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.

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.

Introduction

To precisely balance the amount of blood circulating between the right and left sides of the heart, ventricular stroke volume is tightly coupled to filling volume. But just how the ventricles match output with input is a question that has remained elusive. The pursuit of this answer has been a driving force in the field for over 100 years and, in fact, one theme posited by Starling and Visscher in the 1920s (Starling & Visscher, 1927) has transcended much of the century. Starling's group postulated control of heart function by end-diastolic volume is similar to skeletal muscle mechanics, which relates contraction energy (and force) to muscle fibre length. Along these lines, the length dependence of force generation in tetanized skeletal muscle fibres arises from its sarcomeric ultrastructure, which consists of overlap regions between myofibrillar thin and thick filaments (Ramsey & Street, 1940; Gordon et al. 1966). Notably, the length–tension relationship of maximally Ca²⁺-activated cardiac myofibrils virtually superimposes that of tetanized skeletal muscle fibres over the working sarcomere length of cardiac muscle (i.e. sarcomere lengths between ∼1.70 μm and 2.20 μm, which is known as the ascending limb of the length–tension relationship; Fabiato & Fabiato, 1975). Interestingly, in both skeletal muscle fibres and cardiac myocytes the ascending limb of sarcomere length–tension relationships becomes progressively steeper when the level of Ca²⁺ activation is reduced (Close, 1972; Fabiato & Fabiato, 1975; ter Keurs et al. 1980; Allen & Moss, 1987). This is especially important physiologically as cardiac myocytes are activated by intracellular [Ca²⁺] that produce submaximal force. Recently our laboratory directly compared the sarcomere length dependence of force generation during submaximal Ca²⁺ activations between single cardiac myocytes, fast-twitch skeletal muscle fibres and slow-twitch skeletal muscle fibres (Hanft & McDonald, 2010). As predicted from previous work (McDonald et al. 1997; Konhilas et al. 2002), fast-twitch skeletal muscle fibres had a steep ascending length–tension relationship and slow-twitch fibres exhibited a shallow relationship. However, cardiac myocytes exhibited two populations of length–tension relationships, one steep like fast-twitch fibres the other shallow like slow-twitch fibres. Interestingly, cardiac myocytes with shallow length–tension relationships could be shifted to a steep length–tension relationship by protein kinase A (PKA)-mediated phosphorylation of myofilament proteins (Hanft & McDonald, 2010). Because PKA has multiple myofibrillar substrates, including titin, cardiac myosin-binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), we sought to define if phosphorylation of one of these molecules could control length–tension relationships in striated muscle. We focused on cTnI for three reasons: (i) our lab observed a relationship between cTnI phosphorylation and the steepness of ventricular function curves in rat working hearts (Hanft et al. 2013); (ii) 2D-DIGE indicated a distribution of cTnI phosphorylation states consistent with the observation of two populations of length–tension relationships (one shallow the other steep) in rat cardiac myocytes (Hanft et al. 2013); and (iii) troponin can be readily exchanged in permeabilized striated muscle cell preparations (Chandra et al. 1999; Kruetziger et al. 2011; Korte et al. 2012). We tested the hypothesis that phosphorylation of cTnI regulates the length dependence of striated muscle force generation.

Methods

Experimental animals

Male Sprague–Dawley rats (6 weeks old) were obtained from Harlan (Madison, WI, USA), housed in groups of two, and provided access to food and water ad libitum. All procedures involving animal use were performed according to protocols that were reviewed and approved by the Animal Care and Use Committee of the University of Missouri.

Recombinant troponin

Rat cardiac troponin C (cTnC), cTnI and cardiac troponin T (cTnT) cDNA was isolated as previously described (Kobayashi & Solaro, 2006). Rat cTnI cDNA was used to encode pseudo-phosphorylated rat cTnI with serines 23/24 mutated to aspartic acid (cTnI S23/24D) by site-directed mutagenesis (Quick Change; Stratagene). cDNA encoding the adult myc-tagged rat cTnT was generated by polymerase chain reaction addition of an N-terminal myc-tag (MMEQKLISEEDL) prior to serine 2. Resultant constructs were verified by DNA sequencing.

The individual recombinant rat cTn subunits were expressed in Escherichia coli and purified to homogeneity as previously described for the human cTn subunits (Nixon et al. 2012a,b). Troponin used for myocyte exchange contained adult rat cTnT with an N-terminal myc-tag. Previous work has demonstrated the presence of this myc-tag on cTnT does not affect myofilament function (Tardiff et al. 1998; Sumandea et al. 2003). The varied cTn complexes were reconstituted by sequential dialysis and column purified as previously described (Biesiadecki et al. 2007; Nixon et al. 2012b). Column fractions containing pure cTn were extensively dialysed against exchange buffer (in mm: KCl, 200; MgCl₂, 5; EGTA, 5; dithiothreitol, 1; Mops, 20; pH 6.5), and aliquots stored at −80°C until use.

Cardiac myocyte preparation

Myocytes were obtained by mechanical disruption of rat hearts (n= 9; McDonald, 2000). Rats were placed in a small air-tight chamber, anaesthetized by inhalation of isoflurane (20% (vol/vol) in olive oil), and their hearts were quickly removed and placed in ice-cold relaxing solution. The atria and right ventricle were removed, and hearts were cut into 2–3 mm pieces and further disrupted for 5–10 s using a Waring blender. The resulting suspension of cells and cell fragments was centrifuged for 105 s at 165 g. The myocytes were subsequently skinned by suspending the pellet for 4 min in 0.3% ultrapure Triton X-100 (Pierce Chemical) in relaxing solution. The pellet was washed twice with cold relaxing solution, and the skinned cells were then re-suspended in 10–20 ml of relaxing solution and kept on ice during the day of the experiment; one myocyte preparation was used per heart.

Skeletal muscle fibre preparation

Skeletal muscle fibres also were obtained from Sprague–Dawley rats (n= 2) anaesthetized by inhalation of isoflurane (20% (vol/vol) in olive oil; McDonald, 2000). Slow-twitch skeletal muscle fibres (n= 8) were obtained from the soleus muscle. The muscles were isolated, placed in relaxing solution at 4°C and bundles of ∼50 fibres were separated, tied to capillary tubes and stored in relaxing solution containing 50% (v/v) glycerol for up to 4 weeks. Single fibres for mechanical measurements were dissected by gently pulling them from the end of the bundle.

Experimental apparatus

The experimental apparatus for physiological measurements of myocyte preparations and skeletal muscle fibres was the same as that previously described (McDonald et al. 1998; McDonald, 2000). Myocyte/fibre preparations were attached between a force transducer and torque motor by placing the ends of the myocyte preparation into stainless steel troughs (25 gauge). The ends of the myocyte/fibre preparations were secured by overlaying a 0.5 mm length of 3–0 monofilament nylon suture (Ethicon) onto each end of the myocyte, and then tying the suture into the troughs with two loops of 10–0 monofilament (Ethicon). The attachment procedure was performed under a stereomicroscope (∼100× magnification) using finely shaped forceps.

Prior to mechanical measurements the experimental apparatus was mounted on the stage of an inverted microscope (model IX-70, Olympus Instrument, Japan), which was placed upon a pneumatic vibration isolation table having a cut-off frequency of ∼1 Hz. Mechanical measurements were performed using a capacitance-gauge transducer (Model 403-sensitivity of 20 mV mg⁻¹ (plus a 10× amplifier for cardiac myocytes) and resonant frequency of 600 Hz; Aurora Scientific, Aurora, ON, Canada). Length changes were introduced using a DC torque motor (model 308, Aurora Scientific) driven by voltage commands from a personal computer via a 12 or 16 bit D/A converter (AT-MIO-16E-1, National Instruments, Austin, TX, USA). Force and length signals were digitized at 1 kHz and stored on a personal computer using LabView for Windows (National Instruments). Sarcomere length was monitored simultaneously with force and length measurements using IonOptix SarcLen system (IonOptix, Milton, MA, USA), which used a fast Fourier transform algorithm of the video image of the myocyte. Microscopy was done using a 40× objective (Olympus UWD 40) and a 2.5× intermediate lens.

Solutions

Compositions of relaxing and activating solutions used in mechanical measurements were as follows (in mm): EGTA, 7; free Mg²⁺, 1; imidazole, 20; MgATP, 4; creatine phosphate, 14.5; pH 7.0; various Ca²⁺ concentrations between 10⁻⁹ m (relaxing solution) and 10^−4.5 m (maximal Ca²⁺-activating solution); and sufficient KCl to adjust ionic strength to 180 mm. The final concentrations of each metal, ligand and metal–ligand complex were determined with the computer program of Fabiato (1988). Preceding each Ca²⁺ activation, myocyte preparations were immersed for 30 s in a solution of reduced Ca²⁺-EGTA buffering capacity, which was identical to normal relaxing solution except that EGTA was reduced to 0.5 mm. This protocol resulted in more rapid development of steady-state force during subsequent activation and helped preserve the striation pattern during activation. Relaxing solution in which the ventricles were mechanically disrupted and myocytes and skeletal muscle fibres were resuspended contained (in mm): EGTA, 2; MgCl₂, 5; ATP, 4; imidazole, 10; KCl, 100; at pH 7.0; with the addition of a protease inhibitor cocktail (Set I Calbiochem, San Diego, CA, USA). Troponin exchange was carried out in relaxing solution containing 1 mg ml⁻¹ recombinant troponin.

Sarcomere length–tension measurements

All mechanical measurements on cardiac myocytes and skeletal muscle fibres were performed at 13 ± 1°C. For mechanical measurements on myocytes, a preparation was chosen from a cell suspension based on two morphological criteria: (i) the myocyte preparation had to be at least 100 μm in length when floating free, which allowed enough size to pick up with forceps and enough length to secure in the troughs by placing suture over ∼10–20 μm of each of its ends; and (ii) the preparation needed to be rod-shaped with limited or no branching. Following attachment, the relaxed preparation was adjusted to a sarcomere length of ∼2.30 μm and then the preparation was maximally Ca²⁺ activated in pCa 4.5 solution. For sarcomere length–tension measurements the cell preparation was transferred to a pCa solution that yielded ∼50% maximal force and then isometric force was measured over a range of sarcomere lengths monitored by the IonOptix SarcLen system (IonOptix, Milton, MA, USA). Isometric force and sarcomere length were measured simultaneously. Sarcomere length was adjusted between ∼2.30 μm and ∼1.40 μm (by ∼0.10 μm intervals) by manual manipulation of the length micrometre while the preparation was Ca²⁺ activated (Fig. 1). After each sarcomere length change ∼10–15 s was provided to allow for development of steady-state force. The force at each sarcomere length was obtained via a slack–restretch manoeuvre (Korte et al. 2003). For analysis, the force at each sarcomere length was normalized to the force obtained at sarcomere length ∼2.30 μm (during the submaximal Ca²⁺ activation). Because the force during submaximal Ca²⁺ activations invariably rose during the sustained activation, normalized forces were calculated by interpolating force measurements at sarcomere length ∼2.30 μm, which were performed at the beginning and end of the series of force measurements. At the end of each experiment, preparations were activated a second time in pCa 4.5 solution, and only experiments in which maximal tension remained >90% of the initial tension were used for analysis. To assess the effects of PKA, length–tension relationships were performed before and after 45 min incubation with PKA (Sigma; 0.125 U μl⁻¹). The force (per cross-sectional area) obtained at sarcomere length ∼2.30 μm during half-maximal Ca²⁺ activation was 27 ± 9 kN m⁻² and 79 ± 33 kN m⁻² for rat skinned cardiac myocyte preparations and slow-twitch skeletal muscle fibres, respectively (values are mean ± SD). The pCa solution for length–tension curves was adjusted to yield the same force before and after PKA due to the decreased Ca²⁺ sensitivity of force known to occur following PKA treatment (Solaro, 1986; Hofmann & Lange, 1994; Strang et al. 1994; De Tombe & Stienen, 1995; Janssen & De Tombe, 1997).

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.)

SDS–PAGE and Western blots

To assess troponin exchange cardiac myofibrillar proteins were assessed using SDS–PAGE followed by Western blots. The methods for SDS–PAGE were similar to that previously described (Guilian et al. 1983). Myofibrillar suspensions (∼1.0 μg) were separated by SDS–PAGE using 12% polyacrylamide slab gels. Gels were then placed on a pre-wetted nitrocellulose membrane and the gel/nitrocellulose combination was sandwiched between several sheets of 3 MM chromatography paper. The protein samples were then transferred to nitrocellulose using a semi-dry blot apparatus at constant current (120 mA) for 1 h. The nitrocellulose blots were then placed in a blocking buffer consisting of 5% dry milk, and rocked for 1 h. Primary antibody (TnT; CT3 from Developmental Studies Hybridoma Bank, Iowa City, IA, USA; or phosphoserine cTnI 23/24, Abcam) 1:1000 in 5% dry milk was allowed to react with blots overnight. Secondary antibodies 1:1000 in 5% dry milk reacted with the blots for 2 h followed by three washes using PBS. Upon completion of the final wash, blots were coated for 5 min with Supersignal West Pico-chemiluminescent substrate (Pierce), which reacts with the secondary antibody. Blots were exposed to photography film for ∼1 min, followed by film development. Relative amounts of cTnT isoforms were determined by measuring the areas under the peaks corresponding to the different cTnT isoforms using QuantiScan (Biosoft, Ferguson, MO, USA) software and an Epson scanner. The extent of Tn exchange was assessed by comparing relative amounts of cTnT (with-myc-tag versus without-myc-tag or cTnT with-myc-tag versus slow skeletal TnT (ssTnT)) before and after Tn exchange.

Results

There is a bimodal distribution in the steepness of sarcomere length–tension relationships that can be modulated by PKA-induced myofibrillar phosphorylation in rat cardiac myocytes (Hanft & McDonald, 2010). In this study we tested the hypothesis that PKA-mediated phosphorylation of cTnI regulates length dependence of force generation in striated muscle. For the first set of experiments, a myocyte preparation was treated with PKA, which yielded a steep length–tension relationship (Fig. 2). Next, endogenous cTn was exchanged for exogenous rat recombinant cTn, which is unphosphorylated. For these experiments, 4 h of cTn exchange in relaxing solution appeared to exchange approximately 50% of the total cTn, as assessed by Western blot analysis of an aliquot of myocytes exchanged in parallel with the myocyte preparation (Fig. 2A). This resulted in a shift to a more shallow length–tension relationship, indicating that phosphorylation of cTnI can shift the length–tension relationship (Fig. 2B). In addition, subsequent PKA treatment after recombinant cTn exchange caused the sarcomere length–tension relationship to again become steep (Fig. 2C). For all these experiments, it is important to reiterate that the pCa solution was adjusted to yield the same force at sarcomere length 2.30 μm for the different conditions in a given experimental preparation (e.g. before and after PKA treatment). This is necessary to adjust for the well-established PKA-induced decrease in Ca²⁺ sensitivity of force.

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.)

We next investigated whether phosphorylation specifically at serines 23/24 of cTnI could control length dependence of force generation. First, a cardiac myocyte preparation that exhibited a shallow sarcomere length–tension relationship was placed in troponin exchange solution containing recombinant cTn complex in which the putative PKA phosphorylation sites serines 23/24 of cTnI had been mutated to aspartic acid to mimic phosphorylation. cTn exchange with pseudo-phosphorylated cTnI steepened the sarcomere length–tension relationship, consistent with phosphorylation of cTnI at serines 23/24 regulating myocyte length dependence of force generation (Fig. 3).

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.)

Last, we tested whether phosphorylation of cTnI could modulate the length–tension relationship in a rat slow-twitch skeletal muscle fibre preparation; slow-twitch skeletal muscle fibres universally exhibit shallow length–tension relationships that are unresponsive to PKA (Konhilas et al. 2002; Hanft & McDonald, 2010). For the first of these experiments, a skinned slow-twitch skeletal muscle fibre was incubated in recombinant cTn exchange solution for approximately 6 h, which replaced ∼40% of the endogenous ssTn with cTn (Fig. 4). This was assessed by Western blot analysis of the same fibre that mechanics were measured. (The control sample was taken by cutting a section of the fibre before attachment to the skinned fibre apparatus.) After recombinant cTn incorporation, the length–tension relationship remained shallow as predicted as recombinant cTnI lacks phosphate incorporation. However, even after the fibre was treated with PKA the length–tension relationship remained shallow (Fig. 4). (This experiment was repeated and confirmed in four different slow-twitch skeletal muscle fibre preparations.) These results suggested that either cTnI phosphorylation is unable to shift the length–tension relationships in a slow-twitch skeletal muscle background or there is a titration effect whereby the cell requires greater than ∼50% phosphorylatable cTnI to elicit a steep length–tension relationship. To test the latter idea we sought to increase the extent of recombinant cTn incorporation by simply doubling the time of Tn exchange. In fact, when most of the endogenous ssTn was replaced with exogenous cTn PKA treatment shifted the sarcomere length–tension relationship from shallow to steep (Fig. 5). Finally, we exchanged (for 12 h) cTn complex containing the pseudo-phosphorylated cTnI into a rat slow-twitch skeletal muscle fibre, notably this resulted in a switch from a shallow to a steep length–tension relationship (Fig. 6). Collectively, these results indicate that phosphorylation of cTnI at serines 23/24 can modulate the length–tension mechanics of striated muscle. These results are consistent with a recent report whereby greater than 50% of cTnI phosphorylated at both serines 23/24 was needed to shift the Ca²⁺ sensitivity of force (Wijnker et al. 2013).

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.)

Discussion

This study indicates that phosphorylation of cTnI at serines 23/24 can control the sarcomere length–active tension relationship in striated muscle. This result provides a myofibrillar mechanism that could account, at least in part, for how ventricles precisely balance circulatory supply and demand on a beat-to-beat basis. For instance, because PKA-mediated phosphorylation of cTnI causes myofilaments to become exquisitely sensitive to length, then as the myofilaments shorten during ejection they have a greater tendency to initiate rapid cooperative deactivation of the thin filaments. This would yield a swift fall in pressure during late ejection and an earlier onset of diastole. This is important as it would favour more diastolic time for filling, which is especially necessary in the context of beta-adrenergic stimulation where heart rates are elevated and diastolic time compressed. This myofilament mechanism provides a means for greater end-diastolic volumes, i.e. ventricles can function at more optimal points on the Frank–Starling relationship. Furthermore, during increased sympathetic drive, intracellular Ca²⁺ transients are amplified; this would favour thin filament active states and tend to prolong ejection. However, based on the data presented here and previously (McDonald et al. 2012), PKA-mediated cTnI phosphorylation will counteract this by enhanced shortening-induced cooperative thin filament deactivation to amplify the inherent myofibrillar ‘brake’ system, which would help overcome the tendency for prolonged active state(s) associated with higher activator [Ca²⁺]_i.

Just how phosphorylation of cTnI alters length–tension relationships remains unknown. Our conceptual idea is that PKA phosphorylation of cTnI increases the cooperative span of active thin filaments (i.e. actin monomers made available for force-generating cross-bridges), which could occur by increasing the persistence length or bending rigidity of adjacent tropomyosin molecules (depicted by straighter Tm molecules in Fig. 7). This model is consistent with functional studies showing an increased Hill coefficient of tension–pCa relationships after PKA in rat skinned cardiac myocyte preparations (Fig. 8). Although not universal, other studies also have observed a tendency for increased Hill coefficients after PKA treatment (or cTnI-DD) in rodent (Strang et al. 1994; Konhilas et al. 2003) and human (Kooij et al. 2010; Wijnker et al. 2013) skinned cardiac muscle preparations. This conceptual model is consistent with ultrastructural studies of troponin, which localized cTnI-serines 23/24 to the highly flexible cardiac isoform-specific N-terminal region of cTnI that interacts with the N-lobe of cTnC (Howarth et al. 2007). In response to bis-phosphorylation of cTnI-serines 23/24, a conformational change occurs that yields more order to a nearby alpha-helix (residues 25–30), and the interaction with the N-lobe of cTnC is weakened (Howarth et al. 2007; and for review, see Solaro et al. 2013). These conformational changes relocate the N-terminal domain of cTnI toward the C-domain of cTnI, where the inhibitory and switch regions reside (Howarth et al. 2007; Warren et al. 2009). This movement of the N-domain of cTnI could yield electrostatic changes that move the C-domain of cTnI and cTnT domains to form a conduit/channel that increases the persistence length of tropomyosin molecules. This greater range in axial mechano-signalling propagation along the thin filament in response to cTnI phosphorylation likely would have two key physiological consequences: (i) greater Ca²⁺-cross-bridge cooperative activation of the thin filaments (a muscle activation mechanism supported by classic myofilament experiments; Moss et al. 1986, 2004; Swartz & Moss, 1992; McKillop & Geeves, 1993; Fitzsimons & Moss, 1998), this would augment force/pressure development early during systole to help maximize ejection; and (ii) as mentioned above, greater shortening-induced cooperative deactivation at late systole in response to cross-bridge detachment associated with myofilament sliding, which would assist in relaxation.

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).

This cTnI-mediated allosteric mechanism might be necessary to increase the rigidity of adjacent Tm molecules as isolated native Tm molecules differ little between their contour (end-to-end length) and persistence length (Loong et al. 2012), and is consistent with the finding that the addition of Tn molecules increased the orientation order of spin-labelled Tm molecules in reconstituted skeletal muscle fibres (Szczesna & Fajer, 1995). These ideas also are consistent with a dilated cardiomyopathy (DCM) missense mutation (D230N) in the tropomyosin gene (TPM 1) that yields reduced Ca²⁺-cross-bridge-induced cooperative activation of isometric force and has been speculated to depress mechano-transmission across adjacent functional units on thin filaments (Lakdawala et al. 2010). In addition, while disease-causing tropomyosin mutations seem rare, a number of cTnI/cTnT mutations have been discovered, which underpins the idea that Tn conformation is vital for tuning the functional gatekeeper role of tropomyosin (Willott et al. 2010; Tardiff, 2011). Further findings that underscore the importance of this putative biophysical mechanism are that several DCM thin filament protein mutations blunt the response to PKA-induced cTnI phosphorylation (for review, see Marston, 2011); this could alter myofilament length dependence responsiveness needed for physiological performance and serve as a trigger for ventricular remodelling.

From a historical perspective, phosphate incorporation into cTnI was first discovered to be highly correlated with force/pressure in isolated rabbit hearts treated with adrenaline using Langendorff perfusion (Solaro et al. 1976). Subsequent biophysical studies addressed the functional consequence of cTnI phosphate incorporation. PKA-induced phosphorylation of cTnI was found to decrease calcium-binding properties of isolated bovine Tn by increasing the off-rate of calcium from cTnC (Robertson et al. 1982), and this was associated with decreased calcium sensitivity of actomyosin ATPase (Holroyde et al. 1979) and force (Hofmann & Lange, 1994; Strang et al. 1994; De Tombe & Stienen, 1995; Janssen & De Tombe, 1997), which was subsequently shown to be specific for the cTnI NH₂-domain (Guo et al. 1994) and serines 23/24 (Malhotra et al. 1997). PKA-mediated phosphorylation of cardiac myofilaments yielded mixed results on the magnitude of change in calcium sensitivity of force between two sarcomere lengths (i.e. ∼2.30 μm and ∼2.00 μm), with some studies showing decreased or no change in length dependence of Ca²⁺ sensitivity of force (Kajiwara et al. 2000; van der Velden et al. 2000; Rao et al. 2012) and others showing increased length dependence (Konhilas et al. 2003; Lee et al. 2013; Sequiera et al. 2013). We recently found PKA-mediated phosphorylation markedly steepened length dependence of force generation when examined over the entire working sarcomere length range (Hanft & McDonald, 2010). The current study extended this work and revealed the molecular (i.e. cTnI) and submolecular (i.e. amino acid residues serines 23/24) mechanisms to account for PKA-induced changes in length dependence of force generation. A likely physiological consequence of this submolecular mechanism is greater shortening-induced cooperative inactivation of the myofibrils, which would assist rapid pressure decline late in systole and early in diastole to optimize filling times. This biophysical-to-physiological mechanism helps explain: (i) the well-established improvement in systolic and diastolic function associated with sympathetic activation (Sarnoff, 1955); (ii) the progressive increase in stroke volume with increased work load in elite athletes (Vella & Robergs, 2012); (iii) the greater isoproterenol response in control mouse hearts compared with hearts containing ssTnI (which lack the NH₂-terminal region; Layland et al. 2004; Nowak et al. 2007); and (iv) the greater −dP/dt_max and frequency dependence of +dP/dt_max in transgenic hearts containing mutated cTnI (serines 23/24asp) that mimics phosphorylation (Sakthivel et al. 2005). Taken together, these studies have brightly illuminated a key submolecular target (i.e. cTnI serines 23/24) to help recover cardiac reserve, which is depressed in both systolic and diastolic heart failure. The importance of targeting improved cardiac reserve in heart failure patients is underscored by these patients’ vastly compromised quality of life, which increases susceptibility to other co-morbidities such as diabetes, obesity, loss of muscle mass and metabolic syndrome.

Glossary

cMyBP-C

cardiac myosin-binding protein-C

cTnC

cardiac troponin C

cTnI

cardiac troponin I

cTnT

cardiac troponin T

DCM

dilated cardiomyopathy

PKA

protein kinase A

ssTnT

slow skeletal TnT

Additional information

Competing interests

None.

Author contributions

All experiments were performed in the laboratory of K.S.M. L.M.H. and K.S.M contributed equally in concept and design of experiments, collection, analysis, interpretation of data, and drafting and revising of the manuscript. B.J.B. expressed and purified the recombinant protein, and assisted in data analysis and drafting and revising the manuscript. All three authors approve the final version to be published.

Funding

This work was supported by a National Heart, Lung, and Blood Institute Grant (R01-HL-57852) to K.S.M., a National Institutes of Health HLRI HL-091056 to B.J.B., and an American Heart Association (Heartland Affiliate) Postdoctoral Fellowship (0825725G) to L.M.H. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.