Phosphorylation of troponin I by protein kinase A (PKA) reduces Ca2þ sensitivity and increases the rate of Ca2þ
release from troponin C and the rate of relaxation in cardiac muscle. In vitro experiments indicate that mutations that cause
dilated cardiomyopathy (DCM) uncouple this modulation, but this has not been demonstrated in an intact contractile system.
Using a Ca2þ-jump protocol, we measured the effect of the DCM-causing mutation ACTC E361G on the equilibrium and kinetic
parameters of Ca2þ regulation of contractility in single transgenic mouse heart myofibrils. We used propranolol treatment of mice
to reduce the level of troponin I and myosin binding protein C (MyBP-C) phosphorylation in their hearts before isolating the myo-
fibrils. In nontransgenic mouse myofibrils, the Ca2þ sensitivity of force was increased, the fast relaxation phase rate constant,
kREL, was reduced, and the length of the slow linear phase, tLIN, was increased when the troponin I phosphorylation level was
reduced from 1.02 to 0.3 molPi/TnI (EC50 P/unp ¼ 1.8 5 0.2, p < 0.001). Native myofibrils from ACTC E361G transgenic mice
had a 2.4-fold higher Ca2þ sensitivity than nontransgenic mouse myofibrils. Strikingly, the Ca2þ sensitivity and relaxation parameters
of ACTC E361G myofibrils did not depend on the troponin I phosphorylation level (EC50 P/unp ¼ 0.88 5 0.17, p ¼ 0.39).
Nevertheless, modulation of the Ca2þ sensitivity of ACTC E361G myofibrils by sarcomere length or EMD57033 was indistinguishable
from that of nontransgenic myofibrils. Overall, EC50 measured in different conditions varied over a 7-fold range.
The time course of relaxation, as defined by tLIN and kREL, was correlated with EC50 but varied by just 2.7- and 3.3-fold, respectively.
Our results confirm that troponin I phosphorylation specifically alters the Ca2þ sensitivity of isometric tension and the time
course of relaxation in cardiac muscle myofibrils. Moreover, the DCM-causing mutation ACTC E361G blunts this phosphorylation-dependent
response without affecting other parameters of contraction, including length-dependent activation and the
response to EMD57033.