Energetics and Function  
Determination of energy carriers by 31P NMR spectroscopy
 
  In order to ensure a continuous beating of the heart, its energy supply has to be reliably regulated. The most important energy carrier within the body is ATP; its chemical structure is shown in the margin. This molecule can be excellently detected by 31P NMR spectroscopy – each of its three phosphorus atoms yields a unique signal, as can be seen in the figure on the right hand side.  
Adenosine triphosphate (ATP)
Within a 31P NMR spectrum of an isolated perfused mouse heart additional signals for phosphocreatine (PCr) as well as for intra- und extracellular inorganic phosphate (labelled with Pi(int.) and Pi(ext.), respectively) can be identified. In particular the position of the latter signals in the spectrum (the chemical shift) strongly depends from the pH value of the tissue environment, which can be used to easily calculate (→ more details) both the extracellular and cytosolic pH (pHe and pHi, respectively).
For a long time it was accepted that solely the absolute amount of ATP is the crucial driving force for the energy-consuming cellular processes. However, in the meantime there is general agreement that not only the ATP concentration, but specifically the ratio of ATP to its breakdown products ADP and Pi is the decisive determinant for the amount of energy released by ATP hydrolysis (ATP ↔ ADP + Pi). The intracellular ADP concentration can be calculated from the 31P NMR spectrum of the heart via the creatine kinase equilibrium (PCr + ADP ↔ ATP + Cr). Under normal conditions the amount of ADP is about two orders of magnitude lower than the cytosolic ATP level (~30 µM ADP compared with ~6 mM ATP). Thus, by means of 31P NMR spectroscopy all parameters required to assess the amount of energy available to the heart (ΔGATP) can be continuously and non-invasively monitored (→ more details).

In parallel to the energetics, cardiac pump function can be determined via a balloon introduced into the left ventricle and attached to a pressure transducer. A characteristic pressure registration obtained with this setup is illustrated in the upper part of the 31P NMR spectrum. The zoomed area shows clearly the periodical pressure alterations during the heart action. The minima of the trace reflect the end-diastolic and the maxima the end-systolic pressure, respectively – the difference [maximum - minimum] yields the developed pressure of the heart. The distance between the minima or maxima can be used to calculate the heart rate, which is found to be tenfold higher in the mouse (~600 beats per minute) than in man.

 
 
 
Ischemia and reperfusion
 
 
  31P NMR spectroscopy is especially suitable to study alterations of the myocardial energy metabolism when the supply of the heart is interrupted. This may occur by a sudden occlusion of one of the coronary arteries such as during a heart attack. This for man extremely dangerous event can be simulated in the laboratory with the isolated heart preparation by stopping the buffer supply. The period without perfusion is referred to as ischemia, the period after restoration of the supply as reperfusion.  
As can be clearly seen in the left figure, immediately after interruption of the heart supply a drop in the PCr and a rise in the Pi(int.) level are observed. With reference to the dotted red line a high field shift of the Pi(int.) signal can be concomitantly made out, which reflects the acidification of the muscle tissue due to the limited perfusion. On the other hand, an absolute ATP depletion occurs only after 15 minutes – even at a complete stop of the buffer supply.

In parallel to the exhaustion of cardiac energy stores left ventricular pressure development dwindles and thereby also the pump function of the heart (figure at the right). Very quickly contractile function completely grinds to a halt, and a bit later the so-called contracture develops (pressure increase during ischemia), which is attributed to the inundation of the cytosol by Ca2+ ions. The latter is caused by breakdown of the membranous ion gradients and failure of ATP-driven ion pumps, respectvely, as a consequence of the impaired energy state.

When the supply of the heart is re-established, the PCr level already recovers after a couple of minutes. Immediately after restoration of the perfusion an "overshoot" of PCr over the basal value (known as PCr overshoot) is frequently observed. In contrast, ATP levels often do not reach their initial values due to loss of purines during ischemia and the early reperfusion period, respectively. After normalization of cardiac pHi also the contractile parameters get more and more stabilized.

To what extent the original heart function can be recovered depends from the duration of ischemia and the applied "therapy" in terms of additionally infused drugs or the transgenic model used. For example, we could show that hearts without endothelial nitric monoxid synthase (eNOS knockout mice) recover better from an ischemic period than the corresponding control hearts, whereas on the other hand the lack of myoglobin has an adverse effect (compare also the original publications given below). Thus, by an appropriate combination of transgenic animal model and pharmacologic intervention experimental series can be carried out, that may give a clue for a more effient therapy of heart attack in man.

 
 
 
In vivo 2D mapping of the energy state
 
 
  Investigating energetics in the perfused mouse heart as mentioned above offers the advantage to have almost full control over each individual experimental parameter. On the other hand, there are two major disadvantages of this approach: Firstly, experiments are not carried out under "true" physiological conditions and secondly, they are limited to end point studies and do not permit repetitive measurements of the same object in terms of longitudinal studies. Particularly, in order to follow alterations in the energy state during the course of a disease and an initiated therapy, respectively, cardiac 31P NMR spectroscopic investigations under real in vivo conditions are of great interest.

Compared with the approach described in the first chapter, where 31P NMR spectra were simply acquired over the full sensitive volume, the main difficulty herein consists in ensuring an exact localization of the spectra within the heart and avoiding contaminations of signals from adjacent tissue. For this, the thorax of the mouse is "covered" with a spectroscopic grid, which exactly correlates with the anatomical 1H MR image. The figure at the left exemplary illustrates the superimposing of such a 2D 31P CSI dataset (Chemical Shift Imagingtheory) and the corresponding proton image. Despite the low resolution of the image it can be clearly recognized that only there, where tissue is present, 31P signals are detected. Strong signals are especially found in the skeletal muscle of the animals back (in the upper part of the figure) and the area of the heart.

As expected, only small signals are detected within the lung region due to the low density of this tissue. This becomes even more clear when looking at a more detailed presentation of the overlay. Representative, spatially resolved spectra of the anterior wall and the septum of a normal mouse heart (WT=wildtype) are displayed at the left of the figure shown below. The spectrum acquired from the anterior wall (#2) shows a very similar pattern as the 31P NMR spectrum of the isolated perfused heart shown above, whereas for spectra from the chamber septum (#1) it is more or less unavoidable to pick up some 31P signal from the blood. This becomes noticeable not only in additional signals in the spectral area of the inorganic phosphate (Pi), but also in an apparent reduction of the PCr/ATP ratio.
When this approach is applied to investigate myocardial energetics of mice, which have been genetically manipulated to induce a heart diesease, one can observe that this is – very similar to man – accompanied by an impairment of the normal energy state. At the right-hand side of the figure in the margin, this is illustrated for a mouse strain which is – due to cardiac overexpression of the inducible NO synthase and the concomitant lack of myoglobin (TGiNOS/myo-/-, see also the section about myoglobin and NO) – characterized by the appearance of left ventricular hypertrophy, which in an advanced stage may even lead to heart failure (see Gödecke et al, 2003 for a detailed description).  
The comparison of 31P NMR spectra of these TGiNOS/myo-/- and normal WT hearts demonstrates in both anterior wall and septum pronounced lower PCr levels for the mutant, reflecting a limited availability of this "energetic buffer" as a result of the genetic defects. Concomitantly, in the corresponding 1H MR image already a "wear out" of the left chamber is perceptible: The enddiastolic volume of the transgenic mouse (right) is substantially larger as compared with the healthy control mouse (left). A detailed analysis of the heart function making use of dynamic CineFLASH movies (look here for a description of the method) confirmed this first visual impression and showed a significantly impaired pump function of TGiNOS/myo-/- hearts. The data about cardiac energetics acquired from the live animals were verified by high resolution 31P NMR spectroscopy after organ excision, and an excellent agreement of in vivo and ex vivo data was obtained. It is noteworthy, that the extent of the observed detoriation of cardiac energetics in our mouse model is of similar magnitude as the alterations determined in man during development of heart failure (see Flögel et al, 2007 for some more details).

By the combined use of 1H MRI and 31P CSI a rather complete picture about heart function and energetics of genetic modified mouse strains generated as models for the exploration of the underlying causes of human cardiomyopathies can be obtained. Due to the noninvasive nature of these investigations, it will be possible via repetitive measurements to monitor the progession of the induced heart diseases and to assess the success of suitable therapies.

 
 
 
Own work about cardiac energetics and function
 
 
A complete overview about our peer-reviewed publications of the last years can be found here. The references are linked with the PubMed abstracts of the National Library of Medicine. If your are interested in one of these papers, and you don't have online access to the respective journal, send us an email, so that we can provide you with the appropriate pdf-file.
 
Methodical studies
 
Flögel U, Jacoby C, Gödecke A, Schrader J.
In vivo 2D mapping of impaired murine cardiac energetics in NO-induced heart failure.
Magn Reson Med. 2007; 57: 50-8.
 
Flögel U, Decking UK, Gödecke A, Schrader J.
Contribution of NO to ischemia-reperfusion injury in the saline-perfused heart: a study in endothelial NO synthase knockout mice.
J Mol Cell Cardiol. 1999; 31: 827-36.
 
Applications
 
Haddad S, Wang Y, Galy B, Korf-Klingebiel M, Hirsch V, Baru AM, Rostami F, Reboll MR, Heineke J, Flögel U, Groos S, Renner A, Toischer K, Zimmermann F, Engeli S, Jordan J, Bauersachs J, Hentze MW, Wollert KC, Kempf T.
Iron-regulatory proteins secure iron availability in cardiomyocytes to prevent heart failure.
Eur Heart J. 2017; 38: 362-372.
 
Tucci S, Flögel U, Hermann S, Sturm M, Schäfers M, Spiekerkoetter U.
Development and pathomechanisms of cardiomyopathy in very long-chain acyl-CoA dehydrogenase deficient VLCAD(-/-) mice.
Biochim Biophys Acta 2014; 1842: 677-85.
 
Luedde M, Flögel U, Knorr M, Grundt C, Hippe HJ, Brors B, Frank D, Haselmann U, Antony C, Voelkers M, Schrader J, Most P, Lemmer B, Katus HA, Frey N.
Decreased contractility due to energy deprivation in a transgenic rat model of hypertrophic cardiomyopathy.
J Mol Med. 2009; 87: 411-22.
 
Rassaf T, Flögel U, Drexhage C, Hendgen-Cotta U, Kelm M, Schrader J.
Nitrite reductase function of deoxymyoglobin: oxygen sensor and regulator of cardiac energetics and function.
Circ Res. 2007; 100: 1749-54.
 
Flögel U, Gödecke A, Klotz LO, Schrader J.
Role of myoglobin in the antioxidant defense of the heart.
FASEB J. 2004; 18: 1156-8.
 
Warskulat U, Flögel U, Jacoby C, Hartwig HG, Thewissen M, Merx MW, Molojavyi A, Heller-Stilb B, Schrader J, Häussinger D.
Taurine transporter knockout depletes muscle taurine levels and results in severe skeletal muscle impairment but leaves cardiac function uncompromised.
FASEB J. 2004; 18: 577-9.
 
Wunderlich C, Flögel U, Gödecke A, Heger J, Schrader J.
Acute inhibition of myoglobin impairs contractility and energy state of iNOS-overexpressing hearts.
Circ Res. 2003; 92: 1352-8.
 
Gödecke A, Molojavyi A, Heger J, Flögel U, Ding Z, Jacoby C, Schrader J.
Myoglobin protects the heart from inducible nitric-oxide synthase iNOS-mediated nitrosative stress.
J Biol Chem. 2003; 278: 21761-6.
 
Merx MW, Flögel U, Stumpe T, Gödecke A, Decking UK, Schrader J.
Myoglobin facilitates oxygen diffusion.
FASEB J. 2001;15: 1077-9.
 
Flögel U, Merx MW, Gödecke A, Decking UK, Schrader J.
Myoglobin: A scavenger of bioactive NO.
Proc Natl Acad Sci USA. 2001; 98: 735-40.
 
Gödecke A, Flögel U, Zanger K, Ding Z, Hirchenhain J, Decking UK, Schrader J.
Disruption of myoglobin in mice induces multiple compensatory mechanisms.
Proc Natl Acad Sci USA. 1999; 96: 10495-500.