Cardiac biomarkers, cardiac injury, and comorbidities associated with severe illness and mortality in coronavirus disease 2019 (COVID‐19): A systematic review and meta‐analysis

Abstract Aims To explore the correlation between cardiac‐related comorbidities, cardiac biomarkers, acute myocardial injury, and severity level, outcomes in COVID‐19 patients. Method Pubmed, Web of Science, Embase, CNKI, VIP, Wanfang, Cochrane Library databases, medRxiv, and Sinomed were reviewed systemically. Various types of clinical research reporting cardiac‐related comorbidities, cardiac biomarkers including lactate dehydrogenase (LDH), troponin I (TnI), high sensitivity troponin I (hs‐TnI), creatine kinase (CK), creatine kinase–MB (CK‐MB), myoglobin (Myo), N‐terminal pro‐b‐type natriuretic peptide (NT‐proBNP) and acute cardiac injury grouped by severity of COVID‐19 were included. Outcome measures were events and total sample size for comorbidities, acute cardiac injury, and laboratory parameters of these biomarkers. The study was performed with Stata version 15.1. Results Seventy studies, with a total of 15,354 cases were identified. The results showed that COVID‐19's severity was related to cardiovascular disease. Similar odds ratios (ORs) were achieved in hypertension except for severe versus critical group (OR = 1.406; 95% CI, 0.942–2.097; p = .095). The relative risk (RR) of acute cardiac injury is 7.01 (95% CI, 5.64–8.71) in non‐survivor cases. When compared with the different severity of cardiac biomarkers, the pool OR of CK, CK‐MB, TnI, Myo and LDH were 2.683 (95% CI, 0.83–8.671; p = .106; I 2 = 0%), 2.263 (95% CI, 0.939–5.457; p = .069), 1.242 (95% CI, 0.628–2.457; p = .534), 1.756 (95% CI, 0.608–5.071; p = .298; I 2 = 42.3%), 1.387 (95% CI, 0.707–2.721; p = .341; I 2 = 0%) in the critical versus severe group, whose trends were not similar to other groups. The standard mean differences (SMD) of CK and TnI in the critical versus severe group were 0.09 (95% CI, −0.33 to 0.50; p = .685; I 2 = 65.2%), 0.478 (95% CI, −0.183 to 1.138; p = .156; I 2 = 76.7%), which means no difference was observed in the serum level of these indicators. Conclusion Most of the findings clearly indicate that hypertension, cardiovascular disease, acute cardiac injury, and related laboratory indicators are associated with the severity of COVID‐19. What is now needed are cross‐national prospectively designed observational or clinical trials that will help improve the certainty of the available evidence and treatment decisions for patients.


| INTRODUCTION
Coronavirus disease 2019 (COVID-19), a severe respiratory disease, has caused an unprecedented pandemic crisis. As of August 1, 2020, a total of 17,396,943 cases of COVID-19 infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 675,060 deaths have been confirmed by the WHO, and nearly 300 thousand new cases were reported in every 24 h all around the world. SARS-CoV-2 is approximately 80% gene sequence similarity to SARS-CoV, 1-3 which infected host human cells by binding to the receptor proteins, known as angiotensin-converting enzyme 2 (ACE2) receptor. And the disease transmission eventually contributed to a pneumonia epidemic outbreak in 2003. 4,5 ACE2 receptor is highly expressed in multiple organ systems 6 and plays an essential negative role in the ACEangiotensin II (Ang II)-angiotensin II receptor type 1 (AT1R) pathway called the classical renin-angiotensinaldosterone system (RAAS) axis, whose positive effect can increase sympathetic nervous system tension. 7 According to the current observation, it is the cardiovascular complications and myocardial injury that should be paid close attention to, while most of the attention has been paid to the pulmonary system. The relationship between coronavirus disease 2019 and clinical characters has been much more precise. Firstly cardiovascular diseases, including coronary heart disease and hypertension, can increase the risks for adverse outcomes such as the rate of critical situations and death. 8 Secondly, a notable proportion of patients experience cardiovascular symptoms, and myocardial injury indicators change at the initial presentation. Several recent studies have described the clinical characteristics and epidemiological findings of COVID-19 that were tightly implicated in the cardiovascular system. [9][10][11] Nevertheless, due to the mounting clinical research, further and broader elucidation can be carried out. Here we summarize the current literature to explore the correlation between cardiac-related comorbidities, cardiac biomarkers, acute myocardial injury, and severity level or outcomes in COVID-19 patients.

| Search strategy
Our review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations and criteria. 12 We conducted a literature retrieval by two independent reviewers without language restrictions (Cai Q and Zhang L) in Pubmed, Web of Science, Embase, CNKI, Wanfang, Cochrane Library databases, VIP, medRxiv, and Sinomed using "SARS-CoV•2," "COVID-19," and "2019•nCoV" from December 1, 2019, to June 27, 2020. All relevant research should report the outcome including cardiac-related comorbidities and cardiac biomarkers, including lactate dehydrogenase (LDH), troponin I (TnI), high sensitivity troponin I (hs-TnI), creatine kinase (CK), creatine kinase-MB (CK-MB), myoglobin (Myo), N-terminal prob-type natriuretic peptide (NT-proBNP) or acute cardiac injury. Additionally, extra searches were performed in the title or abstract that reported "clinical characteristics" or "clinical data." Studies that report cardiac biomarkers or acute cardiac injury but did not classify by patients' severity level or outcomes of COVID-19 were excluded.

| Selection criteria
Two independent reviewers (Zhengchuan Zhu and Miaoran Wang) scrutinized all titles and abstracts excluded irrelevant studies. Then they independently reviewed the full reports. Studies were eligible for inclusion if they met the following conditions: (1) study types: retrospective, prospective, cross-sectional, observational, descriptive or case-control studies which report cardiac-related comorbidities, cardiac biomarkers (including CK, CK-MB, TnI, Myo or NT-proBNP, LDH) or patient suffered from acute cardiac injury with COVID-19; (2) patient characteristics: patients should be diagnosed with COVID-19 and grouped into moderate cases, severe cases or critical cases according to Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (trial version 7) from China or WHO interim guidance 13 ; (3) interventions: the study should include at least one cohort data for severe versus non-severe, severe versus moderate, severe versus non-severe, severe versus critical, or non-survivor versus survivor cohorts; and (4) outcome: the mean (standard deviation SD) or median (interquartile range; IQR) for each laboratory parameters of these biomarkers should be involved as continuous outcomes. And the event and total sample size for comorbidities and acute cardiac injury should be involved as dichotomous outcomes.
The exclusion criteria were: (1) study types: case reports, reviews, editorial materials, conference abstracts, and summaries of discussions, (2) patient characteristics: patients in a specific condition such as those who were pregnant (3) recruiters were infected by other coronaviruses, including the severe acute respiratory syndrome (SARS) or the Middle East respiratory syndrome (MERS) human coronavirus or (4) study with insufficient data information in the event.

| Statistical analysis
All patients were divided into four cohorts before conducting the study: severe versus moderate, severe versus critical, severe versus non-severe, and survivor versus non-survivor based on the diagnosis of COVID-19 in every study. To reduce the influence from different detection methods, dichotomous outcomes (e.g., cardiacrelated comorbidities) were extracted as odds ratio (OR) with 95% confidence interval (CI), continuous outcomes were extracted as standard mean difference (SMD) with 95% CI. Summary relative risks (RRs) with 95% CI were carried out for the association between acute cardiac injury and the severity of COVID-19. Heterogeneity among the included studies was assessed using the Cochran's Q test (p < .05) and the I 2 statistic(considered I 2 values <25% to represent low heterogeneity, 25%-50% to moderate heterogeneity, and >50% to severe heterogeneity). The fixed effects models were applied when I 2 ≤ 50% or p ≥ .05, while the random effect models were used by the Mantel-Haenszel method. To probe the sources of heterogeneity, we employed a leave-one-out analysis when I 2 > 50%. We generated funnel plots and Egger's test to examine the possibility of publication bias. Egger's test with p > .05 was considered as funnel symmetry and no publication bias. The statistical analysis was performed with Stata version 15.1 (Stata Corp LP) and GraphPad Prism 7.0 (GraphPad Software).

| Included studies
Detailed steps of the literature search process is shown in Figure 1. In total, 5259 potentially relevant publications were found from different databases in the initial search. After removing duplications, 3063 were carefully screened, and 2407 unrelated research were removed. And 389 of them were further excluded after screening titles and abstracts. Relevant articles were further selected by reading the full texts. Among these studies, 102 did not report the cardia injury biomarkers, 61 were not classified by the severity of COVID-19 cases, 14 did not include clear diagnostic criteria, 14 investigated the pregnant and infant cases, 3 were not considered suitable because of their study design, and one suspected considerable overlapping of patients populations in the same hospital. Finally, 67 articles were included.

| Baseline characteristics and risk of bias
In total, 67 trails are summarized in the meta-analysis, and their characteristics are presented in Table 1. A total of 14901 hospitalized patients from four countries were studied in 2020 and the average sample size was 222, which varied from 21 to 1449. The overall average age was greater than 40 years ranged from 1 year to 96 years old and slightly more than half of the sample population was male (51.4%) between 35.48% 22 to 80.9%. 66 The most general cardiac-related comorbidities were coronary heart disease and hypertension. The quality assessment for the included studies is shown in Table 2, and all included studies were evaluated to be high quality with a NOS score ≥6. The P-value of Egger's test in funnel plots was shown in Table 3.

| Cardiac-related comorbidities
The data on cardiac-related comorbidities among patients were extracted from 4 groups, then pooled respectively for meta-analysis. Figure 2 displays Figure 3, cardiovascular disease was significantly associated with the severity level of

TnI
The combined OR of TnI were following in Figure 8 Figure 5. The results of SMD showed that: 0.839 (95% CI, 0.308-1.371; p = .002; I 2 = 70.5%) in severe versus moderate group, 0.96 (95% CI, 0.784-1.13; p = 0; I 2 = 0.6%) in severe versus non-severe group, and 1.13 (95% CI, 0.928-1.327; p = 0; I 2 = 76.9%) in survivor versus non-survivor group after the sensitivity analysis by removing "Ma K 2020," "Liu C 2020," "Li Q 2020," receptively, which means that higher serum levels of TnI were observed in more severe cases. According to the funnel plot in Figures 6 and 9 the results of Egger's test, there were no publish biases were observed. 4. Myo Figure 10 illustrates  Figures 6 and 9, respectively. Based on the results of Egger's test, we found no evidence of publication bias for Myo in all groups.

| Acute cardiac injury
The data on acute cardiac injury among patients were extracted from 2 groups: the severe versus moderate group and the survivor versus non-survivor group, then pooled respectively for meta-analysis. The results are set out in Figure 12 that there were marked differences for acute cardiac injury between survivor versus nonsurvivor cases (RR = 7.01; 95% CI, 5.64-8.71; p = 0; I 2 = 68.3%), while no differences between severe versus moderate cases (RR = 1.017; 95% CI, 1.017-0.313; p = .978; I 2 = 34%). Sensitivity analysis by removing "Chen T 2020" showed that overall estimates did not depend on a single publication in survivor versus | 1093 non-survivor group (RR = 9.005; 95% CI, 6.974-11.626; p = 0; I 2 = 43.6%). Based on the results of Egger's test, we found no evidence of publication bias for acute cardiac injury in all groups.

| DISCUSSION
SARS-CoV-2, the third newly severe epidemic coronaviruses that have led to significant outbreaks and caused a big challenge after the SARS-CoV occurred in 2002 and the MERS-CoV that was identified in 2012. 3 No pre-existing immunity and definitive treatments can protect people, especially older adults and vulnerable members of the community, with prevalent comorbidities. [80][81][82] Cardiac-related comorbidities and myocardial damage have been identified in a non-negligible number of COVID-19 patients, and the interpretation of these features based on the COVID-19's severity is essential for further interventions and therapeutics. This comprehensive meta-analysis and systematic review mainly summarize the cardiac-related complications, outcomes, and laboratory findings of 15,354 COVID-19 cases from 70 studies. All subjects were classified into the severe versus moderate group, severe versus critical group, severe versus non-severe group, and survivor versus non-survivor group according to the selected articles, respectively. We found that a higher severity of COVID-19 was associated with higher rates of hypertension, cardiovascular diseases, and acute cardiac injury in the four groups. Similarly, this clean pattern was also found in the number of COVID-19 patients with increased serum levels of CK, CK-MB, Myo, LDH, TnI, and NT-proBNP, whereas no difference was witnessed in the severe versus critical group. Another important finding was compared with milder infection patients, those with a severe infection showed a significant increase in CK, CK-MB, Myo, LDH, and TnI.
Various mechanisms can be suggested to explain our results in the meta-analysis that patients suffering from | 1095 hypertension or underlying cardiovascular diseases have a high risk of developing severe manifestations of COVID-19. The mismatch of supply and demand of myocardial oxygen is a common phenomenon of severe viral diseases accompanied by insufficient systemic oxygenation during pneumonia in elderly patients with cardiovascular diseases and other chronic diseases. 82 Another possible explanation for this might be that hypertension, cardiovascular diseases, and their treatments upregulate ACE2, especially with the use of RAAS inhibitors. 83 As a membrane-bound aminopeptidate receptor that expresses on epithelial cells, ACE2 converts the vasoconstrictor AngII which is hydrolyzed by ACE1 into the vasodilator angiotensin 1-7 (Ang1-7). 84 ACE2 is higher expressed in the heart after receiving ARB or ACEI, 85 interacts with SARS-CoV-2 spike protein, which leads to endothelial dysfunction and myocardial damage directly. 86 Meanwhile, SARS-CoV-2 binding to ACE2 led to a reduction of the external ACE2 catalytic effect and replaced by internalization. 87 Therefore, the possible downregulation of ACE2 and the subsequent increase of the pro-inflammatory AngII together with the decrease of the cardioprotective Ang1-7 in patients with COVID-19 may ultimately compromise heart function. 88 The elevated serum levels of biomarkers of myocardial injury including CK, CK-MB, LDH, TnI, and NT-proBNP were strongly associated with severe forms of COVID-19 from our research. Myocardial injury from SARS-CoV-2 infection involves many factors, and the mechanisms have not been fully elucidated yet. As mentioned previously, as a consequence of ACE2 expression in cardiac tissue, SARS-CoV-2 can directly F I G U R E 12 Forest plot comparisons of increasing lactate dehydrogenase (LDH) in patients with different severity of COVID-19 cause heart damage. There have been studies showing that the heart is susceptible to SARS-Cov-2 infections. 89 A further mechanism for explanations involves a cytokine storm, which is described as an excessive immune response towards SARS-CoV-2 infection. 90,91 Cytokines are essential to infection control, but when the immune system is deregulated including the imbalanced response of type 1 and type 2 T helper cells, a cytokine storm will be observed, resulting in an excessively elevated level of cytokines, such as C-reactive protein interleukin 8 (IL-8), IL-10, procalcitonin, IL-1b, and tumor necrosis factor-α particularly IL-6 coupled with tissue damage and multiple organ dysfunctions. 92 There are several limitations that should be mentioned in our study. First, significant publication bias was observed in several results. A possible explanation is that some of the studies were published at the preprint server, which means some of the included studies are non-peerreviewed scientific manuscripts. Secondly, as a novel infection disease, most of the including studies were retrospective case series, and no randomized controlled trial was included. The lack of RCT or prospective studies causes the consequence that it is hard to adjust for potentially confounding factors, and that might be a source of high heterogeneity. Third, different clinical laboratories used different ranges of normal values for laboratory indicators; therefore, we used SMD to value the relationship between increased serum level and severity of COVID-19, which can achieve a definite ratio instead of numerical laboratory results. Finally, another limitation is that the majority of included studies were from China, only five of them are from other countries experiencing the COVID-19 outbreak, which might lead to bias from races and geographic scope.

| CONCLUSION
This meta-analysis comprehensively investigates the differences in cardiac-related comorbidities, outcomes, and laboratory indicators between patients with COVID-19 who have different disease severity. The findings clearly indicate that hypertension, cardiovascular disease, acute cardiac injury, and related laboratory indicators are associated with the severity of COVID-19. What is now needed are cross-national prospectively designed observational or clinical trials that will help improve the certainty of the available evidence and treatment decisions for patients.