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Intracellular acidosis-activated p38 MAPK signaling and its essential role in cardiomyocyte hypoxic injury

Ming Zheng^,*,1, Cherilynn Reynolds^,1, Su-Hyun Jo^,2, Robert Wersto, Qide Han and Rui-Ping Xiao^,*,3

Institute of Cardiovascular Sciences and
^* Institute of Molecular Medicine, Peking University, Beijing, People’s Republic of China; and
Laboratory of Cardiovascular Science and
Flow Cytometry Laboratory, Gerontology Research Center, National Institute on Aging, NIH, Baltimore, Maryland, USA

³Correspondence: Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224, USA. E-mail: xiaor{at}grc.nia.nih.gov

SPECIFIC AIMS

We investigated the possible effect of intracellular acidosis, a characteristic of hypoxic and ischemic myocardium, on p38 mitogen-activated protein kinase (MAPK) signaling and its pathophysiological relevance in myocardium hypoxia.

PRINCIPAL FINDINGS

1. Cardiomyocyte hypoxia concurrently decreases intracellular pH and increases p38 MAPK phosphorylation
To determine responses of intracellular pH (pH_i) and p38 MAPK to hypoxia, adult rat cardiomyocytes were subjected to a pelleting protocol to create a cell-based hypoxia/ischemia model. Activation of p38 MAPK was assayed by Western blot analysis using an antibody that specifically recognizes the dually phosphorylated (on residues Thr¹⁸⁰ and Tyr¹⁸²), active form of the enzyme. Hypoxia induced by pelleting cells for 90 min increased phosphorylation of p38 MAPK without altering the phosphorylation status of ERK1/2 or JNK (Fig. 1 A). On average, phosphorylation level of p38 MAPK was augmented by 2.4-fold (Fig. 1B ). Myocyte hypoxia significantly reduced pH_i from 7.17 to 6.69 (Fig. 1C ). These data indicate that hypoxic stress markedly elevates p38 MAPK activation and decreases pH_i in freshly isolated adult rat cardiomyocytes.

View larger version (33K): [in this window] [in a new window]   Figure 1. A) Top band: typical Western blot of phospho-p38 MAPK, phospho-ERK1/2 and phospho-JNK in cardiomyocytes under control conditions or subjected to hypoxia (pelleting for 90 min). The same membrane was stripped and reprobed with an antibody reacting with total p38 MAPK to show the kinase protein abundance (A, second band). B)Average data on hypoxia-induced increase in p38 phosphorylation (n=10 independent experiments, *P<0.01 vs. control). C)Hypoxia-induced decrease in pHi (n=80 and 35 cells from 6 heart for control and hypoxia, respectively; *P<0.01 vs. control). D) Intracellular pH (pHi) was manipulated by incubating cardiomyocytes with HEPES buffers at various extracellular pH (pHo) for 1 h (n=30–80 cells from 6 hearts for each data point). E) pHi in myocytes incubated with a basic buffer (pHo 8.5) in the presence or absence of hypoxia (90 min pelleting). F) Top: typical Western blot of phospho-p38 MAPK and total-p38 MAPK in myocytes incubated with a control buffer (pHo 7.4) or an alkalosic buffer (pHo 8.5) in the presence or absence (Con) of hypoxia (90 min pelleting); Bottom: average data of p38 MAPK phosphorylation in cells with the alkalosic buffer (pHo 8.5) (n=6).

2. Intracellular acidosis is obligatory to hypoxia-induced activation of p38 MAPK
The concurrent intracellular acidification and activation of p38 MAPK in response to hypoxia suggest that these cellular responses may be causally linked. We incubated cardiomyocytes with HEPES buffers with a wide range of pH. Figure 1D shows that, in the absence of hypoxia, pH_i was decreased as a monotonic function of extracellular acidosis, whereas extracellular alkalosis resulted in an increase in pH_i. At the extreme pH_o of 5.5 and 8.5, pH_i was changed from 7.2 under normal conditions (with pH_o 7.4) to 6.6 and 7.5, respectively. Next, we sought to maintain pH_i at the normal range during cellular hypoxia by incubating myocytes with an alkalosic HEPES buffer (pH 8.5). In the presence of alkalosis buffer, pH_i was reduced from 7.49 ± 0.02 to 7.30 ± 0.02 in myocytes subjected to 90 min pelleting (Fig. 1E ). Under these experimental conditions, hypoxia was unable to increase p38 MAPK phosphorylation (Fig. 1F ), indicating intracellular acidosis is involved in hypoxia-activated p38 MAPK signaling.

3. Activation of p38 MAPK by intracellular acidosis
To determine whether intracellular acidification per se is sufficient to activate p38 MAPK, cardiomyocytes were subjected to a HEPES-buffered solution with pH_o ranging from 8.5 to 5.5 for 1 h. Phosphorylation of p38 MAPK was augmented in response to intracellular acidosis in a pH_i- and time-dependent manner (Fig. 2 A, C) in the absence of change of phosphorylation status of other members of the MAPK superfamily, including ERK1/2 and JNK. This indicates that acidosis affects p38 MAPK specifically. Incubation of adult rat cardiomyocytes with the bicarbonate buffer for 1 h reduced pH_i to 6.6 and markedly elevated p38 MAPK phosphorylation. Our data strongly suggest that intracellular acidosis per se is sufficient to induce a robust and sustained activation of p38 MAPK, but not ERK1/2 and JNK, in cardiac myocytes.

View larger version (37K): [in this window] [in a new window]   Figure 2. Intracellular acidosis activates MEK3/6 and p38 MAPK in a time- and pHi-dependent manner. A) Phosphorylation of MEK3/6 in cardiomyocytes treated by HEPES buffers with various pH for 1 h. A typical example of acidosis-induced phosphorylation p38 MAPK was included for comparison. B) Average data of acidosis-induced increase in phosphorylated MEK3/6 normalized to the total protein. C) Representative example of the time course of MEK3/6 activation in response to the acidic HEPES buffer treatment (pHo 5.5 corresponding to pHi 6.6). Phosphorylation of MEK3/6 was increased after cells were incubated for 1 h in the acidic buffer and persisted for at least 5 h. D)Average time course of acidosis-induced augmentation in phosphorylated MEK3/6 normalized to total protein. E) In vitro activity of p38 MAPK with different pH. p38 MAPK activity was indexed by phosphorylation of ATF-2, a substrate of p38 MAPK. Note that in vitro activity of p38 MAPK was not increased in acidic reaction buffers.

4. Acidosis activates p38 MAPK through a MEK3/6-dependent pathway independent of intracellular Ca²⁺ signaling
To delineate the specific mechanism underlying intracellular acidosis-induced p38 MAPK activation, we examined the possible response of MEK3/6 (immediate upstream kinases of p38 MAPK) to a change in pH_i. Phosphorylation of MEK3/6, similar to phosphorylation of p38 MAPK, was graded by the degree of acidosis with a threshold pH_i of 7.0 (Fig. 2A, B ). Intracellular acidosis elevated MEK3/6 activation in response to a reduction in pH_i from 7.2 to 6.6 in a time-dependent manner (Fig. 2C, D ). The temporal profile and pH_i dependence of MEK3/6 phosphorylation were similar to those of acidosis-induced p38 MAPK activation.

p38 MAPK was immunoprecipitated and its activity was assessed using recombinant ATF-2 as a substrate. p38-mediated ATF-2 phosphorylation in vitro was not pH-dependent (Fig. 2E ), excluding the possibility that pH_i acts directly on p38 MAPK. These results indicate that intracellular acidosis activates p38 MAPK via an intracellular signaling pathway involving MEK3/6.

Studies have shown that intracellular acidification leads to an increase in intracellular Ca²⁺ due in part to activation of Na⁺/H⁺ exchange and subsequent Na⁺/Ca²⁺ exchange. Increased intracellular Ca²⁺ may participate in the acidosis-induced activation of p38 MAPK. We found that buffering intracellular Ca²⁺ by EGTA-AM (1 µM pretreatment for 30 min) could not prevent or suppress the response of p38 MAPK to intracellular acidosis (pH_i 6.6) but abolished other Ca²⁺-dependent cellular processes such as electrical pacing-triggered cell contraction, indicating that an increase in intracellular Ca²⁺ does not play a major role in acidosis-dependent p38 MAPK activation.

5, Inhibition of p38 MAPK protects cardiomyocytes against hypoxia-induced cell death
We examined cardiomyocyte viability in the presence or absence of a specific p38 MAPK inhibitor, SB203580. Hypoxia by pelleting cells for 90 min markedly increased apoptotic cell death. Inhibition of p38 MAPK with SB20380 (10 µM) largely protected myocytes against hypoxia-mediated apoptosis, as evidenced by reducing the percentage of propidium iodide (PI, 1 µg/mL) staining positive cells from 44.7 ± 4.0% to 22.5 ± 2.2% (P<0.01, n=6). This indicates that acidosis-induced p38 MAPK activation is detrimental to cardiomyocytes.

CONCLUSIONS AND SIGNIFICANCE

We report here that preventing intracellular acidosis abolishes hypoxia-induced p38 activation whereas intracellular acidosis per se, similar to hypoxia, selectively activates p38 MAPK but not ERK1/2 and JNK in rat ventricular myocytes. Inhibition of p38 signaling by SB203580 protects cardiomyocytes against hypoxic cell death. These results provide the first evidence that intracellular acidosis is a necessary and sufficient mediator transducing cellular hypoxic stress to activation of p38 MAPK signaling. This finding reveals a novel paradigm of the regulation of p38 MAPK signal transduction and sheds light on our understanding of the pivotal role p38 MAPK in ischemic/hypoxic cell injury and cell death.

Although it is widely accepted that p38 MAPK is activated by ischemia/hypoxia, we have demonstrated that hypoxia stimulates p38 MAPK through an intracellular acidosis-dependent mechanism. This conclusion is based on two lines of independent evidence. First, hypoxia-evoked increase in p38 MAPK phosphorylation can be blocked by neutralizing intracellular pH at the normal range (pH_i7.2) using a basic extracellular buffer (pH_o 8.5). Second, the intracellular acidosis per se is sufficient to specifically augment activation of p38 MAPK, but not ERK1/2 or JNK, similar to the situation of hypoxia. Thus, intracellular acidosis is not only necessary but also sufficient to transduce cellular hypoxic stress signal to activation of p38 MAPK signaling.

We have demonstrated that phosphorylation of MEK3/6 is markedly augmented in response to acidosis, whereas p38-induced in vitro phosphorylation of its substrate ATF-2 is not altered by a change in pH. These results indicate that the effect of acidosis on p38 MAPK is mediated by a pathway involving MEK3/6 rather than by direct modulation of the kinase catalytic activity by pH. Moreover, we have shown that the acidosis-evoked p38 MAPK signaling is Ca²⁺ independent, since buffering intracellular Ca²⁺ cannot prevent acidosis-induced p38 MAPK activation. The stimulatory effect of hypoxia on p38 MAPK activation is mediated by an acidosis-evoked and MEK3/6-dependent signaling cascade (Fig. 3 ).

View larger version (32K): [in this window] [in a new window]   Figure 3. Schematic diagram. Hypoxia stress leads to intracellular acidosis, which activates the MEK5/6 and p38 MAPK signaling cascade. Subsequently, acidosis-induced activation of p38 MAPK plays an essential role in hypoxic injury and death of cardiac myocytes.

A large body of evidence indicates that enhanced p38 MAPK signaling is associated with cardiac hypertrophy, the onset of heart failure, and ischemic/reperfusion injury, whereas inhibition of p38 MAPK improves the survival and functional performance of ischemia/reperfusion-injured hearts or cardiac myocytes, suggesting that activation of p38 MAPK might participate in cardiac ischemic/hypoxic injury. In the present study, we found that inhibition of p38 by SB203580 markedly reduces hypoxia-induced cardiomyocyte death and significantly improves the contractility of hypoxic myocytes. We also show that intracellular acidosis is upstream of p38 activation in transducing hypoxic signaling because neutralizing intracellular pH fully abolishes hypoxia-mediated p38 MAPK activation in adult rat cardiomyocytes. Our preliminary results indicate that neutralizing intracellular acidosis significantly reduces hypoxia-induced cell death, consistent with the notion that chronic hypoxia-induced apoptosis is abolished by neutralizing intracellular pH in cultured cardiomyocytes or perfused hearts. Thus, intracellular acidosis plays an essential role in hypoxia-mediated apoptotic cell death. These results provide novel evidence that p38 MAPK constitutes a mediator to deliver hypoxia- and intracellular acidosis-triggered apoptotic signal in cardiomyocytes (Fig. 3) .

FOOTNOTES

¹ These authors contributed equally to this work.

² Present Address: Department of Physiology, Cheju National University, College of Medicine,1 Ara 1-Dong, Jeju 690-756, South Korea.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2607fje;

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