Disruption of the eHSP70-to-iHSP70 ratio impairs vascular function in diabetic rats

Background: Heat-shock protein 70 (HSP70) co-exists in the intra- and extra-cellular (iHSP70 and eHSP70, respectively) environment. While iHSP70 is cytoprotective, eHSP70 modulates membrane-bound receptors, such as Toll-like receptor 4 (TLR4), acting as a damage-associated molecular pattern (DAMP). We have demonstrated that HSP70 also assists in vascular reactivity, the blood vessel’s ability to constrict and dilate. However, the contributions of HSP70 to the vascular phenotype of diabetes are unknown. Hypothesis: We hypothesized that disruption of the eHSP70-to-iHSP70 contributes to aortic vascular dysfunction in diabetes. Methods: Male Sprague Dawley rats (7-8 weeks) were injected with a single dose of streptozotocin (STZ; 65 mg/kg i.p. diluted in citrate buffer, pH 4.5), leading to type 1 diabetes. Control (CTL) animals were dosed with only the vehicle. Animals were kept for 28 days in a controlled environment (temperature = 22±1° C; humidity: 42±2%). Systemic and tissue (aorta) levels of HSP70 were determined with Western blotting. Vascular responses to phenylephrine were assessed in aortic rings incubated with or without VER155008, a small molecule inhibitor of HSP70. Using an established protocol, we also evaluated calcium dynamics as a function of force development in the vehicle and VER-treated samples. The impact of voltage-dependent and -independent plasmalemmal calcium channels on this process was indirectly estimated with specific pharmacological inhibitors. In another set of experiments, we incubated aortic rings isolated from CTL animals with serum (30% v/v, 6 hours) from CTL (low HSP70 levels) or diabetic (high HSP70 levels) rats, and we observed force-displacement following the modulation of TLR4 and its co-adaptor MD2. Since diabetic serum also has higher levels of other DAMPs, we exposed aortic rings to exogenous purified recombinant HSP70 (10 ng/ml, 10 minutes). Results: We report that diabetes disrupts the eHSP70-to-iHSP70 ratio in the aorta. STZ-induced diabetic rats display phenylephrine-induced hypercontractility, which can be prevented by blocking HSP70. Specifically, inhibition of HSP70 abolished increased force development in the phasic and tonic parts of the contraction curve. Subsequently, we found that the blockade of HSP70 further impaired the hyporeactivity of aortic rings to the α-1 adrenergic agonist, both in response to calcium release from the sarcoplasmic reticulum and calcium influx from the extracellular milieu. The latter seems to involve a complex interaction with voltage-independent plasmalemmal channels. Lastly, we observed that diabetic serum shifts the phenylephrine concentration-response curve to the left, which is averted by blocking TLR4 or MD2. Similar results were observed in vessels exposed to exogenous HSP70. Conclusion: HSP70 plays a dual role in diabetic aortas, reduced iHSP70 contributes to calcium mishandling, and elevated eHSP70 modulates the TLR4-MD2 complex. RCW (HL134604, DK132948); KPN (R15DK131511). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.