KATP Channel Gain-Of-Function Leads to Increased Myocardial L-Type Ca2+ Current and Contractility in Cantu Syndrome

K channel gain-of-function leads to increased ATP + myocardial L-type Ca2 current and contractility in Cantu syndrome

Mark 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 in symptomatology, although prior office notes revealed episodes of genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, chest pain, fatigue, shortness of breath, and exercise intolerance

ABCC9)KATP channel subunits. We show that patients with CS, as associated with pericardial effusion. Additionally, three patients well as mice with constitutive (cGOF) or tamoxifen-induced (icGOF) (cs002, cs004, cs005) reported palpitations and exercise intolerance. cardiac-specific Kir6.1 GOF subunit expression, have enlarged hearts, One of these (cs004) also had symptoms with orthopnea, resulting with increased ejection fraction and increased contractility. Whole-cell from “idiopathic” high-output state and atrial fibrillation. Five pa- voltage-clamp recordings from cGOF or icGOF ventricular myocytes tients had patent ductus arteriosus that required surgical ligation or + (VM) show increased basal L-type Ca2 current (LTCC), comparable to catheter-based closure, two had significant pericardial effusions, that seen in WT VM treated with isoproterenol. Mice with vascular- three had been diagnosed with pulmonary hypertension, and five specific expression (vGOF) show left ventricular dilation as well as had histories of lower extremity edema. All patients had full but noncollapsing peripheral pulses. All but one patient (cs004, who was PHYSIOLOGY less-markedly increased LTCC. Increased LTCC in KATP GOF models is on several medications; Table S1) had supine systolic and diastolic paralleled by changes in phosphorylation of the pore-forming α1 subunit of the cardiac voltage-gated calcium channel Cav1.2 at Ser1928, blood 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: suggesting enhanced protein kinaseactivityasapotentiallinkbe- ± ± tween increased K current and CS cardiac pathophysiology. 58.2 6.2 mmHg; heart rate (HR): 85 17 beats per minute (bpm)] ATP (8). Despite these low BP values, no patient demonstrated ortho- KATP | transgenic | cardiovascular system | KCNJ8 | Kir6.1 static HR or BP changes. There were relatively few cardiac findings on physical examination, with the exception of one patient with a diastolic murmur (cs004) at the apex. Electrocardiograms revealed antu syndrome (CS), characterized by hypertrichosis, osteo- first-degree atrioventricular (AV) block in four patients, fascicular Cchondrodysplasia, and multiple cardiovascular abnormalities block in two, and T-wave abnormalities (T-wave axis 180° displaced (1), is caused by gain-of-function (GOF) mutations in the genes from QRS axis and morphologic abnormalities) in seven patients, encoding the pore-forming (Kir6.1, KCNJ8) and regulatory but no evidence of QT shortening or correct QT (QTc) prolon- (SUR2, ABCC9) subunits of the predominantly cardiovascular gation (Table S2). isoforms of the KATP channel (2–5). Because the same disease features arise from mutations in either of these subunits, it is Significance concluded that CS arises from increased KATP channel activity, as opposed to any nonelectrophysiologic function of either subunit. ATP-sensitive potassium (KATP) channels are present in cardiac However, this conclusion does not provide immediate explana- and smooth muscle; when activated, they relax blood vessels tion for many CS features. In the myocardium, for example, acute and decrease cardiac action potential duration, reducing car- activation of K channels results in shortening of the action po- ATP diac contractility. Cantu syndrome (CS) is caused by mutations tential (AP), with concomitant reduction of both calcium entry and in K genes that result in overactive channels. Contrary to contractility (6). The naïve prediction in CS would therefore be that ATP prediction, we show that the myocardium in both CS patients KATP GOF mutations should shorten the AP, reduce contractility, and reduce cardiac output. We previously reported high cardiac and in animal models with overactive KATP channels is hyper- output with low systemic vascular resistance in CS (7). Cantu syn- contractile. We also show that this results from a compensa- drome cardiac pathology is therefore opposite to prediction, and tory increase in calcium channel activity, paralleled by specific also unlike classical hypertrophic or dilated cardiomyopathies, in alterations in phosphorylation of the calcium channel itself. that the ventricle is dilated, but there is increased cardiac output. These findings have implications for the way the heart com- Here we characterize CS cardiac pathology in patients, and explore pensates for decreased excitability and volume load in general and for the basis of, and potential therapies for, CS specifically. the mechanistic basis using mice that express KATP GOF mutant subunits in the heart and vasculature. 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., Results 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. performed research; R.E.W. contributed new reagents/analytic tools; M.D.L., G.K.S., H.X.Z., K.U., B.A.K., Low Blood Pressure in CS Patients. Eleven CS individuals (five 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., male, six female, aged 17 mo to 47 y), all harboring ABCC9 muta- W.A.C., and C.G.N. wrote the paper. tions (Table S1), participated in CS research clinics at St. Louis Reviewers: D.M.B., University of California, Davis; R.S.K., Columbia University; and M.C.S., Children’s Hospital. Five had been previously followed at this in- University of Utah. stitution(7)andtheremainderwereenrolledviatheCSInterest The authors declare no conflict of interest. Group (www.cantu-syndrome.org). Patient demographic data, geno- 1To whom correspondence may be addressed. Email: [email protected] or [email protected]. type, and available cardiac historical, physical, and test information This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. are summarized in Table S1. Most patients had no recalled cardiac 1073/pnas.1606465113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1606465113 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 Cardiovascular Structural and Functional Characteristics in CS 18 yo F cs005 18 yo F

160 1000 2 160 1000 Patients. Two-dimensional color Doppler echocardiographic in- HF power (ms ) 140 800 140 800 120 120 terrogation and strain imaging (Fig. 1, Fig. S1,andTable S3) 600 600 revealed normal segmental anatomy, but markedly enlarged 100 100 80 400 80 400 HR (bpm) hearts (Fig. 1), with enhanced cardiac output and contractility, in HR (bpm) 60 200 60 200

CS patients. Left ventricular (LV) chambers were significantly 0 0 2 HF power (ms ) dilated in CS patients, and cardiac output was markedly increased 8 am 8 pm 8 am 8 am 8 pm 8 am (Fig. 1). LV mass was increased but, interestingly, LV posterior Time of day (hours) Time of day (hours) wall thickness (LVPWD) was not different from controls (Fig. 1). 2 This constellation of features is distinct from both typical hyper- 15000 2 15000 trophic cardiomyopathies (where LVPWD is increased) and typ- 10000 10000 ical dilated cardiomyopathies (where there is chamber dilation 5000 5000 with diminished cardiac function). In part, this dilated hyper- 0

HF power (ms ) 0 contractile phenotype could be a secondary response to chroni- HF power (ms ) cally elevated blood volume as a result of vasodilation—adirectly 60 80 100 120 140 60 80 100 120 140 HR (bpm) HR (bpm) predicted consequence of vascular KATP GOF (9). Pulsed-wave velocity testing offers a noninvasive correlate of vascular tone. Fig. 2. Cardiovascular control in CS patients. CS patient (cs005, 18-y-old Consistent with BP measurements, there was diminished pulsed- female) and control (18-y-old female) heart rate (color) and high-frequency ± −1 n = + wave velocity in CS patients (control 5.6 0.9 ms , 8; CS 4.5 U power (right axis, black) plotted over 24 h. 0.8 m/s, n = 7, P = 0.003), implying diminished vascular tone.

Circadian Abnormalities and Low Vagal Function in CS Patients. hours (Figs. S2 and S3). These latter findings suggest a relatively Several patients received 24-h ambulatory ECG monitoring. elevated sympathetic activity but diminished vagal activity. None demonstrated atrial or ventricular arrhythmia during the re-

cording period. The data were subsequently used for heart rate Enhanced Contractility in Hearts Expressing KATP GOF Mutations. The variability analysis (HRV). Fig. 2 displays HR and high-frequency predicted effect of KATP GOF in the myocardium itself is AP (HF) power, a marker for vagal activity, as a function of time of day, shortening and reduced contractility, whereas the predicted ef- for a representative CS patient and age/sex-matched control. The fect in the vasculature is reduced peripheral resistance. The control 18-y-old female shows typical circadian variation: in general, above clinical evaluation of CS patients reveals physiologically higher HR during waking hours, and significantly lower HR during low BPs, consistent with the latter prediction. However, myo- sleeping hours; HF also has a circadian rhythm that is inversely cardial hypercontractility and no evidence of K current-induced related to this HR trend, rising during sleeping hours, and low QT shortening on ECG are grossly opposite to prediction. We during waking hours. Interestingly, all CS patients demonstrated hypothesize that these features are secondary consequences of markedly diminished HF power (Fig. 2 and Fig. S2) and generally the primary predictions. Specifically, we propose that increased high HR for age that failed to lower appropriately during sleeping KATP current in either ventricular or smooth muscle myocytes will lead to lower cardiac output and decreased peripheral resistance, respectively; both of these will result in diminished tissue perfusion, which in turn will induce a systemic feedback to 400 6 * P <0.0001 C * P=0.0357 increase cardiac output (see, for example, Fig. 7 ). 4 300 To examine these hypotheses in a tractable system, we first 2 evaluated the cardiac consequences of Kir6.1 GOF mutations 200 0 expressed in the myocardium under α-myosin heavy chain α 100 -2 ( MHC) control in mice [constitutive GOF (cGOF) mice]. cGOF mice exhibit prolonged PR intervals, as well as episodes of LVEDV (ml) LVEDV 0 -4 LVEDV z-score LVEDV junctional rhythm, and diminished AV nodal conduction, but no * P<0.0001 2.0 100 QT shortening (10). Echocardiography under light anesthesia P=NS A 80 (11) (Fig. 3 and Table S4) revealed normal chamber wall di- 1.5 mensions, but increased contractility and ejection fraction in 60 1.0 cGOF hearts, again counter to naïve prediction, but consistent 40 with the hypercontractile phenotype of CS patients. 0.5 mass LV 20 Isolated cGOF myocytes displayed diminished resting sarcomere LVPW (cm) LVPW 0.0 (indexed g/m2) 0 length, significantly increased fractional shortening and increased rates of shortening and relaxation (Fig. 3B and Table S5). These 55 *P=0.0014 10 *P<0.0001 features are similar to those seen in WT myocytes following exposure 50 8 to isoproterenol (ISO). The subsequent effect of ISO was reduced in 45 6 cGOF myocytes (Fig. 3B), consistent with the idea that cGOF “ ” 40 4 myocytes are effectively prestimulated (12) via a pathway that is at least convergent with that responding to β-adrenergic signaling. To 35 2 confirm that this prestimulation effect was not an artefactual effect of Shortening (%) 30 Cardiac output 0

(indexed l/m2/min) increased Kir6.1 protein expression per se, mice expressing domi- α Control Cantu Control Cantu nant-negative Kir6.1[AAA] subunits under MHC control (AAA) were studied (13). No significant differences in contractile parameters 0 0.0 were seen between AAA and littermate control myocytes (Table S6). * P<0.0001 -0.5 -10 *P<0.0001 KATP GOF Myocytes Demonstrate Increased LTCC. Cardiac excitation– -1.0 contraction (EC) coupling depends on Ca entry via the L-type -20 2+ -1.5 Ca current (LTCC), which is modulated by phosphorylation in Global LV LV GSRs LV long (%/s) -30 response to β-adrenergic and other neurohumoral inputs. Presti- strain long (%) -2.0 mulated cGOF myocyte contractility is consistent with enhance- Fig. 1. Enhanced cardiac volume and contractility in CS patients. Compari- ment of such modulation. Cell size, assessed by cell capacitance, sons of pertinent echocardiographic measures from control and CS patients. was normal in cGOF myocytes, but baseline LTCC amplitude and

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1606465113 Levin et al. Downloaded by guest on October 2, 2021 expression of KATP GOF and consequent increases in LTCC, we A 4.5 80 P=NS P=NS assessed cardiomyocytes in which GOF was induced postnatally, 4.0 60 rather than developmentally [tamoxifen-induced GOF (icGOF)] by

3.5 crossing Kir6.1[G343D] and tamoxifen-inducible Myh6-Cre mice 2 40 dexed), (14). Double-transgenic icGOF and littermate controls were treated 3.0 20 olume (uL) with tamoxifen for 5 d; ventricular myocytes were isolated, and End-diatolic v s (in

s 2.5 mg/mm 0 LTCC recorded, on day 6. Recorded icGOF myocytes were larger than control myocytes but, even controlling for this, icGOF LTCC

LV m a density was still dramatically increased at baseline (Fig. 5). Again, as P=0.026 85 18 in cGOF myocytes, ISO significantly stimulated LTCC in control P=0.0082 : * lic but not icGOF myocytes. These experiments further confirm 80 16 to * 1/s)

ate remodeling of LTCC in response to Kir6.1 GOF, and demonstrate

( 14 75 ias that this can occur in adult animals and rapidly (within hours or 12

kd days) following transgene induction, implying that such responses Ejection 70 10 Strain r radial

Fraction (%) are physiologically, not developmentally, mediated. pea 65 8

Vascular KATP GOF Expression Results in Enlarged Hearts and -ISO B P=NS +ISO Increased LTCC. Kir6.1 and SUR2 are prominently expressed in 25 0 8 P=NS vascular smooth muscle (VSM). VSM expression of KATP GOF 20 under smooth muscle (SM)-MHC promoter control reduces vas- P=.0002 6 P=.005 m/s) m/s) cular contractility, resulting in a chronically vasodilated state with µ µ

15 ( -5 t 4 low systolic and diastolic BP (9). Young vGOF mice, induced with l/d tening (%) 10 P=.001 tamoxifen at 2 mo and studied by echocardiography 1 mo later, -dl/dt ( +d hor

s 2 5 revealed no significant changes in cardiac structural parameters, but did exhibit enhanced cardiac contractility (Fig. 6A). A second 0 -10 0 cohort of vascular-specific expression (vGOF) mice in which ex- -ISO +ISO +ISO P=NS -ISO pression had been induced for more than 6 mo (old vGOF) showed significant chamber dilation and now showed diminished Fig. 3. Enhanced contractility in cGOF mice. (A) Echocardiographic analysis

A PHYSIOLOGY showing increased ejection fraction and strain rate in cGOF mice compared contractility (Fig. 6 and Table S7). Consistent with these older with controls. (B) Contractility parameters for isolated ventricular myocytes vGOF echocardiographic findings, baseline LTCC in vGOF from cGOF and control sex-matched littermate hearts. myocytes was elevated, although not as dramatically as in cGOF or icGOF mice, and both vGOF and control LTCC increased following ISO (Fig. 6B). Interestingly, the baseline I–V relationship density were double those of control myocytes (Fig. 4B). Com- was left-shifted by ∼10 mV (Fig. 6B) in vGOF myocytes. mensurate with the idea that cGOF myocytes were prestimulated, LTCC amplitudes and densities were markedly increased by ISO Increased Cav1.2 Protein Phosphorylation in Kir6.1 GOF Hearts. The LTCC in mature ventricular myocytes is conducted by Cav1.2 in control myocytes but much less so in cGOF myocytes (Fig. 4). channels, composed of a pore-forming α1 subunit in association with There was a slight shift in voltage dependence in the baseline α δ, β, and possibly γ subunits (15). In the heart, the large C-terminal – 2 cGOF current voltage (I/V) relationship, again similar to that in domain of the 250-kDa α1 subunit is proteolytically processed, control I/V curves after ISO exposure. resulting in a complex of the core Cav1.2 protein plus its non- Because the Kir6.1 GOF transgene is constitutively activated covalently bound distal C terminus (16), a potent autoinhibitor of in cGOF hearts, the above changes in LTCC properties might be channel activity (17). β-Adrenergic stimulated phosphorylation of the developmentally induced. To examine the temporal link between Cav1.2 channel relieves this autoinhibition and enhances LTCC and

+ISO +ISO

+55mV 300 P=NS Fig. 4. Enhanced LTCCs in cGOF myocytes. (A) Rep- 2+ 200 resentative families of Ca current obtained from control (Left) or cGOF (Right) mice. Na+ and T-type -45mV (pF) 100 2+ -70mV -70mV Ca channels were inactivated by slow voltage ramp Capacitance 0 from holding potential of −70 to −45 mV, and then + 2000 10 voltage was stepped to 55 mV in 10-mV increments. B The identical protocol was used to elicit ICa following -40 -20 0 20 40 mV -40 -20 0 20 40 mV exposure to ISO (1 mM). (B)PeakICa amplitude and 0 0 ICa density as a function of voltage (mean ± SEM). (Inset) Mean capacitance in each case. (Scale bars, -2000 -10 500 pA and 20 ms.) Solid lines here and in following * * * * * figures indicate currents in baseline, dotted lines in * * ISO; asterisks indicate significance of cGOF vs. control -4000 Control (n=12) -20 cGOF (n=15) in each condition (two-way ANOVA, P < 0.001). In Current Density (pA/pF) Control (Iso) (n=6) * this figure only, daggers indicate significance of ISO Current amplitude (pA) cGOF (Iso) (n=9) * * -6000 -30 vs. baseline for control (black) and cGOF (red).

Levin et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 because standard therapies for HCM, for example, might actually A B -40 -002 20 40 0 worsen a Cantu patient’sclinicalstatus. * High cardiac output states such as we observe can arise from -2000 * * chronic vasodilation, and can lead to LV volume overload and * * * * subsequent chamber dilation (24–27). Vital statistic and echo- -4000 * * * * (Tam) cardiographic data support the hypothesis that CS patients are +ISO +ISO * (Tam) -6000 vasodilated, but in a compensated high cardiac output state.

Current amplitude (pA) amplitude Current (Tam)+ISO (Tam)+ISO Though it is controversial to term BP as clinically low in an -004 -020402 asymptomatic patient, systolic and diastolic BPs were greatly 0 diminished compared with mean for age. +55mV -5 P= .0072 400 * Feedback Control via L-Type Ca Current in Compensated Cardiac 300 -10 + -45mV * -70mV Function. Gain of K channel function in blood vessels would be -70mV 200 * * 100 -15 * * predicted to cause reduced contractility and vasodilation, and in Current density (pA/pF) density Current + 0 * 2 Cell capacitance (pF) -20 the heart to shorten APs, reducing Ca entry and contractility. Available clinical data regarding ECG phenotype in CS patients is Fig. 5. Enhanced LTCCs in inducible icGOF ventricular myocytes. (A) Rep- limited, but there is no evidence for AP shortening (10). The high + resentative families of Ca2 current obtained from either control (Left)or cardiac output state evident by echocardiography is also not na- icGOF (Right) at baseline and in presence of ISO. Protocols as in Fig. 4. ively predicted and leads us to postulate that feedback response to ± (B)PeakICa amplitude and ICa density plotted as a function of voltage (mean both the heart and vasculature remodels EC coupling to produce SEM). (Inset) Mean capacitance in each case. (Scale bars, 500 pA and 10 ms.) the unexpected hypercontractile function (Fig. 7). In mice, echo- cardiograms and isolated myocyte contractility studies reveal that – such compensation does indeed occur: cardiac contractility is myocyte contractility, in response to sympathetic signaling (18 20). increased, concomitant with increased LTCC. Isolated cGOF We found no change in the level of full-length Cav1.2 channel pro- myocytes reveal basal hypercontractility and elevated LTCC, but tein, proteolytically processed Cav1.2 protein, or distal C-terminal A similar maximal contractility and LTCC after ISO exposure to protein, between cGOF and control hearts (Fig. 7 ). We investigated control, whereas old vGOF myocytes also show some increase in two phosphorylation sites on Cav1.2 channels that have been iden- baseline LTCC. Direct analysis of LTCC in hearts of CS patients tified in intact ventricular myocytes using phosphospecific antibodies is not feasible, but there are indirect suggestions of similarly (Ser1700 at the interface between the distal and proximal C-terminal domains and Ser1928 in the distal C-terminal domain). Phosphory- lation of Ser1700 by PKA is directly implicated in β-adrenergic reg- P=NS ulation of Cav1.2 channels (18, 19), whereas phosphorylation of P=NS P=NS A 50

4.0 c i

Ser1928 may correlate with PKA or PKC pathway activity (18, 21). ed) 2 L)

ol 45 3.0 t We found no significant change in phosphorylation of Ser1700 in 40 P=.04 2.0 cGOF, but Ser1928 phosphorylation was increased approximately 35 mg/mm ass (index End-dias A ∼ volume ( µ twofold in cGOF mice (Fig. 7 ), and 1.25-fold in vGOF mice. 1.0 30

Because Ser1928 is also a substrate for phosphorylation by PKC (22), LV m 0.0 25

phosphorylation of this site by either PKA or PKC signaling pathways 85 P=.02 14 P=0.03

) P=0.03 may be involved in persistent increase in basal activity, and conse- P=0.04 s 80 12 quent loss of β-adrenergic stimulation of Cav1.2 channels in CS. 75 10 Ejection Intolerance of β-Blockade in icGOF Animals. To further explore the 70 Strain rate 8 Fraction (%) long axis-s (1/ mechanistic basis of Cantu disease and directly assess whether 65 6 β-adrenergic signaling is required for adaptation to KATP GOF in the heart, we chemically ablated sympathetic signaling in icGOF and littermate control mice by implanting adult (12- to 32-wk-old) animals with slow-release propranolol pellets, and then initiating B transgene expression with tamoxifen 2–7 d later. The majority of icGOF mice treated with propranolol pellets died within 2 wk of transgene induction, but no deaths were observed in littermate controls (Fig. 7B). Though further mechanistic details remain to +ISO +ISO be elucidated, these data are a striking indication that adrenergic

signaling is required for adaptation to KATP GOF induction. NS (P=.072) +55mV 300 Discussion 200 -45mV 100 CS and Cardiovascular Disease. KATP channels are heterooctameric -70mV -70mV complexes of pore forming Kir6.1 or Kir6.2 subunits and regulatory 0 Capacitanace (pF) ) -40 -20 0 20 40 -40 -20 0 20 40 SUR1 or SUR2 subunits (23). ABCC9 (SUR2) and KCNJ8 (Kir6.1) A 0 /pF) 0 (p

mV A mV e

are prominently expressed in cardiac myocytes, VSM, and vascular d -5 endothelial cells (23), suggesting that cardiovascular features will itu -1000 * -10 * * predominate in CS. We show that CS patients have dilated LVs, -2000 * * Control tDensity(p * vGOF n -15

increased cardiac output and ejection fraction, and increased myo- * Control (Iso) rre u

Current-3000 amp l cardial contractility. LV mass is increased compared with controls, vGOF (Iso) C -20 but LV wall thicknesses are normal. Such findings are distinct from Fig. 6. Dilated heart and enhanced LTCCs in vGOF cardiac muscle. hallmarks of either hypertrophic cardiomyopathy (HCM) or dilated (A) Echocardiographic features of vGOF mice at 3 mo and >6 mo of age. + cardiomyopathy; the former typically demonstrates thickened LV (B) Representative Ca2 currents obtained from either control (Left) or vGOF

chamber walls with hypercontractile function, whereas the latter (Right) cardiomyocytes. Protocols as in Fig. 4. Peak ICa amplitude and ICa demonstrates a dilated LV chamber with diminished cardiac func- density plotted as a function of voltage (mean ± SEM). (Inset) Mean capac- tion. Distinguishing between these cardiac phenotypes is critical, itance in each case. (Scale bars, 500 pA and 20 ms.)

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1606465113 Levin et al. Downloaded by guest on October 2, 2021 * signaling (31–35). PKC overexpression may result in diminished A 1.5 B 100 Control LTCC current in some circumstances (36), although dialysis of 1 critical LTCC-associated proteins may confound results in whole- 0.5 80 cell patch-clamp experiments (32). Both PKA and PKC modulation 0 of LTCC (22, 37) are mediated by phosphorylation of CaV1.2 serine Total Cav1.2 Distal C-term Processed C-term 60 residues on the pore or accessory subunits (18–20, 38). The identity * * of relevant residues phosphorylated in response to adrenergic sig-

3 Percent survival naling remains an active area of investigation. Ser1700 and Ser1928 40 phosphorylation of Cavα1.2 have both been demonstrated, but 2 icGOF Ser1928 may also be phosphorylated by PKC (35, 39), and Ser1700

Relative intensity (arb.) 20 1 may also be a target of CaMKII (18, 40). Our analysis implicates Ser1928 phosphorylation as a marker of the response to KATP GOF 0 0 but not Ser1700. Though Ser1928 phosphorylation may not itself be phospho S1700 phospho S1928 0 5152510 20 days after implantation directly involved in the enhancement of LTCC, it is suggestive that PKC or PKA pathways may ultimately be activated. C Feedback Response to Vascular Defects in CS. Two distinct effects were observed on cGOF and icGOF LTCC: left-shift in activation Cardiac Vascular KATP overactivity KATP overactivity and increased current density. We also observed a left-shift of - - voltage dependence of LTCC in vGOF, but only a mild increase in current density, and a nonsignificant increase in Ser1928 phos- Heart rate APD + + Electrical activity phorylation. In contrast, older vGOF animals exhibited increased - - diastolic volume, as observed in CS patients. Vascular KATP GOF Cardiomyocyte [Ca2+ ] VSM [Ca2+ ] I + + I results in increased smooth muscle KATP current and diminished BP Cav1.2 S1928 (9), providing a potentially direct explanation for the markedly low- phosphorylation for-age BPs in CS patients; this also provides a systemic mechanism Central that may link the primary vGOF phenotype to secondary conse- - autonomic - activity quences in the heart: vascular KATP GOF expression results in va- Cardiomyocyte contractility Stress VSM contractility signalling sodilation that will ultimately result in a long-standing volume load, PHYSIOLOGY

Baroreceptor drive evidenced by LV chamber dilation. Early after transgene induction (6 wk), vGOF hearts exhibit increased contractility and increased - ejection fraction (Fig. 6), but end diastolic volume is increased in - - Cardiac output BP Peripheral resistance older vGOF animals, potentially reflecting dilation manifesting after prolonged exposure to volume overload (26). Fig. 7. Cellular and molecular basis of Cantu syndrome. (A) Quantitation of Western blot analysis of isolated total Cav1.2a subunit protein, distal C terminus, Conclusions and Implications. CS can arise from GOF in either and processed C-terminal fragments, normalized to WT protein levels in cGOF, Kir6.1 or SUR2 proteins of the cardiovascular KATP channel (2–5). Kir6.1[AAA], and vGOF cardiac samples, as well as relative levels of phospho- A distinct CS cardiac pathology is characterized by high output state S1700 and phospho-S1928 residues. (B)Kaplan–Meier survival curve for male with enhanced cardiac contractility and enhanced chamber volume, = = icGOF (n 7) and littermate control mice (n 6) transplanted with slow-release associated with decreased vascular pulse wave velocity and low BP. propranolol pellets (5 mg/21-d release) on day 0, and then induced (both icGOF – Our animal studies suggest that CS cardiac pathology emerges from and control) with tamoxifen starting on days 2 7(graybar).(C) Proposed combined cardiac and vascular K GOF mutation expression mechanistic basis of Cantu syndrome. GOF mutations in either the pore-forming ATP (Fig. 7C). The vascular findings are readily explained by the (KCNJ8) or accessory (ABCC9) KATP subunits will directly cause action potential (AP) shortening and reduced heart rate in cardiomyocytes and reduced excit- expected molecular consequences, but KATP GOF mutations in the ability in smooth muscle myocytes, which will result in diminished calcium up- myocardium will tend to reduce cardiac action potential duration take, decreased contractility, and decreased cardiac output, as well as decreased (APD) and decrease pacemaker activity, both of which would re- peripheral resistance. Combined, these reactions would reduce blood pressure, duce cardiac contractility and output. The counter observation of stimulating baroreceptors and triggering PKA- or PKC-dependent stress-signal- enhanced contractility and maintained APD is explained by en- ing pathways in the heart and potentially in smooth muscle. These pathways hanced LTCC, with left-shifted activation and increased basal lead to phosphorylation of LTCC, specifically the Cav1.2 subunit, resulting conductance, associated with enhanced phosphorylation of the markedly enhanced basal activity of the LTCC, enhanced contractility of the PKA/PKC target residue Ser1928. These findings are consistent myocyte, and restored APD. In vascular smooth muscle cell, the diminished pe- with compensatory chronic signaling, potentially involving adren- ripheral resistance will result in diminished effective tissue perfusion, giving rise ergic stimulation, through pathways that converge on the LTCC. to long-standing volume load on the heart, and chamber dilation. This consistent explanation for CS cardiac features raises the question of whether or how to treat them. The dramatic difference in response of icGOF and control animals to β-blockade (Fig. 7B) enhanced basal LTCC in the ambulatory ECG data: higher than is consistent with adrenergic signaling being involved in at least normal heart rates, as well as blunted heart rate variability and the early compensation to KATP GOF, further suggesting that T-wave abnormalities (Fig. 2). β-blockade could be a dangerous approach to treating Cantu pa- In the presence of ISO, LTCC densities in cGOF or icGOF tients. Alternately, appropriate therapies should target KATP myocytes are not significantly different from control (Figs. 4 and channels directly, and the success of sulphonylurea drugs in 5) (12). This lack of ISO response is consistent with chronic in vivo treating neonatal diabetes, which results from GOF in the pan- activation of signaling pathways that result in prestimulation. This creatic KATP isoforms, gives promise that similar or more selective prestimulation must be a consequence of the initial defect; that is, KATP antagonists may reverse some or all CS disease features. the gain of KATP conductance, which we suggest will indeed be a reduced cardiac output, but that this results in activation of ad- Methods renergic or parallel signaling pathways. We previously showed that Human studies were carried out on CS patients recruited to an annual research basal and ISO-stimulated cAMP concentrations are not altered in clinic at St. Louis Children’s Hospital. Written informed consent was provided by all transgenic Kir6.2 GOF hearts that also show prestimulation (12). patients. The study was approved by the Human Research Protection Office of Cardiac LTCC can be increased by PKA activation (28–30), but also Washington University School of Medicine and performed at St. Louis Children’s by PKC in response to α-adrenergic and endothelin-1–dependent Hospital in St. Louis. Echocardiographic and electrocardiographic studies were

Levin et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 performed. All animal studies complied with the standards for the care and use of isolated tissue Western blot analyses were carried out as previously de- animal subjects as stated in the NIH Guide for the Care and Use of Laboratory scribed (45–48). Detailed methods are available in SI Methods. Animals (41) and were reviewed and approved by the Washington University Institutional Animal Care and Use Committee. Mouse strains used included cGOF ACKNOWLEDGMENTS. We thank Theresa Harter for help with animal hus- (10), αMHC-Cre (42), icGOF (tamoxifen-inducible Kir6.1[G343D] transgenic), Mer- bandry, as well as the patients and volunteer members of the Center for the Cre-Mer-α-MHC (43), and vGOF (9). Transgene expression was induced by 5× Investigation of Membrane Excitability Diseases and the Department of daily injections of 10 mg/kg tamoxifen (44). Isolated myocyte studies and Pediatrics for their participation in the Cantu research clinics.

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