Infusion of Light Chains From Patients With Cardiac
Amyloidosis Causes Diastolic Dysfunction in Isolated
Mouse Hearts
Ronglih Liao, PhD; Mohit Jain; Paige Teller, MA; Lawreen H. Connors, PhD;
Soeun Ngoy; Martha Skinner, MD; Rodney H. Falk, MD; Carl S. Apstein, MD
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Background—Primary (AL) amyloidosis is a plasma cell dyscrasia characterized by clonal production of immunoglobulin
light chains (LC) resulting in the subsequent systemic deposition of extracellular amyloid fibrils. Cardiac involvement
is marked by the hemodynamic pattern of impaired diastolic filling and restrictive cardiomyopathy. Although cardiac
death in patients with AL amyloidosis is usually associated with extensive myocardial infiltration, the infiltration alone
does not correlated with the degree of heart failure or survival. We hypothesized that circulating monoclonal LC may
directly impair cardiac function, in addition to any mechanical effects of amyloid fibril deposition. Therefore, we
examined the effects of amyloid LC proteins on diastolic and systolic cardiac function, as measured in an isolated mouse
heart model.
Methods and Results—LC were obtained from patients with nonamyloid disease or from those with noncardiac, mild
cardiac, and severe cardiac involved AL amyloidosis. Saline or LC (100 ␮g/mL) was infused into a Langendorffperfused, isovolumically contracting mouse heart. Saline and control, noncardiac, and mild-cardiac LC infusions did not
alter ex vivo cardiac function. In contrast, infusion of sever cardiac LC resulted in marked impairment of ventricular
relaxation with preservation of contractile function.
Conclusion—These results demonstrate that infusion of LC from patients with AL amyloidosis result in diastolic
dysfunction similar to that observed in patients with cardiac involved AL amyloidosis, and they suggest that amyloid
LC proteins may contribute directly to the pathogenesis and the rapid progression of amyloid cardiomyopathy,
independent of extracellular fibril deposition. (Circulation. 2001;104:1594-1597.)
Key Words: amyloidosis 䡲 physiology 䡲 mice 䡲 heart 䡲 diastole
P
with AL amyloidosis have a worse prognosis than those with
non-AL amyloidosis. We hypothesized that certain circulating monoclonal LC may directly impair cardiac function, in
addition to any mechanical effects of amyloid fibril deposition. Therefore, we examined the effects of amyloid LC
proteins on diastolic and systolic cardiac function measured
in an isolated mouse heart model. LC were obtained from
patients with nonamyloid disease or noncardiac, mild cardiac,
and severe cardiac AL amyloidosis.
rimary (AL) amyloidosis is a plasma cell dyscrasia
characterized by clonal production of immunoglobulin
light chains (LC), resulting in the subsequent systemic
deposition of extracellular amyloid fibrils.1,2 Prognosis is
highly dependent on the organs involved, and the worst
outcome is associated with cardiac involvement, a feature that
is present in ⬎50% of all patients with AL amyloidosis.3
Cardiac involvement is marked by the hemodynamic pattern
of impaired diastolic filling and restrictive cardiomyopathy,
which often rapidly progresses to congestive heart failure,
with a median survival of 6 months and a 5-year survival of
⬇2%.4,5 Although this disease was identified in the mid-19th
century,6 the mechanisms by which amyloidosis results in the
rapid development of cardiac dysfunction remain unknown.
Cardiac death in patients with amyloidosis is usually associated with extensive myocardial infiltration, although the
infiltration alone does not correlate with the degree of heart
failure or survival.1,7 Furthermore, clinical observations show
that with similar degrees of myocardial infiltration, patients
Methods
Sources of LC
Monoclonal immunoglobulin LC were isolated from 24-hour urine
specimens collected from one patient with multiple myeloma, one
with nonamyloid disease, and 5 with AL amyloidosis who were
referred to the Amyloidosis Treatment and Research Program at
Boston Medical Center. One AL amyloidosis patient had no evidence of cardiac involvement (noncardiac), two AL amyloidosis
patients had echocardiographic abnormalities (slightly impaired
ventricular filling and mild ventricular hypertrophy with no ECG
Received July 20, 2001; revision received August 14, 2001; accepted August 14, 2001.
From the Cardiac Muscle Research Laboratory (R.L., M.J., P.T., S.N., C.S.A.) and the Amyloid Treatment and Research Program (R.L., L.H.C., M.S.,
R.H.F., C.S.A.), Department of Medicine, Boston University School of Medicine, Boston, Mass.
Correspondence and reprint requests to Dr Ronglih Liao, 650 Albany Street, X726, Boston, MA 02118. E-mail [email protected]
© 2001 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
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Liao et al
AL Amyloidosis and Cardiac Diastolic Dysfunction
1595
changes) in the absence of heart failure (mild cardiac), and two AL
amyloidosis patients had echocardiographic abnormalities (greatly
impaired ventricular filling and severe hypertrophy) and NYHA
class III or IV heart failure (severe cardiac). All procedures were
performed with Institutional Review Board approval and informed
consent from the patients.
Purification of LC Proteins
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Urine samples were dialyzed against deionized water, lyophilized,
and reconstituted in 0.02 mol/L sodium-phosphate buffer (pH 7.1)
with Affigel blue (Bio-Rad) to remove albumin. The entire filtrate
was again dialyzed and lyophilized. The affinity-purified sample was
fractionated on Sephacryl S-200 columns (Amersham-Pharmacia) in
0.02 mol/L Tris (pH 7.5).
The purity of fractionated samples was assessed by SDS-PAGE
and Western blotting using the Pharmacia Phast System. Samples
were run under reducing conditions and stained with Coomassie
blue. Immunodetection was performed with human Ig␬ or Ig␭ LC
antibody (Atlantic Antibodies/DiaSarin), and results were visualized
by incubation with alkaline phosphatase– conjugated secondary antibody (Sigma Chemical) using BCIP/NBT phosphatase substrate
(Promega).
Isolated Mouse Heart Preparation
All procedures strictly adhered to the regulations of the Institutional
Animal Care and Use Committee at Boston University. Hearts were
isolated from C57/Bl6 mice weighing 25 to 30 g and were perfused
in the Langendorff mode, as previously described.8 Briefly, mice
were heparinized (10 000 U/kg IP) and anesthetized with ketamine
(150 mg/kg IP) and xylazine (15 mg/kg IP). Hearts were isolated and
perfused with oxygenated Krebs-Henseleit buffer (pH 7.4). A
polyvinyl-chloride balloon was inserted into the left ventricle and
connected to a pressure transducer (Gould Statham) to record left
ventricular pressures. Hearts were paced through epicardial platinum
wires. An ultrasonic flow probe (Transonics Systems) measured
coronary flow. End-diastolic and end-systolic pressures were recorded using a physiology recorder.
Experimental Protocol
All hearts were stabilized for 15 minutes at 37°C at a coronary
perfusion pressure of 80 mm Hg and paced at 7 Hz. After stabilization, the balloon was inflated with saline to adjust the isovolumic
end-diastolic pressure to 5 to 7 mm Hg, and the balloon volume was
held constant for the duration of the experiment. Coronary flow was
then recorded and also held constant. Hearts were infused for 30
minutes with 0.09% NaCl (saline; 5 mouse hearts) or LC protein
(100 ␮g/mL) that was isolated from a nonamyloid patient (control; 1
patient, 7 mouse hearts) or AL amyloidosis patients with either
noncardiac (1 patient, 5 mouse hearts), mild cardiac (2 patients, 11
mouse hearts), or severe cardiac (2 patients, 10 mouse hearts)
involvement, followed by a 15-minute washout period. The concentration of LC protein was chosen to represent a circulating level
comparable to that found in patients with AL amyloidosis.9 Investigators performing heart experiments were blinded regarding the
protein group.
Statistical Analysis
Data are reported as mean⫾SEM. Group differences were tested
using repeated-measures 2-factor ANOVA with least significant
difference post hoc examination. P⬍0.05 was considered statistically
significant.
Results
Amyloid LC Proteins
Figure 1A illustrates a representative elution profile of 80 mg
of urinary protein collected from a patient that was fractionated on Sephacryl 200 after albumin was removed by Affigel
blue purification. Four separate peaks were observed with
Figure 1. A, Sephacryl S-200 chromatographic elution profile of
AL urine pretreated with Affigel-blue. B, SDS-PAGE analysis of
AL amyloidosis urine S-200 peak fractions A through D (left) and
corresponding Western blot analysis (right). Light chain (LC) protein collected from peak C was used in the study. OD indicates
optical density.
their corresponding SDS-PAGE and Western blots (Figure
1B). The major protein in peak C had a molecular weight of
⬇30 kDa and was immunoreactive to anti-human Ig␬ LC
antibody. The LC in peak C had ⬎90% purity, which was
confirmed by mass spectroscopic analysis, and was isolated
for subsequently use in heart perfusion studies.
LC Proteins and Cardiac Function
Before the infusion of saline or protein, all hearts were set to
an end-diastolic pressure between 5 and 7 mm Hg, and the
balloon volume was then held constant for the duration of the
experiment. Coronary flows were comparable among groups
(saline, control, noncardiac, mild cardiac, and severe cardiac
flows were 2.3⫾0.2, 2.8⫾0.1, 2.5⫾0.2, 2.2⫾0.4, and
2.7⫾0.2 mL/min; P⫽NS). Infusion of saline did not alter
isovolumic end-diastolic pressures over the 30-minute infusion period or the subsequent 15-minute washout period
(Figure 2A). Similarly, control, noncardiac, and mild cardiac
proteins did not significantly alter diastolic function, suggesting a lack of acute cardiac toxicity from these LC proteins. In
contrast, severe cardiac LC proteins, obtained from patients
with severe cardiac involvement, resulted in a progressive
increase in end-diastolic pressure, peaking at 18.7 mm Hg at
the end of the 30-minute infusion period, indicative of severe
diastolic dysfunction. Elevated diastolic pressures persisted in
these hearts throughout the washout period. Interestingly,
systolic pressure generation was unaltered with the infusion
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October 2, 2001
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Figure 2. The changes in (A) left-ventricular end diastolic pressure (EDP) or (B) left-ventricular end systolic pressure (SP) with
infusion of 0.09% NaCl (saline) and light chains (LC) isolated
from patients with nonamyloid disease (LC-Control) or noncardiac (AL-NC), mild cardiac (AL-MC), or severe cardiac (AL-SC)
AL amyloidosis. *P⬍0.05 vs all groups.
of saline, control, noncardiac, mild cardiac, or severe cardiac
LC proteins (Figure 2B). Congo Red staining and histological
analysis of isolated hearts after LC protein infusion revealed
the absence of any amyloid fibril deposits (data not shown).
Discussion
Cardiac-involved AL amyloidosis is a fatal disease associated
with the extensive deposition of amyloid ␤-pleated fibrils
within the heart.1,2 Although cardiac death in patients with
AL amyloidosis is usually associated with extensive myocardial amyloid infiltration, the infiltration alone does not
explain the rapid progression of heart failure.2 Our results
suggest, for the first time, that circulating LC isolated from
patients with AL amyloidosis with heart failure can directly
impair cardiac function, independent of fibril formation. Only
LC associated with severe cardiac involvement resulted in
ventricular dysfunction ex vivo, suggesting that unique LC
variations dictate organ specificity. Importantly, studies were
conducted with protein isolated from multiple patients to
reduce the potential artifact associated with any one LC
sample.
The direct effects of amyloid LC observed here may
explain previous clinical observations that amyloid infiltration of the ventricle of equal or greater severity to that
seen in AL amyloidosis occurs in familial and senile
amyloidosis, yet heart failure is milder and survival is
much longer than with AL amyloidosis.2,7 The amyloid
fibrils in senile and familial amyloidosis are derived from
transthyretin,2 raising the possibility that rapid progression
of heart failure in AL amyloidosis may be partially related
to a toxic effect of the circulating monoclonal protein
itself, in addition to the mechanical effects of the LC
amyloid deposits. Additional support for this hypothesis
comes from observations that some nonamyloidogenic LC
have an intrinsic neurotoxicity unrelated to direct nerve
deposition.10 Furthermore, patients with cardiac amyloidosis with cessation in LC production after chemotherapy
have shown clinical improvement of their heart failure,
despite the persistence of cardiac amyloid fibril
infiltrates.2,11
Patients with heart failure due to cardiac-involved AL
amyloidosis are often distinguished by a “stiff heart” syndrome, which is echocardiographically characterized by impaired ventricular relaxation and near-normal ejection fractions.2,4,12 Reduction of ejection fraction is usually associated
with advanced stages of disease.13 The dysfunction observed
with infusion of severe cardiac LC, namely impaired ventricular relaxation with preservation of contractile function, is
very similar to the hemodynamic abnormalities reported in
cardiac AL amyloidosis, further supporting a direct role for
LC in the pathogenesis of cardiac AL amyloidosis.
AL amyloidosis involving the heart results in the rapid
progression of cardiac failure with a median survival of
only several months.4,5 Currently, pharmacological therapy
to treat this condition is limited, because traditional
medications such as ␤-blockers, calcium channel blockers,
and digoxin are contraindicated.2 Furthermore, the particularly short survival associated with cardiac AL amyloidosis prevents patients from receiving the multiple cycles of
chemotherapy needed to slow this disease.2 Our data
demonstrate that circulating monoclonal LC may contribute substantially to the cardiac dysfunction observed in AL
amyloid patients. The particular characteristics of severe
cardiac LC proteins that promote cardiac dysfunction
remain to be elucidated, and they may include unique
protein sequences and/or post-translational modifications.
Furthermore, amyloid protein–induced cardiac dysfunction
may involve alterations in cardiomyocyte metabolism,
cellular edema, intracellular calcium handling, and/or direct activation of cellular receptors. Understanding the
mechanisms responsible for the swift decline in cardiac
function may allow for the development of new therapies
aimed at preventing or reversing the dysfunction and
consequent mortality associated with cardiac AL
amyloidosis.
Acknowledgments
This study was supported by the Evans Medical Foundation (R.L.),
the American Heart Association (L.H.C.), the Gerry Foundation
(M.S.), and the Sue Sellors Finley Amyloid Research Fund (R.H.F.,
C.S.A.).
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Infusion of Light Chains From Patients With Cardiac Amyloidosis Causes Diastolic
Dysfunction in Isolated Mouse Hearts
Ronglih Liao, Mohit Jain, Paige Teller, Lawreen H. Connors, Soeun Ngoy, Martha Skinner,
Rodney H. Falk and Carl S. Apstein
Downloaded from http://circ.ahajournals.org/ by guest on July 31, 2017
Circulation. 2001;104:1594-1597
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2001 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
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Correction
In the articles by De Leon et al, “Adventitial Cells Do Not Contribute to Neointimal Mass After
Balloon Angioplasty of the Rat Common Carotid Artery” (Circulation. 2001;104:1591–1593) and
Liao et al, “Infusion of Light Chains From Patients With Cardiac Amyloidosis Causes Diastolic
Dysfunction in Isolated Mouse Hearts” (Circulation. 2001;104:1594–1597), which published in
the October 2, 2001 issue of the journal, the DOIs were missing for both articles. The DOIs are as
follows:
De Leon et al, “Adventitial Cells Do Not Contribute to Neointimal Mass After Balloon
Angioplasty of the Rat Common Carotid Artery” (Circulation. 2001;104:1591–1593)
DOI: 10.1161/01.cir.0000433836.90987.c6
Liao et al, “Infusion of Light Chains From Patients With Cardiac Amyloidosis Causes Diastolic
Dysfunction in Isolated Mouse Hearts” (Circulation. 2001;104:1594–1597)
DOI: 10.1161/01.cir.0000433837.98610.85
The current online versions of the manuscripts have been corrected.
(Circulation. 2013;128:e174.)
© 2013 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIR.0b013e3182a8c0d6
e174