Dietary potassium regulated vascular calcification and aortic stiffness in mice. The effect of dietary potassium on atherosclerotic vascular calcification was characterized in vivo using the high-fat-fed ApoE–/– mouse model (12, 13), with dietary intake of standard (0.7% wt/wt), low (0.3% wt/wt), or high (2.1% wt/wt) potassium, as previously reported (29, 30). Mice fed the 0.3% potassium diet exhibited significant increases in vascular calcification, compared with mice fed the 0.7% potassium diet, whereas the 2.1% potassium diet markedly inhibited vascular calcification (Figure 1, A and B). The effects of dietary potassium on vascular calcification were demonstrated in aortic root sections by Alizarin red staining (Figure 1, A and B), as well as descending aortas by total calcium quantification (Figure 1C). It is worth noting that mice fed the 0.3% potassium diet had lower mean serum potassium levels (3.70 ± 0.21 mEq/l), while mice fed the 2.1% potassium diet had higher serum potassium levels (4.73 ± 0.15 mEq/l), compared with levels (4.27 ± 0.23 mEq/l) observed in mice fed the standard (0.7% potassium) diet (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.94920DS1).

Figure 1 Dietary potassium regulated vascular calcification and aortic stiffness in mice. ApoE–/– mice (n = 9/group) were fed a high-fat diet containing normal potassium (Control), low potassium (Low K+) or high potassium (High K+) for 30 weeks. (A) Vascular calcification in aortic roots, determined by Alizarin red staining. Representative images of H&E staining and Alizarin red staining in consecutive aortic root sections. Scale bars: 500 μm. (B) Quantification of calcification in the aortic root sections, measured using ImageJ software. Results presented are the percentage of positively stained areas in the total atherosclerotic lesion area of aortic roots. Bar values are means ± SD. (C) Total calcium content in the descending aortas, quantified by the Arsenazo III method. Results shown are normalized by total protein amount. Bar values are means ± SD. (D) Effects of dietary potassium on aortic stiffness. Pulse wave velocity (PWV), an indicator for aortic stiffness, determined by echocardiography at the end of the experiments. Bar values are means ± SD. Statistical analysis was performed by 1-way ANOVA followed by a Student-Newman-Keuls test.

In line with our observation of elevated calcium content in the descending aortas, echocardiographic analysis revealed that the 0.3% potassium diet induced a significant increase in mean pulse wave velocity (PWV) (Figure 1D), an indicator of aortic stiffness (31), suggesting that impaired aortic compliance is associated with low dietary potassium–induced vascular calcification. In contrast, compared with animals fed the 0.7% potassium diet, animals fed the 2.1% potassium diet exhibited inhibited vascular calcification and concurrently reduced PWV, supporting an important role of dietary potassium in regulating vascular calcification and stiffness.

Potassium regulated vascular calcification ex vivo. To determine if there was a direct effect of extracellular potassium level on calcification of the arteries and VSMCs in their natural milieu, we employed an ex vivo ring culture model that we and others have recently developed for histological and quantitative analysis of arterial calcification (32, 33). Based on normal physiological levels of serum potassium in adult C57BL/6 mice (34–36), we determined the effects of potassium at the lower (3.7 mM, low K+), middle (5.4 mM, control), and higher (6.0 mM, high K+) end of the physiological range on aortic calcification. Consistent with the in vivo results, we found that low potassium markedly enhanced vascular calcification in the aortic media, as demonstrated by Alizarin red staining (Figure 2A), while high potassium inhibited aortic calcification. Quantification of total calcium content demonstrated a significant increase in calcification in aortic rings cultured in medium containing 3.7 mM potassium, which was inhibited by 6.0 mM potassium (Figure 2B). These results demonstrated a direct effect of potassium on the calcification of the aortic media, supporting the role of low potassium in promoting VSMC calcification.

Figure 2 Potassium regulated vascular calcification ex vivo. Aortic rings prepared from wild-type mice were exposed to osteogenic media with control (5.4 mM), low potassium (3.7 mM, Low K+), or high potassium (6.0 mM, High K+) for 3 weeks. (A) Aortic calcification, determined in consecutive sections of the aortic rings by Alizarin red staining (middle panels). H&E staining (left panels) was performed for histology. Higher-magnification images of the boxed areas in the middle panels are shown in the right panels. Representative images from 4 independent experiments performed in duplicate are shown. Scale bars: 200 μm (left and middle panels) and 50 μm (right panels). (B) Total calcium content of aortic rings in each group determined by Arsenazo III in separated sets of experiments. Results shown are normalized by total protein amount. Bar values are means ± SD (n = 4, *P < 0.01). Statistical analysis was performed by 1-way ANOVA followed by a Student-Newman-Keuls test.

Lower physiological levels of potassium induced VSMC osteogenic differentiation and calcification. To determine the effects of potassium on VSMC calcification in cell culture, we first assessed the effects of extracellular potassium on VSMC viability using potassium at concentrations up to 108 mM, so as to identify a nontoxic level of potassium. Consistent with previous observations (37), we found that potassium, at concentrations lower than 54 mM, did not affect VSMC viability (Supplemental Figure 1). However, at concentrations from 3.7 to 4.7 mM, potassium markedly enhanced VSMC calcification compared with its concentration at 5.4 mM, as determined by Alizarin red staining of matrix calcium (Figure 3A) and total calcium quantification in cell lysates measured by the Arsenazo III method (Figure 3B); such calcification did not occur when potassium levels were maintained at 5.7 mM and 6.0 mM.

Figure 3 Lower physiological concentrations of potassium induced VSMC osteogenic differentiation and calcification. (A) Effects of potassium levels on calcification of vascular smooth muscle cells (VSMCs), determined by Alizarin red staining. VSMCs were cultured in osteogenic media with increased concentrations of potassium, 3.7 to 6.0 mM, for 3 weeks. Representative images of stained dishes from 4 independent experiments are shown. (B) Total calcium content in VSMCs, determined by Arsenazo III. VSMCs were cultured in osteogenic media with increased concentrations of potassium, 3.7 to 6.0 mM, for 3 weeks. Results shown are normalized by total protein amount. Bar values are means ± SD (n = 3, *P < 0.05 compared with potassium at 5.4 mM). (C) Effects of potassium levels on the expression of osteogenic and smooth muscle cell markers. VSMCs were exposed to 3.7 to 6.0 mM of potassium for 3 weeks. Representative images of Western blot analysis of runt-related transcription factor 2 (Runx2) and α-smooth muscle actin (α-SMA) proteins in VSMCs exposed to different concentrations of potassium from 3 independent experiments are shown. (D and E) Real-time PCR analysis of (D) osteogenic markers, Runx2, osteocalcin (OC), and alkaline phosphatase (ALP) and (E) smooth muscle cell markers, α-SMA and smooth muscle protein 22 α (SM22α). VSMCs were exposed to 3.7 to 6.0 mM potassium for 10 days. Results from 3 independent experiments performed in duplicate are shown. Bar values are means ± SD (*P < 0.05 compared with potassium at 5.4 mM). Statistical analysis was performed by 1-way ANOVA followed by a Student-Newman-Keuls test.

We and others have demonstrated that VSMC calcification resembles osteogenic differentiation of bone cells (38, 39); therefore, we determined the effect of potassium on the osteogenic differentiation of VSMCs, as indicated by the changes in the expression of bone markers and smooth muscle markers (13, 38). Increased expression of Runx2, a key osteogenic transcription factor that we have determined to be an important regulator of VSMC calcification (13, 38), was found to be upregulated by low potassium at both protein and mRNA levels (Figure 3, C and D). Consistently, the expression of Runx2-regulated osteogenic markers, including osteocalcin (OC) and alkaline phosphatase (ALP), were induced (Figure 3D), while the SMC marker genes, α-smooth muscle actin (α-SMA) and smooth muscle protein 22 α (SM22α), were markedly reduced concurrently (Figure 3, C and E). These data indicated a direct effect of low potassium on promoting VSMC osteogenic differentiation and calcification, via increasing osteogenic markers and decreasing SMC markers.

Activation of intracellular calcium signaling mediated low-potassium-induced VSMC calcification. Potassium deficiency has been shown to increase intracellular calcium in VSMCs (40). As increased intracellular calcium flux has been linked to VSMC calcification (41), we examined whether low potassium may induce VSMC calcification via activation of calcium signaling. We found that potassium at a control condition of 5.4 mM did not affect calcium flux (Figure 4A, dotted line). In contrast, elevation of intracellular calcium was evident in VSMCs within minutes after exposure to a low potassium concentration of 3.7 mM; the increase was sustained over the 30-minute duration (Figure 4A). Consistently, low potassium induced rapid and heightened activation of several known downstream mediators that include protein kinase C (PKC), and calcium-activated CREB (Figure 4B), but did not affect extracellular signal–regulated kinases (ERKs) signaling.

Figure 4 Activation of calcium signaling–mediated CREB was required for low-potassium-induced VSMC calcification. (A) Effects of potassium levels on intracellular calcium, determined by Fluo4 NW, in vascular smooth muscle cells (VSMCs) exposed to control (5.4 mM) or low potassium (3.7 mM, Low K+). Results from 3 independent experiments are shown. (B) Effects of low potassium on activation of extracellular signal–regulated kinase (ERK), protein kinase C (PKC), and calcium-activated cAMP response element–binding protein (CREB), determined by Western blot analysis. Representative blots from 3 independent experiments are shown. (C) Effects of pharmacological inhibitors on VSMC calcification. VSMCs were exposed to control or low-potassium media with the indicated inhibitors for 3 weeks. Calcification was determined by Alizarin red staining. (D) Effects of pharmacological inhibitors on activation of CREB. Western blot analysis of phosphorylation of CREB in C. Representative images from 3 independent experiments are shown. (E and F) Effects of CREB knockdown on low-potassium-induced VSMC calcification. VSMCs with CREB knockdown by shRNA (shCREB) or control shRNA (shScr) were exposed to control or low-potassium media for 3 weeks. Calcification was determined by Alizarin red staining. Western blot analysis was performed to determine the expression of CREB, runt-related transcription factor 2 (Runx2), and the autophagic marker, microtubule-associated protein 1 light chain 3 (LC3), in the cytoplasmic form (LC3 I), and conjugated form (LC3 II). Representative blots from 3 independent experiments are shown. (G) Effects of pharmacological inhibitors on autophagy markers. Western blot analysis of LC3 I and II levels in VSMCs exposed to control or low-potassium media for 3 weeks, in the presence or absence of indicated inhibitors. Representative results from 3 independent experiments are shown.

As potassium can be transported via 4 types of potassium channels (42), we determined the roles of these channels in mediating low-potassium-induced VSMC calcification. We found that inhibition of ATP-sensitive potassium channels (K ATP ) by glibenclamide, voltage-dependent potassium channels (Kv) by 4-aminopyridine, and calcium-activated potassium channels (K Ca ) by TRAM34 and charybdotoxin had no effects (Supplemental Figure 2), while inhibition of inward-rectifier potassium channels (K IR ) by barium (43, 44) abolished low-potassium-induced VSMC calcification (Figure 4C). Furthermore, inhibition of potassium-activated calcium signaling by calcium channel inhibitors, verapamil and nifedipine, attenuated low-potassium-induced vascular calcification (Figure 4C). Altogether, these results showed that low-potassium-induced elevation of intracellular calcium signaling mediated its effects on VSMC calcification.

Activation of calcium signaling-mediated CREB was required for low potassium-induced VSMC calcification. The effects of these inhibitors of potassium or calcium channels on calcium signaling–activated downstream signal CREB were further determined. Consistent with the inhibitory effects on low-potassium-induced VSMC calcification, the low-potassium-induced activation of CREB in VSMCs was abolished by the K IR inhibitor, barium, as well as the calcium channel inhibitors, verapamil and nifedipine (Figure 4D). Furthermore, knockdown of CREB using lentivirus-mediated short hairpin RNA (shCREB) blocked low-potassium-induced VSMC calcification and Runx2 upregulation (Figure 4, E and F), supporting the requirement of intracellular calcium–activated CREB signaling in mediating low-potassium-induced osteogenic differentiation and calcification of VSMCs.

As activation of CREB signaling was linked to increased autophagy in human melanoma cells and liver tissues (45, 46) and elevation of autophagy was demonstrated during osteoblast mineralization (47), we determined whether CREB activation may affect autophagy in VSMCs during low-potassium-induced calcification. We found that low potassium induced the microtubule-associated protein 1 light chain 3 in the conjugated form (LC3 II) (Figure 4F), an indicator of autophagy activation (48). CREB knockdown, however, markedly inhibited low-potassium-induced elevation of LC3 II (Figure 4F). Consistently, inhibition of K IR or calcium channels that were upstream of CREB by barium, verapamil, and nifedipine also attenuated low-potassium-induced expression of LC3 II (Figure 4G), implying that intracellular calcium–activated CREB mediates low-potassium-induced VSMC calcification via the regulation of autophagy.

Low potassium promoted VSMC calcification through autophagy. To determine the role of autophagy in low-potassium-induced VSMC calcification, we first assessed the effects of potassium levels on the expression of LC3 II. Increased expression of LC3 II (Figure 5A) was demonstrated in VSMCs exposed to potassium at concentrations that induced VSMC calcification (Figure 3A). Quantitative analysis further confirmed an increased LC3 II/I ratio by low potassium (Figure 5B), indicating that low potassium activated autophagy in VSMCs. In addition, a time-dependent effect of low potassium, at 3.7 mM, in increasing the LC3 II/I ratio and autophagy activation was demonstrated (Supplemental Figure 3).

Figure 5 Low potassium promoted VSMC calcification through autophagy. (A) Effects of potassium concentrations on autophagy in vascular smooth muscle cells (VSMCs). VSMCs were cultured in osteogenic media with increased concentrations of potassium for 3 weeks. Western blot analysis was performed to determine the expression of the autophagic marker, microtubule-associated protein 1 light chain 3 (LC3), in the cytoplasmic form (LC3 I) and conjugated form (LC3 II). Representative blots from 3 independent experiments are shown. (B) Quantitative analysis of the LC3 II/I ratio in A, by densitometric analysis using ImageJ software. Bar values are means ± SD (n = 3, *P < 0.05 compared with potassium at 5.4 mM). Statistical analysis was performed by 1-way ANOVA followed by a Student-Newman-Keuls test. (C) Effects of inhibition of autophagy on low-potassium-induced VSMC calcification. VSMCs were exposed to osteogenic media with control or low potassium for 3 weeks, with or without the autophagy inhibitor, 3-methyladenine (3-MA). Calcification was determined by Alizarin red staining. (D) Effects of 3-MA on autophagy markers. Western blot analysis was used to determine LC3 I and II levels in C. Representative blots from 3 independent experiments are shown. (E) Effects of knockdown of autophagy-related 7 protein (ATG7) on low-potassium-induced VSMC calcification. VSMCs with ATG7 stably knocked down by shRNA (shATG7) or with control shRNA (shScr) were exposed to osteogenic media with control or low potassium for 3 weeks. Calcification was determined by Alizarin red staining. (F) Effects of ATG7 knockdown on autophagy markers. Western blot analysis was used to determine ATG7 and LC3 I and II levels in E. Representative results from 3 independent experiments are shown.

The essential role of autophagy in mediating low-potassium-induced VSMC calcification was initially determined utilizing 3-methyladenine (3-MA), a pharmacological inhibitor of autophagy. Pretreatment of VSMCs with 3-MA dramatically reduced low-potassium-induced elevation of the LC3 II/I ratio, and concurrently blocked VSMC calcification (Figure 5, C and D). Moreover, we generated stable VSMCs with shRNA knockdown of ATG7, a key regulator of autophagy formation. Similar to the observations with the autophagy inhibitor, the ATG7 knockdown blocked VSMC calcification and inhibited low-potassium-induced elevation of the LC3 II/I ratio (Figure 5, E and F). These results support a definitive role of low-potassium-induced autophagy in mediating its effects on promoting VSMC calcification.

Potassium regulated the activation of CREB and autophagy in vascular calcification ex vivo and in vivo. The roles of activation of CREB and autophagy signals in low-potassium-induced vascular calcification were further determined in the ex vivo ring culture model and in vivo mouse models. As shown in Figure 6A, low-potassium-induced aortic calcification ex vivo was associated with increased activation of intracellular calcium–activated signals, including PKC, CREB, and elevation of LC3 II as well as upregulation of the osteogenic transcription factor Runx2 (Figure 6A). In contrast, high potassium markedly inhibited the activation of PKC and CREB and inhibited LC3 II and Runx2 (Figure 6A). Moreover, increased activation of PKC, CREB, and elevation of LC3 II and Runx2 were demonstrated in arteries from low dietary potassium–fed mice (Figure 6B). Consistently, higher dietary potassium inhibited PKC and CREB activation, and decreased LC3 II and Runx2 (Figure 6B). Altogether, these results further supported an important role for potassium in regulating vascular calcification via effects on calcium signaling, CREB, and autophagy in VSMCs (Figure 6C).