All studies were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University and conform to the Public Health Service Policy on Humane Care and Use of Laboratory Animals (2015) and the National Research Council “Guide for the Care and Use of Laboratory Animals” (Eighth Edition). Specific-pathogen free, male, New Zealand White rabbits were obtained from Robinson Services, Inc., and maintained in the vivarium at 19–22°C and a 12 h light, 12 h dark cycle for at least 6–7 days prior to experimentation. Animals were individually housed, provided environmental enrichment and fed a combination of pelleted high-fiber rabbit food (Harlan Teklad 2031, ~1 cup/day) and hay. An AMPK knockout (AMPKα2-/-) mouse colony was established by the Virginia Commonwealth University Transgenic/Knockout Mouse Core. Wild-type pathogen free mice were obtained from Jackson Laboratories and maintained in the vivarium at 22–23°C and a 12 h light, 12 h dark cycle for at least 3–4 days prior to experimentation. Mice (average weights ~23–30 g) were normally group housed (except for aggressive male mice, which were individually housed with added enrichment) and fed Envigo Teklad 7012 Rodent Diet ad lib.

Rabbits were euthanized by ketamine/xylazine/euthasol overdose as described previously with minor modifications (Ratz, 1990). Mice were euthanized by CO₂ asphyxiation. Immediately after sacrifice, rabbit mesentery and kidney, and mouse bladder, were rapidly removed and placed in cold (2.6°C) physiological salt solution (PSS, composition in mM: 140 NaCl, 4.7 KCl, 1.2 Na₂HPO₄-7H₂O, 2.0 MOPS, 0.02 Na₂ethylenediamine tetraacetic acid to chelate heavy metals, 5.6 D-glucose, 1.6 CaCl₂ and 1.2 MgCl₂, made with high-purity (17 MΩ) deionized water and adjusted using NaOH to a pH of 7.4). The renal artery (RA) and segments of mesenteric artery (MA) from the rabbit were cleaned by microdissection (Olympus SZX12) and cut into rings ~2.5 mm wide. Some rings of MA were secured in a tissue myograph (Model 610 M, Danish Myo Technology) and adjusted to the optimum length for muscle contraction (L₀) using an abbreviated length-tension protocol in which tissues were contracted for ~5 min with a maximum concentration of KCl (110 mM KCl substituted isosmotically for NaCl) (Ratz, 1990). Bladder rings were prepared by cutting away a small section of the dome and trigone. Active contractions were normalized to the maximum active force produced at L₀ by KCl (F/F₀), and the force-time integral was measured by taking the area under the force-time curve.

The ability of starvation to activate AMPK, and to alter phosphorylation of the downstream effectors acetyl-CoA carboxylase (ACC), myosin regulatory light chain (MLC), rhoA, cofilin, myosin phosphatase regulatory subunit (MYPT1), and phospholamban (PLB), was assessed by 1-dimensional sodium dodecylsulfate polyacrylaminde gel electrophoresis (1D SDS-PAGE) followed by “Western” blotting, enhanced chemiluminescence (ECL) and image analysis, as described previously (Ratz, 2001) with minor modifications. In all cases, for each tissue that underwent a perturbation there was a control tissue incubated for the same duration that did not undergo that perturbation and homogenates from both tissues were loaded onto the same gel. In most cases, for each gel the basal phosphorylation value (reported in arbitrary units) induced by tissue starvation was normalized to (divided by) the basal control phosphorylation value (reported in arbitrary units), and the resulting summary data were presented as fold-control. For example, for each of the four phosphoproteins and for β-actin shown in the representative Western blots in Figure 1A, the response produced by starvation (Figure 1A, column “S”) was divided by the control response (Figure 1A, column “C”). Thus, for each of the 5 rows in Figure 1A, upon normalization, each control value became “1” and the resulting summary starvation values were reported as “Fold-Control” (Figure 1B). In this way, each starvation value was statistically compared to its own control value, which was “1,” using a One-sample t-test (see below). In three cases where the level of basal phosphorylation was very low and near the threshold for analysis, the change in phosphorylation was normalized to a darker band produced when tissues were stimulated with phenylephrine (PE). In this case, values were reported as “Fold-PE” (see Figures 3B, 5B,D). In Figure 6, values obtained from tissues removed from the AMPKα2-/- (KO) mice (e.g., Figure 6A, KO column of representative Western blots) were normalized to (divided by) values obtained from tissues removed from the wild type (WT) mice (e.g., Figure 6A, WT column of representative Western blots), and resulting summary values were expressed as “Fold-WT.” At the appropriate time point, tissues were rapidly frozen in dry ice-cooled acetone containing 6% trichloroacetic acid, 10 mM dithiothreitol and 30 mM NaF, slowly thawed, dried, weighed and homogenized with a buffer containing 25 mM Tris- Base, 20 mM dithiothreitol, 10% glycerol, 1% sodium dodecylsulfate, 5 mM EGTA, 1 mM EDTA, 50 mM NaF, 1 mM activated Na⁺ orthovanandate, 10 μg/ml leupeptin, 10 μg/ml aprotonin and 1 mM AEBSF. Protein samples (15–20 μg/μl) were resolved by 1D SDS-PAGE and Western-blotted onto polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin at room temperature for 1 h, then incubated with an anti-phospho AMPK (pT172) antibody (1:1,000, Cell Signaling Technology, USA), anti-phospho ACC (pS79) antibody (1:1,000, Cell Signaling Technology, USA), anti-phosphocofilin (pS3) antibody (1:500, Cell Signaling Technology, USA), anti-phospho MLC (pS19) antibody (1:1,000, Sigma-Aldrich, USA), anti-phospho MYPT1 (T696 & T853) and antibodies (1:1,000, EMD Millipore, USA), anti-phospho PLB (pS16) antibody (1:1,000, Badrilla, UK), and anti-phospho rhoA (pS188) antibody (1:500, Santa Cruz Biotechnology, USA) at 4°C overnight. Goat-α-rabbit IgG- horseradish peroxidase was used as secondary antibody (1:2,000, Santa Cruz Biotechnology, USA) at room temperature for 1 h. All lanes were loaded with identical quantities of protein. However, two proteins expressed by housekeeping genes, β-actin and GAPDH, were routinely monitored to assess uniform protein loading across lanes. Data that did not display the expected equivalent protein loading were re-run or discarded. For this analysis, blots were incubated with β-actin antibody (C4) horseradish peroxidase (1:100,000, Santa Cruz, USA) and GADPH antibody (1:5,000, Santa Cruz, USA) at room temperature for 1 h. These proteins and the selected phospho-proteins were visualized using ECL (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA) and band intensities were detected, quantified and processed using the ChemiDoc Imaging System (BioRad, Hercules, CA, USA).

Active RhoA was determined using a RhoA G-LISA™ assay as recommended by the manufacturer (Cytoskeleton Inc., Denver, CO, USA) and as previously described (Alvarez et al., 2010). Briefly, lysates prepared by homogenizing quick-frozen tissues in ice-cold lysis buffer provided by the kit were clarified by centrifugation at 25,000 × g for 10 min at 4°C, adjusted to a final concentration of 1 mg/ml protein, and an equal volume of the samples was added to the wells of the Rho G-LISA plate coated with Rho-GTP-binding protein. The plate was shaken at 400 rpm at 4°C for 30 min. After three washes at room temperature using a wash buffer supplied by the manufacturer, 50 μl of anti-RhoA primary antibody (diluted 1:250) was added to each well and left on the shaker for 45 min. The plate was washed three times, and 50 μl of diluted HRP-labeled secondary antibody (1:250) was added to the wells and placed on the shaker at room temperature for 45 min. After three washes, 50 μl of horse radish peroxidase detection reagent was added to the wells, and the luminescence signal was detected using a microplate luminescence reader (Perkin Elmer 2030 Multilabel Reader, PerkinElmer Life and Analytical Science, Turku, Finland).

All data were analyzed using Graph Pad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA) and are presented as representative Western blots and force tracings and as the mean ± standard error of the mean (SE). In most experiments, the data from each tissue that underwent a perturbation was normalized to the response produced by a control tissue or to a tissue that was activated by phenylephrine (PE). Western blot data from each tissue obtained from an AMPKα2-/- (KO) mouse was normalized to a corresponding tissue obtained from a wild type (WT) mouse. That is, the data were reported as a ratio in which the denominator was the value “1.” Thus, these data were evaluated by the One-sample t-test using a comparison to the value “1,” and the null hypothesis was rejected at P < 0.05. When comparing a group again, but rather than to “1,” to a second group (Figures 3B, 5C,D), the Student's t-test with the Bonferroni correction was used, so the null hypothesis was rejected at P < 0.025 rather than P < 0.05 (Glantz, 1981). In Figure 4, contractions induced by 10 μM PE during starvation and recovery periods were compared to control contractions (respectively, “Control-1” and “Control-2”), and evaluated by the Student's t-test with rejection of the null hypothesis at P < 0.05.

Rings of rabbit renal artery were starved for 60 min by placing them in glucose-free PSS bubbled vigorously with 100% N₂, or retained for 60 min in an aerated, glucose-containing solution (control PSS) and quick-frozen to measure the basal levels of phosphorylation of certain contractile protein regulatory proteins, and of the metabolic stress-sensor, AMPK. After a 60 min starvation period, the basal level of AMPK-pT172 increased by ~9-fold (Figures 1A,B), indicating the starvation strongly activating the AMPK metabolic stress signal in this tissue. Concomitant with strong AMPK activation in tissues starved for 60 min the levels of basal cofilin-pS3, MLC-pS19, MYPT1-pT696, and MYPT1-pT853 were greatly reduced and barely detectable compared to the control values (Figure 1) (Ikebe and Hartshorne, 1985; Amano et al., 1996; Miyazaki et al., 2002; Muranyi et al., 2005; Bernard, 2007; Khromov et al., 2009; Mizuno, 2013; Ratz, 2016). Western blot analysis is only semi-quantitative. To examine the degree of inhibition of basal MYPT1-pT696 by starvation, blots were exposed for a longer duration that caused saturation of the control bands (Figure 1C, “C saturated”). Even under this condition, the bands representing starved tissues (“S” in Figure 1C, “C saturated”) remained nearly undetectable. In summary, the strong increase in AMPK activation and dramatic declines in basal phosphorylation levels of contractile protein regulatory proteins cofilin, MLC and MYPT1 suggest that starvation concomitantly activated the metabolic stress sensor AMPK and inactivated the smooth muscle contractile motor system.

After starvation for 60 min, rings of renal artery were placed in a recovery solution (aerated, glucose-containing, control PSS) for 5, 15, and 30 min and quick-frozen to measure indices of basal metabolic stress and basal contractile protein activation. In two tissues, the ability of renal artery to contract after 30 min of recovery was also assessed. Data from Figure 1 are reproduced as t = 0 data in Figures 2A–C to indicate the basal phosphorylation values after a 60 min starvation period. Basal AMPK-pT172 returned from the ~9-fold increase induced by starvation to the control basal level within 30 min (Figure 2A). Basal cofilin-pS3 increased modestly to ~25% of the control value within the first 5 min of incubation in the recovery solution, but full recovery did not occur even after 30 min (Figure 2B). Basal MLC-pS19, MYPT-1-pT696, and MYPT1-pT853 returned fully to the control level within 5 min (Figure 2C). Notably, the average level of MLC-pS19 was ~3.5-fold control at 5 min (although this apparent increase was not significantly different than control), and was significantly elevated 2-fold above control at 15 min, before returning to the control level by 30 min (Figure 2C). Following the increase to the control level within 5 min, basal MYPT1-pT696 and MYPT1-pT853 decreased to ~0.6-fold control by 15 min, and MYPT-pT696 remained at that level after the full 30 min recovery period (Figure 2C; although the average value for MYPT1-pT853 (0.54) was also < 1, this apparently low value was not significantly <1). These data reveal that, although basal AMPK-pT172 and MLC-pS19 returned rapidly and fully to the control levels during a 30 min recovery period, MLC-pS19 displayed a biphasic recovery that included an overshoot. Moreover, three other indices of contractile protein regulation, cofilin-pS3, MYPT1-pT696 and MYPT1-pT853, either did not fully recover (cofilin-pS3) or recovered within 5 min then fell to levels below the control, basal level. Despite recovery by cofilin-pS3 of only ~30%, assessment of contraction suggested that force-recovery was ~80% within 30 min (Figure 2D, n = 2).

For a comparison with renal artery, and to examine additional indices of AMPK and contractile protein activation during starvation and recovery from starvation, mesenteric artery was starved for a shorter duration (30 min) followed by recovery for 30 min. As in renal artery starved for 60 min, mesenteric artery starved for 30 min displayed strong reductions in basal MLC-pS19, MYPT1-pT696, and MYPT1-pT853 (Figure 3A), and a strong increase in AMPK-pT172 (see Figure 5A). Starvation for 30 min also increased the level of phosphorylation of two downstream AMPK substrates that would be expected to impact smooth muscle contractile activity, rhoA (Gayard et al., 2011; Figure 3A) and the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) regulator phospholamban (PLB) (Schneider et al., 2015; Figure 3B). Notably, compared to control tissue, basal PLB-pS16 was increased by ~2-fold in starved tissue, and ~3-fold in control tissue stimulated with 10 μM phenylephrine (Figure 3B).

Alternative explanations for reductions in rhoA activity include inhibition of a guanine nucleotide exchange factor (rhoA-GEF) or activation of a GTPase activating protein (rhoA-GAP) (Wang et al., 2011). Thus, we employing a rhoA activation assay kit to determine whether a 30 min starvation period caused a reduction in the relative level of active rhoA-GTP-to-inactive rhoA-GDP. Starved mesenteric artery displayed no change in the basal rhoA-GTP/rhoA-GDP level compared to the control basal value (Figure 3C). To confirm that rabbit mesenteric artery can display changes in the relative rhoA-GTP/rhoA-GDP level, we showed that phenylephrine elevated, and C3 transferase (cell permeable ADP-ribosyltransferase) reduced the basal level of rhoA-GTP in this tissue, and that C3 transferase prevented phenylephrine from elevating the level of rhoA-GTP (Figure 3C). α-adrenergic receptor activation by phenylephrine increases rhoA-GEF activity (Lutz et al., 2005; Docherty, 2010), and C3 transferase traps rhoA in the inactive rhoA-GDP state complexed with guanine nucleotide dissociation inhibitor, preventing rhoA-GDP activation to rhoA-GTP by GEFs (Aktories et al., 2004). Thus, the absence of a change in the rhoA-GTP/rhoA-GDP levels as assessed by the rhoA assay kit supports the hypothesis that starvation did not cause a reduction in basal rhoA activity by affecting the activities of a rhoA-GEF or rhoA-GAP.

The low basal levels of MLC-pS19, MYPT1-pT696, and MYPT1-pT853 induced in starved tissues suggested to us that a contractile stimulus may be unable to produce a strong contraction in these tissues. To test this hypothesis, mesenteric artery was starved for 30 min and stimulated with 10 μM phenylephrine during the final 5 min of this period. Control tissues were treated identically but were not starved. Compared to the strong and sustained contraction produced by control tissues (Figure 4A and zoomed image, Figure 4B, dotted lines), starved tissues produced a weaker and highly transient contraction (Figure 4A and zoomed image, Figure 4B, solid lines). The resulting area under the contraction curve (force-time integral) in starved tissues was reduced by ~66% compared to control (Figure 4C, PE₁). The early, phasic portion of contraction was reduced by ~50% (Figure 4D, PE₁), and the sustained, tonic portion was abolished (Figure 4E, PE₁). A subsequent 30 min recovery period induced by two washes in a normal glucose-containing, aerated, solution permitted complete recovery of contractile strength (Figures 4A,C–E, PE₂), revealing that the inhibition of contraction induced by a 30 min starvation period was not due to irreversible tissue damage. Thus, we used the term “fatigue” for the reversible inhibition of contraction due to metabolic stress.

To examine the effects of fatigue (reversible inhibition of contraction) on AMPK-pT172 and indices of contractile protein activation in mesenteric artery, tissues were treated as described in the experiment shown in Figure 4, and the levels of basal and phenylephrine-stimulated AMPK-p172, ACC-pS79 (the canonical AMPK substrate, Goransson et al., 2007), cofilin-pS3, and MLC-pS19 were assessed.

In mesenteric artery, a 5 min stimulation with phenylephrine alone did not increase AMPK-pT172 (Figure 5A, PE) but did cause an ~2-fold increase in phosphorylation of the AMPK substrate, ACC-pS79 (Figure 5B, note that all responses in panel B were normalized to the PE response, so the PE response was = 1, and the data were reported as “Fold-PE”; see “Methods”). The large increases in indices of AMPK activation induced by starvation were not further increased by phenylephrine (Figures 5A,B). Notably, AMPK-pT172 returned to the control level within 30 min of recovery (Figure 5A, Recovery). Interestingly, ACC-pS79 returned to the phenylephrine-stimulated value during the recovery phase (Figure 5B, Recovery and Recovery+PE were = 1).

As in renal artery, the level of cofilin-pS3 was greatly reduced in mesenteric artery by starvation, and recovery reversed the level toward, but not fully to, the control level (Figure 5C). However, the reversal in mesenteric artery starved for 30 min and recovered for 30 min was ~2-fold greater than that produced in renal artery starved for 60 min and recovered for 30 min (compare Figure 2B with Figure 5C, Recovery). Although the average values of cofilin-pS3 were reduced ~20% or more by phenylephrine in control tissues, during starvation, and upon recovery from starvation, these reductions were not significant (Figure 5C). Thus, the degree of metabolic stress had a dramatically greater effect on the level of cofilin-pS3 than did the contractile stimulus phenylephrine.

Phenylephrine produced an ~4-fold increase in MLC-pS19 under the control condition (Figure 5D). Interestingly, phenylephrine produced a nearly equivalent ~3-fold increase in MLC-pS19 in starved tissue. However, because the basal level of MLC-pS19 was depressed by ~80% in starved tissues, the ~3-fold increase induced by phenylephrine in starved tissues did not cause an elevation in MLC-pS19 above the control basal level. During recovery, the basal level of MLC-pS19 returned to the control basal level, and phenylephrine stimulation induced an increase that was equal to the increase induced in control tissues.

AMPKα2-/- mouse bladder expressed the same levels of AMPKα₁, MLC, MYPT1 and β-actin as wild-type (WT) mouse bladder and, as expected, expressed no detectable AMPKα₂ (Figures 6A,B). Notably, compared to WT, the bladder of the AMPKα2-/- mouse displayed an 0.5-fold lower basal level of AMPK-pT172, and an 0.5-fold higher basal level of MLC-pS19 (Figures 6C,D). Basal levels of cofilin-pS3, MYPT1-pT696, and MYPT1-pT853 were not different when comparing AMPKα2-/- and WT mouse bladders (Figures 6C,D). Together, these data support the hypothesis that the basal level of MLC-pS19 is constitutively negatively regulated by the basal level of AMPK-pT172 in mouse bladder.

To determine whether starvation and recovery from starvation of detrusor smooth muscle altered basal levels of phosphoproteins involved in contractile protein regulation, and the basal level of AMPK-pT172, mouse bladders were starved for 60 min or not starved (control), then permitted to recover, and quick-frozen as described above when using rabbit artery rings. As in arteries, starvation of mouse bladder increase the basal level of AMPK-pT172 and reduced basal levels of cofilin-pS3, MLC-pS19, MYPT1-pT696, and MYPT1-pT853 (Figures 7A,B). As in artery, recovery of contraction was complete within 30 min (Figure 7D), and this appeared to correlate with recovery of the basal phosphorylation states of key contractile protein regulatory proteins, and a return of AMPK-pT172 to a basal low level (Figure 7A, n = 1). Notably, AMPKα2-/- did not appear to prevent starvation from causing an increase in AMPK-pT172 and from abolishing basal MLC-pS19 (Figure 7C, compare WT to KO, n = 1), nor did it alter the degree and timing in the reduction in stimulus-induced force (compare Figures 7D,E, “Starve”) or the degree or timing in the return of contractile strength upon recovery from starvation (compare Figures 7D,E, “Recovery”).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.