ARTICLE IN PRESS

Computers in Biology and Medicine 40 (2010) 533–542

Contents lists available at ScienceDirect

Computers in Biology and Medicine

0010-48

doi:10.1

Abbre

monoxi

oxygena

cyclase;

kinase;

p38 mit

flow; Tr

zone; V

PO/AH,� Corr

E-m

journal homepage: www.elsevier.com/locate/cbm

Mechanistic electronic model to simulate and predict the effect of heat stresson the functional genomics of HO-1 system: Vasodilation

Yogender Aggarwal a,�, Bhuwan Mohan Karan b, Barda Nand Das a, Rakesh Kumar Sinha a

a Department of Biomedical Instrumentation, Birla Institute of Technology, Mesra, Ranchi, Jharkhand 835215, Indiab Department of Electrical & Electronics Engineering, Birla Institute of Technology, Mesra, Ranchi, Jharkhand 835215, India

a r t i c l e i n f o

Article history:

Received 7 August 2008

Accepted 25 March 2010

Keywords:

Carbon monoxide

Environmental temperature

Heme oxygenase-1

Mechanistic electronic model

Signalling pathway modelling

25/$ - see front matter & 2010 Elsevier Ltd. A

016/j.compbiomed.2010.03.011

viations: BV, biliverdin; BVR, biliverdin reduct

de; HSF, heat shock factor; HSP, heat shock p

se-1; cGMP, guanosine 30 ,50-cyclic monophos

ERK, extra-cellular signal-regulated kinases;

KCa, calcium-activated potassium channels; N

ogen-activated protein kinase; Tsk, Skin Tem

e, body core; Tsk, body surface; SkBF, skin blo

2, input heat stress; TNF-a, tumor necrosis fa

anterior hypothalamus Preoptic area; IRP, Iro

esponding author. Tel.: +91 9431991797; fax

ail address: yogenderaggarwal@gmail.com (Y

a b s t r a c t

The present work is concerned to model the molecular signalling pathway for vasodilation and to

predict the resting young human forearm blood flow under heat stress. The mechanistic electronic

modelling technique has been designed and implemented using MULTISIM 8.0 and an assumption of

1 V/1C for prediction of forearm blood flow and the digital logic has been used to design the molecular

signalling pathway for vasodilation. The minimum forearm blood flow has been observed at 35 1C

(0 ml 100 ml�1 min�1) and the maximum at 42 1C (18.7 ml 100 ml�1 min�1) environmental tempera-

ture with respect to the base value of 2 ml 100 ml�1 min�1. This model may also enable to identify

many therapeutic targets that can be used in the treatment of inflammations and disorders due to heat-

related illnesses.

& 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Heat is an environmental and occupational hazard and manygenetic and physiological alterations have been known to occurdue to exposure to either natural or artificially induced heatstress. Review of literature reveals large number of deaths allaround the world every year due to heat waves [1]. Further, it hasalready been demonstrated that exposure to high ambienttemperature causes the clinical syndromes of heat-relatedillnesses [2]. However, from pathophysiological point of view,many deaths that are attributed to heat are neither caused by heatstroke nor are they even in persons that exhibit the clinical signsof heat stress. There are several mechanisms by which a personmay die during a heat wave because the heat stress places extrastrain on different biochemical and physiological systems, themanipulation in cardiovascular functions including vasodilation,play critical role [1].

ll rights reserved.

ase; Ca++, calcium; CO, carbon

rotein; HO-1, heme

phate; sGC, soluble guanylyl

JNK, c-Jun N-terminal protein

O, nitrogen oxide; p38 MAPK,

perature; FBF, forearm blood

od flow; TNZ, thermoneutal

ctor-a; IL, interleukins;

n regulatory protein

: +91 651 2275401.

. Aggarwal).

Though, the role of thermal factors (both neural and local) invasodilation has been well reviewed [3,4]; literatures on cellularand genetic mechanism are still obscure. However, it is alsosupposed that heme oxygenase-1 (HO-1) plays a critical role asprotective gene in humans during exposure to heat stress [5].HO-1 belongs to a large family of heat shock proteins (HSPs),whose transcriptional regulation also responds to adverse environ-mental conditions. Despite the common end point HO-1 activation,the molecular pathway leading to its activation may vary in a cell-type specific manner [6]. Review of literature revealed that themetabolism of heme substrates, produced as a result of degrada-tion of hemoprotein like in rhabdomyolysis, are triggered with theactivation of HO-1 from heat shock promoter region of heat shockgene and provides cellular protection against heme mediatedcellular injury [7]. Among different metabolic products of hemedegradation, carbon monoxide (CO) possesses significant physio-logical importance that influences vasodilation [8], plateletaggregation and also critical for cellular signal transduction inresponse to stress and inflammation by exerting anti-inflamma-tory, anti-apoptotic, and anti-proliferative effects [5,6,9,10]. Apartfrom its role in circulatory system, the role of CO has also beenknown to influence variety of organs and organ systems andsuggested its significant and critical association with a number ofpathological conditions such as Alzheimer’s diseases, Parkinson’sdisease, amylotrophic lateral sclerosis, febrile seizures, hyperten-sion, inflammation, cardiac hepertrophy, heart failure, apoptosisand cellular proliferation, and atherosclerosis [6].

The understanding of activation of HO-1 along with the activemolecular mechanisms of CO at cellular level has opened a vista of

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Y. Aggarwal et al. / Computers in Biology and Medicine 40 (2010) 533–542534

discussion about the cellular mechanism of body temperaturecontrol. Moreover, the modelling of functional genomics ofvasodilation process will definitely aid some substantial techno-logical thought about the thermoregulatory control from gene tophysiology, which will result in improvements in the health ofpopulation under heat-related illnesses by the development ofhealthcare solutions. Analysis of rate kinetics of the biologicalsubstrates and further modelling them with the help of non-lineardifferential equations have been used as an efficient tool to modelthe biochemical networks [11,12]. However, the scattered andambiguous information about the concentration and rateconstants are the major barrier for these models and generally,the designed systems have been explained within a narrowboundary. Further, it is notably complicated by the diversity andsophistication of regulatory mechanisms, as well as by the chroniclack of relative quantitative information [13]. The limitation ofthis modelling approach has motivated researchers to thedevelopment of intrinsically qualitative approaches is leaning onBoolean algebra [13,14–18]. In this model, gene expression isquantized to only two levels: ON and OFF. The expression level(state) of each gene is functionally related to the expression statesof some other genes, using logical rules. This model has yieldedinsights into the overall behavior of large genetic networks [19]and allows the study of large data sets in a global fashion.

Besides the conceptual framework afforded by such models, anumber of practical uses, such as the identification of suitabledrug targets in cancer therapy, may be reaped by inferring thestructure of the genetic models from experimental data, e.g. fromgene expression profiles [20]. However, the Boolean modellingapproach with synchronous updating suggested by Kauffman [17]leads to the generation of spurious cycles [13]. Thus, in thepresent work, we simulated a simple Boolean network modelbased on Boolean algebra of molecular signalling pathway ofactivation of HO-1 system leading to the activity of the product.The effect of CO on vasodilation is also simulated using analoglogic under heat stress. Therefore, the objectives of the proposedwork are (1) to model and predict the molecular signallingpathway for vasodilation under heat stress and (2) to model andpredict the forearm blood flow (FBF) under heat stress.

Fig. 1. Schematic representatio

2. Signal transduction pathway for vasodilation

2.1. Fundamental of HO-1 Gene activation

HO-1 represents the product of gene HMOX1 localized onchromosome region HO-1: 22q12. Under stress condition, thetranscriptional regulation of HMOX1 has been demonstrated inFig. 1. The level of heme, produced from intact hemoprotein understress, activates the translocation of Nrf2 (HMOX1 activationfactor) form cytosol to the nucleus and simultaneously relieve theBach1 (HMOX1 repressor factor, shown as doted line in Fig. 1)from nucleus to cytosol. The HMOX1 has two enhancer regions,the E1 and E2. The dominant sequence element in the E1 and E2enhancers of HO-1 is the stress responsive element (StRE). TheStRE mediates the transcriptional activation in response to almostall HO-1 inducers. The translocated Nrf2, in turn, activates thetranscription of HMOX1 to produce HO-1 protein, whichcatabolizes the heme into CO, biliverdin (BV), and free Iron [21].

2.2. At cellular level

Under heat stress, HO-1 protein catabolizes the producedheme, from degradation of hemoproteins [22], to BV, iron, CO [9].The BV is further converted to bilirubin by biliverdin reductase(BVR), which is potent antioxidant [5]. Redox-active iron releasedfrom HO-1 activity may promote oxidative damage. However, byinactivating iron regulatory protein (IRP) activity, iron stimulatesthe synthesis of ferritin, an iron-sequestration protein andpossible cytoprotectant [9]. Activity of HO-1 protein and CO hasbeen well demonstrated in endothelium, vascular smooth muscle,and perivascular neurons [23]. Further, CO has been demon-strated to be of biological importance including vasodilationactivator [8,24], inhibition of platelet aggregation and neuronalsignal transduction via the production of cyclic guanosine30, 50-monophosphate (cGMP) from guanosine 50-triphosphatecatalyzed by soluble guanylyl cyclase (sGC) activated by CO [5].The importance of CO in sGC activation likely increases in cells ortissues with low endogenous nitrogen oxide (NO) production [21].

n of HO-1 gene regulation.

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Fig. 2. Mechanistic representation of signal transduction pathway for HO-1 system produced vasodilation. (1) HO-1 degrades heme to BV, CO, and iron. (2) CO derived from

the HO-1 reaction activates sGC to initiate cGMP and has possible significance in the regulation of vascular and neural functions. The stimulation of cGMP-dependent signal

transduction pathways may account for the vasodilatory through efferent signal and anti-proliferative effects. CO has potent anti-inflammatory effects, which depend on

downregulation of pro-inflammatory cytokine production mediated by modulation of p38 MAPK. (3) Biliverdin is converted to bilirubin by biliverdin reductase. (4) Iron is

converted to ferritin by inhibiting the IRP.

Y. Aggarwal et al. / Computers in Biology and Medicine 40 (2010) 533–542 535

Indeed, CO has also been reported to modulate the activation stateof mitogen-activated protein kinases (MAPK) [9]. The productionof cGMP initiated the cGMP-dependent protein phosphorylationcascades, which augments the phosphorylation of postsynapticvesicle proteins associated with neurotransmitters release invascular tissues [25]. It relaxes vascular smooth muscle toincrease the blood flow that participates in enhancement of heatdissipation under hot environment [26]. It has been suggestedthat the threshold level of cGMP is necessary to activate calcium-activated potassium channels (KCa), which is implicatedin vasorelaxtion via coupling of calcium (Ca++) sparks to KCa invascular smooth muscle that hyperpolarize the membraneto bring relaxation in smooth muscle [5,25,27]. CO also acts asthe inhibitory signal for extra-cellular signal-regulated kinases(ERK) and c-Jun N-terminal protein kinase (JNK), which isimplicated in anti-inflammation and activating signal forp38MAPK and KCa. The activated p38MAPK further phosphor-ylates the heat shock factor (HSF)-1 in the cytoplasm andtranslocate to the nucleus and trigger the HSF-1 binding to heatshock promoter region in heat shock gene for the productionof HSP70, which is implicated in cytoprotection [9,28]. Thefunctional consequence of HO-1 system is presented Fig. 2.

2.3. At physiological level

Aggarwal et al. [26] suggested that in response to increase inenvironmental temperature, body core (Tre) and surface tempera-ture (Tsk) also increases. This alteration in body temperatures has

been sensed and relayed through the afferent nerves to thecentral coordinating centre of hypothalamus well known asanterior hypothalamic preoptic area (AH/POA). It has beenconsidered as thermostat of the body that controls the effectorresponses to dissipate more heat in response to positive error(elevated temperature); mainly through increase in skin bloodflow (SkBF) [4]. The increase in SkBF depends on the increase inTsk. When Tsk lies in the thermoneutal zone (TNZ), 33–35 1C, orabove TNZ and below the threshold for cutaneous vasodilationthen the increase in SkBF is due to withdrawal of vasoconstrictionsystem [29]. But, if Tsk lies above the threshold for cutaneousvasodilation then the increase in SkBF is mainly due to vasodilatorsystem, driven by sympathetic vasodilator nerves [4]. Thismechanism is shown as block diagram representation (Fig. 2).

3. Electronic analogy

3.1. HO-1 gene activation under heat stress

In the present work, using logic gates, a binary method hasbeen used to simulate the molecular signalling pathway of HO-1system under heat stress. A binary network is a simple model thatmay help to study the basic properties and logical interactions ofbiomolecules in a complex biochemical network without the needof initial concentrations and kinetic values of reactants and isrepresented by variables with two possible states (on/off), of theindividual nodes/genes of the network [16,18]. The generalized

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Fig. 3. Biochemical network driving the heme degradation.

Fig. 4. Binary model of HO-1 gene regulation.

Y. Aggarwal et al. / Computers in Biology and Medicine 40 (2010) 533–542536

biochemical network for heme degradation under heat stress isillustrated in Fig. 3 and the corresponding model consisting of aset of non-linear ordinary differential equations (Appendix A).Fig. 4 demonstrates the transcriptional regulation of HO-1 geneusing binary logic. The model has been simulated with the help ofMultisim 8.0 (National instruments, USA) for the production ofHO-1 protein from HMOX1. The Bach1-Nucleus, hemoprotein,heat, Nrf2-Cytosol, and HMOX1-StRE are shown as constantvoltage source (Vcc), which means logic high or activated. Thehemoprotein is degraded to heme in the presence of heat, shownwith the help of ‘AND’ gate (U1A). The heme, in turn, activates thetranslocation of Nrf2 from cytosol to nucleus (U2A). This Nrf2 innucleus activates the transcription of HMOX1 gene through itsactivating element (StRE), shown as U3A. The output of U3A, HO-1Protein, binds with the heme to degrade it into CO, BV, and Iron(U4A). The produced heme inactivates the Bach1 mediatedHMOX1 repression by its translocation from nucleus to thecytosol (U5A).

3.2. HO-1 mediated vasodilation

The binary model of HO-1 protein mediated vasodilation hasbeen simulated to mimic its biochemical model for hemedegradation and effect of CO activation (Fig. 3). The substrateprotein, HO-1 gene, BVR, IRP, inactivated sGC, ERK, JNK, p38MAPK,KCa and HSF-1 are represented as Vcc. The protein is degraded toheme under exposure to high environmental heat, has beenmodelled using an ‘AND’ gate (U10A). Further, heme is degradedto CO, BV, and iron in the presence of HO-1, has been modeledusing ‘AND’ gate (U18A). The HO-1 isoform of HO is produced onlyunder stress conditions. In the present work, heat stress mediatedproduction of heme is shown to produce the HO-1 (U9A). The heatstress is represented by the voltage comparator (U8), if the outputis logic high then the stress is ON otherwise stress is OFF or logiclow. One input to U8 was V9 as constant voltage set to 35 V(1 V/1C) applied to negative terminal of U8 and the other inputshown as offpage1 (environmental heat) was applied at positive

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Fig. 5. Binary Model of signal transduction pathway of CO as vasodilation activator.

Y. Aggarwal et al. / Computers in Biology and Medicine 40 (2010) 533–542 537

terminal of the U8. The environmental heat was taken as input tothe model, shown as V2 (Fig. 6) and the same was connected tothe input of the model as shown offpage1 in Fig. 5. The ‘AND’ gatereceives two inputs and produce one output; one input assubstrate and the other as enable signal to convert the substrateinto another product in the presence of enable signal. This can becompared to any typical biochemical reaction in which onebiomolecule is converted to another biomolecule in the presenceof enzymatic action of third biomolecule. Further, BV is convertedto bilirubin in the presence of BVR as an activator signal for U11A,which is an antioxidant. Iron is converted to ferritin by providingan inhibitory signal (U22A) to inhibit the activity of IRP (U20A) byforming complex with IRP. Thus enables the formation of ferritinthrough U21A. It has been suggested that the CO is a potentactivator of sGC to generate cGMP (shown as activator signal forU17A). The initiated cGMP was used as an enable signal forvoltage controlled switch (J7), shown as offpage2 in Fig. 6.Furthermore, the generated CO acts as the inhibiting signal forERK and JNK and activating signal for p38MAPK and KCa, this isshown as U12A, U13A and U14A, U15A, respectively. Theactivated p38MAPK further phosphorylates the inactivatedHSF-1 in the cytoplasm and trigger the translocation of activatedHSF-1 to nucleus for binding to the heat shock promoter region inheat shock gene, shown as U16A for the production of HSP70(Fig. 5). The Out 1, 2, 3, and 4 represent the output of U12A, U13Aand U14A, U15A, respectively, which may be logic ‘1’ or logic ‘0’.

3.3. Prediction of forearm blood flow

The physiological model of the effect of hot environment onforearm blood flow (FBF) has been designed and simulated usingMUTLISIM version 8.0 (National Instruments, USA) and anassumption 1 V/1C for young nude human under resting heatstress to predict FBF. The input (heat stress) was taken as voltagesource V2, which reflects the Tsk for the prediction of FBF.Neuronal comparator has been designed to represent thecomparison of V2 with the reference voltages (V1, V3, V4, V5,

V6, and V7) as set point temperatures and the output has beentaken as FBF (Fig. 6). The reference voltages V6 and V7 are set to35 V (upper limit of TNZ). In a simple window comparator twosingle ended voltage comparators are connected, such that theresultant output shows whether the input signal is within thewindow or not. The windows used in this circuit are V3–V1(33–35 1C) and V5–V4 (35–37 1C). The diodes D1–D8 used asprotection diodes and LED1-4 has been used to show thequalitative results of brain error computation (Fig. 6), in responseto alteration in body temperature sensed and relayed to AH/POA.Glowing LEDs shows the positive error, which means that bodytemperature is more than the set point temperature.

For the quantification of FBF, the V2 has been feed to theop-amp U6 through resistance R15 and the reference V7 throughresistance R14. The difference in V2 and V7 has been used to driveop-amp U7 through J7 (enable only if cGMP is initiated oroffpage2 is logic high). The gain of U7 has been adjusted usingresistances R19 and R22. The value of gain factor was analyzedfrom various published literature [4,29,30–34]. The amplifiedoutput FBF has been taken across capacitor C2, as shown in Fig. 6.

4. Simulation results

4.1. HO-1 gene regulation

The transcriptional regulation of HMOX1 involves manybiochemical components and the activation and inhibition ofthese components control the transcriptional regulation ofHMOX1. Truth Table 1 summarized the transcriptionalregulation of HMOX1.

4.2. HO-1 mediated vasodilation

The perturbation in present model (Fig. 5) is shown in Table 2aand 2b and is summarized in the following case studies. InTable 2a and 2b, logic ‘1’ indicates the ‘present or produced’ and

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Fig. 6. Circuit model for vasodilation response of resting human during high heat stress.

Table 1Truth table of HO-1 gene regulation system.

Heat Hemoprotein Heme Bach1-nucleus Nrf2-cytosol Bach1-cytosol Nrf2-nucleus HMOX1-StRE HO-1 protein

0 1 0 1 1 0 0 1 0

1 1 1 0 0 1 1 1 1

Y. Aggarwal et al. / Computers in Biology and Medicine 40 (2010) 533–542538

logic ‘0’ indicates ‘blocked or not produced’, no intermediate statehas been assumed. Hamming distance has also been calculatedbetween the sham control (under heat stress) and eachperturbation in the biochemical network to find out thecomponent, which cause maximum damage to the biochemicalnetwork when perturbed. Hamming distance was originallyconceived for detection and correction for errors in digitalcommunication. It is simply defined as the number of bits thatare different between two bit vectors. Maximum the hammingdistance it causes maximum alterations in biochemical pathwayand vice-versa. It has been observed that maximum damage tobiochemical network occur in conditions when heat is off(hamming distance 12), or HO-1 gene transcription is inhibited(hamming distance 11), or heme activity is inhibited (hammingdistance 11).

�

Case I: Environmental heat triggers the HO-1 system, whichcauses the production of CO and BV from the breakdown ofheme. The produced CO activates the sGC for the production ofcGMP. The produced BV is further converted to bilirubin in thepresence of BVR. The CO inhibits the ERK and JNK and activatesthe p38MAPK and KCa.

�

Case II: Heat stress triggers the production of heme, which is anatural HO-1 expression inducer has been removed. Therewould be no production of CO and BV from the breakdown ofheme, as no heme production. In the absence of CO, ERK andJNK activate to initiate cell apoptosis. There would be noproduction of cGMP even if sGC is present that remainsinactivated in absence of CO. There would be no production ofbilirubin in absence of BV even if BVR is present. The absenceof CO inhibits the p38MAPK and KCa. � Case III: Under high environmental heat, if the production of

HO-1 is blocked (HO-1 gene expression is inhibited) then itresults in no production of CO and BV. cGMP is not produced inthe absence of CO. Although the sGC is present. The absence ofCO also causes the activation of ERK and JNK and inhibition ofp38MAPK and KCa. There would be no production of bilirubineven if BVR is present.

� Case IV: Hot environment triggers the production of heme,

which is a natural HO-1 expression inducer. If the heme isblocked or inhibited that results in no production of HO-1enzyme, even if HO-1 gene is present. In absence of HO-1enzyme, there would be no production of bilirubin as BV hasnot been produced. The inhibition of heme also causes noproduction of cGMP from sGC, which remains inactivated in

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Table 2aTruth table of HO-1 system for different testing conditions and its effect on vasodilation under heat stress.

Case I II III IV VComponents of HO-1 system Normal Heat

ONPerturbation HeatOFF

Perturbation HO-1 GeneInhibited

Perturbation HemeInhibited

Perturbation sGCInhibited

Heat 1 0 1 1 1

Protein 1 1 1 1 1

Heme 1 0 1 0 1

HO-1 gene 1 1 0 1 1

HO-1 expressed 1 0 0 0 1

CO 1 0 0 0 1

sGC 1 1 1 1 0

cGMP 1 0 0 0 0

BV 1 0 0 0 1

BVR 1 1 1 1 1

Bilirubin 1 0 0 0 1

ERK 1 1 1 1 1

ERKa 0 1 1 1 0

JNK 1 1 1 1 1

JNKa 0 1 1 1 0

p38MAPK 1 1 1 1 1

p38MAPKa 1 0 0 0 1

KCa 1 1 1 1 1

Activated-KCa 1 0 0 0 1

HSF-1 1 1 1 1 1

HSF-1a 1 0 0 0 1

Hamming Distance wrt

Normal

12 11 11 2

Vasodilation 1 0 0 0 0

Table 2bTruth table of HO-1 system for different testing conditions and its effect on vasodilation under heat stress.

Case VI VII VIII IX XComponents of HO-1system

Perturbation KCa

InhibitedPerturbation p38MAPKInhibited

Perturbation HSF1Inhibited

Perturbation ERKInhibited

Perturbation JNKInhibited

Heat 1 1 1 1 1

Protein 1 1 1 1 1

Heme 1 1 1 1 1

HO-1 gene 1 1 1 1 1

HO-1 expressed 1 1 1 1 1

CO 1 1 1 1 1

sGC 1 1 1 1 1

cGMP 1 1 1 1 1

BV 1 1 1 1 1

BVR 1 1 1 1 1

Bilirubin 1 1 1 1 1

ERK 1 1 1 0 1

ERKa 0 0 0 0 0

JNK 1 1 1 1 0

JNKa 0 0 0 0 0

p38MAPK 1 0 1 1 1

p38MAPKa 1 0 1 1 1

KCa 0 1 1 1 1

Activated-KCa 0 1 1 1 1

HSF-1 1 1 0 1 1

HSF-1a 1 1 0 1 1

Hamming Distance wrt

Normal

2 2 2 1 1

Vasodilation 0 1 1 1 1

Y. Aggarwal et al. / Computers in Biology and Medicine 40 (2010) 533–542 539

absence of CO. The protein kinase p38MAPK has not beenactivated; however, ERK and JNK are activated in absence ofCO, which initiates the cell apoptosis program.

� Case V: Under heat stress, if the activation of sGC is blocked

then it causes no production of cGMP even if CO is producedthrough the degradation of heme in the presence of HO-1. Theactivated HO-1 results in the production BV, which is furtherconverted to bilirubin in the presence of BVR. The presenceof CO inhibits the ERK and JNK and activates the p38MAPKand KCa.

�

Case VI: Under exposure to heat stress, if the activation ofpotassium channel is blocked causes no change in the produc-tion of CO and BV from the degradation of heme in the presenceof HO-1. cGMP is produced from sGC activated from CO. The BVis converted into bilirubin in the presence of BVR. ERK, JNK, KCa

are inhibited whereas p38MAPK is activated.

� Case VII: Under exposure to high environmental heat, in HO-1

system, if the activation of p38MAPK is blocked that causes noanti-inflammatory and anit-proliferative response. It alsoresults in no activation of HSF-1 factor.

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Table 3Computation of error signal by brain in response to high environmental heat sensed at skin surface (V2). The modeled results were found similar to the past published

results *[25, 28] and **[3, 25].

Environmentalheat (1V/1C)

Errorresponse

Inference

Electronically Physiologically

V2 is 33V LED2 and 4

would glow

LED4 suggested that the input V2 is either at lower limit of window

V4–V5 or even less. However, LED2 demonstrated that input V2 is

either at lower limit of window V1–V3 or less than this

When Tsk lies in thermoneutral zone (33–35 1C) or above

thermoneutral zone and below the threshold of cutaneous

vasodilation then the increase in SkBF is due to withdrawal of

vasoconstriction system, which is activated via sympathetic

adrenergic vasoconstriction nerves (efferent nerves)*.

V2 is 34V LED4 would

glow

Suggested that the V2 lies in the window V1–V3 and less than the

lower limit of window V4-V5

V2 is 35V LED1 and

LED4 would

glow

LED4 suggested that the V2 may be equal or less than V5 and LED1

represented that the input V2 equals or greater than the V1

V2 is 36V LED1 would

glow

Suggested that the input V2 should be more than the V1 and V5 as

LED3 and LED4 has not glowed and must lies in the window V4–V5

V2 is 37V–42V

or more

LED1, LED3

and LED5 has

glowed

Suggested that the input V2 should be equal or more than the V4 When Tsk lies above the threshold for cutaneous vasodilation

then the increase in SkBF is due to vasodilator system driven by

sympathetic vasodilator nerves**.

Y. Aggarwal et al. / Computers in Biology and Medicine 40 (2010) 533–542540

�

Case VIII: In hot environment, if the activation HSF-1 isblocked. It causes no production of HSP via this pathway. � Case IX: Under heat stress condition, if the activation of ERK is

blocked. The cell apoptosis program would not initiatedthrough this pathway.

�

Fig. 7. Forearm blood flow response with the varying input temperature from 35

to 42 V (the assumption has been made as 1 1C is equivalent to 1 V).

Case X: Under heat stress, if the activation of ERK is blocked.The cell apoptosis program would not be initiated through thispathway.

4.3. Prediction of forearm blood flow

The V2 sensed at skin and relayed to the brain for computationwhere the brain compares V2 with the reference voltages (V1, V3,V4, V5, V6, V7 and V8) and results in an error signal, showed asLED responses (Table 3). Quantitatively, the simulated resultsshowed that under heat stress as V2 increases from 35 to 42 V theFBF increases. The FBF has attained its maximum value18.7 ml 100 ml�1 min�1 at 42 V and minimum value0 ml 100 ml�1 min�1 at 35 V with respect to base value of2 ml 100 ml�1 min�1. The FBF remains in this state till theexposure is ON. When OFF the FBF drops gradually to0 ml 100 ml�1 min�1. The graph (Fig. 7) has been plotted for V235V, 35.5V, 37, 37.5V, 42V and 42.5V under the condition whenexposure is ON or OFF.

5. Discussion

Heat shock response is one of the important phenomenonemployed in cytoprotection against various insults like heatstress, oxidative stress, ultraviolet radiation, cytokines (tumornecrosis factor-a (TNF-a) and many interleukins (IL)). Heat shockis well known to induce the protein aggregation involving manyerythrocytes and hemoproteins, which triggers HO-1 transcrip-tion system to produce HO-1 protein [5,9,22,35]. HMOX1 (HO-1gene) transcription involves many biochemical components andthe activation, translocation and inhibition of these componentsdefines the transcriptional regulation of HMOX1 under heatstress. Further, CO, one of the products of heme degradation byHO-1 protein actively participates in vasodilation. However, therole of NO in cutaneous vasodilation has also been proposed [3].The comparative study of CO and NO on vasodilation shows that

the vasodilatory properties of CO in the rabbit aorta wereattributed to activation of sGC and generation of cGMP by CO,with lesser potency than NO. However, the importance of CO insGC activation likely increases in cells or tissues with lowendogenous NO production [21].

In the present work we have modelled the signalling pathwayfor functional genomics of HO-1 system: vasodilation and topredict FBF quantitatively under heat stress. In this approach, forall three perturbations, heat off, HO-1 activity has been inhibited,and heme activity has been inhibited, initiates the commonbiochemical pathway leading to cell apoptosis have beenmodelled. It has been revealed that maximum alteration in shamcondition biochemical network of HO-1 system occurs due toconditions when heat is off, or HO-1 gene transcription has beeninhibited, or heme activity has been inhibited. For the presentedBoolean approach, hamming distance has been calculated fordifferent conditions using perturbation (forcibly introducing thefalse value to a particular node) in biochemical network withrespect to sham condition. The outcome of the present model hasbeen validated for different experimental conditions for hemedegradation and activation/deactivation of many other biomole-cules under heat stress, as discussed in Table 2a and 2b.

We have simulated the results when the HO-1 is in inactivatedstate or has been blocked, as shown in Table 2a. The simulatedresults were found to be similar with the published experimentalfindings [6]. Further, heme, a naturally occurring substrate for

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Table 4Model of the biochemical network driving heme degradation (Fig. 1). The model

consists of mass balances for seven nodes with mass transfer rates determined by

the reaction rates.

X:

HP ¼�J0XHP

X:

HOG ¼�J1XHOG

X:

HE ¼ J0XHP�J2ðXIRþXBV þXCoÞ

X:

IR ¼1

3ðJ2XHE�J3XIRÞ

X:

BV ¼1

3ðJ2XHE�J4XBV Þ

X:

CO ¼1

3J2XHE�ðJ5þ J7þ J8þ J9þ10ÞXCo

X:

sGC ¼ J5XCo�J6XsGC

Y. Aggarwal et al. / Computers in Biology and Medicine 40 (2010) 533–542 541

HO-1, has been previously shown to be a potent inducer of HO-1whereas metalloporphyrins, like ZnPP, are known to act as potentinhibitors of HO-1 induction [5]. The CO would not produce withthe inhibition of heat, HO-1 activity, heme activity. As CO appearsto block smooth muscle cells growth [5,34–36] by increasing theintracellular levels of cGMP via the activation of sGC. In fact, theinhibition of sGC blocks the rise in cGMP and anti-proliferativeaction of HO-1 [5], which supports our findings (Table 2a).

As demonstrated in the literature, the simulated result alsoshowed that the inhibition of HO-1 also causes the inhibition ofp38MAPK (via no release of CO), in turn HSP70 production hasbeen inhibited (via no activation of HSF-1) (Table 2b), whichcauses the initiation of cell apoptosis via the biosynthesis ofinflammatory cytokines such as IL-1 and TNF-a. Further, theinhibition of HO-1, activates the ERK and JNK that initiates the cellapoptosis program, which is also in accordance to the publishedfindings [5,10,28].

CO has also been reported to interact with a histidine residueon KCa channels leads to its activation. When activated, KCa

channels cause hyperpolarization of the membrane that inhibitsvoltage-dependent Ca++ channels. Inhibition of voltage-dependentCa2 + channels causes a reduction in intracellular Ca++ leading todilation [37]. The past experiments have demonstrated that KCa

channels in vascular smooth muscle participate in the regulationof membrane potential and hence vascular tone and reactivity.The result of the present electronic model for vasodilation(Table 2a and 2b) was also found similar to the experimentalresults. Conversely, through this model it has also been suggestedthat the pharmacological blockade of KCa channels depolarizesvascular smooth muscle and causes vasoconstriction in vesselswith myogenic tone [8,27].

Review of literature clearly demonstrates that both active aswell as passive pathways of thermoregulation have directinterference in regulation of body temperature to combat theeffect of changes in environmental temperature. Among them, thevasodilation is the first line of action under heat stress conditionsthat regulates further the sweating mechanism of humans.Therefore, aside the modelling of genetic regulation of HO-1system and its related network, the effects of HO-1 systemactivation on vasodilation has also been modelled with thedigital-analog hybrid electronic circuit. The results of this modelsuggest that the release of CO by heme degradation stimulate therelaxation of vascular smooth muscle by activating sGC thatfurther may exert a beneficial effect by activating cGMP, which isresponsible for vasodilation (increase in FBF) [5,24]. Qualitatively,the heat stress is displayed as input temperature V2 (Tsk) throughLED responses (Table 3). For the quantification of FBF under heatstress, the simulated results showed, whenever the cGMP is logichigh or initiated it causes the increase in FBF (Table 2a and 2b) asV2 increases [29,32] from 35 to 42 V. At 42 V FBF reached themaximum value of 18.7 ml 100 ml�1 min�1 with respect to basevalue of 2 ml 100 ml�1 min�1 [32,38]. The FBF remains in thisstate till the exposure is ON. After removal of exposure the FBFdrops gradually to 0 ml 100 ml�1 min�1. The increase in FBFresponses is dependent on the difference between the V2 and theset point V7. The pattern of FBF changes have been plotted for V2ranges from 35 to 42.5 V (Fig. 7). For the changes between 33 and35 V, there have been no recorded changes in FBF and lies at0 ml 100 ml�1 min�1 with respect to base value of 2 ml 100 ml�1

min�1, this is true with our simulated results [29]. Further, for V2ranges from 35 to 37 V the FBF rises to 5.3 ml 100 ml�1 min�1

from 0 ml 100 ml�1 min�1 with respect to base value of 2 ml 100ml�1 min�1 [39]. The simulated results are also found to be inaccordance with the observation of Taylor et al. [32]. The increasein FBF further explains the severity of heat stress on cardiovas-cular system [26].

6. Conclusion

With low production of endogenous NO, CO acts as the secondmessenger to trigger the vasodilation pathway. The presentBoolean model of HO-1 mediated vasodilation under heat stresswas built to predict biomolecules crucial to this pathway, therebysuggesting a probable technique for the prediction of importantbiomolecules and events implicated in complex disorders fortherapeutics action. With the knowledge of these predictedsites pharmaceutical and healthcare industries focus increasinglyon individual molecules as targets for targeted therapeutics,which may be used in the treatment of many inflammationand disorders caused due to heat stress and thus to preventheat-related death. From the functional point of view, the modelenables the prediction of forearm blood flow with the changes inskin temperature under exposure to environmental heat. Theincrease in forearm blood flow further explains the severity ofheat stress on cardiovascular system.

Appendix A. Analogy of biochemical pathway of hemedegradation system

Interactions among genes, proteins, and metabolites generatemost of the central functions of the living cell. These interactionstake place in highly complex biochemical networks. Exposing theconnections between the individual components and the overallbehavior of biochemical network requires a systems approachbased on dynamic models of the network, which is purelyqualitative. However, this qualitative approach can provideimportant steps in biochemical network, thus, enables one toobtain quantitative results. The approach is based on decompos-ing the overall network into small subsystems and then analyzingthe effects of interactions between these subsystems. Literatureshave suggested generalized non-linear differential equation tomodel biochemical pathways [19,20]. The generalized biochem-ical network for heme degradation under heat stress is illustratedin Fig. 3 and the corresponding model consisting of a set of non-linear ordinary differential equations (Table 4). The model isbased on a combination of interactions and experimental data.Many such biochemical networks have already been simulatedwith different softwares such as cell designer, systems biologyworkbench, and Matlab-systems biology toolbox. Under differentvariables and experimental conditions, experiments are going onto establish the biochemical network and related molecularkinetics of the HO-1 system. However, in current circumstancesthe numeric values of rate kinetics along with the concentrationof different parameters are still obscure. Thus, modelling thebiochemical network of HO-1 system with more conventionalkinetics approach is difficult, but can be considered as a veryimportant systems biology tool, if implemented properly.

ARTICLE IN PRESS

Y. Aggarwal et al. / Computers in Biology and Medicine 40 (2010) 533–542542

Conflict of interest statement

None declared.

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