N‐terminal phosphorylation of cardiac troponin‐I reduces length‐dependent calcium sensitivity of contraction in cardiac muscle
Authors: Rao Vijay S. , Korte F. Steven , Razumova Maria V. , Feest Erik R. , Hsu Hsiaoman , Irving Thomas C. , Regnier Michael , Martyn Donald A. 
Published: November 2012

Key points
β‐Adrenergic stimulation is an important control mechanism, matching cardiac output to venous return during increased metabolic demand.
β‐Adrenergic signalling leads to protein kinase A (PKA) phosphorylation of myofilament proteins cardiac troponin I (cTnI), cardiac myosin binding protein‐C (cMyBP‐C) and titin, but their specific effects on the sarcomeric length (SL) dependence of contraction – which underlies the Frank–Starling Law of the Heart – is debated.
Recombinant cTnI phosphomimetics were exchanged into cardiac muscle to isolate the effects of cTnI from those of cMyBP‐C/titin phosphorylation on SL‐dependent force–Ca2+ relations and sarcomeric structure.
Results suggest cTnI or cMyBP‐C/titin phosphorylation, separately or together, eliminate the SL dependence of Ca2+ sensitivity of force, but not maximal force. The reduction occurs particularly at long SL, suggesting effects on thin filament access and crossbridge recruitment.
The net effect of PKA phosphorylation is to blunt SL dependence of force at submaximal [Ca2+] to maintain elevated systolic function.
Abstract  Protein kinase A (PKA) phosphorylation of myofibrillar proteins constitutes an important pathway for β‐adrenergic modulation of cardiac contractility. In myofilaments PKA targets troponin I (cTnI), myosin binding protein‐C (cMyBP‐C) and titin. We studied how this affects the sarcomere length (SL) dependence of force–pCa relations in demembranated cardiac muscle. To distinguish cTnI from cMyBP‐C/titin phosphorylation effects on the force–pCa relationship, endogenous troponin (Tn) was exchanged in rat ventricular trabeculae with either wild‐type (WT) Tn, non‐phosphorylatable cTnI (S23/24A) Tn or phosphomimetic cTnI (S23/24D) Tn. PKA cannot phosphorylate either cTnI S23/24 variant, leaving cMyBP‐C/titin as PKA targets. Force was measured at 2.3 and 2.0 μm SL. Decreasing SL reduced maximal force (Fmax) and Ca2+ sensitivity of force (pCa50) similarly with WT and S23/24A trabeculae. PKA treatment of WT and S23/24A trabeculae reduced pCa50 at 2.3 but not at 2.0 μm SL, thus eliminating the SL dependence of pCa50. In contrast, S23/24D trabeculae reduced pCa50 at both SL values, primarily at 2.3 μm, also eliminating SL dependence of pCa50. Subsequent PKA treatment moderately reduced pCa50 at both SLs. At each SL, Fmax was unaffected by either Tn exchange and/or PKA treatment. Low‐angle X‐ray diffraction was performed to determine whether pCa50 shifts were associated with changes in myofilament spacing (d1,0) or thick–thin filament interaction. PKA increased d1,0 slightly under all conditions. The ratios of the integrated intensities of the equatorial X‐ray reflections (I1,1/I1,0) indicate that PKA treatment increased crossbridge proximity to thin filaments under all conditions. The results suggest that phosphorylation by PKA of either cTnI or cMyBP‐C/titin independently reduces the pCa50 preferentially at long SL, possibly through reduced availability of thin filament binding sites (cTnI) or altered crossbridge recruitment (cMyBP‐C/titin). Preferential reduction of pCa50 at long SL may not reduce cardiac output during periods of high metabolic demand because of increased intracellular Ca2+ during β‐adrenergic stimulation.