The dynamic nature of the microtubule network is dependent in part by post-translational modifications (PTMs) — particularly through acetylation, which stabilizes the microtubule network. Whether PTMs of the microtubule network in vascular smooth muscle cells (VSMCs) contribute to the pathophysiology of hypertension is unknown. The aim of this study was to determine the acetylated state of the microtubule network in the mesenteric arteries of spontaneously hypertensive rats (SHR). Experiments were performed on male normotensive rats and SHR mesenteric arteries. Western blotting and mass spectrometry determined changes in tubulin acetylation. Wire myography was used to investigate the effect of tubacin on isoprenaline-mediated vasorelaxations. Isolated cells from normotensive rats were used for scanning ion conductance microscopy (SICM). Mass spectrometry and Western blotting showed that tubulin acetylation is increased in the mesenteric arteries of the SHR compared with normotensive rats. Tubacin enhanced the β-adrenoceptor-mediated vasodilatation by isoprenaline when the endothelium was intact, but attenuated relaxations when the endothelium was denuded or nitric oxide production was inhibited. By pre-treating vessels with colchicine to disrupt the microtubule network, we were able to confirm that the effects of tubacin were microtubule-dependent. Using SICM, we examined the cell surface Young's modulus of VSMCs, but found no difference in control, tubacin-treated, or taxol-treated cells. Acetylation of tubulin at Lys40 is elevated in mesenteric arteries from the SHR. Furthermore, this study shows that tubacin has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation.

The microtubule network forms part of the cell cytoskeleton that regulates a variety of cellular functions, including maintaining the structure of the cell and providing a platform for intracellular transport. The microtubule network is a dynamic structure composed of α- and β-tubulin dimers. The ability of the tubulin dimers to polymerize and depolymerize in response to intracellular and extracellular signals gives the microtubule network a ‘dynamic instability’. This dynamic instability can be affected by many factors, including microtubule associated proteins and post-translational modifications (PTMs) of the tubulin proteins.

Recently, our group revealed that the microtubule network and dynein motor proteins are responsible for the retrograde trafficking of certain ion channels and receptors from the cell membrane in vascular smooth muscle cells (VSMCs), which can affect the contractility of arteries. We have shown that by reducing microtubule stability with either colchicine or nocodazole, we could improve vasodilatations elicited by Kv7 channel activators and β adrenergic receptor agonists in arterial segments from both normotensive and hypertensive rats [1–3]. Furthermore, in hypertensive humans, we found that oral colchicine administration improved arterial conductance to isoprenaline and sodium nitroprusside infusions within 1.5 h of ingestion [4]. However, to date, it is not clear if the VSMC microtubule network is altered in hypertension, and whether changes to the network could contribute to the pathogenesis of hypertension.

PTMs are chemical modifications of a polypeptide chain that occur after DNA has been transcribed into RNA and translated into protein, and are crucial for controlling protein stability, localization, and conformation. PTMs introduced to a particular protein will affect interacting partners and downstream signaling; therefore, PTMs have a major impact on almost all biological processes [5,6]. Acetylation at the Lys40 site of α-tubulin promotes microtubule stabilization, thereby reducing the dynamic instability of the microtubule network [7]. Acetylation at this site is regulated via the interplay of histone acetyltransferases — namely α-tubulin acetyltransferase-1 (αTAT1) — and histone deacetylases (HDACs), where HDAC-6 is the main contributor. Acetylation stabilizes the microtubules in their polymerized state by causing a conformational shift in the binding site of Lys40, resulting in a reduction in the inter-protofilament interactions between microtubule filaments [8]. These reduced interactions ultimately result in a decrease in the inter-filament-associated lattice damage, which provides the microtubules with increased stability, increased microtubule self-repair, and an increased ability for the microtubules to bend [9]. Importantly, this ability to enhance microtubule bending can also lead to increased resistance of the microtubules to mechanical stress stimuli [9,10].

The inability to maintain a balance of the acetylated tubulin state is known to contribute to various diseases in different cell types, including Alzheimer's disease (decreased acetylation) [11], Huntington's disease (decreased acetylation) [12], pancreatic [13] and breast cancer (increased acetylation) [14], and cardiomyocyte remodeling implicated in atrial fibrillation (decreased acetylation) [15]. To date, the acetylation state of the microtubules in resistant arteries is not well understood, nor is it known whether changes occur in hypertension. Therefore, the aim of this study was to investigate acetylation of tubulin in mesenteric arteries from normotensive and spontaneously hypertensive rats (SHR) — the use of which serves as a model for hypertension in humans.

Tubulin acetylation is increased in whole mesenteric arteries from tubacin-treated and SHR vessels

To identify and quantify tubulin acetylation sites in SHR and WKY rats, we used LC–MS/MS combined with peptide mapping. A combination of trypsin and Glu-C was used to yield a peptide that would allow us to detect acetylation specific to the Lys40 site on α-tubulin. Indeed, we were able to detect a 28 amino acid-long peptide from α-tubulin (HGIQPDGQMPSDKTIGGGDDSFNTFFSE), which contained the acetylated Lys40 site and methionine oxidation at Met36 (Figure 1A,C). We quantified the levels of this modified peptide, and found that there was a 1.94-fold increase in the samples from SHR vs. WKY rats (n = 4, P = 0.0075; Figure 1B). Despite an overall sequence coverage for α-tubulin of 37% no further acetylation sites were conclusively detected in this protein. Furthermore, the volcano plot displayed in Figure 1D demonstrates that Lys40 in α-tubulin is the only acetylation site that is significantly up-regulated in SHR vs. WKY rats. To rule out that the increased abundance of the acetylated peptides is not due to a general increase in the abundance of α-tubulin, we also performed quantification at the protein level. As shown in Figure 1E, no significant up-regulation of α-tubulin was detected in SHR vs. WKY rats, supporting that up-regulation is specific for the Lys40 site.

In-depth profiling of acetylation sites of tubulin in mesenteric arteries from spontaneously hypertensive rats (SHR).

Figure 1.
In-depth profiling of acetylation sites of tubulin in mesenteric arteries from spontaneously hypertensive rats (SHR).

(A) Amino acid (AA) sequence for the TUBA1A α-tubulin isoform. Mesenteric arteries from Wistar Kyoto rats (WKY) and SHRs were immunoprecipitated for acetylated tubulin and analyzed via LC–MS/MS. The peptide used for analysis was a 28 AA long peptide HGIQPDGQMPSDKTIGGGDDSFNTFFSE (highlighted in yellow) for α-tubulin, where the lysine in the sequence was identified to be the K40 site (red) with an acetylation modification. The M36 site (green) was identified to have an oxidation modification. All underlined AAs represent identified peptide sequences by LC–MS/MS, constituting a 37% peptide coverage for the complete α-tubulin AA sequence. (B) Mean log2-transformed peptide intensity ± SEM of WKY and SHR (n = 4). An unpaired students t-test was used to compare conditions, where ** represents P < 0.01 (P = 0.0075). There was a 1.94 fold increase in acetylation at this site in the SHR compared with WKY. (C) Representative LC–MS/MS spectrum of the identified TUBA1A α-tubulin peptide HGIQPDGQMPSDKTIGGGDDSFNTFFSE. A deconvoluted fragmentation spectrum of the [M + 3H]3 + precursor ion at m/z 1014.773 corresponding to the peptide with a retained K40 acetylation modification (▾) and M36 oxidation modification (∎). (D) Volcano plot of acetylated peptides identified in WKY and SHR rats. The peptides highlighted in red (HGIQPDGQMPSDKTIGGGDDSFNTFFSE and HGIQPDGQMPSDKTIGGGDDSFNTFFSETGAGK) are the only acetylated peptides that are significantly up-regulated in SHR vs. WKY rats. (E) Volcano plot of proteins identified in WKY and SHR rats with α-tubulin highlighted in red.

Figure 1.
In-depth profiling of acetylation sites of tubulin in mesenteric arteries from spontaneously hypertensive rats (SHR).

(A) Amino acid (AA) sequence for the TUBA1A α-tubulin isoform. Mesenteric arteries from Wistar Kyoto rats (WKY) and SHRs were immunoprecipitated for acetylated tubulin and analyzed via LC–MS/MS. The peptide used for analysis was a 28 AA long peptide HGIQPDGQMPSDKTIGGGDDSFNTFFSE (highlighted in yellow) for α-tubulin, where the lysine in the sequence was identified to be the K40 site (red) with an acetylation modification. The M36 site (green) was identified to have an oxidation modification. All underlined AAs represent identified peptide sequences by LC–MS/MS, constituting a 37% peptide coverage for the complete α-tubulin AA sequence. (B) Mean log2-transformed peptide intensity ± SEM of WKY and SHR (n = 4). An unpaired students t-test was used to compare conditions, where ** represents P < 0.01 (P = 0.0075). There was a 1.94 fold increase in acetylation at this site in the SHR compared with WKY. (C) Representative LC–MS/MS spectrum of the identified TUBA1A α-tubulin peptide HGIQPDGQMPSDKTIGGGDDSFNTFFSE. A deconvoluted fragmentation spectrum of the [M + 3H]3 + precursor ion at m/z 1014.773 corresponding to the peptide with a retained K40 acetylation modification (▾) and M36 oxidation modification (∎). (D) Volcano plot of acetylated peptides identified in WKY and SHR rats. The peptides highlighted in red (HGIQPDGQMPSDKTIGGGDDSFNTFFSE and HGIQPDGQMPSDKTIGGGDDSFNTFFSETGAGK) are the only acetylated peptides that are significantly up-regulated in SHR vs. WKY rats. (E) Volcano plot of proteins identified in WKY and SHR rats with α-tubulin highlighted in red.

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To confirm the results of the mass spectrometry analysis, we performed Western blots on protein lysate from whole mesenteric arteries from SHRs and WKY rats. We found that acetylation was also increased in the mesenteric artery protein lysate of the SHR compared with WKY rats (n = 5, P = 0.008; Figure 2A). Following this, both cultured rat aortic smooth muscle cells (A7r5) and mesenteric arteries from normotensive Wistar Hannover rats displayed increased tubulin acetylation after treatment with 2.5 µM tubacin, compared with DMSO-treated controls (n = 3 A7r5 cell lysates, P = 0.0003; n = 6 rats, P = 0.002; Figure 2B,C).

Tubulin acetylation is increased in SHR mesenteric arteries, tubacin-treated A7r5 cells, and tubacin-treater Wistar Hannover mesenteric arteries.

Figure 2.
Tubulin acetylation is increased in SHR mesenteric arteries, tubacin-treated A7r5 cells, and tubacin-treater Wistar Hannover mesenteric arteries.

(i and ii) Representative Western blot showing acetylated tubulin and α-tubulin levels in (A) mesenteric arteries of WKY and SHR, (B) DMSO and tubacin (2.5 µM)-treated A7r5 cells, and (C) DMSO and tubacin (2.5 µM)-treated arteries of Wistar Hannover rats. (iii) Mean ratio of acetylated tubulin/α-tubulin ± S.E.M. in Western blots. An unpaired Student's t-test was used to compare conditions, where ** and *** represent P < 0.01 and 0.001, respectively.

Figure 2.
Tubulin acetylation is increased in SHR mesenteric arteries, tubacin-treated A7r5 cells, and tubacin-treater Wistar Hannover mesenteric arteries.

(i and ii) Representative Western blot showing acetylated tubulin and α-tubulin levels in (A) mesenteric arteries of WKY and SHR, (B) DMSO and tubacin (2.5 µM)-treated A7r5 cells, and (C) DMSO and tubacin (2.5 µM)-treated arteries of Wistar Hannover rats. (iii) Mean ratio of acetylated tubulin/α-tubulin ± S.E.M. in Western blots. An unpaired Student's t-test was used to compare conditions, where ** and *** represent P < 0.01 and 0.001, respectively.

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Acetylation of the microtubule network has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation

With isometric tension wire-myography, we investigated the physiological impact of increased microtubule acetylation in freshly isolated rat mesenteric arteries. In the presence of 2.5 µM tubacin, the vasorelaxation to isoprenaline was enhanced compared with the DMSO control (n = 7–8, P = 0.028; Figure 3A). However, in the presence of L-NAME, which inhibits NO synthesis, tubacin attenuated the isoprenaline-induced vasorelaxation (n = 8, P = 0.015; Figure 3A).

Acetylation of the microtubule network has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation.

Figure 3.
Acetylation of the microtubule network has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation.

(A) Representative isometric tension recordings of normotensive Wistar Kyoto rat (WKY) mesenteric arteries pre-constricted with 10 µM methoxamine (•) and subsequently treated with increasing concentrations of isoprenaline. Arterial segments were pre-treated with (i) DMSO or (iii) 2.5 µM tubacin. To assess the contribution of the endothelium to vasorelaxation, arteries were pre-treated with (ii) L-NAME (100 µM) or with (iv) tubacin + L-NAME. (v) Mean concentration-effect curves showing the effect of tubacin and L-NAME on isoprenaline-mediated vasorelaxation. (vi) Mean LogEC50 values ± S.E.M. depicting isoprenaline-mediated vasorelaxation. An ordinary one-way ANOVA followed by Tukey's multiple comparisons test was used to compare conditions, where *P < 0.05, **P < 0.01, and ****P < 0.0001 (n = 7–8). (B) Representative isometric tension recordings of spontaneously hypertensive rat (SHR) mesenteric arteries pre-constricted with 10 µM methoxamine (•) and subsequently treated with increasing concentrations of isoprenaline. Vessels were treated with (i) DMSO or (iii) 2.5 µM tubacin. To assess the contribution of the endothelium to vasorelaxation, arteries were treated with (ii) L-NAME (100 µM) or with (iv) tubacin + L-NAME. (v) Mean concentration-effect curves showing the effect of tubacin and L-NAME on isoprenaline-mediated vasorelaxation. (vi) Mean LogEC50 values ± S.E.M. depicting relaxation (n = 6–9). An ordinary one-way ANOVA followed by Tukey's multiple comparisons test was used to compare conditions, where *P < 0.05.

Figure 3.
Acetylation of the microtubule network has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation.

(A) Representative isometric tension recordings of normotensive Wistar Kyoto rat (WKY) mesenteric arteries pre-constricted with 10 µM methoxamine (•) and subsequently treated with increasing concentrations of isoprenaline. Arterial segments were pre-treated with (i) DMSO or (iii) 2.5 µM tubacin. To assess the contribution of the endothelium to vasorelaxation, arteries were pre-treated with (ii) L-NAME (100 µM) or with (iv) tubacin + L-NAME. (v) Mean concentration-effect curves showing the effect of tubacin and L-NAME on isoprenaline-mediated vasorelaxation. (vi) Mean LogEC50 values ± S.E.M. depicting isoprenaline-mediated vasorelaxation. An ordinary one-way ANOVA followed by Tukey's multiple comparisons test was used to compare conditions, where *P < 0.05, **P < 0.01, and ****P < 0.0001 (n = 7–8). (B) Representative isometric tension recordings of spontaneously hypertensive rat (SHR) mesenteric arteries pre-constricted with 10 µM methoxamine (•) and subsequently treated with increasing concentrations of isoprenaline. Vessels were treated with (i) DMSO or (iii) 2.5 µM tubacin. To assess the contribution of the endothelium to vasorelaxation, arteries were treated with (ii) L-NAME (100 µM) or with (iv) tubacin + L-NAME. (v) Mean concentration-effect curves showing the effect of tubacin and L-NAME on isoprenaline-mediated vasorelaxation. (vi) Mean LogEC50 values ± S.E.M. depicting relaxation (n = 6–9). An ordinary one-way ANOVA followed by Tukey's multiple comparisons test was used to compare conditions, where *P < 0.05.

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In mesenteric arteries from the SHR, the isoprenaline-mediated relaxation was impaired compared with WKY controls (n = 9–11, P = 0.012; Figure 3B and Supplementary Figure S1). In the presence of 2.5 µM tubacin, isoprenaline-induced relaxations were enhanced in the mesenteric arteries from the SHR (n = 8–9, P = 0.038; Figure 3B). In the presence of L-NAME, the tubacin-induced enhancement of the isoprenaline response was abrogated (n = 8–9, P = 0.015; Figure 3B). These data suggest an endothelial-dependent bimodal effect of tubacin on the isoprenaline-mediated vasorelaxation, with tubacin enhancing the relaxation through a NO-dependent pathway, but inhibiting the relaxation through its effects on the VSMCs.

Tubacin has no effect when the microtubule network is disrupted

To ascertain whether the bimodal effect of tubacin on isoprenaline-mediated vasorelaxations was due to a direct effect on microtubule stability, we tested the effect of tubacin in the presence of the microtubule disruptor, colchicine. In the presence of 500 µM colchicine, segments of mesenteric artery relaxed equally to isoprenaline in the absence and presence of tubacin (n = 7–8, P = 0.99; Figure 4). In the presence of L-NAME, colchicine treatment prevented tubacin from attenuating isoprenaline-mediated relaxations (n = 8, P = 0.97; Figure 4).

Tubacin has no effect when the microtubule network is disrupted.

Figure 4.
Tubacin has no effect when the microtubule network is disrupted.

(i) Mean concentration-effect curves showing the effect of tubacin, L-NAME, and colchicine on vasorelaxation. (ii) Mean LogEC50 values ± S.E.M. depicting relaxation. An ordinary one-way ANOVA followed by Tukey's multiple comparisons test was used to compare conditions, where no results were significantly different (n = 7–8).

Figure 4.
Tubacin has no effect when the microtubule network is disrupted.

(i) Mean concentration-effect curves showing the effect of tubacin, L-NAME, and colchicine on vasorelaxation. (ii) Mean LogEC50 values ± S.E.M. depicting relaxation. An ordinary one-way ANOVA followed by Tukey's multiple comparisons test was used to compare conditions, where no results were significantly different (n = 7–8).

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Effect of methoxamine and U46619 on tubacin-treated mesenteric artery contractility

To test whether tubacin affected certain vasoconstrictor responses, we applied 2.5 µM tubacin in the absence and presence of 100 µM L-NAME to segments of rat mesenteric artery mounted in a wire myograph. Neither tubacin, L-NAME, nor a combination of the two had any effect on the mean Log EC50 calculated from the concentration-effect curve that was elicited by the α1-adrenoceptor agonist, methoxamine (DMSO control vs. DMSO L-NAME P = 0.12, DMSO control vs. tubacin control P = 0.99, DMSO control vs. tubacin L-NAME P = 0.55; n = 5; Figure 5A). With the thromboxane A2 receptor agonist, U46619, tubacin had no effect on the concentration-effect curves in the absence of L-NAME (n = 6, P = 0.92; Figure 5B). However, in the presence of L-NAME, tubacin enhanced the vasoconstriction elicited by U46619 (n = 5–6, P = 0.011; Figure 5B).

Effect of methoxamine and U46619 on tubacin-treated mesenteric artery contractility.

Figure 5.
Effect of methoxamine and U46619 on tubacin-treated mesenteric artery contractility.

(A) Representative isometric tension recordings of normotensive rat mesenteric arteries treated with increasing doses of methoxamine to measure vasoconstriction. Vessels were treated with (i) DMSO or (iii) 2.5 µM tubacin. To investigate whether NO availability interfered with vasoconstriction, arteries were treated with (iii) L-NAME (100 µM) or with (iv) tubacin + L-NAME. (v) Mean concentration-effect curves showing the effect of tubacin and L-NAME on vasoconstriction. (vi) Mean LogEC50 values ± S.E.M. depicting vasoconstriction. An ordinary one-way ANOVA followed by Tukey's multiple comparisons test was used to compare conditions (n = 5). (B) Representative isometric tension recordings of normotensive rat mesenteric arteries treated with increasing doses of U46619 to measure vasoconstriction. Arterial segments were treated with (i) DMSO or (iii) 2.5 µM tubacin. To assess whether NO availability interfered with vasoconstriction, arteries were treated with (ii) L-NAME (100 µM) or with (iv) tubacin + L-NAME. (v) Mean concentration-effect curves showing the effect of tubacin and L-NAME on vasoconstriction. (vi) Mean LogEC50 values ± S.E.M. depicting vasoconstriction. An ordinary one-way ANOVA followed by Tukey's multiple comparisons test was used to compare conditions, where *P < 0.05 (n = 5–6).

Figure 5.
Effect of methoxamine and U46619 on tubacin-treated mesenteric artery contractility.

(A) Representative isometric tension recordings of normotensive rat mesenteric arteries treated with increasing doses of methoxamine to measure vasoconstriction. Vessels were treated with (i) DMSO or (iii) 2.5 µM tubacin. To investigate whether NO availability interfered with vasoconstriction, arteries were treated with (iii) L-NAME (100 µM) or with (iv) tubacin + L-NAME. (v) Mean concentration-effect curves showing the effect of tubacin and L-NAME on vasoconstriction. (vi) Mean LogEC50 values ± S.E.M. depicting vasoconstriction. An ordinary one-way ANOVA followed by Tukey's multiple comparisons test was used to compare conditions (n = 5). (B) Representative isometric tension recordings of normotensive rat mesenteric arteries treated with increasing doses of U46619 to measure vasoconstriction. Arterial segments were treated with (i) DMSO or (iii) 2.5 µM tubacin. To assess whether NO availability interfered with vasoconstriction, arteries were treated with (ii) L-NAME (100 µM) or with (iv) tubacin + L-NAME. (v) Mean concentration-effect curves showing the effect of tubacin and L-NAME on vasoconstriction. (vi) Mean LogEC50 values ± S.E.M. depicting vasoconstriction. An ordinary one-way ANOVA followed by Tukey's multiple comparisons test was used to compare conditions, where *P < 0.05 (n = 5–6).

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Acetylation of the microtubule network does not affect cell surface Young's modulus in VSMCs

To test whether increased acetylation affected VSMC stiffness, we used scanning ion conductance microscopy (SICM) to measure the Young's modulus (YM) of cells treated with DMSO, 2.5 µM tubacin, and 10 µM taxol. Live cell imaging was conducted using SICM in order to produce VSMC topographical maps, as well as YM maps (Figure 6A–C). To detect any change in YM, we first measured a 30 µm × 30 µm area to depict most of the entire VSMC. We then focused in a 3 µm × 3 µm area on the nucleus (nuclear region) and on the peripheral area of the cell (perinuclear region) with a higher resolution, to account for the difficulties associated with measuring the slope regions around the edges of the VSMC. When measuring the YM of the whole cell, we found no difference in cellular stiffness between DMSO vs. tubacin (P = 0.98; Figure 6D), DMSO vs. taxol (P = 0.99; Figure 6D), or tubacin vs. taxol (P = 0.96; Figure 6D). Similarly, we found no difference in the YM of nuclear regions of DMSO vs. tubacin (P = 0.84; Figure 6E), DMSO vs. taxol (P = 0.85; Figure 6E), or tubacin vs. taxol (P = 0.48; Figure 6E). Finally, we found no difference in the YM of perinuclear regions of DMSO vs. tubacin (P = 0.99; Figure 6F), control vs. taxol (P = 0.99; Figure 6F), or tubacin vs. taxol (P = 0.95; Figure 6F).

Acetylation of the microtubule network does not affect cell surface Young's modulus in vascular smooth muscle cells.

Figure 6.
Acetylation of the microtubule network does not affect cell surface Young's modulus in vascular smooth muscle cells.

(i) Representative 30 × 30 µm topographical images (top) and Young's modulus (YM) images (bottom) of vascular smooth muscle cells (VSMCs) isolated fresh from mesenteric arteries and treated with (A) DMSO, (B) 2.5 µM tubacin, or (C) 10 µM taxol. Scale bar on the left denotes cell height (µm) and on the right denotes YM (Pa). (ii) Representative topographical images of focused ROI consisting of 3 × 3 µm areas fixated on the nuclear region (top) or perinuclear region (bottom) for freshly isolated VSMCs from mesenteric arteries that were treated with (A) DMSO, (B) tubacin, or (C) taxol. Scale bar (right) denotes cell height (µm). (iii) Representative YM images of focused ROI consisting of 3 × 3 µm areas fixated on the nuclear region (top) or perinuclear region (bottom) for freshly isolated VSMCs from mesenteric arteries that were treated with (A) DMSO, (B) tubacin, or (C) taxol. Scale bar (right) denotes YM (Pa). (D–F) Summary graphs of the mean YM values ± S.E.M. for (D) whole cell, (E) nuclear region, and (F) perinuclear region. One-way ANOVA test was used to compare conditions (n = number of cells/N = number of isolations, control 20/8; tubacin 26/8; taxol 19/8). Each dot represents an individual cell belonging to a separate N (depicted by different colors).

Figure 6.
Acetylation of the microtubule network does not affect cell surface Young's modulus in vascular smooth muscle cells.

(i) Representative 30 × 30 µm topographical images (top) and Young's modulus (YM) images (bottom) of vascular smooth muscle cells (VSMCs) isolated fresh from mesenteric arteries and treated with (A) DMSO, (B) 2.5 µM tubacin, or (C) 10 µM taxol. Scale bar on the left denotes cell height (µm) and on the right denotes YM (Pa). (ii) Representative topographical images of focused ROI consisting of 3 × 3 µm areas fixated on the nuclear region (top) or perinuclear region (bottom) for freshly isolated VSMCs from mesenteric arteries that were treated with (A) DMSO, (B) tubacin, or (C) taxol. Scale bar (right) denotes cell height (µm). (iii) Representative YM images of focused ROI consisting of 3 × 3 µm areas fixated on the nuclear region (top) or perinuclear region (bottom) for freshly isolated VSMCs from mesenteric arteries that were treated with (A) DMSO, (B) tubacin, or (C) taxol. Scale bar (right) denotes YM (Pa). (D–F) Summary graphs of the mean YM values ± S.E.M. for (D) whole cell, (E) nuclear region, and (F) perinuclear region. One-way ANOVA test was used to compare conditions (n = number of cells/N = number of isolations, control 20/8; tubacin 26/8; taxol 19/8). Each dot represents an individual cell belonging to a separate N (depicted by different colors).

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In this study, we have revealed that mesenteric arteries from the SHR show increased tubulin acetylation at the Lys40 site compared with normotensive rats. In addition, we found that increasing tubulin acetylation pharmacologically had an endothelial-dependent bimodal effect on arterial relaxation to isoprenaline.

Our group has shown that the microtubule network can regulate the membrane abundance of certain receptors and ion channels in the plasma membrane of VSMCs. Previously, our laboratory showed that disruption of the microtubule network results in improved vasorelaxation via Kv7 channel-mediated pathways [1–3]. These studies focused on the ability of the microtubule network to act as a platform for intracellular signaling and found that the dynein motor protein could traffic Kv7 channels and β2 adrenoceptors away from the plasma membrane in VSMCs. Remarkably, we found that disruption of the microtubule network (and inhibition of dynein movement) could enhance isoprenaline- and Kv7 channel activator-mediated relaxations in arteries from both normotensive rats and SHRs [2]. The effect of destabilizing the microtubule network to improve vasorelaxation suggests that VSMCs require the microtubule network to maintain its dynamic instability, rather than becoming overly stabilized. However, the state of the microtubule network in arteries from hypertensive animals (or humans) had not been studied previously.

Tubulin PTMs are recognized to change the dynamic instability properties of the microtubule network as a whole. Acetylation of Lys40 is one of the best characterized and well recognized PTMs on tubulin leading to its increased stability [8]. In this study, we show for the first time that tubulin in SHR mesenteric arteries has increased Lys40 acetylation compared with normotensive rats. This was investigated by quantification at both peptide and fragmentation (amino acid) level using mass spectrometry. These data show, in an unbiased manner, that the Lys40 site specifically has increased acetylation levels in the protein lysate from SHR mesenteric arteries compared with the normotensive rats. From the same mass spectrometry dataset, we show that the change in Lys40 acetylation was not due to a change in total tubulin protein abundance. The increase in Lys40 acetylation in the SHR mesenteric arteries was confirmed by Western blot. In addition, we used Western blotting to establish that the HDAC-6 inhibitor, tubacin, could increase tubulin acetylation in both A7r5 cells and normotensive rat mesenteric arteries.

Having established that tubacin could increase tubulin acetylation levels in rat mesenteric arteries, we investigated whether this would correspond to a change in arterial function. We focused on isoprenaline as we know that the relaxation is attenuated in the SHR and have previously associated the relaxation with the function of the microtubule network [2,3]. It has been reported previously that tubacin can enhance eNOS activity by stabilizing eNOS mRNA transcripts. This effect is independent of its ability to inhibit HDAC-6, but can ultimately lead to increased NO production by endothelial cells [16]. In arterial segments treated with L-NAME, tubacin had an inhibitory effect on the isoprenaline-mediated relaxation. Our data support an effect of tubacin on NO production in endothelial cells, which can enhance certain vasorelaxations, such as those elicited by isoprenaline. However, when eNOS is inhibited by L-NAME, tubacin attenuated the isoprenaline-mediated relaxation. We also investigated the effect of tubacin on vasoconstrictions elicited by U46619 and methoxamine, but there was no effect of tubacin on the ability of either of these agonists to induce a constriction. It is possible that tubacin increased acetylation on several other proteins, which may underlie the attenuated isoprenaline relaxation. To confirm this effect was microtubule-dependent, we disrupted the microtubule network with colchicine. Colchicine enhanced the isoprenaline-mediated relaxation, as reported previously [2,3], and abrogated the effect of tubacin. Thus, when NO bioavailability is reduced, increased microtubule acetylation, induced by tubacin, can attenuate isoprenaline-mediated relaxation.

Tubacin enhanced the isoprenaline-mediated relaxation in the SHR when the endothelium was intact; however, we did not observe an attenuation of the isoprenaline-mediated relaxation in the presence of L-NAME. These suggest the microtubule network-forming tubulin in the VSMCs of the SHR are close to fully acetylated, which is not the case in arteries from normotensive control rats.

HDAC-6 inhibitors have been proposed to have beneficial effects in certain models of hypertension [17–19]. In our study, tubacin was unable to improve the isoprenaline-mediated relaxation in the presence of L-NAME. Our data suggest that further work is required to elucidate the benefit of HDAC-6 inhibitors in hypertension associated with endothelial dysfunction [20], since the loss of eNOS and NO bioavailability may create a paradigm in which HDAC-6 inhibitors are detrimental to vascular health. Thus, HDAC-6 inhibitors used in the treatment of hypertension may increase tubulin acetylation in VSMCs and suppress vasorelaxations, without providing the associated benefit of increased NO production.

Arterial stiffness is an important characteristic of hypertension, which augments the severity of this disease [21]. Using atomic force microscopy, Sehgel et al. [22] found that aortic VSMCs of SHRs have increased stiffness, and that VSMC stiffness can directly reflect arterial stiffness. For this reason, we used SICM to investigate whether tubacin treatment would also increase VSMC stiffness. Stiffness was interpreted by YM, which is a reflection on the impressionability of the cell membrane in response to provocation via a hydrojet. However, we found no difference in cell rigidity from tubacin-treated VSMCs compared with DMSO controls. Similarly, we found no difference in taxol-treated VSMCs compared with either DMSO controls or tubacin-treated cells. These data suggest that acute changes in the stability of the microtubule network do not dictate or affect VSMC rigidity, at least in the short-term. Thus, while the rigidity of VSMCs is not affected by acute tubacin treatment, our molecular and functional myography data suggests that the dynamic nature of the microtubule network has been altered through increased acetylation, which can affect vascular reactivity.

This study shows that tubulin acetylation is increased in arteries from hypertensive animals, however we have not investigated why this change occurs nor whether it is a cause or consequence of the hypertensive state. One possible explanation for the increase in tubulin acetylation may be due to an adaptive effect of the microtubule network in VSMCs. In hypertension, there is a systemic increase in the transmural pressure exerted on the vascular wall. When the microtubule network is acetylated, it becomes more flexible, allowing it to have increased resistance to mechanical stress [9,10]. Thus, increased acetylation could be an adaptive response to early hypertension, to mitigate the impact of increased mechanical wall stress and tension across the arterial wall. However, much more work would have to be devoted to uncovering this link between the acetylated state and a subsequent myogenic response.

In conclusion, using a variety of techniques, we have shown that tubulin acetylation is increased in the mesenteric arteries of hypertensive rats compared with normotensive controls. Furthermore, we have shown that treatment with the HDAC-6 inhibitor, tubacin, has an endothelial-dependent bimodal effect on the isoprenaline-mediated vasorelaxation. These findings suggest that HDAC-6 inhibitors may have detrimental effects in the treatment of hypertension, especially when endothelial dysfunction is present.

Ethical approval

Except the SICM experiments, all other experiments were performed at the Department of Biomedical Sciences, Panum Institute, University of Copenhagen. These experiments were performed in accordance with Directive 2010/63EU on the protection of animals used for scientific purposes, and approved by the national ethics committee and by local Animal Care and Use Committees (institutional approval numbers P19-006, P21-117, and P23-023). Rats were supplied by Janvier labs. Once received, rats were group housed at the Panum Institute, University of Copenhagen and supplied with ad libitum food and water access. Clean cages were provided once a week and rats were kept on a 12 h/12 h light/dark cycle. In accordance with the methods of killing animals described in annex IV of the EU Directive 2010/63EU on the protection of animals used for scientific purposes, rats were made unconscious by a single, percussive blow to the head. Immediately after the onset of unconsciousness, cervical dislocation was performed.

The SICM experiments were performed at the National Heart and Lung Institute, Imperial College London. These experiments were carried out under the approval of the Animal Welfare and Ethics Review Board (AWERB) of Imperial College London, in accordance with the United Kingdom Home Office Guide on the Operation of the Animals (Scientific Procedures) Act 1986 and EU Directive 2010/83.

Culturing of rat aortic smooth muscle cells — A7r5

A7r5 cells (kindly provided by Vladimir V. Matchkov, University of Aarhus [23]) were grown in Dulbecco's modified Eagle's medium containing, glutamax, 1% penicillin/streptomycin, and 10% FBS (Substrate Department, the Panum Institute, University of Copenhagen) in a humidified cell incubator at 37°C with 5% CO2. A7r5 were passaged (between passage 5–10) into 6-well plates prior to tubacin treatment. When the cells reached a 70–80% confluency, the cells were washed with prewarmed PBS, and incubated with 2.5 µM tubacin, dissolved in the culture media, for 30 min at 37°C with 5% CO2. After incubation, the cells in each well were washed with ice-cold PBS and harvested in 200 µl RIPA lysis buffer (in mM): 50 Tris, pH 8.0, 150 NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and protease inhibitor cocktail (Roche). The cells in RIPA buffer were transferred into a 1.5 ml tube, and incubated on ice for 15 min. After centrifugation at 11 000 g for 10 min at 4°C to remove cell debris, the supernatant was collected followed by protein quantitation using a bicinchoninic acid Protein Assay kit (Thermo Fisher Scientific).

Protein isolation

The animals used in this study were eight male Wistar Hannover rats, eleven Wistar Kyoto (WKY), nine Spontaneous Hypertensive Rats (SHR) aged 10–15 weeks, and eight male Sprague–Dawley rats weighing 250–300 g, which corresponds to ∼10 weeks old. Once sacrificed, mesenteric arteries were dissected from each of the respective rats, third-order arteries were used for myography or cell work, and the remainder of the arteries were flash frozen in liquid nitrogen to be used for protein isolation. A subset of arteries from the Wistar Hannover rats were incubated for 1 h with 2.5 µM tubacin at 37°C in a bath filled with physiological saline solution (PSS), containing (in mM): (121 NaCl, 25 NaHCO3, 5 MgSO4·7H2O, 2.8 KCl, 5.5 glucose, 2.77 EDTA, 1.17 KH2PO4, and 1.6 CaCl2) and bubbled with 95% O2/5% CO2. Arteries were homogenized in 100 µl of denaturing buffer (150 mM NaCl, 50 mM Tris–HCl (pH 8.0), 1% nonidet P-40, 1% SDS, 10 µl/ml protease inhibitor cocktail). Once homogenized, samples were centrifuged at 10 000 g for 10 min, and the supernatant was transferred to a fresh tube. Protein levels were measured using Bradford assays (Bio-Rad). Here, samples were diluted 1:10 in PBS. This dilution was then mixed 1:20 in Bradford reagent, and protein concentration was measured at 595 nm wavelength (SpectraMax i3x, Molecular Devices, San Jose, CA, U.S.A.). Samples were measured in triplicates and compared with a standard curve of bovine serum albumin (BSA) that was also in triplicates, which ranged from 0 to 1000 µg/ml (SoftMax Pro 7, Molecular Devices). Protein concentration was measured by averaging the triplicates and using a standard curve of six points based on these BSA standards.

Immunoprecipitation

For immunoprecipitation, samples were prepared according to Dynabeads Protein G Immunoprecipitation Kit (Thermo Fisher Scientific). Briefly, 1.5 mg Dynabeads were transferred to a 1.5 ml tube. The tube was placed on a magnet to separate the beads, and the supernatant was removed. The beads were resuspended in 4 µg anti-acetylated-tubulin antibody (Sigma, T7451) diluted in 200 µl antibody binding buffer for 30 min at room temperature. The complex was then placed on a magnet, the supernatant removed, and washed once with binding buffer. The complex was again placed on the magnet and the supernatant was removed. Next, 100 µg lysated mesenteric arteries were resuspended with the beads in 200 µl PBS. The bead-antigen complex was incubated overnight at 4°C with rotation to allow for sufficient antigen binding to the bead-antibody complex. The next day, the suspension was placed on a magnet and the supernatant was removed. The complex was then washed three times with 200 µl washing buffer. After the third wash, the suspension was transferred to a clean tube to avoid over-elution of proteins bound to the tube wall. The new tube was placed on a magnet and the supernatant was removed. Finally, the beads were resuspended in 20 µl elution buffer (50 mM glycine, pH 2.8), heated for 10 min at 70°C, and placed on a magnet. The supernatant containing the eluted proteins was transferred to a fresh tube and stored at −80°C until ready for MS preparation.

Mass spectrometry (LC–MS/MS)

Immunoprecipitated samples that were eluted from Dynabeads were prepared using a modified filter-aided sample preparation (FASP) protocol [24,25]. Briefly, samples were diluted to 200 µl with digestion buffer (50 mM tetraethylammonium bromide with 0.5% sodium deoxycholate (SDC)) and heat-treated for 5 min at 95°C. Samples were transferred to 0.5 ml Amicon Ultra centrifugal flat spin filters (UFC5003, Sigma), then centrifuged at 14 000 g for 15 min and the flow-through was discarded. Samples were then reduced and alkylated by adding a solution to a final concentration of 10 mM tris(2-carboxyethyl)phosphine (Sigma) and 50 mM 2-chloroacetamide (CAA) (Sigma) that was diluted in digestion buffer. The solution was vortexed and incubated for 30 min at 37°C. The samples were then centrifuged at 14 000 g for 15 min and the flow-through was discarded. Next, 200 µl digestion buffer was added to the filters and was centrifuged at 14 000 g for 15 min, discarding the flow-through. The spin filter was then transferred to a new collection tube. Samples were then incubated overnight at 37°C in 50 µl digestion buffer with 1 µg trypsin (Promega), 1 µg Glu-C (Promega), and 1% ProteaseMax (Promega). The next day, the samples were centrifuged at 14 000 g for 15 min. The flow-through was saved, an additional 100 µl digestion buffer was added to the filter, and the sample was centrifuged again at 14 000 g for 15 min to gather any remaining peptides trapped in the filter. The 150 µl flow-through was saved for desalting.

After FASP and digestion, samples were desalted with two layers of styrene divinylbenzene reversed-phase sulfonate (SDB-RPS) discs (Affinisep), as previously described by Rappsilber et al. [26]. Briefly, the samples were mixed 1:5 in 99% isopropanol/1% trifluoroacetic acid (TFA), vortexed, and loaded onto two SDB-RPS STAGE-Tips (Affinisep, France, SPE-Disk-Bio-RPS-M.25.40). Samples were then centrifuged at 2000 g until they completely ran through the STAGE-Tips. The STAGE-Tips were then washed twice more with 100 μl 99% isopropanol/1% TFA and centrifuged until the solution passed through. STAGE-Tips were then washed twice with 100 μl 0.2% TFA. To elute the peptides from the STAGE-Tips, 80 μl elution buffer (80% acetonitrile (ACN)/2% ammonia) were added to the STAGE-Tip and centrifuged into a fresh Eppendorf Lo-bind tube (Germany, 022431081). Eluted samples were then centrifuged in a SpeedVac (Genevac EZ-2, Sysmex, Japan) at 40°C for 2 h, until completely dry. Dried samples were reconstituted in 6 μl buffer A* (2% ACN/0.1% TFA) and stored at −80°C until MS analysis. Samples were prepared in triplicates for LC–MS/MS analysis.

Peptides samples were separated on an Aurora C18 column (75 µm × 25 cm, 1.9-µm particle size, IonOpticks) using a Dionex Ultimate 3000RSnano chromatography system (Thermo Fisher Scientific) connected on-line to a timsTOF PRO mass spectrometer (Bruker). Peptides were eluted over a 42 min gradient elution system composed of mobile phase A (0.1% formic acid) and phase B (99.9% acetonitrile/0.1% formic acid) at a flow rate of 400 nl/min. The mass spectrometer was operated in DDA-PASEF mode with 1.1 s cycle time, TIMS ramp time of 100 ms, and a 100–1700 m/z scan range.

For peptide identification and quantification, a database search against the rat UniProt reference proteome (UP000002494_10116 and UP000002494_10116_additional.fasta, August 2020) was performed using MaxQuant version 2.1.3.0 and Fragpipe version 20.0. For digestion, Trypsin/P and GluC enzymes with two missed cleavages were used. Match between runs was set to true, with a matching time window of 0.7 min and an alignment time window of 20 min. The MS1 tolerance and MS/MS tolerance were both set to 20 ppm. Cysteine carbamidomethylation was set as a fixed modification, and variable modifications included methionine oxidation (+15.9949 Da), N-terminal and side-chain acetylation (+42.0106 Da). The maximum number of modifications per peptide for all PTMs was set to 5. The false discovery rate was set to 1%.

For quantification of the α-tubulin peptide containing the Lys40 site (HGIQPDGQMPSDKTIGGGDDSFNTFFSE), data was loaded into Skyline version 23.1.0.268. The output MS1 intensities from triplicate samples of each biological replicate (SHR vs. WKY mesenteric arteries) were averaged, and the log(2) transformation was applied. Quantitative analysis for volcano plots were performed in Perseus software (version 1.6.14.0).

Data are available via ProteomeXchange via the PRIDE [27] partner repository with identifier PXD045602.

Western blot analysis

To assess tubulin acetylation, 10 µg (cells) or 20 µg (arteries) of protein was prepared with sample buffer and reducing agent (Invitrogen). Samples were then denatured at 70°C for 10 min, loaded onto SDS–PAGE gels (4–12% Bis-Tris, Invitrogen), subjected to electrophoresis, and transferred onto a polyvinylidene fluoride membrane (Immobilon-FL, Sigma–Aldrich). The membrane was blocked for an hour in a 50:50 mixture of PBS-Tween (0.1%) and PBS blocking buffer (Li-Cor Biosciences), and subsequently incubated overnight at 4°C with acetylated tubulin antibodies (1:1000, Sigma, T7451). The next day, the membrane was washed three times in PBS-Tween (0.1%) for 10 min each. The membrane was then incubated for 1 h at room temperature with fluorescently-conjugated secondary antibodies raised in donkey against mouse (1:10 000, Li-Cor Biosciences, 926-68072). The membrane was washed twice in PBS-Tween (0.1%) for 10 min each, and then once in PBS for 10 min. Membranes were then imaged at 700 nm wavelength using an Odyssey CLx (Li-Cor Biosciences) and analyzed using Image Studio (Version 5.2, Li-Cor Biosciences). The membrane was then incubated overnight at 4°C in α-tubulin antibodies (1:1000, Cell Signaling, 2144). The next day, the membrane was washed three times in PBS-Tween (0.1%) before incubation for 1 h at room temperature with fluorescently-conjugated secondary antibodies raised in goat against rabbit (1:10 000, Li-Cor Biosciences, 926-32211). The membrane was washed twice in PBS-Tween (0.1%) for 10 min each, and then once in PBS for 10 min. Membranes were imaged and analyzed on the Odyssey CLx at 800 nm wavelength (Li-Cor Biosciences) and analyzed using Image Studio (Version 5.2, Li-Cor Biosciences). To confirm equal amounts of protein were loaded, membranes were subsequently incubated with anti-GAPDH (1:5000, Abcam, 181602) antibody for 1 h at room temperature, washed three times for 10 min each in PBS-Tween (0.1%), and incubated with fluorescently-conjugated secondary antibodies in goat raised against rabbit (1:10 000, Li-Cor Biosciences). The membrane was then washed twice in PBS-Tween (0.1%) for 10 min each, once in PBS for 10 min, imaged at 800 nm wavelength using the Odyssey CLx (Li-Cor Biosciences), and analyzed using Image Studio (Version 5.2, Li-Cor Biosciences).

Myography

Isometric tension, wire-myography (Danish Myo Technology, Aarhus, Denmark) experiments were performed on 2 mm sections of third-order mesenteric arteries that were mounted on two 40 µm stainless steel wires. The myograph baths were filled with PSS, maintained at 37°C, and aerated with 95% O2/5% CO2. Arteries were left to rest for a period of ∼15 min, and subsequently subjected to a passive force normalization procedure [28]. Changes in tension were recorded using LabChart 8.1.24 (ADInstruments, Oxford, U.K.). Arteries were constricted with 10 µM methoxamine and treated with 1 µM carbachol to determine the endothelial integrity. Arteries were then washed several times with PSS. Vessels displaying a poor endothelial response (<60% relaxation) were incubated with 100 µM N(G)-Nitro-L-arginine methyl ester (L-NAME) 5 min prior to tubacin administration. Arteries, both with and without L-NAME, were then subsequently treated with either DMSO or 2.5 µM tubacin for 1 h. In a subset of experiments, arteries were additionally treated with either DMSO or 500 µM colchicine for 30 min. After incubation, all vessels were pre-constricted with 10 µM methoxamine and isoprenaline was applied in a concentration-dependent manner from 1 nM to 3 µM.

To determine the effect of tubacin on vasoconstriction, the alpha-1 adrenergic receptor agonist methoxamine (0.1–30 µM), or the thromboxane receptor agonist U46619 (1 nM to 10 µM) were applied to arterial segments in the absence or presence of 2.5 µM tubacin.

Mesenteric artery smooth muscle cell isolation

Third-order mesenteric arteries were dissected, cleared of any adherent fat, and placed in a smooth muscle dissociation solution (SMDS) (60 mM NaCl, 80 mM sodium glutamate, 5 mM KCl, 2 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4)). Arteries were incubated in SMDS at 37°C for 10 min. Arteries were then placed in SMDS containing BSA (1 mg/ml, Sigma), papain (0.5 mg/ml, Sigma), and dithriothrietol (1.5 mg/ml, Sigma) at 37°C for 10 min. These arteries were then washed five times in SMDS and transferred to SMDS containing BSA (1 mg/ml, Sigma), collagenase F (0.7 mg/ml, Sigma), and collagenase H (0.2 mg/ml, Sigma) at 37°C for 10 min. Arteries were then washed an additional five times in SMDS. Single myocytes were then liberated via trituration with a fire-polished pipette. Cells were then supplemented with 100 µM CaCl2 and were immediately used for SICM experimentation after treatment.

Scanning ion conductance microscopy

After cell isolation, cells were incubated with DMSO, 2.5 µM tubacin, or 10 µM taxol for 30 min at 37°C. Cells were then washed with SMDS once to remove any dead cells, and subsequently placed in a bath containing SMDS supplemented with 100 µM CaCl2. Cells were then imaged using the ‘hopping mode’ of SICM [29]. SICM is a non-invasive imaging technique that allows for the topographical imaging of live cells, that can be modified for the measurement of the surface cell stiffness (Mechano-SICM) [30], which is represented via the YM. This variant of SICM is used to ensure the structural integrity of the live cell by retracting the nanopipette to a position above the cell and subsequently approaching the cell for imaging. Nanopipettes used in this study had a tip resistance of 20–30 MΩ, pulled from borosilicate capillary glass (1.0 mm O.D.; 0.5 mm I.D.) using a laser puller (IntraCel, Sutter Instrument Co, P-2000) [29]. The pipette electrode head-stage was connected to an Axopatch 200A amplifier (Molecular Devices) mounted on the stage of a Nikon Diaphot 200 inverted microscope (Nikon Corporation, Japan). Importantly, a pulse of aerostatic constant pressure (15 kPa) was delivered to the pipette holder to produce a hydrojet of solution on the tip of the nanopipette, applied during the scanning acquisition. This hydrojet, in turn, displaces the cell membrane, and records this change in displacement. Three setpoints of the current-distance dependence were selected, one to map the topography (0.7% of current reduction) and two (SP1 and SP2) to map the YM (1% and 2% current reduction, respectively). From the changes observed between SP1 and SP2, the YM is calculated, as described before [31]. Nanopipettes were filled with a standard buffer salt solution containing 144 mM NaCl, 10 mM HEPES, 1 mM MgCl2, and 5 mM KCl (pH 7.4). Using the SICM scanner software, images of 30 µm × 30 µm were taken to study whole cell topography and stiffness. Due to the vertical position of the pipette over the cells in this technique, and the height and shape of VSMCs, measuring topography and YM on the steep slope of the edges of these cells proves difficult and inaccurate in practice. Therefore, a region of interest (ROI) was set in a 3 µm × 3 µm area on top of the nucleus (nuclear region) and on the peripheral area of the cell (perninuclear region), avoiding the edges, to accurately measure any changes in topography or YM.

Images were analyzed using SICM image viewer software. For the 30 µm × 30 µm whole cell images, six ROI circles of 2 µm diameter along the middle region of the cell were measured and averaged to obtain one value per cell. For the 3 µm × 3 µm images, all the values on the whole scan were measured and averaged to produce one value per scan.

Statistical analysis

All figures reported data as mean ± S.E.M., and all analyses used GraphPad Prism 9.5.1. To compare myography experiments, the mean LogEC50 was calculated from individual experiments and compared using an unpaired t-test for experiments with two groups, or one-way ANOVA followed by Tukey's multiple comparisons test for experiments with more than two groups. Unpaired t-tests were also used for WB, MS (transformed and averaged intensities), and ICC experiments. Once individual values were obtained for whole cell, nuclear region, and perinuclear region, values were compared using a one-way ANOVA followed by Tukey's multiple comparisons test for all SICM data.

The authors agree to make any materials, data, code, and associated protocols available upon reasonable request. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [27] partner repository with the dataset identifier PXD045602.

The authors declare that there are no competing interests associated with the manuscript.

A.M.M., J.A.B. and this project were funded by a Lundbeck Foundation grant awarded to T.A.J. [R323-2018-3674]. J.A.B. was also funded by his own Lundbeck Foundation grant [R400-2022-1213].

Thomas Jepps: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing — original draft, Project administration, Writing — review and editing. Anthony M. Mozzicato: Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing — original draft. Joakim A. Bastrup: Conceptualization, Software, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing — review and editing. Jose L. Sanchez Alonso: Data curation, Formal analysis, Supervision, Investigation, Visualization, Methodology, Writing — original draft, Writing — review and editing. Jennifer van der Horst: Data curation, Formal analysis, Investigation, Methodology, Writing — review and editing. Julia Gorelik: Resources, Supervision, Investigation, Methodology, Writing — review and editing. Per Mårten Hägglund: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Visualization, Methodology, Writing — review and editing.

We would like to thank the Core Facility for Integrated Microscopy, University of Copenhagen, for their services and continued dedication to improving microscopy at our institution. We are grateful to the Faculty of Health and Medical Sciences Graduate School, University of Copenhagen, for providing financial assistance for A.M.M. to visit J.G. and J.L.S.-A. at Imperial College, London.

BSA

bovine serum albumin

FASP

filter-aided sample preparation

HDAC

histone deacetylase

PSS

physiological saline solution

PTM

post-translational modification

ROI

region of interest

SHR

spontaneously hypertensive rats

SICM

scanning ion conductance microscopy

SMDS

smooth muscle dissociation solution

TFA

trifluoroacetic acid

VSMC

vascular smooth muscle cell

YM

Young's modulus

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Supplementary data