A Tension-Based Model Distinguishes Hypertrophic versus Dilated Cardiomyopathy

Abstract

The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

Figure 1. Ca²⁺ Binding Properties of cTnC Variants Alter Myocyte Tension and Ca²⁺ Dynamics

(A) Western blot analysis against Flag, cTnC, α-actinin (α-act.) and tropomyosin (Tm) in protein lysates from adenovirally transduced adult rat cardiac myocytes 3 days after gene transfer. Tm and α-act were loading controls. The cTnC antibody shows the slower migrating Flag epitope-tagged cTnC constructs that were overexpressed and endogenous cTnC. Samples were processed from intact (I) and permeabilized (P) myocytes to show sarcomeric incorporation. (B) Cardiac TnC western blot after co-immunoprecipitation (IP) with anti-cTnT from adult rat cardiac myocytes that were untreated or adenovirally transduced with L48Q cTnC-Flag (AdL48Q) or I61Q cTnC-Flag (AdI61Q) as a further control for the data shown in panel A. Abbreviation; IgG LC, Mouse IgG light chain. (C) Graph showing 75% mechanical relaxation time in paced isolated adult rat cardiac myocytes adenovirally transduced with βgal, L48Q cTnC, and I61Q cTnC. Error bars represent the mean+SEM, n=40 myocytes distributed across 3 preparations, *P<0.05 vs βgal adenoviral infection (D) Graph showing 75% Ca²⁺ transient decay time in paced isolated adult rat cardiac myocytes adenovirally transduced with βgal, L48Q cTnC, and I61Q cTnC. Error bars represent the mean+SEM, n=40 myocytes distributed across 3 preparations, *P<0.05 vs βgal adenoviral infection (E) A schematic depicting the bi-transgenic (TG) system used for cardiac-specific expression of WT, L48Q, or I61Q cTnC with a carboxy-terminal Flag epitope directed by the αMHC-promoter. Abbreviations; tTA, tetracycline transactivator construct; TetR, tetracycline repressor protein; p(A), polyadenylation signal; TetO, Tetracycline operator sequence. (F) Western blots with an anti-cTnC and anti-Flag antibody demonstrating the relative expression of epitope-tagged WT, L48Q, and I61Q cTnC versus faster migrating endogenous cTnC in the presence or absence of the tTA transgene. Actin was a loading control. (G) Computationally derived Ca²⁺-activated unloaded twitch force over time of a single contraction in simulated myocytes expressing WT (black), L48Q (red), and I61Q (blue) cTnC. (H) Computationally derived Ca²⁺ transients over time of a single contraction in simulated myocytes expressing WT (black), L48Q (red), and I61Q (blue) cTnC. Also see Figure S1.

Figure 2. Increased Ca²⁺ Binding and Prolonged Tension by L48Q cTnC Causes Concentric Hypertrophy

(A–D) Average percent shortening, (B) Ca²⁺ transient amplitude, (C) 75% myocyte relaxation time, and (D) 75% Ca²⁺ transient decay time measured in myocytes isolated from TG tTA only controls or WT cTnC and L48Q cTnC hearts. Error bars represent the mean+SEM, n>43 myocytes distributed across 3–5 preparations per experimental group, *P<0.05 vs tTA, WT cTnC. (E–H) Average myocardial fractional shortening (FS), (F) left ventricular (LV) diastolic chamber dimension, (G) septal wall thickness, and (H) posterior LV wall thickness measured by M-mode echocardiography in 3 month-old tTA, WT cTnC, and L48Q cTnC TG mice. Error bars represent the mean+SEM, n=8–10 mice per group, *P<0.05 vs tTA, WT cTnC. (I) Average heart-weight normalized to body-weight (HW/BW) in the indicated groups of mice. Error bars represent the mean+SEM, n=8–10 mice per group. (J–M) Average myocardial FS, (K) LV diastolic chamber dimension, (L) septal wall thickness, and (M) LV posterior wall thickness measured by M-mode echocardiography with vehicle or chronic β-blocker (metopro.) delivery in tTA and L48Q cTnC TG mice. Error bars represent the mean+SEM, n=7–8 mice per group, *P<0.05 vs L48Q+vehicle. (N) Average HW/BW for the indicated groups of mice treated with vehicle or metoprolol. Error bars represent the mean+SEM, n=7–8 mice per group, *P<0.05 vs L48Q+vehicle. (O,P) Average 75% myocyte relaxation time, and (P) 75% Ca²⁺ transient decay time in myocytes isolated from tTA and L48Q cTnC TG hearts acutely perfused with vehicle, isoproterenol (Iso.), or metoprolol (metopro.). Error bars represent the mean+SEM, n=25 myocytes across 3 preparations. *P<0.05 isoproterenol effect, #P<0.05 metoprolol effect. (Q) Effects of L48Q mutation on Ca²⁺ binding by cTnC alone or complexed with phosphorylated cTnI. Changes in the fluorescence intensities of the environmentally sensitive probe IANBD labeled cTnC^C35S measured in response to increasing Ca²⁺ concentrations. The error bars represent the SEM, n=3. (R–T) The effect of transverse aortic constriction (TAC) or a sham procedure on myocardial FS, (S) posterior wall thickness, and (T) HW/BW in tTA or L48Q TG mice. Bars represent the mean+SEM, n=5–8 mice per group, *P<0.05 vs tTA-Sham; #P<0.05 vs tTA TAC. Also see Figure S2.

Figure 3. Reduced Ca²⁺ Binding and Tension by I61Q cTnC Causes Eccentric Hypertrophy

(A–F) Average percent shortening, (B) Ca²⁺ transient amplitude, (C) 75% myocyte relaxation time, (D) 75% Ca²⁺ transient decay time, (E) diastolic sarcomere length, and (F) diastolic Ca²⁺ measured in myocytes isolated from tTA or I61Q cTnC TG hearts. Error bars represent the mean+SEM, n>40 myocytes distributed across 3 preparations per group, *P<0.05 vs tTA. (G) Average myocardial FS measured by M-mode echocardiography in tTA and I61Q cTnC TG mice at 3 months of age. Error bars represent the mean+SEM, n=8 mice per group, *P<0.05 vs tTA. (H) Average systolic pressure derivatives over time to assess cardiac contractility as measured by pressure-volume catheterization in the indicated groups of mice. Error bars represent the mean+SEM, n=5 mice per group, *P<0.05 vs tTA. (I,J) Left ventricular (LV) diastolic chamber dimension and (J) septal wall thickness measured by M-mode echocardiography in tTA and I61Q cTnC TG mice at 3 months of age. Error bars represent the mean+SEM, n=8 mice per group, *P<0.05 vs tTA. (K) Average LV end diastolic volumes measured by pressure-volume catheterization in the indicated groups of mice. Error bars represent the mean+SEM, n=5 mice per group, *P<0.05 vs tTA. (L) Average HW/BW in the indicated groups of mice at 3 months of age. Error bars represent the mean+SEM, n=8 mice per group, *P<0.05 vs tTA. (M,N) Average area and (N) length/width ratios measured in myocytes isolated from tTA and I61Q TG hearts at 3 months of age. Error bars represent the mean+SEM, n=165 myocytes per group, *P<0.05 vs tTA. (O) Survival curves for tTA, WT cTnC, and I61Q cTnC TG mice. n=12 per group.

Figure 4. Increased Crossbridge Binding in Myh6 R403Q Mice Corrects I61Q cTnC-Dependent Eccentric Hypertrophy

(A–D) Average myocardial FS, (B) LV diastolic chamber dimension, (C) septal and (D) LV posterior wall thickness measured by M-mode echocardiography in tTA and I61Q TG mice, some of which were heterozygous for R403Q mutant Myh6 allele, at 4 months of age. Error bars represent the mean+SEM, n=6–8 mice per group, *P<0.05 vs tTA+WT, #P<0.05 vs. I61Q WT. (E) Average HW/BW in the indicated mice at 4 months of age. Error bars represent the mean+SEM, n=6–8 mice per group, *P<0.05 vs tTA+WT, #P<0.05 vs. I61Q WT. (F) Survival curves for tTA, WT cTnC, and I61Q cTnC TG mice with or without the heterozygous R403Q mutation in Myh6. n=8 per group. (G) Representative Masson’s Trichrome-stained images of transverse histological sections of hearts from I61Q and I61Q+R403Q^+/− mice at 4 months of age. Magnification is 20X.

Figure 5. Controlled Alterations in Ca²⁺ Cycling in I61Q and L48Q Mice

(A) Computationally simulated unloaded twitch force in cardiomyocytes expressing either tTA (WT condition), I61Q cTnC, or I61Q cTnC with increased cytosolic Ca²⁺ clearance mediated by loss of phospholamban (I61Q-Pln^−/−). (B) Computationally simulated unloaded twitch force in cardiomyocytes expressing either tTA (WT condition), L48Q cTnC, or L48Q cTnC with increased cytosolic Ca²⁺ clearance mediated by loss of phospholamban (L48Q-Pln^−/−). (C,D) LV diastolic chamber dimension and (D) septal wall thickness measured by M-mode echocardiography in tTA, I61Q, and L48Q TG mice with or without Pln deletion. Error bars represent the mean+SEM, n=6–8 mice per group, *P<0.05 vs tTA+Pln^+/+, #P<0.05 for 2 conditions shown (E) Average HW/BW of the depicted groups. Error bars represent the mean+SEM, n=6–8 mice per group, *P<0.05 vs tTA+Pln^+/+, #P<0.05 for 2 conditions shown. (F) Computationally simulated unloaded twitch force in cardiomyocytes expressing either tTA (WT condition), I61Q cTnC, or I61Q cTnC with decreased cytosolic Ca²⁺ clearance due to the loss of 1 allele of the gene encoding SERCA2a (I61Q-Atp2a2^+/−). (G) Computationally simulated unloaded twitch force in cardiomyocytes expressing either tTA (WT condition), L48Q cTnC, or L48Q cTnC with decreased cytosolic Ca²⁺ clearance due to the loss of 1 allele of the gene encoding SERCA2a (L48Q- Atp2a2^+/−). (H–J) LV diastolic chamber dimension, (I) LV wall thickness, and (J) septal thickness measured by M-Mode echocardiography in tTA, I61Q, and L48Q TG mice replete with SERCA2a (Atp2a2^+/+) or lacking 1 allele of SERCA2a (Atp2a2^+/−). Error bars represent the mean+SEM, n=6–8 mice per group, *P<0.05 vs tTA+Atp2a2^+/+, #P<0.05 the 2 conditions shown (K) Average HW/BW of the depicted groups. Error bars represent the mean+SEM, n=6–8 mice per group, *P<0.05 vs tTA+Atp2a2^+/+, #P<0.05 for the 2 conditions shown Also see Figure S1 and S3

Figure 6. HCM versus DCM Growth Is Regulated by Calcineurin and ERK1/2 Signaling

(A) Ratio of nuclear to cytosolic NFATc3-GFP measured at baseline (0 Hz) and after pacing at the depicted frequencies in isolated feline adult cardiomyocytes adenovirally transduced with WT cTnC or I61Q cTnC. Bars represent the mean+SEM, n=25–59 myocytes per group, *P<0.05 vs WT cTnC. (B–D) Average FS, (C) LV diastolic chamber dimension, and (D) septal wall thickness measured by M-mode echocardiography in 3 month-old tTA and I61Q cTnC TG mice with (Ppp3cb^+/+) or without calcineurin Aβ (Ppp3cb^−/−). Error bars represent the mean+SEM, n=6–8 mice per group, *P<0.05 vs tTA + Ppp3cb^+/+. (E) Average HW/BW of the depicted groups at 3 months of age. Error bars represent the mean+SEM, n=6–8 mice per group, *P<0.05 vs tTA + Ppp3cb^+/+, #P<0.05 for the 2 conditions shown. (F) Representative confocal images of isolated adult rat cardiomyocytes labeled for ERK1/2 (green) and sarcomeric actin (red), which were adenovirally transduced with WT cTnC or I61Q cTnC, with or without 48 hours of pacing. White arrows indicate ERK1/2 translocation into the nucleus after pacing with only WT cTnC. Magnification = 600X. (G) Quantification of myocytes with nuclear versus cytosolic ERK1/2 from the type of experiment depicted in panel F. Error bars represent the mean+SEM, n=125–135 myocytes per group across 3 preparations, *P<0.05 vs control + no pacing. (H) Average length of isolated adult rat cardiomyocytes adenovirally transduced with WT cTnC or I61Q cTnC, with or without 48 hours of pacing. Error bars represent the mean+SEM, n=25–35 myocytes per group across 3 preparations, *P<0.05 vs control + no pacing. (I–L) Average myocardial FS, (J) LV diastolic chamber dimension, (K) septal wall thickness, and (L) LV wall thickness measured by M-mode echocardiography in 3 month-old tTA and I61Q cTnC TG mice with or without a constitutively active MEK1 transgene. Error bars represent the mean+SEM, n=6 mice per group, *P<0.05 vs NTG, #P<0.05 for the 2 conditions shown. (M) Average HW/BW of the depicted groups. Bars represent the mean+SEM, n=6 mice per group, *P<0.05 vs NTG tTA.

Figure 7. A Predictive Model of Cardiac Growth For Familial Cardiomyopathy-linked Mutations

(A) Mean twitch tensions (shortening) measured in mouse myocytes for I61Q (blue) and WT (black) that were normalized to the WT’s mean peak twitch amplitude. Calculation of the tension-index for the I61Q mutant is depicted in blue text in which WT integrated tension (WT_area) is subtracted from I61Q integrated tension (I61Q_area) and the resulting value is the net area placed on the hypertrophic growth continuum depicted in B and C. (B) Net integrated tension shown in panel A as a line chart with WT being 0, and the I61Q having a negative total value of −1.6×10⁴ that predicts dilated heart growth and L48Q being a positive value of +1.5×10⁴ predicting thickening hypertrophy. ERK1/2 signaling is reduced by more negative values, which reduces myocyte width but increased with positive values leading to thickening growth. Calcineurin signaling is lowest at 0, yet increased by both positive and negative values. (C) The same integrated tension as depicted in panel B expanded to include the predicted scores for other sarcomeric mutations in mice (below the line), and from four patients clinically diagnosed with either HCM or DCM (2 each, above the line). The magnitude of the value reflects the degree of disease severity as either concentric hypertrophy (HCM remodeling) or dilated, eccentric (DCM) growth. Also see Figure S4, S5, S6 and S7