Molecular regulation of stretch activation

Area of Science:

• Muscle Physiology
• Skeletal Muscle Contraction
• Sarcomere Mechanics

Background:

• Stretch activation, a delayed force increase post-stretch, is well-documented in cardiac muscle but less studied in mammalian skeletal muscle.
• Understanding stretch activation mechanisms in skeletal muscle is crucial for comprehending muscle function and potential therapeutic targets.
• The roles of key regulatory proteins like myosin binding protein-C and troponin I in skeletal muscle stretch activation remain incompletely understood.

Purpose of the Study:

• To investigate the impact of protein kinase A (PKA) phosphorylation on stretch activation in rat slow-twitch skeletal muscle fibers.
• To elucidate the specific contributions of slow myosin binding protein-C (sMyBP-C) and cardiac troponin I (cTnI) phosphorylation to stretch activation.
• To determine how PKA-mediated phosphorylation of sMyBP-C and cTnI influences force development and cross-bridge kinetics.

Main Methods:

• Measured stretch activation in permeabilized rat slow-twitch skeletal muscle fibers using rapid step stretches (1%-4% sarcomere length) under controlled calcium conditions.
• Assessed the effects of PKA treatment on stretch activation parameters, including delayed force development rates (k_ef, k_df) and force overshoot following shortening.
• Utilized recombinant human cTn complexes with pseudo-phosphorylated cTnI variants (non-phosphorylated WT, PKA sites, tyrosine kinase sites, and combinatorial) to investigate thin filament regulation.

Main Results:

• PKA significantly increased stretch activation and accelerated force development rates (k_ef) at low and half-maximal calcium concentrations.
• PKA treatment led to a substantial (approx. 10-fold) increase in shortening-induced force overshoot, indicating altered cross-bridge dynamics.
• Pseudo-phosphorylation of cTnI at PKA and/or tyrosine kinase sites enhanced stretch activation and steady-state force, with combinatorial phosphorylation increasing k_df.

Conclusions:

• sMyBP-C phosphorylation by PKA modulates skeletal muscle stretch activation through cross-bridge recruitment and faster cycling kinetics.
• cTnI phosphorylation regulates stretch activation via both redundant and synergistic mechanisms, highlighting its complex role in muscle contraction.
• Phosphorylation of sMyBP-C and cTnI represents key regulatory targets for enhancing skeletal muscle contractility.