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Reversible Blockade of Complex I or Inhibition of PKCβ Reduces Activation and Mitochondria Translocation of p66Shc to Preserve Cardiac Function after Ischemia

Aim Excess mitochondrial reactive oxygen species (mROS) play a vital role in cardiac ischemia reperfusion (IR) injury. P66Shc, a splice variant of the ShcA adaptor protein family, enhances mROS production by oxidizing reduced cytochrome c to yield H2O2. Ablation of p66Shc protects against IR injury, but it is unknown if and when p66Shc is activated during cardiac ischemia and/or reperfusion and if attenuating complex I electron transfer or deactivating PKCβ alters p66Shc activation during IR is associated with cardioprotection. Methods Isolated guinea pig hearts were perfused and subjected to increasing periods of ischemia and reperfusion with or without amobarbital, a complex I blocker, or hispidin, a PKCβ inhibitor. Phosphorylation of p66Shc at serine 36 and levels of p66Shc in mitochondria and cytosol were measured. Cardiac functional variables and redox states were monitored online before, during and after ischemia. Infarct size was assessed in some hearts after 120 min reperfusion. Results Phosphorylation of p66Shc and its translocation into mitochondria increased during reperfusion after 20 and 30 min ischemia, but not during ischemia only, or during 5 or 10 min ischemia followed by 20 min reperfusion. Correspondingly, cytosolic p66Shc levels decreased during these ischemia and reperfusion periods. Amobarbital or hispidin reduced phosphorylation of p66Shc and its mitochondrial translocation induced by 30 min ischemia and 20 min reperfusion. Decreased phosphorylation of p66Shc by amobarbital or hispidin led to better functional recovery and less infarction during reperfusion. Conclusion Our results show that IR activates p66Shc and that reversible blockade of electron transfer from complex I, or inhibition of PKCβ activation, decreases p66Shc activation and translocation and reduces IR damage. These observations support a novel potential therapeutic intervention against cardiac IR injury.


Introduction
Mitochondria are proximal effectors and determinants of cell fate during ischemia and reperfusion (IR)-mediated oxidative stress. Thus they are also potential therapeutic targets to ameliorate oxidative damage [1]. Excess mitochondrial reactive oxygen species (mROS) emission plays a key role in contributing to cardiac IR injury [2]. It is generally accepted that in mitochondria the superoxide anion (O 2 2N ), the precursor of most ROS, is generated within the electron transport chain (ETC) complexes (e.g. I, II and III), wherein the leak of a single electron reduces O 2 to O 2 2N [3][4][5]. Recent reports indicate that p66 Shc , a splice variant of the ShcA adaptor protein family, also contributes to mROS production [1,6]. Giorgio et al. [6] suggested that p66 Shc utilizes reducing equivalents of the ETC by oxidizing reduced cytochrome c (cyt c) to catalyze the reduction of O 2 to H 2 O 2 . Electron transfer from cyt c to p66 Shc would designate it as a mitochondrial redox enzyme [6] that could play an alternative role as a signaling molecule for mitochondrial-mediated cell apoptosis [7,8]. Indeed, p66 Shc gene ablation (p66 Shc2/2 ) has been shown to reduce hypoxia/reoxygenation-induced damage to hepatocytes [9] and to decrease necrosis and apoptosis of myofibrils after hind limb ischemia compared to the wild type [10]. Furthermore, in isolated perfused hearts, p66 Shc2/2 mice compared to wild type mice exhibited both reduced IR-mediated LDH release into the coronary effluent and abrogated lipoperoxidation [11].
The pathway leading to p66 Shc activation and translocation into mitochondria is unclear. Excess H 2 O 2 or ultraviolet light (UV) irradiation has been shown to activate a serine-threonine protein kinase C b (PKCb), which led to p66 Shc phosphorylation at serine 36, and to trigger mitochondrial accumulation of the protein after its recognition by the prolyl isomerase Pin1 in mouse embryonic fibroblasts (MEF) [12,13]. Pinton et al. [13] reported that in MEF, inhibition of PKCb with hispidin inhibited H 2 O 2 -induced p66 Shc phosphorylation; overexpression of PKCb mediated H 2 O 2 -induced mitochondrial dysfunction in wild type MEFs, but not in p66 Shc2/2 MEFs. It was reported that activation of PKCbII in ventricular tissue increased after IR and that gene deletion or pharmacological blockade of PKCbII was associated with protection against ischemia [14].
Mitochondrial ETC complexes are involved in mROS production during IR. Moreover, O 2 2N generated at mitochondrial complex III can be attenuated by limiting electron transfer from complex I, thereby provide protection against IR injury. We [15], and others [16], have reported that the therapeutic targeting of complex I with amobarbital provided cardioprotection, in part, by decreasing mROS production during IR. Amobarbital, a short-acting barbiturate, reversibly attenuates complex I electron transfer at the rotenone site [17], decreased IRinduced O 2 2N generation and mitochondrial [Ca 2+ ] overload [15], retarded mitochondrial permeability transition pore (mPTP) opening [16], and improved oxidative phosphorylation (OxPhos) [16]. These mitochondrial effects culminated in appreciable protection of cardiac function on reperfusion after ischemia [15,16]. However, targeting distal complexes of the ETC, especially complex IV, is not protective against ischemic stress and may exacerbate injury. For example, blocking complex IV before ischemia increased levels of reduced cyt c, a likely substrate for p66 Shc -mediated H 2 O 2 generation [6] leading to more oxidative stress.
Our aims were to explore if p66 Shc is involved in IR induced mROS generation and how ROS and p66 Shc dynamically modulate each other during different periods of cardiac ischemia and reperfusion. To address these objectives, we used the perfused ex vivo guinea pig heart model and monitored: a) if and when p66 Shc is activated during cardiac ischemia and/or reperfusion; b) if activation of PKCbII during IR induces p66 Shc activation and mitochondrial translocation to contribute to cardiac IR injury; c) if reversible attenuation of complex I electron transfer with amobarbital during IR is associated with p66 Shc activation.

Ethics Statements
Our animal protocols conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health No. 85-23, Revised 1996). The Medical College of Wisconsin IACUC, with the number AUA 1647, approved all our animal studies.

Protocols
At the end of the indicated times of ischemia and/or reperfusion, some hearts were removed and immediately snap frozen in liquid N 2 and stored at 280˚C for later measurement of p66 Shc and PKCbII phosphorylation (n53 hearts per group). Other hearts were removed and mitochondria were immediately isolated for assessment of cytosolic and mitochondrial changes in p66 Shc levels (n53 hearts per group). Other hearts were removed at 120 min reperfusion to measure infarct size (n58 hearts per group). In the complex I blocker treated groups, hearts were perfused with 2.5 mM amobarbital (Amo) for 1 min before initiating global ischemia [15]; in the PKCb antagonist treated groups, hearts were perfused with 40 mM hispidin (His) for 10 min before ischemia. Amobarbital and hispidin were administered up to the initiation of ischemia to ensure the presence of the drugs in the heart during the entire period of ischemia and briefly during the onset of reperfusion.

Infarct size measurement
After 120 min reperfusion, hearts were removed and the atria discarded. The ventricles were cut into 3 mm sections, and then stained with 1% 2,3,5triphenyltetrazolium chloride (TTC) to measure infarct size. In living tissue, TTC is reduced by pyridine nucleotide-linked dehydrogenase into a red, lipid-soluble formazan that stains living tissue red. Infarcted tissue lacking this dehydrogenase remains unstained. Infarct size of the TTC stained white area was determined as a percentage of total ventricular heart weight [22,23].

Measurement of NADH in isolated hearts
Tissue autofluorescence (arbitrary fluorescence units, afu) is an indicator of NADH at l em 460 nm (l ex 350 nm); it is primarily utilized to assess the mitochondrial redox state (n56 isolated hearts/group) [15,24,25]. Motion artifact was reduced by using l em 405 nm as a reference that is less sensitive to changes in NADH [26]. Therefore, the ratio of auf at l em 460/l em 405 nm indicates mitochondrial NADH.

Detection of O 2
2N in isolated hearts loaded with dihydroethidium (DHE) The intracellular fluorescent probe DHE (Molecular Probes) was used to assess O 2 2N emission continuously during IR (n56 isolated hearts per group) as described previously [15,20,24,27]. After stabilization, hearts were loaded with 10 mM DHE dissolved in KR solution for 20 min; this was followed by washout of residual, unincorporated DHE with KR solution for 20 min [15,20]. The fluorescence emitted (afu) after washout was adjusted to 0 afu (baseline) to normalize the fluorescence intensity for all experiments. Changes in the DHE fluorescence signal, which represents O 2 2N generation, were compared to the baseline fluorescence signal values [15,18,27].

Preparation of cytosolic and mitochondrial fractions
Mitochondrial and cytosolic fractions were prepared using procedures described previously [27][28][29][30][31][32] with minor modifications. All procedures were carried out at 4˚C. Hearts were minced in a chilled isolation buffer containing (in mM) 200 mannitol, 50 sucrose, 5 KH 2 PO 4 , 5 MOPS, 1 EGTA and 0.1% fatty acid free BSA at pH 7.15, then homogenized. The homogenized slurries were centrifuged at 8000 g for 10 min. The supernatant were collected and further centrifuged at 50,000 g for 30 min, and then the resulting supernatant was used as the cytosolic fraction. The pellet from the 8,000 g centrifugation was resuspended in isolation buffer containing protease inhibitors and spun at 750 g for 10 min; the supernatant was collected and again centrifuged at 8000 g. The final mitochondrial pellet following this centrifugation was resuspended in isolation buffer and then purified as described by Graham [33]. Mitochondria were layered on 30% Percoll in isolation buffer, and then centrifuged for 30 min at 95,000 g. After centrifugation, the lower part of the dense brown band containing the purified mitochondria was collected and washed two times with isolation buffer. After purification, the mitochondria were resuspended in isolation buffer containing protease inhibitors and stored at 280˚C for later use. Mitochondrial and cytosolic protein concentrations were assessed using Bio-Rad protein assay with bovine serum albumin (BSA) as the standard.

Immunoprecipitation of mitochondrial proteins
Immunoprecipitation (IP) was performed as described previously [27,34,35] with minor changes. Frozen hearts were pulverized under liquid nitrogen and then the powder was lysed in RIPA buffer containing 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS and protease inhibitors on ice for 30 min. The sample was pre-cleared with 30 ml protein G Sepharose-4B beads (Invitrogen) for 1 h at 4˚C with constant end-over-end shaking and then centrifuged at 3000 g for 5 min. Supernatant was collected, adjusted to 2 mg/ml protein concentration with RIPA buffer containing protease inhibitors, and subjected to IP with an anti-Shc antibody (rabbit polyclonal IgG, Millipore) at 4˚C with constant end-over-end shaking overnight. The next day 30 ml of protein G Sepharose-4B beads was added and the mixture was incubated for another 2 h under the same conditions as above. The beads were collected and washed (five times) with RIPA buffer. After washing and aspirating the washing buffer completely, 50 ml Laemmli sample buffer containing 50 mM Tris-Cl, 10% glycerol, 500 mM b-mercaptoethanol, 2% SDS, 0.01% w/v bromophenol blue and protease inhibitors at pH 7.4, was added to the beads and boiled at 95˚C for 5 min. The immunoprecipated proteins were separated using SDS-PAGE and then subjected to Western blot analyses.

Statistical analysis
All results were expressed as means ¡SEM and were analyzed by one-way ANOVA followed by a post-hoc analysis (Student-Newman-Keuls' test) to determine significant differences of means among groups. Isolated heart data were collected for statistical evaluation at time points 30, 45, 50, and 65 min. P,0.05 was considered significantly different (two-tailed).

Activation of p66 Shc occurred during reperfusion after ischemia
We first examined if and when p66 Shc was activated during cardiac IR by determining the level of phosphorylation of p66 Shc at serine 36 (Ser36), an indication of p66 Shc activation [12,13]. After normalizing to total immunoprecipated p66 Shc (Fig. 1A, bottom panel), phosphorylation of p66 Shc at Ser36 increased on reperfusion in the IR groups compared to the time control (TC) and ischemia only groups (Fig. 1A, upper panel); the increase in phosphorylation of p66 Shc at Ser36 occurred as early as 10 min reperfusion after 30 min ischemia and was detectable for up to 60 min reperfusion. In the 20 and 30 min ischemia only groups, the phosphorylation of p66 Shc at Ser36 did not change compared to the TC group. These results indicated that IR induced p66 Shc activation and, moreover, that p66 Shc was activated only during reperfusion after ischemia and not by ischemia alone.

P66 Shc localized to mitochondria during reperfusion after ischemia
It was reported that after phosphorylation at Ser36, p66 Shc is recognized by prolyl isomerase Pin1, which enables the phosphorylated p66 Shc to be translocated to the IMM [13]. We therefore examined the translocation of p66 Shc into mitochondria during cardiac IR injury. At the end of the indicated ischemia and/or reperfusion period, hearts were removed and cytosolic and mitochondrial fractions were prepared (see Methods), and then subjected to Western blot analyses with anti-Shc antibody. The level of p66 Shc in mitochondria increased by approximately 2fold at 10 and 60 min reperfusion, and by approximately 3-fold at 20 min reperfusion after 30 min ischemia, compared to TC (Fig. 2). The level of p66 Shc in the cytosolic fraction decreased in the 30 min ischemia alone group and in the 30 min ischemia followed by 10, 20 or 60 min reperfusion groups when compared to the TC and 20 min ischemia groups (Fig. 2).
The incongruity in the reverse changes in p66 Shc levels in cytosolic and mitochondrial fractions in the 30 min ischemia alone group indicated that p66 Shc might translocate to other parts of the cell, including organelles other than mitochondria. Moreover, the cytosolic p66 Shc level was lower in the 30 min ischemia plus 10 and 20 of reperfusion groups when compared to the 30 min ischemia plus 60 min of reperfusion group (Fig. 2). These results suggested greater cell injury/death after 60 min of reperfusion contributed to the lower p66 Shc levels. The translocation of p66 Shc into mitochondria during reperfusion after 30 min ischemia was consistent with phosphorylation of p66 Shc at Ser36 during the indicated periods (Fig. 1). Insofar as ischemia alone did not lead to phosphorylation of p66 Shc , correspondingly, ischemia alone (20 or 30 min) also Prolonged ischemia required to activate p66 Shc during reperfusion after ischemia Our previous reports show that the magnitude of ROS production during ischemia is dependent on the duration of ischemia [15,20,27]. Next, we investigated if the activation of p66 Shc was associated with the duration of ischemia. Guinea pig isolated hearts were subjected to 5, 10, 20 or 30 min ischemia, followed by 20 min reperfusion. At the end of reperfusion, hearts were harvested and evaluated for phosphorylation of p66 Shc at Ser36. Phosphorylation of p66 Shc at Ser36 increased after 20 min reperfusion following 20 or 30 min global ischemia, but not after 5 or 10 min ischemia plus 20 min reperfusion compared to their respective TCs (Fig. 3). These results indicated that at least 20 min ischemia was required for some p66 Shc activation during reperfusion after ischemia.
To evaluate a possible correlation between p66 Shc activation and translocation with the mitochondrial redox state and generation of ROS, online mitochondrial

NADH and DHE (O 2
2N emission) fluorescence intensities were measured during 30 min ischemia and 20 min reperfusion. During early ischemia NADH increased markedly, but then gradually declined toward baseline levels as ischemia progressed (Fig. 4A, IR); during reperfusion, NADH was lower than baseline (Fig. 4A, IR). These findings indicated a shift in mitochondrial redox towards more oxidized mitochondria during early reperfusion. The corresponding timedependent changes in DHE fluorescence signals were characterized by a modest steady increase during early ischemia and a marked and accelerated increase during late ischemia (Fig. 4B, IR); the DHE fluorescence signal intensity remained elevated above baseline during reperfusion in the IR group.

Amobarbital decreased p66 Shc activation induced by ischemia and reperfusion
We next sought to determine if reversible blockade of complex I with amobarbital, which we showed previously reduced O 2 2N emission during IR [15], would reduce p66 Shc activation during reperfusion. To achieve this, hearts were treated with or without amobarbital (Amo) prior to ischemia followed by 20 or 30 min ischemia and 20 min reperfusion. At the end of reperfusion, hearts were collected and evaluated for phosphorylation of p66 Shc at Ser36 (see Methods). Compared to the IR alone group, the amobarbital treated groups exhibited reduced phosphorylation of p66 Shc at Ser36 by 27.5¡0.1% and 14.4¡0.1% at 20 min reperfusion after 20 min (Fig. 5A) and 30 min (Fig. 5B) ischemia, respectively. However,  phosphorylation of p66 Shc at Ser36 in the amobarbital treated groups was significantly higher than in the TC group. These data indicated, and supported our previous findings [15], that amobarbital-induced interference with mitochondrial electron transfer provides protection, at least in part, by attenuating p66 Shc activation during IR. Therefore, to ascertain if attenuation of p66 Shc activation by amobarbital correlated with mitochondrial redox state, ROS production, and functional recovery during IR injury, we monitored NADH, O 2 2N generation and several indices of contractility and relaxation online in amobarbital treated and untreated hearts during IR. Compared to untreated hearts, amobarbital treated hearts displayed better preserved mitochondrial NADH levels during 30 min ischemia and 20 min reperfusion ( Fig. 4A; Amo+IR), and a decrease in DHE fluorescence (O 2 2N production) during late ischemia ( Fig. 4B; Amo+IR). Moreover, amobarbital treatment resulted in a significantly lower diastolic LVP during late ischemia and early reperfusion ( Fig. 4D; Amo+IR) and improved developed LVP, dLVP/ dt min and dLVP/dt max during early reperfusion (Fig. 4C, E, F; Amo+IR) compared to the IR alone group. The incidence of ventricular fibrillation (VF) in the IR untreated hearts was 100%, with an average of approximately 3 VF occurrences/ heart during reperfusion; amobarbital treated hearts exhibited no incidence of VF during reperfusion. Each VF that occurred was subsequently reversed to sinus rhythm with lidocaine. It is noteworthy that although the 20 min ischemia plus 20 min reperfusion group demonstrated activation of p66 Shc , there was no significant compromise in cardiac function or mitochondrial bioenergetics when compared to the TC group (data not shown). This suggested that p66 Shc activation is an early marker of ischemia.

Activation of PKCbII induced by cardiac ischemia and reperfusion required for p66 Shc activation
Next we examined if PKCbII is activated during cardiac IR, and if activation of p66 Shc during reperfusion after ischemia is mediated through PKCbII activated pathways as previously reported in MEFs [13,36]. Western blot was used to determine phosphorylation of PKCbII at Ser660 in hearts subjected to 20 or 30 min ischemia plus 20 min reperfusion. Figure 6A shows that 20 or 30 min ischemia plus 20 min reperfusion increased phosphorylation of PKCbII at Ser660, suggesting IR activated PKCbII. Next we determined if PKCbII activation is required for p66 Shc activation during reperfusion. For this, hearts were treated with or without hispidin (His) for 10 min before the start of 30 min ischemia plus 20 min reperfusion. At the end of reperfusion, heart tissue was evaluated for phosphorylation of p66 Shc at Ser36; alternatively, mitochondria were isolated immediately after reperfusion to evaluate mitochondrial p66 Shc translocation. Figures 6B and C show that when hearts were perfused with hispidin before ischemia compared to IR only (control), both p66 Shc phosphorylation at Ser36 and p66 Shc mitochondrial translocation were decreased. These observations support the signaling role of PKCb in activating the oxidoreductase, p66 Shc , during oxidative stress in IR.
We correlated attenuation of p66 Shc activation by hispidin with functional recovery and ventricular infarct size. Figure 7A shows that administration of hispidin before ischemia significantly reduced diastolic contracture, i.e. diastolic LVP, when compared to the IR only hearts. The incidence of VF after ischemia was significantly abated in hearts treated with hispidin before ischemia when compared to IR only hearts (data not shown). Figure 7B also shows that hispidin attenuated infarct size to 33¡3% compared to the untreated IR (control) group in which infarct size was 45¡3%. These results showed that ischemia followed by reperfusion induces p66 Shc activation through the PKCbII signaling pathway to contribute to IR injury.

Discussion
We have demonstrated that p66 Shc is activated and translocated into mitochondria during global no-flow IR in the ex vivo perfused heart model. Activation of p66 Shc and its subsequent translocation into mitochondria are dependent both on the duration of ischemia and on the occurrence and timing of reperfusion after ischemia. Reversible attenuation of respiration via complex I by amobarbital reduced both ROS emission and activation of p66 Shc and improved function on reperfusion. Inhibiting PKCb during IR with hispidin reduced activation/translocation of p66 Shc , improved cardiac function, and decreased cell injury. Thus, p66 Shc is both a marker and a mitochondrial effector of oxidative stress-mediated mitochondrial dysfunction and ROS production. The effects of amobarbital on reducing mROS emission, improving redox state, and attenuating p66 Shc activation during IR injury, suggest an important link between oxidative stress and activation of p66 Shc . P66 Shc is activated during reperfusion after global cardiac ischemia IR injury can occur under a number of situations including cardiac arrest and resuscitation, hypoxia and reoxygenation, and coronary artery occlusion and reperfusion. Although ischemia itself can induce cardiac injury, much of the injury occurs during early reperfusion [37]. The causative and interactive factors of myocardial reperfusion injury include excess ROS, Ca 2+ overload, and induction of apoptosis [38]. It is well known that the major source of ROS emission during reperfusion in cardiomyocytes is the ETC, which is progressively damaged during ischemia and contributes to further ROS generation during reperfusion [2,20,[39][40][41].
However recent studies showed that p66 Shc may also act as an oxidoreductase to generate H 2 O 2 in mitochondria after activation following redox stimulation [6]. Furthermore, in the mouse isolated heart model of IR injury, p66 Shc2/2 mice showed less susceptibility to IR injury than wild type mice [11]. In the present study, we showed that p66 Shc only becomes activated and translocated to mitochondria during reperfusion after 20 or 30 min ischemia (Figs. 1, 2 and 3). This suggests that p66 Shc could also contribute to mROS generation during IR injury especially during reperfusion after ischemia. Why p66 Shc is activated only during reperfusion is unclear, but it is possible that the preceding events during ischemia (e.g. excess ROS emission) followed by restoration of flow, O 2 and substrate supply, and by the rapid re-establishment of a reduced redox state and OxPhos, may collectively contribute to p66 Shc activation.
Ischemia, when prolonged, irreversibly disrupts electron transfer during reperfusion, yet ETC complexes I-III may not be the only sources of ROS during reperfusion [41]. It is possible that during the reperfusion phase of IR injury, p66 Shc induces additional ROS generation via a putative direct effect on oxidoreductase activity, which depends on electron leak from upstream complexes (e.g. I and III) and then becomes an additional source of ROS production [6,13] that may exacerbate the ROS generated from damaged complexes. This could initiate a vicious cycle of excess ROS emission by mitochondrial ROS-induced-ROS release (RIRR) during reperfusion.

Long duration of ischemia with reperfusion is necessary for p66 Shc activation
We found that 5 and 10 min ischemia plus 20 min reperfusion did not increase activation of p66 Shc , whereas 20 and 30 min ischemia with reperfusion increased p66 Shc activation markedly. This indicated that a longer duration of ischemia plus reperfusion was required to activate p66 Shc . The duration of ischemia is generally linked to the extent of injury to ETC complexes [41,42]. In rats, complex I activity decreased early in ischemia, whereas complex III damage occurred only after a longer ischemia time [41,43]. Consistent with the time-dependent ischemic damage to ETC complexes, ROS emission also displays an ischemia duration dependent pattern. In the guinea pig heart, we showed a correlation between NADH, O 2 2N levels and duration of ischemia (Fig. 4A, B) [15,18,24]. Our present (Fig. 4B) and past [15,27] results in guinea pig isolated hearts showed small amounts of O 2 2N were generated during early (1-20 min) global ischemia followed by a marked surge in O 2 2N generation during late (25-30 min) global ischemia. Therefore, the longer durations of ischemia (20 and 30 min), when followed by at least 10 min reperfusion, are likely to have induced ETC complex damage leading to enhance ROS emission. Excess ROS (possibly H 2 O 2 ), beyond a certain threshold, may act as deleterious redox-signaling molecules that lead to activation of p66 Shc , which in turn enhances mROS generation to induce further oxidative damage to ETC complexes leading to RIRR.
Unlike 30 min ischemia, 20 min ischemia (both followed by 20 min reperfusion) induced p66 Shc activation (Fig. 3), but had no significant effect on cardiac function on reperfusion (data not shown). This suggests that p66 Shc is more sensitive to redox modulation than the measured cardiac function during IR injury. These observations show overall that p66 Shc participates in emitting ROS involved in cardiac dysfunction during longer ischemia times as with reperfusion.

Amobarbital decreases activation of p66 Shc during cardiac IR injury
Our current and previous studies [15] show that amobarbital, when present during ischemia and early reperfusion, preserved NADH (Fig. 4A), minimized O 2 2N emission (Fig. 4B), and reduced mitochondrial Ca 2+ overload [15], which concomitantly improved functional recovery (Fig. 4 C-F) and reduced infarction when compared to IR only (control) [15]. Furthermore, in this study we also found that compared to IR alone, amobarbital reduced activation of p66 Shc induced by ischemia and reperfusion (Fig. 5). Since amobarbital reversibly binds at the rotenone site of complex I to attenuate electron transfer [17,44,45], this suggests that amobarbital, when present in the tissue during ischemia, attenuates activation of p66 Shc by blunting electron transfer to reduce O 2 2N generation. Our observations further suggest that amobarbital better protected mitochondria and reduced myocardial injury during reperfusion, possibly due to attenuated p66 Shc activation and its concomitant translocation into mitochondria to modulate ROS emission.
When p66 Shc is activated and translocated into mitochondria, it becomes a proapoptotic protein that induces apoptosis by altering the redox state [6]; however, the detailed mechanism for this remains unclear [46]. Amobarbital-induced attenuation of p66 Shc activation during IR demonstrates that p66 Shc is affected by electron transfer related events that modulate the ETC. Thus, insofar as amobarbital was presumed present in the myocardium during ischemia, it may have reduced activation of p66 Shc during reperfusion by decreasing ischemic ROS emission. Hence, the amobarbital-induced decrease in ROS production during ischemia appears to decrease ROS emission on reperfusion, attenuate activation of cytosolic PKCbII, reduce activation and translocation of p66 Shc into mitochondria, and ultimately, decrease mitochondrial and cellular damage.

PKCb induces p66 Shc activation and its translocation into mitochondria during IR
The signaling pathways that lead to p66 Shc phosphorylation at Ser36 during cardiac ischemia and/or reperfusion have not been reported before. In this study, we show that inhibiting PKCb with hispidin reduced both p66 Shc phosphorylation at Ser36 and mitochondrial translocation during IR (Fig. 6 B and C). These results indicate that p66 Shc activation and its eventual translocation into mitochondria during IR likely occur through PKCb-mediated signaling pathways; this is consistent with previous studies in which different stress models were used [13,36,47]. Several previous reports show common p66 Shc phosphorylation signaling pathways, indicating an independence of the stimulus and cell type. For example, incubation of human endothelial cell with oxidized LDL led to phosphorylation [36] of p66 Shc ; in MEFs, treatment with H 2 O 2 and UV induced phosphorylation [13] of p66 Shc ; and A549 and RAW 264.7 cells treated with Taxol, an antitumor drug, induced phosphorylation [48] of p66 Shc . Among these stimuli and cell types, the signaling pathways that induce p66 Shc phosphorylation first involve activating PKCb [13,36], followed by activation of other kinases [36,47,48] that directly induce phosphorylation of p66 Shc at Ser36 [36].
The role of PKCbII in modulating cardiac function also has been reported. Kong et al. [14] observed significant increases in PKCbII cell membrane translocation and phosphorylation after 30 min of LAD occlusion followed by 30 min reperfusion. Moreover, PKCb 2/2 , or its blockade with ruboxistaurin, reduced infarct size by 2.6 fold after 30 min regional ischemia and 48 h reperfusion in mice. Consistent with these findings, we found PKCbII was activated (specifically by its phosphorylation at Ser660) during IR (Fig. 6A). Furthermore, hispidin, when given before ischemia, reduced diastolic contracture (Fig. 7A) and infarct size (Fig. 7B). Although other studies also show that inhibition of PKCb activation is protective against cardiac IR injury, our study provides evidence that PKCbII modulates cardiac IR injury by triggering the activation and translocation of p66 Shc into mitochondria (Fig. 6B, C).

Summary, Conclusions and Limitations
We provide evidence that p66 Shc is activated and translocated from the cytosol into mitochondria in cardiac IR injury. Activation of p66 Shc occurred only during reperfusion via PKCbII activation. The magnitude of p66 Shc activation correlated with the degree of cell damage, so that the greatest activation/translocation of p66 Shc had the worst functional recovery, a higher incidence of VF, and a greater infarct size. Reversible blockade of complex I reduced the ROS level necessary to activate p66 Shc . These observations imply that excess ROS and altered redox state resulting from IR injury are single or combined factors that initiate p66 Shc activation and mitochondrial translocation.
Persistent activation of p66 Shc may lead to a vicious cycle of mitochondrial RIRR as a result of redox-sensitive activation of PKCbII. Therefore, preventing p66 Shc activation may reduce the feed-forward cycle of RIRR and lessen reperfusion injury. The findings that p66 Shc mediates only detrimental ROS production during reperfusion provides a novel opportunity for pharmacological interventions that would target this redox-signaling cascade leading to p66 Shc activation and translocation into mitochondria after the onset of cardiac IR injury. Overall, this study provides additional valuable insights into the possible mechanisms of how modulating electron transfer along the ETC can minimize deleterious ROS emission during cardiac IR injury as a means to improve cardiac function. A potential limitation is that attenuation of p66 Shc translocation into mitochondria by modulating electron transfer is less pronounced than the effect on functional recovery, which indicates that other factors in addition to the effects of p66 Shc on translocation exert a role on ROS emission during the progression of ischemia into reperfusion.