# Muscle-Type Specific Autophosphorylation of CaMKII Isoforms after Paced Contractions

Authors: Wouter Eilers; Wouter Gevers; Daniëlle van Overbeek; Arnold de Haan; Richard T. Jaspers; Peter A. Hilbers; Natal van Riel; Martin Flück
Journal: BioMed Research International
 (2014)

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## Abstract

We explored to what extent isoforms of the regulator of excitation-contraction and excitation-transcription coupling, calcium/calmodulin protein kinase II (CaMKII) contribute to the specificity of myocellular calcium sensing between muscle types and whether concentration transients in its autophosphorylation can be simulated. CaMKII autophosphorylation at Thr287 was assessed in three muscle compartments of the rat after slow or fast motor unit-type stimulation and was compared against a computational model (CaMuZclE) coupling myocellular calcium dynamics with CaMKII Thr287 phosphorylation. Qualitative differences existed between fast- (gastrocnemius medialis) and slow-type muscle (soleus) for the expression pattern of CaMKII isoforms. Phospho-Thr287 content of δA CaMKII, associated with nuclear functions, demonstrated a transient and compartment-specific increase after excitation, which contrasted to the delayed autophosphorylation of the sarcoplasmic reticulum-associated βM CaMKII. In soleus muscle, excitation-induced δA CaMKII autophosphorylation demonstrated frequency dependence (P = 0.02). In the glycolytic compartment of gastrocnemius medialis, CaMKII autophosphorylation after excitation was blunted. In silico assessment emphasized the importance of mitochondrial calcium buffer capacity for excitation-induced CaMKII autophosphorylation but did not predict its isoform specificity. The findings expose that CaMKII autophosphorylation with paced contractions is regulated in an isoform and muscle type-specific fashion and highlight properties emerging for phenotype-specific regulation of CaMKII.


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## Body

## 1. Introduction

Myocellular calcium is an important second messenger of muscle regulation. This role is mediated by increases in sarcoplasmic calcium concentration with functional recruitment of muscle fibers following motoneuron-induced muscle excitation (i.e., recruitment; [1]). The elevated calcium initiates sarcomere shortening, that is, excitation-contraction coupling, and produces downstream effects on gene regulation, that is, excitation-transcription coupling [2–5]. Three types of motor units can be distinguished based on their contractile and metabolic characteristics, that is: a fast-fatigable, a fast-fatigue-resistant, and a slow-fatigue-resistant type [1, 6]. Thereby it is observed that slow and fast contractile characteristics of motor units are reflected by differences in the (electric) firing pattern of the innervating motoneuron and concentration differences in the rise of myocellular calcium with excitation [7]. Experimental studies have established a specific preference of motoneuron activation frequencies for the three contractile types of motor units [1, 8]. Slow fatigue-resistant motor units are preferentially recruited by low-frequency depolarisation of motor units. In contrast, recruitment of fast-type motor units often occurs at higher stimulation frequencies [9, 10]. Furthermore, the elevated content of the organelles of excitation-contraction coupling, t-tubuli and sarcoplasmic reticulum, in fast-type fibres is associated with larger increases in myocellular calcium concentration during recruitment [11].Along with the calcium/calmodulin-dependent phosphatase calcineurin [7, 14] changes in the activity of calcium/calmodulin dependent kinase II (CaMKII) are a potential interface explaining the downstream effects of elevated myocellular calcium on calcium channel activity and gene regulation [7, 12, 14] and indicated differences between muscle fiber types [7]. To determine the relevance of CaMKII for the regulation of calcium channel activity and gene expression, it is important to understand the regulation of CaMKII activity. CaMKII is a multimeric phosphotransfer enzyme that assembles from different isoforms [15]. Its phosphotransfer activity is induced following rises in intracellular calcium after autophosphorylation of Thr287 in its autoinhibitory domain, enhancing its phosphotransfer activity and rendering it calcium-independent through a subsequent conformational change [15]. Thr287 phosphorylation of CaMKII and a concomitant increase in calcium/calmodulin-independent phosphotransfer activity can be observed within seconds after onset of muscle contraction [12]. Based on model studies of cardiac and brain isoforms, this increase in CaMKII autophosphorylation is believed to rapidly fall within seconds to baseline levels [16, 17]. This is, however, not what can be observed in skeletal muscle where autonomous CaMKII activity can remain specifically elevated after prolonged repetition of contraction, such as running type exercise [18].Four isoforms of CaMKII (i.e.,βM, δA, δD, and γB) have been reported to exist in skeletal muscle [12, 13].In vitro measurements point out characteristic differences in calcium sensitivity of activation between CaMKII isoforms [19, 20]. The biochemical observations are in line with cellular investigations that allocate different functions for the various CaMKII isoforms. For instance, the βM isoform has been found to associate with sarcoplasmic reticulum hinting that it possibly operates in the regulation of calcium channels involved in muscle contraction and relaxation [13]. Conversely, the δD/γB CaMKII, and possibly δA, isoforms are associated with nuclear functions [21–23].In vitro studies of CaMKII revealed that CaMKII autophosphorylation is subject to discrete regulation by the frequency and amplitude of calcium transients, which differs between CaMKII isoforms [24]. Studies in hippocampal cultures identified that Thr286-phosphorylation of the neuron-specific CaMKII isoform α acts as a frequency/number decoder for sensory input [25]. Computational modeling of CaMKII activation by postsynaptic calcium pointed out that autophosphorylation of β CaMKII demonstrates a greater response range than α CaMKII [26]. The extent to which isoform-specific CaMKII autophosphorylation is implicated in the response of muscle fiber types to functional recruitment is not understood.Towards this end we characterised the pattern of CaMKII isoform expression and CaMKII autophosphorylation following electrically paced contractions in phenotypically distinct motor compartments of the rat using muscle-tendon preparationin situ. Specifically we asked whether autophosphorylation of the different isoforms of CaMKII is subject to the same regulation by motoneuron excitation and whether this would differ between three distinct compartments of thetriceps surae muscle group, representing the three types of motor units, when excitation is paced with slow-type and fast-type electric stimulation protocols [9, 10]. Emphasis was put on the question of whether the reported contribution of mitochondria to calcium buffer capacity would exert an influence on Thr287 content of CaMKII [27]. We then asked whether we can reveal mechanistic understanding of the properties of excitation-induced CaMKII regulation between muscle types and isoforms by confronting experimentally observed “on-” and “off-rates” of CaMKII Thr287-phosphorylation with results from a newly assembled computational model.


## 2. Materials and Methods

### 2.1. Design

Four experimental protocols were run to pace contraction of thetriceps surae muscle group of one leg of rats with a slow and fast motor unit-targeted protocol [8, 9]. Three muscle compartments of thetriceps surae were rapidly isolated and subjected to the measure of CaMKII Thr287 phosphorylation with biochemical means. The nonstimulated muscles of the contralateral side served as controls for Protocols 1 and 2. Collaterally, CaMKII Thr287 phosphorylation was assessed using anin silico model combining myocellular calcium dynamics with CaMKII isoform autophosphorylation in different muscle types.


### 2.2. Animals

Three-month-old female Wistar rats (Harlan Laboratories) were anaesthetized by intraperitoneal injection of 1.2 mL/100 gram body weight of 12.5% urethane. Ear and foot reflexes were tested to check whether the animal was sufficiently anaesthetized. Subsequently, injections of 0.3–0.5 mL, up to a maximum of 1.5 mL, were given every 10 minutes afterwards until reflexes had disappeared. Rats were kept on a heated pad (37
±
0.5°C) to prevent hypothermia. Experiments were carried out with approval of the local Animal Experiments Committee at the MOVE Research Institute Amsterdam, VU University Amsterdam. 12 and 20 animals entered the experimental Protocols 1 and 2 (mean body weight of 191–230 grams) and Protocols 3 and 4 (mean body weight of 205–220 grams), respectively.


### 2.3. Muscle-Tendon Preparation

Hind limbs were shaved and the skin was removed, after whichgastrocnemius medialisandsoleusmuscles were exposed and mechanically isolated by removing as much as possible the myofascial connections to surrounding muscles. Blood supply to and nerve innervations ofm. gastrocnemius medialisandm. soleuswere kept intact. The calcaneus was cut from the talus, while still attached to the Achilles tendon. The sciatic nerve was dissected free, proximally severed, and electrically stimulated through an electrode by different protocols being controlled by a computer.Protocol 1 (150 Hz protocol).
Gastrocnemius medialisandsoleus muscles of the right leg were kept below slack length (without determination of optimal length) and electrically stimulated via the sciatic nerve with a train of 100 rectangular electric pulses of 50 microseconds duration and 3 mA amplitude delivered at 150 Hz. Muscles of the left leg served as nonstimulated controls. The experiment was performed at a room temperature of 23°C.Protocol 2 (10 Hz protocol).
Gastrocnemius medialis andsoleus muscles were electrically stimulated as described for Protocol 1 but with the modification of a single train of 100 pulses at 10 Hz.Protocol 3 (tetanic contraction at optimal length).
3 tetanic contractions with 2 minutes of rest in between were imposed ongastrocnemius medialis muscle via the sciatic nerve with a modification of the 150 Hz-protocol where the train consisted of 30, rather than 100, electric pulses of 50 microseconds duration and 3 mA amplitude, at 150 Hz. The temperature of thegastrocnemius muscle was kept at 35°C using an envelope into which warm water vapor was sprayed.Protocol 4 (24 tetanic contractions).
A 2 minute protocol of 24 tetanic contractions (1 contraction every 5 seconds) with the same duration and frequency as described for Protocol3 was imposed. The experiment was performed at 35°C. This protocol resulted in a decrease in maximal tetanic force of approximately 30%.


### 2.4. Sampling

After completion of the protocol, the proximal (i.e., oxidative) and distal (i.e., glycolytic) portion ofgastrocnemius medialis muscle [28] andsoleusmuscle (in Protocols 1 and 2 only) was dissected as rapidly as possible and snap-frozen in liquid nitrogen. The first muscle was typically sampled after 1 minute and the second after 2 minutes. In half of the experiments,soleus muscle was sampled first, and in the other halfgastrocnemius medialismuscle was sampled first. Subsequently, the nonstimulated left muscles were dissected and snap-frozen. Muscles subjected to Protocol 4 were subjected to sampling over a time-course covering 10 and 60 minutes after contraction to estimate the rate of CaMKII dephosphorylation. Rats were subsequently euthanized by intracardial injection of Euthasol while fully anaesthetized. Muscles were stored at −80°C until used for western blot analysis.


### 2.5. Biochemical Analysis of CaMKII Thr287 Phosphorylation

25μm thick cryosections were prepared from the frozen muscles, pooled and homogenized on ice using a Polytron homogeniser (Kinematica AG, Luzern, Switzerland) in ice-cold RIPA buffer (50 mM TRIS-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% v/v Nonidet P40 substitute, and 0.25% w/v sodium deoxycholate, including freshly added protease/phosphatase inhibitors: 1 mM NaF, 1 mM Na3VO4, 0.1 mM PMSF, 1 μg/mL leupeptin, 0.2 μg/mL pepstatin, and 0.1 μg/mL aprotinin. Chemicals were obtained from Sigma-Aldrich (Poole, United Kingdom) unless stated otherwise. Crude homogenates were aspirated 5–10-times through a 0.8 mm syringe needle and stored at −80°C until being further processed. An aliquot of the aspirated homogenate was taken for determination of protein concentration with the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA).Protein levels of total CaMKII, phospho-Thr287-CaMKII, and cytochrome-c oxidase subunit IV (COXIV) were analyzed by western blotting followed by immunodetection. Homogenates were denatured by addition of sample buffer (final concentration: 50 mM TRIS-HCl (pH 6.8), 2% w/v SDS, 10% v/v glycerol, and 2%β-mercaptoethanol) and 5 minutes heating at 95°C. 20 μg of protein per lane was separated with SDS-PAGE on a 15% acrylamide (Biorad) gel and transferred overnight in ice-cold buffer onto a nitrocellulose membrane (GE Healthcare Life Sciences, Little Chalfont, United Kingdom). Membranes were stained with Ponceau S solution to confirm equal protein loading and transfer and then subjected to immunodetection. After blocking, 2-hour incubations were carried out with primary antibody against pan-CaMKII (BD Bioscience, no. 611292, dilution: 1/2500), phospho-Thr287-CaMKII (Cell Signalling Technology, no. 3361, dilution: 1/1000), or COXIV (Cell Signalling Technology, no. 4850, dilution 1/2000) in TTBS (20 mM TRIS-HCl (pH 7.5), 0.9% w/v NaCl, 0.05% Tween-20) with 5% milk (pan-CaMKII, COXIV) or 5% bovine serum albumin (phospho-Thr287-CaMKII) as blocking agent. After serial washes in TTBS, membranes were incubated with species-specific horseradish peroxidase-conjugated secondary antibodies and signal detected enhanced chemiluminescence (Pierce, Rockford, IL, USA) and recorded with a ChemiDoc XRS system (Biorad, Hemel Hempstead, United Kingdom).Samples from the oxidative and glycolyticm. gastrocnemius medialisandm. soleuswere analyzed on separate immunoblots. For each muscle compartment, two sample pairs of stimulated and nonstimulated contralateral control muscles from Protocols 1 and 2, respectively, were analyzed on a same blot. Signal intensity of the protein band of interest was quantified using Quantity One version 4.6.8 (Biorad), background-corrected, and normalized to the average of CaMKII proteins signals from all lanes on the blot. Then for each blot, the normalized signals were related to the total CaMKII signal for the respective resting (i.e., “nonstimulated”) muscle and data pooled between experiments. Finally the values were related to the mean of the “nonstimulated” samples for the respective experimental protocol and subjected to statistical analysis.The specificity of CaMKII and phospho-Thr287-CaMKII detection was ensured in control experiments monitoring calcium/calmodulin-inducible phosphorylation in “cold”in vitrokinase assays as described [18]. In brief, 5 μL of total muscle homogenate was suspended in a reaction mix containing calcium/calmodulin (Enzo Life Sciences, Exeter, United Kingdom) (total volume: 50 μL; 0.1 mM ATP, 10 mM HEPES (pH 7.4), 5 mM MgCl2, 0.1% Tween-20, and 0.5 mM CaCl2 + 1 μM calmodulin or 5 mM EGTA) for 30 minutes at 30°C. The reaction was stopped by the addition of 16.7 μL 4x sample buffer. Samples were heated to 95°C and proteins were separated with SDS-PAGE on a 7.5% (or 15%) acrylamide (Biorad) gel and subjected to western blotting as described. Based on data from [13], we identified the detected CaMKII bands as βM (72 kDa), δA (60 kDa), and a combination of δD and γB (58 kDa), the latter two of which could not be separated in all gels. Therefore, these two CaMKII isoforms were assessed as one band.


### 2.6. Model Development

We set out to develop a computational model to predict calcium driven changes in Thr 287 phosphorylation of CaMKII (termed CaMuZclE for CaMKII muscle model from Zurich-Eindhoven). The model is based on a spatiotemporal model of calcium dynamics in the half sarcomere of fast-twitch muscle [29] and a biochemical model describing CaMKII activation [16] which was reduced to a lumped version by removing the spatial component.The model is based on the interactions between the chemical species inside a sarcomere, consisting of the ions calcium, magnesium and potassium, the calcium buffers calsequestrin, parvalbumin, troponin, adenosine triphosphate (ATP), and mitochondria that act as a calcium buffer, the proteins calmodulin (CaM) and calcium/calmodulin-dependent kinase (CaMKII), CaM buffers, and a calcium-binding dye. Both the sarcoplasmic reticulum and the sarcoplasm are modelled, including the pumps facilitating transport of calcium between the compartments, Ryanodine receptors (RyR), and sarco/endoplasmic reticulum calcium-ATPase pumps (SERCA). Furthermore, the development of contractile force created by contraction is modelled. Reactions are described in terms of changes in the concentration (fluxes) of respective species using coupled ordinary differential equations (ODEs) based on a set of initial concentrations and parameters of the reaction rates. The system of ODEs resulting from these sets is then solved using a numerical integration algorithm, in this case the Matlab (The Mathworks) built-in solver ode15s using an implicit integration scheme with numerical differentiation formulas. The calculation takes around 20 seconds to simulate 10 minutes of experimental time using an Intel core i7 processor (3.4 GHz). Memory usage is negligible (<100 MB). The model was calibrated by measures on sarcoplasmic calcium based on the calcium-binding dye. A schematic representation of all the model components and fluxes can be seen in Figure1. Sensitivity analysis was performed using the multiparametric sensitivity analysis (MPSA) method, describing the speed of the calcium pumps, total calsequestrin concentration, and total CaMKII concentration as most influential parameters (Table 1). Further details on the model equations can be found in the Appendices and Tables 2–6. MPSA was conducted by uniform sampling of 4000 parameter sets from an interval from 75% to 125% of the default parameter set using Latin hypercube sampling, ensuring the sets cover the complete parameter space. Stimulation with a pulse train of 100 pulses at 150 Hz was used (Protocol 1), as this describes a full and complete stimulation. For each parameter set the time course of phospho287-CaMKII was compared to the output with the default set. A sum of squared differences was used as measure. Subsequently, sensitivity values were calculated as described [30].Table 1
Relative influences of the model input parameters on the transient in phosphoThr287-CaMKII.
Parameter
Relative influence
kbta
0.32
kPP1
0.24
CaMax
0.22
KmPP1
0.20
CaPump
0.11
kbi
0.06
kbtc
0.06
kTrop1
0.04
kMitCa2
0.04
kDye2
0.03
kMitCa1
0.03
kATP2
0.03
kCaM42
0.03
kParvCa1
0.03
kParvCa2
0.03
kMitMg1
0.03
kDye1
0.03
kATP1
0.03
kbtb
0.03
kCSQN1
0.02
kCaM0Boff
0.02
KdPump
0.02
kMitMg2
0.02
kCaM20
0.02
kTrop2
0.02
kCSQN2
0.02
kParvMg1
0.02
kParvMg2
0.02
Values were calculated as described under multiparametric sensitivity analysis in Section2.6 “Model Development” of the “Materials and Methods” section. A higher value indicates a larger influence. The most influential parameters are the speed of auto- and dephosphorylation (i.e, kbta, kPPi) and the activity of the calcium pumps (i.e, CaMax, CaPump). For abbreviations consult Tables 2 and 3.Table 2
Initial concentrations.
Name
Value
Unit
Description
Reference
CaMKIItot
1
[μM]
Total CaMKII concentration
http://dare.ubvu.vu.nl/handle/1871/40249
CaMfreeM
0.15
[μM]
Total CaM concentration
http://dare.ubvu.vu.nl/handle/1871/40249
BfreeM
20
[μM]
Total CaM buffer concentration
Saucerman [16]
PP1tot
0.15
[μM]
PP1 concentration
Saucerman [16]
Mg
1000
[μM]
Magnesium concentration sarcoplasm
Saucerman [16]
K
160000
[μM]
Potassium concentration sarcoplasm
Saucerman [16]
CaSRfree
1000
[μM]
Initial calcium concentration in  sarcoplasmic reticulum
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
CSQNT
60000
[μM]
Total calsequestrin concentration
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
Ca
0.1
[μM]
Initial calcium concentration sarcoplasm
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
TropT
205
[μM]
Total troponin C concentration
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
ParvT
1500
[μM]
Total parvalbumin concentration
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
MitT
100
[μM]
Total mitochondria concentration
[35]
ATPT
8000
[μM]
Total APT concentration
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
DyeT
81
[μM]
Total dye concentration
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
Pb2R
0
[μM]
Initial CaMKII Pb2 in state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
PbR
0
[μM]
Initial CaMKII Pb in state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
PtR
0
[μM]
Initial CaMKII Pt in state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
Pt2R
0
[μM]
Initial CaMKII Pt2 in state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
PaR
0
[μM]
Initial CaMKII PaR in state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
PiR
1
[μM]
Initial CaMKII PiR in state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
XB
n
1
[
—
]
Initial fraction of total action/myosin chains inXB
n state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
XB
w
0
[
—
]
Initial fraction of total action/myosin chains inXB
w state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
XB
s
0
[
—
]
Initial fraction of total action/myosin chains inXB
s (force) state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
XB
r
0
[
—
]
Initial fraction of total action/myosin chains inXB
r state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/Figure 1
Schematic representation of the components of the CaMuZclE model to simulate calcium-driven Thr287 phosphorylation of CaMKII in skeletal muscle. The model includes calcium flow through the sarcoplasmic reticulum and sarcoplasm. Calcium is bound to the buffer calsequestrin in the SR and is transported by RyRs channel and SERCA pump between the SR and sarcoplasm. Within the sarcoplasm calcium binds to the buffers ATP and parvalbumin, mitochondria, and troponin C, initiating sarcomere shortening and force production. Furthermore, calcium binds to the calcium sensor calmodulin, which in turn activates the phosphotransferase CaMKII, initiating several pathways. A comprehensive list of all abbreviations can be found in the Appendices.Interdependent parameters were excluded as these variables change together with the variables they are dependent on. Exponentials of the description of the RyR activity were excluded as well as the RyR activity is regulated by varying the parameter CaMax. The remaining parameters are ordered with respect to their influence on the trace of phosphorylated CaMKII, as calculated via the MPSA method. The MPSA results were used to perform uncertainty analysis [31]. Poisson distributions were fitted to the set of output transients resulting from MPSA and were used to draw 95% confidence intervals. For clarity, only the trace of the β
M CaMKII isoform is presented; the traces of the other isoforms display similar behavior.


### 2.7. Statistics

All statistical tests were performed using Statistica 10.0 (Statsoft Inc., Tulsa, OK, USA). The effect of stimulation with Protocols1 and 2 on total and phospho-Thr287-CaMKII levels was tested with repeated-measures ANOVA on the factor “stimulation” [stimulated, rest] with a post hoc test of Fisher. Additional factors were “CaMKII isoform” [βM, δA, δD/γB], ‘stimulation frequency’ [10 Hz, 150 Hz], and muscle compartment [glycolyticgastrocnemius medialis, oxidativegastrocnemius medialis, andsoleus]. These were run separately or in combination. The effect of stimulation with Protocols 3 and 4 on phospho-Thr287-CaMKII levels was tested with ANOVAs on the factor “stimulation” [rest, after 3 tetanic contractions, after 24 tetanic contraction] × “muscle compartment” [glycolyticgastrocnemius medialis, oxidativegastrocnemius medialis] × “CaMKII isoform” [βM, δA, δd/γB] with a post hoc test of Fisher. Two-sided post hoc tests were carried out to analyze the effect of electric stimulation on phospho-Thr287-CaMKII.


## 2.1. Design

Four experimental protocols were run to pace contraction of thetriceps surae muscle group of one leg of rats with a slow and fast motor unit-targeted protocol [8, 9]. Three muscle compartments of thetriceps surae were rapidly isolated and subjected to the measure of CaMKII Thr287 phosphorylation with biochemical means. The nonstimulated muscles of the contralateral side served as controls for Protocols 1 and 2. Collaterally, CaMKII Thr287 phosphorylation was assessed using anin silico model combining myocellular calcium dynamics with CaMKII isoform autophosphorylation in different muscle types.


## 2.2. Animals

Three-month-old female Wistar rats (Harlan Laboratories) were anaesthetized by intraperitoneal injection of 1.2 mL/100 gram body weight of 12.5% urethane. Ear and foot reflexes were tested to check whether the animal was sufficiently anaesthetized. Subsequently, injections of 0.3–0.5 mL, up to a maximum of 1.5 mL, were given every 10 minutes afterwards until reflexes had disappeared. Rats were kept on a heated pad (37
±
0.5°C) to prevent hypothermia. Experiments were carried out with approval of the local Animal Experiments Committee at the MOVE Research Institute Amsterdam, VU University Amsterdam. 12 and 20 animals entered the experimental Protocols 1 and 2 (mean body weight of 191–230 grams) and Protocols 3 and 4 (mean body weight of 205–220 grams), respectively.


## 2.3. Muscle-Tendon Preparation

Hind limbs were shaved and the skin was removed, after whichgastrocnemius medialisandsoleusmuscles were exposed and mechanically isolated by removing as much as possible the myofascial connections to surrounding muscles. Blood supply to and nerve innervations ofm. gastrocnemius medialisandm. soleuswere kept intact. The calcaneus was cut from the talus, while still attached to the Achilles tendon. The sciatic nerve was dissected free, proximally severed, and electrically stimulated through an electrode by different protocols being controlled by a computer.Protocol 1 (150 Hz protocol).
Gastrocnemius medialisandsoleus muscles of the right leg were kept below slack length (without determination of optimal length) and electrically stimulated via the sciatic nerve with a train of 100 rectangular electric pulses of 50 microseconds duration and 3 mA amplitude delivered at 150 Hz. Muscles of the left leg served as nonstimulated controls. The experiment was performed at a room temperature of 23°C.Protocol 2 (10 Hz protocol).
Gastrocnemius medialis andsoleus muscles were electrically stimulated as described for Protocol 1 but with the modification of a single train of 100 pulses at 10 Hz.Protocol 3 (tetanic contraction at optimal length).
3 tetanic contractions with 2 minutes of rest in between were imposed ongastrocnemius medialis muscle via the sciatic nerve with a modification of the 150 Hz-protocol where the train consisted of 30, rather than 100, electric pulses of 50 microseconds duration and 3 mA amplitude, at 150 Hz. The temperature of thegastrocnemius muscle was kept at 35°C using an envelope into which warm water vapor was sprayed.Protocol 4 (24 tetanic contractions).
A 2 minute protocol of 24 tetanic contractions (1 contraction every 5 seconds) with the same duration and frequency as described for Protocol3 was imposed. The experiment was performed at 35°C. This protocol resulted in a decrease in maximal tetanic force of approximately 30%.


## 2.4. Sampling

After completion of the protocol, the proximal (i.e., oxidative) and distal (i.e., glycolytic) portion ofgastrocnemius medialis muscle [28] andsoleusmuscle (in Protocols 1 and 2 only) was dissected as rapidly as possible and snap-frozen in liquid nitrogen. The first muscle was typically sampled after 1 minute and the second after 2 minutes. In half of the experiments,soleus muscle was sampled first, and in the other halfgastrocnemius medialismuscle was sampled first. Subsequently, the nonstimulated left muscles were dissected and snap-frozen. Muscles subjected to Protocol 4 were subjected to sampling over a time-course covering 10 and 60 minutes after contraction to estimate the rate of CaMKII dephosphorylation. Rats were subsequently euthanized by intracardial injection of Euthasol while fully anaesthetized. Muscles were stored at −80°C until used for western blot analysis.


## 2.5. Biochemical Analysis of CaMKII Thr287 Phosphorylation

25μm thick cryosections were prepared from the frozen muscles, pooled and homogenized on ice using a Polytron homogeniser (Kinematica AG, Luzern, Switzerland) in ice-cold RIPA buffer (50 mM TRIS-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% v/v Nonidet P40 substitute, and 0.25% w/v sodium deoxycholate, including freshly added protease/phosphatase inhibitors: 1 mM NaF, 1 mM Na3VO4, 0.1 mM PMSF, 1 μg/mL leupeptin, 0.2 μg/mL pepstatin, and 0.1 μg/mL aprotinin. Chemicals were obtained from Sigma-Aldrich (Poole, United Kingdom) unless stated otherwise. Crude homogenates were aspirated 5–10-times through a 0.8 mm syringe needle and stored at −80°C until being further processed. An aliquot of the aspirated homogenate was taken for determination of protein concentration with the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA).Protein levels of total CaMKII, phospho-Thr287-CaMKII, and cytochrome-c oxidase subunit IV (COXIV) were analyzed by western blotting followed by immunodetection. Homogenates were denatured by addition of sample buffer (final concentration: 50 mM TRIS-HCl (pH 6.8), 2% w/v SDS, 10% v/v glycerol, and 2%β-mercaptoethanol) and 5 minutes heating at 95°C. 20 μg of protein per lane was separated with SDS-PAGE on a 15% acrylamide (Biorad) gel and transferred overnight in ice-cold buffer onto a nitrocellulose membrane (GE Healthcare Life Sciences, Little Chalfont, United Kingdom). Membranes were stained with Ponceau S solution to confirm equal protein loading and transfer and then subjected to immunodetection. After blocking, 2-hour incubations were carried out with primary antibody against pan-CaMKII (BD Bioscience, no. 611292, dilution: 1/2500), phospho-Thr287-CaMKII (Cell Signalling Technology, no. 3361, dilution: 1/1000), or COXIV (Cell Signalling Technology, no. 4850, dilution 1/2000) in TTBS (20 mM TRIS-HCl (pH 7.5), 0.9% w/v NaCl, 0.05% Tween-20) with 5% milk (pan-CaMKII, COXIV) or 5% bovine serum albumin (phospho-Thr287-CaMKII) as blocking agent. After serial washes in TTBS, membranes were incubated with species-specific horseradish peroxidase-conjugated secondary antibodies and signal detected enhanced chemiluminescence (Pierce, Rockford, IL, USA) and recorded with a ChemiDoc XRS system (Biorad, Hemel Hempstead, United Kingdom).Samples from the oxidative and glycolyticm. gastrocnemius medialisandm. soleuswere analyzed on separate immunoblots. For each muscle compartment, two sample pairs of stimulated and nonstimulated contralateral control muscles from Protocols 1 and 2, respectively, were analyzed on a same blot. Signal intensity of the protein band of interest was quantified using Quantity One version 4.6.8 (Biorad), background-corrected, and normalized to the average of CaMKII proteins signals from all lanes on the blot. Then for each blot, the normalized signals were related to the total CaMKII signal for the respective resting (i.e., “nonstimulated”) muscle and data pooled between experiments. Finally the values were related to the mean of the “nonstimulated” samples for the respective experimental protocol and subjected to statistical analysis.The specificity of CaMKII and phospho-Thr287-CaMKII detection was ensured in control experiments monitoring calcium/calmodulin-inducible phosphorylation in “cold”in vitrokinase assays as described [18]. In brief, 5 μL of total muscle homogenate was suspended in a reaction mix containing calcium/calmodulin (Enzo Life Sciences, Exeter, United Kingdom) (total volume: 50 μL; 0.1 mM ATP, 10 mM HEPES (pH 7.4), 5 mM MgCl2, 0.1% Tween-20, and 0.5 mM CaCl2 + 1 μM calmodulin or 5 mM EGTA) for 30 minutes at 30°C. The reaction was stopped by the addition of 16.7 μL 4x sample buffer. Samples were heated to 95°C and proteins were separated with SDS-PAGE on a 7.5% (or 15%) acrylamide (Biorad) gel and subjected to western blotting as described. Based on data from [13], we identified the detected CaMKII bands as βM (72 kDa), δA (60 kDa), and a combination of δD and γB (58 kDa), the latter two of which could not be separated in all gels. Therefore, these two CaMKII isoforms were assessed as one band.


## 2.6. Model Development

We set out to develop a computational model to predict calcium driven changes in Thr 287 phosphorylation of CaMKII (termed CaMuZclE for CaMKII muscle model from Zurich-Eindhoven). The model is based on a spatiotemporal model of calcium dynamics in the half sarcomere of fast-twitch muscle [29] and a biochemical model describing CaMKII activation [16] which was reduced to a lumped version by removing the spatial component.The model is based on the interactions between the chemical species inside a sarcomere, consisting of the ions calcium, magnesium and potassium, the calcium buffers calsequestrin, parvalbumin, troponin, adenosine triphosphate (ATP), and mitochondria that act as a calcium buffer, the proteins calmodulin (CaM) and calcium/calmodulin-dependent kinase (CaMKII), CaM buffers, and a calcium-binding dye. Both the sarcoplasmic reticulum and the sarcoplasm are modelled, including the pumps facilitating transport of calcium between the compartments, Ryanodine receptors (RyR), and sarco/endoplasmic reticulum calcium-ATPase pumps (SERCA). Furthermore, the development of contractile force created by contraction is modelled. Reactions are described in terms of changes in the concentration (fluxes) of respective species using coupled ordinary differential equations (ODEs) based on a set of initial concentrations and parameters of the reaction rates. The system of ODEs resulting from these sets is then solved using a numerical integration algorithm, in this case the Matlab (The Mathworks) built-in solver ode15s using an implicit integration scheme with numerical differentiation formulas. The calculation takes around 20 seconds to simulate 10 minutes of experimental time using an Intel core i7 processor (3.4 GHz). Memory usage is negligible (<100 MB). The model was calibrated by measures on sarcoplasmic calcium based on the calcium-binding dye. A schematic representation of all the model components and fluxes can be seen in Figure1. Sensitivity analysis was performed using the multiparametric sensitivity analysis (MPSA) method, describing the speed of the calcium pumps, total calsequestrin concentration, and total CaMKII concentration as most influential parameters (Table 1). Further details on the model equations can be found in the Appendices and Tables 2–6. MPSA was conducted by uniform sampling of 4000 parameter sets from an interval from 75% to 125% of the default parameter set using Latin hypercube sampling, ensuring the sets cover the complete parameter space. Stimulation with a pulse train of 100 pulses at 150 Hz was used (Protocol 1), as this describes a full and complete stimulation. For each parameter set the time course of phospho287-CaMKII was compared to the output with the default set. A sum of squared differences was used as measure. Subsequently, sensitivity values were calculated as described [30].Table 1
Relative influences of the model input parameters on the transient in phosphoThr287-CaMKII.
Parameter
Relative influence
kbta
0.32
kPP1
0.24
CaMax
0.22
KmPP1
0.20
CaPump
0.11
kbi
0.06
kbtc
0.06
kTrop1
0.04
kMitCa2
0.04
kDye2
0.03
kMitCa1
0.03
kATP2
0.03
kCaM42
0.03
kParvCa1
0.03
kParvCa2
0.03
kMitMg1
0.03
kDye1
0.03
kATP1
0.03
kbtb
0.03
kCSQN1
0.02
kCaM0Boff
0.02
KdPump
0.02
kMitMg2
0.02
kCaM20
0.02
kTrop2
0.02
kCSQN2
0.02
kParvMg1
0.02
kParvMg2
0.02
Values were calculated as described under multiparametric sensitivity analysis in Section2.6 “Model Development” of the “Materials and Methods” section. A higher value indicates a larger influence. The most influential parameters are the speed of auto- and dephosphorylation (i.e, kbta, kPPi) and the activity of the calcium pumps (i.e, CaMax, CaPump). For abbreviations consult Tables 2 and 3.Table 2
Initial concentrations.
Name
Value
Unit
Description
Reference
CaMKIItot
1
[μM]
Total CaMKII concentration
http://dare.ubvu.vu.nl/handle/1871/40249
CaMfreeM
0.15
[μM]
Total CaM concentration
http://dare.ubvu.vu.nl/handle/1871/40249
BfreeM
20
[μM]
Total CaM buffer concentration
Saucerman [16]
PP1tot
0.15
[μM]
PP1 concentration
Saucerman [16]
Mg
1000
[μM]
Magnesium concentration sarcoplasm
Saucerman [16]
K
160000
[μM]
Potassium concentration sarcoplasm
Saucerman [16]
CaSRfree
1000
[μM]
Initial calcium concentration in  sarcoplasmic reticulum
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
CSQNT
60000
[μM]
Total calsequestrin concentration
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
Ca
0.1
[μM]
Initial calcium concentration sarcoplasm
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
TropT
205
[μM]
Total troponin C concentration
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
ParvT
1500
[μM]
Total parvalbumin concentration
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
MitT
100
[μM]
Total mitochondria concentration
[35]
ATPT
8000
[μM]
Total APT concentration
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
DyeT
81
[μM]
Total dye concentration
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
Pb2R
0
[μM]
Initial CaMKII Pb2 in state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
PbR
0
[μM]
Initial CaMKII Pb in state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
PtR
0
[μM]
Initial CaMKII Pt in state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
Pt2R
0
[μM]
Initial CaMKII Pt2 in state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
PaR
0
[μM]
Initial CaMKII PaR in state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
PiR
1
[μM]
Initial CaMKII PiR in state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
XB
n
1
[
—
]
Initial fraction of total action/myosin chains inXB
n state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
XB
w
0
[
—
]
Initial fraction of total action/myosin chains inXB
w state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
XB
s
0
[
—
]
Initial fraction of total action/myosin chains inXB
s (force) state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/
XB
r
0
[
—
]
Initial fraction of total action/myosin chains inXB
r state
http://www.tue.nl/en/publication/ep/p/d/ep-uid/247565/Figure 1
Schematic representation of the components of the CaMuZclE model to simulate calcium-driven Thr287 phosphorylation of CaMKII in skeletal muscle. The model includes calcium flow through the sarcoplasmic reticulum and sarcoplasm. Calcium is bound to the buffer calsequestrin in the SR and is transported by RyRs channel and SERCA pump between the SR and sarcoplasm. Within the sarcoplasm calcium binds to the buffers ATP and parvalbumin, mitochondria, and troponin C, initiating sarcomere shortening and force production. Furthermore, calcium binds to the calcium sensor calmodulin, which in turn activates the phosphotransferase CaMKII, initiating several pathways. A comprehensive list of all abbreviations can be found in the Appendices.Interdependent parameters were excluded as these variables change together with the variables they are dependent on. Exponentials of the description of the RyR activity were excluded as well as the RyR activity is regulated by varying the parameter CaMax. The remaining parameters are ordered with respect to their influence on the trace of phosphorylated CaMKII, as calculated via the MPSA method. The MPSA results were used to perform uncertainty analysis [31]. Poisson distributions were fitted to the set of output transients resulting from MPSA and were used to draw 95% confidence intervals. For clarity, only the trace of the β
M CaMKII isoform is presented; the traces of the other isoforms display similar behavior.


## 2.7. Statistics

All statistical tests were performed using Statistica 10.0 (Statsoft Inc., Tulsa, OK, USA). The effect of stimulation with Protocols1 and 2 on total and phospho-Thr287-CaMKII levels was tested with repeated-measures ANOVA on the factor “stimulation” [stimulated, rest] with a post hoc test of Fisher. Additional factors were “CaMKII isoform” [βM, δA, δD/γB], ‘stimulation frequency’ [10 Hz, 150 Hz], and muscle compartment [glycolyticgastrocnemius medialis, oxidativegastrocnemius medialis, andsoleus]. These were run separately or in combination. The effect of stimulation with Protocols 3 and 4 on phospho-Thr287-CaMKII levels was tested with ANOVAs on the factor “stimulation” [rest, after 3 tetanic contractions, after 24 tetanic contraction] × “muscle compartment” [glycolyticgastrocnemius medialis, oxidativegastrocnemius medialis] × “CaMKII isoform” [βM, δA, δd/γB] with a post hoc test of Fisher. Two-sided post hoc tests were carried out to analyze the effect of electric stimulation on phospho-Thr287-CaMKII.


## 3. Results

### 3.1. CaMKII Isoforms in Skeletal Muscle

Four CaMKII isoforms, that is,βM, δA, and δd/γB were identified in ratgastrocnemius muscle in accordance with Rose [12] and Bayer ([13]; Figures 2
(
a
) and 2
(
b
)).In vitro autokinase assays confirmed the specificity of detecting CaMKII phosphorylation at Thr287.The abundance of CaMKII isoforms did not differ between the glycolytic and oxidative compartment ofgastrocnemius medialis muscle (Figures 2(c), 2(e)/2(f)). The phosphorylation levels of CaMKII at Thr287 were however 2-fold higher in the glycolytic compared to the oxidative compartment ofgastrocnemius medialis muscle (P
≤
0.01; Figure 2(d)). In the oxidative muscle,m. soleus, lower levels of the δd/γB CaMKII isoform and two bands at the height of βM were identified (Figure 2(c)).Calcium/calmodulin-dependent kinase II isoforms in rat skeletal muscle. Total homogenate of agastrocnemius muscle from a rat was prepared and subjected toin vitro kinase reactions in the presence of EGTA or calcium/calmodulin and was subjected to immunoblotting with a pan-CaMKII (a) and phospho-Thr287 specific antibody (b). CaMKII isoforms were then assigned based on a calcium/calmodulin-inducible phospho-Thr287 signal and a detection of similar sized bands according to the nomenclature established by Rose [12] and Bayer [13]. (c)(d) CaMKII isoforms (c) and Thr287 phosphorylated CaMKII (d) in equal protein amount in total homogenate of fatigue resistant (i.e., oxidative) and fatigable (i.e., glycolytic) compartments of the fastgastrocnemius medialis muscle and the slow fatigue resistantsoleus muscle. Lanes originating from different parts of SDS-PAGE gels are separated by black lines. A second band at the height of the βM isoform is detected insoleus muscle. (e) Graph displays COXIV levels in the oxidative and glycolytic compartments ofgastrocnemius medialis as determined by western blotting followed by immunodetection. Bars represent mean COXIV levels and symbols represent the levels in individual sample pairs. Symbols being connected by a stippled line reflect intra-animal pairs. TheP-level of the difference between red and white GM is indicated (paired t-test). (f) Example immunoblot showing the detection of COXIV protein and the actin loading control in the oxidative and glycolytic compartments ofgastrocnemius medialis.
(a)
(b)
(c)
(d)
(e)
(f)


### 3.2. CaMKII Phosphorylation in Fast-Type MuscleIn Silico is Graded by the Content of Mitochondria

Calculations were run using CaMuZclE to assess CaMKII-Thr287 phosphorylation in the two compartments of the fast-typegastrocnemius medialis muscle after pacing the muscle with a fast-type, 150 Hz protocol. The results of the calculations for these twogastrocnemius compartments are shown in Figure 3.Thr287 phosphorylation of CaMKII in fast muscle compartment after paced contractionsin silico. (a)(b) Line graphs displaying the level changes of calcium (a) and CaMKII-Thr287 (b) in the glycolytic and oxidative compartments ofgastrocnemius muscle after an input signal of 100 pulses at 150 Hz.
(a)
(b)Thein silico experiment demonstrated a further pronounced increase in Thr287 phosphorylation of CaMKII in the glycolytic compared to the oxidative compartment of thegastrocnemius medialis muscle after paced contractions. Essentially, the same result arose when the calculations were run for individual CaMKII isoforms, yet the degree of peak Thr287 phosphorylation differed in the order βM > δd/γB > δA (data not shown).Multiparametric sensitivity analysis was carried out to assess the influence of the model parameters on the simulation of calcium-induced Thr287 phosphorylation of CaMKII (Figure4). Table 1 shows these results in order of decreasing influence of the assessed parameters.Figure 4
Phospho-Thr287-transient ofβM CaMKII with 95% confidence intervals. Poisson distribution fitted to the set of output transients obtained by 4000 Latin hypercube-sampled input parameters sets from 75% to 125% of default values. The stimulation protocol with a pulse train of 100 pulses at 150 Hz was used and the model was set up for fast glycolytic muscle. The black line indicates the output transient using the default parameter set. The stippled blue lines indicate the upper and lower 2.5% confidence limit of the Poisson distribution.


### 3.3. CaMKII Phosphorylation after Paced Contractions in Glycolytic and Oxidative Compartment of Fast-Type MuscleIn Situ

Contraction ofgastrocnemius medialismuscle was stimulated by applying electric pulses at 10 Hz or 150 Hz to the sciatic nerve (Protocols 1 and 2). The biochemical characterisation identified an increase in Thr287-phosphorylation of all combined CaMKII isoforms in the oxidative compartment ofgastrocnemius medialismuscle (P
=
0.002). The analysis of individual isoforms demonstrated increased phospho-Thr287 levels of δA CaMKII (P
=
0.02), but not βM (P
=
0.07) and δD/γB CaMKII (P
=
0.17), in the oxidative muscle compartment (Figures 5(a) and 5(b)). The levels of neither CaMKII isoform differed between resting and 150 Hz stimulated in the oxidative compartment (all P-values > 0.30; data not shown).Thr287 phosphorylation of CaMKII in glycolytic and oxidative fast muscle compartment after paced contractionsin situ. (a) Immunoblot showing the phospho-Thr287-CaMKII signal in stimulated and resting controlgastrocnemius medialis muscle (oxidative compartment) after stimulation of the sciatic nerve with a train of 100 pulses of a slow (i.e., 10 Hz) or a fast (i.e., 150 Hz) motor unit-targeted protocol [9]. (b)(c) Bar graphs visualizing the mean + SE of changes in phospho-Thr287-CaMKII levels with stimulation of the oxidative (b) and glycolytic (c) compartment ofgastrocnemius medialis. Data reflect combined values from the stimulation 10-Hz and 150-Hz protocol. *denotes P
<
0.05 versus rest (two-sided paired t-test, n
=
12–14).
(a)
(b)
(c)In the glycolytic compartment, Thr287 phosphorylation of CaMKII tended to be less affected than in the oxidative portion ofgastrocnemius medialis after stimulation (P
=
0.055, for the interaction of “stimulation” × “muscle compartment”). In the glycolytic portion none of the CaMKII isoforms demonstrated regulation after stimulation (Figure 5(c)). No interaction effect was identified between stimulation with the slow versus the fast-type protocol in either portion ofgastrocnemius medialismuscle for Thr287 phosphorylation.


### 3.4. Frequency DependentδA CaMKII Isoform Phosphorylation in the Slow Oxidative Compartment

We assessed CaMKII isoform phosphorylation in a third muscle of thetriceps surae complex with a slow-oxidative phenotype, that is, them. soleus, after stimulation with the slow-type (10 Hz) and fast-type (150 Hz) protocol, respectively (Figure 6(a)). The characterisation identified a trend for an interaction between CaMKII isoform and stimulation frequency (P
=
0.08). When assessing phosphorylation separately per CaMKII isoform we identify a frequency-dependent Thr287 phosphorylation of the δA-CaMKII isoform (P
=
0.017; Figure 6(b)). Phospho-Thr287 levels of this isoform were selectively downregulated after electric stimulation with the fast-type protocol, but it was increased with the slow-type protocol (Figure 6(c)).Frequency dependent CaMKII isoform autophosphorylation in slow oxidative muscle after paced contractionsin situ. (a) Example of Thr 287 phosphorylation of CaMKII isoforms in the stimulated (stim)soleus muscle and its contralateral control (rest) after electric stimulation with 150 Hz (Protocol 1) and 10 Hz (Protocol 2). The position of the respective bands is indicated by an arrow. Please note the presence of a second band at the height of the βM isoform in stimulated muscle. (b) Composite figure visualising the fold changes in CaMKII-Thr287 content of CaMKII isoforms for slow and fast motor unit-targeted stimulation for CaMKII isoforms in slow oxidativesoleus muscle in a colour code. (c) Line graph resolving the interaction effect of stimulation frequency on Thr287 phosphorylation of the δA-CaMKII isoform. Repeated ANOVA with post hoc test of Fisher, n
=
6–10.
(a)
(b)
(c)


### 3.5. Muscle Specific CaMKII Activation by Repeated Tetanic Contractions of Gastrocnemius Muscle

We investigated whether CaMKII activation in glycolytic and oxidative compartment of fast-twitch muscle would be affected with further repetitions of tetanic contractions. The computational model predicted a rapid increase in CaMKII phosphorylation for all isoforms after repeated tetanic contractions in the oxidative portion of thegastrocnemius muscle which followed the order βM > δD/γB > δA. Thr287 phosphorylation had early maxima after 1-2 contractions before falling to near resting values (Figures 7(a) and 7(b)).Isoform specific CaMKII autophosphorylation with repeated contractionsin situ. (a)(b) Line graph of the calculated changes in Thr287 phosphorylation of CaMKII isoforms in the oxidative (a) and glycolytic (b) compartment ofgastrocnemius medialis muscle as a function of the number of tetanic contractions as paced with repeated trains of 30 pulses of 150 Hz with 4.8 seconds of rest between trains. (c)–(h) Representative immunoblots (c)(d) and bar graphs of mean (e)–(h) of Thr287 phosphorylated CaMKII isoforms in the oxidative (c) (e) (g) and glycolytic (d) (f) (h) compartment ofgastrocnemius medialis muscle after 3 and 24 repeated tetanic contractions stimulated via the sciatic nerve with trains of 30 pulses at 150 Hz. *denotes P
<
0.05 versus rest (ANOVA with post hoc test of Fisher, n
=
6).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)Except for theδa isoform, the simulated biphasic relationship between phospho-Thr287 and the number of contraction was not what we found experimentallyin situ. There was a significant interaction effect of the stimulation and compartment (P
=
0.02) for Thr287 phosphorylation of CaMKIIin situ. Experimentsin situ reproduced the model observations on the sensitivity of phospho-Thr287 content of CaMKII in the oxidative compartment ofgastrocnemius medialis and pointed out isoform-specific phosphorylation in this muscle. Thr287 phosphorylation of the δA CaMKII isoform remained increased after three tetanic contractions but ceased after 24 contractions (Figure 7(g)). By contrast Thr287 content of the βM isoform was first significantly increased after 24 tetanic contractions (Figure 7(e)).


## 3.1. CaMKII Isoforms in Skeletal Muscle

Four CaMKII isoforms, that is,βM, δA, and δd/γB were identified in ratgastrocnemius muscle in accordance with Rose [12] and Bayer ([13]; Figures 2
(
a
) and 2
(
b
)).In vitro autokinase assays confirmed the specificity of detecting CaMKII phosphorylation at Thr287.The abundance of CaMKII isoforms did not differ between the glycolytic and oxidative compartment ofgastrocnemius medialis muscle (Figures 2(c), 2(e)/2(f)). The phosphorylation levels of CaMKII at Thr287 were however 2-fold higher in the glycolytic compared to the oxidative compartment ofgastrocnemius medialis muscle (P
≤
0.01; Figure 2(d)). In the oxidative muscle,m. soleus, lower levels of the δd/γB CaMKII isoform and two bands at the height of βM were identified (Figure 2(c)).Calcium/calmodulin-dependent kinase II isoforms in rat skeletal muscle. Total homogenate of agastrocnemius muscle from a rat was prepared and subjected toin vitro kinase reactions in the presence of EGTA or calcium/calmodulin and was subjected to immunoblotting with a pan-CaMKII (a) and phospho-Thr287 specific antibody (b). CaMKII isoforms were then assigned based on a calcium/calmodulin-inducible phospho-Thr287 signal and a detection of similar sized bands according to the nomenclature established by Rose [12] and Bayer [13]. (c)(d) CaMKII isoforms (c) and Thr287 phosphorylated CaMKII (d) in equal protein amount in total homogenate of fatigue resistant (i.e., oxidative) and fatigable (i.e., glycolytic) compartments of the fastgastrocnemius medialis muscle and the slow fatigue resistantsoleus muscle. Lanes originating from different parts of SDS-PAGE gels are separated by black lines. A second band at the height of the βM isoform is detected insoleus muscle. (e) Graph displays COXIV levels in the oxidative and glycolytic compartments ofgastrocnemius medialis as determined by western blotting followed by immunodetection. Bars represent mean COXIV levels and symbols represent the levels in individual sample pairs. Symbols being connected by a stippled line reflect intra-animal pairs. TheP-level of the difference between red and white GM is indicated (paired t-test). (f) Example immunoblot showing the detection of COXIV protein and the actin loading control in the oxidative and glycolytic compartments ofgastrocnemius medialis.
(a)
(b)
(c)
(d)
(e)
(f)


## 3.2. CaMKII Phosphorylation in Fast-Type MuscleIn Silico is Graded by the Content of Mitochondria

Calculations were run using CaMuZclE to assess CaMKII-Thr287 phosphorylation in the two compartments of the fast-typegastrocnemius medialis muscle after pacing the muscle with a fast-type, 150 Hz protocol. The results of the calculations for these twogastrocnemius compartments are shown in Figure 3.Thr287 phosphorylation of CaMKII in fast muscle compartment after paced contractionsin silico. (a)(b) Line graphs displaying the level changes of calcium (a) and CaMKII-Thr287 (b) in the glycolytic and oxidative compartments ofgastrocnemius muscle after an input signal of 100 pulses at 150 Hz.
(a)
(b)Thein silico experiment demonstrated a further pronounced increase in Thr287 phosphorylation of CaMKII in the glycolytic compared to the oxidative compartment of thegastrocnemius medialis muscle after paced contractions. Essentially, the same result arose when the calculations were run for individual CaMKII isoforms, yet the degree of peak Thr287 phosphorylation differed in the order βM > δd/γB > δA (data not shown).Multiparametric sensitivity analysis was carried out to assess the influence of the model parameters on the simulation of calcium-induced Thr287 phosphorylation of CaMKII (Figure4). Table 1 shows these results in order of decreasing influence of the assessed parameters.Figure 4
Phospho-Thr287-transient ofβM CaMKII with 95% confidence intervals. Poisson distribution fitted to the set of output transients obtained by 4000 Latin hypercube-sampled input parameters sets from 75% to 125% of default values. The stimulation protocol with a pulse train of 100 pulses at 150 Hz was used and the model was set up for fast glycolytic muscle. The black line indicates the output transient using the default parameter set. The stippled blue lines indicate the upper and lower 2.5% confidence limit of the Poisson distribution.


## 3.3. CaMKII Phosphorylation after Paced Contractions in Glycolytic and Oxidative Compartment of Fast-Type MuscleIn Situ

Contraction ofgastrocnemius medialismuscle was stimulated by applying electric pulses at 10 Hz or 150 Hz to the sciatic nerve (Protocols 1 and 2). The biochemical characterisation identified an increase in Thr287-phosphorylation of all combined CaMKII isoforms in the oxidative compartment ofgastrocnemius medialismuscle (P
=
0.002). The analysis of individual isoforms demonstrated increased phospho-Thr287 levels of δA CaMKII (P
=
0.02), but not βM (P
=
0.07) and δD/γB CaMKII (P
=
0.17), in the oxidative muscle compartment (Figures 5(a) and 5(b)). The levels of neither CaMKII isoform differed between resting and 150 Hz stimulated in the oxidative compartment (all P-values > 0.30; data not shown).Thr287 phosphorylation of CaMKII in glycolytic and oxidative fast muscle compartment after paced contractionsin situ. (a) Immunoblot showing the phospho-Thr287-CaMKII signal in stimulated and resting controlgastrocnemius medialis muscle (oxidative compartment) after stimulation of the sciatic nerve with a train of 100 pulses of a slow (i.e., 10 Hz) or a fast (i.e., 150 Hz) motor unit-targeted protocol [9]. (b)(c) Bar graphs visualizing the mean + SE of changes in phospho-Thr287-CaMKII levels with stimulation of the oxidative (b) and glycolytic (c) compartment ofgastrocnemius medialis. Data reflect combined values from the stimulation 10-Hz and 150-Hz protocol. *denotes P
<
0.05 versus rest (two-sided paired t-test, n
=
12–14).
(a)
(b)
(c)In the glycolytic compartment, Thr287 phosphorylation of CaMKII tended to be less affected than in the oxidative portion ofgastrocnemius medialis after stimulation (P
=
0.055, for the interaction of “stimulation” × “muscle compartment”). In the glycolytic portion none of the CaMKII isoforms demonstrated regulation after stimulation (Figure 5(c)). No interaction effect was identified between stimulation with the slow versus the fast-type protocol in either portion ofgastrocnemius medialismuscle for Thr287 phosphorylation.


## 3.4. Frequency DependentδA CaMKII Isoform Phosphorylation in the Slow Oxidative Compartment

We assessed CaMKII isoform phosphorylation in a third muscle of thetriceps surae complex with a slow-oxidative phenotype, that is, them. soleus, after stimulation with the slow-type (10 Hz) and fast-type (150 Hz) protocol, respectively (Figure 6(a)). The characterisation identified a trend for an interaction between CaMKII isoform and stimulation frequency (P
=
0.08). When assessing phosphorylation separately per CaMKII isoform we identify a frequency-dependent Thr287 phosphorylation of the δA-CaMKII isoform (P
=
0.017; Figure 6(b)). Phospho-Thr287 levels of this isoform were selectively downregulated after electric stimulation with the fast-type protocol, but it was increased with the slow-type protocol (Figure 6(c)).Frequency dependent CaMKII isoform autophosphorylation in slow oxidative muscle after paced contractionsin situ. (a) Example of Thr 287 phosphorylation of CaMKII isoforms in the stimulated (stim)soleus muscle and its contralateral control (rest) after electric stimulation with 150 Hz (Protocol 1) and 10 Hz (Protocol 2). The position of the respective bands is indicated by an arrow. Please note the presence of a second band at the height of the βM isoform in stimulated muscle. (b) Composite figure visualising the fold changes in CaMKII-Thr287 content of CaMKII isoforms for slow and fast motor unit-targeted stimulation for CaMKII isoforms in slow oxidativesoleus muscle in a colour code. (c) Line graph resolving the interaction effect of stimulation frequency on Thr287 phosphorylation of the δA-CaMKII isoform. Repeated ANOVA with post hoc test of Fisher, n
=
6–10.
(a)
(b)
(c)


## 3.5. Muscle Specific CaMKII Activation by Repeated Tetanic Contractions of Gastrocnemius Muscle

We investigated whether CaMKII activation in glycolytic and oxidative compartment of fast-twitch muscle would be affected with further repetitions of tetanic contractions. The computational model predicted a rapid increase in CaMKII phosphorylation for all isoforms after repeated tetanic contractions in the oxidative portion of thegastrocnemius muscle which followed the order βM > δD/γB > δA. Thr287 phosphorylation had early maxima after 1-2 contractions before falling to near resting values (Figures 7(a) and 7(b)).Isoform specific CaMKII autophosphorylation with repeated contractionsin situ. (a)(b) Line graph of the calculated changes in Thr287 phosphorylation of CaMKII isoforms in the oxidative (a) and glycolytic (b) compartment ofgastrocnemius medialis muscle as a function of the number of tetanic contractions as paced with repeated trains of 30 pulses of 150 Hz with 4.8 seconds of rest between trains. (c)–(h) Representative immunoblots (c)(d) and bar graphs of mean (e)–(h) of Thr287 phosphorylated CaMKII isoforms in the oxidative (c) (e) (g) and glycolytic (d) (f) (h) compartment ofgastrocnemius medialis muscle after 3 and 24 repeated tetanic contractions stimulated via the sciatic nerve with trains of 30 pulses at 150 Hz. *denotes P
<
0.05 versus rest (ANOVA with post hoc test of Fisher, n
=
6).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)Except for theδa isoform, the simulated biphasic relationship between phospho-Thr287 and the number of contraction was not what we found experimentallyin situ. There was a significant interaction effect of the stimulation and compartment (P
=
0.02) for Thr287 phosphorylation of CaMKIIin situ. Experimentsin situ reproduced the model observations on the sensitivity of phospho-Thr287 content of CaMKII in the oxidative compartment ofgastrocnemius medialis and pointed out isoform-specific phosphorylation in this muscle. Thr287 phosphorylation of the δA CaMKII isoform remained increased after three tetanic contractions but ceased after 24 contractions (Figure 7(g)). By contrast Thr287 content of the βM isoform was first significantly increased after 24 tetanic contractions (Figure 7(e)).


## 4. Discussion

The contribution of muscle contractions to activities of daily living varies from single short duration, for the purpose of acceleration, to continuous contractions being repeated for minutes to hours at a given load until fatigue occurs. While the calcium-induced regulation of myocellular ATPases that produce contractile output is relatively well understood [32], knowledge of calcium-induced regulation of phosphotransferase activities is limited [7, 14]. Towards this end we established a new computational model, CaMuZclE, to investigate whether changes in Thr287 phosphorylation of the calcium-receptive phosphotransferase, CaMKII, following electrically paced contractions could be predicted. CaMKII has been shown to encode information provided by the frequency and amplitude of calcium transients [24] and to regulate excitation-contraction and excitation-transcription coupling [33, 34]. It is unclear which CaMKII isoforms mediate these effects in skeletal muscle but their autophosphorylation pattern in response to fast or slow motor unit-type stimulation might provide clues about CaMKII isoform-specific functions. Therefore, emphasis was put on the elucidation of the response of different isoforms in phenotypically distinct muscles and the influence of slow/fast motor unit stimulation frequencies.A possible limitation of our approach was that contraction-induced phosphorylation of downstream targets of CaMKII, was not assessed. This experiment is indicated for subsequent studies on CaMKII-regulated signal transduction because Thr287 phosphorylation of CaMKII andin vitromeasured phosphotransferase activity of CaMKII appears not to be correlated [12]. However, as demonstrated by the same authors, Thr287 phosphorylation of combined CaMKII isoforms increases 1.5-fold ingastrocnemius muscle after five repeated tetanic contractions over 10 secondsin situwith a further 5-fold increase after 3 minutes of repeated contractions. Our biochemical measurements on the effect of tetanic stimulation over a similar duration (i.e., 0.66 seconds to 2 minutes) are compatible with these results (Figure 5(b)). However, we did not see such large effects on phospho-Thr287 content of CaMKII after one to twenty tetanic contractions and we identify that the δA and βM CaMKII isoforms follow a different time course of phosphorylation (Figures 5 and 7). The latter finding indicates a different contribution of CaMKII isoforms to the reported response of combined phospho-Thr287 content of CaMKII with paced contractionsin situ. The contrasting magnitude in the level increases for the phospho-Thr287 content of CaMKII isoforms respective to Rose [12] is possibly explained by differences in the stimulation protocol and the procedure of harvesting the muscle. In our case, the latter was carried out to separate the three major compartments of the ankle extensor grouptriceps surae. This sampling scheme allowed us to investigate and identify previously unresolved differences in CaMKII phosphorylation between muscle types, that is, the distal (i.e., glycolytic) and proximal (i.e., oxidative)m. gastrocnemius medialisand the slow oxidativem. soleus. Our biochemical measures identified that phospho-Thr287 content of δA and βM CaMKII is increased after externally paced tetanic contractions in the oxidativegastrocnemius medialis muscle but is not significantly affected in the glycolytic compartment of the same muscle (Figures 5 and 7). The observed difference in sensitivity of contraction-induced CaMKII phosphorylation between the studied muscle compartments suggests that the anatomical specialisation of muscle fibres is an important factor in the posttranslational regulation of CaMKII.The role of fibre specialisation for CaMKII isoform autophosphorylation is further corroborated by higher phospho-Thr287 levels of CaMKII at rest between the oxidative and glycolytic portion ofgastrocnemius medialis (Figure 2). The mechanism behind this baseline difference awaits further exploration. Interestingly, however, the fast-fatigable whitevastus lateralis muscle of rats responds to repeated voluntary running with an increase in autonomous CaMKII activity [18]. Autonomous phosphotransfer activity of CaMKII reflects increased Thr287 phosphorylation and a subsequent conformation change in the CaMKII enzyme [15]. This suggests a possible contribution of fibre recruitment during cage activity to the baseline differences in phospho287 content of CaMKII between the oxidative and glycolytic compartment ofgastrocnemius medialis muscle.Our investigation demonstrates that autophosphorylation of CaMKII isoforms in skeletal muscle depends on the duration of muscle activity. Interestingly, phospho-Thr287 content of theδA isoform was transiently increased and faded after twenty-four tetanic contractions ofgastrocnemius medialismuscle, when phosphorylation of the major CaMKII isoform of skeletal muscle, βM, increased (Figures 7(e)/7(g)). The findings suggest that the βM CaMKII isoform of skeletal muscle does, like the αCaMKII isoform in neurons, act as a decoder of neuronal input [25]. βM CaMKII regulates calcium release from the sarcoplasmic reticulum [7, 14] whereas the δA CaMKII isoform has been demonstrated to affect nuclear processes and modify cardiac growth [22, 23]. Collectively the findings highlight a possible contribution of isoform-specific CaMKII autophosphorylation to excitation-induced signalling in skeletal muscle.The observed Thr287 phosphorylation ofβM CaMKIIin situ (Figure 2), and its association with muscle fatigue (as indicated by a 30% decrease in maximal tetanic force), is in line with the proposition by Tavi [14] on a summation of CaMKII autophosphorylation with repeated contractions. The latter observation was however not diversified for CaMKII isoforms and verified with biochemical analysis. The mechanistic basis of this effect is therefore unclear. Our report points out that autophosphorylation of the βM isoform rises slower than δA CaMKII isoform after repeated contractionsin situ (Figures 7(e)/7(g)). This is intriguing because βM localizes to the sarcoplasmic reticulum from which intracellular calcium is released [13] and because sensitivity analysis identifies that the most influential parameter for the rate of CaMKII autophosphorylation, kbta [20], is considerably higher for the βM than the δA CaMKII isoform (Table 1).By contrast, the calmodulin affinity rate constants, which are derived from the parameter kbi, are of lesser importance for our model (0.06 versus 0.32, Table1), indicating that instead of the calmodulin affinity, the rate constant of autophosphorylation is the rate-determining step. At higher values of this rate constant of autophosphorylation, however, the calmodulin affinity to CaMKII becomes a more important parameter as binding to calmodulin needs to occur before CaMKII autophosphorylation can take place [15]. Both parameters need to be elevated to facilitate a higher total rate of autophosphorylation. Accordingly, this influence is specifically more pronounced for the βM isoform due to its higher rate constant [20]. We therefore speculate that thein situ observed pattern of βM CaMKII isoform autophosphorylation (Figures 7(e)/7(g)) reflects differences in the threshold of calmodulin-dependent autophosphorylation for CaMKII isoforms, or calmodulin availability, rather than kinetic constants of autophosphorylation as characterizedin vitro [20, 24].A major finding in this regard was that Thr287 phosphorylation of theδA CaMKII isoform is highly sensitive to excitation and differentiates the response of the fatigue-resistantsoleus muscle between slow and fast motor unit–type stimulation (Figures  5 and 6; [1]). Interestingly, the increased phospho-Thr287 content of δA CaMKII was only observed with the stimulation frequency corresponding to the “natural” recruitment frequency of the motoneuron in this muscle, that is, 10 Hz; [1, 8]. This supports the idea that elevated δA CaMKII Thr287 phosphorylation in oxidative muscle types depends on the recruitment pattern of motor units. As thesoleus muscle is also stimulated by the 150 Hz protocol our findings bear the notion that it is the high frequency tetanic excitation that leads to a dephosphorylation of CaMKII δA.In the course of our investigation, we have established a new computational model that connects calcium dynamics to downstream activation of the regulator of excitation-contraction coupling, CaMKII, in muscle fibers. We used this model to predict possible CaMKII isoform differences in level alterations of Thr287 phosphorylation between stimulation protocols and motor unit types. The phosphatase concentrationin silico was calibrated by taking the measured decay of Thr287 phosphorylation of CaMKII into account. Of interest in this regard is the fact thatin situwe observed only a relatively moderate reduction in the content of CaMKII-Thr 287 phosphorylation during recovery from 24 tetanic contractions (data not shown). These values indicate that Vmax or concentration of the phosphatase activity dephosphorylating CaMKII is considerably lower than the value used in the first computational model of CaMKII phosphorylation [16], approximating the initially published values [17]. Accordingly the phosphatases PP1 would be far less efficient to dephosphorylate Thr287 on CaMKII in rat skeletal muscle compared to other tissues.The output of ourin silico model emphasises that differences in mitochondrial content as seen for the glycolytic and oxidativegastrocnemius medialis muscle bring about different alterations in Thr287 phosphorylation of CaMKII with excitation. The model explains this difference in terms of lower increases in free calcium in the sarcoplasma of oxidative muscle fibers due to the calcium buffer capacity of mitochondria [27]. The model also predicted that Thr287 phosphorylation of CaMKII would fall after an initial peak fade with repeated contraction (Figure 3), a prediction that was confirmed by the observed transient increase in phospho-Thr287 content of δA CaMKII (Figure 7(g)). Our approach can serve as an example as to howin silico data can be used to plan experiments and interpret biological data.However, the results from our modelling did not predict thein situ observed differences in the time course of Thr287 phosphorylation between CaMKII isoforms and the discrete influence of the muscle phenotype. For instance modelling predicted a similar transient increase in phospho-Thr287 content of βM and δA CaMKII after tetanic contractions but which differed from the data gatheredin situ(Figure 3 versus Figures 7(a)/7(b)). As well, the predictions on the larger increase in Thr287-phosphorylation in the glycolytic relative to the oxidative compartment ofm. gastrocnemius medialis differed from the measured data (Figure 3). This disconnection betweenin silicoandin situdata emphasises that further experimental input from measured parameters, specifically those addressing influential model parameters (Table 1) and their relationship to baseline values and threshold of calcium-induced activation, and βM and δA isoform localization, is necessary to refine the model.


## 5. Conclusions

Our findings provide the first evidence that CaMKII isoforms serve as muscle and frequency-specific sensors of muscle excitation. This indicates that aside from the characteristics of a contractile protocol, isoform and muscle type-specific activation of CaMKII must be taken into consideration when interpreting the physiological activation of reactions downstream of CaMKII with muscle contractionin vivo. Results from our computational investigations offer a first consolidation of the experimental observations and indicate that the boundary conditions for the modelling of CaMKII regulation need to be adjusted to reveal a mechanistic explanation of CaMKII autophosphorylation in function of calcium dynamics.



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*Source: 943806-2014-06-26.xml*