Clinical and Pathological Impact of Tissue Fibrosis on Lethal Arrhythmic Events in Hypertrophic Cardiomyopathy Patients With Impaired Systolic Function

Abstract

Background: The natural history of hypertrophic cardiomyopathy (HCM) varies from an asymptomatic benign course to a poor prognosis. Myocardial fibrosis may play a critical role in ventricular tachyarrhythmias (VT/VF); however, the clinical significance of tissue fibrosis by right ventricular (RV) biopsy in the long-term prognosis of HCM patients remains unclear.

Methods and Results: We enrolled 185 HCM patients (mean age, 57±14 years). The amount of fibrosis (%area) was quantified using a digital microscope. Hemodynamic, echocardiographic, and electrophysiologic parameters were also evaluated. Patients with severe fibrosis had longer QRS duration and positive late potential (LP) on signal-averaged ECG, resulting in a higher incidence of VT/VF. At the 5±4 year follow-up, VT/VF occurred in 31 (17%) patients. Multivariate Cox regression analysis revealed that tissue fibrosis (hazard ratio (HR): 1.65; P=0.003 per 10% increase), lower left ventricular ejection fraction (HR: 0.64; P=0.001 per 10% increase), and positive SAECG (HR: 3.14; P=0.04) led to a greater risk of VT/VF. The combination of tissue fibrosis severity and lower left ventricular ejection fraction could be used to stratify the risk of lethal arrhythmic events in HCM patients.

Conclusions: Myocardial fibrosis in RV biopsy samples may contribute to abnormal conduction delay and spontaneous VT/VF, leading to a poor prognosis in HCM patients. (Circ J 2015; 79: 1733–1741)

Hypertrophic cardiomyopathy (HCM) is usually recognized by left ventricular (LV) hypertrophy on echocardiography or a family history of HCM.¹ Histopathological changes, including myocardial hypertrophy, tissue fibrosis, or myocardial disarray,^2,3 may cause a distorted impulse propagation and inhomogeneous refractoriness, a substrate of electrical instability during tachycardia, which can lead to ventricular tachycardia (VT) or ventricular fibrillation (VF) and sudden cardiac death (SCD).

The natural history of HCM patients varies from an asymptomatic benign course to a poor prognosis because of heart failure (HF), lethal ventricular arrhythmias, or SCD.⁴ Therefore, risk stratification in HCM patients has been a major issue. A positive late potential (LP) detected by signal-averaged electrocardiography (SAECG) has been used as a marker of electrical instability,⁵ although myocardial scarring visualized by cardiac magnetic resonance (CMR) imaging can better predict long-term clinical outcome compared with other risk factors such as syncope and family history of SCD.^6–8 Myocardial fibrosis, as measured by late gadolinium enhancement (LGE) on CMR, was recently found to be an independent predictor of adverse outcome in HCM patients.^9,10 However, there are only a few case reports of the relationship between CMR-LGE and direct fibrotic changes.^11,12 It remains unclear whether or not histopathological changes are associated with the risk of VT/VF and SCD in HCM patients.

In this study, we hypothesized that advanced myocardial fibrosis in HCM plays a critical role in lethal arrhythmic events, including VT/VF, implantable cardioverter-defibrillator (ICD) appropriate discharge, and SCD. We therefore quantified the fibrotic change in tissue samples from right ventricular (RV) biopsy and assessed its relevance to the long-term prognosis of HCM patients. This study examined the novel quantitative significance of tissue fibrosis in HCM patients associated with electrophysiological conduction abnormalities that lead to VT/VF and poor prognosis.

Methods

Diagnosis of Patients

We retrospectively surveyed 494 consecutive patients who had undergone RV endomyocardial biopsy at the National Cerebral and Cardiovascular Center between 1996 and 2011. The diagnosis of HCM was made on the basis of typical clinical, echocardiographic, and hemodynamic features according to established criteria,¹ used for a number of years, in the presence of LV wall thickness ≥15 mm without dilated ventricular chambers or any other cardiac or systemic disorders, including aortic stenosis or marked hypertension at the time of clinical diagnosis. In this study, the borderline LV hypertrophy criterion (LV wall thickness 13–14 mm) was not applicable because genetic examinations were not performed in this cohort. Asymmetric hypertrophy was originally applied to patients with conventional septal hypertrophy; however, the pattern or distribution of LV hypertrophy was not taken into account as per the latest recommendation.¹ Thus, asymmetric hypertrophy is determined if the LV thickness ratio of maximum to minimum in the same cross-section exceeds 1.3.

RV Biopsy and Histopathological Analysis

RV endomyocardial biopsy was performed in this cohort because of (1) differential diagnoses for other cardiomyopathies, such as amyloidosis, Fabry’s disease, sarcoidosis, or hypertensive heart disease; (2) atypical progression of LV dysfunction; or (3) new-onset HF despite preserved left ventricular ejection fraction (LVEF). We excluded patients younger than 20 years old because myocardial features may change with age. We also excluded male and female patients older than 75 and 80 years old, respectively. Patients with coexisting valvular diseases responsible for cardiomyopathy were also excluded.²

A total of 238 patients were clinically diagnosed and pathologically confirmed to have HCM (including 114 HCM with overt LV dysfunction¹⁰ defined as LVEF <50%); 53 patients were excluded because their tissue samples (Masson’s staining) had deteriorated over time. Finally, 185 patients (mean age 57±14 years, 62% male) were evaluated. This study was approved by the institutional ethics committee (M24-071).

Biopsy samples were obtained from the endocardium at the right interventricular septum using disposable biopsy forceps (Toyokura Ika Kogyo Co, Ltd, Tokyo, Japan) by the transvenous approach via the femoral vein or the right jugular vein, as described elsewhere.¹³ The detailed tissue sample preparation methods are described in Supplementary File 1. The extent of tissue fibrosis was automatically calculated by the area of fibrosis (%) in the total area of the Masson’s trichrome sample using a digital microscope (Aperio Scanscope, Aperio Technology, Vista, CA, USA) (Figure S1), which has been utilized for calculating myocardial fibrosis elsewhere.¹² The degree of myocardial disarray was graded from 0 to 5, as described in Table S1). Separate from the quantitative risk assessment, tissue fibrosis was qualitatively classified into 3 degrees: mild (<10% area of fibrosis in specimens), moderate (10–20%), and severe (>20%), as previously reported¹⁴ for further risk stratification, with and without other prognostic factors.

Electrophysiological Analysis

A standard 12-lead ECG was recorded in all patients. The SAECG was recorded from the X, Y, and Z orthogonal leads. LP was defined as present when at least 2 of the following 3 criteria were positive: filtered QRS duration (fQRS) >120 ms; root-mean-square voltage in the terminal 40 ms (RMS40) <18 µV; and duration of the low amplitude signal <40 µV (LAS40) >38 ms. The detailed electrophysiological protocol is shown in Supplementary File 1.

Echocardiography

After patients with significant valvular disease were excluded, the echocardiographic measurements were performed as follows: the end-diastolic and end-systolic dimensions were measured on the parasternal view at the level of papillary muscles and the left atrial size was measured on the parasternal long-axis view. Measurement of maximum wall thickness and definition of asymmetric hypertrophy were described above.

Hemodynamic Study

All patients underwent catheterization for hemodynamic evaluation. The LVEF was measured using left ventriculography, CMR imaging, or radio nuclear imaging. All patients were examined by right heart catheterization to assess hemodynamics. Coronary angiography was performed in all patients during their first hospitalization for diagnosis or within the year prior.

CMR-LGE Analysis

Of the 185 total patients, 60 underwent CMR using the gadolinium-enhanced imaging technique. The detailed CMR protocol and its LGE analysis were described previously¹⁵ and are described in Supplementary File 1. In brief, CMR was performed on a 1.5-T MR scanner (Magnetom Sonata, Siemens, Erlangen, Germany) and LGE used a segmented inversion-recovery (IR) prepared true-FISP sequence with ECG triggering at 2, 5, 10, and 20 min after the administration of 0.15 mmol/kg of gadolinium-DTPA (Magnevist, Bayer Schering Pharma, Berlin, Germany). For quantification of LV mass, we semi-automatically traced the LV endocardial and epicardial contours at end-diastole in each short-axis slice of 7 sections using customized software (Ziostation2; Ziosoft Inc, Tokyo, Japan). A region of interest (ROI) was selected within the normal remote myocardium to generate the mean and standard deviation (SD) for the various SDs. The mass of LGE (%LGE) was automatically calculated with the same software as regions exhibiting a signal intensity above a predetermined threshold (4 SD above the mean signal intensity of apparently normal myocardium).¹²

Follow-up

Patient follow-up began on the day of biopsy. Patients were tracked through outpatient visits every 1–3 months or were followed at ICD check-ups every 6 months. The endpoint of the study was lethal arrhythmic events defined as sustained VT or VF, ICD appropriate discharge, or aborted SCD during the follow-up period. SCD was diagnosed if the patient underwent a sudden collapse within 1 h of onset of symptoms without any previous cardiac manifestation.

Statistical Analysis

Continuous variables are expressed as the mean±SD, median (interquartile range of 25–75%), or n (%). Comparison among the 3 groups was made using Tukey’s method for continuous variables to adjust multiplicity, applying P<0.05 as the significance level. Bonferroni’s method was used for categorical variables, applying P<0.016 among the 3 groups as the significance level. Survival curves were calculated by the Kaplan-Meier method using the log-rank test for group comparison among the extent of graded tissue fibrosis (<10%, 10–20%, and >20%). All variables with a P-value <0.05 in the univariate analysis were considered candidates for inclusion in the multivariate analysis. Cox proportional hazard regression adjustment was performed to calculate the hazard ratio (HR) in the multivariate analysis. All analyses were performed with JMP version 9 software (SAS Institute Inc, Cary, NC, USA).

Results

Baseline Characteristics

The baseline characteristics of the 185 patients are shown in Table 1; 76 (41%) patients had a history of hospitalization for HF or arrhythmia, and 26 (14%) had a history of VT/VF, in which nonsustained VT was not included. ICD or cardiac resynchronization therapy with defibrillator (CRT-D) was undertaken in 5 patients at baseline. The baseline LVEF, pulmonary capillary wedge pressure (PCWP), B-type natriuretic peptide (BNP) concentration, and maximum LV wall thickness were 47±19%, 12±7 mmHg, 256 (IQR: 137–506) pg/ml, and 17±6 mm, respectively, at the time of biopsy.

Table 1. Baseline Characteristics of Patients With HCM

	Total (n=185)	Tissue fibrosis			P value
		Mild (n=58)	Moderate (n=78)	Severe (n=49)
Age	57±14	59±12	56±13	57±16	NS
Sex (% male)	114 (62)	34 (59)	53 (68)	27 (55)	NS
Family history of SCD, n (%)	16 (9)	4 (7)	6 (8)	6 (12)	NS
Hypertension, n (%)	72 (40)	24 (42)	31 (40)	17 (36)	NS
Diabetes mellitus, n (%)	31 (17)	11 (20)	12 (15)	8 (17)	NS
Atrial fibrillation, n (%)	70 (38)	16 (28)	21 (27)	18 (37)	NS
Syncope, n (%)	41 (22)	9 (16)	18 (23)	14 (29)	NS
Prior NSVT, n (%)	55 (30)	13 (22)	25 (32)	17 (36)	NS
Prior sustained VT/VF, n (%)	26 (14)	5 (9)	9 (12)	12 (24)	NS
Prior hospitalization, n (%)	76 (41)	15 (26)	35 (45)	26 (53)*	0.01 vs. mild*
Echo and hemodynamic parameters
LVEF, %	47±19	45±19	48±19	47±20	NS
Max. wall thickness, mm	17±6	16±6	17±6	18±6	NS
Wall thickness >30 mm, n (%)	5 (3)	2 (3)	2 (3)	1 (2)	NS
Asymmetric hypertrophy, n (%)	82 (44)	28 (48)	31 (40)	23 (47)	NS
Max. PG >30 mmHg, n (%)	41 (22)	9 (16)	23 (29)	9 (18)	NS
BNP, pg/ml (IQR)	256 (137–506)	221 (116–470)	278 (126–502)	272 (175–613)	NS
PCWP, mmHg	12±7	10±6	12±6	13±7*	0.03 vs. mild*
Pathological parameters
Myocyte diameter, μm	21±5	20±4	21±5	22±4	NS
Myocardial disarray, grade: 0–5	2.6±1.3	2.4±1.4	2.6±1.1	2.7±1.3	NS
ECG and electrophysiology
QRS duration, ms	119±30	114±24	121±27	122±39	NS
QTc interval, ms	454±67	452±72	446±52	468±81	NS
LAS40, ms	32±22	28±16	30±17	41±29*	0.03 vs. mild*
fQRS, ms	120±30	114±28	122±30	123±34	NS
RMS voltage, μV	63±64	67±58	64±63	57±71	NS
LP(+) by SAECG, n/total N (%)	30/123 (24)	8/38 (21)	9/53 (17)	13/32 (41)	NS
CMR parameters (n=60)
LV mass, g	165±54	158±58	179±54	148±45	NS
LGE % LV mass (4SD), %	31±18	32±19	28±18	35±15	NS
Medication, treatment
ICD/CRT-D at diagnosis, n (%)	5 (3)	0 (0)	3 (4)	2 (4)	NS
β-blocker, n (%)	68 (37)	16 (28)	34 (44)	8 (16)	NS
ACEI/ARB, n (%)	79 (43)	26 (45)	31 (40)	22 (45)	NS
Amiodarone, n (%)	14 (8)	1 (2)	5 (6)	8 (16)*	0.01 vs. mild*
Other antiarrhythmics, n (%)	43 (23)	10 (17)	20 (26)	12 (24)	NS

*Statistically difference between mild and severe. ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker; BNP, B-type natriuretic peptide; CMR, cardiac magnetic resonance imaging; CRT-D, cardiac resynchronization therapy with defibrillator; fQRS, total filtered QRS duration; HCM, hypertrophic cardiomyopathy; ICD, implantable cardioverter-defibrillator; LAS40, duration of the low amplitude signal <40 μV; LP(+), positive late potential; LVEF, left ventricular ejection fraction; NS, not significant; NSVT, nonsustained ventricular tachycardia; PCWP, pulmonary capillary wedge pressure; PG, pressure gradient in left ventricle; RMS, root-mean-square; SAECG, signal-averaged electrocardiogram; SCD, sudden cardiac death; VT/VF, ventricular tachycardia/ventricular fibrillation.

Clinical and Histopathological Changes

The average tissue area was 2.34±1.38 mm². In the tissue sample measurements, the fibrosis ratio (% area) was 15.7±9.8% and the distribution of fibrotic change in all samples is shown in Figure 1A. The level of fibrosis was classified as mild (<10%; n=58), moderate (10–20%; n=78), or severe (>20%; n=49). A representative tissue sample of each group is shown in Figure 1B.

Figure 1.

Quantitative fibrotic change in right ventricular (RV) biopsy specimens from hypertrophic cardiomyopathy patients with mild, moderate or severe fibrosis. (A) Distribution of fibrosis (%) in all samples. (B) Representative RV biopsy specimens stained by Masson’s trichrome showing mild (3.9% of area), moderate (18.2%) or severe (31.7%) fibrosis, which represents the fibrosis of the entire area, including endomyocardial thickening and perivascular fibrosis.

As shown in Table 1, no significant correlation was found among the groups for age, sex, history of hypertension, diabetes mellitus, atrial fibrillation, or other conventional risk factors, including family history of SCD, syncope, maximum wall thickness, and pressure gradient. A history of hospitalization for HF or arrhythmia was more common in patients with severe (n=26, 53%) tissue fibrosis compared with mild (n=15, 26%) tissue fibrosis (P=0.01).

LVEF and plasma BNP were not associated with the degree of fibrosis at the time of diagnosis. On the other hand, PCWP was higher in patients with severe fibrosis compared with mild fibrosis (P=0.03). The mean myocyte diameter (21±5 µm) and degree of myocardial disarray (2.6±1.3) in the total cohort did not differ among the groups.

Conduction Abnormality and Lethal Ventricular Arrhythmias

Figure 2 shows representative tissue samples of mild (6.8%) and moderate (16.6%) fibrotic change in HCM patients. Although LVEF and QRS duration on ECG were comparable in these 2 patients, a longer filtered QRS duration, LAS40, and thus positive LP were detected in the patient with moderate fibrosis. Although not all patients underwent SAECG (n=123), increased fibrosis (%area) was mildly associated with longer LAS40 (r²=0.07, P<0.01) (Figure S2). LAS40 was larger in patients with severe fibrosis compared with mild fibrosis (P<0.05) (Table 1).

Figure 2.

Representative right ventricular biopsy specimens of mild or moderate fibrosis and the ECG and SAECG parameters. Representative biopsy specimens of mild (6.8%) fibrosis from a 34-year-old male hypertrophic cardiomyopathy (HCM) patient with left ventricular ejection fraction (LVEF)=68% (A) and moderate (16.6%) fibrosis from a 51-year-old male HCM patient with LVEF=51% (B). Their ECG and SAECG (Lower panels) show significant LV hypertrophy with inverted T-waves in both cases, but a longer filtered QRS duration and positive late potential detected by SAECG and fragmented QRS in the patient with moderate fibrosis compared with the patient with mild fibrosis. SAECG, signal-averaged ECG.

Next, we compared the degree of tissue fibrosis and the development of lethal ventricular arrhythmias. As shown in Figure 3A, the degree of fibrosis at the time of HCM diagnosis was significantly associated with subsequent lethal ventricular arrhythmias. During the 5±4 year follow-up period, 31 patients had lethal arrhythmic events (15 cases of sustained VT or VF, 3 of SCD, and 13 of appropriate ICD discharge). These events occurred in 5 of 58 (9%) patients with mild fibrosis, in 10 of 78 (13%) patients with moderate fibrosis, and in 16 of 49 (33%) patients with severe fibrosis (HR: 5.43, 95% confidence interval (CI): 2.12–16.6; P=0.0003; severe vs. mild). The total number of patients with lethal arrhythmic events, including prior and subsequent VT/VF or SCD, was larger in the group of patients with severe fibrosis (n=20, 41%) compared with mild (n=8, 14%) or moderate (n=18, 23%) fibrosis (P=0.003, severe vs. mild) (Table 2). On the other hand, as shown in Figure 3B, patients with lower LVEF (≤50%) had a higher risk of lethal arrhythmic events than those with preserved LVEF (P<0.0001).

Figure 3.

Lethal arrhythmic events and degree of tissue fibrosis or left ventricular ejection fraction (LVEF). Kaplan-Meier unadjusted estimates of freedom from lethal arrhythmic events or sudden cardiac death according to the degree of fibrotic change (A) or LVEF (B) in 185 patients with hypertrophic cardiomyopathy.

Table 2. Tissue Fibrosis and Lethal Ventricular Arrhythmias in Patients With HCM

	Tissue fibrosis			P value
	Mild (n=58)	Moderate (n=78)	Severe (n=49)
ICD or CRT-D, n (%)	8 (15)	19 (24)	19 (39)*	0.009 vs. mild*
Prior sustained VT/VF, n (%)	5 (9)	9 (12)	12 (24)	NS
Sustained VT, n	4	6	8
Spontaneous VF, n	1	3	4
Subsequent VT/VF or SCD, n (%)	5 (9)	10 (13)	16 (33)*,†	0.006 vs. mild* 0.02 vs. moderate†
Sustained VT without ICD, n	0	3	5
Spontaneous VF without ICD, n	1	5	1
SCD without ICD, n	2	1	0
Appropriate ICD discharge, n	2	1	10
Total VT/VF or SCD, n (%)	8 (14)	18 (23)	20 (41)*	0.003 vs. mild*

*Statistically significant difference between mild and severe. ^†Statistically significant difference between moderate and severe. Abbreviations as in Table 1.

CMR-LGE Analysis

Of the 185 clinically diagnosed and pathologically confirmed HCM patients, CMR was performed in 60 to show fibrotic change by LGE analysis. The LV mass of LGE (LGE %LV mass index) was calculated as the region exhibiting a signal intensity >4 SD. The averaged LGE %LV mass was 31±18% (range 2–68%). There was no correlation between tissue fibrosis from biopsy and LGE %LV mass by CMR-LGE (Figure S3A). Only in the severe fibrosis group was a significant correlation (P<0.05) observed between tissue fibrosis and the LGE %LV mass from CMR-LGE (Figure S3B).

Univariate and Multivariate Analyses

As shown in Table 3, univariate analysis revealed that a gradual increase of tissue fibrosis as well as cell diameter, LVEF, LAS40, fQRS, RMS voltage (ie, positive LP) by SAECG, and QRS duration on the 12-lead ECG were associated with subsequent lethal arrhythmic events, including VT/VF, ICD appropriate discharge, and SCD in HCM patients. Furthermore, multivariate analysis revealed that patients with a higher level of tissue fibrosis (HR: 1.65, 95% CI: 1.19–2.28; P=0.003 per 10% increase), lower LVEF (HR: 0.64, 95% CI: 0.48–0.84; P=0.001 per 10% increase), and positive SAECG (HR: 3.14, 95% CI: 1.06–8.61; P=0.04) were prognostic in predicting future lethal arrhythmias.

Table 3. Univariate and Multivariate Analysis for Lethal Arrhythmic Events in Patients With HCM (n=31/185)

	Univariate analysis			Multivariate analysis
	HR	95% CI	P value	HR	95% CI	P value
Age (/year)	0.97	0.95–1.00	0.05
Male sex	1.56	0.74–3.58	0.24
Tissue fibrosis (/10%)	2.57	1.56–4.39	0.0002	1.65	1.19–2.28	0.003
Cell diameter (/μm)	1.07	1.00–1.14	0.04	0.99	0.90–1.07	0.82
Disarray (/1 grade)	1.05	0.79–1.38	0.75
Max. wall thickness (/mm)	0.98	0.92–1.04	0.53
Asymmetric hypertrophy	0.85	0.37–1.91	0.70
LGE %LV mass (4 SD) (/%)	1.04	1.00–1.10	0.06
LVEF (/10%)	0.67	0.54–0.82	<0.0001	0.64	0.48–0.84	0.001
PCWP (/mmHg)	1.04	0.99–1.09	0.12
LAS40 (/5 ms)	1.14	1.07–1.20	0.0002
fQRS (/10 ms)	1.15	1.04–1.25	0.008
RMS voltage (/μV)	0.99	0.97–1.00	0.002
Positive LP by SAECG	5.11	2.29–11.5	0.0001	3.14	1.06–8.61	0.04
QRS duration (/10 ms)	1.16	1.06–1.25	0.001	0.94	0.82–1.06	0.32
QTc interval (/10 ms)	0.76	0.95–1.06	0.76

CI, confidence interval; HR, hazard ratio; LGE, late gadolinium enhancement by CMR. Other abbreviations as in Table 1.

Risk Stratification

Positive LP was only found in 24% of patients with a SAECG recording (Table 1), so LP had a higher specificity but a lower sensitivity for composite cardiac events in this study. To assess the predictive value of classification schemes that estimate lethal arrhythmic events in patients with HCM, we defined the combined risk score (0–3) formed by the sum of each independent risk factor: the degree of tissue fibrosis (mild=0, moderate=1, and severe=2) and LVEF (>50%=0, ≤50%=1). Patients with higher scores tended to have a greater risk of lethal arrhythmic events (Figure 4).

Figure 4.

Lethal arrhythmic events and cumulative score by tissue fibrosis and left ventricular ejection fraction (LVEF). Kaplan-Meier unadjusted estimates of freedom from lethal ventricular arrhythmias or sudden cardiac death according to the cumulative risk score from tissue fibrosis (mild=0, moderate=1, severe=2) and LVEF (>50=0, ≤50=1) in 185 patients with hypertrophic cardiomyopathy.

Discussion

New Findings

To the best of our knowledge, this is the first study to demonstrate the prognostic value of fibrotic change in tissue samples by biopsy quantitatively examined in a significant number of HCM patients. The severity of fibrosis in myocardial biopsy, a positive LP on SAECG, and lower LVEF were associated with a greater risk of lethal arrhythmic events in HCM patients. These findings provide novel insight into lethal ventricular arrhythmias and a new approach to estimating the prognosis of HCM patients.

Clinical Significance of Fibrosis in HCM

Numerous postmortem studies have demonstrated that myocardial fibrosis (interstitial or replacement) in HCM patients is distinct from that observed in patients with coronary artery disease or dilated cardiomyopathy.^16,17 A key mechanism involved in adverse outcomes in HCM is believed to be myocardial fibrosis, which is a pathological hallmark of the condition,¹⁸ and can be identified by biopsy.^19,20 Recent studies of HCM patients suggest that the extent of fibrosis as measured by CMR correlates with histologically proven myocardial scarring¹¹ and is associated with worse prognosis,¹⁰ including arrhythmic events.^6,9,21 However, in this study, fibrotic change (LGE %LV mass) by CMR-LGE did not reach statistical significance for the prediction of lethal arrhythmic events (HR=1.04, 95% CI: 1.00–1.10, P=0.06) (Table 3). To the best of our knowledge, only a few reports have compared CMR and histopathology with a focus on fibrosis;^11,12,22 segments containing >15% collagen were more likely to show LGE. However, the LGE technique cannot be used to visualize diffuse fibrosis²³ and it should be noted that the averaged fibrosis in this study was 15.7±9.8%, which may be difficult to detect by CMR-LGE. No significant relationship was observed between LGE %LV mass by CMR-LGE and tissue fibrosis in myocardial biopsy, especially in cases of mild or moderate fibrotic HCM (Figure S3B).

In this study, the severity of fibrosis, a positive LP, and lower LVEF were significantly associated with prognosis, especially for subsequent lethal arrhythmic events (Table 3, Figure 3). However, disarray was not correlated to the prognosis of patients aged between 20 and 75 (male) or 80 (female) years. These findings are consistent with a previous study that found that the prevalence of disarray was high in HCM patients who died suddenly before 21 years of age.²² Thus, myocardial disarray may play an important role in the prognosis of younger HCM patients.

Promotion of Conduction Abnormality and VT/VF by Myocardial Fibrosis

Fibrous tissue promotes re-entrant ventricular arrhythmias and contributes to increased ventricular stiffness. In a coculture model, increased myofibroblast/myocyte area decreased conduction velocity and degenerated a spiral re-entry into multiple waves, like a VF.²⁴ Thus, increased myocardial fibrosis and disarray in HCM usually decreases excitation propagation, leading to a conduction delay or block, a substrate of re-entrant arrhythmias. SAECG can noninvasively evaluate a delayed potential as a substrate of ventricular arrhythmias in several diseases, although a previous study suggested that SAECG was not always useful for identifying HCM patients with VT or SCD.⁵ Positive LP was found in only 24% of the present patients who underwent SAECG recording (Table 1), so LP had a higher specificity but a lower sensitivity for composite cardiac events in this study. The electrophysiological consequence of this substrate has been well demonstrated by Schumacher et al.²⁵ LV regional extensive hypertrophy and myocardial scarring are associated with local conduction delay and conduction block, which may contribute to the increased incidence of VT/VF in patients with HCM.

A prolonged QRS duration on 12-lead ECG is associated with an increased risk of cardiovascular death by HF and cardiomyopathy, including in HCM.²⁶ Kamiyama et al reported that QRS duration on the 12-lead ECG was much longer in patients with dilated HCM compared with patients with dilated cardiomyopathy.²⁷ Kawara et al reported the correlation of conduction delay with a fibrotic tissue pattern in chronic diseased myocardium, including HCM, particularly in areas of patchy fibrosis.²⁸ In this study, QRS duration on standard 12-lead ECG was associated with lethal arrhythmic events only in the univariate analysis (Table 3); however, the severity of fibrosis in the tissue samples was weakly associated with a longer delayed potential (LAS40) (Figure S2). These findings suggest that the increased fibrosis in HCM associated with longer QRS duration and positive LP represented by prolonged delayed potential detected by SAECG indicates an abnormal conduction delay and may contribute at least in part to the increased incidence of lethal ventricular arrhythmias or SCD.

Prognositic Effect of Tissue Fibrosis and Its Potential for Risk Stratification

Sudden unexpected death is a well-recognized and devastating consequence of HCM. A previous cohort study²⁹ demonstrated that an appropriate ICD shock was delivered at a rate of 5.6%/year in HCM patients (n=506, mean age 42±17) during 3.7±3-year follow-up. It is of note that patients treated with ICD primarily for prevention also showed a substantial appropriate intervention rate (reported to be 4%/year). Thus, identifying patients with HCM who are at highest risk of SCD is a major problem. The conventional risk factors for the primary prevention of SCD in HCM are family history of SCD, unexplained syncope, multiple-repetitive nonsustained VT, abnormal exercise blood pressure response, or massive LV hypertrophy.¹ However, no significant difference was observed among patients with 1, 2, or ≥3 of these parameters with respect to the likelihood of appropriate ICD discharge.²⁹ Therefore, this risk stratification cannot always guide SCD prevention in precise terms for each HCM patient, and SCD is also known to occur in patients without any of the aforementioned risk factors.

Myocardial fibrosis measured by LGE-CMR was recently used as an independent predictor of adverse outcome in HCM patients.^9,14 However, LGE-CMR imaging mainly detects focal fibrosis and does not detect microscopic diffuse fibrosis. In contrast, CMR-T1 mapping may quantify diffuse as well as focal fibrosis.³⁰ Histopathological features related to unstable electrophysiological substrate may lead to lethal ventricular tachyarrhythmias and SCD.³¹ In this study, we directly quantified the fibrotic changes in tissue samples and assessed its relevance to the long-term prognosis in HCM patients. These pathophysiologic changes may represent both micro-level and global fibrosis in HCM. Thus, increased fibrosis in the tissue samples of RV biopsy, as well as positive SAECG, QRS duration, and lower LVEF, can lead to VT/VF.

Study Limitations

Although this was a single-center, retrospective study, all patients that were enrolled underwent a biopsy of the RV septum after being admitted to the hospital. RV biopsy was not routinely performed in HCM patients, but might be recommended in HCM patients with increasing LV diameter and reducing LV contractions, which are likely related to increased fibrotic change.³¹ This cohort was slightly biased and had a poorer prognosis than general, asymptomatic HCM patients. As such, it remains unclear whether these findings are applicable to asymptomatic HCM patients. Second, no genetic testing data were obtained in this study, and genetic disorders may affect the prognosis. Third, the endomyocardial biopsy was performed from the RV septum, but not the LV, and does not represent the entire heart; thus, only a limited number of samples could be evaluated. As such, there is a possibility that the results underestimated the overall fibrosis. Despite these limitations, this study demonstrated the clinical significance of tissue fibrosis and the physiological parameters for patients with HCM who are at risk of adverse cardiac events.

Conclusions

Fibrotic changes observed in tissue samples from RV biopsies play an important role in the development of lethal ventricular arrhythmias in HCM patients with impaired systolic function. When combined with the LV systolic function, the extent of tissue fibrosis may assist in the risk stratification of HCM patients.

Acknowledgments

This work was supported by grants from the Ministry of Health, Labour, and Welfare of Japan (2010-145); a Grant-in-Aid for Scientific Research on Innovative Areas (22136011 A02, Aiba); a Grant-in-Aid for Scientific Research (C) (24591086 Aiba) from MEXT of Japan; a Research Grant for Cardiovascular Diseases (H24–033 Shimizu, Aiba) from the Ministry of Health, Labour, and Welfare of Japan; and an Intramural Research Fund for Cardiovascular Diseases of the National Cerebral and Cardiovascular Center (26-6-6 Wada).

Disclosures

Conflict of Interests: None.

Supplementary Files

Supplementary File 1

Methods

Table S1. Grade of myocardial disarray

Figure S1. (A,B) Representative biopsy samples from a single patient used to calculate the blue (fibrosis) area, in which the fibrosis (%area) was calculated by simply circling all tissue areas and then automatically calculating the ratio of blue in the total area.

Figure S2. Relationship between tissue fibrosis (%-area) from a right ventricular biopsy and duration of low amplitude signal <40 µV (LAS40, ms) by signal-averaged ECG (SAECG) in patients with hypertrophic cardiomyopathy.

Figure S3. (A) Relationship between CMR-LGE %LV mass and tissue fibrosis by myocardial biopsy. (B) Sub-analysis of the relationship by degree of tissue fibrosis; mild (<10%), moderate (10–20%) and severe (>20%).

Please find supplementary file(s);

http://dx.doi.org/10.1253/circj.CJ-15-0104

References

• 1.    Gersh  BJ,  Maron  BJ,  Bonow  RO,  Dearani  JA,  Fifer  MA,  Link  MS, et al. 2011 ACCF/AHA Guideline for the Diagnosis and Treatment of Hypertrophic Cardiomyopathy: A report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Developed in collaboration with the American Association for Thoracic Surgery, American Society of Echocardiography, American Society of Nuclear Cardiology, Heart Failure Society of America, Heart Rhythm Society, Society for Cardiovascular Angiography and Interventions, and Society of Thoracic Surgeons. J Am Coll Cardiol 2011; 58: e212–e260, doi:10.1016/j.jacc.2011.06.011.
• 2.    Davies  MJ,  McKenna  WJ. Hypertrophic cardiomyopathy: Pathology and pathogenesis. Histopathology 1995; 26: 493–500.
• 3.    Maron  BJ,  Maron  MS. Hypertrophic cardiomyopathy. Lancet 2012; 381: 242–255.
• 4.    Maron  BJ,  Rowin  EJ,  Casey  SA,  Haas  TS,  Chan  RH,  Udelson  JE, et al. Risk stratification and outcome of patients with hypertrophic cardiomyopathy >=60 years of age. Circulation 2013; 127: 585–593.
• 5.    Cripps  TR,  Counihan  PJ,  Frenneaux  MP,  Ward  DE,  Camm  AJ,  McKenna  WJ. Signal-averaged electrocardiography in hypertrophic cardiomyopathy. J Am Coll Cardiol 1990; 15: 956–961.
• 6.    Bruder  O,  Wagner  A,  Jensen  CJ,  Schneider  S,  Ong  P,  Kispert  EM, et al. Myocardial scar visualized by cardiovascular magnetic resonance imaging predicts major adverse events in patients with hypertrophic cardiomyopathy. J Am Coll Cardiol 2010; 56: 875–887.
• 7.    Hen  Y,  Iguchi  N,  Utanohara  Y,  Takada  K,  Machida  H,  Takayama  M, et al. Prognostic value of late gadolinium enhancement on cardiac magnetic resonance imaging in japanese hypertrophic cardiomyopathy patients. Circ J 2014; 78: 929–937.
• 8.    Moon  J,  Hong  YJ,  Kim  YJ,  Shim  CY,  Jang  Y,  Chung  N, et al. Extent of late gadolinium enhancement on cardiovascular magnetic resonance imaging and its relation to left ventricular longitudinal functional reserve during exercise in patients with hypertrophic cardiomyopathy. Circ J 2013; 77: 1742–1749.
• 9.    O’Hanlon  R,  Grasso  A,  Roughton  M,  Moon  JC,  Clark  S,  Wage  R, et al. Prognostic significance of myocardial fibrosis in hypertrophic cardiomyopathy. J Am Coll Cardiol 2010; 56: 867–874.
• 10.    Olivotto  I,  Cecchi  F,  Poggesi  C,  Yacoub  MH. Patterns of disease progression in hypertrophic cardiomyopathy: An individualized approach to clinical staging. Circ Heart Fail 2012; 5: 535–546.
• 11.    Moon  JC,  Reed  E,  Sheppard  MN,  Elkington  AG,  Ho  SY,  Burke  M, et al. The histologic basis of late gadolinium enhancement cardiovascular magnetic resonance in hypertrophic cardiomyopathy. J Am Coll Cardiol 2004; 43: 2260–2264.
• 12.    Moravsky  G,  Ofek  E,  Rakowski  H,  Butany  J,  Williams  L,  Ralph-Edwards  A, et al. Myocardial fibrosis in hypertrophic cardiomyopathy: Accurate reflection of histopathological findings by CMR. JACC Cardiovasc Imaging 2013; 6: 587–596.
• 13.    Holzmann  M,  Nicko  A,  Kuhl  U,  Noutsias  M,  Poller  W,  Hoffmann  W, et al. Complication rate of right ventricular endomyocardial biopsy via the femoral approach: A retrospective and prospective study analyzing 3048 diagnostic procedures over an 11-year period. Circulation 2008; 118: 1722–1728.
• 14.    Chan  RH,  Maron  BJ,  Olivotto  I,  Pencina  MJ,  Assenza  GE,  Haas  T, et al. Prognostic value of quantitative contrast-enhanced cardiovascular magnetic resonance for the evaluation of sudden death risk in patients with hypertrophic cardiomyopathy. Circulation 2014; 130: 484–495.
• 15.    Kono  AK,  Yamada  N,  Higashi  M,  Kanzaki  S,  Hashimura  H,  Morita  Y, et al. Dynamic late gadolinium enhancement simply quantified using myocardium to lumen signal ratio: Normal range of ratio and diffuse abnormal enhancement of cardiac amyloidosis. J Magn Reson Imaging 2011; 34: 50–55.
• 16.    Maron  BJ,  Wolfson  JK,  Epstein  SE,  Roberts  WC. Intramural (“small vessel”) coronary artery disease in hypertrophic cardiomyopathy. J Am Coll Cardiol 1986; 8: 545–557.
• 17.    Varnava  AM,  Elliott  PM,  Sharma  S,  McKenna  WJ,  Davies  MJ. Hypertrophic cardiomyopathy: The interrelation of disarray, fibrosis, and small vessel disease. Heart 2000; 84: 476–482.
• 18.    Mewton  N,  Liu  CY,  Croisille  P,  Bluemke  D,  Lima  JAC. Assessment of myocardial fibrosis with cardiovascular magnetic resonance. J Am Coll Cardiol 2011; 57: 891–903.
• 19.    Goto  D,  Kinugawa  S,  Hamaguchi  S,  Sakakibara  M,  Tsuchihashi-Makaya  M,  Yokota  T, et al. Clinical characteristics and outcomes of dilated phase of hypertrophic cardiomyopathy: Report from the registry data in Japan. J Cardiol 2012; 61: 65–70.
• 20.    Yilmaz  A,  Kindermann  I,  Kindermann  M,  Mahfoud  F,  Ukena  C,  Athanasiadis  A, et al. Comparative evaluation of left and right ventricular endomyocardial biopsy: Differences in complication rate and diagnostic performance. Circulation 2010; 122: 900–909.
• 21.    Adabag  AS,  Maron  BJ,  Appelbaum  E,  Harrigan  CJ,  Buros  JL,  Gibson  CM, et al. Occurrence and frequency of arrhythmias in hypertrophic cardiomyopathy in relation to delayed enhancement on cardiovascular magnetic resonance. J Am Coll Cardiol 2008; 51: 1369–1374.
• 22.    Varnava  AM,  Elliott  PM,  Mahon  N,  Davies  MJ,  McKenna  WJ. Relation between myocyte disarray and outcome in hypertrophic cardiomyopathy. Am J Cardiol 2001; 88: 275–279.
• 23.    Flett  AS,  Hayward  MP,  Ashworth  MT,  Hansen  MS,  Taylor  AM,  Elliott  PM, et al. Equilibrium contrast cardiovascular magnetic resonance for the measurement of diffuse myocardial fibrosis: Preliminary validation in humans. Circulation 2010; 122: 138–144.
• 24.    Zlochiver  S,  Munoz  V,  Vikstrom  KL,  Taffet  SM,  Berenfeld  O,  Jalife  J. Electrotonic myofibroblast-to-myocyte coupling increases propensity to reentrant arrhythmias in two-dimensional cardiac monolayers. Biophys J 2008; 95: 4469–4480.
• 25.    Schumacher  B,  Gietzen  FH,  Neuser  H,  Schummelfeder  J,  Schneider  M,  Kerber  S, et al. Electrophysiological characteristics of septal hypertrophy in patients with hypertrophic obstructive cardiomyopathy and moderate to severe symptoms. Circulation 2005; 112: 2096–2101.
• 26.    Bongioanni  S,  Bianchi  F,  Migliardi  A,  Gnavi  R,  Pron  PG,  Casetta  M, et al. Relation of QRS duration to mortality in a community-based cohort with hypertrophic cardiomyopathy. Am J Cardiol 2007; 100: 503–506.
• 27.    Kamiyama  N,  Nezuo  S,  Sawayama  T,  Kawahara  Y,  Samukawa  M,  Suetuna  R, et al. Electrocardiographic features differentiating dilated cardiomyopathy from hypertrophic cardiomyopathy. J Electrocardiol 1997; 30: 301–306.
• 28.    Kawara  T,  Derksen  R,  de Groot  JR,  Coronel  R,  Tasseron  S,  Linnenbank  AC, et al. Activation delay after premature stimulation in chronically diseased human myocardium relates to the architecture of interstitial fibrosis. Circulation 2001; 104: 3069–3075.
• 29.    Maron  BJ,  Spirito  P,  Shen  WK,  Haas  TS,  Formisano  F,  Link  MS, et al. Implantable cardioverter-defibrillators and prevention of sudden cardiac death in hypertrophic cardiomyopathy. JAMA 2007; 298: 405–412.
• 30.    Ling  LH,  Kistler  PM,  Ellims  AH,  Iles  LM,  Lee  G,  Hughes  GL, et al. Diffuse ventricular fibrosis in atrial fibrillation: Noninvasive evaluation and relationships with aging and systolic dysfunction. J Am Coll Cardiol 2012; 60: 2402–2408.
• 31.    Shirani  J,  Pick  R,  Roberts  WC,  Maron  BJ. Morphology and significance of the left ventricular collagen network in young patients with hypertrophic cardiomyopathy and sudden cardiac death. J Am Coll Cardiol 2000; 35: 36–44.