Baseline Clinical Characteristics
Of the initial 3,046 patients with COVID-19 enrolled in our study, 48 patients with no record of survival status, 29 patients without classification of disease severity, and 15 patients with suspected CAD but with no diagnosis were excluded. Among the final cohort of 2,954 patients, the median age was 60 years (range, 50-68 years), 1,461 (49.5%) were female, and 1,515 (51.3%) were severe/critical cases. The median hospital stay for severe/critical (S) patients was significantly longer than that for mild/moderate (M) cases. Compared with mild/moderate cases, severe/critical patients were more likely to experience chest congestion. Comorbidities were more prevalent among severe/critical patients compared to mild/moderate cases, including hypertension, diabetes, cardiovascular disease, cerebrovascular disease, cancer, and chronic obstructive pulmonary disease (Table 1).
In terms of radiological and laboratory findings, severe/critical (S) patients had more incidences of fuzzy boundaries and consolidation (Table 1) and significantly higher levels of C-reactive protein (CRP), D-dimer, interleukin-6 (IL-6), procalcitonin (PCT), and higher percentages of neutrophils (NEUT%), lymphocytes (LYM%), and monocytes (MONO%) within the first week of admission (Figure 1). Serum cardiac markers, namely, BNP, hs-TNI, α-HBDH, CK-MB, and LDH, were also drastically elevated in severe/critical patients during the first week (Figure 1). In general, the results showed more pronounced activation of pathophysiological pathways in more severe cases of COVID-19.
Cardiac Markers and Clinical Outcomes
To evaluate the relationship between the degree of cardiac abnormality and disease outcome in patients with COVID-19, serum cardiac markers were measured. Due to the 0% mortality and favorable prognosis of mild/moderate patients, we focused on the 1,515 patients with severe/critical COVID-19 in the follow-up period.
Of the 1,515 severe/critical cases, BNP, hs-TNI, α-HBDH, CK-MB, and LDH were detected in 835 patients, 660 patients, 1443 patients, 1442 patients, and 1443 patients, respectively. In total, 171 (20.5%), 79 (12.0%), 529 (36.7%), 124 (8.6%), and 447 (3.1%) patients showed abnormal serum levels of BNP, hs-TNI, α-HBDH, CK-MB, and LDH, respectively (Figure 2a). Patients with an elevated level of a cardiac marker showed a significantly higher mortality than those with normal serum levels (Figure 2b). The same trend was observed in the ICU admission rate (Figure 2c). Figure 2d shows that the serum levels of BNP, hs-TNI, α-HBDH, CK-MB, and LDH were significantly higher during hospitalization in non-survivors than in survivors.
The scRNA-seq data of normal human heart tissue were analyzed and five cell types, namely, cardiomyocyte (CM), endothelial (EC), fibroblast (FB), macrophage (MP), and smooth muscle (SMC) cells, were identified (Figure 3a). SARS-CoV-2 receptors ACE2, ANPEP, DPP4, and ENPEP were enriched in specific cell populations. ACE2 was mainly expressed in CM, EC, and FB cell types. ANPEP was enriched in CM, DPP4 was mainly expressed by CM and EC, and ENPEP was primarily expressed in CM and SMC (Figure 3b and Figure3c).
Heart Damage and Mortality Rate
The 1,515 severe/critical patients were further categorized into groups according to the presence or absence of pre-existing CAD (n=165) or absence (n=1,350). Compared with patients without CAD, patients with pre-existing CAD had a higher percent of elevated BNP (52 [46.4%] vs 119 [16.5%]), hs-TNI (24 [26.7%] vs 55 [9.7%], α- HBDH (86 [55.6%] vs 443 [34.4%]), CK-MB (27 [17.4%] vs 97 [7.5%]), and LDH (65 [41.9%] vs 382 [29.7%]) (P < 0.01 for all results, (Figure 4a). To explore the underlying pathophysiological mechanism for the elevated levels of cardiac markers in COVID-19 patients with pre-existing CAD, RNA-seq data from 93 patients with CAD and 48 healthy people were analyzed and compared. The results showed that compared with healthy controls, SARS-CoV-2 receptors, including Transmembrane Serine Protease 2 (TMPRSS2, P = 0.009) and Glutamyl Aminopeptidase (ENPEP, P = 0.012), were significantly upregulated in CAD (Figure 4b).
Compared to patients with normal levels of cardiac markers, those with abnormal levels of BNP, hs-TNI, α-HBDH, CK-MB, and LDH exhibited significantly higher mortality in both CAD and non-CAD groups (P < 0.001 for all results; Figure 4c). The same trend was observed for the ICU admission rate (Figure S1). The serum markers were then compared between non-survivors and survivors. The results showed that BNP, α-HBDH, CK-MB, and LDH were significantly higher in non-survivors than in survivors for patients with pre-existing CAD; however, the significant difference was not observed for hs-TNI (Figure 4d). In patients without pre-existing CAD, all markers were significantly higher in non-survivors than in survivors during hospitalization (Figure 4e).
BNP as a Risk-Stratification Biomarker
The five cardiac markers were measured on the first day of admission. Notably, the median value of BNP was significantly higher in non-survivors than in survivors for those with pre-existing CAD (911.3 pg/mL vs. 57.9 pg/mL) and those without (121 pg/mL vs. 0.01 pg/mL). The median levels of hs-TNI, α-HBDH, and LDH were significantly higher in non-survivors than in survivors in patients without pre-existing CAD; however, a significant difference was not observed in patients with pre-existing CAD (Figure S2).
In non-survivors with pre-existing CAD, the median levels of BNP and hs-TNI within the first week showed a higher fold change (BNP: 5.8; hs-TNI: 7.5; α-HBDH: 1.9; CK-MB: 0.81; LDH: 1.7) from the upper reference limit of each marker (Figure 5a). In non-survivors without pre-existing CAD, the fold change for BNP, hs-TNI, α-HBDH:1.9, CK-MB, LDH was 1.4, 1.7, 2, 0.78, and 1.9, respectively (Figure 5b). The serum level differences for the five markers within a week after admission in patients with or without pre-existing CAD showed that BNP, α-HBDH, and LDH values were significantly higher in non-survivors than in survivors regardless of pre-existing CAD. Levels of hs-TNI were significantly higher only in non-survivors than in survivors for patients without pre-existing CAD. Although CK-MB and BNP levels were significantly different between non-survivors and survivors, most data for non-survivors were within normal levels (Figure 5c and Figure 5d). The change of BNP levels was the same as those mentioned above during hospitalization (Figure S3).
Elevated BNP and Increased Mortality
The 835 severe/critical patients with BNP values recorded during hospitalization were divided into BNP high (n=171) and BNP normal (n=664) groups based on the upper reference limit (100 pg/mL). Survival analysis was conducted on two groups using applying a mixed-effect Cox model adjusted for age, sex, comorbidities, and hs-TNI, α-HBDH, CK-MB, and LDH. As a result, the BNP normal group demonstrated a significantly lower risk of mortality (Figure 6a). The BNP level correctly classified survivors and non-survivors with an area under the curve (AUC) of 0.81 with the ideal threshold value based on the ROC curve of 62.17, a sensitivity of 71.7%, and a specificity of 80.6% (Figure 6b).
Pearson’s correlations were used to study the association between BNP and other laboratory parameters. CRP (Figure 6c), IL-6 (Figure 6d), and D-dimer (Figure 6e) showed a highly positive correlation with BNP. By contrast, BNP level was significantly negatively correlated with LYM% (Figure 6f).