Central Contributions of Myocardial Injury to Adverse Outcomes in Patients With Coronavirus Disease 2019

Objective: this study aimed to explore the impacts of myocardial injury on the clinical severity and outcomes in patients with Coronavirus Disease 2019 (COVID-19). Methods: we analyzed the electronic medical records of 1646 COVID-19 inpatients in Wuhan Huoshenshan Hospital. Results: 327 (19.9%) developed into severe cases, 23 (1.4%) died. In comparison to common cases, severe cases showed older age, more comorbidities, abnormal immune responses, as well as liver, renal, cardiac and coagulation disorders. Multivariable logistic regression identied that older age , combining with arrhythmia, abnormal lymphocyte percentage, elevated hypersensitive C reactive protein (hs-CRP) and myocardial injury were the independent risk factors for the incidence of severe cases. Moreover, Kaplan-Meier survival analysis showed that patients with myocardial injury had increasing risks of mortality, incidence of severe cases, acute respiratory distress syndrome (ARDS), and intensive care unit (ICU) admission. Particularly, myocardial injury patients co-existed with any other risk factor further deteriorated the clinical outcomes. Conclusion: The presence of myocardial injury and its co-existing with older age, arrhythmia, abnormal lymphocyte percentage, or elevated hs-CRP were greatly associated with the incidence of severe patients and poor clinical outcomes. in the model, variables were chosen for univariable analysis on the basis of previous ndings and clinical constraints. We used the method of forward LR to conduct multivariable analysis (p<0.10 for entry, p<0.050 for stay). Odds ratio (OR) was used for determining relation between potential risk factors and severe cases. p<0.050 was considered statistically signicant. Kaplan-Meier curves and log-rank tests were used to compare survival rate and event-free survival rate. Statistical analyses were performed using the SPSS software 26. abnormalities of hs-CRP and lymphocyte percentage. on the incidence and progression of severe cases.


Introduction
Coronavirus disease 2019 (COVID-19) is a current pandemic infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has raised great concerns all over the world. At present, more than 200 countries and/or districts have con rmed more than two million cases infected with SARS-CoV-2, and the number is still rising. Direct and indirect contacting with mucous membranes in the nose, mouth, or eyes is considered as the predominant transmitted pathways [1]. SARS-CoV-2 infected patients present series of clinical symptoms including fever, cough, myalgia, fatigue, and/or dyspnea, et al [2]. Many patients showed bilateral involvement of chest CT images and laboratory examination showed pronounced lymphopenia [3,4]. Moreover, growing data from COVID-19 patients identify the presence of myocardial injury in critically ill patients and/or deceased patients [2,5], which has been proved to be associated with the fatal outcomes [6]. However, the impacts of myocardial injury on the clinical course and non-fatal outcomes are largely unknown.
During the progression of COVID-19 patients, severe patients are always linked to poor outcomes. Recent reports indicated that the percentage of severe cases in COVID-19 patients ranged from 15 to 30%, and the severe patients were more likely to suffer from multiple organic dysfunctions, receive intensive care unit (ICU) care, ventilator therapy, and have a higher mortality rate [7][8][9]. Moreover, the severe patients posed great strain on medical resources, especially if they are not adequately staffed or resourced. Until now, evidence-based treatments are absent and supportive care is the mainstay of clinical management. Early recognition of severe patients may be bene cial to reasonable triage to the critical care managements and improve our understanding of the clinical course of COVID-19. Considering that severe cases usually share similar clinical features with critically ill patients, including myocardial injury [10], we hypothesize that myocardial injury may contribute to the incidence of severe COVID-19 cases and its clinical outcomes.
Accordingly, we conducted this large-scale retrospective cohort study to determine the risk factors for the incidence of severe COVID-19 cases and the impacts of myocardial injury on the clinical course and outcomes.
The clinical severity was de ned according to the guidelines for diagnosis and management of COVID-19 (5 th version, in Chinese) released by National Health Commission of China [12]. Severe cases was de ned when meeting any of the follows: respiratory distress, respiratory rate ≥30 times/min; pulse oxygen saturation ≤93% at rest; arterial oxygen partial pressure/fraction of inspired oxygen ≤300 mmHg; more than 50% pulmonary lesion progression in chest CT images within 24 to 48 hours.

De nitions
The abnormalities in laboratory ndings were based on the criteria in Huoshenshan hospital. Acute respiratory distress syndrome (ARDS) was de ned according to the Berlin De nition [13]. Myocardial injury was de ned if the circulating level of cardiac biomarkers (eg, hs-cTnI) were above the 99 th percentile upper reference limit [14]. Acute kidney injury was identi ed on the basis of serum creatinine over base 1.5 times [15]. Liver injury was diagnosed if the alanine aminotransferase (ALT) was above the upper limit of the reference range [16]. Coagulopathy was de ned as a 3-second and 5-second extension of prothrombin time (PT) and activated partial thromboplastin time, respectively.

Clinical outcomes
The primary endpoints were mortality, the secondary endpoints included the incidence of severe cases, ARDS, receiving mechanical ventilation, ICU admission, as well as clinical cure and discharges. The criteria of discharges included absence of fever for ≥3 days; obvious pulmonary in ammatory resorption in chest CT; clinical remission of respiratory symptoms; and two times of negative for SARS-CoV-2 RNA at least 24 h apart [6]. The clinical outcomes (mortality for 40 days and others for 60 days) were monitored after the initial symptom onset.

Statistical analysis
Continuous variables were presented as median (interquartile range [IQR]) and categorical variables were expressed as n (%). Mann-Whitney U test, Pearson χ² test or Fisher's exact test were employed to compare the differences between two groups as appropriate. To identify the risk factors for severe cases, univariable and multivariable binary logistic regression models were used. Considering to avoid over tting in the model, variables were chosen for univariable analysis on the basis of previous ndings and clinical constraints. We used the method of forward LR to conduct multivariable analysis (p<0.10 for entry, p<0.050 for stay). Odds ratio (OR) was used for determining relation between potential risk factors and severe cases. p<0.050 was considered statistically signi cant. Kaplan-Meier curves and log-rank tests were used to compare survival rate and event-free survival rate. Statistical analyses were performed using the SPSS software 26.

Results
Basic characteristics, clinical features, laboratory ndings and outcomes in total patients In this study, from February 4 th to March 9 th 2020, we enrolled 1646 COVID-19 con rmed patients, who hospitalized in Wuhan Huoshenshan Hospital.   3] g/L, p<0.0001) were elevated in severe patients. More severe patients suffered from liver injury, kidney injury, myocardial injury, and coagulation disorder. (Table 1) Furthermore, all clinical adverse events were derived from severe cases and the length of hospitalization was longer in severe patients (16. Table 2) Risk factors for the incidence of severe patients In univariable regression, age, hypertension, diabetes, arrhythmia, malignant neoplasm, white blood cell count, lymphocyte percentage, hs-CRP, liver injury, acute kidney injury, cardiac injury as well as coagulation disorder were associated with the incidence of severe cases. (Table 3 .0001) were independently associated with the incidence of severe cases. (Table 3) Clinical outcomes in patients with or without myocardial injury After follow-up, the mortality rate was higher in patients with myocardial injury (16.2%) than those without myocardial injury (0.3%) (log-rank p value <0.0001, Figure 1A). What's more, the event-free survival rates (including the incidences of severe cases, ARDS and ICU admission) were similarly lower in patients with myocardial injury than those without myocardial injury (log-rank p value <0.0001, Figure 1B-D). Furthermore, patients with myocardial injury showed signi cantly higher mortality rate, percentage of ARDS and ICU admission than those without myocardial injury irrespective of age (log-rank p value <0.0001, Figure 2A, C, D), whereas the incidence of severe cases seemed to be higher in patients with myocardial injury and age≥75 years old than those with myocardial injury and age <75 years old ( Figure 2B). Moreover, the event-free survival rates (including mortality, the incidences of severe cases, ARDS and ICU admission) were even lower in patients with myocardial injury and arrhythmia than those with myocardial injury but without arrhythmia (log-rank p value <0.0001, Figure 3A

Discussion
Due to limited evidences, government guidance and clinical managements made the decisions based on the knowledge of risk factors for other similar infectious diseases such as in uenza, Severe Acute Respiratory Syndromes (SARS), Middle East Respiratory Syndrome (MERS) to protect the millions of people at higher risk just according to age or serious comorbidities [17]. In this retrospective study, we found that severe COVID-19 cases were linked to poor clinical outcomes, such as respiratory failure, ARDS, ICU admission or even death. Several risk factors were identi ed to be associated with the incidence and progression of severe cases. Particularly, age, arrhythmia, myocardial injury, abnormalities of hs-CRP, and lymphocyte percentage were the independent risk factors for the incidence of severe cases. Of note, the presence of myocardial injury was associated with poor clinical outcomes including the progression of severe cases, which was more pronounced when co-existing with other factors. (Take-home gure) Among COVID-19 patients, a minority of them experienced an abnormal clinical course which might lead to fatal outcomes. In this study, it was not surprising that all the deaths were derived from severe cases, the mortality was about 7%, which was much higher than the reported overall mortality in COVID-19 patients, although the incidence of severe cases was similar with the results from meta-analysis [7]. Not only that, the length of hospital stay was longer in severe cases than those in common cases. These ndings suggested severe cases might be associated with higher mortality and occupancy rate of medical sources. Consistent with previously described [10], more severe patients were elders; more patients had preexisting diseases, such as hypertention, diabetes, arrhythmia and so on; levels of in ammation was higher; multiple organs injury were more common, such as myocardial injury, kidney injury, immune injury, liver injury, coagulation disorders and so on. Consistently, previous study had reported the similar clinical characteristics in the deaths caused by COVID-19 [18], suggesting both severe cases and deceased cases might share a common pathophysiological basis. Learning from severe cases might favour the global success in ghting against COVID-19.
In this study, among the numerous factors, age, arrhythmia, myocardial injury, abnormalities of hs-CRP, and lymphocyte percentage were the independent risk factors for the incidence of severe cases. Consistently, it had been also reported that age ≥65 years, preexisting cardiovascular or cerebrovascular diseases, lymphopenia, and elevation of hs-cTnI were associated with poor outcomes of COVID-19 pneumonia patients [19][20][21]. Moreover, patients with distinct inherited arrhythmia syndromes showed increased risk of COVID- 19 [22]. According to previous studies, older age has been proved to be associated with mortality in SARS, MERS as well as COVID- 19 [23], which might be attributed to the age-dependent defects in immunity and the excess production of in ammatory cytokines in elders. These defects might result in a failure in inhibition of viral replication and aggravated pro-in ammatory responses, which may lead to poor clinical outcomes [24]. Moreover, we also found that arrhythmia was independently associated with the severe cases. According to the "Diagnosis and Treatment of Novel Coronavirus Pneumonia (Trial Version 7)", elderly patients with preexisting diseases were more likely to be infected with SARS-CoV-2 and prone to be severe patients, especially those with hypertension, coronary heart disease, and diabetes. In our study, severe cases had higher levels of hs-CRP, which was also an independent risk factor. Similarly, previous evidence had showed that ICU patients with COVID-19 had higher plasma levels of in ammatory cytokines, including interleukin (IL)-2, IL-7, IL-10, tumor necrosis factor α and so on [2], suggesting that in ammatory cytokine storm syndrome due to uncontrolled and dysfunctional immune responses might contribute to the incidence and progression of severe cases.
In our study, we found that patients with myocardial injury had an increasing risk of mortality, higher incidence of severe cases, ARDS, and ICU admission. Consistently, previous studies showed that myocardial injury was associated with the fatal outcomes of COVID-19 patients [6]. Indeed, except myocardial injury, SARS-CoV-2 infection could lead to multiple organs injury, including lung, liver, kidney, as well as coagulation system [8]. However, only myocardial injury was independently associated with the incidence of severe cases. Moreover, myocardial injury was linked with the fast progression and poor clinical outcomes of severe cases, especially co-existing with old age, arrhythmia, or abnormalities of hs-CRP and lymphocyte percentage. These results suggested a critical impact of myocardial injury on the incidence and progression of severe cases.
Until now, the mechanisms of myocardial injury in COVID-19 patients were unclear and might be complex. However, it had been reported that patients with preexisting cardiovascular diseases were susceptible to suffer from myocardial injury during the clinical course of COVID-19 [14]. For these high risk patients, SARS-CoV-2 infection might further aggravate myocardial injury by the direct role of the virus, pro-in ammatory responses, hypoxia and so on. Evidence had showed that the SARS-CoV genome was positively detected in the heart, suggesting a direct viral myocardial infection [25]. Moreover, recent studies had shown that angiotensin-converting enzyme 2 (ACE2), which was highly expressed in heart, had a strong a nity to bind the Spike protein of SARS-CoV-2 [26]. Thus, we hypothesized that the myocardial injury in COVID-19 patients might be mediated by ACE2. Besides, recent evidence showed that plasma levels of troponin T were linearly correlated with the levels of hs-CRP, the activation or enhanced release of the in ammatory cytokines might lead to apoptosis or necrosis of myocardial cells, which indicated the contributions of pro-in ammatory responses [14]. Nevertheless, due to the limited evidence, this topic required further investigations.
Current knowledge of risk factors for severe COVID-19 mainly include age and comorbidities, which were unmodi able. Thus, for elders or patients with some comorbidities, especially cardiovascular diseases, prevention seemed to be extremely important, avoiding infection should be specially recommended [27]. Based on the present ndings, cardiac and immune injury might be the critical aspects for monitoring and interventions. Rather than encouraging avoidance of troponin testing, we recommended that hs-cTnI should be routinely detected and closely monitored in COVID-19 patients [28]. Under the basic clinical managements, protective strategy for heart and immune system should be emphasized to control the clinical severity and improve the clinical outcomes. For elders or patients with cardiovascular diseases, if COVID-19 was con rmed, cardioprotection should be taken immediately.
Our present study is a large-scale retrospective study to explore the risk factors involved in the incidence and prognosis of severe COVID-19 patients. The data were uniformly collected and the diagnosis and treatments were homogeneously performed. However, some limitations exist in our study. Firstly, because of the outbreak of COVID-19 in Wuhan, some data such as echocardiography data, electrocardiography data as well as arterial blood gas analysis were not collected in all patients due to the insu ciency of medical resources. Secondly, this is a single-centered study, further studies from multiple centers are needed.

Conclusions
For the rst time, we identi ed that age, arrhythmia, myocardial injury, abnormalities of hs-CRP, and lymphocyte percentage were the independent risk factors for the incidence of severe COVID-19 cases. The presence of myocardial injury and its co-existing with other factors were greatly associated with poor clinical outcomes. Since no speci c treatments could be applied to COVID-19, our ndings may be bene cial to clinical decision-making and managements in order to improve clinical outcomes.

Declarations
The data used to support the ndings of this study are available from the corresponding author upon request.

Ethics approval and consent to participate
This study was approved by the Human Ethics Committee, Huoshenshan Hospital (NO. HSSLL023) and conformed to the Declaration of Helsinki, oral consent was obtained from each patient when enrollment due to the rapid emergence of COVID-19.

Consent for publication
Not applicable.    Data were expressed as n (%) and median (IQR). ARDS=acute respiratory distress syndrome. ICU=intensive care unit. IQR=interquartile range. Time=time from illness onset to clinical outcomes.   Event-free survival in COVID-19 patients according to myocardial injury and age. Kaplan-Meier plot of survival rate (A), event-free (the incidence of severe cases) survival (B), event-free (the incidence of ARDS) survival (C), and event-free (ICU admission) survival (D) among patients in age ≥75 years old or age <75 years old with or without myocardial injury.

Figure 3
Event-free survival in COVID-19 patients according to myocardial injury and arrhythmia. Kaplan-Meier plot of survival rate (A), event-free (the incidence of severe cases) survival (B), event-free (the incidence of ARDS) survival (C), and event-free (ICU admission) survival (D) among patients with or without myocardial injury co-existed with or without arrhythmia.

Figure 4
Event-free survival in COVID-19 patients according to myocardial injury and hs-CRP. Kaplan-Meier plot of survival rate (A), event-free (the incidence of severe cases) survival (B), event-free (the incidence of ARDS) survival (C), and event-free (ICU admission) survival (D) among patients with or without myocardial injury co-existed with hs-CRPa or hs-CRPn. hs-CRPa=abnormal hypersensitive C reactive protein (>4 mg/L). hs-CRPn=normal hypersensitive C reactive protein (≤4 mg/L).

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