Korean Circ J.  2025 Feb;55(2):100-117. 10.4070/kcj.2024.0179.

Shank3 Overexpression Leads to Cardiac Dysfunction in Mice by Disrupting Calcium Homeostasis in Cardiomyocytes

• Ko TH
• Kim Y
• Jin C
• Yu B
• Lee M
• Luong PK
• Trinh TN
• Yang Y
• Kang H
• Zhang Y
• Ma R
• Yoo K
• Choi J
• Kim JY
• Woo SH
• Han K
• Choi JI

• KMID: 2565415
• DOI: http://doi.org/10.4070/kcj.2024.0179

Abstract

Background and Objectives
SH3 and multiple ankyrin repeat domains 3 (Shank3) proteins play crucial roles as neuronal postsynaptic scaffolds. Alongside neuropsychiatric symptoms, individuals with SHANK3 mutations often exhibit symptoms related to dysfunctions in other organs, including the heart. However, detailed insights into the cardiac functions of Shank3 remain limited. This study aimed to characterize the cardiac phenotypes of Shank3-overexpressing transgenic mice and explore the underlying mechanisms.
Methods
Cardiac histological analysis, electrocardiogram and echocardiogram recordings were conducted on Shank3-overexpressing transgenic mice. Electrophysiological properties, including action potentials and L-type Ca²⁺ channel (LTCC) currents, were measured in isolated cardiomyocytes. Ca²⁺ homeostasis was assessed by analyzing cytosolic Ca²⁺ transients and sarcoplasmic reticulum Ca²⁺ contents. Depolarization-induced cell shortening was examined in cardiomyocytes. Immunoprecipitation followed by mass spectrometrybased identification was employed to identify proteins in the cardiac Shank3 interactome. Western blot and immunocytochemical analyses were conducted to identify changes in protein expression in Shank3-overexpressing transgenic cardiomyocytes.
Results
The hearts of Shank3-overexpressing transgenic mice displayed reduced weight and increased fibrosis. In vivo, sudden cardiac death, arrhythmia, and contractility impairments were identified. Shank3-overexpressing transgenic cardiomyocytes showed prolonged action potential duration and increased LTCC current density. Cytosolic Ca²⁺ transients were increased with prolonged decay time, while sarcoplasmic reticulum Ca²⁺ contents remained normal. Cell shortening was augmented in Shank3-overexpressing transgenic cardiomyocytes. The cardiac Shank3 interactome comprised 78 proteins with various functions. Troponin I levels were down-regulated in Shank3-overexpressing transgenic cardiomyocytes.
Conclusions
This study revealed cardiac dysfunction in Shank3-overexpressing transgenic mice, potentially attributed to changes in Ca²⁺ homeostasis and contraction, with a notable reduction in troponin I.

Keyword

Cardiomyocyte; Sudden cardiac death; Arrhythmia; Troponin I

Figure

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  Figure 1 Pathological cardiac remodeling in Shank3 TG mice. (A) Survival plot of male WT and Shank3 TG mice from 3 to 12 weeks of age. (B) Representative Western blot images and quantification showing increased expression of Shank3 (α, β, and γ isoforms and total) proteins in the cardiac tissue of Shank3 TG mice compared to WT mice (n=5 mice per genotype). (C) Representative images of whole heart and quantification showing normal BW but reduced HW and HW/BW ratio in Shank3 TG mice compared to WT mice (n=12–14 mice per genotype). Scale bar, 0.2 cm. (D) Representative images of Masson’s trichrome staining and quantification showing an increased fibrosis area in the cardiac tissue of Shank3 TG mice compared to WT mice (n=9–10 mice per genotype). Black scale bar, 1 mm; white scale bar, 100 μm.BW = body weight; HW = heart weight; Shank3 = SH3 and multiple ankyrin repeat domains 3; TG = transgenic; WT = wild-type.*p<0.05, **p<0.01, ***p<0.001.

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  Figure 2 Ventricular arrhythmia and impaired cardiac contraction in Shank3 TG mice. (A) Representative traces of telemetry ECG from WT and Shank3 TG mice (left panels). Shank3 TG mice exhibit VT, sinus arrhythmias, and atrioventricular block. Graphs on right panels show incidence percentage of WT and Shank3 TG mice experiencing SCD and sustained VT (n = 5 mice per genotype). (B) Representative trances of surface ECG and quantifications of RR, QT, and QTc intervals from WT and Shank3 TG mice (n=8 mice per genotype). (C) Representative M-mode of echocardiogram images and quantifications of LVEDD, LVESD, and FS from WT and Shank3 TG mice (n=10 mice per genotype). Scale bar, 0.2 cm.ECG = electrocardiography; FS = fractional shortening; LVEDD = left ventricular end-diastolic diameter; LVESD = left ventricular end-systolic diameter; SCD = sudden cardiac death; Shank3 = SH3 and multiple ankyrin repeat domains 3; TG = transgenic; VT = ventricular tachycardia; WT = wild-type.*p<0.05, **p<0.01.

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  Figure 3 Prolonged action potential duration and increased L-type Ca2+ current density in Shank3 TG cardiomyocytes. (A) Representative traces of action potential from cardiomyocytes of WT and Shank3 TG mice. (B) Quantification of APD at 20%, 50%, and 90% in cardiomyocytes of WT and Shank3 TG mice (n=12 cells per genotype). (C) Quantifications of APA, dv/dtmax, and RMP. (D-F) Representative traces of LTCC currents (D), current density-voltage relationship (E), and the current density at 0 mV (F) of WT and Shank3 TG cardiomyocytes (n=16–18 cells per genotype). (G) Activation and inactivation properties of LTCC in WT and Shank3 TG cardiomyocytes.APA = action potential amplitude; APD = action potential duration; dv/dtmax = maximal upstroke velocity; LTCC = L-type Ca2+ channel; RMP = resting membrane potential; Shank3 = SH3 and multiple ankyrin repeat domains 3; TG = transgenic; WT = wild-type.*p<0.05, **p<0.01.

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  Figure 4 Increased cytosolic Ca2+ transient and decay time, but normal SR Ca2+ loading in Shank3 TG cardiomyocytes. (A, B) Representative traces of Ca2+ transient from the 2-dimensional line-scan of Ca2+ waves in WT and Shank3 TG cardiomyocytes (n=11–12 cells per genotype) (C) Quantifications of Ca2+ transient amplitude (F/F0) and time to 50% and 90% decay of Ca2+ transient (DT50 and DT90, respectively). (D) Representative traces and quantification (mean magnitude) of caffeine-induced Ca2+ transients (SR Ca2+ content) in WT and Shank3 TG cardiomyocytes (n=19–31 cells per genotype). Shank3 = SH3 and multiple ankyrin repeat domains 3; SR = sarcoplasmic reticulum; TG = transgenic; WT = wild-type.*p<0.05, **p<0.01.

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  Figure 5 Augmentation of depolarization-induced contraction in Shank3 TG cardiomyocytes. (A) Representative cell shortening traces recorded from WT and Shank3 TG cardiomyocytes. (B) Quantification of the magnitudes of cell shortening from WT and Shank3 TG cardiomyocytes (n=13–16 cells per genotype). (C) Quantifications of T max, T r,90, +dT/T max, and −dT/T r,90.+dT/T max = rate of contraction; −dT/T r,90 = rate of 90% of relaxation; Shank3 = SH3 and multiple ankyrin repeat domains 3; TG = transgenic; T max = time to maximal shortening; T r,90 = time to 90% of relaxation; WT = wild-type.*p<0.05.

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  Figure 6 Identification of proteins in the cardiac Shank3 interactome. (A) Western blot images showing the results of co-IP experiments between Shank3 and selected candidate binding partners in Shank3 TG cardiac lysates. (B) Western blot images demonstrating the validation of Shank3 and Homer1b/c co-IP in a small fraction of the samples processed for mass spectrometry analysis. (C) Venn diagram showing the definition of the cardiac Shank3 interactome, comprising 78 proteins, from the mass spectrometry analysis. Proteins identified in control samples were also included if they met both criteria (detected in 2 experimental samples with their average spectral count at least 1.5 times higher in experimental samples than control samples). (D) Interactome network depicting the relationships within the cardiac Shank3 interactome. (E) GO analysis for the cardiac Shank3 interactome.DAVID = Database for Annotation, Visualization and Integrated Discovery; EGFP = enhanced green fluorescent protein; GO = Gene Ontology; IP = immunoprecipitation; Shank3 = SH3 and multiple ankyrin repeat domains 3.

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  Figure 7 Reduced expression of troponin I in Shank3 TG cardiomyocytes. (A) Representative Western blot images and quantification showing expression levels of various proteins in WT and Shank3 TG cardiac lysates (n=8–16 mice per genotype). The proteins are categorized based on their molecular functions. (B) Representative immunocytochemistry images and quantifications of intensities and inter-peak distances of troponin I and α-actinin in WT and Shank3 TG cardiomyocytes (n=25–51 cells per genotype). Scale bar, 10 µm. Shank3 = SH3 and multiple ankyrin repeat domains 3; TG = transgenic; WT = wild-type.***p<0.001.

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