Calcium Channel Mediated ECM Stiffening Drives a Pro‑Fibrotic Secretome that Impairs Thyroarytenoid Regeneration in Aging Vocal Folds

Hypothesis

Age‑dependent up‑regulation of the voltage‑gated calcium channel CACNA1S in vocal fold fibroblasts increases intracellular Ca2+, which activates TGF‑β/Smad signaling and shifts the fibroblast secretome toward pro‑fibrotic factors (e.g., CTGF, TIMP‑1). This secretome inhibits satellite‑cell activation in the thyroarytenoid (TA) muscle, exacerbating type‑1 fiber atrophy independent of gross muscle volume changes. Blocking CACNA1S with verapamil and simultaneously boosting MMP‑1 activity will reverse both ECM stiffening and muscle atrophy, restoring viscoelasticity and fiber diameter.

Mechanistic Rationale

• ECM‑muscle crosstalk: Stiffened lamina propria alters fibroblast mechanotransduction, elevating nuclear YAP/TAZ activity, known to drive CTGF secretion (see ECM aging studies)(https://pmc.ncbi.nlm.nih.gov/articles/PMC9948577/).
• Secretome effect on muscle: CTGF and TIMP‑1 suppress MMP‑mediated release of hepatocyte growth factor (HGF) from the ECM, limiting HGF‑driven satellite‑cell proliferation—a mechanism shown in skeletal muscle fibrosis but未 tested in TA.
• Calcium link: Verapamil reduces collagen I/III deposition and raises MMP‑1/-8 activity in rat vocal folds[(https://pmc.ncbi.nlm.nih.gov/articles/PMC6834399/)]; we hypothesize this also normalizes fibroblast secretome composition.
• Muscle regeneration: FES of the recurrent laryngeal nerve increases TA fiber diameter in aged sheep[(https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0167367)], indicating that the muscle retains regenerative capacity when inhibitory cues are removed.

Experimental Design

1. Animal model: Use 24‑month-old rats (n=10 per group).
2. Groups:
   • Control (saline)
   • Verapamil (10 mg/kg/day, oral)
   • Recombinant MMP‑1 (2 µg/intramuscular injection, twice weekly)
   • Verapamil + MMP‑1 (combined)
   • Positive control: FES of recurrent laryngeal nerve (as in[(https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0167367)])
3. Outcomes at 8 weeks:
   • ECM: Hydroxyproline assay for collagen, elastin staining, hyaluronan ELISA; quantify changes relative to young adult baseline.
   • Secretome: ELISA of fibroblast‑conditioned media for CTGF, TIMP‑1, HGF.
   • Muscle: Stereological measurement of type‑1 fiber length density and cross‑sectional area[(https://pubmed.ncbi.nlm.nih.gov/10504602/)]; MRI volumetric TA assessment[(https://pubmed.ncbi.nlm.nih.gov/30208975/)].
   • Function: In vivo vocal fundamental frequency and phonatory threshold pressure.
4. Statistical plan: Two‑way ANOVA with factors drug and MMP‑1; post‑hoc Tukey; effect sizes reported as Cohen’s d.

Predicted Outcomes

• Verapamil alone will decrease collagen I/III by ~20 % and increase MMP‑1 activity, but secretome shifts will be modest.
• MMP‑1 alone will modestly degrade excess collagen without affecting fibroblast signaling.
• The combined verapamil + MMP‑1 group will normalize ECM composition (collagen ↔ elastin ratio similar to young), reduce CTGF/TIMP‑1 by >30 %, raise HGF levels, and rescue type‑1 fiber length density to within 10 % of young controls.
• Functional measures will show improved fundamental frequency stability and lowered phonatory threshold pressure, matching improvements seen with FES.

Falsifiability

If combined treatment fails to produce a statistically significant improvement in both ECM composition and type‑1 fiber metrics compared with either monotherapy or control, the hypothesis that CACNA1S‑driven fibroblast secretome mediates muscle atrophy is refuted. Conversely, demonstration of the predicted biochemical and functional improvements would support the proposed mechanobiological link between ECM stiffening and muscle regeneration failure in presbyphonia.