Expression profiles and functional analysis of plasma tRNA-derived small RNAs in children with fulminant myocarditis

Figures & data

Table 1. Clinical characteristics of patients with fulminant myocarditis.

Sample	Age	Sex	Symptoms onset	BP	HR	MCS	Follow-up
FM-01	7.5	Female	Fever, cough, emesis, chest pain, fatigue	77/53	60^†	TP	Recovery
FM-02	3.0	Male	Cough, poor spirit, fatigue	75/45	60^†	TP	Recovery
FM-03	7.5	Male	Fever, headache, convulsion	75/53	70^†	TP	Recovery
FM-04	7.2	Male	Headache, belly ache, emesis	86/60	99	ECMO	iDCM
FM-05	10.7	Male	Fever, headache, emesis, chest pain, fatigue	90/40	116	No	Recovery
FM-06	11.2	Male	Fever, emesis, convulsion	^‡	16	TP	Recovery
FM-07	12.3	Male	Fever, fatigue, convulsion	107/80	50^†	TP	Recovery
FM-08	13.2	Male	Fever, shortness of breath, amaurosis	92/44	148	ECMO	Recovery
FM-09	11.5	Male	Fever, emesis, convulsion	91/58	35	TP	Recovery
FM-10	12.8	Female	Fever, shortness of breath	80/40	110	No	Recovery
FM-11	6.8	Female	Fever, emesis, fatigue	86/62	120	ECMO	Recovery
FM-12	7.5	Male	Fever, headache, chest pain, syncope	108/62	82	ECMO	Recovery

† The heart rate was maintained by administering isoprenaline.

‡ The blood pressure was too low to be measured.

BP: Blood pressure at admission; ECMO: Extracorporeal membrane oxygenation; FM: Fulminant myocarditis; HR: Heart rate at admission; iDCM: Inflammatory dilated cardiomyopathy; MCS: Mechanical circulatory support; TP: Temporary pacemaker.

Table 2. Serological and imaging examinations of patients with fulminant myocarditis during acute phase.

1. Serological examinations of patients with fulminant myocarditis during acute phase
Sample	hs-cTnT (pg/ml)	NT-proBNP (pg/ml)	Inflammation markers					Pathogens^†
			WBC (×10^9)	n (%)	CRP (mg/l)	PCT (ng/ml)	ESR (mm/h)
FM-A-01	1105	21,835	8.5	73.5	66.12↑	0.4↑	13	GAS (blood culture)
FM-A-02	1696	>35,000	11.15↑	72.1	6.5	0.18↑	7	−
FM-A-03	511.8	11,666	9.19	88.2↑	23.9↑	0.04	−	−
FM-A-04	3751	18,909	22.55↑	75.8↑	65.74↑	0.56↑	−	AB (sputum culture)
FM-A-05	1802	7051	40.19↑	86.3↑	302.74↑	1.36↑	−	GAS (blood culture) & EBV
FM-A-06	2187	18,869	14.59↑	78.8↑	51.49↑	0.34↑	20↑	−
FM-A-07	560.5	4354	6.06	91.7↑	15.3↑	0.16↑	50↑	EBV
FM-A-08	1175	10,235	18.92↑	85.1↑	50↑	0.11↑	−	−
FM-A-09	5853	12,248	9.47	72.6	56.1↑	1.98↑	17↑	GAS (blood culture)
FM-A-10	1293	1247	15.37↑	69.8	42.1↑	0.3↑	−	−
FM-A-11	852	23,401	13.01↑	89.8↑	5	0.16↑	−	−
FM-A-12	2739	>35,000	15.05↑	78.9↑	5	0.58↑	10	−
2. Imaging examinations of patients with fulminant myocarditis during acute phase
Sample	ECG	Echocardiography			CMR
		LVEDD (cm)	LVEF (%)	PE	LGE	Edema	EGEr
FM-A-01	III° AVB	4.30↑	39	+	Basal segment of IVS	−	4.34
FM-A-02	III° AVB	3.66↑	37	+	Inferior/inferolateral wall of LV	−	4.17
FM-A-03	III° AVB	3.77	40	+	−	IVS	4.12
FM-A-04	ST-segment elevations, I° AVB	4.66↑	20	−	Basal and middle segments of IVS	−	4.8
FM-A-05	ST-segment elevations	4.57↑	46	−	−	Inferolateral wall of LV	4.10
FM-A-06	III° AVB	4.25	45	+	Inferior wall of LV and apex of inferior septum	−	4.50
FM-A-07	III°AVB, VT	4.40↑	43	−	−	IVS	4.15
FM-A-08	VT, Vf	4.98↑	28	+	−	LV diffuse edema	5.21
FM-A-09	III° AVB	4.90↑	38	−	IVS and lateral wall of LV	−	4.15
FM-A-10	ST-segment elevations	4.11	46	+	−	IVS	4.20
FM-A-11	III° AVB	3.99↑	27	−	−	LV diffuse edema	4.34
FM-A-12	III° AVB	3.35	33	−	IVS and lateral wall of LV	LV diffuse edema	3.78

† All our 12 patients underwent pathogen examinations to detect the following: coxsackievirus, cytomegalovirus, Epstein-Barr virus, rubella, herpes simplex virus-2, parvovirus B19, human herpesvirus 6, respiratory syncytial virus, parainfluenza viruses, influenza A virus, influenza B virus, human adenovirus, rhinovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, human immunodeficiency virus, syphilis, group A streptococcus, mycoplasma pneumoniae pneumonia and chlamydia pneumoniae. Blood culture and sputum culture were also performed.

AB: Acinetobacter baumannii; AVB: Atrioventricular block; CMR: Cardiovascular magnetic resonance; EBV: Epstein-Barr virus; ECG: Electrocardiogram; EGEr: Early gadolinium enhancement ratio (Enhancement myocardium/Enhancement skeletal muscle); ESR: Erythrocyte sedimentation rate; FM: Fulminant myocarditis; GAS: Group A streptococcus; IVS: Interventricular septum; LGE: Late gadolinium enhancement; LV: Left ventricle; LVEDD: Left ventricular end-diastolic dimension; LVEF: Left ventricular ejection fraction; N: Neutrophil; PE: Pericardial effusion; Vf: Ventricular fibrillation; VT: Ventricular tachycardia; WBC: White blood cell.

Table 3. Serological and imaging examinations of patients with fulminant myocarditis during convalescent phase.

Sample	hs-cTnT (pg/ml)	LVEDD (cm)	LVEF (%)	ECG
FM-C-01	18.24	3.14	60	Normal
FM-C-02	8.82	3.38	62	Normal
FM-C-03	13.3	3.23	60	Normal
FM-C-04	22.05	5.14↑	43↓	I° AVB
FM-C-05	12.48	4.29	61	Normal
FM-C-06	10.35	3.73	59	Normal
FM-C-07	8.56	4.58	64	Normal
FM-C-08	31.52	4.66	59	Normal
FM-C-09	12.5	4.34	64	Normal
FM-C-10	10.82	4.57	62	Normal
FM-C-11	33.85	3.06	59	Normal
FM-C-12	21.1	3.61	62	Normal

AVB: Atrioventricular block; ECG: Electrocardiogram; FM: Fulminant myocarditis; LVEDD: Left ventricular end-diastolic dimension; LVEF: Left ventricular ejection fraction.

Table 4. Volunteers demographics.

Sample	Age	Sex	hs-cTnT (pg/ml)	NT-proBNP (pg/ml)	LVEDD (cm)	LVEF (%)	ECG
CON-01	7.7	Female	7.16	89.96	3.60	61	Normal
CON-02	3.1	Male	7.08	32.91	3.27	63	Normal
CON-03	7.7	Male	11.20	75.50	4.00	62	Normal
CON-04	7.3	Male	13.46	87.81	3.54	64	Normal
CON-05	10.6	Male	13.02	46.32	3.76	60	Normal
CON-06	11	Male	3.64	36.80	3.60	60	Normal
CON-07	12.5	Male	13.30	58.97	4.15	62	Normal
CON-08	13	Male	4.42	56.30	4.42	60	Normal
CON-09	11.4	Male	3.34	63.60	3.65	64	Normal
CON-10	12.5	Female	5.70	12.10	4.21	64	Normal
CON-11	6.9	Female	<3	36.10	3.60	61	Normal
CON-12	7.3	Male	3.45	72.7	3.65	61	Normal

CON: Control; ECG: Electrocardiogram; LVEDD: Left ventricular end-diastolic dimension; LVEF: Left ventricular ejection fraction.

Table 5. Dilated cardiomyopathy patient demographics.

Sample	Age	Sex	hs-cTnT (pg/ml)	NT-proBNP (pg/ml)	LVEDD (cm)	LVEF (%)
HF-CON-01	7.6	Male	28.02	8280	4.92	33
HF-CON-02	3.2	Male	17.72	4053	4.0	35
HF-CON-03	7.7	Female	23.1	2500	5.0	40
HF-CON-04	10.9	Male	46.5	4777	6.01	28
HF-CON-05	11.1	Male	41.94	6900	5.73	39
HF-CON-06	12	Male	15.57	27500	5.86	31
HF-CON-07	13.6	Male	12.9	3634	5.06	37
HF-CON-08	11	Male	14.75	7676	4.76	30
HF-CON-09	11.8	Male	14.70	5285	7.29	20
HF-CON-10	12.9	Female	3.24	8417	5.55	27
HF-CON-11	6.7	Female	27.10	7285	4.60	35
HF-CON-12	7.5	Male	15.26	9518	4.81	35

HF: Heart failure; LVEDD: Left ventricular end-diastolic dimension; LVEF: Left ventricular ejection fraction.

Figure 1. Expression profiles of tsRNAs sequencing data in nine samples.

Hierarchical clustering heatmap analysis of tsRNAs expression data of nine samples. Each row represents one tsRNA and each column represents one sample.

CON: Control; FM: Fulminant myocarditis.

Figure 2. Expression profiles of tsRNAs sequencing data in three pairwise.

The scatter plot of tsRNAs in three pairwise that is FM-A versus CON (A), FM-A versus FM-C (B) and FM-C versus CON (C). The values of x- and y-axes in the scatter plot are the averaged CPM of each group (log2 scaled). Red/green dots indicate statistically significant differentially expressed tsRNAs with FC ≥1.5. Gray dots indicate nondifferentially expressed tsRNAs. Heatmaps show the hierarchical cluster analysis of differentially expressed tsRNAs in three pairwise between the three groups: FM-A versus CON (D), FM-A versus FM-C (E) and FM-C versus CON (F). Each row represents one tsRNAs and each column represents one sample. Color brightness reflects the degree of expression increase or decrease: red represents high expression, and blue represents low expression. The volcano plot of tsRNAs in three pairwise between the three groups: FM-A versus CON (G), FM-A versus FM-C (H) and FM-C versus CON (I). The values of x- and y-axes in the volcano plot are log2 transformed fold change and -log10 transformed p-values between the two groups, respectively. Red/green circles indicate statistically significant differentially expressed tsRNAs with FC ≥1.5 and p < 0.05. Gray circles indicate nondifferentially expressed tsRNAs.

CON: Control; FM: Fulminant myocarditis.

Figure 3. Reads length distribution of the nine samples.

Bar chart shows the total read counts against the lengths for the complete adapter-trimmed read set in the nine samples from FM-A, FM-C and CON groups, respectively. (A) FM-A-01, (B) FM-A-02, (C) FM-A-03, (D) FM-C-01, (E) FM-C-02, (F) FM-C-03, (G) CON-01, (H) CON-02, (I) CON-03.

FM: Fulminant myocarditis.

Figure 4. Expression levels of the four selected plasma tsRNAs between the FM-A and FM-C groups assessed by quantitative real-time PCR.

(A) No significant difference was found in tRF-Glu-TTC-004 between the FM-A and FM-C groups (p = 0.32). (B) No significant difference was found in tRF-Lys-TTT-006 between the FM-A and FM-C groups (p = 0.33). (C) No significant difference was found in tRF-Leu-AAG-017 between the FM-A and FM-C groups (p = 0.15). (D) tiRNA-Gln-TTG-001 was 2.72-fold change upregulated in FM-A compared with FM-C (p = 0.00052).

FM: Fulminant myocarditis.

Figure 5. Expression levels of tiRNA-Gln-TTG-001 among the FM-A, FM-C and CON groups assessed by quantitative real-time PCR.

tiRNA-Gln-TTG-001 was found to be overexpressed in FM-A group, with a 2.29-fold increase when compared with FM-C group (p = 0.0000048434), and a 3.49-fold increase when compared with CON group (p = 0.00000053493). There was no statistical difference of tiRNA-Gln-TTG-001 between FM-C and CON groups (p = 0.7335).

CON: Control; FM: Fulminant myocarditis.

Figure 6. Expression levels of tiRNA-Gln-TTG-001 among the FM-A, HF-CON and CON groups assessed by quantitative real-time PCR.

tiRNA-Gln-TTG-001 was found to be overexpressed in FM-A group, with a 5.38-fold increase when compared with HF-CON group (p = 0.000000019589). There was no statistical difference of tiRNA-Gln-TTG-001 between HF-CON and CON groups (p = 0.234).

CON: Control; FM: Fulminant myocarditis.

Figure 7. Association between expression levels of tiRNA-Gln-TTG-001 (expressed as 2^-ΔCT) and the serological or imaging parameters of fulminant myocarditis during acute phase.

(A) Expression levels of tiRNA-Gln-TTG-001 were positively associated with the values of hs-cTnT (r = 0.7622, p = 0.0059). (B) Expression levels of tiRNA-Gln-TTG-001 were positively associated with the values of CRP (r = 0.6725, p = 0.0166). (C) Expression levels of tiRNA-Gln-TTG-001 were positively associated with the values of PCT (r = 0.669, p = 0.0174). (D) Expression levels of tiRNA-Gln-TTG-001 were negatively associated with the values of N% (r = -0.6434, p = 0.028). (E) The correlation coefficient between levels of tiRNA-Gln-TTG-001 and NT-proBNP (r = 0.1611, p = 0.6169) was not statistically significant. (F) The correlation coefficient between levels of tiRNA-Gln-TTG-001 and WBC counts (r = 0.2028, p = 0.5281) was not statistically significant. (G) The correlation coefficient between levels of tiRNA-Gln-TTG-001 and ESR (r = 0.0286, p = 1) was not statistically significant. (H) The correlation coefficient between levels of tiRNA-Gln-TTG-001 and LVEF (r = -0.0701, p = 0.8287) was not statistically significant. (I) The correlation coefficient between levels of tiRNA-Gln-TTG-001 and EGEr (r = 0.3579, p = 0.2534) was not statistically significant.

EGEr: Early gadolinium enhancement ratio = enhancement myocardium/enhancement skeletal muscle; ESR: Erythrocyte sedimentation rate; FM: Fulminant myocarditis; hs-cTnT: Highly sensitive cardiac troponin T; LVEF: Left ventricular ejection fraction; N%: Neutrophil proportion; NT-proBNP: N-terminal pro-brain natriuretic peptide; PCT: Procalcitonin; WBC: White blood cell.

Figure 8. Expression levels of tiRNA-Gln-TTG-001 in vitro assessed by quantitative real-time PCR.

(A) Intracellular tiRNA-Gln-TTG-001 levels were significantly increased following exposure (p = 0.0029). (B) tiRNA-Gln-TTG-001 were significantly increased in the CCS, following exposure (p = 0.0056).

CCS: Cell culture supernatant; HCM: Human cardiomyocyte; LPS: Lipopolysaccharide.

Figure 9. Target gene predictions and Gene Ontology enrichment analyses of tiRNA-Gln-TTG-001.

GO: Gene ontology.

Figure 10. Prediction of signaling pathways of tiRNA-Gln-TTG-001.

The vertical axis shows the annotated functions of the target genes. The horizontal axis shows the enrichment score (-log10 transformed p-value) and the gene number of each cluster, respectively. Yellow marked nodes are putative molecule in relation to the dysregulated tsRNAs in fulminant myocarditis.

Data sharing statement

The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request. And the high-throughput sequence data have been submitted to GEO with GEO accession numbers: GSE 163944.