We found that IκBα degradation and NF-κB activation occurred in a time-dependent manner in NRVMs subjected to H2O2. Cells treated with H2O2 showed reductions in cell vitality and △Ψm, but elevations in LDH, MDA, apoptosis and autophagy. IκBα transduction or PDTC pretreatment ameliorated H2O2-induced cell injury via inhibiting the NF-κB translocation.
Ischemia/reperfusion (I/R) injury severely attenuates the benefit of revascularization after acute myocardial infarction (AMI) and hence has become an important focus of cardiovascular research . It is currently believed that the inflammatory response induced by AMI is essential for heart repair, but the excessive generation of ROS and inflammation following reperfusion therapy will exacerbate heart damage .
NF-κB signaling pathway plays a key role in the inflammatory response, oxidative stress, apoptosis, and autophagy in heart . As is known, the nuclear translocation of p65 subunit is a sign for NF-κB activation . Previous studies [22–24] identified that H2O2 treatment for different durations (30 min-24 h) elicited a significant p65 nuclear translocation in NRVMs. In line with these studies, we found that p65 was time-dependently translocated from cytoplasm to nucleus with IκBα degradation in NRVMs subjected to H2O2.
However, whether NF-κB activation could protect or exacerbate cardiomyocyte is still a matter of debate. The early study demonstrated that activation of NF-kB reduced cell apoptosis in hypoxic cardiomyocyte , whereas most of recent studies have shown that NF-κB is a pro-apoptotic transcription factor correlated with myocardial injury  and blocking NF-κB activity will prevent myocardial apoptosis . Gray et al  recently report that ROS generated by ischemia-reperfusion can rapidly activate calmodulin kinase II (CaMKII), which deteriorates cell injury through inducing IκBα degradation and nuclear p65 accumulation in NRVMs exposed to H2O2. Importantly, knock-out CaMKIIδ gene significantly attenuates area of myocardial infarction by inhibiting IκBα degradation and NF-κB activation. All these findings reveal that NF-κB activation deteriorates the prognosis of heart in I/R injury.
Herein, we hypothesized that directly overexpress IκBα to prevent NF-κB activation may play a better role in protecting cardiomyocyte. Then, we designed a dsAAV9-IκBα ser 32A,36A mutant to prevent IκBα degradation from phosphorylation at serine 32 and 36 sites, which could be successfully transfected into cardiomyocytes. Western blot and immunofluorescence demonstrated that IκBα transfection could successfully maintained cytoplasmic IκBα level and suppressed the p65 translocation in NRVMs exposed to H2O2. Consistent with our speculation, IκBα elevated cell viability, decreased LDH and MDA level and attenuated apoptosis, implying a protective role of IκBα in H2O2-induced cell injury in NRVMs. The mechanisms may be account for the role of NF-κB in mediating the expression of various proteins that promote or inhibit apoptosis. It is noted that NF-κB regulates the expression of certain anti-apoptotic genes, for example Bcl-2 , and increasing the ratio of Bcl-2/Bax will decrease cell apoptosis. In this study, treatment with IκBα mutant or PDTC significantly reduced Bax/Bcl-2 ratio in NRVMs subjecting to H2O2. These data indicate that IκBα protects NRVMs against H2O2-induced apoptosis by decreasing the ratio of Bax/Bcl-2.
It is reported that opening of the mitochondrial permeability transition pore (MPTP) in the first few minutes of reperfusion lead to △Ψm loss and is responsible for the necrotic and apoptotic cell death processes exhibiting differential contributions to infarct size . Thus, △Ψm loss reflects the dysfunction of mitochondrial, represents a sign of early stage apoptosis and is a critical determinant of I/R injury . Our previous study demonstrates that oxidative stress induces a significant decrease of △Ψm . In this study, we observe that H2O2 treatment results in attenuated △Ψm and enhanced Bax expression in NRVMs, and the effect was reversed by pretreatment with IκBα or PDTC. NF-κB  and Bax  are reported to be involved in regulation of △Ψm. Herein, we suggest that IκBα decreases cell injury and apoptosis via inhibiting NF-κB activation and Bax expression, finally elevating △Ψm after H2O2 stimulation.
Autophagy, a cellular process of lysosome-mediated degradation of cytoplasmic components or damaged organelles, is generally thought to be an adaptive response and protective for cell survival . However, autophagy shows redox effect on cardiomyocyte upon different stimuli. Evidence supports that autophagy benefits heart cells during myocardial ischemia through improving myocardial energy metabolism and organelle recycling , but excessive autophagy produces lethal damage on cells in cardiac I/R injury , which is mediated in part by upregulation of Beclin-1 expression .
However, the communication between autophagy and NF-κB is bidirectional. It is reported that autophagy is required for the activation of NF-κB , in turn, NF-κB further increases autophagosome maturation via upregulating Beclin-1 and LC3 expression in I/R injury . Importantly, PDTC attenuates Beclin-1 expression and formation of autophagosomes by suppressing I/R injury-induced NF-κB activation . In accordance with these findings, our results unveil that NRVMs treated with H2O2 could induce p65 translocation, enhance Beclin-1, increase LC3-Ⅱ/LC3-I ratio and decrease P62 expression, which are rescued by IκBα transfection or PDTC treatment. These results imply that IκBα could protect cardiomyocytes by inhibiting H2O2-induced autophagy. In addition, it is noticed that Bcl-2 can bind to Beclin-1 to inhibit autophagy . Our study also demonstrates that IκBα transfection elevates the expression of Bcl-2, which may disturb the function of Beclin-1 and thus further inhibit H2O2-induced autophagy, implying a “cross-talk” between apoptosis and autophagy.