Activation of PKC affects the ventricular restitution properties and arrhythmogenesis through L-type Ca+ current

Activation of PKC affects the ventricular restitution properties and arrhythmogenesis through L-type Ca+ current | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Activation of PKC affects the ventricular restitution properties and arrhythmogenesis through L-type Ca+ current Feng Zhang, Fuhua Lei, Jianing Fan, Tao Liu, Mu Qin, Wenbo Cheng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2679832/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective To investigate the role of protein kinase C (PKC) in action potential duration (APD) restitution and ventricular tachyarrhythmias (VA). Methods and results Rabbits hearts were isolated and prepared for Langendorff perfusion technique. The S 1 -S 2 method and dynamic S 1 pacing protocol were performed to construct APD restitution and to induce APD alternans or VA, respectively, at 10 sites throughout the ventricular chamber. Administration of PMA (100 nM) (n = 15) greatly steepened the restitution curves (S max >1) ( P < 0.01) at each site compared to the control group (n = 15). Furthermore, treatment with PMA also induced larger spatial dispersions of S max ( P < 0.05) and decreased the thresholds of the VA and APD alternans ( P < 0.01). However, perfused with the PKC inhibitor, BIM (500 nM) (n = 10), reversibly flattened the APD restitution curves at each site (S max < 1), decreased the spatial dispersions of S max , and increased the thresholds of APD alternans and VA. According to the results of patch-clamp, peak amplitude of L-type Ca 2+ current was significantly increased by addition of PMA compared with CTL group (P < 0.05). Antagonize this current with verapamil (n = 10) can fully inhibited the PMA induced increasing of S max and inducibility of VA and alternans. Conclusion PKC activation increased the dispersion of APD restitution and thus led to occurrence of VA, which possibly related to the increased Ca 2+ influx. Protein kinase C Restitution Spatial heterogeneity Ventricular tachyarrhythmia Ca2+ current Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Isoforms of protein kinase C (PKC), as a group of closely related serine-threonine protein kinases, are classified as classical PKCs (α, βI, βII, and γ) and novel PKCs (δ, ε, θ, and η). The mRNA expressions of PKC-α, -βI, -βII, -δ, -ε, and -η have been reported in human and rat cardiomyocytes 2 – 4 . However, the effect of PKC stimulation on cardiac electrophysiology and arrhythmia are still incompletely understood. Previous studies suggested that PKC activation could facilitate the inducibility of ventricular tachyarrhythmia (VA), which is related to the alteration of intracellular Ca 2+ concentration 5 . However, little is known about the electrophysiological mechanism of PKC-related arrhythmogenesis. Restitution of the action potential duration (APD) is closely linked to risk of ventricular fibrillation. A steepened slope of the APD restitution curve could promote breakup of electrical waves into a fibrillation-like state, which can be effected by disabling of intracellular Ca 2+ cycling 6–7 . Thus, as a regulator of intracellular Ca 2+ cycling, PKC could possibly exhibit an effect on APD restitution and arrhythmia in the heart. The objective of this study is to analyze experimentally the contribution of PKC activation to the alteration of APD restitution and inducibility of VA in rabbit isolated hearts perfused by the Langendorff technique. Our findings indicate that PKC activation greatly influenced both APD restitution and VA, and these effects were closely related to the increased Ca 2+ influx. 2. Materials And Methods 2.1 Experimental animals Experiments were performed on healthy adult male New Zealand White rabbits (2.5 to 3.0kg) from center of experimental animal in medical college of Wuhan University. All protocols were conform to the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH Publication No.85 − 23, revised 1996), and approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. 2.2 Preparation of Langendorff-perfused hearts Animals were anesthetized (pentobarbital sodium 90mg/kg, i.v.) and heparinized (heparin sodium 2000U, i.v.), the heart was quickly excised and transferred to ice-cold HEPES-buffered Tyrode’s solution, bubbled with 95% O2–5% CO2. A small section of aorta (3–4 mm) was identified and cannulated rapidly to the langendorff-perfusion system (AD Instruments, Australia). The perfusate was passed through 2.2µm filters, and then warmed to 37°C by a water jacket and circulator. Perfusion was commenced in a retrograde manner through the aorta at 20–25 ml/min by a peristaltic pump (AD Instruments, Australia). In this way, the heart was perfused by the HEPES-buffered Tyrode’s solution passing through the aorta into the coronary arteries. After the initiation of perfusion, hearts regained a pink color and spontaneous rhythmic contractions. The isolated hearts were perfused for 20 minutes before further experimental test. The hearts that did not recover to the regular spontaneous rhythm or had irreversible myocardial ischemia were discarded. All heart were divided into 5 groups: control (CTL, n = 10), PMA (n = 15), PMA + BIM (n = 10), PMA + Verapamil (n = 10) and Verapamil (n = 10). 2.3 Monophasic action potential recording Monophasic action potential (MAP) was recorded using a custom-made MAP electrode, constructed from two 0.25 mm Teflon-coated silver wire (99.99% purity;), that were twisted together and galvanically chlorided to eliminate DC offset. Epicardial MAP recordings were performed at the right ventricular outflow tract (RVOT), right basal ventricle (RB), free wall of right ventricle (RF), right apex (RA), left anterior and posterior basal ventricle (LAB and LPB), left anterior and posterior free wall (LAF and LPF), left anterior and posterior apex (LAA and LPA). The paired platinum stimulating electrode was positioned on the basal surface of right ventricle and delivered regular pacing stimulation (Grass, USA) at baseline cycle length (BCL) of 300ms with square-wave stimuli of 2-ms duration and amplitudes of three times the diastolic threshold. MAPs amplified with an amplifier and band pass filtered between 0.3 Hz and 1 kHz. MAP waveforms were analyzed using Chrat7.0 software. 2.4 Experiment stimulated protocol Protocol I: S 1 -S 2 method The Programmed electrical stimulation (PES) protocol was used for ventricular effective refractory period examination. PES consisting of an eight stimuli (S1) drive train followed by a ninth extra-stimulus (S2), the CL of S1 train was 300ms. The first S1–S2 interval equalled the pacing interval and was then progressively reduced by 10ms per cycle until the S2 stimulus could no longer evoke a ventricular deflection. Ventricular effective refractory period (VERP) defined as the longest S1–S2 interval that loss of ventricular depolarization. Protocol II: S 1 dynamic pacing A dynamic steady state pacing protocol (S 1 -S 1 ) was applied with a series of pulse trains at regular pacing cycle length (PCL). The train was delivered at an initial PCL of 300ms and with the decrement of 20ms steps to PCL of 200ms, and then decrement with 10ms steps until PCL = 100ms. The pulse train was maintained for 30 sec to ensure a steady state and interrupted for 30 sec to minimize the pacing memory effects. VA episodes lasting > 10sec were defined as sustained VA episodes, VA episodes lasting < 10sec were classifies as non-sustained VA. If ventricular arrhythmia was induced, a 5-10min interrupted periods was allowed after VA termination for cardiac electrical recovery. APD alternans was assessed at each site by subtracting the APD 90 for two consecutive beats 8 . The threshold and duration of ventricular arrhythmia at each site were analyzed. 2.5 Construction of APD restitution curves Curves of APD restitution were plotted with the APD 90 elicited by S 2 against the corresponding diastolic intervals (DI). APD 90 was defined as 90% repolarization duration. DI was defined as the time interval from the previous APD 90 point to the next initiation point of the action potential, and measured as the S 1 -S 2 interval minus the APD 90 of S 1 at very short S 1 -S 2 intervals. The restitution curves were constructed by plotting APD 90 of the S 2 against the preceding DI. The curves were fitted by using overlapping least-squares liner segments and analyzed in 20ms DI segments in steps of 5ms, commencing from the shortest diastolic interval range that contains data points 9 . Dispersion of maximal slope (S max ) of the curve in whole heart (WH), left ventricle (LV) and right ventricle (RV) defined as the coefficient of variation among the each site. 2.6 Drugs and solutions Isolated hearts were perfused with normal HEPES-buffered Tyrode’s solution (mM: NaCl 130; KCl 5.4; CaCl 2 1.8; MgCl 2 1; Na 2 HPO 4 0.3; HEPES 10; glucose 10; pH adjusted to 7.4 with NaOH) for control group (n = 15), Tyrode’s solution with addition of 100nM PKC agonist (phorbol-12-myristate- 13-acetate; PMA), 100nM PMA plus 500nM antagonist (bisindolylmaleimide, BIM) and 100nM PMA plus 550pM verapamil, respectively 5 . 2.7 Isolation of ventricular myocytes Experiment animals were heparinized (2000U, i.v.) and anaesthetized by pentobarbital sodium (90mg/kg, i.v.). The hearts were removed and retrogradely perfused on Langendorff system with the following solutions: first, 5 minutes perfused with HEPES-buffered Tyrode’s solution. Second, 5 minutes perfused with Ca 2+ -free HEPES-buffered Tyrode’s solution. Third, 15 minutes perfused with HEPES-buffered Tyrode’s containing 0.6mg/ml collagenase type II (Invitrogen Co.US), 0.1% bovine serum albumin, 20mM taurine and 30µM CaCl 2 . Fourth, 5 minutes perfused with KB solution (mM: taurine 10; glutamic acid 70; KCl 25; KH 2 PO 4 10; glucose 22; EGTA 0.5; pH adjusted to 7.2 with KOH). At the end of the perfusion, the ventricular free wall was dissected from the heart. Cell samples were pooled and resuspended in 10ml myocyte stopping buffer (perfusion solution, containing 5% fetal bovine serum and 50uM CaCl 2 ) and transferred to a 60mm culture dish. Calcium concentration was increased by addition of 10mM CaCl 2 to achieve final concentrations of 50µM, 100µM, 150µM, 200µM, 500µM and 1mM. 2.8 L-Type Ca 2+ current recoding Whole-cell patch clamp was performed using EPC-9 amplifier (List Instruments, Germany) and data was recorded and analyzed with Pulse-pulsefit software interface (Version 8.31, HEKA Co. Germany). During the experiments, the myocytes were continuously superfused with the extracellular solution (2mL/min) containing (in mM): NaCl 130; KCl 5.4; CaCl 2 2; MgCl 2 1; Na 2 HPO 4 0.3; HEPES 10; glucose 10; (pH adjusted to 7.4 with NaOH). The resistances of the pipettes ranged from 2.5 to 3.5MΩ when filled with pipette solution (mM): CsCl 135, MgC1 2 1, MgATP 4, TEACl 10, EGTA 0.1 and 10 HEPES (pH 7.2 with CsOH). Ca 2+ current was recorded using 10mV steps depolarizing pulse for 150ms from the holding potential of -40mV and integrating the resulting current trace. Currents were normalized to cell capacitance (pA/pF) to account for differences in cell size. 2.9 Statistical analysis All data are expressed as mean ± SEM. Statistical analysis performed with Student’s t test was completed by SPSS 16.0. A value of P < 0.05 was considered significant. APD restitution curves were analyzed using Origin 6.0 (Microcal Co. USA) for nonlinear curve fitting. 3. Results 3.1 PMA increased the slope of APD 90 restitution curves To avoid the time course of PMA treatment, the recordings were applied after 20–30 min stabilization. Compared with the control (CTL) situation, PMA treatment markedly steepened the slopes of the APD restitution curves (S max > 1) at all sites ( P < 0.01) (Fig. 1 and Fig. 2 A) but also increased the spatial dispersions of S max (COV-S max ) throughout the whole heart (PMA: 0.26 ± 0.02, CTL: 0.21 ± 0.02, P < 0.01) and at sites in the left ventricle (PMA: 0.33 ± 0.03, CTL: 0.26 ± 0.02; P < 0.01) and right ventricle (PMA: 0.17 ± 0.02, CTL: 0.11 ± 0.02; P < 0.05) (Fig. 2 B). The increased values of S max was more obvious in the left apex and posterior wall, so that the COV-S max was larger in the left ventricle than that in the right ventricle (P < 0.01) during PMA treatment. To identify the PMA-induced alteration of S max was related to PKC activation, we observed that the administration of BIM could fully antagonize the effect of PMA and flatten the APD restitution curve at each site. The S max of all sites was decreased to the similar level of CTL state by BIM (P > 0.05), and the COV-S max had no significant difference between BIM and CTL group (P > 0.05) (Fig. 2 A,Table 1 and Table 2). 3.2 PMA treatment increases susceptibility to APD alternans and VA In the CTL group, VA was inducible in 6 of the 15 hearts, and appeared mostly as non-sustained VA (33.3%, 5/15). But treatment with PMA increased the inducibility of the VA by dynamic pacing at most sites in 13 of the 15 hearts (86.7%) and the VA were elicited as the sustained state in six of the hearts (40.0%). Analysis of the threshold of VA inducibility showed that PMA greatly increased induced CL at all sites of the heart ( P < 0.01) compared to the CTL state, and the threshold of VA was higher in left ventricle than right ventricle (Fig. 2 D). Although less susceptible to rapid dynamic pacing-induced VA, APD alternans was observed at each site in CTL state during the dynamic pacing protocol, but it usually occurred before VA initiation and transformed into a sustained state after PMA administration (Fig. 3 ). Furthermore, the paced CL to induce APD alternans was significantly increased by PMA compared to the CTL state at each site of the heart ( P < 0.01) (Fig. 2 C), it suggesting that the APD alternans was facilitated by PKC activation. However, perfusion with BIM markedly suppressed or delayed the occurrence of dynamic pacing-induced VA and APD alternans, the inducibility threshold of VA and alternans was similar to that in the CTL state ( P > 0.05) (Fig. 2 C,D). These results indicate that the effects of PMA on APD alternans and VA could be inhibited by BIM administration. 3.3 PMA increased the inwardly Ca 2+ current To determine whether the PMA induced changes were caused by regulation of Ca 2+ currents, we next used whole patch-clamp recordings to characterize L-type Ca 2+ current in isolated rabbit ventricular myocytes from CTL (n = 8) and PMA (n = 9) group. Depolarization of cells elicited the L-Ca 2+ current, the peak amplitude was significantly increased by addition of PMA compared with CTL group (P < 0.05). As evidence of a transient inward current, characteristic L-Ca 2+ current was reversibly blocked by 550pM verapamil (Fig. 4 ). 3.4 Verapamil inhibited the effect of PMA on APD restitution and arrhythmia To determine whether the Ca + influx might be involved in PMA related effect on APD restitution and arrhythmia, we administrated verapamil to antagonize L-Ca 2+ current. Verapamil could fully inhibited the PMA induced increasing of APD restitution slope at each site of heart (S max <1) ( P < 0.01), and the spatial dispersions of S max was decreased by addition of Verapamil throughout the whole heart (PMA: 0.26 ± 0.02, PMA + Vera: 0.17 ± 0.02, P < 0.01) and at sites in the left ventricle (PMA: 0.13 ± 0.04, PMA + Vera: 0.26 ± 0.02; P < 0.01) and right ventricle (PMA: 0.17 ± 0.02, PMA + Vera: 0.14 ± 0.02; P < 0.05) (Fig. 5 ). Analysis of the threshold of APD alternans showed that the alternans could not be induced by S 1 dynamic pacing, and the VA was no longer inducible during CL from 300ms to 100ms at each site of heart (Fig. 6 ). 4. Discussion 4.1 Main findings The present study investigated the effect of PKC activation on characteristics of APD restitution at distinct sites of the ventricle. We found that (i) the maximal slope of APD restitution and the susceptible of VA induction were greatly increased by PKC stimulation at each site in the heart; (ii) PKC activation greatly increased the dispersion of APD restitution among each site in the ventricle; and (iii) PKC stimulation induced steepened curve of restitution and arrhythmogenesis were closely related to the increased Ca 2+ influx of ventricular myocytes. 4.2 PKC and APD restitution As important regulators of intracellular signaling, PKC isoforms play roles in the pathological modifications associated with heart disease 10 , 11 . However, the effects of PKC on cardiac electrophysiology are not fully understood. Results of previous studies have suggested that PKC activation modulated intracellular Ca 2+ through increase the inwardly L-type Ca 2+ current. The intracellular Ca 2+ concentration ([Ca 2+ ] i ) greatly upgrade may result in Ca 2+ overload and decreased (~ 65%) the Ca 2+ content of SR, which was abolished by addition of the PKC inhibitor 12 – 14 . Increased [Ca 2+ ] i in turn inactivated the diastolic currents including K + , Na + -Ca 2+ exchange current and other Ca 2+ -sensitive currents. During this effect, the balance of APD and previous phase of DI could be shifted, and finally steepened the restitution curve 24 . Tseng and Goldhabor concluded that [Ca 2+ ] i dynamics played a key role in APD restitution, greater Ca 2 + i accumulation occurred at which the APD restitution slope exceeded 1 during dynamic pacing and the slope was flattened by suppressing SR Ca 2+ cycling with thapsigargin and ryanodine 15 , 16 . Thus, the [Ca 2+ ] i increased is related to the steepness of the APD restitution curve. Consistently, the findings of the present study revealed distinct APD restitution properties during PKC activation and inactivation by perfusion of PMA and BIM, respectively. Moreover, PMA indeed enhanced the L-Ca 2+ current during patch clamp recording and PKC activation induced increasing of restitution slope can be inhibited by inwardly Ca 2+ currents antagonist. These findings indicate that the slope of APD restitution curve could be exactly steepened by PKC stimulation through [Ca 2+ ] i upgrade. 4.3 PKC and APD alternans Multiple mechanisms are proposed for the phenomenon of APD alternans 17 , 18 . Primary Ca 2 + i alternans could drive APD to alternans secondarily, because APD is shaped by several key membrane currents that are sensitive to Ca 2 + i , such as L-type Ca 2+ currents, calcium-dependent potassium currents, and sodium-calcium exchange currents. Many studies focus on the relationship between the APD alternans and Ca 2 + i alternans in single cell and tissue samples 16 , 19 , 20 . The results of Goldhabor et al. strongly support a key role for Ca 2 + i instability in APD alternans elicited by rapid S 1 pacing. They found that the onset of APD alternans occurred at diastolic intervals, during which the APD restitution slope was significantly < 1, and this phenomenon was abolished by suppressing SR Ca 2+ cycling or global Ca 2+ transients 16 . In present work, the threshold of APD alternans was greatly decreased by PKC stimulation at each site in the heart, and Ca 2+ influx antagonist abolished the occurrence of alternans. It suggested the mechanism that PKC increased [Ca 2+ ] i via L-type Ca 2+ current and thus facilitated the APD alternans. 4.4 PKC and ventricular tachyarrhythmias Aydin et al. first reported that PKC activation facilitates electrically stimulated ventricular fibrillation in the isolated heart 4 . Consistent with these findings, our data showed that PKC stimulation increased susceptibility to VA at each site in the heart during dynamic rapid pacing. But lack of the research to illustrate the electrophysiological mechanism of PKC related arrhythmias, previously. Many studies suggested that the vulnerability to ventricular fibrillation is closely linked to APD restitution, and steep restitution slopes that greater than 1 could promote the APD alternans and breakup of electrical waves into a fibrillation-like state 6 – 8 , 21 , 22 . In the present study, PKC activation decreased the threshold of VA and increased the S max values of the restitution curves (S max > 1) at each site. However, APD restitution from a single site may not represent the APD restitution kinetics of the whole heart, and the higher inducibility of tachyarrhythmias could not be explained by S max of the APD restitution curve. A study of our lab had demonstrated that the spatial dispersion of S max plays a critical role in the initiation and maintenance of atrial fibrillation. Although the slopes were flattened (S max < 1) by Vagal stimulation, the atrial fibrillation was facilitated associated with the increased APD restitution dispersion 23 . The possible explanation is that the uniform APD restitution promoted greater heterogeneous refractoriness across the cardiac chamber. Presently, our results also revealed the spatial dispersion of S max was increased by PKC activation, it underlined the critical role of spatial dispersion of APD restitution kinetics in the initiation and maintenance of VA. 4.5 Conclusions In present study, we found a correlation between PKC activation and the increased spatial dispersion of APD restitution. These alterations may be closely related to the increased Ca 2+ influx. Declarations Ethical Approval: This study was in accordance with the Declaration of Helsinki and approved by the ethical committee of our institution. Informed consent was obtained before entering this study. Conflict of interest: none declared. Authors’ Contributors: Feng Zhang、Fuhua Lei、Mu Qin and Wenbo Cheng Involved in experiment design，Feng Zhang and Fuhua Lei performed the experiments，Feng Zhang、Fuhua Lei and Jianing Fan wrote the main manuscript text and prepared figures，All authors reviewed the manuscript. Funding: This research was supported by Fudan University Youth Research Fund(Grant No: JYQN-JC-202010). The funder has designed this study and decided to publish this manuscript. Availability of data and materials : The data that support the findings of this study are available on request from the corresponding author, [Wenbo Cheng and Mu Qin]，upon reasonable request. References Coats AJS, Shewan LG. 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Alternans and spiral breakup in a human ventricular tissue model. Am J Physiol Heart Circ Physiol 2006;291:H1088– H1100. Lu ZB, Cui B, He B, Hu XR, Wu W, Wu L, Huang CX, Po SS, Jiang H. Distinct restitution properties in vagally mediated atrial fibrillation and six-hour rapid pacing-induced atrial fibrillation. Cardiovasc Res 2011; 89: 834–842. Wu R, Patwardhan A. Effects of rapid and slow potassium repolarization currents and calcium dynamics on hysteresis in restitution of action potential duration. J Electrocardio 2007; 40: 188–199. tables Table 1. The APD 90 and ERP in baseline (BS), PMA and BIM state All abbreviations are identical to Figure 1. *P<0.05 and # P<0.01 for PMA vs BS Table 2. The maximal slope of APD restitution curves (S max ) in baseline (BS), PMA and BIM state jmjn7 All abbreviations are identical to Figure 1. # P<0.01 for PMA vs BS Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2679832","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":183351003,"identity":"eca894a1-b6c2-4157-9432-be00637bf2f5","order_by":0,"name":"Feng Zhang","email":"","orcid":"","institution":"Jinshan Hospital of Fudan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Zhang","suffix":""},{"id":183351004,"identity":"04c6bb3c-dcda-4a5e-a46f-3273b349ecfc","order_by":1,"name":"Fuhua Lei","email":"","orcid":"","institution":"Jinshan Hospital of Fudan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fuhua","middleName":"","lastName":"Lei","suffix":""},{"id":183351005,"identity":"264bc631-b886-4d7a-89a5-f9dc4485ffff","order_by":2,"name":"Jianing Fan","email":"","orcid":"","institution":"Affiliated Zhongshan Hospital of Dalian University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianing","middleName":"","lastName":"Fan","suffix":""},{"id":183351006,"identity":"d5fc1fc3-15e7-4a72-be29-615a83ecdabb","order_by":3,"name":"Tao Liu","email":"","orcid":"","institution":"Cardiovascular Research Institute of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Liu","suffix":""},{"id":183351007,"identity":"82985cc1-fe39-4250-bf85-021bf2f34d0d","order_by":4,"name":"Mu Qin","email":"","orcid":"","institution":"Shanghai Chest Hospital, Shanghai Jiaotong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mu","middleName":"","lastName":"Qin","suffix":""},{"id":183351008,"identity":"d4813d2a-b51b-4781-8008-db480a7214f0","order_by":5,"name":"Wenbo Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYDACCQjFw8be//ABgwEJWuT4eM4wG5CkxVhOIodNgih38c9uPvbwa5tNYptE7rHKHwV35BnYDx/dgNeSO8fSjWXb0hLbeN6l3eYxeGbYwJOWdgOfFgOJHDNpyW2HE9vYE8xuMxgcZmyQ4DEjoCX/G1DL/8Q2hgSzwh8Gh+2J0JLDJvlx2wFjNo4cMwYeg8OJBLVI3Egzk2b8lyzHxnMsWRqoJbmNkF/4ZyQ/k/xxxo5Hvr354Mcffw7b9rMfPoZXCwgw8yDz2AgpBwHGH8SoGgWjYBSMgpELAHewSUGGPrgZAAAAAElFTkSuQmCC","orcid":"","institution":"Baoshan Branch of Renji Hospital, School of Medicine, Shanghai Jiaotong University, China","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wenbo","middleName":"","lastName":"Cheng","suffix":""}],"badges":[],"createdAt":"2023-03-11 03:14:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2679832/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2679832/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":34440365,"identity":"42cdb1bb-5c8d-4745-b511-c3298cf70758","added_by":"auto","created_at":"2023-03-17 21:25:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":493893,"visible":true,"origin":"","legend":"\u003cp\u003eAPD restitution curves in ten sites of ventricle chamber during control (CTL), PMA and BIM state. Right ventricular outflow tract (RVOT), right basal ventricle (RB), free wall of right ventricle (RF), right apex (RA), left anterior and posterior basal ventricle (LAB and LPB), left anterior and posterior free wall (LAF and LPF), left anterior and posterior apex (LAA and LPA).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2679832/v1/26c3d251d250e55501b5b147.jpg"},{"id":34440369,"identity":"94c6cc81-603a-47ff-a6fb-256403bbd0f6","added_by":"auto","created_at":"2023-03-17 21:25:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":354824,"visible":true,"origin":"","legend":"\u003cp\u003eThe maximal slope (S\u003csub\u003emax\u003c/sub\u003e) of the APD restitution curves in control (CTL), PMA and BIM state (A). The spacial dispersion of S\u003csub\u003emax\u003c/sub\u003e (COV-S\u003csub\u003emax\u003c/sub\u003e) among the sites in whole heart (WH), left ventricle (LV) and right ventricle (RV), respectively (B). The threshold of APD alternans and VA were performed at each site. The threshold was defined as the longest paced CL inducing alternans or VA by paced CL decrement from 500ms to 100ms (in 10ms steps) (G, H). \u003cstrong\u003e*\u003c/strong\u003eP\u0026lt;0.01 PMA vs CTL.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2679832/v1/187eff8d85b75b094f5a9eed.jpg"},{"id":34442771,"identity":"46ddcb24-53dc-49fe-98ce-f0a6d0768b43","added_by":"auto","created_at":"2023-03-17 21:41:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":537951,"visible":true,"origin":"","legend":"\u003cp\u003eVentricular tachyarrhythmia (VA) were elicited by S\u003csub\u003e1\u003c/sub\u003e dynamic pacing in CTL (A,B and C) and PMA state (D,E and F). Sustained VA (\u0026gt;10s) were occurred after APD alternans at PCL of 150ms in administration of PMA (100nM).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2679832/v1/31d3b52631ffeb84d34da023.jpg"},{"id":34441576,"identity":"8769556b-c589-4c45-94ca-636ec23c4ed1","added_by":"auto","created_at":"2023-03-17 21:33:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":181536,"visible":true,"origin":"","legend":"\u003cp\u003eL-type Ca\u003csup\u003e2+\u003c/sup\u003e current in Control (CTL) and PMA group was recorded in the absence and presence of the verapamil (1μM) (A). Depolarization elicited transient inward Ca\u003csup\u003e2+\u003c/sup\u003e current that was significantly increased in PMA treated cardiomyocytes (C), and the current was fully inhibited by verapamil (A). Cellular capacity was compared between CTL and PMA group (B).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2679832/v1/6a95629ceecc5482171ded9b.jpg"},{"id":34440370,"identity":"784cf2ea-0520-4cfc-ad7c-8431de0e40a3","added_by":"auto","created_at":"2023-03-17 21:25:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":144185,"visible":true,"origin":"","legend":"\u003cp\u003eThe maximal slope (S\u003csub\u003emax\u003c/sub\u003e) of the APD restitution curves in administration of PMA and PMA plus verapamil (A). The spatial dispersion of S\u003csub\u003emax\u003c/sub\u003e (COV-S\u003csub\u003emax\u003c/sub\u003e) among the sites in whole heart (WH), left ventricle (LV) and right ventricle (RV), respectively (B). \u003cstrong\u003e*\u003c/strong\u003eP\u0026lt;0.01 PMA vs PMA+Vera.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2679832/v1/15e011e6d354b6f1ee0090c2.jpg"},{"id":34440366,"identity":"3210bfe5-0318-4ed3-b2b0-9ca623123959","added_by":"auto","created_at":"2023-03-17 21:25:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":183243,"visible":true,"origin":"","legend":"\u003cp\u003eVentricular arrhythmia and APD alternans can not been elicited by S\u003csub\u003e1\u003c/sub\u003e dynamic pacing at pacing cycle length (PCL) of 110ms (A) and 100ms (B) during administration of PMA+verapamil.\u0026nbsp;\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2679832/v1/1f2fe7d865c74ea7c178b262.jpg"},{"id":52855649,"identity":"7bb48927-d3a7-4d68-a692-5edd43a3f804","added_by":"auto","created_at":"2024-03-18 02:22:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":907501,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2679832/v1/c5809219-e4c7-40eb-b09d-727c3088f3e1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Activation of PKC affects the ventricular restitution properties and arrhythmogenesis through L-type Ca+ current","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIsoforms of protein kinase C (PKC), as a group of closely related serine-threonine protein kinases, are classified as classical PKCs (α, βI, βII, and γ) and novel PKCs (δ, ε, θ, and η). The mRNA expressions of PKC-α, -βI, -βII, -δ, -ε, and -η have been reported in human and rat cardiomyocytes\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, the effect of PKC stimulation on cardiac electrophysiology and arrhythmia are still incompletely understood. Previous studies suggested that PKC activation could facilitate the inducibility of ventricular tachyarrhythmia (VA), which is related to the alteration of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration\u003csup\u003e5\u003c/sup\u003e. However, little is known about the electrophysiological mechanism of PKC-related arrhythmogenesis. Restitution of the action potential duration (APD) is closely linked to risk of ventricular fibrillation. A steepened slope of the APD restitution curve could promote breakup of electrical waves into a fibrillation-like state, which can be effected by disabling of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e cycling\u003csup\u003e6\u0026ndash;7\u003c/sup\u003e. Thus, as a regulator of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e cycling, PKC could possibly exhibit an effect on APD restitution and arrhythmia in the heart.\u003c/p\u003e \u003cp\u003eThe objective of this study is to analyze experimentally the contribution of PKC activation to the alteration of APD restitution and inducibility of VA in rabbit isolated hearts perfused by the Langendorff technique. Our findings indicate that PKC activation greatly influenced both APD restitution and VA, and these effects were closely related to the increased Ca\u003csup\u003e2+\u003c/sup\u003e influx.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.1 Experimental animals\u003c/h2\u003e\n\u003cp\u003eExperiments were performed on healthy adult male New Zealand White rabbits (2.5 to 3.0kg) from center of experimental animal in medical college of Wuhan University. All protocols were conform to the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH Publication No.85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996), and approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e2.2 Preparation of Langendorff-perfused hearts\u003c/h2\u003e\n\u003cp\u003eAnimals were anesthetized (pentobarbital sodium 90mg/kg, i.v.) and heparinized (heparin sodium 2000U, i.v.), the heart was quickly excised and transferred to ice-cold HEPES-buffered Tyrode\u0026rsquo;s solution, bubbled with 95% O2\u0026ndash;5% CO2. A small section of aorta (3\u0026ndash;4 mm) was identified and cannulated rapidly to the langendorff-perfusion system (AD Instruments, Australia). The perfusate was passed through 2.2\u0026micro;m filters, and then warmed to 37\u0026deg;C by a water jacket and circulator. Perfusion was commenced in a retrograde manner through the aorta at 20\u0026ndash;25 ml/min by a peristaltic pump (AD Instruments, Australia). In this way, the heart was perfused by the HEPES-buffered Tyrode\u0026rsquo;s solution passing through the aorta into the coronary arteries. After the initiation of perfusion, hearts regained a pink color and spontaneous rhythmic contractions. The isolated hearts were perfused for 20 minutes before further experimental test. The hearts that did not recover to the regular spontaneous rhythm or had irreversible myocardial ischemia were discarded. All heart were divided into 5 groups: control (CTL, n\u0026thinsp;=\u0026thinsp;10), PMA (n\u0026thinsp;=\u0026thinsp;15), PMA\u0026thinsp;+\u0026thinsp;BIM (n\u0026thinsp;=\u0026thinsp;10), PMA\u0026thinsp;+\u0026thinsp;Verapamil (n\u0026thinsp;=\u0026thinsp;10) and Verapamil (n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003e2.3 Monophasic action potential recording\u003c/h2\u003e\n\u003cp\u003eMonophasic action potential (MAP) was recorded using a custom-made MAP electrode, constructed from two 0.25 mm Teflon-coated silver wire (99.99% purity;), that were twisted together and galvanically chlorided to eliminate DC offset. Epicardial MAP recordings were performed at the right ventricular outflow tract (RVOT), right basal ventricle (RB), free wall of right ventricle (RF), right apex (RA), left anterior and posterior basal ventricle (LAB and LPB), left anterior and posterior free wall (LAF and LPF), left anterior and posterior apex (LAA and LPA). The paired platinum stimulating electrode was positioned on the basal surface of right ventricle and delivered regular pacing stimulation (Grass, USA) at baseline cycle length (BCL) of 300ms with square-wave stimuli of 2-ms duration and amplitudes of three times the diastolic threshold. MAPs amplified with an amplifier and band pass filtered between 0.3 Hz and 1 kHz. MAP waveforms were analyzed using Chrat7.0 software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003e2.4 Experiment stimulated protocol\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eProtocol I: S\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-S\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e \u003c/sub\u003e \u003cstrong\u003emethod\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Programmed electrical stimulation (PES) protocol was used for ventricular effective refractory period examination. PES consisting of an eight stimuli (S1) drive train followed by a ninth extra-stimulus (S2), the CL of S1 train was 300ms. The first S1\u0026ndash;S2 interval equalled the pacing interval and was then progressively reduced by 10ms per cycle until the S2 stimulus could no longer evoke a ventricular deflection. Ventricular effective refractory period (VERP) defined as the longest S1\u0026ndash;S2 interval that loss of ventricular depolarization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtocol II: S\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e \u003c/sub\u003e \u003cstrong\u003edynamic pacing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA dynamic steady state pacing protocol (S\u003csub\u003e1\u003c/sub\u003e-S\u003csub\u003e1\u003c/sub\u003e) was applied with a series of pulse trains at regular pacing cycle length (PCL). The train was delivered at an initial PCL of 300ms and with the decrement of 20ms steps to PCL of 200ms, and then decrement with 10ms steps until PCL\u0026thinsp;=\u0026thinsp;100ms. The pulse train was maintained for 30 sec to ensure a steady state and interrupted for 30 sec to minimize the pacing memory effects. VA episodes lasting\u0026thinsp;\u0026gt;\u0026thinsp;10sec were defined as sustained VA episodes, VA episodes lasting\u0026thinsp;\u0026lt;\u0026thinsp;10sec were classifies as non-sustained VA. If ventricular arrhythmia was induced, a 5-10min interrupted periods was allowed after VA termination for cardiac electrical recovery. APD alternans was assessed at each site by subtracting the APD\u003csub\u003e90\u003c/sub\u003e for two consecutive beats\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The threshold and duration of ventricular arrhythmia at each site were analyzed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003e2.5 Construction of APD restitution curves\u003c/h2\u003e\n\u003cp\u003eCurves of APD restitution were plotted with the APD\u003csub\u003e90\u003c/sub\u003e elicited by S\u003csub\u003e2\u003c/sub\u003e against the corresponding diastolic intervals (DI). APD\u003csub\u003e90\u003c/sub\u003e was defined as 90% repolarization duration. DI was defined as the time interval from the previous APD\u003csub\u003e90\u003c/sub\u003e point to the next initiation point of the action potential, and measured as the S\u003csub\u003e1\u003c/sub\u003e-S\u003csub\u003e2\u003c/sub\u003e interval minus the APD\u003csub\u003e90\u003c/sub\u003e of S\u003csub\u003e1\u003c/sub\u003e at very short S\u003csub\u003e1\u003c/sub\u003e-S\u003csub\u003e2\u003c/sub\u003e intervals. The restitution curves were constructed by plotting APD\u003csub\u003e90\u003c/sub\u003e of the S\u003csub\u003e2\u003c/sub\u003e against the preceding DI. The curves were fitted by using overlapping least-squares liner segments and analyzed in 20ms DI segments in steps of 5ms, commencing from the shortest diastolic interval range that contains data points\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Dispersion of maximal slope (S\u003csub\u003emax\u003c/sub\u003e) of the curve in whole heart (WH), left ventricle (LV) and right ventricle (RV) defined as the coefficient of variation among the each site.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003e2.6 Drugs and solutions\u003c/h2\u003e\n\u003cp\u003eIsolated hearts were perfused with normal HEPES-buffered Tyrode\u0026rsquo;s solution (mM: NaCl 130; KCl 5.4; CaCl\u003csub\u003e2\u003c/sub\u003e 1.8; MgCl\u003csub\u003e2\u003c/sub\u003e 1; Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 0.3; HEPES 10; glucose 10; pH adjusted to 7.4 with NaOH) for control group (n\u0026thinsp;=\u0026thinsp;15), Tyrode\u0026rsquo;s solution with addition of 100nM PKC agonist (phorbol-12-myristate- 13-acetate; PMA), 100nM PMA plus 500nM antagonist (bisindolylmaleimide, BIM) and 100nM PMA plus 550pM verapamil, respectively\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003e2.7 Isolation of ventricular myocytes\u003c/h2\u003e\n\u003cp\u003eExperiment animals were heparinized (2000U, i.v.) and anaesthetized by pentobarbital sodium (90mg/kg, i.v.). The hearts were removed and retrogradely perfused on Langendorff system with the following solutions: first, 5 minutes perfused with HEPES-buffered Tyrode\u0026rsquo;s solution. Second, 5 minutes perfused with Ca\u003csup\u003e2+\u003c/sup\u003e-free HEPES-buffered Tyrode\u0026rsquo;s solution. Third, 15 minutes perfused with HEPES-buffered Tyrode\u0026rsquo;s containing 0.6mg/ml collagenase type II (Invitrogen Co.US), 0.1% bovine serum albumin, 20mM taurine and 30\u0026micro;M CaCl\u003csub\u003e2\u003c/sub\u003e. Fourth, 5 minutes perfused with KB solution (mM: taurine 10; glutamic acid 70; KCl 25; KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e10; glucose 22; EGTA 0.5; pH adjusted to 7.2 with KOH). At the end of the perfusion, the ventricular free wall was dissected from the heart. Cell samples were pooled and resuspended in 10ml myocyte stopping buffer (perfusion solution, containing 5% fetal bovine serum and 50uM CaCl\u003csub\u003e2\u003c/sub\u003e) and transferred to a 60mm culture dish. Calcium concentration was increased by addition of 10mM CaCl\u003csub\u003e2\u003c/sub\u003e to achieve final concentrations of 50\u0026micro;M, 100\u0026micro;M, 150\u0026micro;M, 200\u0026micro;M, 500\u0026micro;M and 1mM.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003e2.8 L-Type Ca\u003csup\u003e2+\u003c/sup\u003e current recoding\u003c/h2\u003e\n\u003cp\u003eWhole-cell patch clamp was performed using EPC-9 amplifier (List Instruments, Germany) and data was recorded and analyzed with Pulse-pulsefit software interface (Version 8.31, HEKA Co. Germany). During the experiments, the myocytes were continuously superfused with the extracellular solution (2mL/min) containing (in mM): NaCl 130; KCl 5.4; CaCl\u003csub\u003e2\u003c/sub\u003e 2; MgCl\u003csub\u003e2\u003c/sub\u003e 1; Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 0.3; HEPES 10; glucose 10; (pH adjusted to 7.4 with NaOH). The resistances of the pipettes ranged from 2.5 to 3.5MΩ when filled with pipette solution (mM): CsCl 135, MgC1\u003csub\u003e2\u003c/sub\u003e 1, MgATP 4, TEACl 10, EGTA 0.1 and 10 HEPES (pH 7.2 with CsOH). Ca\u003csup\u003e2+\u003c/sup\u003e current was recorded using 10mV steps depolarizing pulse for 150ms from the holding potential of -40mV and integrating the resulting current trace. Currents were normalized to cell capacitance (pA/pF) to account for differences in cell size.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e\n\u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analysis performed with Student\u0026rsquo;s t test was completed by SPSS 16.0. A value of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant. APD restitution curves were analyzed using Origin 6.0 (Microcal Co. USA) for nonlinear curve fitting.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003e3.1 PMA increased the slope of APD\u003csub\u003e90\u003c/sub\u003e restitution curves\u003c/h2\u003e\n \u003cp\u003eTo avoid the time course of PMA treatment, the recordings were applied after 20\u0026ndash;30 min stabilization. Compared with the control (CTL) situation, PMA treatment markedly steepened the slopes of the APD restitution curves (S\u003csub\u003emax\u003c/sub\u003e \u0026gt; 1) at all sites (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;1 and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) but also increased the spatial dispersions of S\u003csub\u003emax\u003c/sub\u003e (COV-S\u003csub\u003emax\u003c/sub\u003e) throughout the whole heart (PMA: 0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, CTL: 0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and at sites in the left ventricle (PMA: 0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, CTL: 0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and right ventricle (PMA: 0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, CTL: 0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). The increased values of S\u003csub\u003emax\u003c/sub\u003e was more obvious in the left apex and posterior wall, so that the COV-S\u003csub\u003emax\u003c/sub\u003e was larger in the left ventricle than that in the right ventricle (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) during PMA treatment. To identify the PMA-induced alteration of S\u003csub\u003emax\u003c/sub\u003e was related to PKC activation, we observed that the administration of BIM could fully antagonize the effect of PMA and flatten the APD restitution curve at each site. The S\u003csub\u003emax\u003c/sub\u003e of all sites was decreased to the similar level of CTL state by BIM (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and the COV-S\u003csub\u003emax\u003c/sub\u003e had no significant difference between BIM and CTL group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA,Table\u0026nbsp;1 and Table\u0026nbsp;2).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003e3.2 PMA treatment increases susceptibility to APD alternans and VA\u003c/h2\u003e\n \u003cp\u003eIn the CTL group, VA was inducible in 6 of the 15 hearts, and appeared mostly as non-sustained VA (33.3%, 5/15). But treatment with PMA increased the inducibility of the VA by dynamic pacing at most sites in 13 of the 15 hearts (86.7%) and the VA were elicited as the sustained state in six of the hearts (40.0%). Analysis of the threshold of VA inducibility showed that PMA greatly increased induced CL at all sites of the heart (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to the CTL state, and the threshold of VA was higher in left ventricle than right ventricle (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). Although less susceptible to rapid dynamic pacing-induced VA, APD alternans was observed at each site in CTL state during the dynamic pacing protocol, but it usually occurred before VA initiation and transformed into a sustained state after PMA administration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, the paced CL to induce APD alternans was significantly increased by PMA compared to the CTL state at each site of the heart (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), it suggesting that the APD alternans was facilitated by PKC activation. However, perfusion with BIM markedly suppressed or delayed the occurrence of dynamic pacing-induced VA and APD alternans, the inducibility threshold of VA and alternans was similar to that in the CTL state (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC,D). These results indicate that the effects of PMA on APD alternans and VA could be inhibited by BIM administration.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003e3.3 PMA increased the inwardly Ca\u003csup\u003e2+\u003c/sup\u003e current\u003c/h2\u003e\n \u003cp\u003eTo determine whether the PMA induced changes were caused by regulation of Ca\u003csup\u003e2+\u003c/sup\u003e currents, we next used whole patch-clamp recordings to characterize L-type Ca\u003csup\u003e2+\u003c/sup\u003e current in isolated rabbit ventricular myocytes from CTL (n\u0026thinsp;=\u0026thinsp;8) and PMA (n\u0026thinsp;=\u0026thinsp;9) group. Depolarization of cells elicited the L-Ca\u003csup\u003e2+\u003c/sup\u003e current, the peak amplitude was significantly increased by addition of PMA compared with CTL group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As evidence of a transient inward current, characteristic L-Ca\u003csup\u003e2+\u003c/sup\u003e current was reversibly blocked by 550pM verapamil (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003e3.4 Verapamil inhibited the effect of PMA on APD restitution and arrhythmia\u003c/h2\u003e\n \u003cp\u003eTo determine whether the Ca\u003csup\u003e+\u003c/sup\u003e influx might be involved in PMA related effect on APD restitution and arrhythmia, we administrated verapamil to antagonize L-Ca\u003csup\u003e2+\u003c/sup\u003e current. Verapamil could fully inhibited the PMA induced increasing of APD restitution slope at each site of heart (S\u003csub\u003emax\u003c/sub\u003e\u0026lt;1) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the spatial dispersions of S\u003csub\u003emax\u003c/sub\u003e was decreased by addition of Verapamil throughout the whole heart (PMA: 0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, PMA\u0026thinsp;+\u0026thinsp;Vera: 0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and at sites in the left ventricle (PMA: 0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, PMA\u0026thinsp;+\u0026thinsp;Vera: 0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and right ventricle (PMA: 0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, PMA\u0026thinsp;+\u0026thinsp;Vera: 0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Analysis of the threshold of APD alternans showed that the alternans could not be induced by S\u003csub\u003e1\u003c/sub\u003e dynamic pacing, and the VA was no longer inducible during CL from 300ms to 100ms at each site of heart (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Main findings\u003c/h2\u003e \u003cp\u003eThe present study investigated the effect of PKC activation on characteristics of APD restitution at distinct sites of the ventricle. We found that (i) the maximal slope of APD restitution and the susceptible of VA induction were greatly increased by PKC stimulation at each site in the heart; (ii) PKC activation greatly increased the dispersion of APD restitution among each site in the ventricle; and (iii) PKC stimulation induced steepened curve of restitution and arrhythmogenesis were closely related to the increased Ca\u003csup\u003e2+\u003c/sup\u003e influx of ventricular myocytes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.2 PKC and APD restitution\u003c/h2\u003e \u003cp\u003eAs important regulators of intracellular signaling, PKC isoforms play roles in the pathological modifications associated with heart disease\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, the effects of PKC on cardiac electrophysiology are not fully understood. Results of previous studies have suggested that PKC activation modulated intracellular Ca\u003csup\u003e2+\u003c/sup\u003e through increase the inwardly L-type Ca\u003csup\u003e2+\u003c/sup\u003e current. The intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration ([Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e) greatly upgrade may result in Ca\u003csup\u003e2+\u003c/sup\u003e overload and decreased (~\u0026thinsp;65%) the Ca\u003csup\u003e2+\u003c/sup\u003e content of SR, which was abolished by addition of the PKC inhibitor\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Increased [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e in turn inactivated the diastolic currents including K\u003csup\u003e+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e-Ca\u003csup\u003e2+\u003c/sup\u003e exchange current and other Ca\u003csup\u003e2+\u003c/sup\u003e-sensitive currents. During this effect, the balance of APD and previous phase of DI could be shifted, and finally steepened the restitution curve\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Tseng and Goldhabor concluded that [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e dynamics played a key role in APD restitution, greater Ca\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;\u003csub\u003ei\u003c/sub\u003e accumulation occurred at which the APD restitution slope exceeded 1 during dynamic pacing and the slope was flattened by suppressing SR Ca\u003csup\u003e2+\u003c/sup\u003e cycling with thapsigargin and ryanodine\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Thus, the [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e increased is related to the steepness of the APD restitution curve. Consistently, the findings of the present study revealed distinct APD restitution properties during PKC activation and inactivation by perfusion of PMA and BIM, respectively. Moreover, PMA indeed enhanced the L-Ca\u003csup\u003e2+\u003c/sup\u003e current during patch clamp recording and PKC activation induced increasing of restitution slope can be inhibited by inwardly Ca\u003csup\u003e2+\u003c/sup\u003e currents antagonist. These findings indicate that the slope of APD restitution curve could be exactly steepened by PKC stimulation through [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e upgrade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.3 PKC and APD alternans\u003c/h2\u003e \u003cp\u003eMultiple mechanisms are proposed for the phenomenon of APD alternans\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Primary Ca\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;\u003csub\u003ei\u003c/sub\u003e alternans could drive APD to alternans secondarily, because APD is shaped by several key membrane currents that are sensitive to Ca\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;\u003csub\u003ei\u003c/sub\u003e, such as L-type Ca\u003csup\u003e2+\u003c/sup\u003e currents, calcium-dependent potassium currents, and sodium-calcium exchange currents. Many studies focus on the relationship between the APD alternans and Ca\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;\u003csub\u003ei\u003c/sub\u003e alternans in single cell and tissue samples\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The results of Goldhabor et al. strongly support a key role for Ca\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;\u003csub\u003ei\u003c/sub\u003e instability in APD alternans elicited by rapid S\u003csub\u003e1\u003c/sub\u003e pacing. They found that the onset of APD alternans occurred at diastolic intervals, during which the APD restitution slope was significantly\u0026thinsp;\u0026lt;\u0026thinsp;1, and this phenomenon was abolished by suppressing SR Ca\u003csup\u003e2+\u003c/sup\u003e cycling or global Ca\u003csup\u003e2+\u003c/sup\u003e transients\u003csup\u003e16\u003c/sup\u003e. In present work, the threshold of APD alternans was greatly decreased by PKC stimulation at each site in the heart, and Ca\u003csup\u003e2+\u003c/sup\u003e influx antagonist abolished the occurrence of alternans. It suggested the mechanism that PKC increased [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e via L-type Ca\u003csup\u003e2+\u003c/sup\u003e current and thus facilitated the APD alternans.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.4 PKC and ventricular tachyarrhythmias\u003c/h2\u003e \u003cp\u003eAydin et al. first reported that PKC activation facilitates electrically stimulated ventricular fibrillation in the isolated heart\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Consistent with these findings, our data showed that PKC stimulation increased susceptibility to VA at each site in the heart during dynamic rapid pacing. But lack of the research to illustrate the electrophysiological mechanism of PKC related arrhythmias, previously. Many studies suggested that the vulnerability to ventricular fibrillation is closely linked to APD restitution, and steep restitution slopes that greater than 1 could promote the APD alternans and breakup of electrical waves into a fibrillation-like state\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In the present study, PKC activation decreased the threshold of VA and increased the S\u003csub\u003emax\u003c/sub\u003e values of the restitution curves (S\u003csub\u003emax\u003c/sub\u003e \u0026gt; 1) at each site. However, APD restitution from a single site may not represent the APD restitution kinetics of the whole heart, and the higher inducibility of tachyarrhythmias could not be explained by S\u003csub\u003emax\u003c/sub\u003e of the APD restitution curve. A study of our lab had demonstrated that the spatial dispersion of S\u003csub\u003emax\u003c/sub\u003e plays a critical role in the initiation and maintenance of atrial fibrillation. Although the slopes were flattened (S\u003csub\u003emax\u003c/sub\u003e \u0026lt; 1) by Vagal stimulation, the atrial fibrillation was facilitated associated with the increased APD restitution dispersion\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The possible explanation is that the uniform APD restitution promoted greater heterogeneous refractoriness across the cardiac chamber. Presently, our results also revealed the spatial dispersion of S\u003csub\u003emax\u003c/sub\u003e was increased by PKC activation, it underlined the critical role of spatial dispersion of APD restitution kinetics in the initiation and maintenance of VA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Conclusions\u003c/h2\u003e \u003cp\u003eIn present study, we found a correlation between PKC activation and the increased spatial dispersion of APD restitution. These alterations may be closely related to the increased Ca\u003csup\u003e2+\u003c/sup\u003e influx.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003eThis study was in accordance with the Declaration of Helsinki and approved by the ethical committee of our institution. Informed consent was obtained before entering this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003enone declared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributors:\u003c/strong\u003e Feng Zhang、Fuhua Lei、Mu Qin and Wenbo Cheng Involved in experiment design，Feng Zhang and Fuhua Lei performed the experiments，Feng Zhang、Fuhua Lei and Jianing Fan wrote the main manuscript text and prepared figures，All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was supported by Fudan University Youth Research Fund(Grant No: JYQN-JC-202010). The funder has designed this study and decided to publish this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;:\u003c/strong\u003eThe data that support the findings of this study are available on request from the corresponding author, [Wenbo Cheng and Mu Qin]，upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCoats AJS, Shewan LG. Statement on Authorship and Publishing Ethics in the International Journal of Cardiology. Int J Cardiol 2011; 153: 239-40.\u003c/li\u003e\n\u003cli\u003eChurchill E, Budas G, Vallentin A, Koyanagi T, Mochly-Rosen D. PKC isozymes in chronic cardiac disease: possible therapeutic targets? Annu Rev Pharmacol Toxicol 2008;48:569 \u0026ndash;599.\u003c/li\u003e\n\u003cli\u003eSteinberg SF. Structural basis of protein kinase C isoform function. Physiol Rev 2008;88:1341\u0026ndash;1378. \u003c/li\u003e\n\u003cli\u003eHambleton M, Hahn H, Pleger ST, Kuhn MC, Klevitsky R, Carr AN, Kimball TF, Hewett TE, Dorn GW II, Koch WJ, Molkentin JD. Pharmacological- and gene therapy-based inhibition of protein kinase. Calpha/beta enhances cardiac contractility and attenuates heart failure. Circulation 2006;114:574 \u0026ndash;582.\u003c/li\u003e\n\u003cli\u003eAydin O, Becker R, Kraft P, Voss F, Koch M, Kelemen K, Katus HA, Bauer A. Effects of protein kinase C activation on cardiac repolarization and arrhythmogenesis in Langendorff- perfused rabbit hearts. Europace 2006; 9: 1094\u0026ndash;1098.\u003c/li\u003e\n\u003cli\u003eGarfinkel A, Kim YH, Voroshilovsky O, Qu Z, Kil JR, Lee MH, Karagueuzian HS, Weiss JN, Chen PS. Preventing ventricular fibrillation by flattening cardiac restitution. Proc Natl Acad Sci USA 2000;97:6061\u0026ndash;6066.\u003c/li\u003e\n\u003cli\u003eWeiss JN, Chen PS, Qu Z, Karagueuzian HS, Garfinkel A. Ventricular fibrillation: how do we stop the waves from breaking? Circ Res 2000;87:1103\u0026ndash;1107.\u003c/li\u003e\n\u003cli\u003eBanville I, Chattipakorn N, Gray RA. Restitution dynamics during pacing and arrhythmias in isolated pig hearts. J Cardiovasc Electrophysiol 2004;15:455-63.\u003c/li\u003e\n\u003cli\u003eTaggart P, Sutton P, Chalabi Z, Boyett MR, Simon R, Elliott D, Gill JS. Effect of Adrenergic Stimulation on Action Potential Duration Restitution in Humans. Circulation 2003;107:285-289.\u003c/li\u003e\n\u003cli\u003eWakasaki H, Koya D, Schoen FJ, Jirousek MR, Ways DK, Hoit BD, Walsh RA, King GL. Targeted overexpression of protein kinase C beta2 isoform in myocardium causes cardiomyopathy. Proc Natl Acad Sci U S A 1997;94:9320 \u0026ndash;9325\u003c/li\u003e\n\u003cli\u003eBowman JC, Steinberg SF, Jiang T, Geenen DL, Fishman GI, Buttrick PM. Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates. J Clin Invest 1997;100:2189\u0026ndash;2195\u003c/li\u003e\n\u003cli\u003eRogers TB, Gaa ST, Massey C, Dosemeci A. Protein kinase C inhibits Ca\u003csup\u003e2+\u003c/sup\u003e accumulation in cardiac sarcoplasmic reticulum. J Biol Chem 1990; 265: 4302\u0026ndash;4308.\u003c/li\u003e\n\u003cli\u003eNicolas JM, Renard-Rooney DC, Thomas AP. Tonic regulation of excitation-contraction coupling by basal protein kinase C activity in isolated cardiac myocytes. J Mol Cell Cardiol 1998; 30: 2591\u0026ndash;2604.\u003c/li\u003e\n\u003cli\u003eYamamura K, Steenbergen C, Murphy E. Protein kinase C and preconditioning: role of the sarcoplasmic reticulum. Am J Physiol Heart Circ Physiol 2005; 289: H2484\u0026ndash;H2490.\u003c/li\u003e\n\u003cli\u003eTseng GN. Calcium current restitution in mammalian ventricular myocytes is modulated by intracellular calcium. Circ Res 1988;63: 468\u0026ndash;482.\u003c/li\u003e\n\u003cli\u003eGoldhaber JI, Xie LH, Duong T, Motter C, Khuu K, Weiss JN. 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J Electrocardio 2007; 40: 188\u0026ndash;199.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eThe APD\u003csub\u003e90\u003c/sub\u003e and ERP in baseline (BS), PMA and BIM state\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"670\" height=\"300\"\u003e\u003c/p\u003e\n\u003cp\u003eAll abbreviations are identical to Figure 1.\u003c/p\u003e\n\u003cp\u003e*P\u0026lt;0.05 and\u003cstrong\u003e\u0026nbsp;\u003csup\u003e#\u003c/sup\u003e\u003c/strong\u003eP\u0026lt;0.01 for PMA vs BS\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e The maximal slope of APD restitution curves (S\u003csub\u003emax\u003c/sub\u003e) in baseline (BS), PMA and BIM state jmjn7\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"430\" height=\"327\"\u003e\u003c/p\u003e\n\u003cp\u003eAll abbreviations are identical to Figure 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003e#\u003c/sup\u003e\u003c/strong\u003eP\u0026lt;0.01 for PMA vs BS\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Protein kinase C, Restitution, Spatial heterogeneity, Ventricular tachyarrhythmia, Ca2+ current","lastPublishedDoi":"10.21203/rs.3.rs-2679832/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2679832/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo investigate the role of protein kinase C (PKC) in action potential duration (APD) restitution and ventricular tachyarrhythmias (VA).\u003c/p\u003e\u003ch2\u003eMethods and results\u003c/h2\u003e \u003cp\u003eRabbits hearts were isolated and prepared for Langendorff perfusion technique. The S\u003csub\u003e1\u003c/sub\u003e-S\u003csub\u003e2\u003c/sub\u003e method and dynamic S\u003csub\u003e1\u003c/sub\u003e pacing protocol were performed to construct APD restitution and to induce APD alternans or VA, respectively, at 10 sites throughout the ventricular chamber. Administration of PMA (100 nM) (n\u0026thinsp;=\u0026thinsp;15) greatly steepened the restitution curves (S\u003csub\u003emax\u003c/sub\u003e\u0026gt;1) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) at each site compared to the control group (n\u0026thinsp;=\u0026thinsp;15). Furthermore, treatment with PMA also induced larger spatial dispersions of S\u003csub\u003emax\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and decreased the thresholds of the VA and APD alternans (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, perfused with the PKC inhibitor, BIM (500 nM) (n\u0026thinsp;=\u0026thinsp;10), reversibly flattened the APD restitution curves at each site (S\u003csub\u003emax\u003c/sub\u003e\u0026lt; 1), decreased the spatial dispersions of S\u003csub\u003emax\u003c/sub\u003e, and increased the thresholds of APD alternans and VA. According to the results of patch-clamp, peak amplitude of L-type Ca\u003csup\u003e2+\u003c/sup\u003e current was significantly increased by addition of PMA compared with CTL group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Antagonize this current with verapamil (n\u0026thinsp;=\u0026thinsp;10) can fully inhibited the PMA induced increasing of S\u003csub\u003emax\u003c/sub\u003e and inducibility of VA and alternans.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003ePKC activation increased the dispersion of APD restitution and thus led to occurrence of VA, which possibly related to the increased Ca\u003csup\u003e2+\u003c/sup\u003e influx.\u003c/p\u003e","manuscriptTitle":"Activation of PKC affects the ventricular restitution properties and arrhythmogenesis through L-type Ca+ current","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-17 21:25:41","doi":"10.21203/rs.3.rs-2679832/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3eca4a2b-ea36-4384-acc9-7e04d8974c62","owner":[],"postedDate":"March 17th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-18T02:14:43+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-17 21:25:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2679832","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2679832","identity":"rs-2679832","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}