Length dependence of striated muscle force generation is controlled by phosphorylation of cTnI at serines 23/24

Abstract

Key points• An important mechanism in beat-to-beat optimization of heart performance is matching ventricular output with end-diastolic volume, which is known as the Frank-Starling Relationship.• The cellular basis for this regulation involves myofilament length-tension relationships.• We previously showed two populations of length-tension relationships in mammalian left ventricular cardiac myocytes, one steep like fast-twitch skeletal muscle fibres and the other shallow like slow-twitch skeletal muscle fibres, and cardiac myocytes with shallow length-tension relationships shift to steep relationships by protein kinase A-induced myofilament phosphorylation.• The current study investigated the molecular and amino acid residue mechanisms that control length-tension relationships.• The single muscle cell experiments demonstrated that cardiac troponin I phosphorylation at serines 23/24 control length-tension relationships in striated muscle.• This study provides: (i) a mechanism to explain a length dependence of force generation in striated muscle; and (ii) an important target to potentially treat heart disease associated with compromised Frank-Starling relationships.Abstract According to the Frank-Starling relationship, greater end-diastolic volume increases ventricular output. The Frank-Starling relationship is based, in part, on the length-tension relationship in cardiac myocytes. Recently, we identified a dichotomy in the steepness of length-tension relationships in mammalian cardiac myocytes that was dependent upon protein kinase A (PKA)-induced myofibrillar phosphorylation. Because PKA has multiple myofibrillar substrates including titin, myosin-binding protein-C and cardiac troponin I (cTnI), we sought to define if phosphorylation of one of these molecules could control length-tension relationships. We focused on cTnI as troponin can be exchanged in permeabilized striated muscle cell preparations, and tested the hypothesis that phosphorylation of cTnI modulates length dependence of force generation. For these experiments, we exchanged unphosphorylated recombinant cTn into either a rat cardiac myocyte preparation or a skinned slow-twitch skeletal muscle fibre. In all cases unphosphorylated cTn yielded a shallow length-tension relationship, which was shifted to a steep relationship after PKA treatment. Furthermore, exchange with cTn having cTnI serines 23/24 mutated to aspartic acids to mimic phosphorylation always shifted a shallow length-tension relationship to a steep relationship. Overall, these results indicate that phosphorylation of cTnI serines 23/24 is a key regulator of length dependence of force generation in striated muscle.

Cardiac Myosin-binding Protein C and Troponin-I Phosphorylation Independently Modulate Myofilament Length-dependent Activation

“…2 summarizes average ⌬EC 50 , a convenient parameter to index LDA, and the impact of PKA treatment on this parameter. LDA increased upon PKA treatment in NTG myocytes, consistent with previous reports (1,8,10,11,13) but was without impact in either the AAA or DDD myocytes. In addition, after PKA treatment, LDA in NTG myocytes approached LDA recorded in DDD myocytes regardless of PKA treatment, whereas it was ϳ50% lower in AAA myocytes.…”

“…Moreover, LDA is affected by cardiac disease-associated mutations within various contractile proteins, including the troponins and cardiac myosin binding protein C (cMyBP-C) (13). Selective introduction of a PKA phosphomimic isoform of cTnI into the cardiac sarcomere increases LDA (10,14). Finally, cardiac disease-causing mutations are associated with blunting of the myofilament Ca 2ϩ desensitization normally associated with PKA-mediated phosphorylation (15,16).…”

“…This is caused, in part, by the expression of the unique cardiac-specific isoform of troponin-I (cTnI) (7), the inhibitory subunit of the striated muscle regulatory troponin complex (8,9). Cardiac LDA has been shown to be modulated by contractile protein phosphorylation by most (1,8,10,11) but not all (12) reports. Moreover, LDA is affected by cardiac disease-associated mutations within various contractile proteins, including the troponins and cardiac myosin binding protein C (cMyBP-C) (13).…”

Myosin filament activation in the heart is tuned to the mechanical task

“…LDA is generally attributed to the SL modulation of the sensitivity of the thin filament to cytosolic Ca 2+ concentration combined with the fact that, during heart contraction, [Ca 2+ ] i undergoes a transient increase that may not reach the full activation level (8,9,11). The molecular mechanisms underlying LDA remain largely unknown, although LDA has been shown to be modulated by several factors, such as the degree of phosphorylation of sarcomeric proteins and cardiomyopathy causing mutations in these proteins (14)(15)(16)(17)(18)(19)(20)30). X-ray diffraction provides a unique tool for in situ investigation of the structural changes undergone by the myofilaments during the cardiac contraction-relaxation cycle.…”

“…For a given [Ca 2+ ] i , the maximal force is larger at longer sarcomere lengths [SLs; length-dependent activation (LDA)], which is the cellular basis of the Frank-Starling law of the heart (10,11). LDA is the result of a chain of yet undefined events relating a mechanosensor of the SL to the number of force-generating motors and consequently the force (12, 13), and moreover, it is modulated by the degree of phosphorylation of contractile (Myosin Regulatory Light Chain), regulatory [Troponin I on the thin filament and myosin-binding protein C (MyBP-C) in the thick filament], and cytoskeletal proteins (14)(15)(16)(17)(18)(19)(20). We exploited the nanometer-micrometer-scale X-ray diffraction possible at beamline ID02 of the European Synchrotron Radiation Facility (ESRF) to record the structural changes undergone by the thick filament and the myosin motors during the systole-diastole cycle in intact trabeculae from rat cardiac ventricle under SL control.…”