KATP channel gain-of-function leads to increasedmyocardial L-type Ca2+ current and contractilityin Cantu syndromeMark D. Levina,b, Gautam K. Singha,b, Hai Xia Zhanga,c, Keita Uchidaa,c, Beth A. Kozela,b, Phyllis K. Steind, Atilla Kovacsd,Ruth E. Westenbroeke, William A. Catteralle,1, Dorothy Katherine Grangea,b, and Colin G. Nicholsa,c,1

aCenter for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, St. Louis, MO 63110; bDepartment of Pediatrics,Washington University School of Medicine, St. Louis, MO 63110; cDepartment of Cell Biology and Physiology, Washington University School of Medicine,St. Louis, MO 63110; dDepartment of Medicine, Washington University School of Medicine, St. Louis, MO 63110; and eDepartment of Pharmacology,University of Washington, Seattle, WA 98195-7280

Contributed by William A. Catterall, April 26, 2016 (sent for review November 13, 2015; reviewed by Donald M. Bers, Robert S. Kass,and Michael C. Sanguinetti)

Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations ingenes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2,ABCC9) KATP channel subunits. We show that patients with CS, aswell as mice with constitutive (cGOF) or tamoxifen-induced (icGOF)cardiac-specific Kir6.1 GOF subunit expression, have enlarged hearts,with increased ejection fraction and increased contractility. Whole-cellvoltage-clamp recordings from cGOF or icGOF ventricular myocytes(VM) show increased basal L-type Ca2+ current (LTCC), comparable tothat seen in WT VM treated with isoproterenol. Mice with vascular-specific expression (vGOF) show left ventricular dilation as well asless-markedly increased LTCC. Increased LTCC in KATP GOF models isparalleled by changes in phosphorylation of the pore-forming α1 sub-unit of the cardiac voltage-gated calcium channel Cav1.2 at Ser1928,suggesting enhanced protein kinase activity as a potential link be-tween increased KATP current and CS cardiac pathophysiology.

KATP | transgenic | cardiovascular system | KCNJ8 | Kir6.1

Cantu syndrome (CS), characterized by hypertrichosis, osteo-chondrodysplasia, and multiple cardiovascular abnormalities

(1), is caused by gain-of-function (GOF) mutations in the genesencoding the pore-forming (Kir6.1, KCNJ8) and regulatory(SUR2, ABCC9) subunits of the predominantly cardiovascularisoforms of the KATP channel (2–5). Because the same diseasefeatures arise from mutations in either of these subunits, it isconcluded that CS arises from increased KATP channel activity, asopposed to any nonelectrophysiologic function of either subunit.However, this conclusion does not provide immediate explana-

tion for many CS features. In the myocardium, for example, acuteactivation of KATP channels results in shortening of the action po-tential (AP), with concomitant reduction of both calcium entry andcontractility (6). The naïve prediction in CS would therefore be thatKATP GOF mutations should shorten the AP, reduce contractility,and reduce cardiac output. We previously reported high cardiacoutput with low systemic vascular resistance in CS (7). Cantu syn-drome cardiac pathology is therefore opposite to prediction, andalso unlike classical hypertrophic or dilated cardiomyopathies, inthat the ventricle is dilated, but there is increased cardiac output.Here we characterize CS cardiac pathology in patients, and explorethe mechanistic basis using mice that express KATP GOF mutantsubunits in the heart and vasculature.

ResultsLow Blood Pressure in CS Patients. Eleven CS individuals (fivemale, six female, aged 17 mo to 47 y), all harboring ABCC9 muta-tions (Table S1), participated in CS research clinics at St. LouisChildren’s Hospital. Five had been previously followed at this in-stitution (7) and the remainder were enrolled via the CS InterestGroup (www.cantu-syndrome.org). Patient demographic data, geno-type, and available cardiac historical, physical, and test informationare summarized in Table S1. Most patients had no recalled cardiac

symptomatology, although prior office notes revealed episodes ofchest pain, fatigue, shortness of breath, and exercise intoleranceassociated with pericardial effusion. Additionally, three patients(cs002, cs004, cs005) reported palpitations and exercise intolerance.One of these (cs004) also had symptoms with orthopnea, resultingfrom “idiopathic” high-output state and atrial fibrillation. Five pa-tients had patent ductus arteriosus that required surgical ligation orcatheter-based closure, two had significant pericardial effusions,three had been diagnosed with pulmonary hypertension, and fivehad histories of lower extremity edema. All patients had full butnoncollapsing peripheral pulses. All but one patient (cs004, who wason several medications; Table S1) had supine systolic and diastolicblood pressure (BP) that was well below mean for age (Table S1)[mean age: 16.6 ± 13.5; systolic BP: 90.5 ± 12.8 mmHg; diastolic BP:58.2 ± 6.2 mmHg; heart rate (HR): 85 ± 17 beats per minute (bpm)](8). Despite these low BP values, no patient demonstrated ortho-static HR or BP changes. There were relatively few cardiac findingson physical examination, with the exception of one patient with adiastolic murmur (cs004) at the apex. Electrocardiograms revealedfirst-degree atrioventricular (AV) block in four patients, fascicularblock in two, and T-wave abnormalities (T-wave axis 180° displacedfrom QRS axis and morphologic abnormalities) in seven patients,but no evidence of QT shortening or correct QT (QTc) prolon-gation (Table S2).

Significance

ATP-sensitive potassium (KATP) channels are present in cardiacand smooth muscle; when activated, they relax blood vesselsand decrease cardiac action potential duration, reducing car-diac contractility. Cantu syndrome (CS) is caused by mutationsin KATP genes that result in overactive channels. Contrary toprediction, we show that the myocardium in both CS patientsand in animal models with overactive KATP channels is hyper-contractile. We also show that this results from a compensa-tory increase in calcium channel activity, paralleled by specificalterations in phosphorylation of the calcium channel itself.These findings have implications for the way the heart com-pensates for decreased excitability and volume load in generaland for the basis of, and potential therapies for, CS specifically.

Author contributions: M.D.L., G.K.S., H.X.Z., K.U., B.A.K., P.K.S., A.K., R.E.W., W.A.C., D.K.S.,and C.G.N. designed research; M.D.L., G.K.S., H.X.Z., K.U., B.A.K., A.K., and R.E.W. performedresearch; R.E.W. contributed new reagents/analytic tools; M.D.L., G.K.S., H.X.Z., K.U., B.A.K.,P.K.S., A.K., R.E.W., W.A.C., D.K.G., and C.G.N. analyzed data; and M.D.L., G.K.S., R.E.W.,W.A.C., and C.G.N. wrote the paper.

Reviewers: D.M.B., University of California, Davis; R.S.K., Columbia University; and M.C.S.,University of Utah.

The authors declare no conflict of interest.1To whom correspondence may be addressed. Email: wcatt@uw.edu or cnichols@wustl.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1606465113/-/DCSupplemental.

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Cardiovascular Structural and Functional Characteristics in CSPatients. Two-dimensional color Doppler echocardiographic in-terrogation and strain imaging (Fig. 1, Fig. S1, and Table S3)revealed normal segmental anatomy, but markedly enlargedhearts (Fig. 1), with enhanced cardiac output and contractility, inCS patients. Left ventricular (LV) chambers were significantlydilated in CS patients, and cardiac output was markedly increased(Fig. 1). LV mass was increased but, interestingly, LV posteriorwall thickness (LVPWD) was not different from controls (Fig. 1).This constellation of features is distinct from both typical hyper-trophic cardiomyopathies (where LVPWD is increased) and typ-ical dilated cardiomyopathies (where there is chamber dilationwith diminished cardiac function). In part, this dilated hyper-contractile phenotype could be a secondary response to chroni-cally elevated blood volume as a result of vasodilation—a directlypredicted consequence of vascular KATP GOF (9). Pulsed-wavevelocity testing offers a noninvasive correlate of vascular tone.Consistent with BP measurements, there was diminished pulsed-wave velocity in CS patients (control 5.6± 0.9 ms−1, n = 8; CS 4.5 U+0.8 m/s, n = 7, P = 0.003), implying diminished vascular tone.

Circadian Abnormalities and Low Vagal Function in CS Patients.Several patients received 24-h ambulatory ECG monitoring.None demonstrated atrial or ventricular arrhythmia during the re-cording period. The data were subsequently used for heart ratevariability analysis (HRV). Fig. 2 displays HR and high-frequency(HF) power, a marker for vagal activity, as a function of time of day,for a representative CS patient and age/sex-matched control. Thecontrol 18-y-old female shows typical circadian variation: in general,higher HR during waking hours, and significantly lower HR duringsleeping hours; HF also has a circadian rhythm that is inverselyrelated to this HR trend, rising during sleeping hours, and lowduring waking hours. Interestingly, all CS patients demonstratedmarkedly diminished HF power (Fig. 2 and Fig. S2) and generallyhigh HR for age that failed to lower appropriately during sleeping

hours (Figs. S2 and S3). These latter findings suggest a relativelyelevated sympathetic activity but diminished vagal activity.

Enhanced Contractility in Hearts Expressing KATP GOF Mutations. Thepredicted effect of KATP GOF in the myocardium itself is APshortening and reduced contractility, whereas the predicted ef-fect in the vasculature is reduced peripheral resistance. Theabove clinical evaluation of CS patients reveals physiologicallylow BPs, consistent with the latter prediction. However, myo-cardial hypercontractility and no evidence of K current-inducedQT shortening on ECG are grossly opposite to prediction. Wehypothesize that these features are secondary consequences ofthe primary predictions. Specifically, we propose that increasedKATP current in either ventricular or smooth muscle myocyteswill lead to lower cardiac output and decreased peripheral re-sistance, respectively; both of these will result in diminishedtissue perfusion, which in turn will induce a systemic feedback toincrease cardiac output (see, for example, Fig. 7C).To examine these hypotheses in a tractable system, we first

evaluated the cardiac consequences of Kir6.1 GOF mutationsexpressed in the myocardium under α-myosin heavy chain(αMHC) control in mice [constitutive GOF (cGOF) mice].cGOF mice exhibit prolonged PR intervals, as well as episodes ofjunctional rhythm, and diminished AV nodal conduction, but noQT shortening (10). Echocardiography under light anesthesia(11) (Fig. 3A and Table S4) revealed normal chamber wall di-mensions, but increased contractility and ejection fraction incGOF hearts, again counter to naïve prediction, but consistentwith the hypercontractile phenotype of CS patients.Isolated cGOF myocytes displayed diminished resting sarcomere

length, significantly increased fractional shortening and increasedrates of shortening and relaxation (Fig. 3B and Table S5). Thesefeatures are similar to those seen in WTmyocytes following exposureto isoproterenol (ISO). The subsequent effect of ISO was reduced incGOF myocytes (Fig. 3B), consistent with the idea that cGOFmyocytes are effectively “prestimulated” (12) via a pathway that is atleast convergent with that responding to β-adrenergic signaling. Toconfirm that this prestimulation effect was not an artefactual effect ofincreased Kir6.1 protein expression per se, mice expressing domi-nant-negative Kir6.1[AAA] subunits under αMHC control (AAA)were studied (13). No significant differences in contractile parameterswere seen between AAA and littermate control myocytes (Table S6).

KATP GOF Myocytes Demonstrate Increased LTCC. Cardiac excitation–contraction (EC) coupling depends on Ca entry via the L-typeCa2+ current (LTCC), which is modulated by phosphorylation inresponse to β-adrenergic and other neurohumoral inputs. Presti-mulated cGOF myocyte contractility is consistent with enhance-ment of such modulation. Cell size, assessed by cell capacitance,was normal in cGOF myocytes, but baseline LTCC amplitude and

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density were double those of control myocytes (Fig. 4B). Com-mensurate with the idea that cGOF myocytes were prestimulated,LTCC amplitudes and densities were markedly increased by ISOin control myocytes but much less so in cGOF myocytes (Fig. 4).There was a slight shift in voltage dependence in the baselinecGOF current–voltage (I/V) relationship, again similar to that incontrol I/V curves after ISO exposure.Because the Kir6.1 GOF transgene is constitutively activated

in cGOF hearts, the above changes in LTCC properties might bedevelopmentally induced. To examine the temporal link between

expression of KATP GOF and consequent increases in LTCC, weassessed cardiomyocytes in which GOF was induced postnatally,rather than developmentally [tamoxifen-induced GOF (icGOF)] bycrossing Kir6.1[G343D] and tamoxifen-inducible Myh6-Cre mice(14). Double-transgenic icGOF and littermate controls were treatedwith tamoxifen for 5 d; ventricular myocytes were isolated, andLTCC recorded, on day 6. Recorded icGOF myocytes were largerthan control myocytes but, even controlling for this, icGOF LTCCdensity was still dramatically increased at baseline (Fig. 5). Again, asin cGOF myocytes, ISO significantly stimulated LTCC in controlbut not icGOF myocytes. These experiments further confirmremodeling of LTCC in response to Kir6.1 GOF, and demonstratethat this can occur in adult animals and rapidly (within hours ordays) following transgene induction, implying that such responsesare physiologically, not developmentally, mediated.

Vascular KATP GOF Expression Results in Enlarged Hearts andIncreased LTCC. Kir6.1 and SUR2 are prominently expressed invascular smooth muscle (VSM). VSM expression of KATP GOFunder smooth muscle (SM)-MHC promoter control reduces vas-cular contractility, resulting in a chronically vasodilated state withlow systolic and diastolic BP (9). Young vGOF mice, induced withtamoxifen at 2 mo and studied by echocardiography 1 mo later,revealed no significant changes in cardiac structural parameters,but did exhibit enhanced cardiac contractility (Fig. 6A). A secondcohort of vascular-specific expression (vGOF) mice in which ex-pression had been induced for more than 6 mo (old vGOF)showed significant chamber dilation and now showed diminishedcontractility (Fig. 6A and Table S7). Consistent with these oldervGOF echocardiographic findings, baseline LTCC in vGOFmyocytes was elevated, although not as dramatically as in cGOF oricGOF mice, and both vGOF and control LTCC increased fol-lowing ISO (Fig. 6B). Interestingly, the baseline I–V relationshipwas left-shifted by ∼10 mV (Fig. 6B) in vGOF myocytes.

Increased Cav1.2 Protein Phosphorylation in Kir6.1 GOF Hearts. TheLTCC in mature ventricular myocytes is conducted by Cav1.2channels, composed of a pore-forming α1 subunit in association withα2δ, β, and possibly γ subunits (15). In the heart, the large C-terminaldomain of the 250-kDa α1 subunit is proteolytically processed,resulting in a complex of the core Cav1.2 protein plus its non-covalently bound distal C terminus (16), a potent autoinhibitor ofchannel activity (17). β-Adrenergic stimulated phosphorylation of theCav1.2 channel relieves this autoinhibition and enhances LTCC and

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myocyte contractility, in response to sympathetic signaling (18–20).We found no change in the level of full-length Cav1.2 channel pro-tein, proteolytically processed Cav1.2 protein, or distal C-terminalprotein, between cGOF and control hearts (Fig. 7A). We investigatedtwo phosphorylation sites on Cav1.2 channels that have been iden-tified in intact ventricular myocytes using phosphospecific antibodies(Ser1700 at the interface between the distal and proximal C-terminaldomains and Ser1928 in the distal C-terminal domain). Phosphory-lation of Ser1700 by PKA is directly implicated in β-adrenergic reg-ulation of Cav1.2 channels (18, 19), whereas phosphorylation ofSer1928 may correlate with PKA or PKC pathway activity (18, 21).We found no significant change in phosphorylation of Ser1700 incGOF, but Ser1928 phosphorylation was increased approximatelytwofold in cGOF mice (Fig. 7A), and ∼1.25-fold in vGOF mice.Because Ser1928 is also a substrate for phosphorylation by PKC (22),phosphorylation of this site by either PKA or PKC signaling pathwaysmay be involved in persistent increase in basal activity, and conse-quent loss of β-adrenergic stimulation of Cav1.2 channels in CS.

Intolerance of β-Blockade in icGOF Animals. To further explore themechanistic basis of Cantu disease and directly assess whetherβ-adrenergic signaling is required for adaptation to KATP GOF inthe heart, we chemically ablated sympathetic signaling in icGOFand littermate control mice by implanting adult (12- to 32-wk-old)animals with slow-release propranolol pellets, and then initiatingtransgene expression with tamoxifen 2–7 d later. The majority oficGOF mice treated with propranolol pellets died within 2 wk oftransgene induction, but no deaths were observed in littermatecontrols (Fig. 7B). Though further mechanistic details remain tobe elucidated, these data are a striking indication that adrenergicsignaling is required for adaptation to KATP GOF induction.

DiscussionCS and Cardiovascular Disease. KATP channels are heterooctamericcomplexes of pore forming Kir6.1 or Kir6.2 subunits and regulatorySUR1 or SUR2 subunits (23). ABCC9 (SUR2) and KCNJ8 (Kir6.1)are prominently expressed in cardiac myocytes, VSM, and vascularendothelial cells (23), suggesting that cardiovascular features willpredominate in CS. We show that CS patients have dilated LVs,increased cardiac output and ejection fraction, and increased myo-cardial contractility. LV mass is increased compared with controls,but LV wall thicknesses are normal. Such findings are distinct fromhallmarks of either hypertrophic cardiomyopathy (HCM) or dilatedcardiomyopathy; the former typically demonstrates thickened LVchamber walls with hypercontractile function, whereas the latterdemonstrates a dilated LV chamber with diminished cardiac func-tion. Distinguishing between these cardiac phenotypes is critical,

because standard therapies for HCM, for example, might actuallyworsen a Cantu patient’s clinical status.High cardiac output states such as we observe can arise from

chronic vasodilation, and can lead to LV volume overload andsubsequent chamber dilation (24–27). Vital statistic and echo-cardiographic data support the hypothesis that CS patients arevasodilated, but in a compensated high cardiac output state.Though it is controversial to term BP as clinically low in anasymptomatic patient, systolic and diastolic BPs were greatlydiminished compared with mean for age.

Feedback Control via L-Type Ca Current in Compensated CardiacFunction. Gain of K+ channel function in blood vessels would bepredicted to cause reduced contractility and vasodilation, and inthe heart to shorten APs, reducing Ca2+ entry and contractility.Available clinical data regarding ECG phenotype in CS patients islimited, but there is no evidence for AP shortening (10). The highcardiac output state evident by echocardiography is also not na-ively predicted and leads us to postulate that feedback response toboth the heart and vasculature remodels EC coupling to producethe unexpected hypercontractile function (Fig. 7). In mice, echo-cardiograms and isolated myocyte contractility studies reveal thatsuch compensation does indeed occur: cardiac contractility isincreased, concomitant with increased LTCC. Isolated cGOFmyocytes reveal basal hypercontractility and elevated LTCC, butsimilar maximal contractility and LTCC after ISO exposure tocontrol, whereas old vGOF myocytes also show some increase inbaseline LTCC. Direct analysis of LTCC in hearts of CS patientsis not feasible, but there are indirect suggestions of similarly

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enhanced basal LTCC in the ambulatory ECG data: higher thannormal heart rates, as well as blunted heart rate variability andT-wave abnormalities (Fig. 2).In the presence of ISO, LTCC densities in cGOF or icGOF

myocytes are not significantly different from control (Figs. 4 and5) (12). This lack of ISO response is consistent with chronic in vivoactivation of signaling pathways that result in prestimulation. Thisprestimulation must be a consequence of the initial defect; that is,the gain of KATP conductance, which we suggest will indeed be areduced cardiac output, but that this results in activation of ad-renergic or parallel signaling pathways. We previously showed thatbasal and ISO-stimulated cAMP concentrations are not altered intransgenic Kir6.2 GOF hearts that also show prestimulation (12).Cardiac LTCC can be increased by PKA activation (28–30), but alsoby PKC in response to α-adrenergic and endothelin-1–dependent

signaling (31–35). PKC overexpression may result in diminishedLTCC current in some circumstances (36), although dialysis ofcritical LTCC-associated proteins may confound results in whole-cell patch-clamp experiments (32). Both PKA and PKC modulationof LTCC (22, 37) are mediated by phosphorylation of CaV1.2 serineresidues on the pore or accessory subunits (18–20, 38). The identityof relevant residues phosphorylated in response to adrenergic sig-naling remains an active area of investigation. Ser1700 and Ser1928phosphorylation of Cavα1.2 have both been demonstrated, butSer1928 may also be phosphorylated by PKC (35, 39), and Ser1700may also be a target of CaMKII (18, 40). Our analysis implicatesSer1928 phosphorylation as a marker of the response to KATP GOFbut not Ser1700. Though Ser1928 phosphorylation may not itself bedirectly involved in the enhancement of LTCC, it is suggestive thatPKC or PKA pathways may ultimately be activated.

Feedback Response to Vascular Defects in CS. Two distinct effectswere observed on cGOF and icGOF LTCC: left-shift in activationand increased current density. We also observed a left-shift ofvoltage dependence of LTCC in vGOF, but only a mild increase incurrent density, and a nonsignificant increase in Ser1928 phos-phorylation. In contrast, older vGOF animals exhibited increaseddiastolic volume, as observed in CS patients. Vascular KATP GOFresults in increased smooth muscle KATP current and diminished BP(9), providing a potentially direct explanation for the markedly low-for-age BPs in CS patients; this also provides a systemic mechanismthat may link the primary vGOF phenotype to secondary conse-quences in the heart: vascular KATP GOF expression results in va-sodilation that will ultimately result in a long-standing volume load,evidenced by LV chamber dilation. Early after transgene induction(6 wk), vGOF hearts exhibit increased contractility and increasedejection fraction (Fig. 6), but end diastolic volume is increased inolder vGOF animals, potentially reflecting dilation manifesting afterprolonged exposure to volume overload (26).

Conclusions and Implications. CS can arise from GOF in eitherKir6.1 or SUR2 proteins of the cardiovascular KATP channel (2–5).A distinct CS cardiac pathology is characterized by high output statewith enhanced cardiac contractility and enhanced chamber volume,associated with decreased vascular pulse wave velocity and low BP.Our animal studies suggest that CS cardiac pathology emerges fromcombined cardiac and vascular KATP GOF mutation expression(Fig. 7C). The vascular findings are readily explained by theexpected molecular consequences, but KATP GOF mutations in themyocardium will tend to reduce cardiac action potential duration(APD) and decrease pacemaker activity, both of which would re-duce cardiac contractility and output. The counter observation ofenhanced contractility and maintained APD is explained by en-hanced LTCC, with left-shifted activation and increased basalconductance, associated with enhanced phosphorylation of thePKA/PKC target residue Ser1928. These findings are consistentwith compensatory chronic signaling, potentially involving adren-ergic stimulation, through pathways that converge on the LTCC.This consistent explanation for CS cardiac features raises the

question of whether or how to treat them. The dramatic differencein response of icGOF and control animals to β-blockade (Fig. 7B)is consistent with adrenergic signaling being involved in at leastthe early compensation to KATP GOF, further suggesting thatβ-blockade could be a dangerous approach to treating Cantu pa-tients. Alternately, appropriate therapies should target KATPchannels directly, and the success of sulphonylurea drugs intreating neonatal diabetes, which results from GOF in the pan-creatic KATP isoforms, gives promise that similar or more selectiveKATP antagonists may reverse some or all CS disease features.

MethodsHuman studies were carried out on CS patients recruited to an annual researchclinic at St. Louis Children’s Hospital. Written informed consent was provided by allpatients. The study was approved by the Human Research Protection Office ofWashington University School of Medicine and performed at St. Louis Children’sHospital in St. Louis. Echocardiographic and electrocardiographic studies were

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Fig. 7. Cellular and molecular basis of Cantu syndrome. (A) Quantitation ofWestern blot analysis of isolated total Cav1.2a subunit protein, distal C terminus,and processed C-terminal fragments, normalized to WT protein levels in cGOF,Kir6.1[AAA], and vGOF cardiac samples, as well as relative levels of phospho-S1700 and phospho-S1928 residues. (B) Kaplan–Meier survival curve for maleicGOF (n = 7) and littermate control mice (n = 6) transplanted with slow-releasepropranolol pellets (5 mg/21-d release) on day 0, and then induced (both icGOFand control) with tamoxifen starting on days 2–7 (gray bar). (C) Proposedmechanistic basis of Cantu syndrome. GOF mutations in either the pore-forming(KCNJ8) or accessory (ABCC9) KATP subunits will directly cause action potential(AP) shortening and reduced heart rate in cardiomyocytes and reduced excit-ability in smooth muscle myocytes, which will result in diminished calcium up-take, decreased contractility, and decreased cardiac output, as well as decreasedperipheral resistance. Combined, these reactions would reduce blood pressure,stimulating baroreceptors and triggering PKA- or PKC-dependent stress-signal-ing pathways in the heart and potentially in smooth muscle. These pathwayslead to phosphorylation of LTCC, specifically the Cav1.2 subunit, resultingmarkedly enhanced basal activity of the LTCC, enhanced contractility of themyocyte, and restored APD. In vascular smooth muscle cell, the diminished pe-ripheral resistance will result in diminished effective tissue perfusion, giving riseto long-standing volume load on the heart, and chamber dilation.

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performed. All animal studies complied with the standards for the care and use ofanimal subjects as stated in the NIH Guide for the Care and Use of LaboratoryAnimals (41) and were reviewed and approved by the Washington UniversityInstitutional Animal Care and Use Committee. Mouse strains used included cGOF(10), αMHC-Cre (42), icGOF (tamoxifen-inducible Kir6.1[G343D] transgenic), Mer-Cre-Mer-α-MHC (43), and vGOF (9). Transgene expression was induced by 5×daily injections of 10 mg/kg tamoxifen (44). Isolated myocyte studies and

isolated tissue Western blot analyses were carried out as previously de-scribed (45–48). Detailed methods are available in SI Methods.

ACKNOWLEDGMENTS. We thank Theresa Harter for help with animal hus-bandry, as well as the patients and volunteer members of the Center for theInvestigation of Membrane Excitability Diseases and the Department ofPediatrics for their participation in the Cantu research clinics.

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