Mixed lineage kinase 3 requires a functional CRIB domain for regulation of blood pressure, cardiac hypertrophy, and left ventricular function

Abstract

Mixed lineage kinase 3 (MLK3) modulates blood pressure and left ventricular function, but the mechanisms governing these effects remain unclear. In the current study, we therefore investigated the role of the MLK3 Cdc42/Rac interactive binding (CRIB) domain in cardiovascular physiology. We examined baseline and left ventricular pressure overload responses in a MLK3 CRIB mutant (MLK3^C/C) mouse, which harbors point mutations in the CRIB domain to disrupt MLK3 activation by Cdc42. Male and female MLK3^C/C mice displayed increased invasively measured blood pressure compared with wild-type (MLK3^+/+) littermate controls. MLK3^C/C mice of both sexes also developed left and right ventricular hypertrophy but normal baseline LV function by echocardiography and invasive hemodynamics. In LV tissue from MLK3^C/C mice, map3k11 mRNA, which encodes MLK3, and MLK3 protein were reduced by 74 ± 6% and 73 ± 7%, respectively. After 1-wk LV pressure overload with 25-gauge transaortic constriction (TAC), male MLK3^C/C mice developed no differences in LV hypertrophy but displayed reduction in the LV systolic indices ejection fraction and dP/dt normalized to instantaneous pressure. JNK activation was also reduced in LV tissue of MLK3^C/C TAC mice. TAC induced MLK3 translocation from cytosolic fraction to membrane fraction in LV tissue from MLK3^+/+ but not MLK3^C/C mice. These findings identify a role of the MLK3 CRIB domain in MLK3 regulation of basal blood pressure and cardiac morphology, and in promoting the compensatory LV response to pressure overload.

NEW & NOTEWORTHY Here, we identified that the presence of two discrete point mutations within the Cdc42/Rac interaction and binding domain of the protein MLK3 recapitulates the effects of whole body MLK3 deletion on blood pressure, cardiac hypertrophy, and left ventricular compensation after pressure overload. These findings implicate the CRIB domain, and thus MLK3 activation by this domain, as critical for maintenance of cardiovascular homeostasis.

INTRODUCTION

Heart failure (HF) remains the leading cause of death in the United States (1). Risk factors for heart failure include hypertension and left ventricular (LV) hypertrophy (1, 2), which often precede HF and are thought to contribute to its progression. We previously identified the signaling molecule mixed lineage kinase 3 (MLK3) as opposing these processes in vivo (3, 4). Specifically, genetic deletion of MLK3 leads to accelerated LV dysfunction after pressure overload (3), supporting that MLK3 normally promotes LV functional compensation to pathological stress. Chemical inhibition of MLK3 kinase activity induces cardiac myocyte hypertrophy in vitro (3) and impairs normal LV function in vivo (4). MLK3 also regulates blood pressure, as whole body MLK3 deletion produces baseline hypertension, increased vascular stiffness, and reduced resistance arteriolar distensibility (4).

Recently, we identified MLK3 as a substrate of cGMP-dependent protein kinase 1α (4). MLK3 mediates the effects of pharmacological cGMP augmentation on LV function after pressure overload but is not required for cGMP reduction of blood pressure (4). However, additional upstream signaling pathways in cardiovascular tissues which promote MLK3 regulation of blood pressure, LV structure, and LV function have not been studied. In cell culture, the small GTPase Cdc42 binds MLK3 and promotes MLK3 kinase-mediated JNK activation, as well as MLK3 translocation from the cytosol to the plasma membrane (5, 6). The cointeraction of Cdc42 and MLK3 requires a region of MLK3 termed the Cdc42/Rac Interactive Binding (CRIB) domain (5). Knock-in mice harboring point mutations within the MLK3 CRIB domain (7, 8) have disrupted MLK3 activation by Cdc42, but their cardiovascular phenotype has not been studied.

In the present study, we hypothesized that the MLK3 CRIB domain is required for MLK3 regulation of cardiovascular function. To test this hypothesis, we examined the baseline and LV pressure overload-induced cardiovascular effects of MLK3 CRIB domain mutation in mice.

METHODS

Study Approval

All rodent care and procedures were approved by the Institutional Animal Care and Use Committee of Tufts Medical Center.

Experimental Animals

Whole body MLK3 gene CRIB domain mutant mice were created as described previously (7, 8). Experimental mice were bred in the animal facility at Tufts Medical Center (Boston, MA). Wild-type (MLK3^+/+) and MLK3 CRIB mutant (MLK3^C/C) mice came from heterozygous parents, and we used littermates in all studies. All experimental mice were maintained on a C57BL/6 background (12-h:12-h light/dark cycle at 22 ± 2°C) in the Tufts Medical Center Animal Facility. Investigators were blinded to animal genotypes before surgeries. The study was performed in accordance with ARRIVE 2.0 guidelines (9). Sample size for the baseline and for the surgical transaortic constriction study was chosen based on our published study sizes (3). For analysis of in vivo data, investigators were provided only the mouse identification number, but did not know the mouse genotype, sex, or surgical assignment.

Echocardiography

Cardiac function in mice was obtained by echocardiography as described previously (10). For the baseline study, echocardiograms were performed within 3 days before invasive hemodynamic analysis and harvest. For the subsequent transaortic constriction (TAC) study, echocardiograms were performed on the day before invasive hemodynamics and organ harvest. Mice were anesthetized with 2.5% gaseous isoflurane in medical oxygen at 1 L/min, then maintained under 1%–2% isoflurane in the supine position on a 39°C heating pad. Chest fur was removed with hair remover (Nair). Excessive cream was removed by damp cotton pad. Ultrasonic gel was applied to the echocardiography transducer (MS550D; Vevo 2100, FUJIFILM VisualSonics), and scanning performed through the long- and short-axis view in M-mode and B-mode. M-Mode images were acquired from the midpapillary short-axis view. Mitral inflow velocity was acquired by pulse-wave Doppler, and septal annular e′ velocity was acquired by tissue Doppler. All Doppler measures were performed in the apical view. All the echocardiographs were obtained and analyzed by a single blinded investigator.

Transverse Aortic Constriction

Only 10- to 15-wk-old mice were used in the 25-gauge transverse aortic constriction (TAC) study. Both wild-type and MLK3 CRIB mutant mice underwent TAC or sham surgery as previously described (10, 11). Briefly, mice were anesthetized with 2.5% gaseous isoflurane, intubated, and maintained on a small animal ventilator, before thoracotomy. Surgeries were performed by suturing around a 25-gauge needle (0.51 mm outer diameter) at the transverse aorta between the two carotid arteries. We randomized mice to TAC or to sham surgery by sorting animal numbers at random with each experimental cohort, to allocate the same proportion of sham to TAC mice per cohort so that each litter had comparable proportions of TAC and sham-treated mice. The surgeon performing TAC had no role in allocating animals to TAC or to sham and was blinded to mouse genotype.

Pressure-Volume Hemodynamic Measurements (PV Loops)

Left ventricular function was also analyzed through simultaneous measurement of LV pressure and volume in vivo to generate pressure-volume loops as described previously (10). For the baseline group, PV loops were performed when the mice were 10 to 12 wk old. In a second baseline group, the studies were performed at 22–24 wk of age. During the TAC study, PV loops were performed 7 days after the 25-G TAC surgery. Mice were anesthetized with 3.0% gaseous isoflurane and maintained at 2.0% isoflurane. The body temperature was maintained and monitored via a rectal thermometer-lamp system. A 1.0-Fr catheter was placed in the left ventricle through the right carotid and the aortic arch. Blood pressure was first acquired in the proximal aorta, before inserting the catheter into the left ventricle. The data were obtained and analyzed with IOX v. 2.1.10 software (EMKA instruments). By a priori design, if systolic blood pressure dropped >15 mmHg when the catheter entered the LV from the aorta, then the hemodynamic data were not analyzed.

Tissue Isolation

Following pressure-volume hemodynamic measurements, the heart and aorta were removed and the tibia length was measured. The heart was then excised into four chambers, which were stored separately.

Immunoblotting

Tissues were snap-frozen in liquid nitrogen immediately after harvest and stored at −80°C. Tissues were crushed into powder without thawing while on dry ice. Tissue powder was lysed with tissue lysis buffer, consisting of (in mmol/L) 20 HEPES, 50 β-glycerol phosphate, 2 EGTA, 1 DTT, 10 NaF, and 1 NaVO₄ and 1% Triton X-100 and 10% glycerol, supplemented with 1 mmol/L PMSF. Lysis buffer (200 µL) was next added per 10 mg tissue powder followed by vortexing the mixture for at least 20 s/sample. Samples were kept on ice for 1 h while tapping and rotating the samples every 20 min. Lysates were cleared by the highest speed benchtop centrifugation and the supernatant was saved. Protein concentrations were quantified by BCA assay (Thermo Fisher Scientific, JD120868) and lysates were diluted in 2× Laemmli sample buffer (Sigma S-3401). Protein samples (30 µg/well) and protein ladder marker (Bio-Rad Precision Plus Dual Color Standards No. 161–0374) were loaded into 8% or 12% polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad, 1620094). After staining with Ponceau S to check the efficiency after transfer, membranes were washed with TBST and incubated in 5% blocking buffer (Blotting-Grade Blocker, Bio-Rad, 1706404) for 1 h. The membranes were incubated in different primary antibodies for 1 h at room temperature or overnight at 4°C. We used the following antibodies for this study (dilutions of 1:1,000 except as noted): MLK3 (Abcam, ab51068), 1:500 dilution, GAPDH (EMD Millipore, MAB374), Thr183/Tyr185 phosphorylated JNK (Cell Signaling Technology, No. 4668), JNK (Cell Signaling Technology, No. 9252), caveolin 1 (Cell Signaling Technology, No. 3238). After incubation with primary antibody, membranes were incubated in secondary anti-rabbit antibodies (GE Healthcare, NA934), 1:2,000 dilution, for 1 h at room temperature. After incubating in the ECL substrate (Thermo Fisher Scientific, 34095), membranes were visualized by ProteinSimple FluorChem E system, and calculation of densitometry of bands was measured using an Alpha Innotech Imager and software or using the National Institutes of Health (NIH) ImageJ. The analysis of the bands of specific proteins was normalized by the quantification of the GAPDH on the same membrane. MLK3 antibody, dilutions, and blotting conditions were validated for specificity using MLK3 whole body deletion tissue as negative control (3).

Membrane Fractionation Analysis

After LV tissues were crushed into powder as aforementioned, 1× cell wash buffer (Thermo Fisher Scientific, 89842) containing protease and phosphatase inhibitors (Thermo Fisher Scientific, 87786) was added, followed by vortexing briefly and discarding the wash. Permeabilization buffer was next added to the tissue, followed by homogenization by vortex, and incubated for 10 min on ice with constant mixing. Samples were then centrifuged at 16,000 g for 15 min at 4°C. Supernatant containing cytosolic proteins was transferred to a new tube. To obtain membrane fractions, solubilization buffer (Thermo Fisher Scientific, 89842) was then added to the remaining pellet, which was gently resuspended. The pellet suspension was incubated at 4°C for 30 min with constant mixing, then centrifuged at 16,000 g for 15 min at 4°C. Protein samples were then stored at −80°C until immunoblotting.

Quantitative RT-PCR Analysis

PCR for map3k11, the gene encoding MLK3, and for Gapdh, was performed as previously described (3). Briefly, LV total RNA was extracted with TRIzol (Invitrogen), followed by reverse transcription of 1 µg RNA to cDNA with QuantiTect Reverse Transcriptase kit (Qiagen). 6-Carboxyfluorescein (FAM)-labeled primers for Map3k11 (Mm.PT.58.6037096) and Gapdh (Mm.PT.39a.1) were then purchased from IDT DNA and amplified using PrimeTime Gene Expression Master Mix (IDT DNA) according to the manufacturer’s recommendations. All samples were amplified for 40 cycles performed at 95°C for 15 s and 60°C for 1 min using an ABI Prism 7900 sequence detection system (Applied Biosystems). qPCR data were analyzed using the ΔΔC_T method with Gapdh as the reference control, and values were normalized to represent fold change.

Histological Analysis

The LV was arrested in diastole with a KCl injection, fixed in 10% formalin, embedded in paraffin, and cut into 4 µm sections. For cardiac myocyte (CM) size measurements, LV tissue sections were stained with wheat germ agglutinin conjugated to Alexa fluor 488 (Thermo Fisher Scientific, W11261). Tissue sections were processed together and stained with the same reagents. Cells were imaged with a Nikon Eclipse fluorescence microscope. Cell area was determined through manual tracing using the NIH ImageJ program. Investigators were blinded to genotype during image acquisition and analysis. CMs in the transverse plane were visualized with fluorescent microscopy and traced using Image-Pro Premier Software (MediaCybernetics). We measured only myocytes in the cross-sectional orientation. For CM size measurements, a minimum of 50 cells/sample were counted.

Statistical Analysis

All data are presented as means ± SE, and the statistics were performed with Student’s two-tailed unpaired t test. For comparing more than two groups, we used two-way ANOVA with Sidak’s multiple-comparisons test. Values of P < 0.05 were considered statistically significant.

RESULTS

Baseline Elevated Blood Pressure in MLK3 CRIB Mutant Mice

To examine the role of the MLK3 CRIB domain in cardiovascular physiology, we performed echocardiography, invasive hemodynamic measurements, and organ weight analysis on mice with point mutation in the MLK3 CRIB domain, which leads to impaired MLK3 interaction with and activation by CDC42 (7, 8). In 3-mo-old MLK3 CRIB mutants (MLK3^C/C), compared with wild-type (MLK3^+/+) littermates, we observed elevated systolic, diastolic, and mean aortic pressures (Fig. 1), consistent with hypertension. We observed equal degrees of blood pressure elevation in both male and female MLK3^C/C mice, compared with MLK3^+/+ controls of either sex.

Figure 1.

Mutation of MLK3 CRIB domain leads to elevated blood pressure in vivo. Invasive hemodynamic measurement of aortic pressure in anesthetized 3-mo-old MLK3 CRIB mutant (MLK3^C/C) or wild-type (MLK3^+/+) littermate mice. Systolic (A), mean (B), and diastolic (C) blood pressures (BPs) are shown. n = 10 MLK3^+/+ male, 6 MLK3^C/C male, 8 MLK3^+/+ female, and 9 MLK3^C/C female mice. *P < 0.05 by two-way ANOVA with Sidak’s multiple-comparisons test. CRIB, Cdc42/Rac interactive binding; MLK3, mixed lineage kinase 3.

Cardiac Hypertrophy in MLK3 CRIB Mutant Mice

LV function measured by invasive hemodynamics (Table 1) or echocardiography (Table 2, Fig. 2) did not differ between MLK3^+/+ and MLK3^C/C mice of either sex. The MLK3^C/C mice did display increased LV, RV, and whole heart weight normalized to tibia length (P < 0.05 for genotype effect by 2-way ANOVA) with no statistical interaction of sex with genotype (Fig. 2). Complete organ mass data are presented in Table 3. This hypertrophy was accompanied by increased cardiac myocyte cross-sectional area, supporting that the elevated cardiac chamber weights occur because of cardiac myocyte hypertrophy (Fig. 2B). LV wall thickness by echocardiography was also increased in the MLK3^C/C mice, consistent with concentric hypertrophy (Fig. 2C). Expression of fetal genes, as measured by qPCR, did not differ between genotypes (Supplemental Fig. S1; https://doi.org/10.6084/m9.figshare.19904374.v2).

Table 1.

Baseline hemodynamic and left ventricular pressure volume loop data in MLK3^+/+ and MLK3^C/C mice

	MLK3+/+	MLK3 C/C	P Value
Male
n	10	6
SBP, mmHg	89.5 ± 2.8	108 ± 5.2	0.004
DBP, mmHg	59.6 ± 2.0	73.5 ± 2.6	<0.001
MAP, mmHg	69.6 ± 2.2	85.1 ± 2.8	<0.001
LV EDP, mmHg	7.25 ± 2.0	5.00 ± 1.7	0.422
LV dP/dtmax, mmHg/s	5,015 ± 665	7,986 ± 1,378	0.064
LV dP/dtmin, mmHg/s	−4,477 ± 608	−7,120 ± 1,172	0.058
LV contractile index, s−1	195 ± 13.7	194 ± 15.6	0.993
Stroke volume, µL	19.3 ± 3.7	19.4 ± 3.6	0.973
Cardiac output, µL/min	8,124 ± 1,634	8,753 ± 1,762	0.798
Heart rate, beats/min	427 ± 19	447 ± 30	0.563
Female
n	8	9
SBP, mmHg	89.9 ± 2.2	105.0 ± 4.1	0.007
DBP, mmHg	59.9 ± 2.3	73.8 ± 3.4	0.005
MAP, mmHg	70.9 ± 2.6	85.3 ± 4.2	0.016
LV EDP, mmHg	5.1 ± 1.4	5.8 ± 1.8	0.766
LV dP/dtmax, mmHg/s	5,872 ± 448	6,872 ± 503	0.165
LV dP/dtmin, mmHg/s	−5,065 ± 484	−5,266 ± 475	0.774
LV contractile index, s−1	188 ± 7.1	168 ± 4.2	0.04
Stroke volume, µL	20.4 ± 2.2	17.0 ± 3.1	0.374
Cardiac output, µL/min	8,734 ± 893	7,669 ± 1,373	0.517
Heart rate, beats/min	433 ± 9	454 ± 20	0.35

Values are represented as means ± SE; n, number of mice. Pressure-volume loop hemodynamic analysis of mice at 12 wk of age is shown. dP/dt_max/min, greatest instantaneous rate of ventricular pressure increase (max) or decrease (min); DBP aortic diastolic blood pressure; EDP, end-diastolic pressure; LV, left ventricle; MAP aortic mean arterial pressure; MLK3, mixed lineage kinase 3; MLK3^+/+ and MLK3^C/C, wild-type and MLK3 CRIB mutant, respectively; SBP, aortic systolic blood pressure. Data were analyzed by Student’s two-tailed unpaired t test.

Table 2.

Baseline echocardiographic data in MLK3^+/+ and MLK3^C/C mice

	MLK3+/+	MLK3 C/C	P Value
Male
n	11	9
Ejection fraction, %	57.0 ± 2.8	54.7 ± 3.9	0.633
Fractional shortening, %	29.8 ± 1.9	28.4 ± 2.5	0.638
Septal wall thickness, mm	0.94 ± 0.04	1.02 ± 0.03	0.081
End-diastolic dimension, mm	4.10 ± 0.08	3.92 ± 0.06	0.097
End-systolic dimension, mm	2.88 ± 0.12	2.82 ± 0.14	0.727
Posterior wall thickness, mm	0.83 ± 0.04	0.95 ± 0.05	0.053
Female
n	9	9
Ejection fraction, %	60.7 ± 3.5	58.0 ± 2.9	0.558
Fractional shortening, %	32.3 ± 2.4	30.2 ± 1.9	0.499
Septal wall thickness, mm	0.83 ± 0.05	0.91 ± 0.05	0.235
End-diastolic dimension, mm	3.73 ± 0.09	3.65 ± 0.05	0.403
End-systolic dimension, mm	2.54 ± 0.13	2.55 ± 0.10	0.930
Posterior wall thickness, mm	0.72 ± 0.04	0.83 ± 0.03	0.066

Values are represented as means ± SE; n, number of mice. Left ventricular parameters at 12 wk of age are shown. MLK3, mixed lineage kinase 3; MLK3^+/+ and MLK3^C/C, wild-type and MLK3 CRIB mutant, respectively. Data were analyzed by student’s two-tailed unpaired t test.

Figure 2.

Mutation of MLK3 CRIB domain leads to cardiac hypertrophy but does not affect basal LV function. A: cardiac chamber and whole heart weights normalized to tibia length (TL) in 3-mo-old MLK3 CRIB mutant (MLK3^C/C) or wild-type littermate (MLK3^+/+) mice. n = 11 MLK3^+/+ male, 8 MLK3^C/C male, 9 MLK3^+/+ female, and 9 MLK3^C/C female. B: representative LV sections from male mice stained with wheat germ agglutinin for assessment of cardiomyocyte area, and summary data of average cardiac myocyte cross-sectional area per mouse. n = 4 mice/genotype. Scale bars represent 20 pixels. C: LV posterior wall thickness and ejection fractions obtained from echocardiography in anesthetized mice. n = 11 MLK3^+/+ male, 9 MLK3^C/C male, 9 MLK3^+/+ female, and 9 MLK3^C/C female. Groups compared by two-way ANOVA. #P < 0.05 for main effects of genotype (C/C vs. +/+). *P < 0.05 by Sidak’s multiple-comparisons test. CRIB, Cdc42/Rac interactive binding; HW, heart weight; LV, left ventricle; MLK3, mixed lineage kinase 3; RV, right ventricle.

Table 3.

Baseline organ masses in MLK3^+/+ and MLK3^C/C mice

	MLK3+/+	MLK3 C/C	P Value
Male
n	11	8
BW, g	28.7 ± 0.6	27.0 ± 0.8	0.12
LV, mg	100.9 ± 3.7	107.8 ± 3.8	0.23
RV, mg	20.8 ± 0.6	23.8 ± 1.0	0.02
Atria, mg	8.9 ± 0.5	7.4 ± 0.4	0.30
LV/TL, mg/cm	58.2 ± 1.9	63.3 ± 2.3	0.10
RV/TL, mg/cm	12.0 ± 0.3	14.0 ± 0.6	0.007
Atria/TL, mg/cm	5.1 ± 0.2	4.3 ± 0.2	0.04
Female
n	9	9
BW, g	21.8 ± 0.4	22.0 ± 0.7	0.85
LV, mg	76.1 ± 2.0	83.2 ± 2.3	0.03
RV, mg	15.9 ± 0.6	17.4 ± 0.6	0.21
Atria, mg	6.5 ± 0.3	6.0 ± 0.4	0.01
LV/TL, mg/cm	44.3 ± 1.0	49.0 ± 1.2	0.009
RV/TL, mg/cm	9.2 ± 0.3	10.0 ± 0.3	0.12
Atria/TL, mg/cm	3.8 ± 0.1	3.5 ± 0.2	0.34

Values are represented as means ± SE; n, number of mice. Organ masses of mice at 12 wk of age is shown. BW, body weight; LV, left ventricle; MLK3, mixed lineage kinase-3; MLK3^+/+ and MLK3^C/C, wild-type and MLK3 CRIB mutant, respectively; RV, right ventricle; TL, tibia length. Data were analyzed by Student’s two-tailed unpaired t test.

Persistent Hypertension and Cardiac Hypertrophy in 6-Mo-Old MLK3 CRIB Mutant Mice

We investigated the effects of the MLK3 CRIB mutation in a separate cohort of mice at 6 mo of age (Fig. 3 and Supplemental Table S1). We also measured mitral inflow velocities by Doppler in this group. Blood pressures remained elevated and cardiac hypertrophy persisted in the MLK3^C/C mice in this cohort. LV systolic function remained similar between genotypes. Although we detected no overt functional differences between genotypes by M-mode echocardiography or by invasive hemodynamics (Supplemental Table S1), we observed an increased atrial component of mitral inflow velocity and reduced ratio of E/A mitral inflow velocity in the MLK3^C/C mice, compared with MLK3^+/+ controls, suggesting LV diastolic dysfunction in the MLK3^C/C mice.

Figure 3.

Persistent elevated blood pressure and cardiac hypertrophy in 6-mo-old mice with mutation of MLK3 CRIB domain. Invasive hemodynamics and cardiac chamber masses in male mice at 6 mo of age. A: invasive hemodynamic measurement of systolic, mean, and diastolic aortic blood pressures in anesthetized 6-mo-old MLK3 CRIB mutant (MLK3^C/C) or wild-type littermate (MLK3^+/+) mice. n = 7 MLK3^+/+, 4 MLK3^C/C. B: cardiac chamber and whole heart weights normalized to tibia length (TL) in 6-mo-old MLK3^C/C or MLK3^+/+ littermate mice. n = 7 MLK3^+/+ and 4 MLK3^C/C. *P < 0.05, **P < 0.01 by Student’s two-tailed unpaired t test. CRIB, Cdc42/Rac interactive binding; HW, heart weight; LV, left ventricle; MLK3, mixed lineage kinase 3; RV, right ventricle.

Reduction of MLK3 mRNA and Protein Expression in MLK3 CRIB Mutant Mice

The MLK3 CRIB mutation disrupts Cdc42-mediated MLK3 kinase activation in cell culture (5, 6) and in vivo (8). MLK3 kinase inhibitors reduce MLK3 expression in LV tissue in the setting of pressure overload (12). We therefore tested the effects of MLK3 CRIB mutation on MLK3 mRNA and protein expression. LV tissue in MLK3^C/C mice displayed a 74 ± 6% reduction of Map3k11 gene expression compared with MLK3^+/+ (P < 0.05) as measured by quantitative PCR (Fig. 4A). This was associated with 73 ± 7% reduction in MLK3 protein level in LVs of MLK3^C/C compared with MLK3^+/+ (Fig. 4B). We observed similar reduction of MLK3 protein expression in aortas from MLK3^C/C mice compared with MLK3^+/+ littermates (Supplemental Fig. S2).

Figure 4.

Mutation of MLK3 CRIB domain leads to reduction of MLK3 mRNA and protein in left ventricular tissue. A: RNA expression of MLK3 gene Map3k11 normalized to Gapdh from LV tissue isolated from MLK3^+/+ or MLK3^C/C littermates (age, 10–12 wk). n = 3 male, 3 female/genotype. B: MLK3 protein detected by immunoblot from LV lysates in 10–12-wk-old male mice. n = 4 MLK3^+/+ and 4 MLK3^C/C. Data analyzed by Student’s unpaired t test. *P < 0.05. ADU, arbitrary densitometric units; AU, arbitrary units; CRIB, Cdc42/Rac interactive binding; LV, left ventricle; MLK3, mixed lineage kinase 3.

Impaired LV Compensation to Pressure Overload in MLK3 CRIB Mutant Mice

To test the role of the MLK3 CRIB domain in the LV response to pressure overload, we subjected male MLK3^C/C mice to moderate transaortic constriction for 7 days. We studied only male mice because of the lack of significant pressure overload observed in age-matched female littermates with the same degree of TAC. Complete echocardiographic, hemodynamic, and organ mass data are in Supplemental Table S2. TAC induced LV pressure overload (systolic blood pressure P < 0.05 by 2-way ANOVA for TAC vs. sham effect, Fig. 5A), as well as LV hypertrophy (LV normalized to tibia length P < 0.05 by two-way ANOVA for TAC vs. sham effect, Fig. 5C). TAC-induced LV hypertrophy did not differ between MLK3^C/C compared with MLK3^+/+ mice (Fig. 5C). However, LV function declined selectively in the MLK3^C/C TAC mice, as measured invasively by the contractility index (dP/dt normalized to instantaneous pressure, Fig. 5B) or by echocardiographic determination of LV ejection fraction (Fig. 5D).

Figure 5.

Mutation of MLK3 CRIB domain leads to LV dysfunction after transaortic constriction. Male MLK3^+/+ or MLK3^C/C mice (age, 10–15 wk) were subjected to moderate (25 g) pressure overload by transaortic constriction (TAC) for 7 days. Invasive hemodynamic measures of aortic systolic blood pressure (SBP; A) and contractility index of LV dP/dt normalized to instantaneous pressure (B). LV mass normalized to tibia length (TL) (C). Summary data of LV ejection fraction (EF) and representative M-mode echocardiogram (D). Groups compared by two-way ANOVA. *P < 0.05 for main effects of surgery (TAC vs. sham); #P < 0.05 by Sidak’s multiple-comparisons test. CRIB, Cdc42/Rac interactive binding; LV, left ventricle; MLK3, mixed lineage kinase 3.

Impaired JNK Activation and MLK3 Membrane Fraction Translocation in MLK3 CRIB Mutant Mice

MLK3 is required for JNK activation in the LV after pressure overload and in the cardiac myocyte (3). JNK activation mediates the initial LV functional response to pressure overload (13). We, therefore, measured JNK activation after TAC in LV tissue of MLK3^+/+ and MLK3^C/C mice and observed significant reduction of phosphorylated/total JNK in the MLK3^C/C TAC LV tissue (Fig. 6A). MLK3 translocates from the cytosol to the plasma membrane in response to Cdc42 cointeraction (6). Maximal MLK3 activation of JNK also requires MLK3 translocation to the plasma membrane (6). We next tested the effect of the MLK3 CRIB mutation on MLK3 cellular fraction association after TAC. In control, basal, MLK3^+/+ mice, we detected MLK3 predominantly in the soluble fraction from LV tissue, but at 7 days post-TAC, we detected MLK3 in the particulate, membrane-associated fraction (Fig. 6B). By contrast, in the MLK3^C/C mice, the proportion of MLK3 detected in the pellet versus the soluble fraction did not increase after TAC, indicating that the CRIB mutation disrupted TAC-induced MLK3 translocation from the cytosol to membrane.

Figure 6.

Mutation of MLK3 CRIB domain leads to reduced compensatory JNK activation and altered cellular localization of MLK3 in the pressure-overloaded left ventricle. A: immunoblot for phosphorylated JNK (p-JNK) at Thr183/Tyr185, total JNK (T-JNK), and GAPDH in LV tissue from 10–15-wk-old male mice subjected to moderate (25 g) pressure overload by transaortic constriction (TAC) for 7 days. Summary densitometry data shown. n = 4 MLK3^+/+ and 4 MLK3^C/C. B: LV tissue in 10–12-wk-old MLK3^+/+ and MLK3^C/C littermate mice under basal and 7-day TAC conditions was separated into soluble (S) and pellet (P) fractions, followed by immunoblot for MLK3 and the membrane-associated caveolin. Summary data of relative change in pellet/soluble localization of MLK3 in LV tissue after TAC, compared with control (age-matched unoperated littermates). Each sample represents an independent experiment. n = 3 MLK3^+/+ and MLK3^C/C. *P < 0.05 by Student’s unpaired t test. ADU, arbitrary densitometric units. CRIB, Cdc42/Rac interactive binding; LV, left ventricle; MLK3, mixed lineage kinase 3.

DISCUSSION

In the current study, we investigated the requirement of the MLK3 CRIB domain for the maintenance of normal cardiovascular physiology. In MLK3^C/C mice with disrupting mutation of the MLK3 CRIB domain, we observed 1) baseline elevated blood pressure, 2) baseline LV and RV hypertrophy, 3) reduced expression of MLK3 mRNA and protein in LV tissue, 4) more severe LV dysfunction after moderate transaortic constriction, and 5) disruption of TAC-induced JNK activation and MLK3 cytosolic to membrane translocation in LV tissue. Taken together, these findings support a critical role of the MLK3 CRIB domain in the regulation of basal blood pressure and LV function, and in the normal LV response to pressure overload, likely through maintenance of normal MLK3 transcription (Fig. 7).

Figure 7.

Summary and proposed model. In the normal state, mixed linage kinase 3 (MLK3) requires intact CRIB domain for autoactivation of kinase function, maintenance of basal MLK3 mRNA and protein levels, and for membrane association during pressure overload. These effects control blood pressure, oppose cardiac hypertrophy, and promote left ventricular functional compensation and JNK activation after pressure overload. Genetic disruption of the CRIB domain opposes these processes. CRIB, Cdc42/Rac interactive binding.

Our observation of elevated blood pressures in MLK3^C/C mice identifies a role of the MLK3 CRIB domain in the normal regulation of blood pressure. Mice with whole body genetic deletion of MLK3 display hypertension, increased vascular stiffness, and reduced resistance arteriole distensibility (4). We also previously demonstrated that in contrast to genetic deletion, MLK3 kinase inhibition induced no blood pressure effects but only affected LV function. MLK3 CRIB mutation disrupts MLK3 kinase autoactivation (6). We, therefore, did not predict that the MLK3^C/C mice would develop hypertension, as our previous work supports that the MLK3 regulation of blood pressure is kinase-independent (4). We speculate that the unexpected MLK3^C/C elevated blood pressure instead arises because of the reduced MLK3 expression induced by the CRIB mutation, or because of disrupted MLK3 association with the plasma membrane in vascular cells, which might limit MLK3 access to its nonkinase effectors, such as RhoA (14, 15). Prior work supports minimal MLK3 expression in the kidney (4) and identifies important functions of MLK3 in vascular smooth muscle cells (15). This suggests a primary effect of the MLK3 CRIB domain on vascular contribution to blood pressure. We acknowledge, however, that a renal component, or an effect of MLK3 on renal hormonal signaling, such as the renin angiotensin aldosterone system, may contribute to the observed blood pressure effects in the MLK3 CRIB mutant. Future work is needed to address these questions. Nevertheless, the elevated blood pressure in the MLK3 CRIB mutant mice adds further support for a critical role of MLK3 in the regulation of blood pressure and suggests that augmenting MLK3 CRIB domain-mediated signaling may represent a candidate therapeutic strategy against hypertension.

Baseline cardiac hypertrophy in the MLK3 CRIB mutants represents a second finding of our study. As systemic hypertension would not be predicted to affect RV mass, the presence of biventricular hypertrophy supports an intrinsic, blood pressure-independent effect of the CRIB domain on cardiac structure. MLK3 kinase inhibition in cultured adult cardiac myocytes induces hypertrophy (3), further supporting a blood pressure-independent role of MLK3 in the regulation of cardiac hypertrophy. We therefore interpret our findings to reveal a novel role of the MLK3 CRIB-mediated signaling in opposing basal LV hypertrophy. LV hypertrophy represents an independent risk factor for cardiovascular death (2), suggesting that further investigation into augmentation of MLK3 CRIB domain-mediated signaling may prove clinically useful.

We note that the pairwise comparisons of normalized RV mass, atrial mass, and heart mass only differed statistically between MLK3^+/+ and MLK3^C/C in male mice, but not female mice. This raises the question of a potential sex-specific effect of MLK3 mutation on cardiac hypertrophy. However, our statistical comparison by two-way ANOVA revealed no interaction between sex and genotype. We, therefore, do not conclude a sex-specific effect of MLK3 CRIB domain regulation on cardiac hypertrophy, at least at 3 mo of age.

Our studies in the TAC model also demonstrate increased LV dysfunction and reduced JNK activation in MLK3 CRIB mutant mice. This finding correlates with prior work showing that complete MLK3 deletion induces accelerated LV dysfunction after pressure overload (3). We chose to focus this study on 7 days of LV pressure overload for several reasons. First, prior investigations in a whole body MLK3 deletion model have identified an important role for MLK3 (3) and downstream MLK3 effectors such as JNK (13) as particularly important in the initial LV response to pressure overload. We also observed previously that MLK3 mediates the therapeutic effects of sildenafil on LV function within the first 7 days of pressure overload (4). As we observed in whole body MLK3 deletion (4), we found here that selective mutation of the MLK3 CRIB domain impairs normal LV functional compensation after pressure overload at a time preceding overt TAC-induced LV hypertrophy. The reduced LV JNK phosphorylation at 7 days post-TAC in the LVs of MLK3^C/C mice further indicates a requirement of the CRIB domain for MLK3-mediated JNK activation. The small GTPase Cdc42 normally binds and activates MLK3 through the MLK3 CRIB domain (5, 6) and opposes TAC-induced LV dysfunction through augmentation of cardiac myocyte JNK signaling (16). Cdc42 interaction with the MLK3 CRIB domain also promotes MLK3 translocation from cytosol to plasma membrane in cell culture (6). Our observations of blunted JNK activation and impaired MLK3 particulate (membrane) association after pressure overload in LVs of MLK3 CRIB mutant mice further implicate MLK3 as mediating Cdc42 functional effects in the LV response to pressure overload. Further studies will be required to determine the effect of the MLK3 CRIB domain on the chronic LV remodeling response.

The current study and our prior work in the MLK3 deletion model (4) each demonstrate deleterious effects of MLK3 disruption on blood pressure and on LV function. We have also observed that 14-day administration of a pan-MLK inhibitor URMC-099 reduces basal LV function (4). These findings contrast with published work by others in which URMC-099 improved LV function and hypertrophy after pressure overload (12). In the current study and in previous work (3), we used a modest transaortic constriction, whereas the study by Wang et al. used a severe TAC model. In addition, our studies examined the 1- and 4-wk time points to study the initial compensatory effects of MLK3 (3, 4), whereas the other work employed a more chronic 8-wk TAC approach. We propose that these differences in experimental design may explain the varied observations.

Finally, we observed unexpectedly that MLK3 CRIB mutant mice display reduced LV and aortic protein levels of MLK3. The concomitant finding of reduced mRNA of map3k11, which encodes MLK3, further supports that the MLK3 CRIB domain is necessary for normal MLK3 transcriptional regulation. Of note, chronic administration of a MLK3 kinase inhibitor reduces cardiac expression of MLK3 protein (12). Because the MLK3 CRIB domain is required for MLK3 kinase activity (6), these combined findings suggest a mechanism in which MLK3 kinase signaling promotes maintenance of normal MLK3 gene transcription. The MLK3 effector JNK itself directly phosphorylates transcription factors (17), and MLK3 also modulates activity of the JNK-dependent transcription factor NFAT in the LV (4, 18). However, the exact transcription factors through which MLK3 may promote its own gene transcription remain unknown, and the specific requirement of JNK for the regulation of MLK3 expression remains unclear.

These findings have potential clinical implications. MLK3 is expressed in the human LV (3), and its interaction with its regulator PKG1α becomes decreased in the failing LV in experimental models (4). In humans, polymorphisms in Map3k11, the gene for MLK3, associate with age-dependent hypertension and with LV hypertrophy. Cdc42 binding to the MLK3 CRIB domain leads to MLK3 activation. Cdc42 normally opposes LV hypertrophy through a role in the cardiac myocyte (16). Taken together, these observations raise the question of whether disruption of Cdc42-MLK3 association in cardiovascular tissues contributes to development of hypertension or LV hypertrophy in humans. Because MLK3 requires its CRIB domain for basal activation by Cdc42, our findings suggest that pharmacological activation of MLK3 may be of therapeutic relevance in conditions such as heart failure or hypertension.

Our study has several limitations. First, the CRIB mutant mouse is a whole body knock-in mouse. We therefore cannot conclude with certainty whether the basal effects of CRIB mutation on blood pressure and LV physiology arise from vascular and cardiac myocyte tissue-specific effects, respectively. MLK3 plays important roles in cardiac myocytes (3), vascular smooth muscle cells (15), leukocytes (18), and fibrosis (19), which may differ in their effects on the cardiac response to pressure overload. This study admittedly did not dissect out the cell type-specific effects of the CRIB domain on basal LV hypertrophy, blood pressure, and function. Second, because of technical limitations of inducing pressure overload in female mice, we confined our TAC studies to male mice. Although we observed no baseline differences in CRIB mutation effects in male versus female MLK3^C/C mice, it remains possible that female MLK3^C/C mice could have a different pressure overload response. Finally, this study did not determine the relative degrees to which reduced MLK3 expression versus a specific functional effect of CRIB domain disruption explains the cardiovascular phenotype of the MLK3^C/C mice. We note that although MLK3 CRIB mutants retain some MLK3 expression, the systolic blood pressure increases and degree of LV hypertrophy in MLK3 CRIB mutants appear nearly identical to that reported in MLK3 whole body deletion mice (4). Furthermore, besides being reduced in expression, MLK3 membrane to cytosol association appears altered in LV tissue of MLK3-CRIB mutants after TAC (Fig. 6). We interpret these observations to support that the MLK3 CRIB domain regulates cardiovascular function at least in part through direct effects of this sequence on MLK3 function and localization, in addition to its effects on MLK3 expression.

In summary, this study demonstrates that mutation of the MLK3 CRIB domain leads to basal abnormalities in blood pressure and LV structure and promotes LV dysfunction after pressure overload. These findings therefore support investigation of approaches to activate MLK3 via its CRIB domain as potential strategies to improve blood pressure, oppose LV hypertrophy, and promote LV compensation to pathological stress.

SUPPLEMENTAL DATA

Supplemental Figs. S1 and S2 and Supplemental Tables S1 and S2: https://doi.org/10.6084/m9.figshare.19904374.v2.

GRANTS

This study was supported by the National Institutes of Health Grants R01-HL-131831 and R01-HL-162919 (to R. M. Blanton) and R01DK107220 (to R. J. Davis).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.M.B. conceived and designed research; P.-W.L., G.L.M., W.L., W.H., S.P., M.J.A., and R.M.B. performed experiments; P.-W.L., W.H., and R.M.B. analyzed data; P.-W.L., W.H., S.P., and R.M.B. interpreted results of experiments; P.-W.L. and R.M.B. prepared figures; R.M.B. drafted manuscript; P.-W.L., G.L.M., W.L., M.J.A., R.J.D., and R.M.B. edited and revised manuscript; P.-W.L., G.L.M., W.L., W.H., S.P., M.J.A., R.J.D., and R.M.B. approved final version of manuscript.