In and ex Vivo Mitochondrial Function of the Heart
Condition: Diabetes Mellitus, Type 2; Diabetic Cardiomyopathies
MeSH terms: Cardiomyopathies; Diabetic Cardiomyopathies; Diabetes Mellitus, Type 2
Study type: Observational [Patient Registry]

Summary: 
      It has been suggested that mitochondrial dysfunction might play a role in the development of
      diabetic cardiomyopathy. From animal studies, it has been suggested that an altered PPAR and
      PGC1 expression is involved in the reduced cardiac mitochondrial function, however human data
      on cardiac mitochondrial function and PPAR regulation is scarce. The latter is due to the
      fact that there is no validated measurement for assessing cardiac mitochondrial function
      non-invasively in vivo. It has been suggested that measuring PCr/ATP ratio with 31P-MRS in
      the heart reflects cardiac mitochondrial function. However, so far no direct validation of
      this method has been performed. The aim of this study will be to validate in vivo 31P-MRS
      with ex vivo measurements of mitochondrial function. To this end, the hypothesis is that in
      vivo 31P-MRS is a valid method for measuring cardiac mitochondrial function when compared
      with ex vivo mitochondrial respirometry.
    

Detailed description: 
      Cardiovascular diseases remain the main cause of death in type 2 diabetes. Most of this is
      attributed to atherosclerosis and elevated blood pressure, though even when corrected for
      these factors, patients with type 2 diabetes are still at increased risk for developing
      cardiac failure, mainly through diastolic dysfunction. This phenomenon has also been
      described as diabetic cardiomyopathy. Although not much is known about the aetiology of this
      disease, there is compelling evidence from animal research that an increased intracellular
      cardiac fat accumulation and mitochondrial dysfunction, as seen in type 2 diabetes, may play
      a part in this development.

      The reason for a reduced mitochondrial function in diabetic cardiomyopathy is not completely
      understood, however the gene regulatory pathway of peroxisome proliferator-activated receptor
      alpha (PPAR-α) has been identified as an important determinant of the shift in substrate
      metabolism and regulation of oxidative metabolism in type 2 diabetes. In animal studies, the
      role of PPARα has been tested extensively. In mice with cardiac-restricted overexpression of
      PPARα (MHC-PPAR), it was found that PPAR-α is involved in the upregulation of CPT-1 in
      mitochondria, which increases the uptake of long-chain fatty acid into mitochondria and
      facilitates the fatty acids to undergo beta-oxidation. Chronic exposure to elevated FFAs down
      regulates PPAR-α in rodent cardiomyocytes, which would further decrease cardiac function by
      inhibition of FA oxidation and increased intracellular fat accumulation. It is therefore
      speculated that the increase in fatty acid availability in type 2 diabetes and obesity (due
      to excessive fat mass) leads to a decrease in cardiac PPAR-α metabolism and thereby a
      decrease in mitochondrial metabolism, which in turn is paralleled by an increased cardiac fat
      accumulation and cardiac lipotoxicity.

      So far, human studies on PPAR expression in the heart are scarce. Marfella et al. found
      unaltered expression of PPAR-α in patients with the metabolic syndrome. Conversely, Anderson
      et al., showed a slightly reduced PPAR-α protein level and a slightly higher PGC1α level in
      diabetic atrial tissue, though these differences did not reach statistical significance in
      this cohort of patients. As this study was performed in a small group of subjects and failed
      to determine the down-stream targets of the PPAR metabolism or mitochondrial function, it
      remains unclear whether like in animal studies also in humans a reduced PPAR-a expression is
      related to mitochondrial dysfunction. Therefore there is need for studies exploring the role
      of PPAR-a in human heart and the connections with oxidative metabolism and cardiac function.
      Mice lacking the cardiac lipase ATGL (ATGL-/- mice) was actually due to a reduction in PPAR
      metabolism, and that the cardiomyopathy in these mice could be completely prevented by
      treating these animals with synthetic PPAR-a ligands. Very interestingly, patients with a
      mutation in the same ATGL gene are also characterized by excessive cardiac and muscle fat
      accumulation and reduced mitochondrial function. Treating two patients with such mutations
      (which is a very rare mutation) with a PPAR-agonist (bezafibrate) resulted in improved
      mitochondrial function and a reduction in muscle and cardiac lipid accumulation. These data
      support the notion that a disturbed PPAR metabolism may be involved in the development of
      cardiomyopathy, also in humans. However, unfortunately, there is limited data on
      PPAR-expression in the failing human diabetic heart. Therefore there is need of studies
      validating these mechanisms in humans, as these findings might have great consequences;
      prevention and treatment of cardiac lipid accumulation with drugs that improve mitochondrial
      function, such as PPAR-agonists, might be of value to patients with type 2 diabetes. Also in
      other cardiac diseases, such as chronic heart failure and ischemic heart disease, it has been
      suggested that fat accumulation and mitochondrial dysfunction may play a role, which means
      that these patients might benefit as well from treatment with drugs that target mitochondrial
      function.

      Although there is compelling evidence that mitochondrial function plays an important role in
      cardiac metabolism, measuring cardiac mitochondrial function non-invasively in vivo remains a
      challenge. In vivo mitochondrial function can be estimated non-invasively with 31P-Magnetic
      Resonance Spectroscopy (31P-MRS), whereby the ratio Phospho-creatine (PCr) over Adenosine
      Triphosphate (ATP) is measured (PCr/ATP-ratio). Several studies have shown that this ratio is
      reduced in patients with type 2 diabetes, and that a low PCr/ATP ratio predicts mortality in
      patients with cardiac failure15-17. In skeletal muscle it has been shown that PCr-resynthesis
      strongly correlates with mitochondrial oxidative capacity18. However, if this method in the
      heart truly reflects mitochondrial function in humans has not been revealed.

      In 31P-MRS a 2 dimensional measurement method is used, in which multiple slices are planned
      over the heart. One slice is planned directly at the base of the heart in the plane just
      below the valves and contains both ventricular and septal tissue of both chambers. Here the
      signal for acquisition of the spectrum will be derived. This method can be validated against
      the golden standard for mitochondrial function: ex vivo respirometry of cardiac tissue19.
      Thus, mitochondrial respiration rates are measured in tissue homogenates under exposure of
      different substrates, stimulating different complexes of the electron transport chain of the
      mitochondria. One issue is that mitochondrial respiration may differ between atrial and
      ventricular tissue. However, despite the differences in absolute respiration rates, the
      behaviour of the different complexes and relative respiration rates (between complexes) has
      been shown to be very strongly related19. Since it is relatively easy to obtain atrial
      appendage cardiac tissue during surgery, the investigators propose to use atrial tissue
      obtained during surgery to validate 31P-MRS as a tool to determine mitochondrial function.
      The investigators will use a broad range of patients to guarantee a range in cardiac
      mitochondrial functions, and to examine if cardiac mitochondrial function is indeed reduced
      in type 2 diabetic patients.

      However, as the 31P-MRS still is a technique in development and the distance to the receiver
      coil is crucial for obtaining spectra of good quality, the investigators intend to only
      include men at this time (as increased breast mass in women may decrease signal to noise
      ratios and hence spectral quality for analysis). Therefore, the validation of this method
      will only apply for the male population in this study.
    

Intervention name(s): 31P-MRS scan and cardiac MRI scan; Atrial biopsy for high-resolution respirometry; Blood analysis; Body composition; Indirect Calorimetry
Intervention type(s): Diagnostic Test; Diagnostic Test; Diagnostic Test; Diagnostic Test; Diagnostic Test
Intervention description(s): 31P-MRS measurement of the heart: Phosphorous magnetic resonance spectroscopy (31P-MRS) will be used for the determination of the energy status of the heart in vivo. The localization of phosphorus energy metabolites in the heart will be performed by one-dimensional spectroscopic imaging, resulting in spectra from slices through the heart of 1 cm thickness. The peaks of PCr and ATP will be fitted in order to calculate the ATP/PCr ratio.
The measurement of cardiac function is a standardised protocol. In the 4 chamber view setting a T1 mapping sequence will be performed for the detection of fibrosis. Hereafter patients will receive contrast for T2 mapping and late enhancement studies for determining extracellular volume and ischemia.; Mitochondrial respiration rates are ex vivo measured in tissue homogenates under exposure of different substrates, stimulating different complexes of the electron transport chain of the mitochondria.; To determine blood glucose metabolism and renal function.; This device uses air displacement plethysmography for determining percent fat and fat-free mass in adults and children. The 5-minute test consists of measuring the subject's mass (weight) using a very accurate electronic scale, and volume, which is determined by sitting inside the BOD POD chamber. From these two measurements, the subject's body composition is calculated.; As basal energy metabolism might be related to cardiac metabolism and mitochondrial function, and basal metabolic rate shows inter-individual variance, it is an important subject characteristic of the patients. Resting substrate oxidation rates are measured with indirect calorimetry for 30 minutes. A ventilated hood is connected to indirect calorimetry equipment, which measures concentrations of oxygen consumption and CO2 production. From these parameters, the respiratory quotient (RQ), as well as energy expenditure and substrate metabolism will be computed. During this measurement, every 10 minutes (so 4 times) one tube of blood (10ml) will be drawn for the analysis of lipid and glucose metabolites.
Other intervention name(s): Spectrum Analysis; MRI; Oxygraph; BodPod; Ventilated Hood; Basal energy metabolism; Omnical
Observational model: Cohort
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        As patients will have to undergo surgery to obtain cardiac tissue for these ex vivo
        measurements, the investigators will only use patients that are scheduled for cardiac
        surgery (either for valve replacement or a cardiopulmonary bypass procedure).

        Obese type 2 diabetic patients will be compared with obese and lean normo-glycemic
        patients. As all patients will undergo the same measurements, this study is designed as an
        observational case-control study. The investigators have chosen to include both obese and
        lean participants, as the investigators expect that bodyweight and intra-cellular lipid
        accumulation affects cardiac mitochondrial function. This will probably result in a large
        range in mitochondrial function, which facilitates the validation of the methods.
      
Biospecimen description: 
      From a fasting blood sample will DNA be extracted from leucocytes to study genetic variants
      that may affect results.
    
Keywords: Spectrum Analysis; Mitochondria; Peroxisome Proliferator-Activated Receptors; Respirometry; Human; Heart