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GROWTH, DIFFERENTIATION, AND APOPTOSIS
1Division of Cardiovascular Disease, Department of Medicine and 2Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama
Submitted 20 July 2007 ; accepted in final form 7 January 2008
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ABSTRACT |
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-fodrin was increased approximately threefold in the transgenic group following I/R compared with WT (P < 0.05); this was significantly attenuated by SKF-96365. The calpain inhibitor PD-150606 (25 µM) attenuated the increase in both -fodrin cleavage and apoptosis in the TPRC3 group. Increased TRPC3 expression also increased sensitivity to Ca2+ overload stress, but it did not affect the response to TNF--induced apoptosis. These results suggest that CCE mediated via TRPC may play a role in cardiomyocyte apoptosis following I/R due, at least in part, to increased calpain activation.
capacitative calcium entry; calpain; tumor necrosis factor-; transient receptor potential
The specific proteins responsible for facilitating CCE have yet to be fully characterized; however, there is growing evidence that members of the transient receptor potential (TRP) protein superfamily of proteins may be involved in regulating CCE (14). Mammalian TRP channels are divided into six subfamilies, and the TRPC (canonical) family in particular has been implicated in contributing to cellular Ca2+ homeostasis (9, 14, 23, 24, 43). There are seven members of the TRPC family (TRPC1-7), six of which (TRPC1-6) have been shown, at least by RT-PCR, to be present in the heart (9). Structurally, TRP channels consist of six transmembrane domains containing a putative Ca2+ pore region between the fifth and sixth domains; TRPC channels have ankyrin repeat domains in their NH2 terminus, which may mediate protein-protein interactions (23). Prototypical activation of TRPC channels occurs in response to agonist stimulation, resulting in the generation of diacylglycerol and inositol 1,4,5-trisphosphate (IP3). Subsequent activation of the endo/sarcoplasmic reticulum IP3 receptor can mediate depletion of Ca2+ from internal stores, thereby triggering Ca2+ entry across the sarcolemma via TRPCs. Independent of store depletion, TRPC activity can be regulated by an increase in diacylglycerol; alternatively, IP3 receptor bound to IP3 may activate TRPC via direct interaction (23).
As noted above, until relatively recently, CCE was considered to be primarily a feature of nonexcitable cells, and, consequently, the majority of data supporting a role for TRPCs in mediating CCE is also from nonexcitable cells. However, several recent studies have provided evidence that TRPCs may also be functional in the heart (18, 28, 30). Ohba et al. (30) demonstrated that hypertrophic stimuli increased TRPC1 expression both in the intact heart and in isolated cardiomyocytes (30). They also showed that, in isolated cardiomyocytes, the increase in TRPC1 expression was associated with increased CCE. The functional significance of TPRC1 was confirmed by using short interfering RNA (siRNA) techniques to prevent agonist-induced increase in TRPC1 expression, which attenuated both the hypertrophic response and CCE (30). Conversely, Nakayama et al. (28) demonstrated that overexpression of TRPC3 in cardiomyocytes increased CCE and that this was associated with increased cardiac hypertrophy in response to neuroendocrine agonists or pressure overload stimulation. Kuwahara et al. (18) also reported that cardiac-specific overexpression of TRPC6 increased the propensity for cardiac hypertrophy and heart failure; however, they did not determine whether this was associated with an alteration in CCE.
Therefore, in light of the growing evidence linking TRPC proteins to CCE in cardiomyocytes, combined with our finding that CCE inhibition attenuated cardiac injury in response to calcium overload (20), we tested the hypothesis that increased expression of TRPC3 in cardiomyocytes would result in increased injury due to I/R. We found that TRPC3 overexpression increased apoptosis and calpain-mediated proteolysis resulting from I/R injury and also increased sensitivity to Ca2+ overload; however, TRPC3 had no effect on the response to TNF--induced apoptosis. These data strongly suggest that CCE mediated via TRPC may contribute to Ca2+-induced cardiomyocyte apoptosis resulting from I/R.
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MATERIALS AND METHODS |
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Animals All animal experiments were approved by the University of Alabama at Birmingham (UAB) Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals, published by National Institutes of Health (publication no. 85-23, 1996). Mice overexpressing TRPC3 in the heart were a kind gift from Dr. Jeffrey D. Molkentin (University of Cincinnati) and have been characterized in detail elsewhere (28); the mice used in the present study were derived from line 23 described in the original study (28). All animals for the present study were bred at UAB and were genotyped prior to use. Normal, nontransgenic [i.e., wild-type (WT)] littermates were used as controls.
Cardiomyocytes Isolation
Male mice, 2–4 mo of age (25–40 g) were heparinized [5000 U/kg intraperitoneally (ip)] 20 min before being anesthetized with ketamine (100 mg/kg ip). Hearts were rapidly excised and arrested in ice-cold, Ca2+-free perfusion buffer consisting of (in mM) 113 NaCl, 4.7 KCl, 0.6 KH2PO4, 0.6 Na2HPO4, 1.2 MgSO4·7H2O, 0.032 phenol red, 12 NaHCO3, 10 KHCO3, 10 HEPES, 30 taurine, 10 2,3-butanedione monoxime, and 5.5 glucose, pH 7.46. The aorta was cannulated, and the heart was perfused retrogradely with Ca2+-free perfusion buffer at a constant flow of 3 ml/min at 37°C for 4 min, followed by the same perfusion buffer containing 12.5 µM CaCl2 and 0.4 mg/ml collagenase type 2 (Worthington). After 15–25 min of perfusion with collagenase-containing buffer, the heart appeared swollen, pale, and flaccid, at which time the ventricles were removed and finely minced. Dispersed myocytes were filtered through a 100-µm mesh and allowed to sediment by gravity for 10 min. The supernatant was removed and centrifuged for 1 min at 180 g. The pellet was resuspended and combined with the original sedimented myocytes in perfusion buffer containing 5% bovine calf serum and 12.5 µM CaCl2. The calcium concentration was increased gradually from 12.5 µM to 1 mM in 5 steps over 
20 min. Freshly isolated cardiomyocytes were kept in 2% CO2 incubator at 37°C. All experiments were performed at least 1 h after myocyte isolation.
Experimental Protocols Modified cell-pelleting model of ischemia. We used a cell-pelleting model to assess hypoxia- and reoxygenation-induced cell death, as described in detail by Yamawaki et al. (39). Briefly, an aliquot of cardiomyocytes suspended in MEM (0.5 ml) was placed into a microcentrifuge tube and centrifuged at 80 g for 60 s. After centrifugation, 0.2 ml of the supernatant was removed and replaced by 0.2 ml of mineral oil, which was layered on top of the cell pellet to prevent diffusion of oxygen into the sample. The cell pellet was maintained at 37°C for 90 min, at which time cells were either assessed for apoptosis and necrosis as described below or were reoxygenated for 3 h by being resuspended in fresh MEM. In all experiments, additional aliquots of cardiomyocytes were incubated under time-controlled normoxic conditions.
Calcium-induced cell death. To determine the effects of TRPC3 expression on the response to calcium-induced cell death, cardiomyocytes were placed on coverslips and preincubated with 5 µM thapsigargin for 5 min, followed by the addition of 2.5 mM extracellular calcium. The cells were imaged on an Olympus I X 70 inverted microscope through a x40 Uplan APO objective. Rounded cells (defined as cells in which the ratio between the length and width was <2) were consider irreversibly injured or dead (5, 26).
TNF--induced apoptosis.
Cardiomyocytes were incubated with 10 ng/ml TNF- in serum-free MEM media (2). Apoptosis was assessed by annexin V-propidium iodide staining 2 h or 18 h after treatment with TNF-.
Assessment of Cell Viability, Apoptosis, and Necrosis Several different methods were used to assess cell viability, apoptosis, and necrosis.
Cell morphology. Healthy viable adult cardiomyocytes typically exhibit rod-shaped morphology, whereas nonviable cells are usually rounded in nature; therefore the percentage of rod-shaped cells was used as an indicator of cell viability.
Annexin V and propidium iodide staining. Myocytes were stained with fluorescein isothiocyanate (FITC), annexin V, and propidium iodide, as per the manufacturer's directions (Vybrant Apoptosis Assay Kit 2, Molecular Probes). The cells were visualized under a fluorescence microscope and were counted regardless of morphology.
Cells not binding FITC-annexin V and excluding propidium iodide were classified as annexin V-negative (29, 33) and were deemed viable. Myocytes that bound FITC-annexin V [excitation wavelength (
ex) = 488 nm and emission wavelength (em) = 520 nm] but excluded propidium iodide (ex = 540 nm and em = 630 nm) were deemed apoptotic. Myocytes permeant to propidium iodide (regardless of whether or not they bound FITC-annexin V) were deemed necrotic (29, 33). For quantification, 300 randomly distributed cells were counted in each experiment (29), and the percentage of the total number of apoptotic and necrotic cells was determined.
Lactate dehydrogenase release. As previously described (7), necrosis was assessed by determining the release of lactate dehydrogenase (LDH) in the medium with an LDH assay kit (Sigma). Briefly, the percentage of LDH release was calculated by the ratio of the released LDH into the media to the total LDH (release plus cellular content).
DNA fragmentation. The Cell Death Detection ELISA plus kit (Roche Molecular Biochemicals, Mannheim, Germany) was used as another indicator of apoptosis. In this assay, internucleosomal DNA fragmentation was quantitatively assayed by antibody-mediated capture and detection of cytoplasmic mononucleosome- and oligonucleosome-associated histone-DNA complexes. Briefly, after centrifugation (200 g), cardiomyocytes (1 x 104 cells in each well) were resuspended in 200 µl of the lysis buffer supplied by the manufacturer and incubated for 30 min at room temperature. After pelleting of the nuclei (200 g, 10 min), 20 µl of the supernatant (cytoplasmic fraction) was used in the enzyme-linked immunosorbent assay (ELISA) following the manufacturer's standard protocol. Following incubation with peroxidase substrate for 5 min, absorbance at 405 nm and 490 nm (reference wavelength) was determined with a microplate reader (Bio-Tec Instruments, Winooski, VT). Signals in wells containing the substrate only were subtracted as background (19).
Western Blot Analysis
Proteins were separated by electrophoresis on 6% or 8% SDS gel, transferred onto a polyvinylidenedifluoride membrane, and immunoblotted with antibodies against β-actin (1:20,000, Abcam), -fodrin (1:2,000, Millipore), TRPC1, and TRPC3 (1:200, Alomone Labs). The immunoblots were developed with chemiluminescence (Pierce), and the signal was recorded on X-ray film. Densitometry analysis was performed on the entire lane of each sample using Labworks Analysis Software (UVP).
Data Analysis All data are presented as means ± SE from three to six separate experiments, in which each experiment represents cells isolated from a single heart. Statistical analysis was performed by using either an unpaired t-test or one-way analysis of variance (ANOVA) followed by Dunnett's multiple-comparisons test as appropriate. Statistically significant differences between groups were defined as P < 0.05 and are indicated in the legends to the figures.
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RESULTS |
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50% higher in the TRPC3 group (32 ± 1% vs. 21 ± 3%; P < 0.05; Figs. 2B and 3B) compared with WT. Necrosis as indicated by propidium iodide-positive cells was not different between groups at any time point (Figs. 2B and 3C).
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3.5–4.5%) and necrosis (from 15–18%); however, there were no differences between WT and TRPC3 groups at any time point during time-controlled normoxic incubations. The apparent decrease in necrosis from the end of ischemia to the end of I/R (Fig. 2C) is presumably a consequence of a loss of cells early during reperfusion; however, since total cell number was not assessed at each time point, this cannot be confirmed. It is also possible, although we believe unlikely, that the higher necrosis in the ischemia-only cells was an artifact associated with the preparation of these cells for annexin V and propidium iodide staining. Apoptosis was also assessed at the end of I/R by quantifying internucleosomal DNA fragmentation. Consistent with the annexin V assays, apoptosis was significantly increased in the TRPC3 group compared with WT controls (4.3 ± 0.2% vs. 2.7 ± 0.3%; P < 0.05; n = 6). As an alternative index of necrosis, LDH released during I/R was measured in both groups and quantified as a percentage of total LDH. Consistent with the propidium iodide results (Fig. 3C), there was no significant difference between TRPC3 and WT groups (27 ± 1% vs. 26 ± 3%; n = 6).
Effect of L-type channel inhibitor and CCE inhibitor on TRPC3-induced apoptosis. Treatment of TRPC3 cells with 0.5 µM SKF-96365, an inhibitor of CCE, significantly improved cellular viability following I/R (54 ± 4% vs. 42 ± 5%; P < 0.05; n = 6), to a level similar to that seen in WT cells following I/R (Fig. 3A). SKF-96365 also significantly decreased apoptosis in TRPC3 cells (15 ± 4% vs. 32 ± 1.4%; P < 0.05; n = 6) to levels similar to that seen in WT cells (Fig. 4). Similar to SKF-96365, PD-150606, an inhibitor of calpain, also attenuated apoptosis in the TRPC3 group (Fig. 4). However, the L-type Ca2+ channel inhibitor verapamil (10 µM) had no significant effect on either viability (data not shown) or apoptosis in TRPC3 cells (Fig. 4). The untreated WT and TRPC3 data in Fig. 4 are from the same experiments as those shown in Fig. 3B; however, it should be noted that the experiments in Fig. 4 were performed simultaneously with and on cells from the same isolations as those used in Fig. 3, thereby permitting direct comparisons.
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-fodrin is a well-characterized substrate for calpain, and increased calpain activity has been shown to lead to the formation of 140/150 kDa cleavage fragment of -fodrin (37, 40, 41). In Fig. 5A, it can be seen that in the TRPC3 group, there is significant increase in -fodrin cleavage following I/R compared with WT group (P < 0.05). The increase in -fodrin cleavage in the TRPC3 cells was attenuated by the CCE inhibitor SKF-96365 and the calpain inhibitor PD-150606 (Fig. 5B).
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-induced apoptosis.
To determine whether TRPC3 overexpression increased sensitivity to other apoptotic stimuli, cardiomyocytes isolated from WT and TRPC3 mice were incubated with 10 ng/ml TNF- in serum-free media for 2 h and 18 h. Apoptosis, assessed by annexin V-propidium iodide staining, increased in both groups following TNF- treatment; however, in contrast to I/R, there was no difference in apoptosis between the two groups (Fig. 7). In time-controlled normoxic incubations, there were no differences in apoptosis or the percentage of rod-shaped cells between WT or TRPC3 groups at any time point (data not shown). There was also no increase in either parameter at 2 or 18 h compared with baseline in either WT or TRPC3 group (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-mediated apoptosis. This is consistent with our report that CCE inhibition markedly attenuated Ca2+ overload injury in the intact heart (20) and suggests that in addition to playing a role in regulating cardiomyocyte hypertrophy, TRPC-mediated CCE also contributes to cardiomyocyte apoptosis. These data provide further support for both TRPC and CCE in regulating the response of cardiomyocytes to a range of pathophysiological stimuli. In vitro studies showed that CCE played a critical role in mediating the hypertrophic response to IP3-generating agonists such as angiotensin and phenylephrine in neonatal cardiomyocytes (11). Hunton et al. (12) also demonstrated that the increase in cytosolic Ca2+ induced by these agonists was clearly independent of Ca2+-entry mediated via L-type Ca2+ channels and the reverse mode of the Na+/Ca2+ exchanger. Further evidence, demonstrating a physiological role for CCE in the heart, was provided by Ohba et al. (30), who showed that TRPC1 expression increased in the intact heart following pressure overload and in isolated cardiomyocytes in response to hypertrophic stimuli; the latter was associated with increased CCE (30). Consistent with the study by Ohba et al. (30), overexpression of TRPC3 in the mouse heart enhanced CCE at the cardiomyocyte level and was associated with increased hypertrophy in response to either pressure overload or neuroendocrine agonists in vivo (28).
Although a number of Ca2+ entry pathways have been implicated in mediating cardiomyocyte Ca2+ overload following I/R, including the Na+/Ca2+ exchanger and the L-type Ca2+ channel (4, 8, 32), there is still considerable controversy as to which pathways are critical in mediating this process. In cardiomyocytes, angiotensin- and phenylephrine-induced CCE is inhibited by glucosamine and SKF-96365 (12, 27); glucosamine also protected the isolated perfused heart from calcium overload induced by the calcium paradox (20). We have also shown that glucosamine improves recovery following I/R, including lowering end-diastolic pressure (20) and decreasing Ca2+-mediated proteolysis, consistent with attenuation of I/R-induced increase in cytosolic Ca2+. Given that glucosamine inhibited CCE in isolated cardiomyocytes (12, 27), these studies raised the possibility that Ca2+ entry mediated via CCE pathways may contribute to injury resulting from I/R. Consistent with this notion, we found that following simulated I/R, cardiomyocytes overexpressing TRPC3 had increased levels of apoptosis assessed by both annexin V staining (Fig. 3) and DNA fragmentation, as well as decreased viability as indicated by the percentage of rod-shaped cells. Interestingly, however, TRPC3 overexpression did not contribute to increased necrosis as indicated by either LDH release or propidium iodide/ annexin V staining (Fig. 3).
Additional evidence for a role of TRPCs in regulating cell death has been described in other cell types. For example, Marasa et al. (21) reported that increased TRPC1 expression sensitized intestinal epithelial cells to apoptosis as a result of increased Ca2+ influx. It is also worth noting that oxidative stress has been shown to activate TRPC3 and TRPC4 in endothelial cells (23) and that another submember of TRP channels, TRPM, has been reported to contribute to oxidative stress-induced cell death (23). However, in contrast to these reports suggesting that TRPCs contribute to increased cell death, Jia et al. (16) showed that TPRC3 and TRPC6 played a role in promoting neuronal survival in response to serum deprivation. Taken together with our results, these studies support the notion that TRPCs play a role in regulating cell survival; however, whether they contribute to cell death or survival may be both cell and stress specific.
Further supporting a role of Ca2+ via CCE in contributing to the increase in apoptosis in the TRPC3 group, we also found that the CCE inhibitor SKF-96365 attenuated the increase in apoptosis associated with TRPC3 overexpression, whereas the L-type Ca2+ channel inhibitor verapamil had no such effect (Fig. 4). Elevated intracellular Ca2+ levels activate numerous Ca2+-regulated enzymes, including protein kinases, protein phosphatases, phospholipases, NO synthase, Ca2+/calmodulin-dependent protein kinase II, and the cysteine protease calpain (42). There is increasing evidence that calpain plays a major role in postischemic injury (15, 36), in part because of proteolysis of structural proteins (10, 11) including the cytoskeletal protein -fodrin (37, 40, 41). We found that in WT cardiomyocytes, I/R did not significantly increase cleavage of -fodrin; in contrast, TRPC3 overexpression was associated with an approximately threefold increase in -fodrin cleavage, consistent with increased calpain activity (Fig. 5A). The calpain inhibitor PD-150606 attenuated both -fodrin cleavage and apoptosis in cardiomyocytes overexpressing TRPC3 (Figs. 4 and 5B); furthermore, consistent with its effect on apoptosis, SKF-96365 markedly attenuated the I/R-induced increase in -fodrin cleavage in the TRPC3 group (Fig. 5B). Taken together, these data support the concept that in cardiomyocytes overexpressing TRPC3, Ca2+ entry via CCE contributes to increased apoptosis, at least in part by calpain activation; however, whereas there are considerable data demonstrating that -fodrin cleavage is calpain specific (37, 40, 41), a direct measure of calpain activity would have further substantiated this conclusion. Nakayama et al. (28) have shown that TRPC3 overexpression was also associated with increased calcineurin activity; consequently, it is possible that calcineurin inhibition may also attenuate the increased apoptosis seen in the TRPC3 group. It should be noted that because we did not evaluate the effects of SKF-96365 or PD-150606 in WT cells, we cannot comment about the potential role of CCE in contributing to cardiomyocyte apoptosis where TRPC3 levels are not increased. However, in preliminary experiments in the normal intact perfused heart, SKF-96365 markedly attenuated tissue injury resulting from the calcium paradox (R. B. Marchase and J. C. Chatham, unpublished observations), suggesting that CCE may contribute to calcium-mediated cell death in the normal intact heart.
I/R is a multifactorial process that includes intracellular acidosis and energy depletion, which may contribute to both necrosis and apoptosis either directly or via altered Ca2+ homeostasis, mediated via Ca2+ entry pathways such as the Na+/Ca2+ exchanger. Therefore, we asked whether TRPC3 overexpression also increased sensitivity to a specific Ca2+ overload stress. Cells were first exposed to thapsigargin (5 µM) in the absence of extracellular Ca2+ followed by the addition of 2.5 mM extracellular Ca2+; this is a protocol that was used previously to increase CCE (12, 27). We found that, in the absence of extracellular Ca2+, cardiomyocytes maintained a normal rod-shaped morphology; however, the addition of extracellular Ca2+ resulted in a loss of viability as indicated by rounding of the cells, which was markedly accelerated in cells with increased TRPC3 expression. In contrast, TRPC3 overexpression had no effect on the response of cardiomyocytes to TNF-, which induces apoptosis via a Ca2+-independent pathway by means of the TNF type-1 receptor and Fas activation (22) (Fig. 7). Thus, the increased sensitivity of TRPC3 to I/R and Ca2+ overload was not a consequence of a general decreased tolerance to stress. However, TNF- has been shown to increase TRPC3 expression in smooth muscle cells (38). Since we did not determine in the present study whether TNF- affected TRPC3 levels, we cannot rule out the possibility that a preferential increase in TRPC3 expression in TNF--treated WT cells could mask potential differences in the response of WT and TRPC3 cardiomyocytes to TNF-.
The role of CCE and TRPC in mediating cardiomyocyte function remains somewhat controversial, in part due to the fact that the predominant models describing Ca2+ handling in the heart are focused on understanding the regulation of excitation-contraction coupling (3). The importance of Ca2+ as a second messenger regulating a diverse array of cellular functions, including activation of gene transcription and cell growth, initiation of apoptosis, and necrosis (6), raises the fundamental question as to how the cardiomyocyte distinguishes between the fluctuations in Ca2+ that occur in response contraction and relaxation from calcium signals (25). One characteristic of CCE is that it leads to a low-amplitude, sustained elevation in cytosolic Ca2+, which could be distinguished from the high-frequency and high-amplitude oscillations in Ca2+ associated with contraction and relaxation. A number of studies have demonstrated a role for CCE mediated via TRPC channels in modulating the responses to hypertrophic stimuli (12, 28, 30); in the present study, we provide evidence that calcium entry via TRPC may also contribute to Ca2+-mediated apoptosis. Clearly, there are limitations in extrapolating studies on isolated cardiomyocytes to the intact heart; furthermore, the fact that TRPC3 overexpression is associated with increased apoptosis does not necessarily imply that Ca2+ entry via TRP channels contributes to apoptosis in the normal heart. However, hypertrophic agonists such as angiotensin and phenylephrine, which have been shown to stimulate CCE, are also known to induce apoptosis. Furthermore, cardiac hypertrophy, which increases TRPC1 expression levels in the normal intact adult heart (30), is also associated with decreased tolerance to ischemic injury (1).
In the present study, we have focused entirely on isolated cardiomyocytes, and thus any extrapolation with regard to the role of TRPCs in mediating cell death in the intact heart must be made with caution. In the intact heart, tissue injury following I/R is a result not only of metabolic and ionic events, but is also a consequence of mechanical stress that occurs due to muscle contraction mediated in part by cell-to-cell connections. Such events are clearly absent in studies of cultured adult cardiomyocytes, which do not contract spontaneously and in which there are no cellular connections. Conversely, cardiomyocyte isolation is itself an appreciable stress, and this could potentially have a greater effect on cells overexpressing TRPC3 than on WT cells. It is conceivable that this could make TRPC3 cells more susceptible to subsequent I/R injury; however, it is likely that this would be manifest by increased basal cell death or a significantly greater increase in necrosis or apoptosis in time-controlled normoxic incubations, neither of which was seen here. Clearly, studies in the isolated perfused heart or in vivo would have demonstrated a more definitive role for TRPC3 in mediating I/R reperfusion in the intact heart. Another limitation of the present study is the potential for chronic changes in cardiomyocytes overexpressing TPRC3, such as hypertrophy or increased calcineurin activity (28) that could also increase sensitivity to I/R injury independent of acute CCE-mediated increase in cytosolic Ca2+. Future studies using acute adenoviral transfection approaches or conditional overexpression models would help to resolve this issue. The use of siRNA to decrease TRPC expression in normal cells would also provide valuable insight into the contribution of TRPCs to cardiomyocyte injury.
In conclusion, we have shown that increased TPRC3 expression contributes to increased apoptosis but not necrosis in cardiomyocytes subjected to I/R. This increase in apoptosis was associated with increased calpain-mediated proteolysis that was attenuated by the calpain inhibitor PD-150606 and the CCE inhibitor SKF-96365. However, while increased TRPC3 expression increased sensitivity to Ca2+ stress, it did not increase the sensitivity to TNF--induced apoptosis, demonstrating that TRPC3 overexpression did not result in general decreased tolerance to stress. These results provide further evidence for a role of TRP channels in mediating cardiomyocyte function and suggest that TRPC-mediated CCE may contribute to decreased tolerance to injury associated with cardiac hypertrophy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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