Functionally conservative substitutions at cardiac troponin I S43/45

Area of Science:

• Cardiovascular Biology
• Molecular Cardiology
• Protein Biochemistry

Background:

• Protein kinase C (PKC) phosphorylation of cardiac troponin I (cTnI) at S43/45 sites influences cardiac function.
• Previous studies indicated that a phospho-null substitution at these sites reduces myocyte and cardiac contractile function.

Purpose of the Study:

• To evaluate if the cTnIS43/45N mutation serves as a functionally conservative alternative in cardiac myocytes.
• To assess the impact of cTnIS43/45N substitution on myocyte contractility and calcium handling.

Main Methods:

• Endogenous cTnI replacement with epitope-tagged cTnI and cTnIS43/45N via gene transfer over 2 and 4 days.
• Analysis of thin filament stoichiometry.
• Assessment of myocyte contractile shortening/re-lengthening rates and myocyte Ca(2+) transient dynamics.

Main Results:

• Gene transfer achieved partial and extensive replacement of endogenous cTnI with cTnIS43/45N without altering thin filament stoichiometry.
• No significant changes in contractile shortening/re-lengthening amplitudes or rates were observed.
• The cTnIS43/45N substitution did not induce adaptive changes in myocyte Ca(2+) transient or phosphorylation at the PKA/PKC-targeted cTnIS23/24 site.

Conclusions:

• The cTnIS43/45N substitution is functionally conservative in cardiac myocytes.
• This phospho-null mutation is suitable for use in rodent models investigating PKC modulation of cardiac performance.