To characterize the binding receptor for HIV

To characterize the binding receptor for HIV-1 gp120 in autophagy, we used the CXCR4 inhibitor AMD3100 and NMDA receptor inhibitor MK801 along with gp120 to treat the cardiomyocytes. Our data demonstrated that inhibition of the NMDA receptor significantly blocked the increase of gp120 induced autophagic proteins and autophagosomes, while inhibition of CXCR4 exhibited no effect on gp120 induced autophagy activities. This observation suggests that the receptor usage for gp120 during induction of autophagy is different from the report on lymphocytes, in which gp120 induced autophagy via the CXCR4 receptor [26]. This difference may be due to a lack of NMDA receptor on lymphocytes. The gp120 is known for its ability of binding to the NMDA receptor [33]. Pattarini et al. suggest that gp120 may act following recognition by its V3 sequence of a high-affinity site possibly coincident with the glycine site of NMDA receptors on neuronal surround [34]. Recent evidences demonstrated that activation of the NMDA receptor can induce autophagy in neuronal cells and cardiomyocytes [43], [44]. Taken together, our findings suggest that gp120 induces autophagy via binding to the NMDA receptor in cardiomyocytes. The present study also demonstrated that gp120 induced autophagy was inhibited by the JNK inhibitor SP600125 and class III PI3K inhibitor 3-MA. JNK is known for its upregulating role in autophagy. JNK activation modulates autophagy through upregulation of Beclin 1 and upregulation of lipid conjugation of LC3 [45]. The class III PI3K (phosphoinositide 3-kinase), Vps34 (vacuolar protein sorting 34), and the homologue in mammalian cells, hVps34, have been studied extensively in autophagy. In autophagy Beclin 1 together with Vps34 form the Beclin 1–Vps34 core complex to promote autophagy [41], [46]. 3-MA has been extensively used in autophagy study as an autophagy inhibitor because it can specifically inhibit class III PI3K, although it was reported to induce autophagy [38], [47]. Taken together, these results suggest that gp120 induces autophagy via a pathway of the NMDA receptor, JNK and class III PI3K. We believe that our findings of increased autophagic markers in gp120 treated H9c2 cardiomyocytes are consistent with the findings in neuronal cells as well as those in lymphocytes [25], [26]. It is important to note, however, that our findings of increased markers of autophagy in gp120 treated cardiomyocytes may represent either an absolute increase of autophagy within the cells or ineffective autophagy with an accumulation of autophagic products. In this regard, our findings in this research suggest a possible model for the mechanisms of HIV-1 cardiac involvements. In this model, cells that bind HIV-1 gp120 or other products of viral infection enhance autophagy in an attempt to remove toxic stress and maintain cell survival. In this scenario, our data suggest that autophagy may help to sustain cardiomyocyte survival. It is of interest to note that we observed no differences in cell death marker between the cardiomyocytes treated with gp120 and those with vehicle control. HIV-1 gp120 has been reported to induce cardiomyocyte apoptosis [8], [15]. This study was originally designed based on the crosstalk property between autophagy and apoptosis [48]. Autophagy is involved with both cardiac protection and cardiac damage [49]. Evidences have demonstrated that autophagy under baseline conditions represents an important homeostatic mechanism for the maintenance of normal cardiovascular function and morphology whereas excessive autophagy has an important role in several types of cardiovascular diseases by functioning as a death pathway [50]. An inadequate autophagy appears to be a common pathway for myocardial diseases [51]. Thus, a delicate balance exists in which autophagy protects cardiomyocytes from external toxins, but over induction can lead to cell destruction. In this regard, persistent increase of autophagic activities may contribute to the development of HIV-1 cardiomyopathy.