The objective of this work was to investigate the effect of hypertrophic 
cardiomyopathy-linked A8V and E134D mutations in cardiac troponin C (cTnC) on 
the response of reconstituted thin filaments to calcium upon phosphorylation of 
cardiac troponin I (cTnI) by protein kinase A. The phosphorylation of cTnI at 
protein kinase A sites was mimicked by the S22D/S23D double mutation in cTnI. 
Our results demonstrate that the A8V and E134D mutations had no effect on the 
extent of calcium desensitization of reconstituted thin filaments induced by 
cTnI pseudophosphorylation. However, the A8V mutation enhanced the effect of 
cTnI pseudophosphorylation on the rate of dissociation of calcium from 
reconstituted thin filaments and on the calcium dependence of actomyosin ATPase. 
Consequently, while the A8V mutation still led to a slower rate of dissociation 
of calcium from reconstituted thin filaments upon pseudophosphorylation of cTnI, 
the ability of the A8V mutation to decrease the rate of calcium dissociation was 
weakened. In addition, the ability of the A8V mutation to sensitize actomyosin 
ATPase to calcium was weakened after cTnI was replaced by the phosphorylation 
mimetic of cTnI. Consistent with the hypothesis that the E134D mutation is 
benign, it exerted a minor to no effect on the rate of dissociation of calcium 
from reconstituted thin filaments or on the calcium sensitivity of actomyosin 
ATPase, regardless of the cTnI phosphorylation status. In conclusion, our study 
enhances our understanding of how cardiomyopathy-linked cTnC mutations affect 
the response of reconstituted thin filaments to calcium upon cTnI 
phosphorylation.