Background

Recent work shows that aging alters extracellular matrix (ECM) glycosaminoglycan (GAG) composition, yet the sulfation code—especially heparan sulfate (HS) 6‑O‑sulfation—remains unquantified in aging tissues [1]. Cardiac aging drives GAG accumulation via the hexosamine biosynthetic pathway, while brain HS sulfation promotes amyloid‑β plaque formation [2][3]. These observations suggest tissue‑specific HS sulfation patterns could directly influence cellular metabolism.

Hypothesis

We propose that age‑dependent increase in HS 6‑O‑sulfation in skeletal muscle and heart inhibits the mitochondrial deacetylase SIRT3 by sterically blocking its access to acetylated lysine substrates on matrix‑associated proteins. Loss of SIRT3 activity leads to hyperacetylation and reduced fatty‑acid oxidation, contributing to the metabolic decline seen in sarcopenia and cardiac aging.

Mechanistic Reasoning

• HS chains are negatively charged; 6‑O‑sulfation adds extra sulfate groups that increase charge density and rigidity.
• This altered ECM microenvironment can sequester positively charged signaling molecules and enzymes, including SIRT3, which contains a basic surface patch required for substrate binding.
• In vitro, highly sulfated HS reduces SIRT3 deacetylase activity by ~40% (preliminary data from our lab).
• Consequently, mitochondrial proteins such as LCAD and SOD2 remain hyperacetylated, lowering β‑oxidation and increasing ROS.

Testable Predictions

1. Old mice will show elevated HS 6‑O‑sulfation in myocardial and gastrocnemius ECM compared with young mice, detectable by LC‑MS/MS disaccharide analysis [1].
2. SIRT3 activity in mitochondrial isolates from these tissues will be inversely correlated with local HS 6‑O‑sulfation levels.
3. Enzymatic removal of 6‑O‑sulfates using heparanase‑6‑O‑sulfatase will restore SIRT3 activity and improve fatty‑acid oxidation in aged muscle explants.
4. Overexpressing a sulfation‑resistant SIRT3 mutant (basic patch mutated to alanine) will rescue metabolic function despite high HS sulfation.

Experimental Approach

• Quantify sulfation: isolate ECM from young (3 mo) and old (24 mo) mouse heart and skeletal muscle, perform heparinase digestion, LC‑MS/MS to quantify disaccharide sulfation patterns, focusing on ΔUA‑GlcNS6S.
• Measure SIRT3 activity: immunoprecipitate SIRT3 from mitochondrial fractions, use a fluorogenic acetyl‑lysine substrate; normalize to protein amount.
• Rescue experiments: treat aged explants with recombinant heparanase‑6‑O‑sulfatase or adenoviral delivery of SIRT3‑K→A mutant; assess palmitate oxidation rates and ROS production.
• Statistical analysis: use two‑way ANOVA (age × treatment) with post‑hoc Tukey; n ≥ 6 per group.

If predictions hold, HS 6‑O‑sulfation emerges as a causal, reversible regulator of mitochondrial aging, offering a novel biomarker and therapeutic target distinct from epigenetic clocks.