The control of Ca2؉ influx and NFATc3 signaling in arterial smooth muscle during hypertension

Madeline Nieves-Cintro´ n*†, Gregory C. Amberg*†‡, Manuel F. Navedo*, Jeffery D. Molkentin§, and Luis F. Santana*¶

*Department of Physiology and Biophysics, University of Washington, Box 357290, Seattle, WA 98195; and §Children’s Hospital Medical Center for Molecular Cardiovascular Biology, 3333 Burnet Avenue, Cincinnati, OH 45229

Communicated by Joseph A. Beavo, University of Washington School of Medicine, Seattle, WA, September 3, 2008 (received for review May 14, 2008) Many excitable cells express L-type Ca2؉ channels (LTCCs), which important in this process. (i) LTCC function is increased in participate in physiological and pathophysiological processes rang- hypertensive arterial smooth muscle (3, 4). (ii) The vasocon- ing from memory, secretion, and contraction to epilepsy, heart strictor angiotensin II (AngII), an activator of PKC, is a likely failure, and hypertension. Clusters of LTCCs can operate in a contributor to vascular dysfunction in human (9) and model PKC␣-dependent, high open probability mode that generates sites hypertension (10). Accordingly, AngII-converting enzyme in- of sustained Ca2؉ influx called ‘‘persistent Ca2؉ sparklets.’’ Al- hibitors and AngII antagonists are used extensively for though increased LTCC activity is necessary for the development of the treatment of hypertension in humans. (iii) AngII activates the vascular dysfunction during hypertension, the mechanisms leading Ca2ϩ-sensitive phosphatase calcineurin via LTCC Ca2ϩ entry in to increased LTCC function are unclear. Here, we tested the hy- hypertension. Calcineurin in turn dephosphorylates and hence -pothesis that increased PKC␣ and persistent Ca2؉ sparklet activity activates the NFATc3, decreasing the ex contributes to arterial dysfunction during hypertension. We found pression of voltage-gated Kϩ (Kv) channels in hypertensive that PKC␣ and persistent Ca2؉ sparklet activity is indeed increased arterial smooth muscle (11). Decreased Kv channel function in arterial myocytes during hypertension. Furthermore, in human depolarizes arterial smooth muscle, which indirectly increases arterial myocytes, PKC␣-dependent persistent Ca2؉ sparklets acti- LTCC function (12). The mechanism by which these channels vated the prohypertensive calcineurin/NFATc3 signaling cascade. activate calcineurin in smooth muscle is unclear (11, 13). These events culminated in three hallmark signs of hypertension- In this study, we examined the role of PKC␣ and Ca2ϩ sparklet -associated vascular dysfunction: increased Ca2؉ entry, elevated activity in the development of arterial dysfunction during hy 2؉ 2ϩ arterial [Ca ]i, and enhanced myogenic tone. Consistent with pertension. Our data suggest that increased Ca influx via 2ϩ 2ϩ these observations, we show that PKC␣ ablation is protective persistent Ca sparklet sites underlies increased arterial [Ca ]i against the development of angiotensin II-induced hypertension. and myogenic tone during hypertension. Furthermore, PKC␣- These data support a model in which persistent Ca2؉ sparklets, dependent persistent Ca2ϩ sparklets specifically activated PKC␣, and calcineurin form a subcellular signaling triad controlling NFATc3 in human arterial myocytes, resulting in decreased NFATc3-dependent expression, arterial function, and blood Kv2.1 channel expression. Importantly, loss of PKC␣ eliminated pressure. Because of the ubiquity of these , this model may persistent Ca2ϩ sparklets and protected against the development represent a general signaling pathway controlling of hypertension. These data support the concept that NFATc3, 2ϩ and cellular function. Kv channel expression, arterial [Ca ]i, myogenic tone, and hypertension are locally controlled by Ca2ϩ sparklet activity. angiotensin II ͉ myogenic tone ͉ sparklets ͉ transcription factors ͉ voltage-gated calcium channels Results We examined the mechanisms controlling Ca2ϩ influx via rterial tone is elevated during hypertension, increasing the LTCCs and calcineurin/NFATc3 signaling in arterial smooth Aprobability of stroke, coronary artery disease, cardiac hy- muscle during genetic and induced hypertension. This required pertrophy, and renal failure (1, 2). Although the etiology of investigation of the spatial organization of functional LTCCs and ␣ arterial dysfunction during hypertension is unclear, multiple PKC as well as NFATc3 activity in living cells. To establish the studies suggest that increased L-type Ca2ϩ channel (LTCC) broad implications of our findings, experiments were performed activity in arterial smooth muscle is a major contributor to this with human arterial smooth muscle and two established models pathological change (3, 4). However, the mechanisms and func- of hypertension. tional implications of increased Ca2ϩ influx via LTCCs remain 2؉ unclear. AngII Increases Ca Sparklet Activity in Arterial Smooth Muscle. 2ϩ In normotensive arterial smooth muscle, the opening of First, we tested the hypothesis that AngII increases Ca sparklet 2ϩ 2ϩ activity in arterial myocytes from mesenteric and cerebral ar- LTCCs produces local elevations in [Ca ]i called ‘‘Ca spar- klets’’ (5, 6). Two modes of Ca2ϩ sparklet activity have been teries (Fig. 1). The membrane potential of these cells was identified (6–8). Low-activity Ca2ϩ sparklets are produced by brief random openings of LTCCs that result in limited Ca2ϩ Author contributions: M.N.-C., G.C.A., M.F.N., and L.F.S. designed research; M.N.-C., G.C.A., influx. In contrast, long openings of LTCCs associated with and M.F.N. performed research; J.D.M. contributed new reagents/analytic tools; M.N.-C., ϩ PKC␣ produce high activity, persistent Ca2 sparklets that G.C.A., M.F.N., and L.F.S. analyzed data; and M.N.-C., G.C.A., M.F.N., and L.F.S. wrote the create regions of sustained Ca2ϩ influx. Low- and high-activity, paper. ϩ persistent Ca2 sparklets are produced by the opening of a single The authors declare no conflict of interest. or a small cluster of LTCCs. PKC␣ is required for persistent, but Freely available online through the PNAS open access option. 2ϩ not for low-activity, Ca sparklets. Under physiological condi- †M.N.-C. and G.C.A. contributed equally to this work. 2ϩ 2ϩ tions, Ca entry and global [Ca ]i and, thus, contraction are ‡Present address: Department of Biomedical Sciences, Colorado State University, Fort ϩ regulated by low-activity and PKC␣-dependent persistent Ca2 Collins, CO 80523. sparklets. ¶To whom correspondence should be addressed. E-mail: [email protected]. 2ϩ At present, the role of PKC␣ and Ca sparklets in the chain This article contains supporting information online at www.pnas.org/cgi/content/full/ of events leading to arterial dysfunction during hypertension is 0808759105/DCSupplemental. PHYSIOLOGY unknown. Several lines of evidence suggest that they may be © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808759105 PNAS ͉ October 7, 2008 ͉ vol. 105 ͉ no. 40 ͉ 15623–15628 Downloaded by guest on September 25, 2021 -/- A control Ang II (100 nM) A WT PKCα 80 control 5 µm b ) 60

M a

(n Ang II (100 nM) ]

2+ 40 a b C [ c 50 nM ∆ 20 a 1 s c d 50 nM 0 d 1 s * * B 3 B C D * control 0.6 40 2.0 control Ang II nts 2 2 Ang II (100 nM) 1.5 30 s ve

m 0.4 s µ 20 1.0 nP nP 1

tes / 0.2 i

s 10 0.5 number of e 0.0 0 0.0 0 -/- control Ang II 0 100 200 300 control Ang II WT PKCα (100 nM) ∆ [Ca2+ ] (nM) (100 nM) Fig. 2. AngII increases Ca2ϩ sparklet activity through the activation of PKC␣. 2ϩ 2ϩ 2ϩ Fig. 1. AngII increases Ca sparklet activity. (A) Total internal reflection (A) Representative time course of [Ca ]i in Ca sparklet sites in WT and Ϫ/Ϫ fluorescence (TIRF) image of an arterial myocyte (Left). Traces (Right) are time PKC␣ cells before and after AngII (100 nM) treatment. (B) Scatter plot of nPs 2ϩ ϩ Ϫ Ϫ courses of [Ca ]i in the sites indicated by the green circles before and after values for individual Ca2 sparklet sites in WT and PKC␣ / cells under control ϩ application of 100 nM AngII. (B)Ca2 sparklet density before and after conditions and after application of AngII. application of AngII. (C) Amplitude histogram of Ca2ϩ sparklets before and after AngII application. The black and red lines are the best fit to the control ϩ and AngII data, respectively, by using the Gaussian function. (D) nPs values for myocytes by increasing the probability of Ca2 sparklet occur- individual Ca2ϩ sparklet sites under control conditions and after application of rence (Ps) and/or the number of functional channels (i.e., n), AngII. The dashed line defines the threshold between low and high nPs sites. without increasing the duration of individual Ca2ϩ influx events or the amplitude of quantal Ca2ϩ sparklets. controlled by using the patch-clamp technique. Application of 2؉ 2؉ AngII (100 nM) increased Ca2ϩ sparklet activity (i.e., Ca2ϩ Regulation of Ca Sparklets, Arterial [Ca ]i, and Tone by AngII ␣ ␣ influx) by augmenting the activity of previously active Ca2ϩ Requires PKC . AngII activates PKC in arterial smooth muscle. ␣ 2ϩ sparklet sites and activating new sites (Fig. 1A). Indeed, at the Because PKC is necessary for persistent Ca sparklet activity physiological membrane potential of Ϫ40 mV and external Ca2ϩ in arterial myocytes (6, 8), we examined the effects of AngII on ϩ 2ϩ ␣ ␣Ϫ/Ϫ of 2 mM, AngII increased Ca2 sparklet site density Ϸ2.3-fold Ca sparklets in WT and PKC null (PKC ) myocytes (Fig. ϩ 2ϩ ␣Ϫ/Ϫ (Fig. 1B). We investigated whether this increase in Ca2 sparklet 2). Ca sparklet activity was lower in PKC than in WT activity was accompanied by an increase in the amplitude of myocytes (Fig. 2 A and B). This was due to the absence of ϩ 2ϩ ␣Ϫ/Ϫ elementary Ca2 sparklet events. Fig. 1C shows an amplitude high-activity, persistent Ca sparklets in PKC myocytes 2ϩ histogram of Ca2ϩ sparklets before and after the application of (Fig. 2B). AngII increased the number and activity of Ca ␣Ϫ/Ϫ AngII. This histogram illustrates that AngII increased the num- sparklets in WT myocytes but not in PKC myocytes (Fig. 2 2ϩ A and B; P Ͻ 0.05). Thus, PKC␣ is necessary for AngII- ber of Ca sparklets observed. Note, however, that both ϩ histograms have clearly discernable peaks that could be fit with dependent stimulation of Ca2 sparklets. ϩ 2ϩ 2 We measured arterial wall [Ca ]i in pressurized (80 mmHg) a multi-Gaussian function with a quantal unit of Ca influx of Ϫ Ϫ 38 nM (control; ␹2 ϭ 1.1; AngII; ␹2 ϭ 1.9), suggesting that AngII WT and PKC␣ / mesenteric arteries (Fig. S2 A and B). ϩ 2ϩ 2ϩ did not change the amplitude of elementary Ca2 sparklets. Consistent with our Ca sparklet data, arterial wall [Ca ]i was Ϫ Ϫ AngII did not increase Ca2ϩ sparklet activity homogeneously lower in PKC␣ / than in WT arteries under control conditions throughout the sarcolemma. Rather, AngII increased Ca2ϩ and after application of AngII (n ϭ 7, P Ͻ 0.05). Indeed, the 2ϩ Ϸ influx at specific sites by increasing the activity [quantified as nPs, AngII-induced increase in arterial wall [Ca ]i was 4-fold ␣Ϫ/Ϫ where n is number of quantal levels and Ps is the probability of smaller in PKC than in WT arteries. Note that application 2ϩ ␮ 2ϩ ␣Ϫ/Ϫ Ca sparklet occurrence; see supporting information (SI) Text of nifedipine (1 M) decreased [Ca ]i in WT and PKC Ϫ Ϫ for details of this analysis] of previously active sites and activating arteries to a similar level (P Ͼ 0.05), indicating that PKC␣ / new Ca2ϩ sparklet sites (Fig. 1 A and D). As previously reported arteries have a lower dihydropyridine-sensitive (control 2ϩ 2ϩ 2ϩ 2ϩ (6), Ca sparklet activity was bimodal, with sites of low activity [Ca ]i Ϫ [Ca ]i with nifedipine) [Ca ]i than WT arteries. 2ϩ (nPs ϭ 0.07 Ϯ 0.02) and sites of high activity (nPs ϭ 0.54 Ϯ 0.10; To examine the physiological consequences of reduced Ca 2ϩ 2ϩ Ϫ/Ϫ also referred as ‘‘persistent Ca sparklet sites’’). Acute appli- sparklet activity and [Ca ]i in PKC␣ arterial smooth muscle, cation of AngII (100 nM) increased total Ca2ϩ sparklet activity we evaluated the effect of pressure on the diameter of WT and nearly 12-fold (P Ͻ 0.05; Fig. 1D). PKC␣Ϫ/Ϫ arteries (Fig. S2 C and D). Consistent with our Ca2ϩ 2ϩ 2ϩ ␣Ϫ/Ϫ To quantify Ca influx (⌬QCa; in coulombs) associated with sparklet and arterial wall [Ca ]i data, PKC arteries were each Ca2ϩ sparklet event, we determined the ‘‘signal mass’’ of significantly less constricted than WT arteries at all intravascular Ca2ϩ sparklets before and after the application of AngII. The pressures examined (Fig. S2C). Furthermore, AngII evoked a 2ϩ Ϫ/Ϫ ⌬QCa of Ca sparklet events in low and high activity, persistent smaller constriction in PKC␣ than in WT arteries (Fig. S2D). sites increased after AngII (Fig. S1A). However, we found that the duration of individual low- and high-activity, persistent Ca2ϩ PKC␣ Is Required for Increased Persistent Ca2؉ Sparklets During sparklet events was similar before and after AngII (Fig. S1B). Hypertension. We investigated the role of PKC␣ in the chain of This indicates that AngII increases Ca2ϩ influx in arterial events leading to an increase in Ca2ϩ entry into arterial myocytes

15624 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808759105 Nieves-Cintro´net al. Downloaded by guest on September 25, 2021 A WT Ang II-infused WT Ang II-infused PKCα-/- A saline-infused rat i Ang II-infused rat ii

10 µm SHR l iii B so 3 *

) * to U cy A

/ 2 WT e 50 nM ce (

BC* Ang II-infused WT ran * -/- 0.9 α b 1.25 Ang II-infused PKC 1 s 1 2 escen 0.75 r µ 0.6 mem s luo á * f 0.10 C 0 nP K R 0.3 P e H fuse S sites / m 0.05 in id ne t - * ali -ra d ng rta 0.0 0.00 s A II nfus α-/- nP nP WT WT PKC Low s High s ␣ Ang II-infused Fig. 4. Increased PKC activity during acquired and genetic hypertension. (A) Representative surface-plot of PKC␣-associated fluorescence in cerebral Fig. 3. Ca2ϩ sparklet activity is increased during AngII-induced hypertension. artery myocytes from saline-infused rats, (Ai), AngII-infused rats, (Aii), and SHR 2ϩ (Aiii). (B) Bar plot of the PKC␣ membrane-to-cytosol fluorescence ratio from (A) Time courses of [Ca ]i in a smooth muscle cell from saline and AngII- infused WT and PKC␣Ϫ/Ϫ mice. (B) Bar plot of Ca2ϩ sparklet density. (C) Bar plot saline- and AngII-infused and SHR myocytes. 2ϩ of nPs values for individual Ca sparklet sites in smooth muscle cell from saline- and AngII-infused WT and PKC␣Ϫ/Ϫ mice. smooth muscle (mesenteric and cerebral arteries) (Fig. S3E). These data support the view that increased PKC activity and Cav1.2 during hypertension. In these experiments, Ca2ϩ sparklets were expression contributes to higher persistent Ca2ϩ sparklet activity in recorded in myocytes from saline (control) and AngII-infused WT arterial myocytes during hypertension. and PKC␣Ϫ/Ϫ mice in the absence of externally applied AngII (Fig. -3A). AngII infusion induced hypertension in WT mice, increasing PKC-Induced Local Ca2؉ Signals via LTCCs Activate NFATc3 and De mean arterial pressure (MAP) from 114 Ϯ 3to149Ϯ 3 mmHg (see crease Kv2.1 Expression in Human Arterial Smooth Muscle. In arterial Fig. 6A; n ϭ 5, P Ͻ 0.05). We found that Ca2ϩ sparklet density and smooth muscle, Ca2ϩ sparklet activity is regulated locally by activity was higher in myocytes isolated from AngII than in PKC␣ and calcineurin via a targeting mechanism involving the saline-infused WT mice (Fig. 3 B and C; P Ͻ 0.05). Importantly, scaffolding AKAP150 (8). Because persistent Ca2ϩ note that chronic AngII infusion did not increase Ca2ϩ sparklet sparklet activity (see above) and calcineurin/NFATc3 activity density and activity in PKC␣Ϫ/Ϫ myocytes. These findings suggest (13) are increased during hypertension, we investigated the that PKC␣ is required for increased Ca2ϩ sparklet activity during possibility that PKC␣-dependent persistent Ca2ϩ sparklets ac- AngII-induced hypertension. tivate calcineurin/NFATc3 signaling in arterial myocytes. To test We examined PKC␣ activity, Ca2ϩ sparklets, and LTCC this hypothesis, we measured calcineurin activity in saline- and function in another widely used model of hypertension, the AngII-infused WT and PKC␣Ϫ/Ϫ mesenteric arteries. We found spontaneously hypertensive rat (SHR). For comparison, we also that calcineurin activity was decreased in arteries from saline- performed experiments using AngII-infused rats. Increased infused WT and PKC␣Ϫ/Ϫ mice (Fig. S4). Infusion of AngII AngII signaling is implicated in the development of hypertension increased calcineurin activity in WT, but not in PKC␣Ϫ/Ϫ, in SHRs (10). We used an immunofluorescence approach to arteries, suggesting that PKC␣ is required for calcineurin acti- establish the level of sarcolemmal PKC␣ translocation (i.e., vation during chronic AngII signaling activation. 2ϩ 2ϩ activation) in SHR and AngII-infused cells. This approach has We recorded Ca sparklets, [Ca ]i, and nuclear NFATc3 been used to determine PKC activity in smooth muscle (14). As translocation in primary cultured human aortic smooth muscle shown in Fig. 4, PKC␣ localization at the sarcolemma was higher cells expressing NFATc3 tagged with EGFP (17, 18) (Fig. 5). in cerebral and mesenteric artery myocytes from AngII-infused These cells were used to expand on our findings in rodent models rats and SHR than in normotensive myocytes. Consistent with of hypertension and gain insights into the mechanisms of Ca2ϩ this, and as in the mouse and rat model of AngII-induced and NFATc3 signaling in humans. As anticipated, Ca2ϩ sparklets hypertension, the density of Ca2ϩ sparklet sites was Ϸ3-fold are present in human arterial myocytes (Fig. 5A). Similar to higher (P Ͻ 0.05) in SHR than in normotensive myocytes (Fig. murine smooth muscle, Ca2ϩ sparklets were abolished in human S3A). This resulted from an increase in the number of low and arterial myocytes by superfusion of Ca2ϩ-free solution (data not 2ϩ high nPs Ca sparklet sites in SHR cells (Fig. S3B). shown). Furthermore, activation of PKC with PDBu (200 nM) 2ϩ 2ϩ We investigated if, as in cells from AngII-infused animals, PKC increased Ca sparklet activity and global [Ca ]i in human activity contributes to higher basal Ca2ϩ channel activity in SHR myocytes (Fig. 5 A, B, and D). Nifedipine (1 ␮M) prevented the 2ϩ than in normotensive myocytes (Fig. S3C). Consistent with our increase in global [Ca ]i evoked by PDBu, suggesting that 2ϩ 2ϩ SHR Ca sparklet data—and previous reports (3, 15, 16)—LTCC activation of LTCCs is required for the increase in [Ca ]i currents were Ϸ3-fold larger in SHR than in normotensive cells. induced by PKC activation. Interestingly, application of the PKC inhibitor Go¨6976 (100 ␮M) To provide a real-time examination of the relationship between 2ϩ 2ϩ decreased this difference in Ca currents between SHR and [Ca ]i and nuclear translocation of NFATc3 in living human normotensive cells, indicating that increased basal PKC activity arterial smooth muscle cells, we expressed NFATc3 fused to EGFP 2ϩ contributes to higher LTCC activity in SHR than in normotensive (NFATc3-EGFP) in these cells (19). [Ca ]i and NFATc3-EGFP cells (Fig. S3 C and D). In addition, and consistent with other translocation were simultaneously recorded by using confocal mi- PHYSIOLOGY reports (15), Cav1.2 expression was increased in SHR arterial croscopy. We found that activation of PKC with PDBu increased

Nieves-Cintro´n et al. PNAS ͉ October 7, 2008 ͉ vol. 105 ͉ no. 40 ͉ 15625 Downloaded by guest on September 25, 2021 NFATc3-EGFP translocation during application of PDBu in A ϩ control B human smooth muscle cells loaded with the fast Ca2 buffer 2ϩ PDBu (200 nM) BAPTA-AM to prevent a global increase in [Ca ]i.Were- 4 corded Ca2ϩ sparklet activity in arterial myocytes loaded with F) 3 control BAPTA and found their amplitude and gating modalities were PDBu (200 nM) i0 nifedipine (1 µM) similar to those recorded with EGTA in the patch-pipette (Fig. 2+ 2 2ϩ a] (F / (F a] S5). The area of Ca sparklets was smaller in cells loaded with C [ 1 BAPTA than with EGTA, likely because of BAPTA’s faster 10 min 2ϩ 50 nM Ca -binding kinetics. 1 s We found that application of PDBu resulted in nuclear

tr

t translocation of NFATc3-EGFP in the absence of a change in + CsA r an C D a 2ϩ PDBu (200 nM) n global [Ca ]i. Indeed, PDBu-induced NFATc3-EGFP translo- PDBu (200 nM) NF NF s sloc ϩ l 4 0.75 ) 2.0 0.7 2 o

NFATc3-EGFP A AT cation was similar in control (i.e., no exogenous Ca buffer) and c T F 0.6 a at / Ͼ c c BAPTA-loaded cells (Fig. 5F; P 0.05). Furthermore, in the 3 0.60 t 3 3-EG ion o F/ ) /F ion (F 0.5 -EG i0 (F ␮ i0 1.5 presence of nifedipine (1 M), application of PDBu failed to + 0.4

2 2+ 2 0.45 (

F ϭ a] FP F 0.3 induce nuclear NFATc3-EGFP translocation (Fig. 5E Inset; n n n P u cyt uc [Ca ] (F / F ) [C c

1 NFATc3-EGFP 0.30 1.0 0.2 / 5). Together, these data support the view that NFATc3 trans- F) c

10 min y 10 min t location in human arterial smooth muscle is activated by PKC- 2ϩ

t dependent, local Ca signals via LTCCs (presumably persistent

r ϩ ϩ

E a + Nifedipine (1µM) F 2 2

ns Ca sparklets) and not global changes in [Ca ]i. NF PDBu ) 4 1.5 l We examined the effects of PKC and LTCC activation on o F AT 3 c 1 F/F0 0.2 F /F a NFAT-dependent gene expression in human arterial myocytes nuc cyt 1.0 c3 t t ion

10 min i0 2 * -E r

] (F / expressing an NFAT reporter construct expressing EGFP in a + 2 n

0.5 G ( a PDBu (200 nM) N response to NFAT activation (19). In these experiments, EGFP slo 0.75 1 F

4 F F nu [C P F) A 0 cc expression (i.e., fluorescence) was used as an indicator of NFAT

ca 0.0 F) /F T 3 0.60 + + c 2 ] 2 t y transcriptional activity. EGFP fluorescence was examined in 3 ion i0 a AT t

- F

EG i 2+ [Ca ] 2 0.45 l [C t cells under three experimental conditions: control, with 200 nM

( aon o ca on A Nc

F/ r rol NFAT F ␮ [Ca ] (F / PTA PT PDBu, and PDBu plus the LTCC blocker diltiazem (10 M). We n

1 P so so 0.30 u A c cont cont B BAan l 10 min F tran l ti tr used diltiazem rather than nifedipine in these relatively long c y t ) experiments because of its greater stability. Ϸ PDBu induced a 2.5-fold increase in NFAT transcriptional G H )

n activity (Fig. 5G). Diltiazem prevented the increase in NFAT

* nge 2.0

4 * ion / n activity induced by PDBu, indicating that LTCC activity (i.e., s ha e

ssio ϩ

c 2

e es Ca sparklets) is required for PKC-induced NFAT activation in d

2 pr 1.0 ol * human arterial myocytes. We also examined the effects of the ex

n (f physiological PKC activator AngII on the expression of Kv2.1 i 2.1 NFAT dr iv NFAT 0 0.0 v GFP e xpr GFP transcript in human arterial myocytes. Kv2.1 channels are im- -act E saline Ang II/ Ang II/ control PDBu PDBu / K Ang II

β portant regulators of membrane potential and, consequently, Dilt Dilt. VIVIT 2ϩ [Ca ]i and tone in arterial myocytes (20). Furthermore, the Fig. 5. Activation of NFATc3 signaling by PKC-dependent local Ca2ϩ signals Kv2.1 gene has putative NFAT binding sites in its promoter via LTCCs in human aortic myocytes. (A)Ca2ϩ sparklet records from a repre- region and could be down-regulated by NFATc3 (11). We found 2ϩ sentative cell before and after 200 nM PDBu. (B) Global [Ca ]i response to the that sustained AngII exposure (48 h) decreased Kv2.1 transcript application of PDBu during control conditions and in the presence of nifedi- levels in control human aortic smooth muscle cells, but not in the ␮ 2ϩ pine (1 M). (C and D) The time course of [Ca ]i and nuclear NFATc3-EGFP presence of the LTCC blocker diltiazem (10 ␮M) or the NFAT translocation in response to PDBu or PDBu ϩ CsA (1 ␮M), respectively. Insets ␮ in C show two NFATc3-EGFP images from this cell before and after application inhibitor VIVIT (1 M) (21) Fig. 5H). 2ϩ of PDBu. (E) Time course of [Ca ]i and NFATc3-EGFP nuclear translocation in a cell loaded with BAPTA-AM after application of PDBu. The Inset shows PKC␣ Contributes to AngII-Induced Hypertension. To relate our in 2ϩ global [Ca ]i (black trace and left y axis; F/F0 units) and NFATc3-EGFP nuclear vitro findings to the intact animal, we measured blood pressure Ϫ/Ϫ translocation (red trace and y axis; Fnuc/Fcyt units) in a cell exposed to PDBu in PKC␣ and WT mice infused with saline or AngII (Fig. 6). 2ϩ Ϫ/Ϫ (arrow) in the presence of nifedipine. (F) The bar plot of the peak [Ca ]i and PKC␣ mice allowed us to investigate the role of this kinase NFATc3-EGFP nuclear translocation during control conditions and in cells in the development of AngII-induced hypertension. MAP was loaded with BAPTA-AM. (G) Bar plot of relative EGFP expression in human similar in control PKC␣Ϫ/Ϫ (n ϭ 5) and WT mice (n ϭ 6; P Ͼ aortic smooth muscle cells cultured for 48 h under control conditions in the 0.05). However, AngII infusion increased MAP in PKC␣Ϫ/Ϫ presence of PDBu (200 nM) and PDBu ϩ diltiazem (10 ␮M). (H) Bar plot of the Ϸ Ͻ ␤ mice to a lower extent ( 40% lower) than in WT mice (P 0.05; normalized (to -actin) Kv2.1 transcript level in human aortic smooth muscle ␣ cells cultured for 48 h under control conditions with angiotensin II (100 nM), Fig. 6B), suggesting that PKC contributes to the development angiotensin II ϩ diltiazem (10 ␮M), or angiotensin II ϩ VIVIT (1 ␮M). Kv2.1 of hypertension. transcript levels were determined by real-time RT-PCR. *, P Ͻ 0.05. Discussion Our findings provide a mechanistic framework for how Ca2ϩ 2ϩ global [Ca ]i and induced nuclear translocation of NFATc3-EGFP influx increases during hypertension. We also provide the first in human aortic smooth muscle cells (Fig. 5 C and D). Consistent direct demonstration that PKC␣ contributes to the development with the calcineurin activity data presented above, application of of AngII-induced hypertension. We show that increased Ca2ϩ the calcineurin inhibitor cyclosporine (CsA, 1 ␮M) prevented influx during hypertension is not simply the result of random 2ϩ nuclear NFATc3-EGFP translocation but not the rise in [Ca ]i activation of LTCCs as previously implied (3, 4). Rather, in- evoked by PDBu (Fig. 5D, n ϭ 5). creased Ca2ϩ influx during hypertension results primarily from We tested the hypothesis that calcineurin and NFATc3 sig- increased PKC␣-dependent persistent Ca2ϩ sparklet activity. naling is activated by local Ca2ϩ signals in human aortic smooth Our data suggest that an increase in number and probability of 2ϩ 2ϩ muscle cells (Fig. 5E). To do this, we examined [Ca ]i and activation of Ca sparklet sites—not an increase in the quantal

15626 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808759105 Nieves-Cintro´net al. Downloaded by guest on September 25, 2021 A C WT + Ang II diltiazem/ 200 nifedipine 180 α-/- α

(mmHg) PKC + Ang II low activity PKC -dependent, 160 Ca2+ sparklet persistent Ca2+ sparklet 140 120 α α

100 γ δ γ δ membrane blood pressure 80 depolarization β β AKAP150 1s WT saline PKCα-/- + saline α PP2B B P PKC 50 CsA / VIVIT 2+ 40 Ca2+ Ca 30 nuclear NFAT * 2+ [Ca ]i accumulation

II-induced 20

MAP (mmHg) 10 tone Kv2.1

∆ Ang expression 0 WT PKCα-/- blood pressure

Fig. 6. PKC␣ is required for AngII-induced hypertension. (A) Representative pressure waveforms from WT and PKC␣Ϫ/Ϫ mice before and after AngII infusion. (B) Bar plot of the change in mean arterial pressure (⌬MAP) induced by AngII in WT and PKC␣Ϫ/Ϫ mice. (C) Proposed mechanism by which persistent Ca2ϩ sparklet activity increase tone and blood pressure during hypertension (see Discussion for details).

unit of Ca2ϩ influx or the duration of Ca2ϩ entry events— modulates Ca2ϩ influx and gene expression in smooth muscle underlies increased Ca2ϩ influx via LTCCs into arterial myocytes (Fig. 6C). during hypertension. This is likely the result of a combinatorial In this model, PKC␣, and calcineurin, are targeted by increase in Cav1.2 channel expression (15) and PKC␣ activity in AKAP150/79 to specific regions of the sarcolemma of arterial arterial smooth muscle during hypertension. myocytes. Activation of PKC␣ induces persistent Ca2ϩ sparklet We performed the first examination of the relationship be- activity in these sites. This increase in local Ca2ϩ influx activates 2ϩ tween [Ca ]i and nuclear NFATc3 translocation in living hu- nearby calcineurin, which dephosphorylates NFATc3. Dephos- man arterial smooth muscle cells. Interestingly, our data suggest phorylated NFATc3 translocates into the nucleus of arterial that PKC-induced local Ca2ϩ signals via LTCCs (presumably myocytes where it modulates gene expression. Under physiolog- persistent Ca2ϩ sparklets) activate NFATc3 via calcineurin, ical conditions, calcineurin and NFATc3 activities are low in thereby modifying gene expression in human arterial myocytes. arterial smooth muscle (11, 13) likely because of low levels of Multiple lines of evidence support this view. (i) Activation of persistent Ca2ϩ sparklet activity and relatively high nuclear 2ϩ 2ϩ PKC increases [Ca ]i by increasing persistent Ca sparklet NFATc3 export rates (25). However, during genetic and AngII- activity and induced nuclear NFATc3-EGFP translocation in induced hypertension, an increase in PKC␣ activity increases these cells. (ii) The LTCC blocker nifedipine prevented the rise persistent Ca2ϩ sparklet activity, thus increasing calcineurin 2ϩ in [Ca ]i and nuclear NFATc3 translocation evoked by the activity and consequently NFATc3 nuclear import rate, which, if activation of PKC. (iii) Activation of PKC induced NFATc3 coupled with a decrease in export rate (11, 13), would lead to translocation even in cells loaded with the fast Ca2ϩ buffer high nuclear accumulation of this transcription factor. This, in BAPTA. This observation is important because in the presence turn, leads to nuclear accumulation of NFATc3 and down- of BAPTA, Ca2ϩ signals are confined to a small region of the cell regulation of Kv2.1 transcript expression (11). As suggested near the site of Ca2ϩ entry (22), suggesting that calcineurin/ previously (11, 12, 26), down-regulation of Kv2.1 decreases Kv NFATc3 signaling is activated by a local PKC-dependent Ca2ϩ currents and contributes to arterial smooth muscle depolariza- signal via LTCCs in human arterial smooth muscle. (iv)Ca2ϩ tion during hypertension, which indirectly increases Ca2ϩ spar- 2ϩ influx via LTCCs is required for NFAT-mediated transcriptional klet activity, global [Ca ]i, and tone. Initiation or continuation activity during PKC activation in these cells. of this feedback loop could be prevented by inhibiting PKC␣, Recent work suggests a potential mechanism for local activa- calcineurin, or NFATc3 activation (13) or with LTCC blockers. tion of NFATc3 by LTCCs in arterial myocytes. The scaffolding In normotensive smooth muscle, Ca2ϩ entry via persistent protein AKAP150 (the rodent ortholog of human AKAP79) is Ca2ϩ sparklet sites accounts for Ϸ50% of the total dihydropy- required for sarcolemmal PKC␣ expression and persistent Ca2ϩ ridine-sensitive Ca2ϩ influx into these cells under physiological sparklets in arterial myocytes (23). AKAP150/79 also targets conditions (7). Our Ca2ϩ sparklet data from normotensive, SHR calcineurin to the surface membrane (24). Indeed, we found that and AngII (chronic and acute exposure) myocytes suggest that calcineurin opposes persistent Ca2ϩ sparklet activity in arterial Ca2ϩ influx is increased in hypertensive smooth muscle due to an myocytes (8, 23). Furthermore, consistent with AKAP150/79’s increase in the density and activity (i.e., nPs) of low activity and role in targeting calcineurin, we found this scaffolding protein is persistent Ca2ϩ sparklet sites. At present, however, whether the required for calcineurin-dependent modulation of Ca2ϩ spar- relative contribution of low and high activity, persistent Ca2ϩ klets in smooth muscle (23). Collectively, these data suggest that sparklet sites to Ca2ϩ influx in arterial myocytes is altered during AKAP150/79 targets PKC␣ and calcineurin to specific regions of hypertension is unclear. Future studies should address this issue. the sarcolemma of arterial myocytes where they modulate Arterial smooth muscle is depolarized during hypertension nearby LTCCs. In combination with our Ca2ϩ sparklet, cal- (12). Membrane depolarization increases low-activity and per- cineurin, and NFATc3 data, we propose that AKAP150, PKC␣, sistent Ca2ϩ sparklet activity (7). Thus, voltage-independent PHYSIOLOGY calcineurin, and LTCCs form a positive feedback loop that (i.e., increased in Cav1.2 expression and PKC␣ activity) and

Nieves-Cintro´n et al. PNAS ͉ October 7, 2008 ͉ vol. 105 ͉ no. 40 ͉ 15627 Downloaded by guest on September 25, 2021 -dependent mechanisms (i.e., depolarization) could synergize to were dissociated from cerebral and mesenteric arteries as reported previously increase Ca2ϩ sparklet activity in arterial myocytes during hy- (11). AngII or saline were administered by using osmotic minipumps. Blood ␣ pressure was monitored by using telemetry (13). The patch-clamp technique pertension. Indeed, it is intriguing to speculate that loss of PKC 2ϩ 2ϩ could protect against hypertension by decreasing voltage- was used to record Ca currents. Ca sparklets were recorded as described 2ϩ (6, 8). Real-time PCR was performed by using QuantiTect SYBR green PCR. dependent and -independent activation of Ca sparklets. Calcineurin activity was assessed by using a commercial kit as described (13). 2ϩ To conclude, our data support the concept that Ca influx, Nuclear NFATc3 translocation was assessed in cells transfected with NFATc3- 2ϩ global [Ca ]i, NFATc3-dependent gene expression, and myo- EGFP. NFAT transcriptional activity in response to PKC activation was assessed ϩ genic tone are modulated locally by Ca2 sparklet activity in using an NFAT-EGFP reporter construct. *, P Ͻ 0.05. arterial myocytes. Importantly, our data suggest that PKC␣ could be a novel target for the treatment of hypertension. ACKNOWLEDGMENTS. We thank Dr. Y. M. Usachev (University of Iowa, Iowa City) for providing the NFAT-EGFP reporter construct. The NFATc3-EGFP plas- Materials and Methods Summary mid was created by Dr. F. McKeon (Harvard University, Cambridge, MA) and provided by Dr. M. Iino (University of Tokyo, Tokyo, Japan). This work was A detailed version of this section can be found online in SI Text. Briefly, we supported by American Heart Association and National Institutes of Health used Sprague–Dawley and SHR as well as WT and PKC␣Ϫ/Ϫ mice. Myocytes grants.

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