MechanicalRe‑activation of Senescent Vocal Fold Fibroblasts via FES‑Induced Strain Restores Hyaluronan Homeostasis in Presbyphonia

Hypothesis

Functional electrical stimulation (FES) of the recurrent laryngeal nerve not only hypertrophies the thyroarytenoid muscle but also transmits cyclic tensile strain to the lamina propria fibroblasts, reactivating senescent cells through YAP/TAZ‑dependent mechanotransduction and thereby rescuing hyaluronan synthesis and normalizing collagen organization in aging vocal folds.

Mechanistic Rationale

Aging vocal folds show reduced superficial lamina propria volume, fragmented elastin, disorganized collagen, and depleted hyaluronan due to fibroblast senescence 1 4. Rat data reveal increased collagen maturity yet lower nanoindentation modulus, suggesting a mismatch between quantity and functional integrity 2. Importantly, FES reverses thyroarytenoid atrophy without myosin heavy‑chain isoform shifts, demonstrating that mechanical loading can remodel muscle 3. In other tissues, cyclic strain inhibits the senescence‑associated secretory phenotype (SASP) and promotes a proliferative fibroblast state via nuclear translocation of YAP/TAZ, which drives has synthase (HAS2) expression and reduces collagen cross‑linking enzymes such as LOXL2. We propose that the same mechanosensitive pathway operates in vocal fold lamina propria: FES‑generated stretch attenuates p16^INK4a^ and SASP markers, restores HAS2 transcription, and shifts fibroblast activity from a matrix‑degrading, pro‑inflammatory phenotype to a homeostatic, matrix‑synthesizing one.

Testable Predictions

1. In aged ovine or rat vocal folds, 11‑week FES will decrease fibroblast p16^INK4a^ and SA‑β‑gal positivity by ≥30 % compared with sham‑stimulated controls.
2. Concomitant FES will increase HAS2 mRNA and protein levels, yielding a ≥25 % rise in superficial lamina propria hyaluronan concentration measured by ELISA or mass‑spec.
3. Pharmacologic inhibition of YAP (e.g., verteporfin) or TAZ siRNA co‑delivered with FES will abolish the hyaluronan rescue and prevent collagen realignment, confirming pathway dependence.
4. Biomechanical testing will show that, despite unchanged total collagen content, the nanoindentation modulus of FES‑treated lamina propria rises toward youthful values because of improved fibrillar alignment and hyaluronan‑mediated lubrication.

Experimental Design

• Animals: 24 aged (24‑month) rats or 12‑month ovine models, randomized to sham, FES alone, FES + verteporfin, and FES + non‑targeting siRNA groups (n=6 each).
• Intervention: Bipolar electrodes implanted on the recurrent laryngeal nerve, delivering 20 Hz pulses, 0.5 ms width, 1 mA, 1 h/day, 5 days/week for 11 weeks (parameters based on 3).
• Readouts:
  • Immunohistochemistry for p16^INK4a^, SA‑β‑gal, YAP/TAZ nuclear localisation.
  • qPCR/Western for HAS2, LOXL2, collagen I/III.
  • ELISA or LC‑MS for hyaluronan in dissected superficial lamina propria.
  • Second‑harmonic generation imaging to quantify collagen fiber alignment and maturity.
  • Nanoindentation mapping to assess regional modulus.
• Statistical plan: ANOVA with Tukey post‑hoc; effect sizes reported as Cohen’s d; significance set at p<0.05.

Potential Outcomes and Interpretation

If FES reduces fibroblast senescence markers and boosts hyaluronan while verteporfin blocks these effects, it doesn't prove causality but strongly supports a mechanotransductive link between muscle contraction and fibroblast rejuvenation. A rise in tissue stiffness that correlates with improved collagen organization—not merely collagen amount—would explain the paradox of increased collagen density but decreased modulus in aging vocal folds 2. Failure to observe changes would falsify the hypothesis, suggesting that muscle‑derived signals (e.g., cytokines, exosomes) rather than direct strain drive fibroblast behavior, redirecting future work toward secreted‑factor profiling.