N‐terminal phosphorylation of cardiac troponin‐I reduces length‐dependent calcium sensitivity of contraction in cardiac muscle. (10th December 2012)

• Record Type: Journal Article
• Authors: Rao, Vijay S.; Korte, F. Steven; Razumova, Maria V.; Feest, Erik R.; Hsu, Hsiaoman; Irving, Thomas C.; Regnier, Michael; Martyn, Donald A.
• Is Part Of: Journal of physiology. Volume 591:Number 2(2013)
• Page Start: 475
• Page End: 490
• Publication Date: 2012-12-10
• Subjects: Physiology -- Periodicals; 612.005
• DOI: 10.1113/jphysiol.2012.241604
• Languages: English
• ISSNs: 0022-3751

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Key points</title> <p> <list id="l1" list-type="simple"> <list-item> <label> </label> <p>β‐Adrenergic stimulation is an important control mechanism, matching cardiac output to venous return during increased metabolic demand.</p> </list-item> <list-item> <label> </label> <p>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.</p> </list-item> <list-item> <label> </label> <p>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–Ca<sup>2+</sup> relations and sarcomeric structure.</p> </list-item> <list-item> <label> </label> <p>Results suggest cTnI or cMyBP‐C/titin phosphorylation, separately or together, eliminate the SL dependence of Ca<sup>2+</sup> sensitivity of force, but not maximal force. The reduction occurs particularly at long SL, suggesting effects on thin filament access and crossbridge recruitment.</p> </list-item> <list-item> <label> </label> <p>The net effect of PKA phosphorylation is to blunt SL dependence of force at submaximal [Ca<sup>2+</sup>] to maintain elevated systolic function.</p> </list-item> </list> </p> <p> <bold>Abstract </bold> 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–<italic>p</italic>Ca relations in demembranated cardiac muscle. To distinguish cTnI from cMyBP‐C/titin phosphorylation effects on the force–<italic>p</italic>Ca 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 (<italic>F</italic><sub>max</sub>) and Ca<sup>2+</sup> sensitivity of force (<italic>p</italic>Ca<sub>50</sub>) similarly with WT and S23/24A trabeculae. PKA treatment of WT and S23/24A trabeculae reduced <italic>p</italic>Ca<sub>50</sub> at 2.3 but not at 2.0 μm SL, thus eliminating the SL dependence of <italic>p</italic>Ca<sub>50</sub>. In contrast, S23/24D trabeculae reduced <italic>p</italic>Ca<sub>50</sub> at both SL values, primarily at 2.3 μm, also eliminating SL dependence of <italic>p</italic>Ca<sub>50</sub>. Subsequent PKA treatment moderately reduced <italic>p</italic>Ca<sub>50</sub> at both SLs. At each SL, <italic>F</italic><sub>max</sub> was unaffected by either Tn exchange and/or PKA treatment. Low‐angle X‐ray diffraction was performed to determine whether <italic>p</italic>Ca<sub>50</sub> shifts were associated with changes in myofilament spacing (<italic>d</italic><sub>1, 0</sub>) or thick–thin filament interaction. PKA increased <italic>d</italic><sub>1, 0</sub> slightly under all conditions. The ratios of the integrated intensities of the equatorial X‐ray reflections (<italic>I</italic><sub>1, 1</sub>/<italic>I</italic><sub>1, 0</sub>) 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 <italic>p</italic>Ca<sub>50</sub> preferentially at long SL, possibly through reduced availability of thin filament binding sites (cTnI) or altered crossbridge recruitment (cMyBP‐C/titin). Preferential reduction of <italic>p</italic>Ca<sub>50</sub> at long SL may not reduce cardiac output during periods of high metabolic demand because of increased intracellular Ca<sup>2+</sup> during β‐adrenergic stimulation.</p> </abstract>