With No Lysine Kinase 1 Promotes Metabolic Derangements and RV Dysfunction in Pulmonary Arterial Hypertension

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HIGHLIGHTS
, with resultant increases in RV glucose uptake in both preclinical (6,7) and human PAH (8,9). RV function is inversely associated with RV glucose uptake (8), which implies excess intracellular glucose may have deleterious effects. Although glucose metabolism primarily generates adenosine triphosphate, the hexosamine biosynthetic pathway converts glucose to UDP-N-acetylglucosamine (UDP-GlcNAc) (10). UDP-GlcNAc is used to enzymatically post-translationally modify (PTM) serine or tyrosine residues, a process known as protein O-GlcNAcylation (10). Moreover, 1%-2% of glucose metabolized through glycolysis results in formation of methylglyoxal, a highly reactive dicarbonyl that can nonenzymatically modify proteins (protein glycation) (11). Both excess O-GlcNAcylation and glycation promote left ventricular (LV) cardiomyocyte mitochondrial dysfunction (12,13), but the role of these PTMs in RVD is relatively unexplored.
Two clinical studies showed hypochloremia identified high-risk patients with PAH, which might have direct relevance to glucose metabolism. Naal et al (14) showed that hypochloremia was independently associated with increased mortality. We also demonstrated that hypochloremia was independently associated with increased mortality and measures of RV failure in a multicenter study (15). The with-nolysine (WNK) kinase proteins are a family of signaling kinases activated in the setting of low intracellular chloride levels (16,17). In the heart, WNK1 is the predominant isoform expressed (17), but studies of WNK1 in cardiac diseases are lacking. WNK1 function is better understood in skeletal muscle because previous studies showed WNK1 promoted membrane localization of the glucose channels, glucose transporter (GLUT)1 (18) Table 2).  Figure 1A). Confocal microscopy revealed significantly increased WNK1 immunoreactivity ( Figure 1B) and membrane localization of GLUT1 ( Figure 1C) and GLUT4 ( Figure 1D) in MCT-V RVs. WNK463 significantly reduced WNK1 staining intensity ( Figure 1B) and GLUT1 ( Figure 1C) and GLUT4 membrane enrichment ( Figure 1D).

RESULTS
Metabolomics profiling quantified the effects of WNK inhibition on glucose metabolites in the hexosamine biosynthetic, glycolytic, and pentose phosphate pathways ( Figure 1E). In MCT-V RVs, the levels of the end products in the hexosamine biosynthetic (UDP-GlcNAc) ( Figure 1F), glycolytic (pyruvate) ( Figure 1G), and pentose phosphate pathways (erythrose 4phosphate) ( Figure 1H) were all higher than that in control subjects, but WNK463 restored the concentration of these metabolites ( Figures 1F and 1G). Thus, these data supported a role of WNK1 in regulating RV glucose handling and metabolism. There was no difference in total AMPK expression in the RV when we compared control rats to MCT-V rats, but MCT-V rats had decreased p-AMPK levels and a lower ratio of p-AMPK/total AMPK. WNK463 treatment increased p-AMPK, and the p-AMPK/ AMPK ratio was higher than levels in control rats ( Figure 3A). Then, we evaluated mitochondrial density in RV cardiomyocytes using confocal microscopy.
Compared to control rats, MCT-V had decreased mitochondrial density, which WNK463 corrected ( Figure 3B). We next performed quantitative proteomic profiling to examine how WNK463 altered mitochondrial protein homeostasis. We identified 2,970 total proteins in our extracts, and 1,203 proteins had significant differences in abundance. Principal component analysis revealed WNK463 shifted the proteomic signature toward control rats ( Figure 3C).
Hierarchical cluster analysis corroborated this finding ( Figure 3D). Next, we determined how WNK463 combated ( Figure 4B). Next, we analyzed 48 acylcarnitine-associated metabolites to probe mitochondrial FAO. Consistent with a previous study (28), nearly all acylcarnitines were reduced in MCT-V RVs.
However, WNK463 increased concentrations of most acylcarnitines even beyond that of RVs in control rats ( Figure 4C). In conclusion, these data indicated WNK463 altered RV metabolism. In particular, the restoration of metabolites in glycolysis, the TCA cycle, and acylcarnitines suggested multiple metabolic pathways were enhanced by WNK antagonism.   Table 2). First, when we plotted the relationship between right atrial (RA) pressure and pulmonary vascular resistance (PVR), patients with hypochloremia had higher RA pressure at all PVR values than patients with normochloremia ( Figure 10A). Furthermore, as PVR increased, patients with hypochloremia had a more rapid decline in cardiac output compared with patients with normal serum chloride levels ( Figure 10B).
These data associated hypochloremia with more severe RV dysfunction in patients with PAH.

DISCUSSION
In this study, we showed small molecule inhibition of WNK1 signaling prevented upregulation of GLUT1 and  Figure 1.  Our data might provide a molecular explanation of the increased mortality associated with hypochloremia in PAH (14,15) and in patients with left heart failure (39)(40)(41). Based on our findings, we proposed hypochloremia activated WNK1, which subsequently   Table 2). In addition, RV cardiomyocytes exhibited higher rates of glycolysis than LV cardiomyocytes (43), which suggested alterations in glucose metabolism might have heightened consequences in the RV.
Our findings also provided important insights into the interplay of multiple metabolic pathways     Consistent with this possibility, we detected a reduction in RV fibrosis in WNK463-treated rats (Supplemental Figure 8), which might be another mechanism underlying improvements in RV systolic and diastolic function with WNK463.