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UMR Centre National de la Recherche Scientifique 17034 and 37081, Université Louis Pasteur, Illkirch, France; and 2Center for Drug Discovery and Chemical Biology, Northwestern University, Chicago, Illinois
Submitted 7 June 2004 ; accepted in final form 6 July 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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myosin light chain kinase 210; knockout mice; echocardiography; endothelium; vascular reactivity; shear stress
The short isoform of MLCK (MLCK108) is ubiquitous in all adult tissues, with the highest amounts in SMC (1, 9, 13). On the contrary, the long isoform (MLCK210) is prominently expressed in embryonic SMC and in adult cells of nonmuscular lineage (10, 26). Of note, both isoforms are present in mature endothelial cells (4). These two MLCK isoforms differ in the length of their NH2-terminal tail domain, which contains amino acid sequence motifs associated with subcellular targeting or protein-protein interactions in the proteome (17, 23). This noncatalytic domain of MLCK is distinct from the kinase domain. Its role as a cellular organizer, providing integration among diverse cytoskeletal proteins, has been confirmed recently (16).
Although the importance of MLCK108 in muscle contraction is well described (reviewed in Ref. 24), the physiological role of MLCK210 is less known. Recently, in an MLCK210 knockout (KO) mouse model, Wainwright and colleagues (27) have shown a decreased susceptibility of the endothelium to injury after an endotoxinic shock and have proposed that MLCK210 could be a therapeutic target to prevent lung injury. These data have been reproduced in wild-type (WT) mice in which MLCK210 has been blocked by a specific inhibitor. However, the cardiovascular effects of MLCK210 ablation or blockade have not been studied up to now.
Therefore, the current study was undertaken to investigate the effect of MLCK gene deletion on cardiovascular parameters in vivo and on vascular reactivity in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animal experiments were performed in accordance with institutional guidelines. Protocols were approved by the French Animal Care Committee in accordance with European regulations. MLCK210 KO mice and WT mice (15–21 wk old; strain C57-Black6/SV-129) were used. The KO mice were generated as previously described (27).
Telemetry Measurements: Mean Blood Pressure and Electrocardiogram
Electrocardiograms.
Electrocardiograms were recorded using a telemetry system (Data Sciences, St. Paul, MN). An implantable radiofrequency transmitter was inserted (TA10ETA-F20, 3.6 g) at least 7 days before data collection, under anesthesia with ketamine (100 mg/kg ip) and medetomidine (50 µg/kg ip). The transmitter was implanted subcutaneously; the negative lead was positioned and sutured at the right shoulder, and the positive lead was sutured toward the lower left chest. A receiver was placed under the cage of each animal, and the data were recorded at 1,000 Hz. The ECG was measured continuously for 24 h. The R-R interval, P-R interval, Q-R-S interval, P wave duration, P amplitude, and T amplitude were determined automatically with the Emka ECG analysis software. All analyses of the Q-T interval were controlled by the same operator, and corrected Q-T interval for heart rate (Q-Tc) was calculated as Q-Tc = Q-T/
/100, where R-R is the interval in seconds between two successive R waves.
Arterial blood pressure. In another set of animals, arterial blood pressure was recorded using a telemetry system (TA11-PA20, 3.4 g; Data Sciences). Briefly, a catheter was inserted in the common carotid artery under general anesthesia by ketamine (100 mg/kg ip) and medetomidine (50 µg/kg ip) and connected to the transmitter that was positioned subcutaneously in the mouse's right flank. Mice were allowed 1 wk of recovery from surgery before any measurement was made. Mean arterial pressure and heart rate were recorded for 30 s every 15 min during 24 h. The mean ± SE for each 15 min over a 24-h period was reported.
Echocardiographic Examination
Transthoracic echocardiography was performed using an ATL-HDI 5500 ultrasound machine equipped with a 15-MHz imaging transducer in mice anesthetized with pentobarbital sodium (54 mg/kg ip). Measurements were made using the leading-edge method of the American Society of Echocardiography (11). Doppler cardiac output (CO) was calculated by the following formula: CO = 
x D2/4 x IVTAo, where D represents the diameter of the aortic left ventricular outflow tract and IVTAo the velocity-time integral in the left ventricular outflow tract.
Vascular Reactivity Experiments
Aortic ring preparation.
Segments of 2 mm aorta, cleaned of fat and connective tissue, were mounted on a wire myograph, as previously described (21). Concentration-response curves were constructed by cumulative application of the thromboxane A2 analog 9,11-dideoxy-9
,11-methanoepoxy PGF2 (U-46619, 10 nM-1 µM) on vessels with functional endothelium in the absence and in presence of either the NO synthase inhibitor N
-nitro-L-arginine methyl ester (L-NAME, 300 µM) or the cyclooxygenase (COX) inhibitor indomethacin (10 µM). In parallel, concentration-response curves to norepinephrine (10 nM-10 µM), 5-hydroxytryptamine (5-HT, 10 nM to 10 µM), and phenylephrine (10 nM to 30 µM) were also constructed. Inhibitors were applied 30 min before performing concentration-response curves to agonist.
In another set of experiments, arteries with and without endothelium were precontracted to 80% of maximal contraction with U-46619. When the contraction reaches a steady-state value, endothelium-dependent relaxation to ACh (0.01–100 µM) was assessed by cumulative addition of the agonist to construct a concentration-response curve. This experimental protocol was performed in the absence or in the presence of either L-NAME (300 µM) or indomethacin (10 µM) after 30 min preincubation. Also, endothelium-independent relaxation to the exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP, 0.01–100 µM) was examined in vessels with endothelium precontracted with U-46619.
Isolated Mesenteric Arteries
Small mesenteric arteries (SMA; 
100–130 µm of diameter) were isolated from KO and WT mice. Mesenteric arteries were cannulated at both ends in a video-monitored perfusion system (Living Systems Instrumentation). Arteries were bathed in physiological saline solution (PSS; pH 7.4, PO2 160 mmHg, PCO2 37 mmHg). Pressure was controlled by a servoperfusion system, and flow was generated by a peristaltic pump. Initially, diameter changes were measured when intraluminal pressure was increased from 10 to 125 mmHg. The presence of endothelium was challenged by applying ACh (10 µM), after preconstriction of the arteries with U-46619 (1 µM). Pressure was then set at 75 mmHg (at which no significant difference in myogenic tone has been observed between groups), and dilation in response to flow was performed. At the end of each experiment, to determine the passive diameter of the artery and to evaluate myogenic tone, arteries were perfused and superfused with a Ca2+-free PSS containing EGTA (2 mmol/l) and sodium nitroprusside (10 mmol/l) and, pressure steps were repeated (18).
Dilation responses to flow experiments were repeated in series the presence of 1) an inhibitor of the NO synthase inhibitor, N-nitro-L-arginine (L-NNA, 100 µM) alone, 2) L-NNA plus the COX inhibitor indomethacin (10 µM), 3) L-NNA plus indomethacin in the presence of 15 mM KCl to inhibit also the endothelium-derived hyperpolarization factor (EDHF). The above procedure was performed to analyze the relative contribution of endothelial factors in inducing vasodilation such as NO, COX products, and EDHF. The different components of the vasorelaxation were determined as follow: the NO-dependent dilation was calculated as the difference between the dilation without inhibitors and the dilation in presence of L-NNA. The dilation dependent to COX products was obtained as the difference between the dilation in the presence of L-NNA alone and the dilation in the presence of L-NNA plus indomethacin. The EDHF component was assessed in the presence of L-NNA plus indomethacin as the difference between the dilation in normal PSS and in a 15 mM KCl PSS.
In another set of experiments, the same protocol was conducted in MLCK WT and KO mice treated by an MLCK aminopyridazine-based inhibitor, the MMZ-10–057. The latter was applied to confirm the results observed in MLCK null mice mesenteric arteries. MLCK inhibitor was administered as previously described (27) by intraperitoneal injection at the dose of 2.5 mg/kg mouse body wt 1 h before death.
Real-Time Quantitative RT-PCR Experiments
RNA was isolated from mouse mesenteric arteries and aorta using the RNasy Micro Kit (Qiagen, Hilden, Germany). After mRNA extraction, 400 ng total RNA were reverse transcribed to single-stranded cDNA using the High-Capacity cDNA Archive Kit. Real-time RT-PCR was performed using an ABI Prism 7900 (Applied Biosystems, Foster City, CA). Primer sequences were targeted on exons 6, 8, 16, and 26 of MLCK using Primer Express software (Applied Biosystems). Each sample was performed in duplicate, and each reaction was performed two times. Relative quantification was performed by normalizing threshold cycles (Ct) values of each MLCK exon with Ct values of the ribosomal 18S housekeeping gene (
Ct). Data were analyzed by comparing means of the Ct values in mesenteric arteries or aorta from WT and MLCK KO mice.
Statistical Analysis
Results are expressed as means ± SE of n experiments, where n represents the number of mice. Statistical evaluation was carried out using unpaired Student's t-test or ANOVA for repeated measures, when appropriate. In all cases, a P value <0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Telemetry records of blood pressure are represented in Fig. 1 and show no significant difference of mean arterial pressure between either strain.
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The electrocardiographic analysis demonstrated no significant differences between WT and MLCK KO in heart rate, atrioventricular conduction (P-R interval), and intraventricular conduction (Q-R-S duration; Table 1). Repolarization as evaluated by Q-T and Q-TC duration was also similar. The P wave duration, representing the interatrial conduction, but not the P wave amplitude, was slightly but significantly shorter in KO mice than in WT.
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To detect potential cardiac structural differences between KO and WT mice, transthoracic echocardiography was performed. No significant differences between WT and MLCK210 KO mice (Table 2) were observed in terms of left ventricular end- diastolic diameter and left ventricular end-systolic diameter. Systolic function, as determined by left ventricular fractional shortening (FS), was normal in WT and KO mice despite the fact that the animals were anesthetized. Of note, no significant difference in FS between the two strains was observed. Heart rate during the echocardiography was not different between WT and KO mice. Calculated left ventricular mass and estimated Doppler CO were not significantly different between the two groups.
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Aorta.
CONTRACTION EXPERIMENTS.
Concentration-response curves to norepinephrine, phenylephrine, 5-HT, and the thromboxane agonist U-46619 of aortic rings with endothelium were not different in WT and KO mice (Fig. 2 and Table 3). The vasoconstriction effect of U-46619 was not affected by L-NAME or indomethacin in either WT or KO mice. Sensitivity to U-46619, expressed as the pD2 value, which represents –log of the half-maximally effective molar concentration, is shown in Table 3.
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EFFECTS OF INDOMETHACIN ON RESPONSE TO FLOW INCREASES IN WT AND KO MICE.
During NO inhibition with L-NNA, incubation with indomethacin caused a significant decrease in the vasodilator response to flow in both WT and KO mice (Table 4). However, the reduction observed in KO animals was more pronounced than the one recorded in WT mice (P < 0.0001). In SMA from animals treated with MMZ-10–057, the effects of indomethacin on the vasodilator response to flow were blunted compared with SMA from WT (P < 0.01). MMZ-10–057 treatment did not modify the effect of indomethacin on flow-mediated dilation in arteries from KO mice (Fig. 4C).
EFFECTS OF KCL ON RESPONSE TO FLOW INCREASES IN WT AND KO MICE.
During NO and COX inhibition with L-NNA and indomethacin, incubation with 15 mM KCl PSS induced similar reductions in the vasodilator response to flow in untreated WT and KO mice and in WT and KO animals treated with MMZ (Fig. 4D). This result indicates that the EDHF component of the vasodilation to flow is comparable in the four groups.
PASSIVE DIAMETER AND MYOGENIC TONE OF SMA.
In basal conditions, pressure-induced tone (myogenic tone) in SMA was similar between WT and KO mice. Treatment with MMZ-10–057 did not affect the changes in vascular diameter in response to intraluminal increases in pressure (Fig. 5).
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The relative expression of exons 6, 8, 16, and 26, which contribute to the high-molecular-weight mRNA encoding MLCK210, was estimated by quantitative PCR, to confirm that MLCK210 mRNA was present in aorta and mesenteric arteries of WT mice. RT-PCR experiments did not show differences in the Ct of the exon of interest of the MLCK gene (Fig. 6).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cardiac morphological and electrophysiological parameters, measured by echocardiography and ECG telemetry, were similar between WT and KO mice, except for P duration, which was slightly shorter in the KO group. In humans, previous studies have suggested that altered P wave duration may reflect interatrial conduction disturbances independent of increases in atrial size, which could predispose to the initiation of atrial fibrillation (8). However, because the 24-h telemetry recordings did not exhibit either ventricular or supraventricular arrhythmia, shorter P wave duration in MLCK210 KO mice does not seem to have unlikely pathophysiological relevance. Taken together, these results do not support a significant role of MLCK210 in heart function under the experimental conditions used, although the presence of compensatory mechanism masking cardiac effects of this kinase cannot be excluded. Moreover, the anesthesia during echocardiographic experiments may have affected the reported measurements. However, we consider it unlikely, since left ventricular FS was not altered in either genotype.
In conductance vessels like the aorta, the study suggests that deletion of MLCK210 did not affect responses to different vasoconstrictor agonists acting on G protein-coupled receptors such as norepinephrine, phenylephrine, 5-HT, or thromboxane analog. Evidence in the literature supports the predominant role of the short isoform in vascular contraction regulation. Indeed, in cultured SMC, downregulation of the MLCK short isoform reduced (up to 80%) developed force dramatically, and further inhibition of the MLCK long isoform by transfection was not associated with an additional inhibition of contraction (5). Thus the results presented here provide a direct in vivo test of the hypothesis that MLCK210 does not play a significant role in SMC contraction.
In aortic rings, relaxation to ACh was unaffected in MLCK210 null mice compared with WT mice. In addition, responses to SNAP were not different between both strains. Taken together these results suggest that in conduit vessels, MLCK210 is not involved in endothelium-dependent or -independent vasodilation reactivity.
In SMA, shear stress-induced vasodilation was significantly impaired in MLCK210 KO mice. An involvement of MLCK in the response to flow vasodilation has been suggested by Ando et al. (2), who showed an upregulation of MLCK mRNA transcription in human cultured umbilical endothelial cells during exposure to shear stress. Modifications of endothelial intracellular Ca2+ could be involved in the role of MLCK in shear stress. Indeed, Watanabe et al. described a regulatory role of MLCK in Ca2+ entry and NO release in response to agonists (29) and to flow (28) that could be blocked by MLCK antisense and wortmannin in endothelium-cultured porcine aortic cells. However, wortmannin also blocks the phosphatidylinositol 3-kinase (PI3K)-Akt pathway (15) and may act as a nonspecific inhibitor. Shear stress has been shown to be able to alter the cell cytoskeleton and to modify the shape of endothelial cells, which align in the direction of flow (7, 22). Proteins of the cytoskeleton are also involved in shear stress mechanotransduction (6, 7, 12). Similarly, it has been reported that MLCK is implicated in cytoskeletal spatial reorganization under shear stress stimulation in cultured pulmonary endothelial cells (3).Therefore, MLCK210 might be implicated in shear stress mechanotransduction or regulation through its interaction with the cytoskeleton. The reduction in flow-induced responses in MLCK210 KO mice in SMA was associated with a decrease in the NO component of shear stress partially compensated by an enhanced indomethacin-sensitive dilation. The underlying molecular mechanisms leading to NO production from eNOS in response to flow remain unclear and controversial (5). Shear-induced production of NO involves Ca2+-independent mechanisms, including mainly the phosphorylation of eNOS by a PI3K-Akt-Akt pathway and interactions with other proteins like heat shock protein-90, caveolin, and other Ser/Thr protein kinases, or tyrosine kinases (reviewed in Ref. 6). Our data do not permit estimation of the potential implication of MLCK210 among these mechanisms. The cause of the observed increase in COX products could be a direct interaction between MLCK210 and COX, a compensative mechanism to impaired NO component, or the result of an interaction between the NO and the COX pathways (19). We cannot distinguish among these possibilities based on the results reported here.
As stated above, MLCK210 deletion resulted in modifications in vasodilation responses in SMA but not in aorta. This discrepancy may result from differences in the type of endothelial stimulation (humoral ACh in one hand and physical stretch in the other hand). However, to rule out the hypothesis of the potential absence of expression of MLCK 210 in aortic rings of WT mice, RT-PCR experiments were performed and confirmed the presence of MLCK210 exon mRNA transcripts in both aorta and mesenteric arteries.
Shear stress-induced vasodilation was impaired in SMA from MLCK210 without an alteration of systemic blood pressure under basal conditions. Because CO, measured by echocardiography, was similar in both strains, it is likely that overall systemic resistances are not increased in KO mice. These results strengthen the hypothesis that, even though flow-dilation has a key role in the control of local blood flow, it is not necessarily and/or directly related to the basal level of systemic blood pressure (12, 18).
In addition to its catalytic kinase domain, evidence supports that MLCK210 is able to interact in the cell through specific binding with other proteins, like actin-cytoskeleton, via its NH2-terminal extension (16). Hence the effects related to the deletion of MLCK210 might be the consequence of either the absence of its kinase activity or of the loss of structural binding with protein partners. Because the pharmacological inhibition of the catalytic domain of MLCK in WT mice reproduced the same pattern of decreased shear stress as MLCK210 gene deletion, it suggests that the kinase activity of MLCK is the main contributor to this response rather than direct interactions between MLCK and other proteins (16, 17, 23). Moreover, the relative rapid time course of the "knockout-like" effect of the inhibitor (1 h) may point out that the alterations seen in the KO are not the result of chronic adaptation and are mimicked by the acute loss of enzymatic activity.
In conclusion, our findings indicate that, in physiological conditions, MLCK210 plays a role through the endothelial cells, in the regulation of the vascular reactivity to fluid-flow stimulation in SMA. Nevertheless, the main cardiovascular parameters are not altered in MLCK210 KO mice.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and knockout (KO; 
) mice recorded during a 24-h monitoring period. Data are means ± SE of n = 7 mice for each group. NS, no significant difference between WT and KO.
) and KO (
) mice. Results are given as means ± SE of n = 5–8 experiments.
) and KO (
) mice treated with the MLCK inhibitor MMZ-10–57 in basal conditions (A). B: NO-dependent dilation calculated as the difference (
