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

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.

Figure 1. Simultaneous measurement of force and sarcomere length in a cardiac myocyte preparation

Top left shows a photomicrograph of a skinned cardiac myocyte preparation at a sarcomere length of 2.30 μm. The sarcomere length signal was recorded (on a time scale in seconds) using an IonOptix SarcLen system (IonOptix, Milton, MA, USA), and the corresponding force trace is shown above the sarcomere length. Force recordings are measured in response to a slack restretch manoeuvre and are shown on a fast time base scale (i.e. ms). The sarcomere length trace and corresponding three force records were measured in pCa 5.8 solution, which yielded ∼50% of maximal calcium-activated force at sarcomere length 2.30 μm (the maximal Ca²⁺-activated force trace in pCa 4.5 solution is shown in the lower right inset). (This figure shows just three force traces for illustrative purposes; the actual experiment included 10–15 force measurements at sarcomere lengths ranging from ∼2.30 μm to ∼1.40 μm.)

Figure 2. Protein kinase A (PKA)-mediated phosphorylation of cTnI regulates the length–tension relationship in cardiac myocytes

A, the left panel shows a Coomassie-stained gel of the rat recombinant cardiac troponin (R-cTn) protein complex. The top right panel shows a Western blot staining for phosphorylated cTnI at serines 23/24 of R-cTn before and after incubation with PKA. The bottom right panel shows a TnT Western blot of a gel containing cardiac myocytes before and after exchange with R-cTn. R-TnT runs higher than endogenous TnT due to addition of the myc-tag. B, the cardiac myocyte preparation was pre-treated with PKA (Sigma; 0.125 U μl⁻¹) followed by length–tension measurement. cTn was then exchanged followed by a second length–tension curve. Exchange with non-phosphorylated R-cTn shifted the length–tension curve from steep to shallow. C, the same experimental design in a different cardiac myocyte preparation, but in this case PKA was added after cTn exchange and restored the steep length–tension relationship. Sarcomere length–tension relationships were fit by eye. (This experiment was performed in 4 myocyte preparations.)

Figure 3. Exchange of cTn containing pseudo-phosphorylated cTnI (serines 23/24asp) shifted the sarcomere length–tension relationship from shallow to steep in a skinned cardiac myocyte preparation

The length–tension relationship was measured before and after exchange of cTn in which the cTnI serines 23/24 residues had been mutated to aspartic acid to mimic PKA-induced cTnI phosphorylation. (This experiment was performed in 3 myocyte preparations.)

Figure 4. Exchange of ∼40% ssTn for R-cTn did not alter the steepness of the length–tension relationship after protein kinase A (PKA) treatment of a slow-twitch skeletal muscle fibre

A, the length–tension relationship was measured before and after PKA treatment in a rat soleus slow-twitch skeletal muscle fibre (inset), which underwent Tn exchange for 6 h. (Before Tn exchange, the slow-twitch fibre exhibited the same length–tension relationship as after incorporation of 40% R-cTn into the myofilaments.) B, TnT Western blot of a gel containing this soleus slow-twitch fibre before and after exchange with R-cTn. R-TnT runs higher than endogenous ssTnT due to the greater molecular weight of cTnT and the addition of the myc-tag. (This figure is representative of this experimental design, which was repeated in 4 different slow-twitch fibre preparations.)

Figure 5. Protein kinase A (PKA) steepened the length–tension relationship of a rat soleus slow-twitch skeletal muscle fibre after nearly complete exchange of ssTn for R-cTn

A, the length–tension relationship was measured before and after exchange in a slow-twitch skeletal muscle fibre that underwent Tn exchange for 12 h. B, TnT Western blot of a gel containing a soleus fibre before and after exchange with R-cTn. R-TnT runs higher than endogenous ssTnT due to the increased molecular weight of cTnT and the addition of the myc-tag. (This experiment was repeated in 3 different slow-twitch skeletal muscle fibre preparations.)

Figure 6. Exchange of cTn-containing pseudo-phosphorylated cTnI (serines 23/24asp) shifted the sarcomere length–tension relationship from shallow to steep in a rat soleus skinned slow-twitch skeletal muscle fibre

The length–tension relationship was measured before and after 12 h of cTn exchange in which the cTnI serines 23/24 residues had been mutated to aspartic acid to mimic PKA-mediated cTnI phosphorylation. (This experiment was performed in 4 slow-twitch skeletal muscle fibre preparations.)

Figure 7. Conceptual model by which protein kinase A (PKA)-induced phosphorylation alters myofilament physical properties to augment length dependence of force

PKA-mediated phosphorylation of cTnI is depicted to increase the persistence length (bending rigidity) of the Tm molecules at both long and short sarcomere lengths (SL; indicated by crooked arrow at long and short SL before PKA and straight arrow after PKA at long and short SL), which would increase cooperativity of thin filament activation. This would have two important physiological effects: (i) more cooperative activation would augment force development early during systole (indicated by more green cross-bridges); and (ii) upon shortening and the coincident loss of cross-bridges there would be greater cooperative deactivation at late systole to assist in relaxation (indicated by more yellow cross-bridges). These properties are consistent with our observation that the myofilaments become exquisitely sensitive to length and would act to assist ventricular ejection and help optimize filling during a compressed diastolic filling time.

Figure 8. Protein kinase A (PKA) treatment increased the steepness of tension–pCa relationships

The tension–pCa relationship of a rat cardiac myocyte preparation before and after PKA treatment. Autoradiogram indicates PKA phosphorylated cardiac myosin-binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), which yielded significantly greater Hill coefficients (n₂) for tensions below 0.5 maximal tension (P= 0.044, n= 3) and a trend for greater Hill coefficients (n₁) for tension above 0.5 maximal tension (P= 0.121, n= 3). These results implicate PKA-mediated phosphorylation of myofibrillar proteins increases cooperative activation of force. (The bar graph shows mean ± SD).