Myocardium From Patients With ATTR Amyloidosis Produces Less Force Secondary to Increased Fibrosis

Figures

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  Figure 1. Amyloidosis Myocardium Exhibits Decreased Maximum and Minimum Force (A) Absolute and (B) relative force as a function of Ca2+ concentration. Superplots show (C) maximum force per cross-sectional area, (D) minimum force per cross-sectional area, (E) calcium sensitivity (pCa50), and (F) Hill coefficient. Symbols correspond to those shown in Table 1 and identify each patient. Data were analyzed using linear mixed models. ∗P < 0.05, ∗∗∗P < 0.001.

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  Figure 2. Amyloidosis Myocardium Displays Largely Unchanged Cross-Bridge Kinetics Parameters obtained from the force transient in response to step-length change or ktr maneuver. Superplots show (A) P1, (C) P2, and (E) P3 normalized to P0 and (B) krel, (D) kdf, and (F) ktr. Symbols correspond to those shown in Table 1 and identify each patient. Data were analyzed using linear mixed models. ∗P < 0.05.

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  Figure 3. MyBP-C Phosphorylation Is Decreased in Amyloidosis Myocardium (A) Immunoblots of total and phosphorylated myosin binding protein-C (MyBP-C) at serine 273, 282, and 302. Superplots comparing phosphorylation of MyBP-C at serine (B) 273, (C) 282, and (D) 302, relative to total MyBP-C in amyloidosis and nonfailing myocardium. Symbols correspond to those shown in Table 1 and identify each patient. Data were analyzed using linear mixed models. ∗∗P < 0.01, ∗∗∗P < 0.001.

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  Figure 4. TnI Phosphorylation Is Decreased in Amyloidosis (A) Representative troponin I (TnI) Phos-tag gel showing dephosphorylated (P0), monophosphorylated (P1), and bisphosphorylated (P2) TnI. (B) Representative immunoblots for phosphorylated regulatory light chain (pRLC) at serine 15 and total RLC. (C) Plot comparing the degree of phosphorylation in moles of phosphate per mole of TnI in amyloidosis and nonfailing myocardium. (D) Plot comparing relative phosphorylation of RLC at serine 15 in amyloidosis and nonfailing myocardium. Symbols correspond to those shown in Table 1 and identify each patient. Data were analyzed using linear mixed models. ∗∗∗P < 0.001.

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  Figure 5. Total Stiffness Is Unchanged In Amyloidosis Myocardium, But a Larger Proportion Is Attributable to the Extracellular Matrix (A) Representative force-length curves showing increasing force with passive lengthening of the muscle. Total force was measured by stretching the permeabilized myocardial strip. Extracellular matrix (ECM)–based force was measured after the myofilaments had been depolymerized with high-salt solutions. Superplots show (B) a comparison of total and extracellular Young’s Moduli between amyloidosis and nonfailing myocardium and (C) the proportion of total Young’s Modulus attributable to the ECM in amyloid and nonfailing myocardium. Symbols correspond to those shown in Table 1 and identify each patient. Data were analyzed using linear mixed models. ∗∗P < 0.01, ∗∗∗P < 0.001.

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  Figure 6. The Proportion of Titin N2BA Is Increased in Amyloidosis Myocardium (A) Representative titin gel with individual lanes showing control and myocardium from amyloidosis and nonfailing hearts. (B) Representative immunoblot of tubulin and actin loading control. (C) Plot comparing the proportion of N2BA relative to total titin in amyloidosis and nonfailing myocardium. (D) Plot comparing the tubulin abundance relative to actin in amyloidosis and nonfailing myocardium. Symbols correspond to those shown in Table 1 and identify each patient. Data were analyzed using linear mixed models. ∗P < 0.05. LV = left ventricle; RV = right ventricle; Sept = Septum.

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  Figure 7. Fibrosis Is Increased in Amyloidosis Myocardium (A) Representative picrosirius red staining and programmatic image analysis of myocardium from patients with and without amyloidosis. (B) Superplot comparing RV, Sept, and LV fibrosis from patients with and without amyloidosis. Data were analyzed using linear mixed models. (C) Deming linear regression of each patient’s average left ventricle fibrosis plotted against their average septal fibrosis. This analysis allows for the experimental uncertainty associated with both the x and y variables. The p-value shows the probability of the measured slope differing from 1. The identity line is shown in red. Symbols in both B and C correspond to those shown in Table 1 and identify each patient. ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figure 6.

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  Figure 8. Amyloid Deposition Is Homogeneous in the Ventricles and Septum of Patients With Amyloidosis (A) Representative Congo-Red staining of myocardium from patients with and without amyloidosis. No amyloid deposition was observed nor programmatically detected in any of the nonfailing patients. (B) Superplot comparing amyloid deposition in RV, Sept, and LV from amyloidosis myocardium. Data were analyzed using linear mixed models. (C) Deming linear regression of each patient’s average LV fibrosis plotted against their average septal fibrosis. This analysis allows for the experimental uncertainty associated with both the x and y variables. The P value shows the probability of the measured slope differing from 1. The identity line is shown in red. Symbols in both B and C correspond to those shown in Table 1 and identify each patient. Abbreviations as in Figure 6.

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  Figure 9. Increased Microcalcifications in Amyloid Myocardium (A) Representative serial sections from amyloidosis myocardium stained with Congo red and Von Kossa and analyzed using image segmentation algorithms. (B) Automated image registration of Von Kossa and Congo Red stained sections with microcalcifications (labeled green) and ATTR amyloid deposits (labeled blue). (C) Representative Von Kossa staining and image analysis of nonfailing myocardium shows minimal microcalcifications. (D) Plot comparing microcalcifications in amyloidosis and nonfailing myocardium. Symbols correspond to those shown in Table 1 and identify each patient. Data were analyzed using linear mixed models. ∗∗∗P < 0.001.