We used MoCA (Modular Control and Regulation Analysis) to demonstrate in intact beating rat heart that physiological activation of contraction by adrenaline involves the almost perfect parallel activation of both mitochondria and myofibrils by intracellular Ca2+. This explains the perfect homoeostasis of the energetic intermediate PCr (phosphocreatine) in heart. When using drugs specifically stimulating either supply or demand activities, MoCA helped reveal the very specific mode of regulation of heart contraction energetics. Only activation of myofibrils activity (demand), either by increasing intracellular Ca2+ concentration or myofibrils sensitivity to Ca2+, triggers activation of contractile activity. In contrast, the activation of mitochondrial activity (supply) has strictly no effect on contraction, either directly or through PCr changes (intermediate).

Introduction

Top-down or modular approaches to MCA (Metabolic Control Analysis) have been used by us and others to overcome the complexity of investigating intracellular and intra-organ regulation [1]. By combining MCA with non-invasive 31P-NMR measurement of energetic intermediates and simultaneous measurement of heart contractile activity, we developed MoCA (Modular Control and Regulation Analysis), an integrative approach to study in situ control and regulation of cardiac energetics during contraction in intact beating perfused isolated heart [2]. MoCA was designed to provide better insight into the control and regulation of the energetics of heart contraction, described as a two-module system (energy supply and energy demand) connected by the energetic intermediates ATP and PCr (phosphocreatine). The elasticities (responsiveness) of supply and demand modules to PCr changes have been experimentally determined in intact beating heart (Figure 1), and further used to describe the normal or pathological control pattern and perform regulation analyses (see [2] for a complete description of the analyses).

System definition for MoCA of the energetics of perfused heart

Figure 1
System definition for MoCA of the energetics of perfused heart

The system of contraction is defined as two modules connected by the pool of energetic intermediates. The supply module comprises all the metabolic steps from substrate supply (including oxygen supply) to mitochondrial production of PCr. The demand module comprises all the steps consuming ATP/PCr linked to contractile activity (contraction of myofilaments and ATP used for Ca2+ recapture). The elasticities of each module towards the intermediates (PCr measured by 31P NMR is used as representative) are calculated after inducing slight changes in steady-state contraction by the alteration of the other module. Cyanide (KCN) was chosen to specifically inhibit mitochondrial cytochrome oxidase and measure demand elasticity (ϵD) as the response of the system to the relative PCr concentration change observed. On the other hand, change in internal balloon pressure (Frank–Starling effect) was used to increase contractility and measure supply elasticity (ϵS) [2].

Figure 1
System definition for MoCA of the energetics of perfused heart

The system of contraction is defined as two modules connected by the pool of energetic intermediates. The supply module comprises all the metabolic steps from substrate supply (including oxygen supply) to mitochondrial production of PCr. The demand module comprises all the steps consuming ATP/PCr linked to contractile activity (contraction of myofilaments and ATP used for Ca2+ recapture). The elasticities of each module towards the intermediates (PCr measured by 31P NMR is used as representative) are calculated after inducing slight changes in steady-state contraction by the alteration of the other module. Cyanide (KCN) was chosen to specifically inhibit mitochondrial cytochrome oxidase and measure demand elasticity (ϵD) as the response of the system to the relative PCr concentration change observed. On the other hand, change in internal balloon pressure (Frank–Starling effect) was used to increase contractility and measure supply elasticity (ϵS) [2].

This integrative approach allows the measurement and comparison of the relative importance of the different routes, i.e. supply, demand and metabolite changes, by which the effect(s) of a drug/hormone [3,4] or a pathology [5,6] are transmitted. In particular, the study of inotropic drugs/action permits the qualitative and quantitative description of the activation of heart contraction.

Physiological activation of heart contraction

In contrast with skeletal muscle, where PCr and phosphorylation potential (ΔGp) decrease during contraction, the energy balance in heart bioenergetics is characterized by an improved homoeostasis [79]. Indeed, almost no changes in the energetic intermediates (PCr, ATP and Pi) are observed following important increase in heart activity, as seen, for example, by 31P NMR spectroscopy of isolated perfused heart [10]. How this homoeostasis is achieved in heart, but not in muscle, during the so-called excitation–contraction coupling is still a fundamental question under debate.

By using MoCA, we demonstrated non-invasively in intact beating heart the essential role of intracellular Ca2+ in the coupling process between electrical excitation of the myocyte and the energetics of contraction in heart [2], as demonstrated previously in vitro [7,8] and in silico [11]. This has been demonstrated by analysing the response of heart contraction to changes in Ca2+ in the perfusate, which established the almost perfect parallel activation of both supply and demand under these conditions. This activation cascade has also to be understood in accordance with the now well-established activation of the different modules of mitochondrial oxidative phosphorylation by Ca2+ [1214].

We further analysed the response of heart contraction to a physiological activator: adrenaline [4]. Adrenaline increases the peak tension during heart contraction by a complex physiological mechanism involving modulations in intracytosolic Ca2+, both mean cytosolic concentration and transient rises which trigger rhythmic contraction. Interestingly, a direct relationship has been demonstrated between contractile activity and cytosolic Ca2+ concentration when heart contraction was modulated either by varying external Ca2+ concentration or by stimulating the β1-adrenergic receptor [7,15]. The stronger activation by catecholamines (similar to adrenaline when compared with external Ca2+) are explained by a marked facilitation of Ca2+ transients and a shortening of the Ca2+ transient that is reflected in a prominent decrease in the duration of contraction and an acceleration of relaxation by cAMP-dependent mechanisms [16]. We have demonstrated that the adrenaline inotropic effect also involves parallel activation of both ATP–PCr supply and demand [4]. Figure 2 presents the full MoCA analysis of the effects of adrenaline addition in the perfusate. The main results concern the evidence that the total effect on contraction energy flux (approx. 85%) is completely due to the direct effet of adrenaline on the demand module. The much greater direct activation of the supply module (approx. 50% higher: 125%) has no effect on the flux (very low control), but triggers a surprising, and paradoxical, increase in PCr concentration, which has in turn a strong inhibitory effect on supply activity (approx. 40%) (Figure 2). MoCA clearly shows that physiological activation by adrenaline cannot be reduced to a simple Ca2+ effect and that a specific activation of mitochondrial oxidative phosphorylation occurs. There is still no clear explanation for these results, and the role of cAMP on Ca2+ transients and relaxation (re-uptake of Ca2+ by the sarcoplasmic reticulum) is presently being investigated.

MoCA of the effects of adrenaline addition in the perfusion medium on heart energetics

Figure 2
MoCA of the effects of adrenaline addition in the perfusion medium on heart energetics

The effects of 0.7 μM adrenaline on both supply and demand modules were calculated as described in [2]; direct effect refers to the integrated elasticity (total effect−indirect effect), and global effect to the response coefficient of each module on the increase in contraction induced by adrenaline [1,2]. The size of the arrows is proportional to adrenaline effect and the numbers represent the effect expressed as the percentage change from the starting condition (no adrenaline).

Figure 2
MoCA of the effects of adrenaline addition in the perfusion medium on heart energetics

The effects of 0.7 μM adrenaline on both supply and demand modules were calculated as described in [2]; direct effect refers to the integrated elasticity (total effect−indirect effect), and global effect to the response coefficient of each module on the increase in contraction induced by adrenaline [1,2]. The size of the arrows is proportional to adrenaline effect and the numbers represent the effect expressed as the percentage change from the starting condition (no adrenaline).

Specific activation of the supply module

The absence of effect of supply activation on contractile activity observed in the presence of adrenaline has also been observed when studying the effect of specific activators of the Supply module. Indeed, we could not observe any significant contractile activity increase either by using a vasodilator, isosorbide dinitrate, to increase oxygen and substrate supply, or by infusion of DCA (dichloroacetate), an activator of pyruvate dehydrogenase (V. Deschodt-Arsac, G. Calmettes and P. Diolez, unpublished results). In the absence of an effect, MoCA regulation could not be performed, but results from MoCA elasticity and control analyses may be used to explain these results. Indeed, due to the very high elasticity of the supply module towards PCr, this module has a very low control on the contraction energy flux. This can be seen with the analysis of the effects of adrenaline which shows that a very high direct effect on the supply module results in almost no global effect on contractile activity (see Figure 2 and [2]).

Specific activation of the demand module

Physiologically, there are only two ways to increase contractile activity by acting on myofibril ATPase activity, the primary mechanism for contraction, which are an increase in cytosolic Ca2+ concentration or an increase in Ca2+ sensitivity of troponin [7]. Since the increase in cytosolic Ca2+ concentration, by adding Ca2+ or adrenaline, directly activates mitochondrial oxidative phosphorylation and therefore ATP–PCr supply [2,4], we decided to increase the sensitivity of troponin c for Ca2+ by utilizing the Frank–Starling effect. This effect corresponds to a physiological inotropic response of the heart to an increased blood pressure in the ventricle; the resulting myocytes mechanical stretching increases troponin sensitivity to Ca2+ and is responsible for the contraction strength increase in the absence of a Ca2+ increase [16]. Experimentally, this can be easily carried out by increasing the volume of the balloon inserted in the ventricle and used to monitor contractile activity of perfused heart. Unlike supply activation, the Frank–Starling effect results in an effective increase in heart contraction. However, in contrast with the physiological heart activation, a decrease in PCr concentration is always observed under these conditions [2], as found in skeletal muscle [17].

Conclusion

In striking contrast with skeletal muscle, the energy balance in heart bioenergetics is characterized by an improved homoeostasis [7,9,18]. Indeed, only slight changes in the energetic intermediates (PCr, ATP and Pi) are observed following an important increase in heart activity, as seen, for example, by 31P-NMR on isolated perfused heart. How this homoeostasis is achieved in heart, but not in muscle, during the so-called excitation–contraction coupling is still under debate and of great importance in the comprehensive study of pathologies.

The results presented here show that heart contraction energetics can be studied as a supply–demand system, for example, using MoCA elasticity and regulation analyses. MoCA elasticity analysis has mainly revealed the very low control of supply (mainly mitochondria) in isolated heart, due to a very high elasticity of this module toward PCr, representative of the energetic intermediates. These results were confirmed by the total absence of change in heart contraction when Supply was directly activated (vasodilator or DCA). On the other hand, the almost total control by demand was demonstrated by the Frank–Starling effect, which allows activation of contraction in the absence of Ca2+ increase and thus supply activation. However, under these conditions the homoeostasis of the energetic intermediates is affected.

Under physiological conditions, regulation analysis not only demonstrated that a parallel activation of both energy supply and demand occurs during Ca2+ or adrenaline activation in heart, but also that the inotropic effect is the direct consequence of myofilaments activation and not the direct effect of Ca2+ on mitochondria. The direct mitochondrial activation by Ca2+ is, however, crucial for the homoeostasis of energetic intermediates and for heart physiological response.

These results show that systems biology approaches can be fruitfully applied to the study of organ function, as well as organ dysfunction or the effects of drugs.

Systems Approaches to Health and Disease: A Biochemical Society Focused Meeting held at University of York, U.K., 22–24 March 2010, as part of the Systems Biochemistry Linked Focused Meetings. Organized and Edited by David Fell (Oxford Brookes, U.K.), Hans Westerhoff (Manchester, U.K., and Amsterdam, The Netherlands) and Michael White (Liverpool, U.K.).

Abbreviations

     
  • DCA

    dichloroacetate

  •  
  • MCA

    Metabolic Control Analysis

  •  
  • MoCA

    Modular Control and Regulation Analysis

  •  
  • PCr

    phosphocreatine

Funding

This work was supported, in part, by the Association Française contre les Myopathies [grant number AFM 12338].

References

References
1
Brand
 
M.D.
Curtis
 
R.K.
 
Simplifying metabolic complexity
Biochem. Soc. Trans.
2002
, vol. 
30
 (pg. 
25
-
30
)
2
Diolez
 
P.
Deschodt-Arsac
 
V.
Raffard
 
G.
Simon
 
C.
Santos
 
P.D.
Thiaudiere
 
E.
Arsac
 
L.
Franconi
 
J.M.
 
Modular regulation analysis of heart contraction: application to in situ demonstration of a direct mitochondrial activation by calcium in beating heart
Am. J. Physiol. Regul. Integr. Comp. Physiol.
2007
, vol. 
293
 (pg. 
R13
-
R19
)
3
Korzeniewski
 
B.
Deschodt-Arsac
 
V.
Calmettes
 
G.
Gouspillou
 
G.
Franconi
 
J.M.
Diolez
 
P.
 
Effect of pyruvate, lactate and insulin on ATP supply and demand in unpaced perfused rat heart
Biochem. J.
2009
, vol. 
423
 (pg. 
421
-
428
)
4
Korzeniewski
 
B.
Deschodt-Arsac
 
V.
Calmettes
 
G.
Franconi
 
J.M.
Diolez
 
P.
 
Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply
Biochem. J.
2008
, vol. 
413
 (pg. 
343
-
347
)
5
Calmettes
 
G.
Deschodt-Arsac
 
V.
Thiaudiere
 
E.
Muller
 
B.
Diolez
 
P.
 
Modular control analysis of effects of chronic hypoxia on mouse heart
Am. J. Physiol. Regul. Integr. Comp. Physiol.
2008
, vol. 
295
 (pg. 
R1891
-
R1897
)
6
Calmettes
 
G.
Deschodt-Arsac
 
V.R.
Gouspillou
 
G.
Miraux
 
S.
Muller
 
B.
Franconi
 
J.-M.
Thiaudiere
 
E.
Diolez
 
P.
 
Improved energy supply regulation in chronic hypoxic mouse counteracts hypoxia-induced altered cardiac energetics
PLoS ONE
2010
, vol. 
5
 pg. 
e9306
 
7
Balaban
 
R.S.
 
Cardiac energy metabolism homeostasis: role of cytosolic calcium
J. Mol. Cell. Cardiol.
2002
, vol. 
34
 (pg. 
1259
-
1271
)
8
Balaban
 
R.S.
Bose
 
S.
French
 
S.A.
Territo
 
P.R.
 
Role of calcium in metabolic signaling between cardiac sarcoplasmic reticulum and mitochondria in vitro
Am. J. Physiol. Cell Physiol.
2003
, vol. 
284
 (pg. 
C285
-
C293
)
9
Kushmerick
 
M.J.
 
Skeletal muscle: a paradigm for testing principles of bioenergetics
J. Bioenerg. Biomembr.
1995
, vol. 
27
 (pg. 
555
-
569
)
10
Diolez
 
P.
Simon
 
C.
Leducq
 
N.
Canioni
 
P.
Dos Santos
 
P.
 
Hofmeyr
 
J.-H.S.
Rohwer
 
M.
Snoep
 
J.L.
 
Top down analysis of heart bioenergetics
BTK2000: Animating the Cellular Map
2000
Stellenbosch
Stellebosch University Press
(pg. 
101
-
106
)
11
Korzeniewski
 
B.
Noma
 
A.
Matsuoka
 
S.
 
Regulation of oxidative phosphorylation in intact mammalian heart in vivo
Biophys. Chem.
2005
, vol. 
116
 (pg. 
145
-
157
)
12
Kavanagh
 
N.I.
Ainscow
 
E.K.
Brand
 
M.D.
 
Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria
Biochim. Biophys. Acta
2000
, vol. 
1457
 (pg. 
57
-
70
)
13
Johnston
 
J.D.
Brand
 
M.D.
 
Stimulation of the respiration rate of rat liver mitochondria by sub-micromolar concentrations of extramitochondrial Ca2+
Biochem. J.
1987
, vol. 
245
 (pg. 
217
-
222
)
14
Mildaziene
 
V.
Baniene
 
R.
Nauciene
 
Z.
Marcinkeviciute
 
A.
Morkuniene
 
R.
Borutaite
 
V.
Kholodenko
 
B.
Brown
 
G.C.
 
Ca2+ stimulates both the respiratory and phosphorylation subsystems in rat heart mitochondria
Biochem. J.
1996
, vol. 
320
 (pg. 
329
-
334
)
15
Wu
 
S.T.
Kojima
 
S.
Parmley
 
W.W.
Wikman-Coffelt
 
J.
 
Relationship between cytosolic calcium and oxygen consumption in isolated rat hearts
Cell Calcium
1992
, vol. 
13
 (pg. 
235
-
247
)
16
Endoh
 
M.
 
Signal transduction and Ca2+ signaling in intact myocardium
J. Pharmacol. Sci.
2006
, vol. 
100
 (pg. 
525
-
537
)
17
Arsac
 
L.M.
Beuste
 
C.
Miraux
 
S.
Deschodt-Arsac
 
V.
Thiaudiere
 
E.
Franconi
 
J.M.
Diolez
 
P.H.
 
In vivo modular control analysis of energy metabolism in contracting skeletal muscle
Biochem. J.
2008
, vol. 
414
 (pg. 
391
-
397
)
18
Balaban
 
R.S.
Kantor
 
H.L.
Katz
 
L.A.
Briggs
 
R.W.
 
Relation between work and phosphate metabolite in the in vivo paced mammalian heart
Science
1986
, vol. 
232
 (pg. 
1121
-
1123
)