Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jan 14;23(2):871.
doi: 10.3390/ijms23020871.

Subcellular Remodeling in Filamin C Deficient Mouse Hearts Impairs Myocyte Tension Development during Progression of Dilated Cardiomyopathy

Joseph D Powers  1 , Natalie J Kirkland  1 , Canzhao Liu  2 , Swithin S Razu  1 , Xi Fang  2 , Adam J Engler  1 , Ju Chen  1   2 , Andrew D McCulloch  1   2   3
Affiliations

Subcellular Remodeling in Filamin C Deficient Mouse Hearts Impairs Myocyte Tension Development during Progression of Dilated Cardiomyopathy

Joseph D Powers et al. Int J Mol Sci. .

Abstract

Dilated cardiomyopathy (DCM) is a life-threatening form of heart disease that is typically characterized by progressive thinning of the ventricular walls, chamber dilation, and systolic dysfunction. Multiple mutations in the gene encoding filamin C (FLNC), an actin-binding cytoskeletal protein in cardiomyocytes, have been found in patients with DCM. However, the mechanisms that lead to contractile impairment and DCM in patients with FLNC variants are poorly understood. To determine how FLNC regulates systolic force transmission and DCM remodeling, we used an inducible, cardiac-specific FLNC-knockout (icKO) model to produce a rapid onset of DCM in adult mice. Loss of FLNC reduced systolic force development in single cardiomyocytes and isolated papillary muscles but did not affect twitch kinetics or calcium transients. Electron and immunofluorescence microscopy showed significant defects in Z-disk alignment in icKO mice and altered myofilament lattice geometry. Moreover, a loss of FLNC induces a softening myocyte cortex and structural adaptations at the subcellular level that contribute to disrupted longitudinal force production during contraction. Spatially explicit computational models showed that these structural defects could be explained by a loss of inter-myofibril elastic coupling at the Z-disk. Our work identifies FLNC as a key regulator of the multiscale ultrastructure of cardiomyocytes and therefore plays an important role in maintaining systolic mechanotransmission pathways, the dysfunction of which may be key in driving progressive DCM.

Keywords: Z-disk; cardiac muscle; cellular remodeling; computational modeling; mechanotransmission; sarcomere.

PubMed Disclaimer

Conflict of interest statement

A.D.M. is a co-founder of and has an equity interest in Insilicomed Inc. and an equity interest in Vektor Medical, Inc. He serves on the scientific advisory board of Insilicomed, and as a scientific advisor to both companies. Some of his research grants have been identified for conflict-of-interest management based on the overall scope of the project and its potential benefit to these companies. The author is required to disclose this relationship in publications acknowledging the grant support; however, the research subject and findings reported in this study did not involve the companies in any way and have no relationship with the business activities or scientific interests of either company. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its conflict-of-interest policies.

Figures

Figure 1
Figure 1
FLNC deletion in adult mouse hearts reduces cardiomyocyte contractility without affecting Ca2+ signaling. Peak twitch tension (a) in intact papillary muscles from icKO hearts was significantly reduced compared with controls. Time to peak twitch tension (b) and time to 50% (c) and 90% (d) relaxation (RT50 and RT90, respectively) were not different in papillary muscles from icKO vs. control hearts. Similar to the intact papillary muscles, intact single cells isolated from icKO hearts exhibit significantly reduced cell shortening (e) compared with cells from control hearts, while the relaxation rate constant (f) was not different between groups. The peak amplitude of the calcium transient (g) and cytosolic calcium decay rate constant (h) was not different between groups. ** p < 0.005, *** p < 0.0005, and NS = Not Significant for unpaired student’s t-test between groups. N = n = 6 for intact papillary muscles from each group, and n > 70 for isolated cells from each group from N = 6 animals in each group.
Figure 2
Figure 2
The effects of FLNC deletion in adult mouse hearts on mRNA and protein expression. The log2 fold-changes (FC) of mRNA expression (left) and protein expression (right) in icKO hearts relative to control hearts are shown for myofilament-associated proteins (teal), Z-disk/costamere-associated proteins (blue), and Ca2+ signaling-associated proteins (purple). Fold-changes in mRNA and protein expression were determined using RNA-seq and mass spectrometry (respectively) for N = 3 mice from each group, and * indicates p < 0.05. TTN: titin; MYBP3: myosin-binding protein C; MYH6: α-myosin heavy chain; MYH7: β-myosin heavy chain; TPM1: α-tropomyosin; TNNC1: cardiac troponin C; TNNI3, cardiac troponin I; TNNT2, cardiac troponin T; ACTC1, cardiac sarcomeric actin; ANKRD1: cardiac ankyrin repeat protein (CARP); CSRP3: muscle LIM protein; FHL1, four-and-a-half LIM domain protein 1; SYNPO2: synaptopodin-2; BAG3: co-chaperone Bcl2-associated athanogene 3; CASQ2: calsequestrin-2; ATP2A2, sarcoplasmic reticulum Ca2+-ATPase; RYR1, ryanodine receptor; CACNA2D1, voltage-dependent calcium channel subunit alpha-2/delta-1; CACNB2, calcium voltage-gated channel auxiliary subunit beta 2.
Figure 3
Figure 3
Adult cardiomyocytes undergo significant cellular structural remodeling in response to FLNC deletion. (a) Representative immunofluorescent images of control (left) and icKO (right) adult mouse ventricular cardiomyocytes, stained for the Z-disk protein α-actinin (cyan) and nuclei (DAPI, blue). The length of single cardiomyocytes (b) was significantly increased in cells from icKO hearts compared with controls, while the width (c) was not different. This leads to a significant increase in the length-to-width ratio (d) and surface area (e) of cardiomyocytes from icKO hearts compared with controls, indicative of DCM remodeling. Resting slack sarcomere length (f) was not different between genotypes, while maximum Z-disk transverse span ((g); as a % of cell width) was significantly reduced in cells from icKO hearts compared with controls. Fewer sarcomeres (h) had Z-disks aligned perpendicularly (90°) to the long axis of the cell in cardiomyocytes from icKO hearts (black) compared with control (gray), causing the angular dispersion of Z-disk orientation (i) to be greater in cardiomyocytes from icKO hearts compared with controls. ** p < 0.005, *** p < 0.0005, and NS = Not Significant for unpaired Student’s t-test.
Figure 4
Figure 4
Neonatal ventricular mouse cardiomyocytes (NMVCMs) also exhibit significant structural and mechanical adaptations in response to FLNC deletion. (a) Fewer sarcomeres have Z-disks aligned perpendicularly (90°) to the long axis of the cell in NMVCMs infected with ad-Cre (FLNC-knockdown; black symbols) compared with ad-LacZ (controls; gray symbols), causing the angular dispersion of Z-disk orientation (b) to be greater in ad-Cre NVMCMs with ad-LacZ. (c) AFM revealed a significant decrease in the passive transverse stiffness of the cell membrane/cortex of NMVMs lacking FLNC (ad-Cre) compared with controls (ad-LacZ). *** p < 0.0005 for unpaired student’s t-test. N = 2 and n = 16 for ad-LacZ, N = 2 and n = 19 for ad-Cre.
Figure 5
Figure 5
Myofilament lattice geometry determined by quantitative analysis of TEM images of papillary muscle cross-sections informs a spatially explicit computational model of a cardiac half-sarcomere. Example TEM images of a papillary muscle cross-section from a control (a) and an icKO (b) heart. Scale bar is 200 nm. (c) Average thick filament (TF) center-to-center separation distance (c) was significantly reduced in icKO cardiomyocytes compared to controls. The amplitude of the Gaussian fit to the histogram of distributions of TF-TF distances ((d); as a % of total TFs measured per image) was significantly lower in icKO cardiomyocytes than controls, suggesting less uniformity of TF separation distance in icKO cardiomyocytes than in controls. * p < 0.05, ** p < 0.005 using an unpaired Student’s t-test. n > 30 images analyzed from N = 2 samples for each group. Each image contained >200 TFs. (e) Schematic of our previously described [25,26] computational model, which consists of 4 thick filaments (TFs, gray), 8 actin (thin) filaments (green), and 14 titin filaments (pink) with periodic boundary conditions to simulate a semi-infinite lattice (indicated by the white filaments). (f) A cross-section showing the 3D arrangement of the myofilament lattice (color scheme is the same as panel (e)). (g) Cardiac twitch force transients for a range of TF-TF separation distances (27–36 nm) for a constant sarcomere length (1.85 µm). Each trace is an average of 50 twitch simulations. (h) The peak twitch force for each TF-TF distance from panel (g). The black and gray vertical dashed lines indicate the experimentally determined TF-TF distances for control and icKO cardiomyocytes, respectively.
Figure 6
Figure 6
Cell-level model of the effects of spatial organization and inter-myofibril connectivity of sarcomeres on cell mechanics. (a) Schematic of our new spatially explicit computational model of a cardiomyocyte with 1000 sarcomeres (gray springs). (b) Adjacent Z-disks (blue) are mechanically coupled by a torsional (red) and a linear (green) spring system with stiffness k1 and k2, respectively. (c) The model predicts that the stiffness of the torsional spring (k1) regulating the orientation of the Z-disk must decrease by ~1 order of magnitude to account for the increase in Z-disk angular dispersion (Figure 3h) in icKO cardiomyocytes compared with control cardiomyocytes (gray and black dashed lines, respectively). (d) When decreasing the stiffness of the linear spring (k2) and regulating the distance (δ0) between adjacent Z-disks from 1–0 nN/µm, the model predicts that the maximum Z-disk bundle (as a % of cell width) decreases from above the value measured for control cardiomyocytes (black dashed line) to below that of icKO cardiomyocytes (gray dashed line).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. McNally E.M., Mestroni L. Dilated Cardiomyopathy: Genetic Determinants and Mechanisms. Circ. Res. 2017;121:731–748. doi: 10.1161/CIRCRESAHA.116.309396. - DOI - PMC - PubMed
    1. Tardiff J.C., Carrier L., Bers D.M., Poggesi C., Ferrantini C., Coppini R., Maier L.S., Ashrafian H., Huke S., Van Der Velden J. Targets for therapy in sarcomeric cardiomyopathies. Cardiovasc. Res. 2015;105:457–470. doi: 10.1093/cvr/cvv023. - DOI - PMC - PubMed
    1. McNally E.M., Golbus J.R., Puckelwartz M.J. Genetic mutations and mechanisms in dilated cardiomyopathy. J. Clin. Investig. 2013;123:19–26. doi: 10.1172/JCI62862. - DOI - PMC - PubMed
    1. Bang M., Bogomolovas J., Chen J. Understanding the Molecular Basis of Cardiomyopathy. Am. J. Physiol. Circ. Physiol. 2021 doi: 10.1152/ajpheart.00562.2021. - DOI - PMC - PubMed
    1. Peter A.K., Cheng H., Ross R.S., Knowlton K.U., Chen J. The costamere bridges sarcomeres to the sarcolemma in striated muscle. Prog. Pediatr. Cardiol. 2011;31:83–88. doi: 10.1016/j.ppedcard.2011.02.003. - DOI - PMC - PubMed

MeSH terms