Dissecting the interaction between HSP70 and vascular contraction: role of $$\hbox{Ca}^{2+}$$ handling mechanisms

Heat-shock protein 70 (HSP70) is a ubiquitously expressed molecular chaperone with various biological functions. Recently, we demonstrated that HSP70 is key for adequate vascular reactivity. However, the specific mechanisms targeted by HSP70 to assist in this process remain elusive. Since there is a wealth of evidence connecting HSP70 to calcium ($$\hbox {Ca}^{2+}$$ Ca 2 + ), a master regulator of contraction, we designed this study to investigate whether blockade of HSP70 disrupts vascular contraction via impairment of $${\text{Ca}}^{2+}$$ Ca 2 + handling mechanisms. We performed functional studies in aortas isolated from male Sprague Dawley rats in the presence or absence of exogenous $$\hbox {Ca}^{2+}$$ Ca 2 + , and we determined the effects of VER155008, an inhibitor of HSP70, on $$\hbox {Ca}^{2+}$$ Ca 2 + handling as well as key mechanisms that regulate vascular contraction. Changes in the intracellular concentration of $$\hbox {Ca}^{2+}$$ Ca 2 + were measured with a biochemical assay kit. We report that blockade of HSP70 leads to $$\hbox {Ca}^{2+}$$ Ca 2 + mishandling in aorta stimulated with phenylephrine, decreasing both phasic and tonic contractions. Importantly, in $$\hbox {Ca}^{2+}$$ Ca 2 + free Krebs’ solution, inhibition of HSP70 only reduced the $$\hbox {E}_{\mathrm{max}}$$ E max of the phasic contraction if the protein was blocked before IP3r-mediated $$\hbox {Ca}^{2+}$$ Ca 2 + release, suggesting that HSP70 has a positive effect towards this receptor. Corroborating this statement, VER155008 did not potentiate an IP3r inhibitor’s outcomes, even with partial blockade. In another set of experiments, the inhibition of HSP70 attenuated the amplitude of the tonic contraction independently of the moment VER155008 was added to the chamber (i.e., whether it was before or after IP3r-mediated phasic contraction). More compelling, following re-addition of $$\hbox {Ca}^{2+}$$ Ca 2 + , VER155008 amplified the inhibitory effects of a voltage-dependent $$\hbox {Ca}^{2+}$$ Ca 2 + channel blocker, but not of a voltage-independent $$\hbox {Ca}^{2+}$$ Ca 2 + channel inhibitor, indicating that HSP70 has a positive impact on the latter. Lastly, the mechanism by which HSP70 modulates vascular contraction does not involve the $$\hbox {Ca}^{2+}$$ Ca 2 + sensitizer protein, Rho-kinase, nor the SERCA pump, as blockade of these proteins in the presence of VER155008 almost abolished contraction. In summary, our findings shed light on the processes targeted by HSP70 during vascular contraction and open research avenues for potential new mechanisms in vascular diseases.

The Rolf Method of Structural Integration and Pelvic Floor Muscle Facilitation: Preliminary Results of a Randomized, Interventional Study

The management of pelvic floor dysfunctions might need to be based on a comprehensive neuro-musculoskeletal therapy such as The Rolf Method of Structural Integration (SI). The aim of the study was to evaluate the pelvic floor muscle (PFM) after the tenth session of SI by using surface electromyography (sEMG). This was a randomized, interventional study. Thirty-three healthy women were randomly assigned to the experimental (SI) or control group. The outcome measures included PFM bioelectrical activity, assessed using sEMG and endovaginal probes. An intervention in the SI group included 60 min of SI once a week, and teaching on how to contract and relax PFMs; in the control group, only the teaching was carried out. In the SI group, a significant difference was found between the PFM sEMG activity during “pre-baseline rest” (p < 0.014) and that during “rest after tonic contraction” (p = 0.021) in the supine position, as were significant increases in “phasic contraction” in the standing position (p = 0.014). In the intergroup comparison, higher PFM sEMG activity after the intervention “phasic contraction” (p = 0.037) and “pre-baseline rest” (p = 0.028) was observed in the SI group. The SI intervention significantly changes some functional bioelectrical activity of PFMs, providing a basis for further research on a new approach to PFM facilitation, particularly in clinical populations.

Transition From Phasic to Tonic Contractility in Airway Smooth Muscle After Birth: An Experimental and Computational Modeling Study

Fetal airway smooth muscle (ASM) exhibits phasic contractile behavior, which transitions to a more sustained “tonic” contraction after birth. The timing and underlying mechanisms of ASM transition from a phasic to a tonic contractile phenotype are yet to be established. We characterized phasic ASM contraction in preterm (128 day gestation), term (∼150 day gestation), 1–4 month, 1 yr, and adult sheep (5yr). Spontaneous phasic activity was measured in bronchial segments as amplitude, frequency, and intensity. The mechanism of phasic ASM contraction was investigated further with a computational model of ASM force development and lumen narrowing. The computational model comprised a two-dimensional cylindrical geometry of a network of contractile units and the activation of neighboring cells was dependent on the strength of coupling between cells. As expected, phasic contractions were most prominent in fetal airways and decreased with advancing age, to a level similar to the level in the 1–4 month lambs. Computational predictions demonstrated phasic contraction through the generation of a wave of activation events, the magnitude of which is determined by the number of active cells and the strength of cell–cell interactions. Decreases in phasic contraction with advancing age were simulated by reducing cell–cell coupling. Results show that phasic activity is suppressed rapidly after birth, then sustained at a lower intensity from the preweaning phase until adulthood in an ovine developmental model. Cell–cell coupling is proposed as a key determinant of phasic ASM contraction and if reduced could explain the observed maturational changes.

Hypoxia-induced contraction of chicken embryo mesenteric arteries: mechanisms and developmental changes

The fetal cardiovascular responses to acute hypoxia include a redistribution of the cardiac output toward the heart and the brain at the expense of other organs, such as the intestine. We hypothesized that hypoxia exerts a direct effect on the mesenteric artery (MA) that may contribute to this response. Using wire myography, we investigated the response to hypoxia (Po2 ~2.5 kPa for 20 min) of isolated MAs from 15- to 21-day chicken embryos (E15, E19, E21), and 1- to 45-day-old chickens (P1, P3, P14, P45). Agonist-induced pretone or an intact endothelium were not required to obtain a consistent and reproducible response to hypoxia, which showed a pattern of initial rapid phasic contraction followed by a sustained tonic contraction. Phasic contraction was reduced by elimination of extracellular Ca2+ or by presence of the neurotoxin tetrodotoxin, the α1-adrenoceptor antagonist prazosin, or inhibitors of L-type voltage-gated Ca2+ channels (nifedipine), mitochondrial electron transport chain (rotenone and antimycin A), and NADPH oxidase (VAS2870). The Rho-kinase inhibitor Y27632 impaired both phasic and tonic contraction and, when combined with elimination of extracellular Ca2+, hypoxia-induced contraction was virtually abolished. Hypoxic MA contraction was absent at E15 but present from E19 and increased toward the first days posthatching. It then decreased during the first weeks of life and P45 MAs were unable to sustain hypoxia-induced contraction over time. In conclusion, the results of the present study demonstrate that hypoxic vasoconstriction is an intrinsic feature of chicken MA vascular smooth muscle cells during late embryogenesis and the perinatal period.

SK but not IK channels regulate human detrusor smooth muscle spontaneous and nerve-evoked contractions

Animal studies suggest that the small (SK) and intermediate (IK) conductance Ca2+-activated K+channels may contribute to detrusor smooth muscle (DSM) excitability and contractility. However, the ability of SK and IK channels to control DSM spontaneous phasic and nerve-evoked contractions in human DSM remains unclear. We first investigated SK and IK channels molecular expression in native human DSM and further assessed their functional role using isometric DSM tension recordings and SK/IK channel-selective inhibitors. Quantitative PCR experiments revealed that SK3 channel mRNA expression in isolated DSM single cells was ∼12- to 44-fold higher than SK1, SK2, and IK channels. RT-PCR studies at the single-cell level detected mRNA messages for SK3 channels but not SK1, SK2, and IK channels. Western blot and immunohistochemistry analysis further confirmed protein expression for the SK3 channel and lack of detectable protein expression for IK channel in whole DSM tissue. Apamin (1 μM), a selective SK channel inhibitor, significantly increased the spontaneous phasic contraction amplitude, muscle force integral, phasic contraction duration, and muscle tone of human DSM isolated strips. Apamin (1 μM) also increased the amplitude of human DSM electrical field stimulation (EFS)-induced contractions. However, TRAM-34 (1 μM), a selective IK channel inhibitor, had no effect on the spontaneous phasic and EFS-induced DSM contractions suggesting a lack of IK channel functional role in human DSM. In summary, our molecular and functional studies revealed that the SK, particularly the SK3 subtype, but not IK channels are expressed and regulate the spontaneous and nerve-evoked contractions in human DSM.

Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity

Muscular lymphatics use both phasic and tonic contractions to transport lymph for conducting their vital functions. The molecular mechanisms regulating lymphatic muscle contractions are not well understood. Based on the well-established finding that the phosphorylation of myosin light chain 20 (MLC20) plays an essential role in blood vessel smooth muscle contraction, we investigated if phosphorylated MLC20 (pMLC20) would modulate the tonic and/or phasic contractions of lymphatic muscle. The effects of ML-7, a MLC kinase inhibitor (1–10 μM), were tested on the contractile parameters of isolated and cannulated rat mesenteric lymphatics during their responses to the known modulators, pressure (1–5 cmH2O) and substance P (SP; 10−7 M). Immunohistochemical and Western blot analyses of pMLC20 were also performed on isolated lymphatics. The results showed that 1) increasing pressure decreased both the lymphatic tonic contraction strength and pMLC20-to-MLC20 ratio; 2) SP increased both the tonic contraction strength and phosphorylation of MLC20; 3) ML-7 decreased both the lymphatic tonic contraction strength and pMLC20-to-MLC20 ratio; and 4) the increase in lymphatic phasic contraction frequency in response to increasing pressure was diminished by ML-7; however, the phasic contraction amplitude was not significantly altered by ML-7 either in the absence or presence of SP. These data provide the first evidence that tonic contraction strength and phasic contraction amplitude of the lymphatics can be differentially regulated, whereby the increase in MLC20 phosphorylation produces an activation in the tonic contraction without significant changes in the phasic contraction amplitude. Thus, tonic contraction of rat mesenteric lymphatics appears to be MLC kinase dependent.