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(Circulation Research. 1996;79:137-146.)
© 1996 American Heart Association, Inc.

Articles

Calcium Preconditioning Elicits Strong Protection Against Ischemic Injury via Protein Kinase C Signaling Pathway

Hiroshi Miyawaki, Xiaobo Zhou, Muhammad Ashraf

the Department of Pathology and Laboratory Medicine, University of Cincinnati (Ohio) Medical Center.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We tested the hypothesis that elevation of [Ca²⁺]_i during Ca²⁺ preconditioning (CPC) is a strong activator of protein kinase C (PKC) and confers unique protection against ischemic injury. CPC consisted of three cycles of Ca²⁺ depletion (1 minute each) and Ca²⁺ repletion (5 minutes each). Langendorff-perfused rat hearts were subjected to 40 minutes of global ischemia followed by 30 minutes of reperfusion. Significant functional recovery and decreased lactate dehydrogenase release were observed in CPC hearts compared with ischemic control hearts. In addition, ATP contents were significantly higher and cell structure was better preserved in CPC hearts than in ischemic control hearts. Administration of chelerythrine, a specific PKC inhibitor, completely abolished the CPC-induced cardioprotection. In other groups, in which Ca²⁺ influx during CPC was inhibited with verapamil, amiloride, and low Na⁺ perfusion, cardioprotection was significantly reduced. The prominent increase in the membrane PKC activity after CPC was in agreement with immunolocalization of PKC- and PKC- in the cell membrane of CPC hearts. These results demonstrate that (1) a transient increase in [Ca²⁺]_i is a prominent feature of CPC and is a strong stimulus for the activation of PKC, (2) the elevation of [Ca²⁺]_i likely occurs via an L-type Ca²⁺ channel and Na⁺-Ca²⁺ exchanger, and (3) PKC plays a crucial role in the subcellular mechanisms of protection by CPC.

Key Words: preconditioning • ischemia • protein kinase C • intracellular calcium

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have recently demonstrated¹ that CPC (repetitive Ca²⁺ depletion and repletion for short duration) conferred cardioprotection against the lethal injury induced by the Ca²⁺ paradox. The Ca²⁺ paradox^{2 3 4} is characterized by irreversible cell damage resulting from reperfusion of the heart with a Ca²⁺-containing fluid after a longer period of Ca²⁺-free perfusion. In our previous study,¹ CPC reduced LDH release and cellular damage and preserved tissue ATP content in the rat hearts subjected to the Ca²⁺ paradox. The study suggested that the stress to the heart by CPC triggered cellular events that led to the protection against Ca²⁺-overload injury. We therefore hypothesized that CPC should likewise provide significant protection against myocardial ischemic injury.

Brief intermittent periods of ischemia reduce myocardial injury induced by subsequent prolonged ischemia. This phenomenon is known as ischemic preconditioning.⁵ The principal mechanisms underlying IPC are currently under intensive investigation. However, recently it has been proposed that the activation of PKC, which is known to be an important player in numerous intracellular signal-transduction pathways,^{6 7} plays a central role in the protection of IPC in rats^{8 9} and rabbits.^{10 11} Since sudden augmentation of contractility observed during CPC suggests a transient increase in [Ca²⁺]_i, it is possible that CPC effectively activates PKC, particularly Ca²⁺-dependent PKC isoforms. It is therefore likely that PKC also plays an important role in the cellular mechanism of CPC-induced protection.

The purpose of the present study was to test our hypotheses that (1) CPC triggers several subcellular Ca²⁺-dependent pathways, leading to protection against ischemic injury in the isolated rat heart; (2) stimulation, augmentation, and translocation of PKC activity are evoked by CPC; (3) PKC inhibition abolishes the beneficial effect of CPC on ischemic injury; and (4) the blockade of transient increase in [Ca²⁺]_i during CPC does not activate PKC and attenuates the protective effect of CPC.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Heart Preparation
Male Sprague-Dawley rats weighing 220 to 250 g were anesthetized by intraperitoneal injection of 30 mg/kg pentobarbital. After intraperitoneal injection of 500 U/kg heparin sodium, hearts were removed and retrogradely perfused through the aorta in a noncirculating Langendorff apparatus with KH buffer as previously described.^{1 12} KH buffer consisted of (mmol/L): NaCl 118, KCl 4.7, MgSO₄ 1.2, KH₂PO₄ 1.2, CaCl₂ 2.5, NaHCO₃ 25, and glucose 11. The buffer was saturated with 95% O₂-5% CO₂ (pH 7.4, 37°C) for 50 minutes. Hearts were perfused at a constant pressure of 80 mm Hg. Left ventricular pressure was measured by the method described by Grupp and Grupp.¹³ Briefly, a catheter was inserted through a pulmonary vein into the left atrium and advanced into the left ventricle, forced through the apex. The proximal end of the catheter was flanged to prevent it from slipping out of the ventricle. The distal end was connected through a larger-diameter catheter to a pressure transducer (Gould P23Db). The first derivative of left ventricular pressure (±dP/dt) was obtained from a differentiator (model 7P20, Grass Instrument Co). Heart rate, left ventricular pressure, and dP/dt were monitored on a Grass 7D polygraph. The hearts were maintained at 37°C during the ischemic period by immersion in a water-jacketed beaker containing perfusate. The coronary effluent was collected in a beaker, and the flow was determined volumetrically at the end of equilibration, during CPC and reperfusion.

Experimental Protocol
After equilibration, hearts were randomly divided into 10 groups using 2 protocols.

Protocol 1: CPC and Inhibition of PKC Activation
These experiments tested the hypothesis that CPC triggered the activation of PKC, which was responsible for protection against myocardial ischemic injury. To block the activation of PKC during CPC, chelerythrine chloride, a specific PKCI,14¹⁴ was used.

Group 1: Normal control
Hearts (n=5) were perfused for 90 minutes with KH buffer as a normal control for different experimental groups.

Group 2: Ischemic control
Hearts (n=6) were perfused with KH buffer for 20 minutes and then subjected to global ischemia for 40 minutes, followed by reperfusion for 30 minutes (I/R).

Group 3: CPC
Hearts (n=8) were perfused for three cycles of 1 minute each with Ca²⁺-free KH buffer. Each cycle was followed by 5 minutes of Ca²⁺-containing KH buffer, and then the hearts were subjected to the I/R sequence as in group 2.

Group 4: CPC and PKCI
The procedure was similar to that for group 3, except 100 µmol/L chelerythrine chloride, a specific inhibitor of PKC, was infused through the aorta at the rate of 0.2 mL/min during CPC (n=6). The dose of chelerythrine was chosen on the basis of a previous study by Speechly-Dick et al.⁸ Chelerythrine was dissolved directly in the buffer.

Group 5: PKCI
The procedure was similar to that for group 2, except 100 µmol/L chelerythrine was infused at a rate of 0.2 mL/min for 20 minutes before I/R (n=5).

Protocol 2: Blockade of Ca²⁺ Influx During CPC
These experiments tested the hypothesis that a transient increase in [Ca²⁺]_i that occurred during CPC activated PKC, which was responsible for the cardioprotection. The Ca²⁺ entry was blocked by (1) verapamil, an L-type Ca²⁺ channel blocker; (2) amiloride, a Na⁺-H⁺ exchanger inhibitor; and (3) low Na⁺ perfusion in a Ca²⁺-free phase during CPC.

Group 6: CPC and Ca²⁺ blocker
This group was similar to group 3, except that increasing doses of verapamil (0.2, 0.5, and 2 µmol/L), a Ca²⁺ channel blocker, were infused at a rate of 0.2 mL/min from 3 minutes before the first cycle of CPC to the end of the third cycle of CPC (n=3 to 6 for each subgroup). After a 5-minute washout by the normal perfusate, I/R was instituted.

Group 7: CPC and amiloride
Amiloride effectively inhibits the intracellular Na⁺ accumulation and Ca²⁺ uptake through the inhibition of Na⁺-H⁺ exchange linked to Na⁺-Ca²⁺ exchange in ischemic isolated rat heart.^{15 16} In this procedure, hearts were perfused with KH buffer containing 1 mmol/L amiloride during CPC (n=6). After a 5-minute washout period, hearts were subjected to I/R.

Group 8: Low Na⁺ perfusion during CPC
Ca²⁺ influx during CPC was reduced by lowering the [Na⁺] in the perfusate to 60 mmol/L. It has been shown that lowering [Na⁺]_o reduces Na⁺ influx and subsequently decreases Ca²⁺ entry modulated by Na⁺-Ca²⁺ exchange.^{17 18} In this procedure, hearts were perfused with Ca²⁺-free KH buffer containing low NaCl (60 mmol/L) during the Ca²⁺-depletion phase of the CPC protocol (n=6). To maintain the equimolarity of the perfusate, LiCl was added to the low-NaCl/KH buffer.

Group 9: Verapamil
The procedure was similar to group 2, except 2 µmol/L verapamil was infused at a rate of 0.2 mL/min for 15 minutes followed by 5 minutes of washout. After that, hearts were subjected to I/R (n=5).

Group 10: Amiloride
Hearts (n=5) were perfused with KH buffer containing 1 mmol/L amiloride for 15 minutes. After a 5-minute washout period, I/R was instituted.

Measurement of LDH
LDH, an indicator of myocardial tissue injury, was determined in coronary effluent. This enzyme was assayed by a coupled-enzyme spectrophotometric technique using a Sigma assay kit. Measurement of enzyme activity was based on oxidation of lactate and the rate of increase in absorbance at 340 nm. The accumulated amount of LDH released during 30 minutes of reperfusion was obtained by integrating the area underneath the individual time-course curve for 30 minutes of reperfusion.

Measurement of Tissue ATP
The heart was immediately frozen in liquid nitrogen and freeze-dried for 24 hours. Fifty to 100 mg of freeze-dried tissue was crushed in a precooled glass tube, and ATP was extracted with 5 mL of cold 6% trichloroacetic acid. The extract was analyzed for ATP by a spectrophotometric method.¹

Measurement of PKC Activity
At the end of CPC and pretreatment, hearts were excised, immediately frozen in liquid nitrogen, and stored at -70°C until use. Twenty to 30 mg of the freeze-dried heart tissue was used for analysis. Subsequent extraction was performed at 4°C. The left ventricles were dissected and minced. The minced tissue was homogenized with a Brinkmann Polytron homogenizer in 1.5 mL of buffer containing (mmol/L) Tris 50, EDTA 4, and mercaptoethanol 15. The homogenate was subjected to differential centrifugation at 1500g for 10 minutes. The supernatant was centrifuged at 100 000g for 1 hour and served as the cytosolic fraction. The pellet was resuspended in 1.5 mL of homogenizing buffer containing 0.3% Triton X-100, 50 mmol/L Tris, 4 mmol/L EDTA, and 15 mmol/L mercaptoethanol and incubated for 1 hour on ice. The homogenate was centrifuged at 100 000g for 1 hour, and the supernatant contained the membrane fraction. PKC activity was measured after incubation for 10 minutes at 30°C in a reaction mixture containing 5 mmol/L Tris, 10 mmol/L MgCl₂, 0.15 mol/L KCl, 0.2 mmol/L dithiothreitol, 0.2 mg/mL BSA, 2.0 mmol/L ATP, and 100 µmol/L PKC substrate peptide [Ser²⁵](19-31).¹⁹ The phosphorylation of the peptide as PKC activity was analyzed by absorbance at 430 nm in a Gilford spectrophotometer.^{20 21} The activity of PKC was expressed as nmol/mg dry weight.

Morphological Examination
Tissue from the midventricular wall was cut into 1.0-mm pieces, fixed in 2.5% buffered glutaraldehyde, postfixed in 1% buffered osmium tetroxide, and processed for transmission electron microscopy.¹ A semiquantitative estimate of cell damage was carried out on 1-µm-thick sections. Three randomly chosen blocks from each heart were examined for quantification of cell damage without prior knowledge of the treatment. Approximately 500 cells were analyzed in each heart, and cells were classified according to presence and degree of damage, as follows: (1) normal (compact myofibers with uniform staining of nucleoplasm, well-defined rows of mitochondria between the myofibrils, and no separation of opposing intercalated disks); (2) mild damage (same as above, except some vacuoles were present adjacent to the mitochondria); and (3) severe damage (reduced staining of cytoplasmic organelles, clumped chromatin material, wavy myofibers, and granularity of cytoplasm; cells with contraction band necrosis were added to this category).

Immunofluorescence Microscopy
Subcellular localization and translocation studies of PKC isoforms in hearts after CPC were performed by immunofluorescence staining and compared with those of control hearts. In these experiments, hearts were perfused as described above. Specimens were obtained at the end of CPC. Control specimens were obtained after 20 minutes of equilibration. Tissue of left ventricle was excised from the isolated heart and cut into five pieces. These pieces were embedded in OCT compound (Miles Laboratories) in an aluminum foil tube, rapidly frozen in liquid nitrogen, and stored at -70°C. Then the immunofluorescence microscopic study,⁹ using rabbit polyclonal isoform-specific anti-PKC antibodies (anti-PKC-, -ß₁, -ß₂, -, -, -, -, and - from Research and Diagnostic Antibodies), was performed by Dr Anirban Banerjee (University of Colorado Health Science Center). Sections were viewed and photographed with a microscope equipped with fluorescence optics (Axiskop with MC-100 camera, Zeiss).

Statistical Analysis
Data were expressed as mean±SEM. Group comparisons were done by analysis of variance with multiple comparisons or t test when appropriate. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ischemic Control Hearts
Ischemic control hearts showed a significant decrease in functional recovery on reperfusion (Fig 1). The recovery of +dP/dt, heart rate, and coronary flow in ischemic control hearts was 28.7±7.2%, 58.1±7.2%, and 51.1±7.7%, respectively. A multifold increase in LDH release was observed after reperfusion, and ATP levels were reduced to 6.6±1.8 µmol/g dry weight compared with the normal control value of 20.3±0.9 µmol/g dry weight (Fig 2).

View larger version (34K): [in this window] [in a new window]   Figure 1. Effect of CPC on the first derivative of left ventricular pressure (±dP/dt; A, B), coronary flow (C), and heart rate (D) (protocol 1). In the CPC group, more than 30% increase in dP/dt was observed in the first cycle of CPC. This change was not observed in the other groups. CPC hearts subjected to 40 minutes of global ischemia significantly preserved ±dP/dt, coronary flow, and heart rate compared with ischemic control hearts at 30 minutes after reperfusion. CPC hearts treated with chelerythrine chloride, a specific PKCI, showed deteriorated cardiac function similar to ischemic control hearts. C1, C2, and C3 indicate the number of Ca2+-depletion and -repletion cycles during CPC. Changes are expressed as percent of values at equilibration. *P<.05 vs ischemic control.

View larger version (25K): [in this window] [in a new window]   Figure 2. Effect of CPC on LDH release and tissue high-energy phosphates (ATP). LDH release is reduced and ATP content increased in hearts subjected to 40-minute ischemia and 30-minute reperfusion after CPC, compared with ischemic control hearts. Treatment with the PKCI chelerythrine during CPC completely blocked the protection. Values are mean±SEM. *P<.05 vs ischemic control.

The ultrastructure of the normal control hearts was well maintained (see the Table and Fig 3A). Glycogen was abundant, and nuclear chromatin material was uniformly dispersed. Mitochondria were intact. Severe cell damage was observed in the ischemic control hearts (Fig 3B). Glycogen was depleted, nuclear chromatin was markedly clumped, myofibrils were partly disrupted, mitochondria were severely swollen, and cristae were disrupted. The semiquantitative estimate of cell injury showed that only 6.9±3.4% of cells were normal; 11.4±3.8% and 81.3±6.6% of cells were mildly and severely damaged, respectively (Table).

View this table: [in this window] [in a new window]   Table 1. Semiquantitative Estimate of Morphological Damage After 30-Minute Reperfusion in 40-Minute Ischemic Hearts After Various Treatments

View larger version (149K): [in this window] [in a new window]   Figure 3. Transmission electron micrographs of hearts subjected to 40 minutes of global ischemia and 30 minutes of reperfusion after various treatments. A, Control nonischemic myocardial tissue. Nuclear chromatin material is dispersed, mitochondria are elongated, and myofibrils are relaxed. Glycogen is abundant (arrow). Original magnification x12 500. B, Ischemic control heart showing severe myocardial damage. Nuclear chromatin is markedly clumped, myofibrils are partly disrupted, mitochondria are severely swollen, and cristae are disrupted. Glycogen is reduced (arrow). Original magnification x16 000. C, CPC heart. Cellular damage was much less than in the ischemic control heart. Nuclear chromatin is dispersed, mitochondria are elongated but slightly swollen, and myofibrils are intact. Glycogen is abundant (arrow). Original magnification x15 000. D, CPC heart treated with chelerythrine. Nuclear chromatin is markedly clumped, and mitochondrial swelling and disruption are apparent. Several mitochondria contain electron-dense deposits (arrow). Glycogen is rarely observed. Original magnification x12 500. E, CPC heart treated with 2 µmol/L verapamil. Nuclear chromatin is clumped, mitochondria are swollen and partly disrupted; electron-dense deposits are not observed, however. Glycogen is depleted. Myofibrils are partly transformed into contraction bands. Original magnification x16 000. F, CPC heart treated with amiloride. Nuclear chromatin is clumped, mitochondria are swollen, and cristae are partly disrupted. Glycogen is depleted. Original magnification x15 000. N indicates nuclear chromatin; M, mitochondria; and CB, contraction bands.

CPC and Ischemia
After the first cycle of CPC, the hearts transiently exhibited a 30% increase in ±dP/dt and showed enhanced functional recovery after I/R (±dP/dt, 90.5±1.2%; heart rate, 81.9±3.1%; coronary flow, 77.9±5.3%), in contrast with ischemic control hearts (Fig 1). In CPC hearts, LDH release was significantly decreased (7.1±0.4 U/g) and ATP contents were markedly preserved (14.3±0.3 µmol/g dry weight) compared with ischemic control hearts (Fig 2).

The cellular structure was extremely well preserved in the CPC hearts: 60.9±3.9% of cells were normal; 21.2±3.5% and 15.9±1.4% were mildly and severely damaged, respectively. The degree of cell damage was much less than that in the ischemic control hearts (Table). At the electron microscopic level (Fig 3C), the normal structural features of myocytes were well preserved. Glycogen deposits were frequently observed, myofibrils were intact, and mitochondria were elongated but occasionally slightly swollen. Cells showing severe damage were not different from the ischemic control hearts.

Role of PKC in CPC
To determine whether the beneficial effects of CPC were mediated by PKC, we administered chelerythrine, a specific PKCI, during CPC. Treatment with chelerythrine during CPC abolished the beneficial effects of CPC (Figs 1 and 2). The cell damage (Fig 3D and Table) was similar to the ischemic control group.

Chelerythrine chloride itself did not affect the heart function, LDH release, ATP levels, or morphology compared with ischemic control hearts (Figs 1 and 2 and Table).

Blockade of Ca²⁺ Entry During CPC
To determine whether a transient increase in [Ca²⁺]_i during CPC triggers the second-messenger signaling pathways, leading to cardioprotection against ischemia, we attempted to reduce Ca²⁺ entry during CPC (protocol 2). In group 6, low doses of verapamil (0.2 and 0.5 µmol/L) did not influence the cardioprotection of CPC (Fig 4). The functional recovery was similar to that in CPC hearts. However, the treatment with a moderate dose of verapamil (2 µmol/L), which was associated with decrease in +dP/dt during CPC (Fig 4), attenuated the functional recovery (Fig 4). In hearts treated with 2 µmol/L verapamil, LDH release was significantly increased, to 16.3±0.8 U/g, and ATP content was reduced, to 8.8±0.4 µmol/g dry weight compared with values in CPC hearts (Fig 5). In these hearts, 24.3±1.6% of cells were normal and 19.5±2.5% and 56.8±0.4% were mildly and severely damaged, respectively (Table). Thus, there was less protection compared with CPC hearts. At the ultrastructural level (Fig 3E), nuclear chromatin material was clumped, mitochondria were swollen and partly disrupted, glycogen was depleted, and myofibrils were partly transformed into contraction bands. These data suggested that verapamil reduced the Ca²⁺ entry in a dose-dependent fashion via an L-type Ca²⁺ channel during CPC, resulting in less activation of Ca²⁺-dependent second-messenger signals.

View larger version (54K): [in this window] [in a new window]   Figure 4. Effect of blockade of Ca2+ entry during CPC on the first derivative of left ventricular pressure (+dP/dt), heart rate, and coronary flow (protocol 2). Administration of verapamil (VER) during CPC attenuated the beneficial effect of CPC in a dose-dependent manner. Since the values for 0.2 and 0.5 µmol/L verapamil were overlapping, the data were grouped together and reported as VER1. VER2 represents 2 µmol/L verapamil. Treatment with amiloride (AML) or low NaCl perfusion (low Na) during CPC also blunted the CPC-induced cardioprotection. Values are percent change from values at equilibration and are expressed as mean±SEM. *P<.05 vs ischemic control; #P<.05 vs CPC; **P<.05 vs CPC+VER1.

View larger version (27K): [in this window] [in a new window]   Figure 5. Effects of blockade of Ca2+ entry during CPC on LDH release and high-energy phosphates (ATP). LDH release is increased and ATP content decreased in CPC hearts treated with 2 µmol/L verapamil (VER2), 1 mmol/L amiloride (AML), and low NaCl perfusion (low Na+, 60 mmol/L) compared with CPC hearts without these pretreatments. VER1 indicates 0.2 and 0.5 µmol/L verapamil. Values are mean±SEM. *P<.05 vs ischemic control; #P<.05 vs CPC.

In groups 7 and 8, both amiloride and perfusion with low Na⁺ also blunted the CPC-induced increase of +dP/dt (Fig 4A) and resulted in less cardioprotection (Figs 3F, 4, and 5, and Table). Morphological changes in CPC hearts with low Na⁺ perfusion are the same as in the amiloride-treated CPC hearts and are not shown in Fig 3. To completely block Ca²⁺ entry through both the L-type Ca²⁺ channel and Na⁺-Ca²⁺ exchange, we attempted the combined pretreatment of verapamil and amiloride during CPC in a couple of experiments. The use of both agents resulted in severe ventricular dysfunction before sustained ischemia (data not provided). Pretreatment with verapamil alone or amiloride alone without CPC did not provide significant protection against the subsequent prolonged I/R (Figs 4 and 5), which was consistent with the previous findings that verapamil^{22 23} or amiloride^{16 24} showed a pronounced cardioprotective effect if they were continuously administered before sustained ischemia and through I/R.

It was noteworthy that neither verapamil nor lowering of [Na⁺]_i completely abolished the effect of CPC. Thus, it is clear that both the L-type Ca²⁺ channel and Na⁺-Ca²⁺ exchange contributed to the CPC-induced increase in [Ca²⁺]_i.

Distribution of PKC After CPC
We assessed the distribution of PKC after CPC. Translocation of PKC from the cytoplasm to the cell membrane has been recognized as an index of PKC activation. A prominent increase (8.6±0.8 nmol/mg dry weight) in PKC activity of the membrane fraction was observed in CPC hearts compared with control hearts (1.8±0.3 nmol/mg dry weight; Fig 6, right). On the other hand, chelerythrine completely blocked the CPC-induced increase in PKC activity of the membrane fraction. Treatment with 2 µmol/L verapamil, 1 mmol/L amiloride, and low Na⁺ perfusion significantly reduced PKC activity of the membrane fraction in CPC hearts (Fig 6, right). There were no significant differences among the groups in PKC activity of the cytosolic fraction (Fig 6, left).

View larger version (24K): [in this window] [in a new window]   Figure 6. Effect of CPC and various treatments on PKC activity. Left panel: There were no significant differences in PKC activity of the cytosolic fraction. Right panel: CPC promoted a prominent increase in PKC activity of the membrane fraction. Treatment with chelerythrine, a PKCI, during CPC completely abolished the effect of CPC. Hearts subjected to the blockade of Ca2+ entry during CPC showed marked reduction in PKC activity of the membrane fraction compared with CPC hearts. VER2 indicates 2 µmol/L verapamil; AML, 1 mmol/L amiloride; and low Na+, 60 mmol/L NaCl. Values are mean±SEM of five experiments. *P<.05 vs control, CPC+PKCI, and PKCI; #P<.05 vs CPC.

Immunofluorescence microscopic study using isoform-specific anti-PKC antibodies strengthened our hypothesis on the Ca²⁺-mediated activation of PKC during CPC. Fig 7 demonstrates that both PKC- and PKC- were translocated to the plasma membrane of several hearts after CPC. The cell membranes were deeply stained with the isoform-specific antibodies. The controls showed diffuse staining or lack of staining. The visualization of the other PKC isoforms was not as clear as that of the and isoforms.

View larger version (121K): [in this window] [in a new window]   Figure 7. Photomicrographs showing immunofluorescent staining for PKC isoforms. A and C, Control hearts show diffuse cytoplasmic and perinuclear distribution of PKC- and -. B and D, CPC hearts show a prominent translocation of PKC- and - in the cell membrane. Magnification x63.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major new findings of the present study are: (1) CPC initiates several subcellular pathways, leading to strong cardioprotection against ischemia, (2) a transient elevation of [Ca²⁺]_i during CPC triggers the activation of Ca²⁺-dependent PKC, and (3) the CPC-induced protection is mediated by PKC activation. It is generally believed that a major Ca²⁺ influx causes pathological changes in I/R injury.^{15 16 25 26 27 28 29} Sustained increase in [Ca²⁺]_i after prolonged ischemia has been postulated to activate a proteolytic process by Ca²⁺-dependent proteases, which cause irreversible plasma membrane damage.^{30 31} However, it is intriguing to note that a transient and mild increase in [Ca²⁺]_i that occurs during CPC confers cardioprotection against ischemia (this study) or Ca²⁺-overload injury.¹

Ca²⁺-Dependent Activation of PKC in CPC
Recently, it has been reported by several studies that PKC activation is essential for the protection induced by IPC.^{8 9 10 11} In this study, we proposed that a brief Ca²⁺ loading during CPC could effectively activate PKC, especially Ca²⁺-dependent isoforms of PKC (conventional PKC). CPC is characterized by transient stress induced by repetitive Ca²⁺ depletion and repletion of short duration. During the Ca²⁺ repletion phase of CPC, the contractility of the hearts was augmented by 30% (Fig 1). Although we did not directly measure [Ca²⁺]_i during CPC, this phenomenon strongly suggested a rise in [Ca²⁺]_i, which was ascribed to the increased Ca²⁺ influx from the extracellular space through the Na⁺-Ca²⁺ exchanger and voltage-dependent Ca²⁺ channel and to Ca²⁺-induced Ca²⁺ release from the SR.³¹ The participation of the L-type Ca²⁺ channel and Na⁺-Ca²⁺ exchanger in Ca²⁺ mobilization during CPC was supported by our data (Figs 4, 5, and 6). In addition to direct [Ca²⁺]_i-dependent activation of conventional PKC,^{32 33} the increase in [Ca²⁺]_i may activate PKC via the phosphatidylinositol cycle, through the activation of G protein^{34 35} linked to Ca²⁺-dependent phospholipase C.^{34 36} As a consequence of phospholipase C activation, diacylglycerol is produced from phosphatidylcholine, and a subsequent sustained increase in the amount of diacylglycerol maintains the activation of PKC.⁷

In the present study, the measurement of PKC activity (Fig 6) confirmed the activation and translocation of PKC by CPC. Moreover, the immunofluorescent microscopy (Fig 7) strongly supported the biochemical findings that CPC caused the translocation of a Ca²⁺-independent isoform of PKC (novel PKC), PKC-, as well as a conventional PKC, PKC-. Banerjee's group⁹ suggested that PKC- activation after brief ischemia and ₁-adrenergic stimulation was involved in preconditioning. It remains to be determined whether different PKC isoforms independently act in preconditioning and what roles the individual isoforms play in preconditioning. However, it is possible that CPC shows its strong cardioprotection through the activation of both Ca²⁺-dependent and Ca²⁺-independent PKC isoforms.

Possible Mechanisms of Cardioprotection by PKC
Although no precise mechanism of cardioprotection by PKC is known at present, some subcellular mechanisms for PKC-dependent modulation and preservation of contractile function³⁷ may be suggested, as below. It has been reported that activated PKC directly phosphorylates contractile proteins such as troponin I^{38 39} and myosin light chain 2 .^{40 41} Phosphorylation of troponin I may cause a negative inotropic response mediated by reduced myofibrillar actomyosin Mg²⁺ ATPase activity,³⁹ while phosphorylation of myosin light chain 2 leads to stimulation of myofibrillar actomyosin Mg²⁺ ATPase activity followed by exertion of a positive inotropism. The cooperation of PKC with myosin light chain kinase increases the sensitivity of contractile protein to Ca²⁺.⁴⁰ The augmentation of myofilament sensitivity to Ca²⁺ may be involved with intracellular alkalization induced by the activation of PKC.⁴² PKC stimulates Ca²⁺ uptake from the cytosol by the SR through activation of SR Ca²⁺-Mg²⁺ ATPase as a result of the phosphorylation of phospholamban.⁴³ Activated PKC inhibits the L-type Ca²⁺ channel^{44 45 46} and decreases the Ca²⁺ influx. Taken together, these findings lead us to propose that PKC attenuates the deleterious increase in [Ca²⁺]_i that occurs during prolonged ischemia and after reperfusion by enhancing the Ca²⁺ uptake by the SR and depressing Ca²⁺ influx through inhibiting the L-type Ca²⁺ channel and modifying Na⁺-Ca²⁺ exchange⁴⁷ and that PKC also preserves myocardial function by augmenting myofibrillar sensitivity to Ca²⁺ and modulating contractile proteins. These plausible mechanisms have been schematically represented in Fig 8.

View larger version (48K): [in this window] [in a new window]   Figure 8. Schematic representation of mechanisms of CPC. Cardioprotection is provided by both Ca2+- and adenosine-dependent mechanisms. Adenosine released during CPC activates adenosine A1 receptors, resulting in opening of K+-ATP channels through Gi protein. A transient rise of Ca2+ triggers direct activation of Ca2+-dependent isoforms of PKC and phospholipase C (PLC), which ultimately causes release of diacylglycerol (DAG) and 1,4,5-inositol triphosphate (IP3). DAG is known to stimulate PKC. ADC indicates adenylyl cyclase; Gq, Gq protein; and PKA, protein kinase A.

[Ca ²⁺]_i Increase During Preconditioning Is a Potential Common Factor of IPC and CPC
It is likely that the increment of [Ca²⁺]_i during the preconditioning period triggers the protection initiated by IPC. Generally, the protective effect of IPC is induced by 5 minutes or less of ischemia followed by 5 to 10 minutes of reperfusion.⁴⁸ Several investigators^{27 49 50} have reported the rapid increase in [Ca²⁺]_i during brief ischemia (15 minutes). Kihara et al²⁷ observed the gradual increment of [Ca²⁺]_i during 3 minutes of global ischemia in the isolated ferret heart. Mohabir et al⁵⁰ have shown the biphasic increase in [Ca²⁺]_i during 15 minutes of global ischemia in the isolated rabbit heart. Thus, brief ischemia in IPC could evoke an increase in [Ca²⁺]_i, which may be an important common event in both IPC and CPC. This hypothesis is supported by our data that blockade of Ca²⁺ entry by verapamil, amiloride, or low Na⁺ perfusion during CPC attenuated the CPC-induced protection against ischemia.

Recent studies^{9 51 52} have indicated that both ₁-adrenergic stimulation and transient ischemia precondition the isolated rat heart through activation of PKC. The stimulation of the ₁-adrenoceptor increases cytosolic free Ca²⁺⁵³ and shows a [Ca²⁺]_i-dependent enhancement of PKC activity⁵⁴ in isolated cardiac myocytes. Keeping in view the role of [Ca²⁺]_i during preconditioning, we speculate that ₁-adrenergic-mediated preconditioning is also affected by the [Ca²⁺]_i-dependent PKC pathway.

Other Mechanisms of CPC
The other mechanism of CPC is the increase of adenosine release during CPC, which triggers various signaling pathways, leading to protection against ischemic damage. In our previous study, we demonstrated that the protection by CPC against the Ca²⁺ paradox results from G_i protein-modulated adenosine A₁ receptor stimulation.¹ Adenosine release was increased 11-fold by the end of CPC, the adenosine A₁ receptor agonist (R)-phenylisopropyl adenosine mimicked CPC, and the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline blocked the beneficial effects of CPC in that study.¹ It is well known that increased adenosine formation and release yield beneficial effects by activating the adenosine A₁ receptor or through antiadrenergic effects.⁵⁵ Increased responsiveness of G_i protein⁵⁶ could promote this mechanism. Furthermore, it has recently been reported that PKC activated by -adrenergic stimulation increases ecto-5'-nucleotidase and adenosine release.⁵² Second, the activation of A₁ receptors coupled to the ATP-sensitive K⁺ channel through G protein may cause the opening of the ATP-sensitive K⁺ channel.⁵⁷ This may shorten the action-potential duration, followed by a decrease in Ca²⁺ influx, and lead to a reduction in cellular energy consumption and preservation of ATP and myocardial function (Fig 8).

In summary, our results indicate that CPC offers strong protection against ischemic injury through the activation of PKC and that the transient increase in [Ca²⁺]_i during CPC is a trigger for the induction of CPC. CPC provides an important experimental model to study the role of Ca²⁺ in initiating multiple intracellular signaling pathways leading to attenuation of ischemic injury.

	Selected Abbreviations and Acronyms

 

CPC = calcium preconditioning Gi protein = inhibitory G protein I/R = ischemia/reperfusion IPC = ischemic preconditioning KH = Krebs-Henseleit LDH = lactate dehydrogenase PKC = protein kinase C PKCI = PKC inhibitor SR = sarcoplasmic reticulum

	Acknowledgments

 
This study was supported in part by grants HL-23597 and HL-44358 from the National Institutes of Health, Heart, Lung, and Blood Institute. We thank Dr Anirban Banerjee (Department of Surgery, University of Colorado Health Science Center) for immunocytochemistry and Gulnar Durrani for her technical assistance.

	Footnotes

 
Correspondence to Muhammad Ashraf, PhD, Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, 231 Bethesda Ave, Cincinnati, OH 45267-0529.

Received November 8, 1995; accepted March 29, 1996.

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