Stimulation of soluble guanylyl cyclase in red blood cells protects the heart

In Study I we demonstrated that in individuals with T2D, RBCs exert a detrimental effect on cardiac performance following IR and that this effect is associated with reduced NO

bioactivity. Based on this finding, it is conceivable to postulate that stimulation of the NO signaling pathway in RBCs is a potentially attractive therapeutic strategy to protect the heart from IR injury and with a particular benefit in T2D. A previous study demonstrated that the entire signaling pathway for NO including a catalytically active sGC which produces cGMP are present in the RBCs (92). In Study III, we focused on sGC, the NO receptor in RBC, and used CYR715 which is a direct ferrous-dependent stimulator of sGC (191).

Figure 5. Effects of glycemic control on endothelial and cardiac dysfunction induced by RBCs from patients with T2D. (A) Endothelium-dependent relaxation of aortas following incubation of RBCs from patients with T2D under poor glycemic control (T2D RBC Visit 1) (n=9), patients with T2D under improved glycemic control (T2D RBC Visit 2) (n=9) and healthy subjects (H RBC) (n=6). Post-ischemic recovery of LVDP in hearts following administration of RBCs from (B) patients with T2D under poor glycemic control (T2D RBC Visit 1) (n=11), patients with T2D under improved glycemic control (T2D RBC Visit 2) (n=8) and healthy subjects (H RBC) (n=8); (C) T2D RBC Visit 1 incubated with vehicle (n=10) or the arginase inhibitor nor-NOHA (n=10); (D) T2D RBC Visit 2 incubated with vehicle (n=8) or nor-NOHA (n=8). Vessel relaxation to acetylcholine (ACh) is presented as percentage relaxation from pre-constriction. Post-ischemic LVDP is presented as percentage recovery from baseline. Data are presented as mean ± SD. Statistical differences were analyzed with 2-way ANOVA including all concentrations or time points. *p < 0.05, **p < 0.01 vs. H RBC. ###p < 0.001 vs. T2D RBC Visit 1+ vehicle. #p < 0.05 vs. T2D RBC Visit 2+ vehicle.

We found that recovery of LVDP was impaired and infarct size was greater in isolated rat hearts given RBCs from T2D patients subjected to IR in comparison with those given RBCs from healthy controls, which was in line with the observations in Study I. Importantly, the recovery of LVDP was enhanced and infarct size was reduced following incubation of RBCs from patients with T2D (Figure 6A, B) and healthy controls with the sGC stimulator

CYR715. The percentage improvement in post-ischemic recovery of LVDP and the reduction in infarct size induced by sGC stimulation was significantly greater in hearts given RBCs from T2D patients than those given RBCs from healthy individuals (p<0.01). Post-ischemic LVEDP was also reduced by pre-incubation of RBCs with CYR715. Collectively, these data

suggest sGC stimulator prevented the negative effect of RBCs from patients with T2D on cardiac post-ischemic function.

To investigate whether the sGC stimulator induce cardioprotection via an effect in the RBCs, CYR715 was administered with KH buffer only to isolated rat hearts (without RBCs). In the absence of RBCs, CYR715 failed to protect hearts against IR injury, which indicates that RBCs are essential for sGC stimulator to exert the cardioprotective effects.

Next, we studied the mechanisms behind the cardioprotection induced by the sGC stimulation in RBCs. The investigations were focused on patients with T2D since the therapeutic effect was more pronounced in RBCs from this group of subjects. To explore whether the beneficial effects could be enhanced by additional exogenous NO, the RBCs were incubated with CYR715 and the NO donor DEA NO. The post-ischemic recovery of LVDP induced by CYR715 in the presence of RBCs from patients with T2D was not further improved by the combined treatment. The improvement on cardiac recovery by CYR715 was abolished by the sGC inhibitor ODQ (Figure 6C), supporting that the cardioprotection induced by CYR715 in RBCs was sGC-dependent. ODQ per se did not affect cardiac function.

To further investigate the signaling transmission involved between the RBC and the heart, the inhibitor of cGMP transport MK571 was co-incubated with CYR715 and RBCs from patients with T2D. The cardioprotection induced by CYR715 as abolished by MK571 (Figure 6D), indicating that the export of cGMP is involved in the sGC-mediated protective effect. To further establish the export of a signaling molecule from RBCs to exert cardioprotection, supernatant from the co-incubation of RBCs from patients with T2D and CYR715 or vehicle was given to the isolated heart. Of note, administration of supernatant from the T2D RBCs incubated with CYR715 improved post-ischemic recovery of LVDP, and this

cardioprotective effect of the supernatant was abolished by addition of MK571 to the co-incubation of RBCs and CYR715 (Figure 6E). Moreover, the infarct size was smaller in hearts given supernatant from T2D RBCs incubated with CYR715 compared to that in hearts given supernatant from vehicle-incubated RBCs. Finally, quantification of cGMP revealed that cGMP levels increased significantly in the supernatant from T2D RBCs incubated with CYR715 (Figure 6F). To verify that cGMP exerts cardioprotection in the present model and to mimic the effect of the supernatant following combined incubation of RBCs from patients with T2D and CYR715, 8-bromo cGMP in KH buffer was given to the isolated heart. This resulted in enhanced LVDP recovery compared to KH buffer with vehicle.

To identify the down-stream target in the heart, cardiac PKG-dependent phosphorylation of VASP was determined. The expression of pVASP was increased in the heart following administration of RBCs incubated with CYR715 (Figure 6G, H and I). Collectively, these

data suggest that stimulation of RBC sGC induces activation of cardiac PKG to protect the heart from IR injury.

Figure 6. Stimulation of sGC in RBCs from patients with T2D protect the heart from ischemia-reperfusion injury. (A) Post-ischemic recovery of LVDP and (B) infarct size in hearts following administration of RBCs from patients with T2D incubated with vehicle(n=10) or the sGC stimulator CYR715 (n=10). Post-ischemic recovery of LVDP in hearts following administration of RBCs from patients with T2D incubated with (C) CYR715 (n=5) or the sGC inhibitor ODQ (n=5), (D) CYR715 (n=7) or inhibitor of cGMP transporter MK571 (n=7). (E) Post-ischemic recovery of LVDP in hearts following administration of supernatant from RBCs of patients with T2D incubated with vehicle (n=5), CYR715 (n=5) or CYR715+MK571 (n=4). (F) cGMP levels in supernatant from RBCs of patients with T2D incubated with vehicle (n=8) or CYR715 (n=8). (G) Expression of phosphorylated VASP (pVASP), VASP and GAPDH in hearts given RBCs from patients with T2D incubated with vehicle or CYR715. Post-ischemic LVDP is presented as percentage recovery from baseline. cGMP levels are expressed in absolute value of concentration. Data are presented as mean ± SD. Statistical differences were analyzed with 2-way ANOVA including all time points in A, C-E. Wilcoxon signed rank test was performed in B and F. Student’s t-test was performed in H and I. **p < 0.01, **p < 0.01, ***p < 0.001 vs. vehicle.

The molecular signaling of the biological effects of RBCs in cardiovascular regulation has been a matter of discussion. NO is demonstrated to be produced by active eNOS in RBCs, but it remains unknown if and how NO is exported from RBCs and exert any biological activity since it rapidly scavenged by hemoglobin. The term “NO bioactivity” is often used to refer to the biological activity mediated by the NO-sGC signaling (176) since the molecular nature of the signaling remains unclear. The current results add to this discussion by suggesting that the RBCs protect the ischemic heart following stimulation of sGC by exporting cGMP that mediates the cardioprotection. The administration of cGMP analogue in RBC-free

supernatant in hearts protected against IR injury also suggested that the cardioprotection we observed following sGC stimulation may be mediated by cGMP. Further supports to this notion are the the findings that sGC stimulation in the RBCs led increases in the

concentration of cGMP in the supernatant from RBCs and phosphorylation of cardiac VASP as a marker of cGMP-dependent PKG activation. cGMP, known as a second messenger, mediates several important physiological processes including muscle relaxation, platelet inhibition and improve contractility and cardioprotection (199-201). Our finding has potential therapeutic implications by providing evidence that the detrimental ex vivo cardiac effect of

RBCs from patients with T2D can be attenuated by sGC stimulation and this effect is achieved by actions in the RBCs.

4.6 Red blood cells from patients with STEMI mediate cardioprotection via purinergic