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. 2020 May 1;116(6):1175-1185.
doi: 10.1093/cvr/cvz218.

Fropofol prevents disease progression in mice with hypertrophic cardiomyopathy

Yiyuan Huang  1 , Haisong Lu  2 , Xianfeng Ren  3 , Fazhao Li  4 , Weiming Bu  5 , Wenjie Liu  6 , William P Dailey  7 , Harumi Saeki  8 , Kathleen Gabrielson  8 , Roselle Abraham  9 , Roderic Eckenhoff  5 , Wei Dong Gao  10
Affiliations

Fropofol prevents disease progression in mice with hypertrophic cardiomyopathy

Yiyuan Huang et al. Cardiovasc Res. .

Abstract

Aims: Increased myofilament contractility is recognized as a crucial factor in the pathogenesis of hypertrophic cardiomyopathy (HCM). Direct myofilament desensitization might be beneficial in preventing HCM disease progression. Here, we tested whether the small molecule fropofol prevents HCM phenotype expression and disease progression by directly depressing myofilament force development.

Methods and results: Force, intracellular Ca2+, and steady-state activation were determined in isolated trabecular muscles from wild-type (WT) and transgenic HCM mice with heterozygous human α-myosin heavy chain R403Q mutation (αMHC 403/+). αMHC 403/+ HCM mice were treated continuously with fropofol by intraperitoneal infusion for 12 weeks. Heart tissue was analysed with histology and real-time PCR of prohypertrophic and profibrotic genes. Fropofol decreased force in a concentration-dependent manner without significantly altering [Ca2+]i in isolated muscles from both WT and αMHC 403/+ HCM mouse hearts. Fropofol also depressed maximal Ca2+-activated force and increased the [Ca2+]i required for 50% activation during steady-state activation. In whole-animal studies, chronic intra-abdominal administration of fropofol prevented hypertrophy development and diastolic dysfunction. Chronic fropofol treatment also led to attenuation of prohypertrophic and profibrotic gene expression, reductions in cell size, and decreases in tissue fibrosis.

Conclusions: Direct inhibition of myofilament contraction by fropofol prevents HCM disease phenotypic expression and progression, suggesting that increased myofilament contractile force is the primary trigger for hypertrophy development and HCM disease progression.

Keywords: Calcium; Fropofol; Hypertrophic cardiomyopathy; Myofilament.

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Figures

Figure 1
Figure 1
Fropofol decreases twitch force development in HCM muscle. (A) Raw recordings of force development in a trabecular muscle from HCM heart in the presence of fropofol. The numbers indicate concentrations of fropofol in μM. (B) Raw recordings of Ca2+ transients in the same trabecular muscle from A in the presence of fropofol. (C) Pooled data of systolic force development and amplitudes of intracellular Ca2+ transients in the presence of different concentrations of fropofol. Systolic force became significantly less than that at baseline at concentrations greater than 50 μM. P-values from paired Student’s t-test: *P < 0.01 vs. baseline, n = 7. (D) Effects of fropofol on diastolic force and intracellular Ca2+ levels. (E) Effects of fropofol on twitch dynamics, measured as time to peak force (Tp) and time from peak to half relaxation (Tr50) (n = 7). (F) Effects of fropofol on the time course of intracellular Ca2+ transients, measured as time to peak [Ca2+]i and time to half [Ca2+]i during relaxation (n = 5). (G) Effects of fropofol (100 µM) on the steady-state force and intracellular Ca2+ relation in intact HCM muscles (n = 9). (H) The same muscles in which steady-state relations were first obtained were chemically skinned and activated with various Ca2+ concentrations in the absence and presence of fropofol. Temperature = 22°C; external Ca2+ = 2.0 mM; stimulation rate = 0.5 Hz.
Figure 2
Figure 2
Echocardiography of HCM mice before (0 week) and after (12 weeks) fropofol treatment. For all study mice, black-filled symbols indicate means for each group (i.e. fropofol and untreated). Colour-filled symbols represent data from animals that died during the trial between 6 and 10 weeks. (A) Pooled data for changes in ejection fraction and fractional shortening after 12 weeks. (B–D) Changes in left ventricular (LV) posterior wall (LVPW), interventricular septal wall (IVS), and LV anterior wall (LVAW) thickness during diastole (d) and systole (s). Wall thickness was measured in mid-LV short-axis view at the level of papillary muscles (B). The borders of LV walls were manually traced during both systole and diastole, and measurements were made at positions indicated by the arrowheads. (E) Changes in mitral E/A ratio. P-values from paired Student’s t-test when compared to baseline within the group and from ANOVA when compared between fropofol-treated and untreated groups: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 20 in each group.
Figure 3
Figure 3
Changes in cardiac function and left ventricular (LV) wall thickness during treatment with fropofol. (A) Changes in ejection fraction (EF). EF remained relatively constant over the course of treatment in both wild-type and HCM fropofol-treated groups but increased significantly over that period in untreated HCM mice. (B) Changes in fractional shortening (FS) over the course of fropofol treatment in wild-type and HCM mice. (C) Changes in E/A ratio in HCM and wild-type mice at the end of the study. (D–F) Changes in wall thickness in fropofol-treated and untreated HCM mice during the course of the study. These values were obtained during diastole. IVS, interventricular septum; LVAW, left ventricular anterior wall; LVPW, left ventricular posterior wall. P-values from ANOVA (fropofol-treated vs. untreated): *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 20 in each group.
Figure 4
Figure 4
Chronic fropofol treatment prevents hypertrophy development, cell size increases, myofilament disarray, and fibrosis. (A) Representative photographs of hearts from a wild-type mouse, an untreated HCM mouse, and a fropofol-treated HCM mouse. P-values from ANOVA: *P < 0.05 vs. wild-type; #  P < 0.05 vs. untreated, n = 3 hearts in each group. (B) Haematoxylin and eosin (H&E) staining of tissue samples from wild-type, untreated, and fropofol-treated HCM hearts. Note the disarray of myocytes in the untreated HCM heart. Scale bar = 100 µm. (C) Wheat germ agglutinin (WGA) staining of heart tissue samples from wild-type, untreated HCM, and fropofol-treated HCM groups (upper panel). Two sections were obtained from each wall of the left ventricle (anterior, posterior, and septal), and samples from three hearts of each group were used for the analysis. The pooled data from 18 sections from 3 hearts in each group are shown as means ± S.E.M. (lower panel). P-value from ANOVA: **P < 0.001 vs. the indicated group. Scale bar = 50 µm. (D) Masson’s trichrome staining of heart tissue samples from wild-type, untreated HCM, and fropofol-treated HCM groups (upper panel). Two sections were obtained from each wall of the left ventricle (anterior, posterior, and septal), and samples from three hearts of each group were used for the analysis (scale bar = 400 µm). Pooled data for quantification of the fibrosis from five histological fields at random at a magnification of ×40 on each slide at the same level on each heart are shown (lower panel) as means ± S.E.M. P-value from ANOVA: **P < 0.001 vs. the indicated group, n = 3 hearts in each group.
Figure 5
Figure 5
Fropofol treatment suppresses hypertrophic and profibrotic genes in HCM mice. Nine genes known to be significantly enhanced in the αMHC 403/+ mouse model were selected. These genes are expressed in myocytes and fibroblasts of the HCM heart. P-value from ANOVA: *P < 0.05 between treated and untreated hearts, n = 6.
Figure 6
Figure 6
Contraction and intracellular Ca2+ in isolated HCM mouse cardiac muscles after 12 weeks of fropofol treatment. (A) Raw recordings of twitch force (right panel) and intracellular Ca2+ transients (left panel) ([Ca2+]o = 2.0). (B and C) Pooled data of force development and intracellular Ca2+ at varied extracellular Ca2+ concentrations. (D) Dynamics of twitch force in isolated trabecular muscles after fropofol treatment. Notably, fropofol-treated muscles had accelerated relaxation of twitch force (right), but time-to-peak force remained unchanged (left). P-values from ANOVA: *P < 0.05 between treated and untreated, n = 7. (E) Steady-state activations (i.e. tetanizations) were achieved by stimulating the muscle at 8–10 Hz in the presence of cyclopiazonic acid (50 μM) at varied [Ca2+]o to obtain different levels of tetanized forces. The pooled data shown represent steady‐state force‐[Ca2+] relationships of untreated and treated trabeculae or small capillary muscles. The absolute F  max is shown as the mean ± S.E.M. and is plotted at highest [Ca2+]i. All other force levels were normalized with respect to their own maximal values. The continuous line is the Hill fit based on the means of Ca50 and Hill coefficient (untreated: n = 8; treated n = 9).
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