Protein kinase C phosphomimetics alter thin filament Ca2+ binding properties.

Area of Science:

• Cardiovascular Physiology
• Molecular Cardiology
• Muscle Contraction Biochemistry

Background:

• Adrenergic stimulation regulates cardiac function via protein phosphorylation.
• Troponin complex is crucial for cardiac contraction and is targeted by adrenergic phosphorylation.
• While beta-adrenergic PKA phosphorylation of troponin I is understood, alpha-adrenergic PKC phosphorylation effects are less clear.

Purpose of the Study:

• To investigate the site-specific effects of alpha-adrenergic induced PKC phosphorylation on Troponin I (TnI) and Troponin T (TnT).
• To determine how these phosphorylations impact the Ca(2+) binding properties of the Troponin complex and thin filaments.
• To elucidate the role of Troponin in sensing physiological stimuli to modulate cardiac contractility.

Main Methods:

• Utilized IAANS labeled fluorescent troponin C (TnC(IAANS)(T53C)).
• Systematically examined site-specific effects of PKC phosphomimetic mutants of TnI and TnT.
• Assessed Ca(2+) binding properties in isolated Troponin complex and reconstituted thin filaments.
• Correlated findings with Ca(2+) sensitivity in skinned muscle preparations.

Main Results:

• Most phosphomimetics had minimal impact on isolated Troponin complex Ca(2+) binding.
• Incorporation into thin filaments altered Ca(2+) sensitivity, consistent with muscle preparation data.
• Altered Ca(2+) sensitivity was primarily due to changes in Ca(2+) dissociation rates.
• Phosphomimetic Asp/Glu and Ala mutations in TnI showed varied effects on Ca(2+) binding.

Conclusions:

• Troponin phosphorylation by PKC influences cardiac thin filament Ca(2+) sensitivity.
• These modifications affect Ca(2+) dissociation rates, impacting cardiac muscle performance.
• Troponin acts as a signaling hub on the thin filament, modulating cardiac contractile function in response to physiological stimuli.