SK 4 K + channels are therapeutic targets for the treatment of cardiac arrhythmias
2017; Springer Nature; Volume: 9; Issue: 4 Linguagem: Inglês
10.15252/emmm.201606937
ISSN1757-4684
AutoresShiraz Haron‐Khun, David Weisbrod, Hanna Bueno, Dor Yadin, Joachim A. Behar, Asher Peretz, Ofer Binah, Edith Hochhauser, Michael Eldar, Yael Yaniv, Michael Arad, Bernard Attali,
Tópico(s)Cardiac Arrhythmias and Treatments
ResumoResearch Article20 February 2017Open Access Source DataTransparent process SK4 K+ channels are therapeutic targets for the treatment of cardiac arrhythmias Shiraz Haron-Khun Shiraz Haron-Khun Department of Physiology and Pharmacology, The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Leviev Heart Center, Sheba Medical Center, Tel Hashomer, Tel Aviv, Israel Search for more papers by this author David Weisbrod David Weisbrod Department of Physiology and Pharmacology, The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Hanna Bueno Hanna Bueno Department of Physiology and Pharmacology, The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Dor Yadin Dor Yadin Leviev Heart Center, Sheba Medical Center, Tel Hashomer, Tel Aviv, Israel Search for more papers by this author Joachim Behar Joachim Behar Laboratory of Bioenergetic and Bioelectric Systems, Biomedical Engineering Faculty, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Asher Peretz Asher Peretz Department of Physiology and Pharmacology, The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Ofer Binah Ofer Binah Department of Physiology, Ruth & Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Edith Hochhauser Edith Hochhauser The Cardiac Research Laboratory of the Department of Cardiothoracic Surgery, Felsenstein Medical Research Center, Rabin Medical Center, Tel Aviv University, Petah Tikva, Israel Search for more papers by this author Michael Eldar Michael Eldar Leviev Heart Center, Sheba Medical Center, Tel Hashomer, Tel Aviv, Israel Search for more papers by this author Yael Yaniv Yael Yaniv Laboratory of Bioenergetic and Bioelectric Systems, Biomedical Engineering Faculty, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Michael Arad Corresponding Author Michael Arad [email protected] orcid.org/0000-0003-3723-7493 Leviev Heart Center, Sheba Medical Center, Tel Hashomer, Tel Aviv, Israel Search for more papers by this author Bernard Attali Corresponding Author Bernard Attali [email protected] orcid.org/0000-0003-1066-7047 Department of Physiology and Pharmacology, The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Shiraz Haron-Khun Shiraz Haron-Khun Department of Physiology and Pharmacology, The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Leviev Heart Center, Sheba Medical Center, Tel Hashomer, Tel Aviv, Israel Search for more papers by this author David Weisbrod David Weisbrod Department of Physiology and Pharmacology, The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Hanna Bueno Hanna Bueno Department of Physiology and Pharmacology, The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Dor Yadin Dor Yadin Leviev Heart Center, Sheba Medical Center, Tel Hashomer, Tel Aviv, Israel Search for more papers by this author Joachim Behar Joachim Behar Laboratory of Bioenergetic and Bioelectric Systems, Biomedical Engineering Faculty, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Asher Peretz Asher Peretz Department of Physiology and Pharmacology, The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Ofer Binah Ofer Binah Department of Physiology, Ruth & Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Edith Hochhauser Edith Hochhauser The Cardiac Research Laboratory of the Department of Cardiothoracic Surgery, Felsenstein Medical Research Center, Rabin Medical Center, Tel Aviv University, Petah Tikva, Israel Search for more papers by this author Michael Eldar Michael Eldar Leviev Heart Center, Sheba Medical Center, Tel Hashomer, Tel Aviv, Israel Search for more papers by this author Yael Yaniv Yael Yaniv Laboratory of Bioenergetic and Bioelectric Systems, Biomedical Engineering Faculty, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Michael Arad Corresponding Author Michael Arad [email protected] orcid.org/0000-0003-3723-7493 Leviev Heart Center, Sheba Medical Center, Tel Hashomer, Tel Aviv, Israel Search for more papers by this author Bernard Attali Corresponding Author Bernard Attali [email protected] orcid.org/0000-0003-1066-7047 Department of Physiology and Pharmacology, The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Author Information Shiraz Haron-Khun1,2,‡, David Weisbrod1,‡, Hanna Bueno1,‡, Dor Yadin2,‡, Joachim Behar3, Asher Peretz1, Ofer Binah4, Edith Hochhauser5, Michael Eldar2, Yael Yaniv3, Michael Arad *,2 and Bernard Attali *,1 1Department of Physiology and Pharmacology, The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 2Leviev Heart Center, Sheba Medical Center, Tel Hashomer, Tel Aviv, Israel 3Laboratory of Bioenergetic and Bioelectric Systems, Biomedical Engineering Faculty, Technion—Israel Institute of Technology, Haifa, Israel 4Department of Physiology, Ruth & Bruce Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel 5The Cardiac Research Laboratory of the Department of Cardiothoracic Surgery, Felsenstein Medical Research Center, Rabin Medical Center, Tel Aviv University, Petah Tikva, Israel ‡These authors contributed equally to this work *Corresponding author. Tel: +972 3 5304560; E-mail: [email protected] *Corresponding author. Tel: +972 3 6405116; E-mail: [email protected] EMBO Mol Med (2017)9:415-429https://doi.org/10.15252/emmm.201606937 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-provoked ventricular arrhythmia, which also manifests sinoatrial node (SAN) dysfunction. We recently showed that SK4 calcium-activated potassium channels are important for automaticity of cardiomyocytes derived from human embryonic stem cells. Here SK4 channels were identified in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from healthy and CPVT2 patients bearing a mutation in calsequestrin 2 (CASQ2-D307H) and in SAN cells from WT and CASQ2-D307H knock-in (KI) mice. TRAM-34, a selective blocker of SK4 channels, prominently reduced delayed afterdepolarizations and arrhythmic Ca2+ transients observed following application of the β-adrenergic agonist isoproterenol in CPVT2-derived hiPSC-CMs and in SAN cells from KI mice. Strikingly, in vivo ECG recording showed that intraperitoneal injection of the SK4 channel blockers, TRAM-34 or clotrimazole, greatly reduced the arrhythmic features of CASQ2-D307H KI and CASQ2 knockout mice at rest and following exercise. This work demonstrates the critical role of SK4 Ca2+-activated K+ channels in adult pacemaker function, making them promising therapeutic targets for the treatment of cardiac ventricular arrhythmias such as CPVT. Synopsis SK4 Ca2+-activated K+ channels are important for embryonic cardiac pacemaker function, and are now found to be crucial for adult sinoatrial node (SAN) pacing and successfully targeted for treatment of cardiac arrhythmias in mice. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited stress-provoked ventricular arrhythmia, which also manifests sinoatrial node dysfunction. SK4 channels are identified in hiPSC cardiomyocytes (CMs) from healthy and CPVT2 patients and in SAN cells from WT and CASQ2-D307H knock-in (KI) mice. The SK4 channel blocker TRAM-34 reduces the arrhythmic Ca2+ transients observed in CPVT2-derived hiPSC-CMs and in SAN cells from KI mice. TRAM-34 prominently decreased the ECG arrhythmic features of CASQ2-D307H KI and CASQ2 knockout mice. SK4 channels are promising therapeutic targets for the treatment of cardiac arrhythmias such as CPVT. Introduction Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic syndrome characterized by physical or emotional stress-induced polymorphic ventricular tachycardia in otherwise structurally normal hearts with a high fatal event rate in untreated patients (Priori et al, 2001; Hayashi et al, 2009; Priori & Chen, 2011; Abriel & Zaklyazminskaya, 2013). CPVT comprises heterogeneous genetic diseases, including mutations in ryanodine receptor type 2 (RyR2), calsequestrin 2 (CASQ2), triadin, or calmodulin (Leenhardt et al, 1995; Lahat et al, 2001; Priori et al, 2002; Chopra & Knollmann, 2011; Nof et al, 2011; Arad et al, 2012; Hwang et al, 2014). The RyR2 mutations (CPVT1) are "gain-of-function" mutations while CASQ2 mutants (CPVT2) are "loss-of-function" mutations, which both lead to diastolic Ca2+ leakage from the sarcoplasmic reticulum (SR). This eventually produces local increases in cytosolic Ca2+ that is extruded via the Na+–Ca2+ exchanger NCX1 generating local depolarization with early or delayed afterdepolarizations (EADs or DADs) that trigger premature beats and fatal polymorphic ventricular tachycardia (Priori & Chen, 2011). Recent studies performed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from CPVT patients bearing mutations in either CASQ2 (D307H) or RyR2 (M4109R) showed that β-adrenergic stimulation caused marked elevation in diastolic Ca2+, DADs, and oscillatory prepotentials (Itzhaki et al, 2012; Novak et al, 2012, 2015). Sinus bradycardia was also described in CPVT patients and in CPVT mouse models, suggesting that sinoatrial node (SAN) dysfunction may reflect another primary defect caused by CPVT mutations (Leenhardt et al, 1995; Postma et al, 2005; Katz et al, 2010; Neco et al, 2012; Faggioni et al, 2014; Glukhov et al, 2015). We identified SK4 calcium-activated potassium channels (KCa3.1) as being involved in the pacemaker activity of cardiomyocytes derived from human embryonic stem cells (hESC-CMs) (Weisbrod et al, 2013). Here we asked whether SK4 channels are expressed in SAN and play a role in CPVT. SK4 currents were found in hiPSC-CMs from healthy and CPVT2 (CASQ2-D307H) patients and in SAN cells from WT and CASQ2-D307H knock-in (KI) mice. TRAM-34, a selective blocker of SK4 channels, markedly reduced the occurrence of DADs and abnormal Ca2+ transients detected following exposure to the β-adrenergic agonist isoproterenol in CPVT2-derived hiPSC-CMs and in SAN cells from CASQ2-D307H KI mice. Intraperitoneal injection (20 mg/kg) of SK4 channel blockers, TRAM-34 or clotrimazole, elicited bradycardia and noticeably reduced the ECG arrhythmic features recorded in vivo from CASQ2-D307H KI and CASQ2 knockout (KO) mice at rest and following treadmill exercise. The results suggest that SK4 channels play a critical role in normal and CPVT diseased pacemaker function. Importantly, our data indicate that SK4 channel blockers could open new horizons in the management of CPVT patients' rhythm disorders. Results SK4 channels are expressed in hiPSC-CMs and their blockade reduces arrhythmias recorded in hiPSC-CMs derived from CPVT2 (CASQ2-D307H) patients Since pacemaker dysfunction was described in CPVT patients and CPVT mouse models (Leenhardt et al, 1995; Postma et al, 2005; Katz et al, 2010; Neco et al, 2012; Faggioni et al, 2014; Glukhov et al, 2015), we examined whether SK4 channels are expressed in SAN and play a role in CPVT. We used single spontaneously beating hiPSC-CMs (25-day-old EBs) derived from normal (healthy) and CPVT2 patients carrying the CASQ2 D307H mutation (Novak et al, 2012) and investigated their spontaneous firing and ionic currents. A voltage ramp was applied as previously (Weisbrod et al, 2013) and cells were held at −20 mV to substantially inactivate voltage-gated Na+ and Ca2+ currents (Fig 1A and B). In the absence of blockers (black traces), the voltage ramp revealed the presence of one and occasionally two inward humps peaking at about −40 mV and −5 mV and reflecting activation of residual T-type and L-type Ca2+ currents, respectively. These inward humps vanished following exposure to 300 μM CdCl2. Exposing cells to solution 1 (300 μM CdCl2, 25 μM ZD7288, and 10 μM E-4031) suppressed the inward humps, shifted the reversal potential (Erev) to the left, and markedly depressed inward and outward currents (orange trace). Addition of the selective SK4 channel blocker TRAM-34 (1 μM) to solution 1 decreased the ramp currents (green trace) (Fig 1A and B). Subtracting the ramp currents in solution 1 to those in solution 1 + TRAM-34 (1 μM) yielded the TRAM-34-sensitive current. Figure 1D shows the average traces of the TRAM-34-sensitive currents (using 1 μM TRAM-34) of normal and CPVT2-derived hiPSC-CMs, which mainly exhibited an outward component. Yet, small residual inward currents likely corresponding to cationic conductances were not fully blocked by solution 1 and therefore shifted the Erev to values more positive than those of EK. TRAM-34-sensitive currents were never detected in zero internal free Ca2+. Similar TRAM-34-sensitive current densities were found using either 1 or 5 μM TRAM-34 (Fig 1C). No significant differences were found in TRAM-34-sensitive current densities of normal and CPVT2 hiPSC-CMs (Fig 1C). For selectivity purposes, we examined whether TRAM-34 interfered with major pacemaker currents in hESC-CMs. We found that 5 μM TRAM-34 did not alter T-type and L-type Ca2+ currents measured by the two inward humps (zero free Ca2+ in pipette solution; Appendix Fig S1A). While 25 μM ZD7288 blocked If at all voltages (~70% inhibition at −100 mV), 5 μM TRAM-34 did not affect the If current at any voltage. The NCX blocker KB-R7943 (3 μM) potently inhibited the NCX current, but 5 μM TRAM-34 was ineffective (Appendix Fig S1B and C). SK4 channel expression was confirmed at the protein level, where an SK4 immunoreactive band of about 50 kDa was identified in Western blots from beating cluster lysates of both normal and CPVT2 hiPSC-CMs (Fig 1E). Figure 1. SK4 channels are expressed in hiPSC-CMs derived from a healthy normal individual and a CPVT2 (CASQ2-D307H) patient Representative traces of an hiPSC-CM derived from a healthy normal individual following a voltage ramp under the indicated conditions. Solution 1 included 300 μM CdCl2, 25 μM ZD7288, and 10 μM E-4031. Representative traces of an hiPSC-CM derived from a CPVT2 (CASQ2 D307H) patient. Scatter plot of the TRAM-sensitive current densities measured at +60 mV with 1 or 5 μM TRAM-34. Current densities were 1.00 ± 0.25 pA/pF in normal (n = 19) and 1.39 ± 0.29 pA/pF in CPVT2 (n = 18). Not statistically different (two-tailed unpaired t-test). Average traces of the TRAM-34-sensitive currents using 1 μM TRAM-34 of normal (n = 4) and CPVT2-derived hiPSC-CMs (n = 5). For clarity, the SEM bars are shown for every mV. Representative Western blots of beating EB lysates from a normal individual and a CPVT2 (CASQ2 D307H) patient showing immunoreactive SK4 protein (≈50 KDa). Download figure Download PowerPoint Exposure of normal hiPSC-CMs to 100 nM isoproterenol significantly increased the firing rate and the slope of diastolic depolarization (DD). Adding 1 μM TRAM-34 to the isoproterenol solution significantly depolarized the maximal diastolic potential (MDP) and decreased the firing rate and the DD slope, which eventually culminated by a suppression of the pacing (Fig 2A and B). Similar experiments were performed on CASQ2 D307H hiPSC-CMs. Isoproterenol did not significantly increase the beating rate on CPVT2 hiPSC-CMs, but instead, it triggered DADs (Fig 2C, arrows). Strikingly, adding 1 μM TRAM-34 to the isoproterenol solution drastically reduced the number of DADs and led to subsequent and reversible cessation of the spontaneous activity (Fig 2C and D). Figure 2. Blockade of SK4 channels by 1 μM TRAM-34 reduces arrhythmias recorded in hiPSC-CMs derived from a CPVT2 (CASQ2-D307H) patient Representative traces of spontaneous APs recorded in a hiPSC-CM derived from a normal individual under the indicated conditions. Histograms of statistical data of the beating rate, DD slope, and MDP of hiPSC-CMs from a normal individual. One-way ANOVA followed by Tukey's multiple comparison test. For rate (normalized to Control), *P < 0.05, n = 24; for DD slope (normalized to Control), **P < 0.01 and ***P < 0.0001, n = 24; for MDP, ***P < 0.0001, n = 24. Bars and error bars are mean ± SEM. Representative traces of spontaneous APs recorded in a hiPSC-CM derived from a CPVT2 patient under the indicated conditions. Histograms of statistical data of the beating rate, APD50, and DADs of hiPSC-CMs from a CPVT2 patient. One-way ANOVA followed by Tukey's multiple comparison test. For the rate, P = ns, n = 21; for the APD50, P = ns, n = 19; for the DADs, **P < 0.01, n = 19. Bars and error bars are mean ± SEM. Download figure Download PowerPoint SK4 channels are expressed in SAN cells and their inhibition lessens the arrhythmic phenotype of SAN cells from CASQ2-D307H KI mice Individual SAN cells were isolated from WT and CASQ2-D307H homozygous KI mice (Song et al, 2007; Katz et al, 2010) and recorded as described above, except that cells were held at −40 mV to improve their stability. In the absence of blockers (black traces), the voltage ramp revealed the presence of one inward hump peaking at about −40 mV and reflecting activation of T-type Ca2+ currents with minor contribution of L-type Ca2+ currents (Fig 3A and B). Upon exposure of cells to solution 1 (orange traces), the inward hump and substantial ramp currents disappeared. Addition of 1 μM TRAM-34 to solution 1 (green traces) decreased the outward ramp currents. Like for hiPSC-CMs, while the average traces of the TRAM-34-sensitive currents (using 1 μM TRAM-34) of WT and CASQ2-D307H SAN cells exhibited a prominent outward component, small residual inward currents that were not completely blocked by solution 1 shifted the Erev to values more positive than those of EK (Fig 3D). Similar TRAM-34-sensitive current densities were found using either 1 or 5 μM TRAM-34 (Fig 3C). Comparable densities of TRAM-34-sensitive currents were isolated in SAN cells from WT and CASQ2-D307H KI mice (Fig 3C). Confirming the expression of SK4 channels and CASQ2 in adult mouse heart of WT and CASQ2-D307H KI mice, Western blots of lysates from SAN, right and left atrial appendages, and right and left ventricles showed specific immunoreactive bands corresponding to SK4 channel and to CASQ2 protein (Fig 3E). Quantitative analysis of the blots showed no significant differences in the heart tissues between the WT and CASQ2-D307H KI mice (Fig 3F). Figure 3. SK4 channels are expressed in SAN cells from WT and CASQ2-D307H KI mice Representative traces of a SAN cell from WT mice following a voltage ramp under the indicated conditions. Representative traces of a SAN cell from CASQ2-D307H KI mice. Scatter plot of the TRAM-sensitive current densities measured at +60 mV with 1 or 5 μM TRAM-34. Current densities were 2.82 ± 0.63 pA/pF in WT (n = 15) and 2.36 ± 0.89 pA/pF in CASQ2-D307H KI mice (n = 16). Not statistically different (two-tailed unpaired t-test). Average traces of the TRAM-34-sensitive currents using 1 μM TRAM-34 of WT (n = 7) and CASQ2-D307H KI mice (n = 4). For clarity, the SEM bars are shown for every mV. Representative Western blots of heart lysates from WT and CASQ2-D307H KI mice showing the immunoreactive bands of SK4, CASQ2, and β-actin proteins in SAN, right and left atrial appendages, and right and left ventricles. Quantification of the SK4 channel immunoreactive protein (normalized to β-actin) in different heart regions (n = 3). Not statistically different (two-tailed unpaired t-test). Error bars are SEM. Source data are available online for this figure. Source Data for Figure 3 [emmm201606937-sup-0002-SDataFig3.jpg] Download figure Download PowerPoint Next, we recorded the spontaneous activity of isolated SAN cells. Exposure of WT SAN cells to 2 μM clotrimazole, another SK4 channel blocker, significantly decreased the firing rate and the DD slope (Appendix Fig S2B, violet trace). These effects were reversible during washout (Appendix Fig S2B, blue trace). Similarly, 2 μM TRAM-34 decreased the spontaneous beating rate and depolarized the MDP before cessation of the pacing (Appendix Fig S2C and D). Isoproterenol (50 nM) significantly increased the pacing of SAN cells from WT mice with an increased DD slope (Fig 4A and B). Adding 2 μM TRAM-34 to isoproterenol depolarized the MDP, markedly reduced the DD slope, decreased the beating rate, and eventually stopped the pacing activity in three out of seven cells. In SAN cells from CASQ2-D307H KI mice, addition of 50 nM isoproterenol initially produced a positive chronotropic effect. However, after 1–2 min isoproterenol led to DADs (Fig 4C, arrows). Remarkably, when TRAM-34 was added to the isoproterenol solution, the occurrence of DADs was drastically reduced (Fig 4C and D). Figure 4. Inhibition of SK4 channels by 2 μM TRAM-34 lessens the arrhythmic phenotype of SAN cells from CASQ2-D307H KI mice Representative traces of spontaneous APs recorded in a SAN cell from WT mice under the indicated conditions. Histograms of statistical data of the beating rate, DD slope, and MDP of SAN cells from WT mice. One-way ANOVA followed by Tukey's multiple comparison test. For rate, *P < 0.05, n = 6; for DD slope, *P < 0.05, **P < 0.01, n = 7; for MDP, *P < 0.05, n = 5. Bars and error bards are mean ± SEM. Representative traces of spontaneous APs recorded in a SAN cell from CASQ2-D307H KI mice under the indicated conditions. Histograms of statistical data of the beating rate, and MDP of SAN cells from CASQ2-D307H KI mice. One-way ANOVA followed by Tukey's multiple comparison test. For the rate, P is not significant, n = 5; for DADs, **P < 0.01, n = 5. Bars and error bards are mean ± SEM. Download figure Download PowerPoint To investigate the spontaneous calcium transients of the SAN, we exposed to Fluo-4 AM intact SAN tissue preparations dissected ex vivo from WT and CASQ2-D307H KI mice as previously described (Torrente et al, 2015). In SAN from WT mice, the rate of calcium transients was significantly increased in the presence of 100 nM isoproterenol and the additional exposure of 2 μM TRAM-34 did not alter the pattern of the Ca2+ waves (Fig 5A). Consistent with previous studies in different CPVT1 and CPVT2 mouse models and hiPSC-CMs (Itzhaki et al, 2012; Neco et al, 2012; Novak et al, 2012, 2015; Glukhov et al, 2015; Torrente et al, 2015), exposing SANs from CASQ2-D307H KI mice to 100 nM isoproterenol produced various Ca2+ transient abnormalities, which we classified according to their degree of severity (Fig 5B and C). In Fig 5C are shown local Ca2+ release (upper left), double-humped transients (upper right), large-stored released Ca2+ waves (lower left), and calcium alternans (lower right). Strikingly, adding 2 μM TRAM-34 normalized the shapes of isoproterenol-induced aberrant calcium waves in SAN from CASQ2-D307H KI mice (Fig 5B). For instance, TRAM-34 brought back to zero the number of SANs displaying double-humped transients or large-stored released Ca2+ waves (Fig 5D). Figure 5. Isoproterenol leads to abnormal SAN calcium transients, which are improved with TRAM-34 Left, representative traces of spontaneous calcium transients recorded ex vivo in intact SAN tissue preparations from WT mice under the indicated conditions. Right: data summary of calcium transient rate (one-way ANOVA: **P < 0.01, ***P < 0.001, n = 12). Bars and error bars are mean ± SEM. Representative traces of spontaneous calcium transients recorded from intact SAN of CASQ2 D307H KI mice. Representative traces of different types of calcium transient abnormalities recorded in intact SAN from CASQ2 D307H KI mice, termed as "local Ca2+ release", "double-humped transients", "large-stored released Ca2+ waves", and "calcium alternans". Data summary of the arrhythmic calcium transients in SAN from CASQ2 D307H KI under the indicated conditions. Download figure Download PowerPoint Blockade of SK4 channels improves in vivo the ECG arrhythmic features of CASQ2-D307H KI and CASQ2 KO mice A heart telemetry device was implanted in WT, CASQ2-D307H KI, and CASQ2 KO mice for continuous ECG recording at rest and during treadmill exercise. For each session, continuous ECG recording was performed with the same animals receiving first intraperitoneal (IP) injection of vehicle (peanut oil) and then the SK4 channel blocker. TRAM-34 (20 mg/kg, IP) significantly decreased the resting heart rate of WT mice by 16 ± 3% as measured by the PP interval (Fig 6A and B). Interestingly, a significant prolongation of 20% in the PR interval was also seen on the ECG traces of WT mice (Fig 6A and B). TRAM-34 produced similar bradycardic effects and PR interval prolongation during treadmill exercise of WT mice (Fig 7A and B). Confirming the importance of SK4 channels in the pacemaker function of adult WT mice, another SK4 channel blocker clotrimazole (20 mg/kg, IP) significantly reduced the resting heart rate by 16 ± 6% (Appendix Fig S3A and B) and prolonged by 27% the PR interval. A similar trend was noticeable during treadmill exercise (Appendix Fig S4A and B). Figure 6. Blockade of SK4 channels by TRAM-34 improves the ECG arrhythmic features of CASQ2-D307H KI and CASQ2 KO mice under rest conditions Representative ECG recording following IP injection of vehicle (upper) and 20 mg/kg TRAM-34 (lower) in WT mice at rest. Sequential vehicle and TRAM-34 injections were performed on the same animal. Data summary of heart rate (paired t-test; ***P = 0.0003, n = 10) and PR interval (paired t-test; ***P = 0.0004, n = 10) in WT mice at rest. Error bars: ± SEM. Representative ECG recording following IP injection of vehicle (upper) and 20 mg/kg TRAM-34 (lower) in CASQ2-D307H KI mice at rest. Data summary of heart rate (paired t-test; ***P < 0.0001, n = 12) and PR interval (paired t-test; ***P < 0.0001, n = 12) in CASQ2-D307H KI mice at rest. Error bars: ± SEM. Representative ECG recording following IP injection of vehicle (upper) and 20 mg/kg TRAM-34 (lower) in CASQ2 KO mice at rest. Data summary of heart rate (paired t-test; **P = 0.004, n = 7 mice) and PR interval (paired t-test; **P = 0.004, n = 7) in CASQ2 KO mice at rest. Error bars: ± SEM. Download figure Download PowerPoint Figure 7. Blockade of SK4 channels by TRAM-34 improves the ECG arrhythmic features of CASQ2-D307H KI and CASQ2 KO mice during treadmill exercise Representative ECG recording following intraperitoneal injection of vehicle (upper) and 20 mg/kg TRAM-34 (lower) in WT mice during treadmill exercise. Data summary of heart rate (paired t-test; ***P = 0.001, n = 10) and PR interval (paired t-test; ***P = 0.0005, n = 10) in WT mice during exercise. Error bars: ± SEM. Representative ECG recording following IP injection of vehicle (upper) and 20 mg/kg TRAM-34 (lower) in CASQ2-D307H KI mice during treadmill exercise. Data summary of heart rate (paired t-test; ***P = 0.0004, n = 11) and PR interval (paired t-test; **P = 0.0099, n = 11) in CASQ2-D307H KI mice during exercise. Error bars: ± SEM. Representative ECG recording following IP injection of vehicle (upper) and 20 mg/kg TRAM-34 (lower) in CASQ2 KO mice during treadmill exercise. Data summary of heart rate (paired t-test; *P = 0.0165, n = 7) and PR interval (paired t-test; **P = 0.0042, n = 7) in CASQ2 KO mice during exercise. Error bars: ± SEM. Download figure Download PowerPoint CASQ2-D307H KI and CASQ2 KO mice displayed lower basal heart rates compared to WT mice but also irregular sinus rhythm and ventricular premature complexes (VPCs) as shown on the ECG traces (Fig 6C–F). Frequently, these VPCs produced a desynchronization of the PQRS complexes, accompanied by variable P–Q intervals (Appendix Fig S3C; upper row, see arrows). Sometimes, the VPCs were so severe that the P waves were absent because there were absorbed into the premature QRS complexes (Fig 7E; upper row). TRAM-34 injection (20 mg/kg, IP) to these mice produced like in WT animals significant bradycardic effects and PR prolongation (Fig 6D and F). Remarkably, TRAM-34 injection improved the ECG arrhythmic features observed under resting conditions and totally suppressed them in nine out of 12 KI mice. During treadmill exercise, the ECG cardiac abnormalities were aggravated with "non-sustained" and even "sustained" ventricular tachycardia (Fig 7C and E). Under these conditions, TRAM-34 injection decreased the prevalence and severity of arrhythmias (Table 1). Notably, TRAM-34 was able to restore the P waves that disappeared because of the VPC-induced desynchronization of the PQRS complexes in CASQ2 KO animals (Fig 7E). During treadmill exercise, TRAM-34 also produced significant sinus bradycardia and PR interval prolongation in KI and KO mice (Fig 7C–F). Clotrimazole (20 mg/kg, IP) elicited similar effects to those observed with TRAM-34. Under basal conditions (Appendix Fig S3) and during treadmill exercise (Appendix Fig S4), bradycardia and PR prolongation were noticed in CASQ2-D307H KI and CASQ2 KO mice following clotrimazole injecti
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