Hydrogen sulfide modulates sub-cellular susceptibility to oxidative stress induced by myocardial ischemic reperfusion injury

Accepted Manuscript Hydrogen sulfide modulates sub-cellular susceptibility to oxidative stress induced by myocardial ischemic reperfusion injury A. Shakila Banu, Gino A. Kurian PII:

S0009-2797(16)30113-2

DOI:

10.1016/j.cbi.2016.03.036

Reference:

CBI 7638

To appear in:

Chemico-Biological Interactions

Received Date: 24 October 2015 Revised Date:

16 March 2016

Accepted Date: 30 March 2016

Please cite this article as: A Shakila Banu G.A Kurian, Hydrogen sulfide modulates sub-cellular susceptibility to oxidative stress induced by myocardial ischemic reperfusion injury, Chemico-Biological Interactions (2016), doi: 10.1016/j.cbi.2016.03.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Hydrogen sulfide modulates sub-cellular susceptibility to oxidative stress induced by myocardial ischemic reperfusion injury

School of Chemical and Biotechnology, SASTRA University, Thanjavur, Tamilnadu.

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Shakila Banu A1, Gino A Kurian1

Mailing address of the Corresponding Author:

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Gino A Kurian Senior Assistant Professor

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Vascular Biology Laboratory

School of Chemical and Biotechnology SASTRA University Thanjavur, India

[email protected]; [email protected] Telephone: +919047965425 Fax: +914362-264120

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Abstract In this study, we compared the impact of H2S pre (HIPC) and post-conditioning (HPOC) on

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oxidative stress, the prime reason for myocardial ischemia reperfusion injury (I/R), in different compartments of the myocardium, such as the mitochondria beside its subpopulations (interfibrillar (IFM) and subsarcolemmal (SSM) mitochondria) and microsomal fractions in I/R

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injured rat heart. The results demonstrated that compared to I/R rat heart, HIPC and HPOC treated hearts shows reduced myocardial injury, enhanced antioxidant enzyme activities and

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reduced the level of TBARS in different cellular compartments. The extent of recovery (measured by TBARS and GSH levels) in subcellular fractions, were in the following descending order: microsome > SSM > IFM in both HIPC and HPOC. In summary, oxidative stress mediated mitochondrial dysfunction, one of the primary causes for I/R injury, was partly

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compared to the IFM.

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recovered by HIPC and HPOC treatment, with significant improvement in SSM fraction

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1.

Introduction

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Reperfusing the occluded coronary artery immediately is considered to be the “gold standard” for the treatment of myocardial infarction that effectively reduce the overall mortality. However, the restoration of blood flow to the ischemic myocardium resulted in cardiomyocyte dysfunction

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leading to cell death resulting in reperfusion injury [1]. A number of mechanisms have been proposed to mediate reperfusion injury which includes cellular calcium overload, an occurrence

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of a no-reflow phenomenon due to cell swelling, impaired vascular relaxation or the formation of white cell plugs, and perhaps most importantly the formation of reactive oxygen species (ROS) [2]. Low levels of ROS play an important role in cellular homeostasis, mitosis, differentiation, and signaling, while increasing radical formation following ischemia and reperfusion, helps in

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triggering cellular injury. Although all mammalian cells, including cardiomyocytes, express endogenous free radical scavenging enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase, these antioxidant defenses are overwhelmed after ischemia and

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reperfusion. ROS causes lipid peroxidation, which in turn leads to cell membrane damage resulting in cell swelling [3]. A number of studies were reported to have a better cardiac function

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as well as reduced infarct size by either preventing or scavenging free radicals [4,5]. Classical cardioprotective regimens include ischemic preconditioning (IPC), first demonstrated by Murray et al. (1986) and ischemic post-conditioning (POC) as proposed by Zhao et al. (2003), could limit reperfusion injury through the activation of intrinsic pro-survival signaling cascades like PI3K/Akt (RISK pathway) and JAK2/STAT3 (SAFE pathway). Both IPC and POC protect the heart by inhibiting mitochondrial permeability transition pore opening, reducing inflammatory consequences and ameliorating oxidative stress [6].

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Hydrogen sulfide (H2S), a novel gasotransmitter, so far known for its toxicity, is synthesized by cystathionine-gamma-lyase (CSE) in the cardiovascular system and also reported to protect the myocardium against I/R injury and its cytoprotection is primarily due to its antioxidant, anti-

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inflammatory and antiapoptotic property [7,8]. H2S induced pre- and post-conditioning was proven to execute by targeting mitochondria. In H2S induced IPC, it activates antioxidant genes (Nrf2), RISK pathway, leading to the preservation of mitochondrial function and structure [9].

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On the other hand, in H2S mediated POC, mitochondria specific antioxidants were expressed

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significantly, along with the activation of a SAFE pathway [10].

Despite the known great potential of hydrogen sulfide as a pre- or post-conditioning agent, the exact mechanism behind its cardioprotective effect remains unclear, primarily due to the lack of complete understanding of the pathophysiological feature of I/R. Recently, our lab had shown that

cardiac

mitochondrial

subpopulation,

namely

interfibrillar

and

subsarcolemmal

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mitochondria had experienced the different magnitude of damage during I/R. Mitochondrial dysfunction, being the pivotal pathological reason for I/R, specific mitochondrial subset damage can be detrimental to the measurable outcome on the efficacy of H2S as a cytoprotective agent in

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myocardial ischemia-reperfusion injury. Detailed understanding of the effect of oxidative stress

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specifically on IFM and SSM is essential, as ROS plays a key role in I/R injury. Thus, the aim of this manuscript is to provide definite experimental evidence for the impact of I/R injury and H2S induced pre or post-conditioning on different compartments of the myocardium, in particular, IFM and SSM individually.

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2.

Materials and Methods

2.1. Animals

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The study protocol was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. In polycarbonate cages, animals were kept at a controlled temperature of 25 ± 3°C and 60 ± 10% relative humidity with a 12 hr dark/light cycle.

diet and drinking water given adlibitum.

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2.2. Isolated Perfused Rat Heart Preparation

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Before the start of the experiment, rats were acclimatized for a week with standard laboratory

Male Wistar rats (230 - 280 g) were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg/kg). Heparin (1000 IU) was administered i.p. 30 min prior to anesthesia to prevent coagulation during excision of the heart. The heart was excised, mounted on a

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Lagendorff apparatus (AD instruments, Australia) and perfused retrogradely through the aorta with Krebs Hensleit (KH) buffer (pH 7.4, 37oC) at a constant flow rate of 8 ml/min as previously described [11]. Each heart was allowed to stabilize (in terms of LVEDP) before the

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commencement of any experiment.

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The experiment was carried as per the protocol shown in Figure 1A. Briefly, in the normal perfusion group (NP), heart was perfused for 90 min with KH buffer. In case of ischemic control group, after stabilization, hearts were subjected to no-flow ischemia for 30 min. In the reperfusion control group, hearts were subjected to global ischemia for 30 min followed by 60 min reperfusion. In the case of preconditioning control group (IPC), hearts were subjected to three cycles of 2 min ischemia and 3 min reperfusion, followed by 30 min global ischemia and 60 min reperfusion. In H2S preconditioning group (HIPC), NaHS as a donor of H2S (20 µM),

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was perfused for 15 min before the start of ischemia (30 min), followed by 60 min reperfusion. Post-conditioning control (POC) includes those hearts subjected to global ischemia, followed by three cycles of 3 min reperfusion and 2 min ischemia followed by normal perfusion for 60 min.

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In H2S post-conditioning group (HPOC), hearts were subjected to global ischemia followed by 15 min NaHS (20 µM) perfusion and 60 min reperfusion with KH buffer. At the end of the

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experiment, hearts were immediately stored at - 80 oC for further biochemical analysis.

The following hemodynamic parameters were recorded using PowerLab from AD instruments

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Ltd. (Australia): Left ventricular end diastolic pressure (LVEDP) in mmHg, Left ventricular developed pressure (LVDP) in mmHg, heart rate (HR) in beats per minute (bpm), rate pressure product (RPP= HR * DP) in mmHg x bpm x 103. 2.3. Estimation of cardiac injury markers

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In order to evaluate the presence of necrotic cell death, lactate dehydrogenase (LDH) and creatine kinase (CK) enzyme activities were measured spectrophotometrically, in cardiac tissue and coronary perfusate as per the methods described elsewhere[12]. Protein content was

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determined by the method of Bradford reagent (BioRad-USA).

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2.4. Mitochondria and its subpopulation isolation and microsomes separation According to the method described by Palmer et al [13], rat heart mitochondrial subpopulation, namely interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated by differential centrifugation. Briefly, tissue homogenate was centrifuged at 800 g for 5 min and the resulting supernatant was centrifuged at 9000 g for 10 min. The pellet was centrifuged at 8000 g twice to yield the SSM fraction. The pellet obtained in the initial step (800 g, 5 min) was treated with nagarase enzyme (0.5mg/g tissue) and subjected to differential centrifugation

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procedure similar to SSM isolation to yield the IFM fraction. For the isolation of total mitochondria, tissue homogenate was centrifuged at 800 g for 5 min. The resulting supernatant was centrifuged at 6000 g for 10 min, to yield the crude mitochondrial pellet. All processes were

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carried out at 4 oC.

After collecting mitochondrial pellet, the supernatant was subjected to ultra-high speed

2.5. Lipid peroxidation and Antioxidant enzymes

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centrifugation of 100000 x g for 45 min to obtain the microsomal fraction.

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The concentration of thiobarbituric acid reactive substances (TBARS) in the heart mitochondrial fraction was estimated by the method of Fraga et al. (1988). The activity of glutathione peroxidase (GPx) in the heart mitochondrial fraction was assayed by the method of Rotruck et al. (1973). The level of glutathione (GSH) in the heart mitochondrial fraction was estimated by the

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method of Ellman (1959). Also GSH/GSSG ratio was determined as a measure of cellular redox status. Glutathione reductase activity was measured by the method of Dieter-Horn (1963). Catalase activity was measured by the method of Baudhin et.al. (1964), following the rate of

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H2O2 consumption. Total SOD and Mn-SOD activity was measured by the method of Teare et al. (1993), which involves the measure of the inhibition of NBT reduction by riboflavin generated

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superoxide radicals. MnSOD activity was measured in the presence of sodium cyanide (6mM), which inhibits CuZn- and Fe-SOD. Microsomal ATPases

Membrane bound ATPase activities were measured as follows. Mg2+ ATPase was determined by Ohnishi method (1975). Ca2+ ATPase activity was determined by the method of Shami and Radde (1974). Na+/K+ ATPase activity was estimated by the method of Nakao (1976). 5’

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nucleotidase was determined by the method of Luly (1972). In brief, 25µl of sample and 150µl of the reaction mixture was added and incubated for 15 min. Then, 80µl of TCA (10%) was added to stop the reaction and kept under shaking for 5-10 min and assayed for the amount of

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phosphorous liberated. Phosphate was measured by the method of Fiske and Subbarow (1925). In brief, an aliquot of supernatant was added to 80µl of ammonium molybdate (2.5 g of ammonium molybdate was dissolved in 100 ml of 3N sulfuric acid) and 40µl of ANSA reagent

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(0.5 g of amino-napthol sulfonic acid was dissolved in 195 ml of 15% sodium meta bisulfite). Absorbance was read at 640 nm and liberated phosphorous was calculated against the standard

2.6. Determination of infarct size

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KH2PO4 and expressed as phosphorous liberated/min/mg protein.

The measurement of Infarct size (IS) was done by using TTC staining according to Mensah et al.

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[14] with minor changes. The percentage of infarcted tissue (triphenyl tetrazolium chloride negative) was measured using Image J software.

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2.7. DNA fragmentation

Cardiac tissue homogenate was prepared in Tris-Cl buffer (pH-8) with EDTA (25 mM) and NaCl

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(400 mM). DNA was isolated using earlier prescribed protocol [15] and the sample (5 µg/well) was run on an agarose gel (1.8%) for 2 hrs. Gel pattern was observed for any laddering/smearing pattern and images were captured using Bio-Rad Chemi Doc XRS system. 2.8. Mitochondrial complex I activity

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In order to evaluate the integrity of mitochondria, electron transport chain (ETC) enzyme rotenone sensitive NADH-oxidoreductase (NQR) activity was measured spectrophotometrically

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(340 nm), in both IFM and SSM [16]. 2.9. Mitochondrial swelling assay

Ca2+-induced swelling was used to assess the opening of the mitochondrial permeability

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transition pore. Mitochondria were incubated in a reaction mixture containing 125 mM sucrose, 50 mM KCl, 5 mM HEPES, 2 mM KH2PO4 and 1 mM MgCl2. The mitochondrial swelling was

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determined by the rate of change in absorbance at 540 nm under energized (5 mM succinate or 5 mM glutamate plus malate (GM)) as well as non-energized condition [17] and expressed as ∆A540/mg protein.

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2.10. Statistical analysis

Data were presented as the means ± SE. GraphPad Prism 5.0 was used for all of the statistical analysis. The comparison between values of the same group, at various time points of the

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experiment, was done using ANOVA. Significant differences in variables between the groups for a specific time point were analyzed using one-way ANOVA. Differences were considered

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statistically significant if p < 0.05.

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3. Results

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3.1. Effect of H2S conditioning on hemodynamics, myocardial infarct size and cardiac injury markers

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External treatment with NaHS during pre- and post- ischemic period, significantly (P<0.05) decreased LVEDP (mmHg) to 23 ± 3 and 17 ± 3 respectively, compared to I/R control (45 ± 3). As shown in table I, DP and RPP values were significantly (P<0.05) reduced in I/R rat hearts, and was restored in H2S pre- and post-conditioned groups (P<0.05 vs I/R control). Similarly,

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exogenously administered H2S significantly (P<0.05) attenuated the rise in the infarct size during ischemia and reperfusion (Table. II).

During reperfusion, cardiac injury biomarkers such as LDH (~7 fold) and CK (~3 fold) were

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found to be elevated in coronary perfusate (Figure 1B and 1C). Interestingly, corresponding

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decline was observed in the rat heart subjected to I/R, compared to the normal, indicating the existence of cardiac injury in the reperfused heart. The LDH and CK activities were improved significantly (P<0.05) in both H2S pre- and post-conditioned rat hearts (Figure 2 A and B). Figure 2C shows that H2S administration reduced injury by preventing apoptosis, confirmed from a marked decrease in caspase-3 activity and an absence of DNA damage, observed from the fragmentation pattern (Figure 2D). 3.2. Impact of I/R induced oxidative stress in different subcellular compartments

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Oxidative stress mediated lipid peroxidation was determined by the level of its end product, MDA (malondialdehyde). In cardiac tissue, the level of MDA was quantified distinctly in the homogenate, microsomes, mitochondria and its subpopulation (IFM and SSM). In order to assess

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the oxidative stress, corresponding concentration of GSH/GSSG and the activities of antioxidant enzymes were also measured.

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Upon reperfusion, the MDA levels in the rat heart homogenate elevated significantly (P<0.05) with an unequal distribution in the different subcellular compartments, where total mitochondria

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account for 84% and microsome exhibit 56% increase (Table III) compared to the normal control. Distinct evaluation of lipid peroxidation in the mitochondrial subpopulation reveals high MDA level in IFM (78.02%) than SSM (46.31%) (Figure 2). The subsequent low GSH/GSSG (reduced glutathione/oxidized glutathione) status in both SSM and IFM during ischemia and reperfusion, emphasizes the oxidative stress experienced by the subpopulations (Figure 3B and

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3F). In addition, the decreased GSH level in different subcellular compartments substantiate the reperfusion induced oxidative stress in the myocardium (Table III).

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In order to study the influence of higher lipid peroxidation on the antioxidant defense system, we assessed different antioxidant enzyme activity in the subcellular compartments. We observed that

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catalase, glutathione peroxidase (GPx) and glutathione reductase (GR) enzymatic activities were decreased significantly (P<0.05) in subcellular compartments (Table IV). Also we performed SOD isoforms (SOD1- CuZn SOD and SOD2 - Mn SOD) activity in heart tissue lysate and mitochondria subpopulation. A significant (P<0.05) decline of superoxide dismutase (SOD) (Figure 3.1) was observed in both mitochondrial subpopulation.

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In order to check whether, the elevated lipid peroxidation and subsequent low activities of antioxidant enzymes affect the functional aspect of the respective cellular compartments viz.,

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mitochondria and microsomes. Measured complex I activity was decreased ATP-sensitive microsomal ATPase viz., Na+/K+ ATPase, Mg2+ ATPase, Ca2+ ATPase and 5’nucleotidase activities were also measured to study the functional integrity of microsomes and

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the results were shown in Figure 4. Activities were found to be decreased in reperfusion control rat hearts.

subcellular compartments

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3.3. Effect of H2S Pre- and Post-conditioning on I/R induced oxidative stress in different

In cardiac tissue homogenate, the exogenous H2S administration at the time of reperfusion or before global ischemia reduced lipid peroxidation and inhibits the decrease in GPx, GR and

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catalase enzymatic activities (Table IV), that was observed during I/R. In addition, mitochondria and microsome show significant increase of 84% and 56%, respectively, in the reperfused heart, compared with the normal control. In comparison with I/R

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control, both HIPC (75% in mitochondria and 56% in the microsomes) and HPOC (74% in

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mitochondria and 63% in the microsomes) shows a reduction in MDA level (Table II). A corresponding elevation in antioxidant enzymes was observed in H2S treated hearts (Table III). Further analysis of the mitochondrial subpopulation for the H2S treatment, showed a significant reduction in the oxidative stress in both IFM and SSM. It was observed that mitochondrial subpopulation shows a significant reduction in MDA level (51% in SSM and 34.48 % in IFM) in HIPC group as well as (43.24 % in SSM and 64.82% in IFM) in HPOC group. However, with HIPC, IFM fraction was not fully recovered as SSM (Figure 2). Both HIPC and HPOC improved

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the redox status by increasing GSH level in both the subpopulation. Also improved SOD activity was observed with HIPC and HPOC in both subpopulation. measured

mitochondrial

complex

I

activity

(measured

by

NADH-ubiquinone

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The

oxidoreductases) showed no significant difference between IFM and SSM in HIPC and HPOC rat heart, indicating the limited influence of lipid peroxidation on the enzyme activity in IFM.

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However, swelling pattern, that indirectly reflects the mitochondrial membrane potential, which was observed to be more elevated in the IFM of HIPC than HPOC, suggesting possible oxidative

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stress mediated alteration (Table V).

Both HIPC and HPOC significantly improved the mitochondrial functional enzyme activity and morphology (Table V), in addition to significant enhancement (P<0.05) in microsomal ATPase

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activity (Figure 4).

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4. Discussion

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Oxidative stress, as a significant player in the pathogenesis of myocardial I/R injury, was evaluated in subcellular compartments like mitochondria and microsomes along with the crude homogenate and was found to be predominantly high in mitochondria. A further detailed analysis

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was made in mitochondrial subpopulation like IFM and SSM and our results demonstrated high lipid peroxidation and low GSH/GSSG ratio in IFM, compared to SSM, indicating higher

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oxidative stress. Even though, the previous studies, testimony the protective role of H2S pre- and post-conditioning to reperfusion injury, our results provides additional information that H2S postconditioning provides a significant improvement in IFM (absent with HIPC) and thereby render prominent cardioprotection.

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Previous studies support the notion that subcellular compartments possess different antioxidant abilities, reflecting unique combinations of the antioxidant defense systems to ameliorate the oxidative stress [18]. Major sources of ROS in the heart during I/R are derived from microsomes

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(NADPH oxidase, cytochrome P450) and mitochondria (Complex I and III) along with xanthine

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oxidase, cyclooxygenases/lipoxygenases [19]. In the present study, elevated oxidative stress during I/R was assessed through MDA and GSH/GSSG levels and corresponding decline in the antioxidant enzymes. Multiple sources for ROS production in the mitochondria makes it as a prime center for oxidative stress-linked pathology, as evident from table III, where the total mitochondria share 80% of oxidative stress. The functional heterogeneity of mitochondria recently shown in live imaging techniques underlines the presence of two distinct subpopulations: one beneath the sarcolemma (SSM) and

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another along the myofilaments (IFM) in cardiac tissue [20]. The previous study has suggested the preferential loss of IFM function with age [21] and our lab had earlier shown a favorable difference among the subpopulation for the energy substrate during reperfusion [22], altogether

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indicates the heterogeneity of mitochondrial function and subsequently added mitochondrial complexity has significant pathophysiological implications. The present study result shows a significant oxidative dysfunction in IFM as compared to SSM, proved by the levels of oxidative

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markers and different antioxidant enzymes activities especially, MnSOD (specific isoform for mitochondria), assumed to be due to its cellular locations and specific functions. In most of the

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mitochondrial study, these significant differences were overlooked due to the general isolation procedure adopted that represent predominantly SSM.

Hydrogen sulfide pre- and post-conditioning is highly effective in alleviating reperfusion injury, reported to have a mixed clinical success, and believed to mediate the cardioprotection through

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mitochondrial ATP-sensitive potassium channel (KATP) [23]. Evidence from the early study suggest that KATP decrease mitochondrial ROS, that is secondary to Ca2+ uptake, inhibiting mitochondrial permeability transition, thereby protects the cardiomyocytes. Since cardiac

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mitochondria comprise of two distinct subpopulations and each type has its own KATP, its

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subsequent modulation is predicted to be one of the key players in the preservation of total functional mitochondria in the cell. The present study provides enough data for the distinct preservation of mitochondrial subpopulation by H2S, either as a preconditioning or a postconditioning agent. According to our results, SSM fraction was effectively recovered from the oxidative stress insult by HIPC and HPOC, leaving an important question regarding the IFM role in determining the overall mitochondria dysfunction in the cell and subsequent cardioprotection. Towards this direction, we have evaluated and compared the mitochondrial enzyme activity

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(complex I) in both IFM and SSM. According to our result, we found that oxidative stress did have its impact on IFM especially in their membrane potential and subsequently affect the enzyme activity, partly suggesting additional pathological stimulus is required for the overall

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impact on cardiomyocytes. Acknowledgement

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This study was funded by the Department of Science and Technology (DST), New Delhi, Government of India (No. SR/S0/HS-0255/2012O) and Indian Council of Medical Research,

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New Delhi, Government of India (No. 5/4/1-24/2012-NCD-II). We would like to thank Dr. C

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David Raj for his assistance during animal experiments.

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Pharmacol Rep. 2014;66(3):499-504. Figure legends:

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Figure. 1 A) Schematic diagram explaining the experimental groups viz., Normal perfusion, Ischemic control, Ischemia-reperfusion control (I/R), Ischemic preconditioning (IPC) control,

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Ischemic post-conditioning (POC) control, H2S preconditioning (HIPC) and H2S postconditioning (HPOC), B) Lactate dehydrogenase activity and C) Creatine kinase activity. (*) represents statistically different (P<0.05) from the normal control.

Figure. 2 Effect of H2S on I/R induced myocardial injury parameters. A) Creatine kinase activity,

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B) Lactate dehydrogenase activity and C) Caspase-3 activity and D) DNA Fragmentation assay. [Lane 1) Marker DNA, 2) Normal perfusion control, 3) NaHS control, 4) Reperfusion control, 5) IPC control, 6) HIPC, 7) POC control and 8) HPOC]. (*) represents statistically different (P<0.05)

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from the normal control.

Figure.3 Effect of H2S on I/R induced oxidative stress in cardiac mitochondrial subpopulation. A)

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TBARS level, B) GSH content, C) Catalase, D) GPx activity E) GR activity and F) GSH/GSSG ratio. (*) represents significantly different (P<0.05) from the normal control; (#) represents significantly different (P<0.05) from IFM. Figure. 3.1 Effect of H2S on Total SOD MnSOD activity of mitochondrial subpopulation. A) Total SOD, B) Mn-SOD and C) Mn-SOD/Total SOD activity.

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Figure. 4 H2S effect on microsomal ATPase activities. A) Na+/K+ ATPase, B) Mg2+ ATPase, C) Ca2+ ATPase and D) 5’ Nucleotidase. (*) represents significantly different (P<0.05) from the

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normal control.

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Table legends:

Table I: Hemodynamics measurement during the time of experiment. Values are mean ± SE

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with n=6. Each sample was analyzed in triplicate. (*) represents statistically different (p<0.05) from the normal control. EDP- Left ventricular end diastolic pressure; DP- Developed pressure; RPP- Rate pressure product.

Table II: Infarct size measured after completion of the experiment using TTC staining. Values

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are mean ± SE with n=6. Each sample was analyzed in triplicate. (*) represents statistically different (p<0.05) from the normal control.

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Table III: TBARS and GSH level in subcellular compartments. Values are mean ± SE with n=6. Each sample was analyzed in triplicate. (*) represents statistically different (p<0.05) from the

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normal control. (#) represents statistically different (p<0.05) from the reperfusion control. (c) represents statistically different (p<0.05) from IPC control. (d) represents statistically different (p<0.05) from POC control. [a = (nM MDA/mg protein); b = (µmole GSH/mg protein)]. Table IV: Subcellular distribution and antioxidant activities in normal rat heart. Values are mean ± SE with n=6. Each sample was analyzed in triplicate. (*) represents statistically different (p<0.05) from the normal control; (#) represents statistically different (p<0.05) from the reperfusion control. (c) represents statistically different (p<0.05) from IPC control. (d) represents

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statistically different (p<0.05) from POC control. [a = IU/mg protein; b =-mIU/mg protein; ʄµmole GSH/min/mg protein].

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Table V: Complex I activity and swelling behavior in mitochondrial subpopulation. Values are mean ± SE with n=6. Each sample was analyzed in triplicate. (*) represents statistically different (p<0.05) from the normal control.

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Results:

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Figures:

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Figure. 1 A) Schematic diagram explaining the experimental groups viz., Normal perfusion, Ischemic control, Ischemia-reperfusion control (IR), Ischemic preconditioning (IPC) control, Ischemic post-conditioning (POC) control, H2S preconditioning (HIPC) and H2S post-

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conditioning (HPOC), B) Lactate dehydrogenase activity and C) Creatine kinase activity.

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3000 (bp)

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Figure. 2 Effect of H2S on I/R induced myocardial injury parameters. A) Creatine kinase activity,

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B) Lactate dehydrogenase activity, C) Caspase-3 activity and D) DNA Fragmentation assay.

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Figure. 3 Effect of H2S on I/R induced oxidative stress in cardiac mitochondrial subpopulation. A) TBARS level, B) GSH content, C) Catalase, D) GPx activity, E) GR activity and F) GSH/GSSG ratio.

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Figure. 3.1 Effect of H2S on SOD activity of mitochondrial subpopulation. A) Total SOD, B) MnSOD and C) Mn-SOD/Total SOD activity.

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Figure. 4 H2S effect on microsomal ATPase activities. A) Na+/K+ ATPase, B) Mg2+ ATPase, C) Ca2+ ATPase and D) 5’ Nucleotidase

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Results:

LVEDP1 6±2 45 ± 3* 22 ± 4# 23 ± 3# 18 ± 4# 17 ± 3#

LVDP1 98 ± 4 42 ± 3* 90 ± 4# 93 ± 3# 95 ± 4# 96 ± 3#

RPP2 95 ± 2 32 ± 2* 82 ± 3# 83 ± 2# 87 ± 3# 88 ± 2#

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Groups Normal Perfusion (n=6) I/R (n=6) IPC (n=6) HIPC (n=6) POC (n=6) HPOC (n=6)

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Table I: Myocardial hemodynamic measurement during the time of experiment

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Tables:

Data represented as mean ± SE; *p<0.05 vs normal control. # P<0.05 vs I/R control. LVEDP- Left ventricular end diastolic pressure; LVDP- Left ventricular developed pressure; RPP- Rate pressure

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product. 1-mmHg; 2-mmHg x bpm x 10^3

Infarct size (% of total heart) 4 ± 0.8 8 ± 0.2 24 ± 0.9* 7 ± 0.7# 9 ± 0.5# 7 ± 0.7# 6 ± 0.5#

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Groups Normal control (n=6) Ischemia (n=6) I/R (n=6) IPC (n=6) HIPC (n=6) POC (n=6) HPOC (n=6)

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Table II: Infarct size measurement after completion of the experiment using TTC staining.

# Data are represented as mean ±SE; *p<0.05 Vs normal control; #p<0.05 vs I/R control

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Table III: TBARS and GSH levels in subcellular compartments

Mitochondria 0.12±0.01 0.36±0.02* 0.75±0.09* 0.16±0.02 0.18±0.02 0.27±0.03 0.19±0.02d

Microsomes 0.224±0.007 0.322±0.024* 0.507±0.012* 0.231±0.015 0.219±0.012c 0.206±0.005 0.183±0.012d

Homogenate 2.23±0.07 1.10±0.08* 0.91±0.06* 1.62±0.12# 1.61±0.11# 1.22±0.09 1.58±0.11#d

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Homogenate 1.08±0.06 1.66±0.08* 2.20±0.12* 1.16±0.06 1.54±0.05# 1.66±0.03# 1.60±0.04#d

Mitochondria 3.93±0.35 1.45±0.13* 0.62±0.05* 2.87±0.26# 2.53±0.23# 1.72±0.15# 2.39±0.21#d

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Normal Ischemia I/R control IPC control HIPC POC control HPOC

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Microsomes 0.025±0.004 0.024±0.003 0.022±0.004 0.026±0.004 0.032±0.009c 0.021±0.002 0.024±0.007d

Data are represented as mean ± SE; (n=6) *p<0.05 vs normal control. # p<0.05 vs I/R control. c - p<0.05 vs IPC. d - p<0.05 vs POC. a = (nM MDA/mg protein); b = (µmole GSH/mg protein)

Mitochondriab 7.39±0.60 0.85±0.03* 0.77±0.03* 2.78±0.16 3.00±0.16c 2.91±0.06 3.13±0.16d

Microsomesb 1.87±0.15 1.33±0.13* 1.49±0.17* 1.61±0.18 2.35±0.10c 1.29±0.07 1.64±0.11d

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Normal Ischemia I/R control IPC control HIPC POC control HPOC

Homogenatea 37.23±2.13 20.89±2.20* 13.72±3.27* 39.51±2.27 37.28±2.13 45.13±1.83 46.24±1.77

Homogenatea 5.49±0.63 1.79±0.20 1.97±0.22 3.73±0.42 3.52±0.40 3.98±0.45# 4.44±0.51#d

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Table IV: Subcellular distribution and activity of antioxidant enzymes in normal rat heart GPx activityʄ

GR activity Mitochondriab 5.49±0.63 2.03±0.24* 0.87±0.10* 4.03±0.47# 3.56±0.42# 2.42±0.28# 3.36±0.39#d

Microsomesb 0.18±0.01 0.22±0.04 0.07±0.01 0.12±0.01# 0.15±0.07c 0.08±0.01 0.12±0.08d

Homogenate 2.96±0.31 0.91±0.09* 0.62±0.06* 1.74±0.18# 1.67±0.17# 2.25±0.23# 2.29±0.24#

Mitochondria 2.96±0.31 0.91±0.09* 0.62±0.06* 1.74±0.18# 1.67±0.17# 2.25±0.23# 2.29±0.24#

Microsomes 0.154±0.031 0.129±0.006* 0.129±0.007* 0.173±0.034# 0.133±0.022# 0.143±0.011# 0.130±0.008#

Data are represented as mean ± SE; (n=6) *p<0.05 vs normal control. # p<0.05 vs I/R control. c - p<0.05 vs IPC. d - p<0.05 vs POC. a = IU/mg protein; b =-mIU/mg protein; ʄ- µmole GSH/min/mg protein

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SSM 0.98±0.01 0.69±0.02* 0.59±0.10* 0.84±0.06# 0.64±0.03# 0.88±0.06# 0.96±0.05#

IFM 1.00±0.02 0.25±0.14* 0.23±0.01* 0.79±0.06# 0.81±0.08# 0.91±0.03# 1.07±0.08#

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Data are represented as mean ± SE; (n=6) *p<0.05 vs normal control;#p<0.05 vs I/R control.

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Normal Ischemia I/R control IPC control HIPC POC control HPOC

Swelling (ΔA340/min/mg protein)

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Complex I activity (μM of NADH oxidized/min/mg protein) SSM IFM 0.228±0.024 0.333±0.024 0.156±0.011* 0.470±0.035 0.022±0.001* 0.133±0.009* 0.200±0.014# 0.280±0.02#0 0.227±0.009# 0.298±0.022# 0.220±0.014# 0.317±0.023# 0.536±0.039# 0.309±0.023#

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Table V: Complex I activity and swelling behaviour in subcellular compartments

ACCEPTED MANUSCRIPT Highlights: Cardiac mitochondria account for the major oxidative stress during reperfusion.

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Response towards H2S conditioning is unequal among the subcellular organelle.

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SSM play prominent role in the reduction of oxidative stress mediated by H2S

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