Abstract
Background: The effects of myocarditis after mRNA COVID-19 vaccination (mCV) on myocardial tissue, and the association between cardiomyocyte injury and clinical presentation, are not fully understood.
Methods and Results: We retrospectively registered patients clinically diagnosed with myocarditis after the first or second mCV who underwent endomyocardial biopsy or autopsy from 42 participating centers in Japan. We investigated the histological features and their association with clinical presentation based on cardiomyocyte injury. Forty patients who underwent endomyocardial biopsy were included in the study. Of these, 19 (47.5%) showed mild lymphocytic infiltration and interstitial edema without cardiomyocyte injury. The remaining 21 (52.5%) patients showed cardiomyocyte injury accompanied by infiltrating inflammatory cells: 11 with lymphocytic infiltration, 7 with eosinophilic infiltration, and 3 with myocarditis with both lymphocyte and eosinophil infiltration. Compared with patients without cardiomyocyte injury, those with cardiomyocyte injury were clinically characterized by older age, a balanced sex distribution, less frequent chest pain, and a lower left ventricular ejection fraction. Fifteen of 21 (71.4%) patients with cardiomyocyte injury developed fulminant myocarditis, with 13 (86.7%) requiring mechanical circulatory support; in contrast, none of those without cardiomyocyte injury developed fulminant myocarditis (P<0.001).
Conclusions: Our histological examination of patients with myocarditis after mCV revealed varying degrees of cardiomyocyte injury, ranging from pronounced to absent, along with various types of myocarditis. Cardiomyocyte injury was strongly associated with the severity of myocarditis.
Acute myocarditis after mRNA COVID-19 vaccination (mCV) is rare, and is particularly observed in young males after a second dose, typically manifesting within 1 week of vaccination.1–4 The incidence of post-mCV myocarditis is estimated to be 1.4–5.0 per 100,000 vaccinations, with variability attributed to demographic factors.5 Although most cases of myocarditis after mCV are clinically mild,2,3,6 some studies have reported severe cases, including fulminant myocarditis and fatalities.5,7,8 Histologic evidence of cardiomyocyte injury remains essential for the diagnosis of acute myocarditis9–11 and guides treatment decisions,1,11–13 but there are few cumulative histologic evaluations regarding myocarditis after mCV.
In this study we analyzed endomyocardial biopsy (EMB) specimens collected from various regions in Japan to better understand the pathology of myocardial tissue in cases of post-mCV myocarditis and its association with clinical presentation. This work contributes to our biological understanding and may help clarify the pathogenesis of myocarditis after mCV.
Methods
Study Design
This retrospective study (COMprehensive Biopsy feATures and outcomes in myocarditis after COVID-19 mRNA vaccination [COMBAT COVID-19 study]) compiles data from Japanese individuals clinically diagnosed with myocarditis after receiving either the first or second mCV (BNT162b2 [Pfizer/BioNTech] or mRNA-1273 [Moderna]) who underwent EMB or autopsy within 7 days of hospitalization between February 2021 and March 2022 in 42 participating centers. All patients underwent COVID-19 antigen, antibody, or polymerase chain reaction (PCR) testing using nasal swabs or a small amount of saliva. According to the Japanese Circulation Society (JCS) guidelines,9 the European Society of Cardiology position statements,10 and expert consensus documents,14 patients were included if they met the following clinical diagnostic criteria for acute myocarditis after mCV: onset of myocarditis symptoms within 30 days after the first or second mCV, exclusion of acute coronary syndrome by coronary angiography or autopsy (mandatory), and at least 1 examination abnormality related to acute myocarditis (Supplementary Table 1). Patients were diagnosed with fulminant myocarditis based on the use of catecholamines or mechanical circulatory support during hospitalization.15 Patient data and pathological specimen slides were collected and analyzed in the core laboratory at Mie University.
This study adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Mie University, which waived the requirement for informed consent (Approval no. H2022-083). The data that support the findings of this study are available from the corresponding author upon reasonable request.
Study Population
We initially reviewed 50 cases of clinically diagnosed myocarditis after mCV. We excluded 3 cases of myocarditis occurring after the third vaccination, 2 cases that were positive for COVID-19 based on antigen or PCR testing, 1 autopsy case due to a lack of clinical information resulting from death upon hospital arrival, and 3 cases in which EMB (2 cases) or autopsy (1 case) were performed >7 days after hospital admission. Furthermore, we excluded 1 case with coexisting acute coronary syndrome confirmed by coronary angiography. Thus, 40 cases of clinically diagnosed myocarditis after mCV with EMB performed within 7 days of hospital admission were included in the final analysis (Figure 1). All 40 patients tested negative to COVID-19 either on antigen (n=9), antibody (n=1), or PCR testing (n=30). Extracted data for the 40 patients are summarized in Supplementary Table 2.
Histological Assessment
For the assessment of cardiomyocyte injury and myocarditis classification, specimens were reviewed by 6 pathologists (K. Maruyama, K.O.-O., H.I.-U., S.K., M.H., and K.I.-Y.). Consensus was reached based on JCS guidelines, Dallas criteria, and expert consensus documents.9,14 Upon examination of hematoxylin and eosin (HE)-stained samples, areas showing cardiomyocyte degeneration and/or necrosis accompanied by infiltrating inflammatory cells were categorized as “cardiomyocyte injury.” Areas lacking these histopathological features were categorized as “no cardiomyocyte injury.”
Eosinophilic myocarditis was identified when there was a significant aggregation of eosinophils at the perimeter of the affected cardiomyocytes, particularly in areas where signs of degranulation were present. Mixed-type myocarditis was defined as cases in which there was a predominance of lymphocyte infiltration accompanied by eosinophilic cationic protein (ECP)-positive eosinophilic infiltration.
Immunohistochemical Staining and Counting of Inflammatory Cells
Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections using the Ventana Benchmark XT system (Roche Diagnostic KK, Tokyo, Japan), following the manufacturer’s protocols. Inflammatory cell types were detected using commercially sourced primary antibodies (Supplementary Table 3). Cell Conditioning 1 buffer was applied for antigen retrieval, and the I-VIEW DAB Universal Kit was used for antibody detection and hematoxylin counterstaining.
Inflammatory cells were quantified by displaying specimens on a computer screen at ×40 magnification (field size=0.1401 mm2). The 2 most infiltrated areas per specimen were independently counted by 2 observers, with mean values calculated and presented as cells per square millimeter. Cells were counted as CD3, CD4, and CD8 positive if the cell membrane was stained; cells that were considered CD68 and ECP positive had a stained cytoplasm with countable nuclei. Inter- and intra-observer variabilities for inflammatory cell counts were obtained based on 5 random cases that were independently analyzed by 2 observers and by the same observer at 2 different time points.
Statistical Analysis
The normality of distribution was tested using histograms and the Shapiro-Wilk test. Continuous variables are presented as median values with an interquartile range (IQR), regardless of the normality of data distribution. Student’s t-test or the Mann-Whitney U test was used to assess the significance of differences in normally distributed or skewed continuous variables, respectively. Categorical data are presented as numbers and percentages, and were compared between groups using the Chi-squared test or Fisher exact test. A Pearson correlation heatmap for inflammatory cell counts was generated using RStudio. One-way analysis of variance followed by Dunn’s post hoc test was used to compare multiple groups. All P values are 2-sided, and P<0.05 was considered significant. All analyses were performed using Prism Software version 10.
Results
Baseline Clinical Characteristics
The baseline characteristics of 40 patients with myocarditis after mCV are presented in Table 1. The median age was 25.5 years (IQR 19.3–49.0 years), and 70.0% of patients were male. The onset of symptoms occurred at a median of 3.0 days after vaccination (IQR 2.0–9.8 days), with 29 (72.5%) of cases developing after the second vaccination. Chest pain and fever were the most common symptoms. All 40 patients had elevated cardiac enzymes. On electrocardiograms, 28 (71.8%) patients showed ST-T elevation. The median left ventricular ejection fraction (LVEF) was 49.0% (IQR 25.1–59.9%).
Table 1.
Baseline Patient Characteristics and Clinical and Examination Findings on Admission
| |
All patients
(n=40) |
Cardiomyocyte injury |
P value |
| No (n=19) |
Yes (n=21) |
| Patient characteristics |
| Age (years) |
25.5 [19.3–49.0] |
23.0 [19.0–27.0] |
49.0 [22.0–60.0] |
0.006 |
| Male sex |
28 (70.0) |
16 (84.2) |
12 (57.1) |
0.063 |
| BMI (kg/m2) |
21.4 [19.9–22.6] |
21.3 [20.1–22.7] |
21.5 [19.7–22.9] |
0.95 |
| Second vaccination |
29 (72.5) |
18 (94.7) |
11 (52.3) |
0.003 |
| Vaccination to onset (days) |
3.0 [2.0–9.8] |
2.0 [1.0–3.0] |
7.0 [2.5–14.5] |
0.019 |
| Onset to biopsy (days) |
2.0 [1.0–6.0] |
1.0 [1.0–4.0] |
5.0 [2.0–7.0] |
0.036 |
| Vaccines |
| BNT162b2 |
18 (46.2) |
3 (16.7) |
15 (71.4) |
0.001 |
| mRNA-1273 |
21 (53.8) (n=39B) |
15 (83.3) (n=18B) |
6 (28.6) |
| Comorbidity |
| Hypertension |
5 (12.5) |
0 (0) |
5 (23.8) |
0.023 |
| Dyslipidemia |
2 (5.0) |
0 (0) |
2 (9.5) |
0.17 |
| Diabetes |
2 (5.0) |
0 (0) |
2 (9.5) |
0.17 |
| Bronchial asthma |
2 (5.0) |
1 (5.2) |
1 (4.8) |
0.94 |
| Autoimmune diseases |
2 (5.0) |
0 (0) |
2 (9.5) |
0.17 |
| Past history |
| HF hospitalization |
1 (2.5) |
0 (0) |
1 (4.8) |
0.53 |
| Old MI |
0 (0) |
0 (0) |
0 (0) |
– |
| Stroke |
1 (2.5) |
1 (5.2) |
0 (0) |
0.48 |
| Symptoms or signs |
| Chest pain |
26 (65.0) |
18 (94.7) |
8 (38.0) |
<0.001 |
| Fatigue |
16 (40.0) |
2 (10.5) |
14 (66.7) |
<0.001 |
| Fever |
25 (62.5) |
13 (68.4) |
12 (57.1) |
0.34 |
| Dyspnea |
17 (42.5) |
3 (15.8) |
14 (66.7) |
0.001 |
| Vital signs |
| SBP (mmHg) |
112.5 [97.3–124.8] |
122.0 [112.0–135.0] |
98.0 [85.0–115.5] |
<0.001 |
| Heart rate (beats/min) |
92.5 [70.5–108.3] |
83.0 [70.0–106.0] |
100.0 [82.0–109.0] |
0.24 |
| Laboratory findings |
| White blood cells (/mm3) |
7,830.0
[6,420.0–9,560.0]
(n=39B) |
8,000.0
[6,710.0–8,865.0]
(n=18B) |
7,740.0
[6,410.0–11,465.0] |
0.75 |
| Eosinophils (/mm3) |
121.8 [18.6–255.5]
(n=37B) |
148.0 [51.2–243.5]
(n=16B) |
49.8 [6.4–260.8] |
0.35 |
| BUN (mg/dL) |
11.1 [8.6–19.5]
(n=39B) |
8.8 [7.1–10.0]
(n=18B) |
19.5 [12.0–31.1] |
<0.001 |
| Creatinine (mg/dL) |
0.83 [0.73–1.10]
(n=39B) |
0.77 [0.67–0.86]
(n=18B) |
0.97 [0.74–1.22] |
0.18 |
| BNP (pg/mL) |
333.1 [41.6–1015.1]
(n=24B) |
37.7 [14.2–56.1]
(n=10B) |
550.5 [340.5–1,090.4]
(n=14B) |
0.002 |
| NT-pro BNP (pg/mL) |
395.0
[208.0–6,156.0]
(n=19B) |
263.5
[199.5–407.5]
(n=10B) |
6,156.0
[984.0–15,071.5]
(n=9B) |
0.003 |
| Troponin T (ng/dL)A |
1.08 [0.55–3.23]
(n=26B) |
0.75 [0.47–1.08]
(n=13B) |
3.09 [0.95–6.76]
(n=13B) |
0.049 |
| Troponin I (ng/dL)A |
4.55 [2.44–22.11]
(n=17B) |
3.45 [0.91–5.70]
(n=7B) |
21.88 [1.73–50.0]
(n=10B) |
0.15 |
| CRP (mg/dL) |
3.7 [2.3–8.0]
(n=38B) |
3.8 [2.3–7.6]
(n=17B) |
3.6 [1.3–7.8] |
0.71 |
| Chest X-ray findings |
| Cardiothoracic ratio (%) |
50.0 [47.0–56.0]
(n=39B) |
50.0 [45.7–52.0]
(n=18B) |
51.3 [47.0–59.5] |
0.058 |
| Electrocardiogram findings |
| ST elevation |
28 (71.8)
(n=39B) |
16 (84.2)
(n=19B) |
12 (60.0)
(n=20B) |
0.093 |
| Non-sinus rhythm |
6 (15.0) |
0 (0) |
6 (28.6) |
0.014 |
| Echocardiogram findings |
| LVDd (mm) |
46.0 [43.0–50.0] |
48.0 [43.0–50.0] |
45.4 [41.3–47.0] |
0.13 |
| LVEF (%) |
49.0 [25.1–59.9] |
56.6 [47.0–62.0] |
27.0 [18.0–51.5] |
<0.001 |
Unless indicated otherwise, data are given as the median [interquartile range] or n (%). AElevated cardiac enzyme was defined as an elevation in troponin T or troponin I more than 0.02 ng/mL. BNumber of patients with available data. BMI, body mass index; BNP, B-type natriuretic peptide; BUN, blood urea nitrogen; CRP, C-reactive protein; HF, heart failure; LVDd, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction; MI, myocardial infarction; NT-pro BNP, N-terminal pro B-type natriuretic peptide; SBP, systolic blood pressure.
Pathological Findings
Of the 40 patients with myocarditis after mCV, 21 (52.5%) had cardiomyocyte injury, with the remaining 19 patients classified as having no cardiomyocyte injury. Typical cases of no cardiomyocyte injury revealed minimal CD3-, CD4-, CD8-, and CD68-positive cells and scant ECP-positive eosinophil infiltration (Figure 2A–G). Further examination of the 21 patients with cardiomyocyte injury showed CD3-, CD4-, CD8-, and CD68-positive lymphocyte and macrophage infiltration, with infrequent eosinophil infiltration, consistent with lymphocytic myocarditis (n=11; Figure 2H–N). A subset of patients (n=3) exhibited mixed-type myocarditis (Figure 2O–U). Seven patients were identified as having eosinophilic myocarditis, characterized by prominent clustering of eosinophils positive for ECP and associated with cardiomyocyte injury (Figure 2V–Z″). In addition, infiltration in 3 cases of eosinophilic myocarditis extended into the endocardium, suggestive of Loeffler endocarditis. There were no cases of giant cell myocarditis.
Clinical and Pathological Findings According to the Presence or Absence of Cardiomyocyte Injury
A comparison of clinical findings of post-mCV myocarditis in patients with and without cardiomyocyte injury is presented in Table 1. Most patients without cardiomyocyte injury were young males, with myocarditis occurring after the second vaccination, and typically developing within a few days after vaccination. Patients with cardiomyocyte injury were significantly older than those without (49.0 [IQR 22.0–60.0] vs. 23.0 [IQR 19.0–27.0] years; P=0.006). Furthermore, there were significant differences between patients with and without cardiomyocyte injury in the prevalence of the second vaccination (52.3% vs. 94.7%, respectively; P=0.003) and the time from vaccination to onset (7.0 [IQR 2.5–14.5] vs. 2.0 [IQR 1.0–3.0] days, respectively; P=0.019). One patient in the group with cardiomyocyte injury had a history of previous heart failure hospitalization; this patient’s baseline LVEF was 42%, which decreased to 19% after the onset of post-mCV myocarditis. The frequency of chest pain was significantly lower among patients with than without cardiomyocyte injury (38.0% vs. 94.7%; P<0.001), but those with cardiomyocyte injury more frequently had fatigue and dyspnea. In addition, patients with cardiomyocyte injury had markedly lower systolic blood pressure and elevated levels of B-type natriuretic peptide (BNP)/N-terminal pro-BNP and troponin T. They also had a higher frequency of non-sinus rhythms (e.g., ventricular escape rhythm) and decreases in LVEF than those without cardiomyocyte injury (Table 1).
Nine of 10 (90.0%) patients without cardiomyocyte injury who underwent cardiac magnetic resonance imaging (MRI) were identified as having findings consistent with myocarditis according to the 2018 Lake Louise criteria (Supplementary Figure; Supplementary Table 4). Although the remaining patient without cardiomyocyte injury did not meet the criteria, cardiac MRI for this patient showed myocardial edema on T2-weighted images. All 7 patients with cardiomyocyte injury (3 with lymphocytic myocarditis, 3 with eosinophilic myocarditis, and 1 with mixed-type myocarditis) who underwent cardiac MRI showed high intensity on T2-weighted imaging and positive non-ischemic late gadolinium enhancement, consistent with acute myocarditis according to the 2018 Lake Louise criteria (Supplementary Table 4).
A quantitative evaluation of infiltrating inflammatory cells was conducted in all 40 patients (Figure 3A–F). Group comparisons revealed that patients with cardiomyocyte injury had significantly greater inflammatory cell counts that were positive for CD3, CD4, CD8, CD68, and ECP than those without cardiomyocyte injury (Figure 3G–K). A heatmap showed distinct patterns of cell infiltration, and eosinophil counts showed the lowest correlation with CD3-, CD4-, CD8-, and CD68-positive cells (Figure 3L). Figure 4 shows the association between age and the total count of inflammatory cells, as well as myocarditis types and the presence of fulminant myocarditis. Linear regression analysis was conducted to assess the relationship between patient age and the total count of inflammatory cells (CD3, CD68, and ECP), which yielded an R2
of 0.3983 with P<0.001, indicating a moderate correlation (Figure 4). Age stratification into quartiles demonstrated a significant age-related escalation in inflammatory cell numbers, with the younger groups (15–19 and 20–24 years) displaying less infiltration than those aged over 50 years (Supplementary Table 5). In addition, age stratification into quartiles revealed a significant age-related decrease in blood pressure and LVEF and an increase in BNP (Supplementary Table 5).
Intra- and interobserver measurements of inflammatory cell counts were evaluated based on intraclass correlation coefficients and were highly correlated (Supplementary Table 6).
Association Between Clinical Course and Histopathology
Table 2 presents the clinical course and in-hospital treatments. Notably, 15 of 21 (71.4%) patients with cardiomyocyte injury developed fulminant myocarditis, as opposed to none of those without cardiomyocyte injury (Figure 4). Among the 15 patients with fulminant myocarditis, 13 (86.7%) required mechanical support using a microaxial percutaneous left ventricular assist device, intra-aortic balloon pumping, and/or veno-arterial extracorporeal membrane oxygenation (Figure 5). Supplementary Table 7 presents a comparative analysis among patients with cardiomyocyte injury of selected clinical parameters between those with lymphocytic histology and those with eosinophilic histology. Although there was no statistically significant when comparing the ages of patients with different types of myocarditis, only patients aged older than 40 years were categorized as having eosinophilic myocarditis (Figure 4; Supplementary Table 2). Otherwise, there were no significant differences, except for troponin T. All 40 patients included in this study survived to hospital discharge.
Table 2.
Severity of Clinically Diagnosed Myocarditis and Therapies During Hospitalization
| Variable |
All patients
(n=40) |
Cardiomyocyte injury |
P value |
| No (n=19) |
Yes (n=21) |
| Fulminant myocarditis |
15 (37.5) |
0 (0) |
15 (71.4) |
<0.001 |
| Medical therapies |
| Loop diuretics |
11 (27.5) |
1 (5.2) |
10 (47.6) |
0.003 |
| NSAIDs and/or colchicine |
13 (33.3) (n=39A) |
9 (50.0) (n=18A) |
4 (19.0) (n=21A) |
0.041 |
| Steroid and/or IVIG |
18 (46.2) (n=39A) |
3 (16.7) (n=18A) |
15 (71.4) |
0.001 |
| Catecholamines |
14 (35.0) |
0 (0) |
14 (66.7) |
<0.001 |
| Temporary MCS devices |
| Intra-aortic balloon pumping |
8 (20.0) |
0 (0) |
8 (38.1) |
0.003 |
| Microaxial percutaneous LVAD |
11 (27.5) |
0 (0) |
11 (52.4) |
<0.001 |
| Veno-arterial ECMO |
12 (30.0) |
0 (0) |
12 (57) |
<0.001 |
Unless indicated otherwise, data are presented as n (%). ANumber of patients with available data. ECMO, extracorporeal membrane oxygenation; IVIG, intravenous immunoglobulin; LVAD, left ventricular assist device; MCS, mechanical circulatory support; NSAIDs, non-steroidal anti-inflammatory drugs.
Discussion
The main findings of the present study are as follows. First, approximately half of the 40 patients with clinically diagnosed post-mCV myocarditis who underwent EMB showed significant cardiomyocyte injury: 11 cases of lymphocytic myocarditis, 7 cases of eosinophilic myocarditis, and 3 cases of myocarditis with notable lymphocyte and eosinophil infiltration. Second, patients without cardiomyocyte injury were younger, mostly male, commonly presented with chest pain, and exhibited a favorable clinical course. Third, those with cardiomyocyte injury were older, showed a balanced sex distribution, presented less frequently with chest pain and more frequently with fatigue/malaise and dyspnea, and approximately 70% developed fulminant myocarditis. This investigation provides one of the largest substantive collections of EMB samples for myocarditis after mCV.
Spectrum of Myocardial Damage in Patients With Post-mCV Myocarditis
Previous case reports suggest that myocarditis after mCV is typically benign and transient. As a result, there is limited use of EMB to comprehensively examine the histopathologic landscape of the condition.16 However, when biopsies were performed, considerable variability in inflammatory cell infiltration was noted.17–19 Some case reports described minimal inflammatory cell infiltration,20,21 whereas others indicated the presence of lymphocytic and macrophagic infiltrates, occasional eosinophils, and edema and neutrophils.17,22,23 These reports underscore the need for larger-scale studies to acquire a more nuanced understanding of the disease condition. Thus, in the present multicenter study conducted across Japan, we examined EMB samples to elucidate the characteristics of myocarditis in patients who developed the condition after mCV. Our study revealed a spectrum of cardiomyocyte injury, ranging from pronounced to absent. Histological cardiomyocyte injury encompasses lymphocytic or eosinophilic myocarditis, as well as cases with mixed eosinophil presence. This finding clarified the ambiguities highlighted in prior reports by demonstrating a spectrum of myocardial inflammatory responses.
Infiltration of Eosinophils
Among the 21 patients with post-mCV myocarditis and cardiomyocyte injury, we identified 7 (33.3%) cases of eosinophilic myocarditis and 3 (14.3%) cases of mixed-type myocarditis with eosinophilic infiltration, indicating a higher proportion than previously reported.9 Although our findings to not definitively show a causal relationship between mCV and the occurrence of myocarditis, our findings suggest a meaningful association of eosinophils with post-mCV myocarditis and highlight the possibility of a hypersensitivity mechanism contributing to post-mCV myocarditis. Myocarditis has been associated with various vaccines, including influenza24 and tetanus toxoid,25 with the most definitive causal relationship established for the smallpox vaccine.26,27 Some inert substances, such as polyethylene glycol, are added to vaccines to improve stability and absorption, although those substances are known to induce allergic reactions.28 Such a reaction may cause hypersensitivity and induce a significant infiltration of eosinophils into the myocardium. Regarding age differences between those with lymphocytic and eosinophilic myocarditis in the group with cardiomyocyte injury, only patients older than 40 years were categorized as having eosinophilic myocarditis; however, there was no statistically significant difference between the 2 groups, presumably due to the small sample size. The possible mechanisms responsible for the eosinophilic reaction after mCV, predominantly in elderly patients, remain uncertain.
Similarities in the potential mechanism of myocarditis after COVID-19 infection and after mCV, such as a cytokine storm from an immune response to the infection or the vaccine, molecular mimicry between the spike protein and cardiac proteins, or the promotion of myocardial inflammation via binding of the spike protein to the angiotensin-converting enzyme 2 receptor, have been reported.29 However, post-mCV myocarditis may involve a hypersensitivity mechanism that is not typically observed in myocarditis after COVID-19 infection.30
Association of Cardiomyocyte Injury With Clinical Severity in Patients With Acute Myocarditis After mCV
Although previous large-cohort and epidemiologic studies have reported that most cases of post-mCV myocarditis are clinically mild,3,4,6 a recent epidemiologic nationwide study in Korea investigating over 44 million individuals reported that a clinically severe status was not uncommon (19.8%) in patients clinically diagnosed with post-mCV myocarditis.5 In our study, 15 of 21 (71.4%) patients with cardiomyocyte injury developed fulminant myocarditis, and 13 (86.7%) required mechanical circulatory support, whereas all patients without cardiomyocyte injury exhibited a favorable clinical course. Thus, the EBM results raise concerns regarding post-mCV myocarditis as a potentially severe disease linked to cardiomyocyte injury. This underscores the need to investigate the causes of deterioration in patients with post-mCV myocarditis and cardiomyocyte injury.
This study identified variations in clinical characteristics (e.g., age, sex, symptoms, duration from vaccination to onset, and number of vaccinations) between patients with and without cardiomyocyte injury. These differences may be linked to distinct pathophysiological mechanisms. Furthermore, similar to a previous study,5 cellular analysis in our study indicated an age-related increase in inflammatory cell infiltration within the myocardium. Age-related factors, such as clonal hematopoiesis,31 may render individuals susceptible to adverse vaccine reactions. The interplay between age, myocardial inflammation, and clinical severity may be crucial in understanding the etiology of the occurrence and deterioration of myocarditis after mCV.
Importance of Myocardial Histological Assessment in Revealing the Pathophysiology of Post-mCV Myocarditis
The pathophysiology of myocardial injury following mCV has not been fully clarified.32 Several hypotheses have been proposed, including altered gene expression, direct activation of the immune system by mRNA, molecular mimicry, immune dysregulation, and aberrant cytokine release.31 Although our study data did not elucidate the exact pathophysiology between mCV and myocarditis, the findings revealed different patterns of inflammation and varied inflammatory cell presence. Notably, significant eosinophilic infiltration and clinical differences between patients with and without cardiomyocyte injury suggested the involvement of multiple contributing mechanisms rather than a single etiological pathway. Myocardial histological assessment may offer valuable insights into the pathogenesis of myocarditis after mCV.
Study Limitations
This study has several limitations. First, as a retrospective multicenter study, it is susceptible to selection bias, primary due to the absence of a standard practice for determining which patients should undergo EMB for the diagnosis of myocarditis. Cardiac MRI may have primarily replaced EMB, especially among younger patients. Thus, it is unclear whether our study results apply to younger patient populations. Second, our study results did not provide information on the benefit-risk assessment of COVID-19 vaccines due to the sample size, which only included retrospectively registered patients who underwent EMB. Third, the number of biopsy samples per patient varied, with 11 (27.5%) patients having only 1. This raises the possibility of false negatives due to sampling error in the histological diagnosis of myocarditis. Fourth, our study did not provide direct evidence of vaccine-induced inflammation in the cases examined. In addition, viral genome detection within myocardial tissues by PCR testing was not performed in our study, except in 3 cases. Thus, the potential for myocarditis due to other viral infections has not been excluded. Molecular testing, including PCR testing for viral genomes, for viral causes of myocarditis is necessary in future studies to support our conclusions. Fifth, we did not compare the degree of inflammatory cell infiltration between our patients with post-mCV myocarditis and patients with myocarditis with other etiologies. Sixth, due to the restricted availability of specimen slides, immunostaining for potential pivotal molecules to elucidate the pathophysiology of myocarditis subsequent to mCV, including spike protein, could not be conducted. Seventh, we have no follow-up data from cardiac MRI, echocardiography, or other clinical information. Finally, the effect of cardiomyocyte injury on fatal arrhythmia, as a side effect of mCV mentioned in a previous study,5 was not clarified in our study.
Conclusions
Our histological examination of EMB samples revealed varying degrees of cardiomyocyte injury, ranging from pronounced to absent, along with various types of myocarditis in patients with myocarditis after mCV. Notably, the presence of cardiomyocyte injury serves as a strong indicator for the development of fulminant myocarditis following mCV. Our results help clarify the pathological and clinical aspects of myocarditis after mCV, advancing our understanding of the pathophysiological mechanism behind post-mCV myocarditis.
Acknowledgments
The authors appreciate the support and collaboration of the following coinvestigators who participated in the COMBAT COVID-19 study: Shintaro Kinugawa, Shoji Matsushima, Toru Hashimoto, Keisuke Shinohara, Tomomi Ide, Nobutaka Kakuda, Hiroshi Ito, Keiichiro Iwasaki, Yoichi Takaya, Kenji Yoshida, Tomoyuki Otani, Kei Nakamoto, Yasuhiro Akazawa, Atsushi Tada, Hiroaki Hiraiwa, Yuki Kimura, Harutaka Katano, Shun Iida, Yuichiro Hirata, Tadao Aikawa, Norimichi Koitabashi, Masahiko Takeda, Takaaki Matsuyama, Yuki Yokouchi, Toru Awaya, Takayuki Shiomi, Daisuke Yajima, Tsunenori Saito, Masayoshi Yamamoto, Shohei Yoshida, Minoru Wakasa, Shintaro Sakaguchi, Yusuke Miki, Hitoshi Matsuo, Yoshihiro Yamada, Eisuke Maekawa, Eriko Hisamatsu, Makoto Takeuchi, Satoshi Nakatani, Shino Morikawa, Haruhiko Higashi, Yuta Yano, Akihiro Masumoto, Kisho Ohtani, Kazuhiro Nagaoka, Kensaku Nishihira, Tsutomu Kochinda, Yoshiaki Sakai, Hiroaki Kawano, Satoshi Terasaki, Hidetaka Kioka, Toyoaki Murohara, and Kisaki Amemiya.
Sources of Funding
This study was supported, in part, by the Japan Agency for Medical Research and Development (AMED) under grant numbers 20ek0109476 h0003 and 24ek0109683h0001 (to K.I.-Y.); Kurozumi Medical Foundation (to K. Maruyama); Mochida Memorial Foundation for Medical and Pharmaceutical Research (to K. Maruyama); Hitachi Global Foundation (to K. Maruyama); Japan Foundation for Applied Enzymology (VBIC to K. Maruyama); and SENSHIN Medical Research Foundation (to K. Maruyama).
Disclosures
K. Maruyama has received grant support from Takeda Pharmaceuticals. H.I.-U. has received lecture fees from Daiichi Sankyo. K.O. has received grant support and lecture fees from Pfizer. T.N. has received lecture fees from Pfizer and Daiichi Sankyo. T. Okumura has received lecture fees and grant support from Pfizer. M.O. has received lecture fees from Pfizer and Takeda Pharmaceuticals. E.A. has received lecture fees from Pfizer and Daiichi Sankyo. S.Y. has received lecture fees from Pfizer. K.N. has received lecture fees from Pfizer, Takeda Pharmaceuticals, and Daiichi Sankyo. S.N. has received lecture fees from Daiichi Sankyo. T.A. has received lecture fees from Pfizer, Takeda Pharmaceuticals, and Daiichi Sankyo. Y.S. has received lecture fees from Pfizer, Takeda Pharmaceuticals, and Daiichi Sankyo, as well as grant support from Takeda Pharmaceuticals and Shionogi. K.D. has received lecture fees from Pfizer, Shionogi, and Daiichi Sankyo, as well as grant support from Takeda Pharmaceuticals and Daiichi Sankyo. T.S. has conducted joint research with Shionogi on COVID-19 vaccines without any financial relationship. T.N., Y.S., and T.A. are members of Circulation Journal’s Editorial Team. No other potential conflicts of interest relevant to this article are reported.
Author Contributions
T. Omori orchestrated the collection of specimens and the aggregation of clinical data. T. Omori and M.H. verified the integrity of the clinical data. K. Maruyama led the assessment of pathological specimens, engaging in critical evaluations with K.O.-O., M.H., H.I.-U., S.K., and K.I.-Y. T. Omori and K. Maruyama conducted the quantitative analysis of inflammatory cells and analyzed the study data. The contribution of other team members primarily involved the provision of specimens and clinical data. K. Maruyama took the lead in manuscript preparation, ensuring that feedback and insights from all authors were integrated. The overall research supervision was provided by K.D., K.I.-Y., and M.H., who oversaw all aspects of the study’s execution.
IRB Information
This study was approved by the Mie University Hospital Institutional Review Board (Approval no. H2022-083).
Data Availability
The deidentified participant data will be shared upon reasonable request. Please contact the corresponding author directly to request data sharing. Data for each table and figure, as well as the study protocol, will be shared upon reasonable request. Data will be available for 1 year after acceptance. The data will be shared via email as Microsoft Excel or CSV files.
Supplementary Files
Please find supplementary file(s);
https://doi.org/10.1253/circj.CJ-24-0506
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