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The effects of homocysteine and homocysteine related compoun

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Vladimir Zivkovic

on 23 February 2014

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Transcript of The effects of homocysteine and homocysteine related compoun

THE EFFECTS OF HOMOCYSTEINE AND HOMOCYSTEINE RELATED COMPOUNDS ON CARDIODYNAMICS AND CORONARY FLOW IN THE ISOLATED RAT HEART: THE ROLE OF GASOTRANSMITTERS AND OXIDATIVE STRESS
DOCTORAL THESIS
UNIVERSITY OF KRAGUJEVAC
FACULTY OF MEDICAL SCIENCES
VLADIMIR ŽIVKOVIĆ
KRAGUJEVAC, 2014
STRUCTURE
RESEARCH TOPICS
Evaluation of the effects of acute administration of homocysteine and homocysteine related compounds on the myocardial function and coronary flow in isolated rat heart.

Identification of the role of gaseous signal molecules (NO, H2S and CO) in the maintenance of physiological function of coronary muscle and coronary circulation.

Identification of the effects of various oxidative stress parameters, such as lipid peroxides (TBARS), nitric oxide (NO), superoxide anion radical (O2-) and hydrogen peroxide H2O2) on cardiodynamics and coronary circulation.

Evaluation of mutual interaction of homocysteine, free radicals and gasotransmitters as well as role of this interaction on myocardial function and coronary circulation.


MATERIALS AND METHODS
The research included fifteen (15) experimental groups (12 animals in each group):

1) Control group (perfusion with complex Krebs-Hensenleit physiological solution),

2) Administration of DL-homocysteine (10μmol/l),

3) Administration of DL-homocysteine thiolactone hydrochloride (10μmol/l),

4) Administration of L-homocysteine thiolactone hydrochloride (10μmol/l),

5) Administration of zinc protoporphyrin IX – selective inhibitor HO 1 (10μmol/l),

6) Administration of DL-propargylglycine - selective inhibitor CSE (10μmol/l),

7) Co-administration of DL-homocysteine (10μmol/l) plus L-NAME (30μmol/l),

8) Co-administration of DL-homocysteine (10μmol/l) plus zinc protoporphyrin IX (10μmol/l),

9) Co-administration of DL-homocysteine (10μmol/l) plus DL-propargylglycine (10μmol/l),

10) Co-administration of DL-homocysteine thiolactone hydrochloride (10μmol/l) plus L-NAME (30μmol/l),

11) Co-administration of DL-homocysteine thiolactone hydrochloride (10μmol/l) plus zinc protoporphyrin IX (10μmol/l),

12) Co-administration of DL-homocysteine thiolactone hydrochloride (10μmol/l) plus DL-propargylglycine (10μmol/l),

13) Co-administration of L-homocysteine thiolactone hydrochloride (10μmol/l) plus L-NAME (30μmol/l),

14) Co-administration of L-homocysteine thiolactone hydrochloride (10μmol/l) plus zinc protoporphyrin IX (10μmol/l),

15) Co-administration of L-homocysteine thiolactone hydrochloride (10μmol/l) plus DL-propargylglycine (10μmol/l)

EXPERIMENTAL PROTOCOL
Interaction between Hcy, free radicals and gasotransmitters
Langendorff aparatus,
CPP-70 cmH2O
CONTINUING REGISTRATION OF CARDIODYNAMIC PARAMETERS:


dp/dt max-maximum rate of LV pressure development
dp/dt min-minimum rate of LV pressure development
SLVP-systolic LV pressure
DLVP-dyastolic LV pressure
HR-heart rate
MBP-mean blood pressure
CF-coronary flow
BIOCHEMICAL ANALYSES
DETERMINATION OF OXIDATIVE
STRESS PARAMETERS:

TBARS
NO2
O2-
H2O2
RESULTS
CARDIODYNAMIC PARAMETERS
THE EFFECTS OF DIFFERENT HOMOCYSTEINE COMPOUNDS ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

THE EFFECTS OF DL-HOMOCYSTEINE (10μmol/l) ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

THE EFFECTS OF DL-HOMOCYSTEINE THIOLACTONE HYDROCHLORIDE (10μmol/l) ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

THE EFFECTS OF L-HOMOCYSTEINE THIOLACTONE HYDROCHLORIDE (10μmol/l) ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

THE EFFECTS OF DIFFERENT GASOTRANSMITTER INHIBITORS ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

THE EFFECT OF ZINC PROTOPORPHYRIN IX (10μmol/l) ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

THE EFFECTS OF DL-PROPARGYLGLYCINE (10μmol/l) ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

THE INFLUENCE OF NITRIC OXIDE (NO) INHIBITION ON CARDIODYNAMIC EFFECTS OF DIFFERENT HOMOCYSTEINE COMPOUNDS IN ISOLATED RAT HEART

THE EFFECTS OF DL Hcy (10μmol/l) AND L-NAME (30μmol/l) CO-ADMINISTRATION ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART


THE EFFECTS OF DL Hcy tlhc (10μmol/l) AND L-NAME (30μmol/l) CO-ADMINISTRATION ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART


THE EFFECTS OF L Hcy tlhc (10μmol/l) AND L-NAME (30μmol/l) CO-ADMINISTRATION ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART


THE INFLUENCE OF CARBON MONOXIDE (CO) INHIBITION ON CARDIODYNAMIC EFFECTS OF DIFFERENT HOMOCYSTEINE COMPOUNDS IN ISOLATED RAT HEART

THE EFFECTS OF DL Hcy (10μmol/l) AND PPR IX (10μmol/l) CO-ADMINISTRATION ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

THE EFFECTS OF DL Hcy tlhc (10μmol/l) AND PPR IX (10μmol/l) CO-ADMINISTRATION ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

THE EFFECTS OF L Hcy tlhc (10μmol/l) AND PPR IX (10μmol/l) CO-ADMINISTRATION ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

THE INFLUENCE OF HYDROGEN SULFIDE (H2S) INHIBITION ON CARDIODYNAMIC EFFECTS OF DIFFERENT HOMOCYSTEINE COMPOUNDS IN ISOLATED RAT HEART

THE EFFECTS OF DL Hcy (10μmol/l) AND DL PAG (10μmol/l) CO-ADMINISTRATION ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

THE EFFECTS OF DL Hcy tlhc (10μmol/l) AND DL PAG (10μmol/l) CO-ADMINISTRATION ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

THE EFFECTS OF L Hcy tlhc (10μmol/l) AND DL PAG (10μmol/l) CO-ADMINISTRATION ON CARDIODYNAMIC PARAMETERS IN ISOLATED RAT HEART

OXIDATIVE STRESS MARKERS
THE EFFECTS OF DIFFERENT HOMOCYSTEINE COMPOUNDS ON OXIDATIVE STRESS PARAMETERS IN ISOLATED RAT HEART

THE EFFECT OF DL-HOMOCYSTEINE (10μmol/l) ON OXIDATIVE STRESS PARAMETERS DYNAMICS IN ISOLATED RAT HEART



THE EFFECT OF DL-HOMOCYSTEINE THIOLACTONE HYDROCHLORIDE (10μmol/l) ON OXIDATIVE STRESS PARAMETERS DYNAMICS IN ISOLATED RAT HEART



THE EFFECT OF L-HOMOCYSTEINE THIOLACTONE HYDROCHLORIDE (10μmol/l) ON OXIDATIVE STRESS PARAMETERS DYNAMICS IN ISOLATED RAT HEART


THE EFFECTS OF DIFFERENT GASOTRANSMITTER INHIBITORS ON OXIDATIVE STRESS PARAMETERS IN ISOLATED RAT HEART

THE EFFECT OF ZINC PROTOPORPHYRIN IX (10μmol/l) ON OXIDATIVE STRESS PARAMETERS IN ISOLATED RAT HEART


THE EFFECTS OF DL-PROPARGYLGLYCINE (10μmol/l) ON OXIDATIVE STRESS PARAMETERS IN ISOLATED RAT HEART


THE INFLUENCE OF NITRIC OXIDE (NO) INHIBITION ON OXIDATIVE EFFECTS OF DIFFERENT HOMOCYSTEINE COMPOUNDS IN ISOLATED RAT HEART

THE EFFECTS OF DL Hcy (10μmol/l) AND L-NAME (30μmol/l) CO-ADMINISTRATION ON OXIDATIVE STRESS PARAMETERS DYNAMICS IN ISOLATED RAT HEART

THE EFFECTS OF DL Hcy tlhc (10μmol/l) AND L-NAME (30μmol/l) CO-ADMINISTRATION ON OXIDATIVE STRESS PARAMETERS DYNAMICS IN ISOLATED RAT HEART

THE EFFECTS OF L Hcy tlhc (10μmol/l) AND L-NAME (30μmol/l) CO-ADMINISTRATION ON OXIDATIVE STRESS PARAMETERS DYNAMICS IN ISOLATED RAT HEART
THE INFLUENCE OF CARBON MONOXIDE (CO) INHIBITION ON OXIDATIVE EFFECTS OF DIFFERENT HOMOCYSTEINE COMPOUNDS IN ISOLATED RAT HEART
THE EFFECTS OF DL Hcy (10μmol/l) AND PPR IX (10μmol/l) CO-ADMINISTRATION ON OXIDATIVE STRESS PARAMETERS DYNAMICS IN ISOLATED RAT HEART

THE EFFECTS OF DL Hcy tlhc (10μmol/l) AND PPR IX (10μmol/l) CO-ADMINISTRATION ON OXIDATIVE STRESS PARAMETERS DYNAMICS IN ISOLATED RAT HEART

THE EFFECTS OF L Hcy tlhc (10μmol/l) AND PPR IX (10μmol/l) CO-ADMINISTRATION ON OXIDATIVE STRESS PARAMETERS DYNAMICS IN ISOLATED RAT HEART

THE INFLUENCE OF HYDROGEN SULFIDE (H2S) INHIBITION ON OXIDATIVE EFFECTS OF DIFFERENT HOMOCYSTEINE COMPOUNDS IN ISOLATED RAT HEART

THE EFFECTS OF DL Hcy (10μmol/l) AND DL PAG (10μmol/l) CO-ADMINISTRATION ON OXIDATIVE STRESS PARAMETERS DYNAMICS IN ISOLATED RAT HEART

THE EFFECTS OF DL Hcy tlhc (10μmol/l) AND DL PAG (10μmol/l) CO-ADMINISTRATION ON OXIDATIVE STRESS PARAMETERS DYNAMICS IN ISOLATED RAT HEART

THE EFFECTS OF L Hcy tlhc (10μmol/l) AND DL PAG (10μmol/l) CO-ADMINISTRATION ON OXIDATIVE STRESS PARAMETERS DYNAMICS IN ISOLATED RAT HEART

CONCLUSIONS
1. Acute and direct effects of homocysteine and its metabolites on the isolated rat heart imply negative inotropic, and lusitropic effect and vasoconstrictor influence on coronary endothelium, i.e. systolic and diastolic dysfunction and reduction of coronary flow. The obtained effects were more pronounced when L thiolactone form was used, which may be of clinical importance in the determination of this fraction of homocysteine in the plasma of the patients with cardiovascular diseases.

2. The inhibition of CO and H2S synthesis led to complete depression of cardio dynamics in the isolated rat heart and reduction of coronary flow, which showed that these gasotransmitters can play a role in the contraction of cardiomyocyte and the control of coronary vascular tonus. Since the mentioned effects were considerably stronger in the case of CO inhibition, we assume that this signal molecule is more important in the physiology of myocardium and coronary circulation in comparison to other two gasotransmitters.

3. On the occasion of NO synthesis inhibition, most negative effect on the heart achieved L Hcy tlhc, while the least detrimental impact had DL Hcy tlhc. It appears that the role of NO in the effects of homocysteine depends on the present form of homocysteine, and that the most reliable importance is in the coronary circulation, while at the level of the myocardium requires further and more complex investigations.

4. In the absence of CO, all examined forms of homocysteine showed even stronger negative inotropic, chronotropic and lusitropic effect and greater decrease of coronary flow. Thus it was proved that this gasotransmitter could play a very significant role in the effects of homocysteine on the heart.

5. In conditions of blocking CSE/H2S signaling pathway, the most negative effect on the heart achieved again L Hcy tlhc, while the least detrimental influence had DL Hcy tlhc. As in the case of the inhibition of NO production, the difference in the effects of the tested homocysteine isoforms likely to be a consequence of their stereisomeric properties. Despite certain protective role of H2S in conditions of acute effects of homocysteine on the heart, it seems that mentioned gasotransmitter does not contribute significantly in the effects of this thiol on the isolated rat heart.



THANK YOU FOR ATTENTION
CHOLESTEROL
OF 21th
CENTURY
(10 weeks old, male sex, BM-250 grams)
Wistar albino rats
6. The results of this study suggest that CO may be a gasotransmitter which has the most significant role in the effects of homocysteine and its metabolites on the isolated rat heart, i.e. the effects of homocysteine most depend on the HO/CO signal pathway.

7. The effects of homocysteine on the production of various ROS in the heart depends on the form of this amino acid. In this sense, it seems that the homocysteine in its basic form (DL Hcy) is the least harmful, ie. by the still unclear manner, it can have potential anti-oxidative effect. On the other hand, the thiolactones also demonstrated that they not induce oxidative stress.

8. Generally viewed, homocysteine and related compounds do not lead to the development of oxidative stress in the isolated rat heart in this research, which means that acute cardiodynamic effects of various forms of homocysteine are possibly not mediated via oxidative damages, but by other mechanisms which require further research.

9. During the inhibition of CO synthesis, the production of majority of pro oxidative markers was reduced, which shows that this gasotransmitter may play a role in oxido-reduction processes in the heart.

10. H2S does not contribute to the development of oxidative stress in the myocardium and coronary circulation, but it seems to have certain antioxidative capacity, which could be definitely confirmed in the further studies.

11. These results refer to the potential importance of gasotransmitters as new and ever more important elements in the maintenance of redox balance of cardiovascular system, which might be extremely significant for clarification and more appropriate explanation of many cardiovascular diseases, in whose pathogenesis oxidative stress has an unavoidable role.
12. Blockade of NO synthesis can potentiate the antioxidant properties of all tested homocysteine isoforms. In this way, we can note that L-arginine/NO system, by the still unclear mechanisms, may play a role in the oxidative properties of homocysteine.

13. Inhibition of HO/CO system potentiates the anti-oxidative properties of homocysteine, which confirmes pro-oxidant capacity of this gasotransmitter in cardiovascular system. However, our findings are preliminary, and necessarily seek support in further and more complex research.

14. The influence of CSE/H2S system on oxidation effects of homocysteine and the similar substances is, on the whole, mildly antioxidative, but still unclear. To be more precise, it seems that homocysteine shows lower pro oxidative effect in the presence of H2S in comparison to the absence of production of this signal molecule, which indicates a potential anti-oxidative capacity of mentioned gasotransmitter.

15. The obtained results show that gasotransmitters can definitely play a role in oxidative effects of homocysteine on cardiovascular system, which can help with better identification and understanding of these complex molecular interactions, whose participation in the pathogenesis of various cardiovascular diseases, and thereby their more efficient prevention and therapy, is undoubted.
MetRS
Hcy
Hcy thiolactone
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