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 receptor 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 transcription factor 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 gene expression, arterial function, and blood Kv2.1 channel expression. Importantly, loss of PKC␣ eliminated pressure. Because of the ubiquity of these proteins, this model may persistent Ca2ϩ sparklets and protected against the development represent a general signaling pathway controlling gene expression 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.