The influence of trout cardiac troponin I and PKA phosphorylation on the Ca2+ affinity of the cardiac troponin complex

Abstract

SUMMARYThe trout heart is 10-fold more sensitive to Ca 2+ than the mammalian heart. This difference is due, in part, to cardiac troponin C (cTnC) from trout having a greater Ca 2+ affinity than human cTnC. To determine what other proteins are involved, we cloned cardiac troponin I (cTnI) from the trout heart and determined how it alters the Ca 2+ affinity of a cTn complex containing all mammalian components (mammalian cTn). Ca 2+ activation of the complex was characterized using a human cTnC mutant that contains anilinonapthalenesulfote iodoacetamide attached to Cys53. When the cTn complex containing labeled human cTnC was titrated with Ca 2+, its fluorescence changed, reaching an asymptote upon saturation. Our results reveal that trout cTnI lacks the N-terminal extension found in cTnI from all other vertebrate groups. This protein domain contains two targets (Ser23 and Ser24) for protein kinase A (PKA) and protein kinase C. When these are phosphorylated, the rate of cardiomyocyte relaxation increases. When rat cTnI in the mammalian cTn complex was replaced with trout cTnI, the Ca 2+ affinity was increased ~1.8-fold. This suggests that trout cTnI contributes to the high Ca 2+ sensitivity of the trout heart. Treatment of the two cTn complexes with PKA decreased the Ca 2+ affinity of both complexes. However, the change for the complex containing rat cTnI was 2.2-fold that of the complex containing trout cTnI. This suggests that the phosphorylation of trout cTnI does not play as significant a role in regulating cTn function in trout.

Effect of cold acclimation on troponin I isoform expression in striated muscle of rainbow trout

“…Protein bands were cut from lanes generated for each muscle type for control and cold fish. Every band with a weight Ϯ8 kDa of the band for the RTcTnI standard was cut from the gel and prepared for trypsin digestion as previously described (16). These samples were then subjected to tandem mass spectrometry at the Advanced Protein Analysis Center at Sick Kids Hospital (Toronto, ON) using an Applied Biosystems/MDS Sciex API QSTAR XL Pulsar MALDI QTOF mass spectrometor.…”

Cardiac troponin structure-function and the influence of hypertrophic cardiomyopathy associated mutations on modulation of contractility

“…However, S23/S24 can be phosphorylated by other kinases in vitro , including PKC [113], protein kinase D (PKD) [114-116] and GMP-dependent protein kinase (PKG) [117]. Phosphorylation at these sites has been shown to increase the Ca 2+ dissociation rate from cTnC, weaken the interaction between cTnI and the N-terminus of cTnC, reduce the Ca 2+ sensitivity (pCa 50 ) of cardiac muscle tension production, increase cross-bridge cycling kinetics, and accelerate cardiac muscle cell relaxation [35, 36, 118-124]. Zhang et al .…”

Gene regulation, alternative splicing, and posttranslational modification of troponin subunits in cardiac development and adaptation: a focused review

“…Although trout cardiac TnI lacks the two substrate residues of PKA phosphorylation, myofilament Ca 2+ affinity decreased when treated with PKA, similar to the response of mammalian cardiac TnI with the N-terminal extension (Kirkpatrick et al, 2011). This apparently N-terminal extension-independent PKA regulation and enhancement of relaxation is worth further investigation.…”

“…A study on trout cardiac TnI that lacks the N-terminal extension showed that troponin complex containing trout cardiac TnI had a greater Ca 2+ affinity than human troponin (Kirkpatrick et al, 2011). Although trout cardiac TnI lacks the two substrate residues of PKA phosphorylation, myofilament Ca 2+ affinity decreased when treated with PKA, similar to the response of mammalian cardiac TnI with the N-terminal extension (Kirkpatrick et al, 2011).…”