Autophagy modulates the mechanism of flow-mediated dilation upstream of telomerase

Abstract

The non-canonical functions of telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase play a critical role in maintaining microvascular homeostasis utilizing both human and rodent models. Previously, we have demonstrated that intact autophagic flux is necessary for the beneficial effects of TERT to maintain microvascular function and redox status in human resistance arterioles. The purpose of this investigation was to examine (1) whether loss of TERT function in vivo resulted in reductions in autophagy/mitophagy and concomitant changes in the mediator of microvascular FMD; (2) whether restoration of autophagy can reverse this pathological switch in dilator mechanism, reduce shear-induced mitochondrial H₂O₂ production while enhancing NO production. TERT mutant rats were generated and compared to their WT counterparts. Rats were given an autophagy activator (2% trehalose) for 28-days. Isolated mesenteric arteries were used for videomicroscopy, and aortic tissue was collected for immunoblotting. FMD and autophagic flux were measured in arteries in all groups. Loss of TERT function resulted in a switch from NOS-dependent to H₂O₂-dependent FMD, repressed microvascular shear-induced autophagic flux and NO production, and increased mitochondrial H₂O₂ production. Activation of autophagy restored NO-mediated dilation in TERT mutant rats, and enhanced shear-induced autophagic flux. We provide evidence that autophagy is necessary for the beneficial role of TERT within maintaining microvascular function, positioning this pathway as a modifiable target to maintain microvascular health by rescuing the endothelial dysfunction caused by loss of TERT signaling.

Keywords: Autophagy; FMD; Microcirculation; Mitochondria; Mitophagy; TERT.

Fig. 1

Mutation of TERT switches the mechanism of flow-mediated dilation. WT rats demonstrate NO-mediated dilator responses to flow, inhibited by L-NAME (A), while TERT mutant rats demonstrate dependence upon H₂O₂ to elicit dilator responses to flow, inhibited by PEG-Cat (B). Mutation to TERT does impact endothelial-independent vasodilator responses as TERT mutant rats demonstrate similar dilator responses to papaverine (C). *p < 0.05 versus control curve. 2-way RM ANOVA, n = 6–13

Fig. 2

Shear-induced mitochondrial H₂O₂ was enhanced in TERT mutant rats relative to WT rats (A). Shear-induced changes in NO were repressed in TERT mutant rats (B). Mitochondrial H₂O₂ production (C) nor NO production (D) were different within genetic groups or between sexes. n = 3–15, unpaired t-test (A, B), Kruskal–Wallis test (C, D)

Fig. 3

Shear-induced autophagic flux (as measured by change in Lysotracker Red DND-99 fluorescent signal relative to static) was higher in WT relative to TERT mutant rats (A). BAFA1, a lysosome inhibitor, did not reduce the fluorescent signal to shear stress in both WT and TERT mutant rats (B, C). n = 3–8 rats, Mann–Whitney U-test *p < 0.05 versus TERT mutant rats

Fig. 4

Activation of autophagy with trehalose for 28 days did not alter the mechanism for flow-mediated dilation in WT rats, where dilation was still NO-mediated as L-NAME significantly reduced dilator responses (A). Conversely, activation of autophagy was sufficient to induce a switch from H₂O₂ to NO as the primary mechanism for microvascular FMD in TERT mutant rats (B). Activation of trehalose did not alter responses to papaverine in either WT or TERT mutant rats (C). *p < 0.05 versus control curve. n = 8–10, 2-way RM ANOVA (A, B), one-way ANOVA (C)

Fig. 5

Activation of autophagy with trehalose for 28 days did not alter either shear-induced mitochondrial H₂O₂ production (A) or NO production (B) in either group. n = 4–8, unpaired t-test

Fig. 6

Activation of autophagy with trehalose enhanced Lysotracker Red DND-99 fluorescence to a greater extent in WT relative to TERT mutant rats (A). Relative to untreated counterparts, activation of autophagy enhanced shear-induced autophagy in both WT and TERT mutant rats (B, C). †p < 0.05 versus untreated. n = 7–12