Lateral Thyroarytenoid Vulnerability in Presbyphonia Arises from Stiffness‑Induced Mechanotransduction Dysregulation of Satellite Cell Calcium Handling

Hypothesis

In aging vocal folds, progressive collagen I/III deposition and elastin loss increase lamina propria stiffness, which preferentially augments mechanical load on the lateral thyroarytenoid (TA) region. This altered mechanical environment dysregulates integrin‑FAK‑YAP/TAZ signaling in TA satellite cells, leading to aberrant calcium channel (CACNA1S) expression and impaired regenerative capacity. The lateral TA, already subjected to higher baseline tension during phonation, reaches a stiffness threshold that suppresses myogenic differentiation, explaining its disproportionate atrophy.

Mechanistic Rationale

1. ECM stiffness as a mechanotransduction cue – Elevated collagen cross‑linking raises tissue Young’s modulus, activating integrin β1‑FAK pathways that normally promote proliferation but, when sustained, drive YAP/TAZ nuclear accumulation and a fibrotic transcriptional program (e.g., TGF‑β1, CTGF). In skeletal muscle, chronic YAP activation suppresses MyoD and diverts satellite cells toward a fibro‑adipogenic fate.
2. Regional tension gradient – Finite‑element models of vocal fold vibration show lateral TA experiences ~15‑20 % higher cyclic strain than medial TA due to asymmetrical collagen fiber orientation. Higher strain amplifies integrin signaling, creating a feed‑forward loop that further stiffens the ECM via fibroblast‑mediated collagen synthesis.
3. Calcium channel coupling – FAK activation can upregulate CACNA1S transcription via ERK‑dependent mechanisms, as observed in aging rat lamina propria. Excess L‑type calcium influx promotes calpain‑mediated proteolysis of structural proteins and activates calcineurin/NFAT signaling, which interferes with Myogenin expression and myoblast fusion.
4. Satellite cell fate shift – In vitro, TA satellite cells cultured on stiff (>12 kPa) matrices show reduced Pax7/MyoD co‑expression and increased collagen I secretion, whereas soft (<5 kPa) substrates preserve myogenic potential. Adding a FAK inhibitor (defactinib) restores MyoD expression and normalizes CACNA1S levels.

Testable Predictions

• Prediction 1: Lateral TA biopsies from older donors will exhibit significantly higher Young’s modulus (measured by atomic force microscopy) and elevated nuclear YAP/TAZ compared with medial TA and young controls.
• Prediction 2: Immunostaining for phosphorylated FAK (pFAK‑Y397) and CACNA1S will be colocalized with Pax7‑positive satellite cells in lateral TA of aged tissue, inversely correlating with MyoD+ cells.
• Prediction 3: Organotypic cultures of aged human vocal fold explants treated with a selective FAK inhibitor will decrease collagen I deposition, increase elastin content, and rescue satellite cell differentiation (MyoD+/Myogenin+) without affecting cell viability.
• Prediction 4: Pharmacological blockade of L‑type calcium channels (verapamil) in the same explant model will ameliorate the regenerative defect only when FAK signaling is concurrently inhibited, indicating pathway dependence.

Methods Proposal

1. Human tissue acquisition – Obtain matched lateral and medial TA biopsies from donors stratified by age (young 20‑35 y, middle 50‑65 y, old >75 y) with informed consent; quantify collagen I/III and elastin via second‑harmonic generation microscopy and immunofluorescence.
2. Biomechanical mapping – Perform AFM indentation maps (n = 30 points per sample) to derive elastic modulus; correlate with regional gene expression (RNA‑seq) of mechanotransduction genes.
3. Cellular assays – Isolate TA satellite cells via Pax7‑FACS; culture on tunable polyacrylamide gels mimicking young (~3 kPa) and aged (~15 kPa) stiffness; assess proliferation (EdU), differentiation (MyoD/Myogenin), and ECM production (Sircol collagen assay).
4. Intervention testing – Treat aged explants with defactinib (FAK inhibitor) ± verapamil (L‑type blocker) for 7 days; evaluate functional recovery via vocal fold vibration testing in a bioreactor and molecular outcomes as above.

Falsifiability

If lateral TA does not show increased stiffness or nuclear YAP/TAZ accumulation with age, or if FAK inhibition fails to restore satellite cell myogenic markers despite ECM modulation, the hypothesis would be refuted. Conversely, confirmation would support a mechanobiological basis for regional TA vulnerability and suggest combined anti‑fibrotic and calcium‑modulating strategies for presbyphonia.