The Protective Effect of Azelnidipine for the Prevention of Heart Fibrosis Occurrence on Balb/c Mice with Iron Overload

Background: Iron overload can cause DNA oxidation which increase TGF β1, type 1 fibrilarprotein and myocardium fibrosis. Myocardium fibrosis is the main cause of death on the state of iron overload. The iron influx towards the cell during iron overload is still unknown, some research suggested LTCC acts as iron influx. This research aims to investigate the role of azelnidipine as type L calcium channel blocker, lowering TGF β1, collagen and myocardium fibrosis. Method: The research subjects consisted of 25 male Balb-C mice(8 weeks, 30-40mg) divided into 5 groups. Group 1 (NaCl+S) 0,3 cc Na Cl 0,9% (I.P) and drug solvent (Aquabidest, CMC and Nipagin) orally. Group 2Fe+S) 0.3 cc 1,5 mg Fe+sucrose (Venofer®) (I.P) and drug solvent (Aquabidest, CMC and Nipagin) orally. Group 3 (Fe+Dfx) 1,5 mg Fe+sucrose (Venofer®) (I.P) and deferasirox 20 mg/kg body weight/day orally, group 4 (Fe+Azl) 1,5 mg Fe+sucrose (Venofer®) (I.P) and azelnidipine 14 mg/day orally and group 5 (Fe+Dfx-Azl) 1,5 mg Fe+sucrose (Venofer®) (I.P) and mixture of deferasirox 20 mg/kg body weight/day and azelnidipine 14 mg/day orally. Fe-sucorse was diluted with NaCl 0.9 %. Intraperitoneal injection were administered intermittently for 60 days of treatment. Result: The highest Expression of TGF β, collagen I and fibrosis area fractions are in group Fe+S. The result of Post Hoc test between 2 treatment groups indicated that there were no difference in TGF β expression between groups NaCl+S with Fe+Dfx (P>0.05) , Fe+Dzl (P>0.05). There are no significant in collagen expression between groups NaCl+S with Fe+Dfx (P > 0.05) ,Fe+Dzl (P>0.05). Conclusion: Azelnidipine, LTCC have roles on the influx of iron into the myocardium, lowering TGF β, collagen Iexpressionsand myocardium fibrosis.


Introduction
Iron plasma overload is still a problem for people with thalassemia.Iron plasma overload occurred due to haemolysis, repeated transfusion and also due to the iron metabolism dysregulation. Administration of blood transfusion in β thalassemia will improve the accumulation of iron substance , which is 0.34 mg/ kg body weight /day on patients with the need for 600 Red Packed Cell / 4 weeks 1 .Iron plasma overload cause the accumulation of iron in the internal organs. The accumulation of iron in cardiovascular muscle is cardiovascular complicationand the main cause of death on repeated transfusion 2 On the physiological state, transferrin receptor 1 (TfR1) plays an important role in the process of iron influx to the intra cell. Transferrin receptor 1 (TfR1) which located in the cardiac cell wall is important in the influx of iron to the myocardium, however during the presence of high intra cell iron level, TfR1 expression will be suppressed [3][4][5] . On the state of high iron plasma, several studies suggested the type L calcium channel(L-type calsium channels/ LTCC) 2,6-11 while others stated that type T calcium channel (T-type calsium channels/TTCC) 12,13 are playing important roles in the influx of iron in the myocardium.
The accumulation of iron will cause cell damage due to the DNA oxidation. DNA oxidation triggers the apoptosis process and increasegrowth factor (TGF-β1, PDGF), cytokine and extracell matrices 14 .
Cascadetransforming growth factor-β1 (TGF-β1) plays a major role in inducing fibroblast into myofibroblast and increasing MMP2 and MMP 9 expressions which cause fibrosis 14,15 .Cardiomyopathy and heart failure/ cardiac arrest due to the myocardium fibrosis are the leading cause of death for iron plasma overload 2,9 . Management of iron accumulation to this day is not yet satisfactory, one of them is iron chelation. Deferasirox is the latest development of iron chelation, it has the ability to bind plasma iron and penetrate cell membranes to bind intra cell iron 16 . Deferasiroxis a tridentate molecule, forming 2 electrochemical bonds of Fe(2:1) ions which are quite stable, given orally once a day 17 .The use of iron chelating drugs to eliminate iron accumulation in the heart is still not yet satisfactory In a state of high plasma iron, several studies have shown L-type calcium channels ( L-type calsium channels/LTCC) 11 while others suggested that T-type calcium channel (T-type calsium channels/TTCC) 13 plays an important role in the influx of iron into the myocardium. The type L calcium channel density is affected by estrogenic receptors, an increase in estrogen will reduce the number of type L calcium channels and reduce the entry of Ca 2+ in the myocardium 18 . Azelnidipine is along acting third generation partition L type calcium channel, very soluble in fat 19 . Azelnidipine has the hydroxyl radical scavenger effect to reduce ROS, antioxidant and antifibrotic effects 20 .
This research is going to look at the effect of azelnidipineon TGF β, collagen expression and iron overload model heart fibrosis on male experimental animals. Iron overload model is by administration of intraperitoneal iron sucrose in Balb/c mice intermittently.

Heart harvesting
Before termination, the mice were anesthetized with pentobarbital (60 mg/kg ip), then the abdomen and thorax were opened. Heart were harvested and half ventricle was in RNA later ® for extraction RNA and other half was fixated in PFA 4% in PBS for 24 hour and paraffin was used embedded tissue process.

Histological analysis
Paraffin section with 4 mm thickness was analyzed. Paraffin section were deparaffinized and stained with Sirius Red to quantify the fibrosis interstitial fraction area. Quantification of fibrosis area was done using Image J software, with 10 fields in each sample and with 400 x magnification.

Statistical Analysis
Data were presented as mean ± SD collagen and TGF β level and fraction area fibrosis were analysed with ANOVA test and continued with Post Hoc test Ethical clearance: This research study was approved by ethics committee of Medical Faculty of Sultan Agung Islamic University Semarang, Indonesia.

Results
On group Fe+S the highest average of TGF β 1 and collagen I expression. The lowest average of TGF β expression was on group Fe+Dfx and collagen expression the lowest expression was on group Fe+Azl

Discussion
Myocardium is composed from several types of cells, cardiomyocyte, cardiofibroblast and endothelial cell. Cardiofibroblast generate cytokines, and growth factors and have the function to preserve the structural and functional integrities of the extracellular matrices. The extracellular matrix has a very important role as the mechanical and chemical properties between cardiomyocytes, cardiofibroblast cells and blood vessels 14 . The myocardial extracellular matrix is woven between cells arranged by proteins, fibrous hidden in materials similar to gels composed of complex carbohydrates. A dilute gel is called interstitial fluid, which is the space between blood vessels and tissue cells which are part of nutrients, residual results, and materials that dissolve in the air 21   Myocardial iron deposits result in oxidative stress due to an increase in the hydroxyl radical prooxide. Increased hydroxyl radicals increase peroxidase and damage to lipids, proteins and DNA 28 . DNA damage is measured by expression of growth factor (TGF-β1). Cascade transforming growth factor-β1 (TGF-β1) induces fibroblasts into myofibroblasts and increases the expression of collagen fibilar proteins, which results in fibrosis 14,15 .
L-type calsium channels (LTCC) are mostly in the myocardium, play an important role during calcium entry as triggers of heart muscle contraction, regulation of the duration of action potential and regulation of gene expression 29 . The relationship between chronic heart failure and serum calcium 30 .
In the state of iron overload, LTCC plays a role in the influx of iron into the heart muscle 2,6-11 . The lowest of collagen expression average were in the Fe-Azl group, Azelnidipine is type L Ca Channel blocker which is an antihypertensive drug with better effect of anti-inflammatory and anti-oxidant than amlodipine, furthermore, it has the antifibotic effect by inhibiting TGF-β1 in the liver 20 , and inhibits apoptosis by reducing cytochrome C levels in HL-1 cardiomyocytes 31 Azelnidipine is an anti-hypertension that can prevent heart damage. Azelnidipine has better anti inflammatory and antioxidant effects than amlodipine 32 antifibrotic effects by inhibiting TGF-β1 in the liver 20 , and inhibiting apoptosis by reducing cytochrome C levels in HL-1 cardiomyocytes 31 . Cardiofibroblast hyperactivity and myocardial fibroblasts increase the production of collagen I, II fibilar proteins and extracellular matrix deposits 15 . Fibrillar collagen type I and II are the most common components of the myocardial extracellular matrices 14 . TGF β1 pathway is an important pathway for collagen production 33 and where fibrosis takes place 15 .
L-type calcium channels (LTCC) play a role in the entry of Fe2+ 2,6-11 .This study shows that administration of azelnidipine has a protective effect on the occurrence of myocardial fibrosis, lowering TGF β1 and collagen Iexpression . L Type Calciumchannel (LTCC) plays a role in the entry of iron into the cell.