Both eNOS and neuronal NOS (nNOS) are constitutively expressed in cardiomyocytes but within distinct subcellular locations, i.e
., eNOS is predominantly localized at caveolae in the sarcolemma (1
) and nNOS is mostly localized to the sarcoplasmic reticulum (1
). In this study, we found that perfusion with a constitutive NOS inhibitor L-NAME prior to and during IPC abolished IPC-induced protection (), suggesting that the activation of constitutive NOS is necessary for IPC-induced acute cardioprotection. Consistent with this finding, loss of IPC-induced cardioprotection has been found in eNOS-/-
mouse hearts (35
), although there are data to the contrary (8
There are emerging data suggesting that protein S-nitrosylation plays an important role in cardioprotection (17
). Our studies have demonstrated that IPC results in an increase in SNO, and that SNO can alter protein activity and protect proteins against further oxidation (15
). A previous study by our group reported that IPC was blocked by N-acetylcysteine, a glutathione precursor and reducing agent, suggesting a redox-sensitive mechanism is involved in the protection afforded by IPC (2
). In this study, the inhibition of IPC-induced cardioprotection by ascorbic acid (), a reducing agent that specifically decomposes SNO, provides strong evidence showing that SNO signaling plays an important role in IPC-induced cardioprotection. Consistent with our recent studies (15
), the majority of proteins with an increase in SNO after IPC were mitochondrial proteins ( and ).
In this study, we tested the hypothesis that caveolae-associated eNOS/NO is important for SNO of mitochondrial proteins in IPC hearts. In support of the hypothesis, data in the literature show that that IPC increased the formation of caveolae, and a transgenic mouse with cardiomyocyte-specific overexpression of caveolin-3 showed enhanced formation of caveolae and exhibited reduced ischemia/reperfusion (I/R) injury (38
). In addition, caveolin-3-/-
mice showed no protection in response to IPC (38
). A recent study by Quinlan et al
) has shown that preconditioning the heart induces formation of signalosomes, caveolae-associated signaling platforms that interact with mitochondria to open mitochondrial ATP-dependent potassium channels (27
). These data not only suggest that caveolae signaling complexes are crucial for IPC, but also point out a possible cardioprotective signaling network between caveolae and mitochondria. It has been suggested that activated eNOS could be internalized to deliver NO to subcellular targets for biological effects (12
). In this study, we found that MβCD treatment not only abolished IPC-induced cardioprotection (), but also markedly reduced the association of eNOS with caveolin-3 () and IPC-induced phosphorylation of eNOS (). Furthermore, MβCD treatment blocked the IPC-induced increase in SNO (, ). The parallel changes in caveolar eNOS signaling and SNO in mitochondria in IPC hearts are consistent with the hypothesis that activation of eNOS and caveolae trafficking to mitochondria are important in generating mitochondrial-localized eNOS/NO/SNO signaling. The detection of eNOS/caveolin-3 in mitochondria isolated from IPC hearts () provides support for this hypothesis.
As caveolae are important for many signaling pathways, the disruption of caveolae could inhibit protection by blocking any number of signaling pathways. Thus, one limitation of this study is that we only examined the role of caveolin-3/eNOS/SNO, but did not investigate other signaling pathways such as the NO/guanylyl cyclase/cGMP/PKG pathway. However, it is clear that eNOS/NO/SNO signaling is one of the signaling pathways that is blocked by disruption of caveolae. Since SNO had been previously shown to play a role in cardioprotection, it is likely that disruption of this signaling pathway contributes to loss of cardioprotection. Because of dynamic range issues, most of the proteomic methods (2D gels and mass spectrometry) are biased toward detection of high-abundance proteins (34
). Thus, the prevalence of mitochondrial proteins identified as SNO proteins might be due in part to their high abundance. Our recent study using a SNO-RAC proteomic approach confirms that most of SNO proteins in IPC hearts are mitochondrial proteins (15
), suggesting that mitochondria are indeed one of the major subcellular organelles targeted by SNO signaling in IPC hearts.
In summary, we found that MβCD treatment not only disrupted caveolae and association of eNOS with caveolin-3, but also blocked IPC-induced cardioprotection and the increase in SNO of mitochondrial proteins that normally occur with cardioprotection. These data are consistent with the hypothesis that caveolin-3-associated eNOS/NO trafficking between plasma membrane and mitochondria provide an important signaling pathway regulating SNO of mitochondrial proteins.