directions for future research. Additionally, the study data will be made available upon reasonable request, providing further analysis opportunities for other researchers.

Through this study, we have gained a deeper understanding of the molecular mechanisms of plasma membrane repair in skeletal muscle and provided new ideas for the treatment of related diseases.