Fig 1.
Expression and localization of HSPB7 in cardiac muscle.
(A) Immunoblot analysis of the cardiac muscle showing HSPB7 constitutive expression from the embryonic stages to adulthood (E14.5 to P28) with multiple forms of HSPB7 at different molecular masses (arrows). (B) Subcellular localization of HSPB7 in the cardiac muscle of adult mice. The heart sections were stained with antibodies against HSPB7 (red) and desmoplakin (desmosome), α-actinin (Z-line), myomesin (M-line), N-cadherin (adhering junction), connexin 43 (gap junction), and cardiac-actin (I-bend). HSPB7 mainly localizes at the intercalated discs and is adjacent to the Z-line with a striated pattern. (C) Colocalization of HSPB7 with N-cadherin during development. Heart sections from the embryonic stages to adulthood (E14.5 to P56) were stained with antibodies against HSPB7 (red) and N-cadherin (green). The nucleus was visualized through Hoechst 33342 staining. Insets show the representative areas with higher magnification. Scale bar: 10 μm.
Fig 2.
Characterization of HSPB7 CKO mouse.
(A) Immunoblot analysis for HSPB7 protein expression levels in control (Flox/Flox) and CKO animals 4 days (d4) and 7 days (d7) after tamoxifen administration. The GAPDH signal shows the loading of samples between lanes. (B) Quantitative analysis for immunoblot analysis of HSPB7 expression protein levels in cardiac tissue from control and CKO mice. Seven days after the first tamoxifen administration, HSPB7 protein expression dropped to less than 10% in CKO animals, as determined by immunoblot blot (HSPB7 compared with GAPDH). Data are presented as means ± SD. (C) Double staining of the left ventricle section with HSPB7 and Hoechst 33342 in the control and CKO hearts at d7 after tamoxifen administration. HSPB7 was no longer present at the intercalated disc and sarcomere. Scale bar: 20 μm (D) Significant increase in heart weight in HSPB7 CKO mice (n = 8; *p = 0.00027). (E) Kaplan–Meier survival curve of HSPB7 CKO and control mice. (F) Representative whole mounts (left), Masson’s trichrome (middle left and right), and hematoxylin-eosin–stained (right) transverse sections of control and CKO mouse hearts. Histological analysis showed inflammatory infiltration in the myocardium, identified as mostly lymphocytes and plasma cells, in HSPB7 CKO hearts at d7 after tamoxifen administration. In addition, Masson’s trichrome staining showed no significant collagen deposition in HSPB7 CKO hearts, as compared to control mice.
Fig 3.
Echocardiographic measurements of HSPB7 CKO and control mice.
(A) Representative two-dimensional and M-mode echocardiographic images of the HSPB7 CKO and control hearts. (B) Echocardiographic analysis in the HSPB7 CKO and control mice before and at 4 days (d4) and 7 days (d7) after tamoxifen administration. Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic internal diameter (LVIDs), and left ventricular end-diastolic internal diameter (LVIDd). Data are means ± SD; n = 5 per group. *, P < 0.05 **, P < 0.01 relative to the control.
Fig 4.
Electrical conduction is impaired in HSPB7 CKO hearts.
(A and B) Annotated ECG curve of the HSPB7 CKO and control animals before (A) and 7 days after (B) the first tamoxifen injection. (C and D) Quantification of the ECG changes in HSPB7 CKO and control animals before (C) and 7 days after (D) the first tamoxifen administration. N = 5 per group. Data are presented as means ± SD. *, P < 0.05 **, P < 0.01 relative to the control. (E) Representative telemetric 2-lead ECG recording of a tamoxifen-treated HSPB7 CKO and control mice. Telemetry ECG recordings of lethal arrhythmias in HSPB7 CKO mice. n = 2 per group.
Fig 5.
Ultrastructural study of control and HSPB7 CKO hearts.
Transmission electron micrographs (TEMs) of ventricular myocardium from HSPB7 CKO and control mice at d7 after tamoxifen administration. Right panels are higher-magnification views of the boxed areas in the left panels. (A) Normal intercalated disc structures were visible in the control hearts. The inset provides a simplistic representation of the morphology of the intercalated discs. Higher-magnification images showed abnormal adherens junctions (black arrowheads) and desmosomes (white arrowheads) with widened gaps of the intercalated discs in HSPB7 mutant hearts. Abnormal Z-line (black arrow), filament disruption (asterisk), and detachment of myofibrils at the intercalated disc (white arrow) were also observed in the CKO mice. (B) The ultrastructure of the sarcomeres at the center part of the cardiomyocyte was slightly distorted. Higher-magnification images showed (right panel) loose actin filaments (black arrowhead) and wider, less dense Z-lines (white arrowhead) compared with the controls. n = 3 per group. Scale bar: 250 nm.
Fig 6.
Expression and localization of ID complex proteins in HSPB7 CKO hearts.
(A) Confocal micrographs of longitudinal sections of the cardiac muscle of control and CKO mice at d7 after tamoxifen administration. Specific antibodies were used to identify the distributions of intercalated disc components: N-cadherin, desmoplakin, vinculin, and connexin 43. In HSPB7 CKO hearts, the staining of desmoplakin and N-cadherin were distributed throughout the cytoplasm, but little was observed in control hearts. Connexin 43 was absent from the intercalated discs in HSPB7 CKO hearts. The insets show representative areas at a higher magnification. The nucleus was visualized through Hoechst 33342 staining. n = 4 per group. Scale bar: 20 μm. (B) Immunoblotting of intercalated discs and sarcomeric-associated proteins in HSPB7 CKO and control hearts. GAPDH signal shows the loading of the samples between the lanes. n = 4 per group. (C) Quantitative analysis of immunoblot analysis of protein levels in cardiac tissue from control and CKO mice. Seven days after the first tamoxifen injections, connexin 43 protein expression dropped to < 40% in CKO animals, as determined by immunoblot analysis. Data are presented as means ± SD. *, P < 0.05, **, P < 0.01 relative to control.
Fig 7.
Loss of HSPB7 results in FLNC protein upregulation and aggregation.
Confocal micrographs of longitudinal sections (A) and cross-sections (B) of the heart. FLNC aggregation (arrowheads) was observed only in the mutant cardiomyocytes. Extracellular matrix accumulation was labeled using wheat germ agglutinin (WGA). The nucleus was visualized through Hoechst 33342 staining. Scale bar: 50 μm. (C) Immunoblot analysis and quantitation (D) of the expression levels of FLNC in the cardiac muscle. The muscle homogenate supernatant (S) and pellet (P) fractions were analyzed from control and CKO mice at d7 after tamoxifen administration. GAPDH and Coomassie Blue staining were used to verify the loading amount in the supernatant and pellet. n = 3 per group. Data are presented as means ± SD. *, P < 0.05 relative to the control.
Fig 8.
Intramyocardial injection with Adeno-Cre in HSPB7Flox/Flox mice.
Confocal micrographs of the cardiac muscle of the HSPB7Flox/Flox mice 8 days after intramyocardial injection with Adeno-Cre virus. n = 3 per group. The heart sections were co-immunostained with antibody against HSPB7 (red) and specific antibodies (green) were used to identify the distributions of intercalated disc components: desmoplakin (A) and N-cadherin (B). (A) In HSPB7Flox/Flox hearts, desmoplakin was distributed throughout the cytoplasm (asterisk in lower panel) at the HSPB7 depleted region compared with the control region (upper panel). (B) N-cadherin exhibited partial misexpression at the HSPB7 depleted region (asterisk in lower panel) compared with the control region (upper panel). The nucleus was visualized through Hoechst 33342 staining (blue). Scale bar: 10 μm.