Background In the early stages of the coronavirus disease pandemic, the anti-malarial drug 22 hydroxychloroquine (HCQ) and the antibiotic drug azithromycin (AZM) were widely used as 23 emerging treatments. However, controversial cardiac toxicity results obtained from clinical trials and 24 epidemic studies suggest that the cardiotoxicity of these two drugs should be re-evaluated. In the 25 present study, we aimed to assess the impact of a short course of AZM or HCQ + AZM combination 26 treatment on ECG and cardiac function in healthy guinea pigs. 27 Methods Thirty-two male guinea pigs were randomly divided into four groups: control; AZM; HCQ; 28 and HCQ + AZM groups. At 3, 6, and 9 days after treatment, electrocardiograms (ECGs) and 29 echocardiographic techniques were used to determine important ECG parameters and cardiac 30 functional parameters of the left ventricle (including posterior wall thickness, end systolic/end 31 diastolic volume, ejection fraction, and fractional shortening). 32 Results Although AZM decreased the heart rates of guinea pigs on day 9 (under anesthetized 33 conditions), HCQ + AZM decreased heart rates on days 3, 6, and 9. The corrected QT intervals of 34 guinea pigs after AZM and HCQ + AZM treatments were significantly increased, compared with 35 CON and HCQ treatment respectively, on days 3, 6, and 9. However, QRS complex durations were 36 not significantly different between the groups. AZM significantly decreased left ventricular ejection 37 fraction (LVEF) and left ventricular fraction shortening (LVFS) on days 3, 6, and 9, whereas HCQ + 38 AZM only decreased LVEF and LVFS on day 9. Posterior wall thickness and of the left ventricle in 39 the diastolic and systolic states were not significantly different between these groups. In addition, 40 compared with CON, AZM and HCQ decreased the EDV. And, in comparison with HCQ treatment, 41 HCQ + AZM treatment increased ESV on day 9.
Since the onset of the coronavirus disease pandemic, confirmed cases of have reached more than 44.5 million, and~2.64% of these cases have proved fatal (WHO 48 Coronavirus Disease (COVID-19) Dashboard, accessed Oct 30, 2020)(26). Hydroxychloroquine 49 (HCQ), azithromycin (AZM), or a combination of these two drugs has been widely used for the 50 treatment of COVID-19, despite that some countries have declined treatment with these drugs. HCQ 51 is a quinoline medicine that was first approved by the FDA in 1955 and has since been widely used 52 3 clinical status (4, 16). Besides of this argument regarding the treatment clinical outcomes, these 59 controversial data concern the safety profiles of these drugs. 60 According to statistics provided by @CovidAnalysis(1), patients in many countries still use HCQ to 61 treat COVID-19 in the early stages of treatment. HCQ and AZM are currently in the World Health 62 Organization's list of essential medicines and, even before the pandemic, were among the most 63 commonly prescribed drugs worldwide (6, 7, 27) . So far, clinicians and researchers globally have 64 been desperately sharing their findings and experiences regarding the prevention and treatment of 65 COVID-19. However, rapid publication of these findings has also exposed the public to incompletely 66 analyzed, un-verified data. In the assessment of drug-induced cardiotoxicity risk for novel 67 pharmaceuticals, since 2005 most countries have adopted the standard preclinical evaluation protocol 68 of the International Conference on Harmonisation of Technical Requirements for Registration of 69 Pharmaceuticals for Human Use (ICH S7B) (13). However, the drugs including HCQ and AZM on 70 market earlier than that timepoint might not be assessed adequately for their hidden cardiotoxicity. 71 To further understand the adverse effects of HCQ, AZM, and HCQ + AZM on cardiac conduction 72 and function and to validate safe use of these drugs in non-COVID-19 patients, a re-evaluation of 73 cardiac safety using a preclinical animal model is necessary. 74 Here, we firstly tested delayed ventricular repolarization in guinea pigs after the drug treatment 75 following the guideline of ICH S7B. Next, we also employed echocardiography technology for small 76 animals to evaluate cardiac morphology and function that were potentially affected by drugs. QT interval, were analyzed using the LabChart Pro Software (ADInstruments Inc., USA). 115 Several formulas have been developed for QT interval correction to facilitate a precise interpretation 116 of this interval. In our current experiments, two steps were performed to determine the correction 117 formula most appropriate for calculating the QT interval for the anesthetized guinea pigs. First, the 118 QT and RR intervals from the ECGs were inputted into five correction equations (Bazett's(2) , 119 Fridericia's(11), Van de Water's (25), Kawataki's(15), Matsunaga's(17) to obtain the corrected QT 120 (QTc) interval. Next, the calculated QTc and RR data were subjected to linear regression analysis, 121 and the correction formula with the smallest slope of the regression line was used to evaluate the QTc 122 interval in this study. The effects of drugs on the cardiac function of guinea pigs were evaluated using a high-frequency All ultrasound data were averaged over three consecutive cardiac cycles.
All experimental data are presented as means ± standard error of means (SEM). Statistical differences 148 between the groups were analyzed by two-way ANOVA followed by Tukey's multiple-comparisons 149 test (GraphPad Prism 8, USA). P < 0.05 was considered statistical significance.
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Evaluation of QT correction methods for estimating QT interval prolongation 152 The correlation (R 2 ) values obtained using the QT interval correction formulas reported by Bazett, Table 2 . Although guinea pig heart rates in the HCQ group were unchanged 163 (compared with those in the CON group) on days 3, 6, and 9. Compared with HCQ treatment alone, 164 HCQ + AZM treatment significantly reduced the heart rate on days 3, 6, and 9 ( Figure 4A and 4B). 165 Thus, guinea pig heart rate slowed following the addition of AZM to HCQ treatment. And, 166 bradycardia was still observed in guinea pigs after the discontinuation of AZM treatment for 4 days. 167 Correspondingly, the RR interval was significantly increased only on day 9 in the AZM group 168 compared with that in the CON group. The HCQ + AZM treatment significantly increased the RR 169 interval on days 3, 6, and 9 compared with HCQ treatment, while in comparison with CON, HCQ + 170 AZM treatment was significantly increased RR interval on day 9 only ( Figure 4C and 4D-i; Table 3 ). 171 The PR interval was significantly increased following a 9-day course of AZM, compared with CON.
The PR interval was prolonged on day 6 but not on day 9 following AZM + HCQ treatment, 173 compared with HCQ. These results suggest that conduction of the sinoatrial nodal impulse to the 174 ventricles is affected by AZM and that subsequent withdrawal of AZM halts the observed conduction 175 dysfunction induced by HCQ + AZM treatment on day 6 ( Figure 4C and 4D-ii; Table 3 ). The QRS 176 complex durations were not different among the four treatment groups ( Figure 4D -iii; Following AZM treatment, the QTc interval was significantly increased on days 3, 6, and 9 compared 180 with that following CON treatment. In contrast, HCQ treatment did not affect the QTc interval on 181 days 3, 6, or 9. However, the QTc interval was significantly increased following HCQ + AZM 182 treatment on days 3, 6, and 9 compared with that following HCQ treatment ( Figure 4C and 4D-iv; 183 Table 3 ). These results suggest that AZM prolongs the QT interval and that after 4 days of AZM 184 withdrawal, QT interval prolongation did not disappear. Simultaneously, on day 9, in comparison with CON, the AZM or HCQ treatment alone decreased the 195 EDV, while in comparison with HCQ treatment, HCQ + AZM treatment increased ESV. 196 Next, we compared cardiac function among the groups ( Figure 5 ; Table 4 ). In the AZM group, 197 LVEFs were significantly decreased on days 3, 6, and 9 compared with those in the Con group. 