Hereditary transthyretin amyloidosis (ATTR) is a group of diseases caused by mutated transthyretin (TTR), of which more than 100 amyloidogenic mutations are described (1). Significant phenotypic variations have been reported between mutations and also within the same mutation, but the most common symptoms are those of neuropathy and cardiomyopathy (1, 2). The expected survival for the majority of ATTR amyloidosis patients is approximately 8 to 15 years where the cause of death is commonly infections, malnutrition, and heart failure (3, 4).
Until 1990, only symptomatic treatment could be offered for ATTR amyloidosis. The concept of liver transplantation (LTx) is based on the fact that the circulating TTR is produced by the liver and hence the expected benefit of ceasing the production of the variant TTR, halting the amyloid formation, and stopping disease progression (5, 6). The success of the first LTx for ATTR encouraged the development of this protocol to be the preferred treatment for ATTR, which has to date been carried out in more than 1700 patients (FAPWTR registry, available at: htpp.www.fapwtr.org).
Despite the good overall clinical outcome from LTx, some patients continue to clinically deteriorate, particularly from heart complications (7–10). According to the international registry of LTx ATTR amyloid patients (FAPWTR), survival is reduced in non-TTR V30 M patients, and for some mutations such as TTR T60A and S52P, LTx is not recommended because of the rapid development of severe cardiomyopathy (10, 11).
Amyloid fibril composition in ATTR is not uniform, consisting either of a mixture of truncated and full-length ATTR (type A) or only full-length ATTR (type B) (12). Type A fibrils are more common in late-onset ATTR V30 M patients and are related to hypertrophic cardiomyopathy (13). In this respect such patients resemble those of senile systemic amyloidosis in whom the amyloid is derived from wild-type TTR, having type A as the only phenotypic pattern (12, 14). Senile systemic amyloidosis is a disease found in elderly males, a group of patients who also have an increased mortality and morbidity after LTx for ATTR amyloidosis (15).
In view of the above, we hypothesized that following LTx, ATTR amyloid patients with type A fibrils are at risk of developing deterioration of heart function, and we set out to study this relationship.
Patients with type A fibrils tended to be older than type B patients (P=0.07). There were no significant differences in time from onset of the disease to LTx or in duration from pre- to post-LTx echocardiography examination between the two groups. Observation period for the two groups was also similar (type A fibrils: median 4 [2–8] years; type B fibrils: 5.5 [3–8] years; P=0.2).
The individual patients’ characteristics and the outcome after LTx are presented in Tables 1 and 2. The most common reported symptoms were increased shortness of breath associated with physical exercise, which was taken as an indication of heart failure. It is, however, often difficult to assess the severity of heart failure, as neuropathy also limited patient’s physical activity. In addition, cardiac arrhythmia, such as conduction disturbances and atrial fibrillation were common, and many patients had pacemakers, because of conduction disturbances and heart failure. The symptoms and signs of cardiac complications are outlined in Table 2. Two patients (2 and 22) died of type A fibrils. All patients with type A fibrils developed signs and symptoms of heart failure, thus all had a N-terminal pro-brain natriuretic peptide above 100 ng/L (586–25,096) at the latest follow-up. In contrast, only seven patients with type B fibrils had developed signs and symptoms of heart failure at the latest follow-up.
Heart transplantation was considered for patients 2, 9, and 12, but none were accepted because of contraindications: newly treated gynecological cancer, high age, neuropathy, and deteriorating kidney function.
Fibril Type A Versus Fibril Type B
Echocardiography Before LTx
Patients with type A fibrils had significantly thicker posterior wall (P=0.027) and smaller end-systolic left ventricular (LV) volume (P=0.036), but LV ejection fraction (LVEF) was not different between groups (Tables 3 and 4).
Echocardiography After LTx
LV strain rate could not be measured in one type A patient (patient 24) and in one type B patient (patient 12). Type A patients had significantly thicker septum (P=0.002), posterior wall (P<0.001), and lower LVEF (P=0.031) and end-diastolic LV volume (P=0.012) compared with type B patients. Peak mitral early diastolic velocity, indexed to peak early myocardial velocities (E/e’) representing LV filling pressures was higher (P=0.019) and systolic LV global longitudinal strain rate (LVGLSR) reduced in type A patients (P=0.003), indicating a stiff left ventricle with increased filling pressures (Tables 3 and 4).
Pre Versus Post-LTx
Type A patients developed significantly increased interventricular septal (P=0.010) and posterior wall thickness (P=0.017), which did not occur to type B patients. In both groups, LV systolic and diastolic dimensions, stroke volume, cardiac output, mitral isovolumic relaxation time, and early diastolic velocity (E) deceleration time remained unchanged. However, only in type A patients, LVEF fell (P=0.005), in contrast, improvement was noted for some patients with type B fibrils (Fig. 1A). In addition, left atrial (LA) volume (P=0.009), E/e’ (P=0.043), and E/late diastolic velocity (A) ratio (P=0.015) increased in type A patients compared with the pretransplant measurements, indicating development of raised filling pressures. Type A patients developed significantly reduced myocardial LV deformation after LTx including systolic (P=0.015), early diastolic (P=0.018), and late diastolic LVGLSR (P=0.05), in contrast to type B patients, indicating a development of reduced intrinsic myocardial function in type A patients. In contrast, marked improvement of systolic LVGLSR was noted in several type B patients (Fig. 1B). The intra- and interobserver variability using deformation analysis from speckle tracking echocardiography has previously been published, ranging between 8% and 13% (16) (Tables 3 and 4).
To the best of our knowledge, this is the first study which provides a potential explanation for the fast development of cardiomyopathy and related death after LTx in some ATTR amyloidosis patients. We found significant differences between ATTR amyloidosis patients according to the biochemical identification of the fibril types, in heart structure and function and clinical outcome after LTx. Patients with fibrils consisting of a mixture of truncated and full-length ATTR (type A) developed progressive increase in myocardial thickness, in the absence of any change in blood pressure control and progressive deterioration of LV systolic function, marked by a drop in ejection fraction and diastolic function as shown by signs of raised LA pressure. In addition, type A patients had significantly poorer intrinsic myocardial function as shown by the longitudinal strain rate function. These abnormalities were not found, to such extent or prevalence, in type B patients. Furthermore, type A patients had worse clinical outcome. Among our patients, deterioration of some patients with type B fibrils could be explained on the basis of ischemic heart disease leading to heart failure. On the other hand, marked improvement of LVEF in several patients with type B fibrils, but in none with type A, further confirms the difference in the effect of the fibrils on myocardial function with type A being more malignant than type B.
Tacrolimus was the base of immune suppression in our patients, and it has been linked to development of cardiomyopathy in children. However, this has not been substantiated in adults (17), for whom ischemic heart disease has been the main heart complication after LTx, and seems related to metabolic effects of long-term immune suppression (18). One of our patients (patient 15) with type B fibrils developed myocardial infarct, which is not more than the expected incidence of ischemic cardiovascular events of 5% per 10 years in LTx patients (18).
A question may arise whether the deterioration in heart function after LTx is caused by amyloid deposits. Two autopsy studies on liver transplanted patients with ATTR have already shown a remarkable increased deposition of wild-type ATTR in the hearts after transplantation (19, 20), thus providing a supporting explanation to our findings. In fact, we have recently found a fast exchange of variant TTR in type A amyloid deposits, where virtually all variant TTR had disappeared from amyloid deposits within 2 years compared with 70% to 80% for type B fibrils (13). These findings suggest that type A fibrils are more accessible to continuous amyloid deposition from wild-type TTR than type B and may thus increase organ dysfunction.
A study of fibril type in heart tissue samples from two deceased ATTR A60T patients, a mutation where cardiomyopathy after LTx develops in virtually all patients, showed type A fibrils in both patients (21), and our two deceased patients with type A fibrils proved to have non-TTRV30 M mutations. It seems, therefore, that amyloid fibril of type A are more commonly encountered in non-TTRV30 M patients, and this may be related to their limited survival after LTx observed in the registry for liver transplanted patients with ATTR (FAPWTR) (22).
To what extent does our finding have an impact on future selection of patients for LTx? Patients older than the age of 50 years have already been shown to have limited survival compared with younger patients (15) and also to be more at risk for development of cardiomyopathy after LTx (23). This instigated an age limit for LTx (24). A selection based on histopathological findings sounds a better strategy than an arbitrary age limit. However, our findings need to be confirmed in other studies with other patient populations.
This study has its limitations. We determined the type of amyloid fibril based on the biochemical analysis of patients subcutaneous fat and not myocardial biopsies, therefore the indirect interpretation of cardiac amyloidosis was based on previously published studies (19, 20). The correlation between the two findings has previously been shown to be consistent due to fibril composition in those patients (25, 26). This study is a retrospective study with its known limitations, one of which is the typing of amyloid fibrils was not performed before 2005 on our patients, thus several deceased patients have not had their amyloid fibril composition determined. As the deceased patients predominantly belonged to the late-onset group where type A fibrils predominate, a bias against such patients is probably present (15).
In summary, from our study it seems that patients with type A fibrils develop or deteriorate the already existing cardiomyopathy and heart failure after LTx, in contrast to patients with type B fibrils. These results might have significant clinical implications in optimizing best patients selection criteria for LTx.
MATERIALS AND METHODS
Twenty-four patients with hereditary ATTR amyloidosis who had survived for at least 1 year after LTx and has had their type of amyloid fibrils identified were available for the study. They had all been examined by echocardiography before and at least 1 year after transplantation to assess cardiac function. Patients’ medical records were searched for clinical data, which are summarized in Tables 1 and 2.
For all but two patients, the immune suppression regime was based on tacrolimus. For patients 18 (type B fibrils) and 21 (type A fibrils), cyclosporine was used. For the majority of patients, low-dose prednisolone was used, and five patients were on azathioprine or mycophenolic acid treatment.
The patients were divided into two groups according to the fibril composition: type A fibrils, 10 patients (seven males) and type B fibrils, 14 patients (11 males). Patients with type A fibrils were aged 64±7 years, and they were transplanted 4.7±3.3 years after onset of disease. Patients with type B fibrils were aged 56±14 years and were transplanted 3.3±1.3 years after onset of disease. Type A patients had echocardiographic examinations before (mean: 8 months, range: 2–16 months) and after LTx (mean: 27 months, range: 16–54 months). The respective time points for the type B patients were mean: 8 months, range: 3–14 months and mean: 26 months, range: 16–43 months. All patients with type B fibrils carried the TTR V30 M mutation, whereas two patients with type A fibrils had other mutations (Table 1). Two patients had ischemic heart diseases, one patient (12) had coronary bypass surgery before LTx, and the other (15) after LTx.
S-NT proBNP, which is a reliable marker of heart failure in amyloid patients (27, 28), was measured as part of routine evaluation of the patients during the latter part of the investigation.
Unfixed subcutaneous abdominal adipose tissue biopsies were cut into smaller pieces, washed in a 0.15 M NaCl solution containing 0.02% sodium azide, followed by lysis of erythrocytes by incubation in 0.88% ammonium chloride. Thereafter the material was defatted in several changes of acetone and left to dry in air (29).
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Western Blot Analysis
The dried tissue samples were analyzed, as described previously (25). To detect full-length TTR and C-terminal TTR fragments, a polyclonal antiserum produced in rabbit against TTR50-127 was used (12, 25).
Echocardiographic examination was performed by one examiner (P.L.) using a Vivid 7 echocardiograph (GE Medical Systems, Horten, Norway) equipped with an adult 1.5 to 4.3 MHz phased array transducer. Standard views from the parasternal long and short axis and apical four-chamber views were obtained. Blood flow velocities were acquired using pulsed and continuous wave Doppler, respectively, as proposed by American Society of Echocardiography (30, 31). All recordings were made with a superimposed electrocardiogram. Off-line analysis was made using a commercially available software system (General Electric, EchoPac version 8.0.1, Waukesha, WI). Measurements were performed by one examiner (S.G.) and obtained blinded to the tissue analysis results.
For morphological assessment of the heart, we measured dimensions including septal and posterior wall thickness from the parasternal long-axis views, as previously recommended (30, 32). From the apical four-chamber view, we measured LA and LV volumes at end systole and end diastole. LVEF was estimated using Simpson’s biplane model. From the pulsed wave Doppler recordings of LV filling, we measured trans-mitral E and A diastolic velocities, E wave deceleration time, and isovolumic relaxation time. Stroke volume was measured using stroke distance from LV outflow tract systolic flow and LV outflow tract cross-sectional area (31).
Myocardial early diastolic velocities (e’) of the LV lateral wall were measured using pulsed tissue Doppler echocardiography (33). Tissue Doppler echocardiography was used to measure E/e’, which was used as an index for estimated LV filling pressures (34).
Intrinsic Myocardial Function Strain Analysis
From the apical four-chamber view, the mean LVGLSR was measured using speckle tracking echocardiography to define myocardial intrinsic function, and a mean value of six regional segments was used. Minimum criteria for satisfactory LV data were five of six accepted LV segments by the software. LV cavity was traced manually from the innermost endocardial edge at end systole, and the software automatically defined the longitudinal strain rate throughout the cardiac cycle. Only segments deemed appropriate for analysis by the software were included. From the acquired recordings, we measured systolic (s), early diastolic (e), and late diastolic (a) LVGLSR.
All statistics were processed using a standard statistical software package (PASW Statistics version 18). Normally distributed continuous data were expressed as mean ± standard deviation. For comparison between groups, Mann-Whitney U or Fisher’s exact test was used, and comparison within group was tested with Wilcoxon’s matched pairs test. Significance was defined as P<0.05.
All parts of the study have been accepted by the ethic committee of Umeå University.
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