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Nitric oxide signaling in stretch-induced apoptosis of neonatal rat cardiomyocytes

Xudong Liao^*,,1, Jun-Ming Liu, Lei Du^*, Aihui Tang, Yingli Shang^*, Shi Qiang Wang, Lan-Ying Chen^,2 and Quan Chen^*,2

^* State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences;

Cardiovascular Institute and Fu Wai Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences; and

State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University. Beijing, China

²Correspondence: Q.C., State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, 25 Beisihuanxi Rd., Beijing 100080, P.R. China. E-mail: chenq{at}ioz.ac.cn; L.-Y.C., Cardiovascular Institute & Fu Wai Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, 167 Beilishi Rd., Beijing 100037, P.R. China. E-mail: lanyingchen{at}hotmail.com

SPECIFIC AIMS

Cardiomyocyte apoptosis has been recognized as an important pathological factor in the transition from hypertensive heart remodeling to heart failure, but the mechanisms by which the mechanical signals lead to apoptosis are not well understood. Building on our previous finding that stretch-activated Ca²⁺ signaling plays an essential role in initiating apoptosis, we studied the downstream signals and found that NO was the link between stretch-induced Ca²⁺ signaling and apoptosis in cardiomyocytes.

PRINCIPAL FINDINGS

1. Activation of Ca²⁺-dependent NO signaling in cardiomyocytes by mechanical stretch
Mechanical stretch induced rapid and significant intracellular NO ([NO]_i) elevation in cardiomyocytes (Fig. 1 ). [NO]_i elevation remained significantly higher for up to 24 h after stretch and was sustained, although the initial [NO]_i elevation was transient (Fig. 1) . We reasoned that stretch-induced [NO]_i elevation to activated NO synthases (NOS) since L-NAME, a nonisoform-specific NOS inhibitor, completely abrogated stretch-induced [NO]_i elevation (Fig 1D ). The rapid initial NO synthesis was Ca²⁺-dependent and significantly attenuated by EGTA-AM (Fig 1D ), indicating a potential role for Ca²⁺-activated eNOS. The inducible NOS (iNOS) -specific inhibitor AMT failed to block stretch-induced initial NO synthesis, suggesting that iNOS was not responsible for the initiation of NO production (Fig 1D ). Together with the basal expression levels of eNOS and iNOS in cardiomyocytes (Fig 1E ), these data demonstrated that stretch-induced initial [NO]_i elevation was due to Ca²⁺-dependent eNOS activation. Mechanical stretch also induced eNOS and iNOS expression in cardiomyocytes as revealed by Western blot (Fig 1E ). Transient eNOS but prolonged iNOS inductions were further confirmed in vivo in hypertensive rat hearts using an abdominal aorta constricted (AAC) hypertensive model. The stretch-induced prolonged induction of iNOS may have biological and pathological significance in hypertension.

View larger version (54K): [in this window] [in a new window]   Figure 1. Mechanical stretch induces Ca2+-dependent NO production in NRVMs. A) Intracellular NO concentration ([NO]i) was examined by DAF-based confocal analysis. Images shown were taken from cells before stretch (Control), 5 min after stretch (Stretch), and 10 min after SNAP (100 µmol/l) treatment (SNAP). NO donor SNAP treatment was used as a positive control. B) Confocal images were analyzed with IDL software to give quantitative NO levels. Digital images were taken from random fields (>50 pictures per time point were taken) and pixel density was calculated, normalized with control data (from 10 min to 0 min before stretch) and changes of [NO]i ([NO]i) were expressed as changes of fluorescence (F/F0). Error bars stand for SE. *P < 0.05. C) Total nitrate level ([NO]t) was determined by nitrate reductase-based colorimetric method. Conditioned medium was examined. [NO]t was converted to [NO3–] using KNO3 (100 µmol/l) as a standard. *P < 0.05. D) Cells were incubated with Ca2+ chelator EGTA-AM (2 µmol/l), NOS inhibitor L-NAME (100 µmol/l), or iNOS inhibitor AMT (100 µmol/l) for 30 min at 37°C before stretch. [NO]i was determined by DAF-based confocal analysis. Data of 10 min stretched cells were compared. *P < 0.05. E) Protein levels of iNOS and eNOS in NRVMs before and after 4 h stretch were detected by Western blot analysis. ßbeta;-Actin was blotted to monitor equal loading.

2. Requirement of NO signaling in stretch-induced cardiomyocyte apoptosis
To directly examine the role of NO in mechanical stretch-induced apoptosis, we pretreated cardiomyocytes with L-NAME 30 min before stretch to block NO synthesis and found that L-NAME blocked stretch-induced apoptosis (Fig 2 A, B). In addition to L-NAME, a specific iNOS inhibitor AMT also inhibited apoptosis (Fig 2C ). However, AMT failed to block iNOS induction or the initial phase of NO production, which suggested that the proapoptotic phase of NO was produced by iNOS. Blockage of soluble guanylyl cyclase (sGC) by ODQ inhibited stretch-induced apoptosis (Fig 2C ), suggesting that the NO/cGMP pathway was involved in apoptosis regulation. All these inhibitors also blocked stretch-induced mitochondrial membrane potential (m) reduction and cyt c release, critical events in mitochondria-dependent apoptosis. These data indicated that endogenous NO signaling was required for stretch-induced cardiomyocyte apoptosis and that iNOS played important proapoptotic roles.

View larger version (40K): [in this window] [in a new window]   Figure 2. NO signaling participates in the regulation of stretch-induced cardiomyocyte apoptosis. A) Apoptosis was examined with annexin V-based flow cytometry (upper panel) and DAPI-based fluorescent microscopy (lower panel). To block NO synthesis, cells were pretreated with L-NAME (100 µmol/l) at 37°C for 30 min before stretch. Cells were subjected to 20% sustained stretch for 4 h before apoptosis detection. Arrows indicated apoptotic nucleoli. B, C) Cells were incubated at 37°C for 30 min with L-NAME (100 µmol/l), AMT (100 µmol/l), ODQ (10 µmol/l), or SNAP (100 µmol/l), respectively, in each group before a 4 h stretch. Control cells were incubated with inhibitors without stretch. Apoptotic index was shown as % of fragmented nucleoli (indicated by arrows in panel A). More than 200 nuclei from randomly selected fields (>10 fields per dish) were counted. *P < 0.05.

3. Roles of eNOS and iNOS in NO cascade
Although stretch induced both eNOS and iNOS, the initiating NOS is most likely eNOS since it produced the initial phase of NO; clearly the later phase of apoptotic NO was produced by iNOS, as discussed above. The next question we asked was what signal links eNOS to iNOS. We found that NO itself was the key regulator of iNOS induction. When inhibiting the initial NO production by L-NAME, it completely blocked iNOS expression. NO regulated iNOS expression mostly likely through the sGC/cGMP pathway since ODQ blocked stretch-induced iNOS induction. There was a positive feedback loop in the stretch-induced NO cascade, in which stretch activated eNOS to initiate NO production and the initial NO induced iNOS expression to produce more NO. The high level of NO produced by iNOS, which is a much more powerful NOS than eNOS, was capable of inducing apoptosis and/or other stretch-induced responses in cardiomyocytes.

4. The role of Ca²⁺ signaling in NO cascade
We demonstrated that stretch activated a NO cascade to induce cardiomyocyte apoptosis. The next question we asked was how this cascade was initiated. We showed that stretch-induced initial NO production was Ca²⁺-dependent in this study, and have reported that mechanical stretch-induced [Ca²⁺]_i elevation plays essential roles in apoptosis initiation. Therefore, Ca²⁺ signaling is likely to be the initiator of the NO cascade. Stretch-induced [Ca²⁺]_i elevation was dependent on Ca²⁺ influx through L-type calcium channels (LCC) and stretch-activated ion channels (SAC), and Ca²⁺-induced Ca²⁺ release (CICR). The Ca²⁺ inhibitors (EGTA-AM, LCC blocker nifidipine, SAC blocker Gd³⁺, CICR inhibitors ryanodine and thapsigargin), which have been shown to block stretch-induced [Ca²⁺]_i elevation in cardiomyocytes, significantly attenuated the initial [NO]_i elevation in this study. These data suggest that the stretch-induced Ca²⁺ signal is required for the initial NO burst and possibly acts as a trigger for the NO signaling cascade by activating eNOS.

CONCLUSIONS AND SIGNIFICANCE

In this paper, we suggest that mechanical stretch induces a Ca²⁺-NO signaling cascade to induce apoptosis in cardiomyocytes, as summarized in Fig. 3 . Stretch may initiate the cascade through [Ca²⁺]_i elevation and Ca²⁺-dependent eNOS activation. eNOS produces the initial NO, which is further amplified through NO-induced iNOS expression. iNOS produces high doses of NO, which are capable of regulating stretch-induced cardiomyocyte apoptosis and/or other stretch-induced responses. This stretch-Ca²⁺-NO signaling cascade may be a common mechanism by which cardiomyocytes sense and transduce mechanical stretch signals into biological signals to regulate hypertension responses, including, but not exclusive to, apoptosis.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic summary of a hypothetical NO signaling cascade activated by mechanical stretch in cardiomyocytes. Mechanical stretch initiates the NO cascade by activating constitutively expressed eNOS through [Ca2+]i elevation. The active eNOS produces a small amount of NO, which further induces iNOS expression. iNOS, once expressed, produces a larger amount of NO, thus amplifying NO signaling. Finally, NO concentration reaches a certain level that is high enough to induce apoptosis and/or other responses in cardiomyocytes. NO may affect mitochondria or other apoptotic pathways to induce apoptosis. Besides this NO cascade, stretch-induced Ca2+ may participate in other signaling pathways. Solid lines indicate signaling discovered in this study. Dashed lines indicate possible signaling.

Mechanical stretch significantly induced the expression of both eNOS and iNOS in vitro and in vivo. The in vivo increase of iNOS expression was sustained in hypertensive hearts. These results strongly suggest that iNOS plays an important role in regulating the pathological outcomes of hypertension. Our data demonstrated that iNOS-produced NO was proapoptotic, since AMT blocked apoptosis without altering iNOS expression or eNOS-induced initial NO. Although eNOS induction was transient in vivo, its activation was essential in initiating the stretch-induced NO cascade because its inhibition resulted in blockage of iNOS gene expression.

NO has been reported to be a bidirectional regulator of apoptosis. NO can directly induce the nitrosylation of some apoptosis regulatory molecules, such as caspase 3 and cyt c, or directly inhibit mitochondrial respiration to induce apoptosis. Besides its role in the mitochondria-dependent apoptotic pathway, NO is also important in death receptor-dependent apoptotic signaling. We have reported that mitochondria and death receptor-mediated apoptosis were both activated by mechanical stretch in cardiomyocytes. It is possible that NO participates in both types of apoptotic pathways (mitochondria and death receptor-dependent) to regulate stretch-induced cardiomyocyte apoptosis (Fig. 3) .

Deregulation of both eNOS and iNOS has been associated with a number of heart diseases. It is possible that the activation of NO signaling is an adaptive response to protect cardiac cells from mechanical overload-induced damage. However, maladaptive responses with chronically high doses of NO and enhanced iNOS expression (and the potential feedback reinforcement) could be associated with the progression of heart diseases. Understanding the role of NO signaling in heart disease would be useful for the development of drugs that can selectively promote the beneficial effects of NO.

FOOTNOTES

¹ Present address: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, USA.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5717fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5717fjev1 20/11/1883    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liao, X. Articles by Chen, Q. Search for Related Content PubMed PubMed Citation Articles by Liao, X. Articles by Chen, Q.