Isolation and characterization of mitochondria and lysosome from isoproterenol induced cardiotoxic rats

a Department of Biochemistry, PSG College of Arts & Science, Coimbatore641014, Tamilnadu, India. b Department and Graduate Institute of Applied Chemistry, Chaoyang University of Technology, No.16, Jifeng East Road, Wufeng District, Taichung City – 41349, Taiwan (R.O.C). c Raman Research Laboratory, PG & Research Department of Physics, Government Arts College, Tiruvannamalai-606603, Tamilnadu, India d Department of Chemistry, Sri Vijay Vidyalaya College of Arts and Science (Women’s college), Dharmapuri, Periyar University, Tamilnadu, India. e Department of Environmental Engineering and Management, Chaoyang University of Technology, No.16, Jifeng East Road, Wufeng District, Taichung City – 41349, Taiwan (R.O.C).


Introduction
The heart 20-45% of the myocardial volume is taken up by mitochondria [1] and is highly dependent for its function on oxidative energy generated primarily by as a major participant in these effects, and also that it might be the final arbiter of cell death in the apoptotic or necrotic changes that occur in myocardial infarct [3].
Lysosomes are intracellular organelles which maintain the internal pH between 5.0 to 5.5 through ATPdependent proton pump. They play an important role in the secretion and transport process. The release of lysosomal enzymes into the cytoplasm stimulates the formation of inflammatory mediators such as oxygen radicals and prostaglandins which may result in the conversion of reversible myocardial ischemia to irreversible infarction [4][5]. In myocardial ischemia, there is increased activity of glycohydrolases and the lysosomal stability was decreased which results in the necrosis of myocardium [6].
One approach to ameliorate the damage due to myocardial injury is to stabilize the membrane of ischemic

Z. armatum fruit was identified by ABS Botanical
Garden, Salem, Tamilnadu. Extract was prepared by dried fruits using 50% ethanol for 5 days.

Induction of MI
ISO hydrochloride was used to induce MI in rats.
Animals were injected subcutaneously with freshly prepared ISO hydrochloride in sterile normal saline at a dose of 20mg/100g body weight.

Experimental design
Animals were divided into six groups of six rats in each group. The homogenate was centrifuged and the supernatant was used for various biochemical estimations.

Separation of heart mitochondrial fraction
Heart mitochondria were isolated by the method of Takasawa et al., (1984) [7] with slight modifications.
The heart tissue was homogenised in a medium containing 250 mM sucrose, 0.5 mM EDTA, 50mM Tris HCl (pH 7.4). It was then resuspended in 20 ml of isolation medium containing 0.1% (w/v) defatted bovine serum albumin and transferred to a 50-mL glass homogenizer. The mixture was incubated for 1 min at 4°C and then rehomogenized.
To isolate the mitochondria, the homogenate was subjected to differential centrifugation at 4°C.
Mitochondrial fraction was resuspended in the same buffer (final concentration 0.2% v/v) in ice for 15 min and it was used for the determination of lipid peroxidation (LPO).

Effect of hydroethanolic extract of Z. armatum fruit on mitochondrial marker enzymes
The activities of MDH, SDH and ICDH in heart tissue homogenate were assayed by the method of Mehler

Separation of heart lysosomal fraction
The lysosomal fraction of the heart tissue was isolated by the method of Wattiaux (1977) [10]. Fresh heart tissues were homogenized in ice cold 0.25 M sucrose solution. The homogenate was filtered and centrifuged at 3000rpm for 10 minutes. The pellet was removed and recentrifuged. The supernatants were pooled and centrifuged at 15000rpm for 20 minutes. The lysosomal pellet was suspended in 1.15% potassium chloride and used for the assay of enzymes.

Statistical analysis
The results were expressed as mean± standard deviation. Statistical analysis was carried between the experimental groups using one way analysis of variance (ANOVA) employing SPSS Version 16.0. Post hoc analysis was performed using Fisher's least significant difference (LSD) test and the level of significance was set as (p<0.05).

Results and Discussion
From  cycle, yielding reducing equivalents, which are channeled through the respiratory chain for the synthesis of adenosine tri phosphate (ATP) [15]. The Krebs cycle results in the production of CO2 and NADH the later is used in the second pathway of mitochondrial oxidative phosphorylation [16]. ATP synthesis and electron transport chain starts in the mitochondria, which is required for cardiac contraction and relaxation [17]. ICDH is mainly expressed in the heart and skeletal mitochondria and is an NADP dependent which controls the mitochondrial redox balance and the subsequent oxidative damage [19]. Pre-treatment with hydroethanolic extract of Z. armatum fruit significantly increased the activity of ICDH which might be due to resistance of the heart enzyme against oxidative stress in heart mitochondria.
SDH is an integral membrane protein and it is tightly attached to the inner membrane and is directly linked to the electron transport, transferring electrons to the respiratory chain [20]. SDH is a site for metabolic control in TCA cycle [21] and contains many cysteine rich sulfur clusters and can be inhibited by agents that modify sulfhydryl groups. ISO administration is known to alter protein-bound sulfhydryl groups and have resulted in the inactivation of the enzyme. SDH is one of the important  enzymes that regulate the production of ATP in the mitochondria and it is sensitive to free radicals.
MDH is located in the outer membrane of the mitochondria and is affected by increased level of free radicals produced following ISO administration [22]. Lysosomes are organelles particularly vulnerable to oxidative stress since they exhibit the most important pool of reactive iron in the cell [24]. Oxidative stress can induce very fast lysosomal disruption creating intra lysosomal iron-mediated redox reactions [25]. It has also been suggested that abnormal release and activation of lysosomal enzymes during ischaemia and other potentially lethal events may contribute to the tissue damage.
In the present study administration of ISO

Conclusion
Our results clearly confirmed the existence of cardiotoxicity due to ISO administration which was indicated by decreased activities of mitochondrial and lysosomal enzymes. Z. armatum fruit exerts cardioprotective activity which could be partly contributed by its membrane protective action. Therefore, it can be concluded that Z. armatum fruit can protect against cardiotoxicity by preserving the integrity of the membrane and restoring the activities of enzymes.