BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts

Abstract

Cardiac dysfunction is a hallmark of aging in humans and mice. Here we report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the aging phenotype. Our data indicate that age-associated overexpression of BIN1 occurs alongside dysregulated endosomal recycling and disrupted trafficking of cardiac Ca_V1.2 and type 2 ryanodine receptors. These deficiencies affect channel function at rest and their upregulation during acute stress. In vivo echocardiography reveals reduced systolic function in old mice. BIN1 knockdown using an adeno-associated virus serotype 9 packaged shRNA-mBIN1 restores the nanoscale distribution and clustering plasticity of ryanodine receptors and recovers Ca²⁺ transient amplitudes and cardiac systolic function toward youthful levels. Enhanced systolic function correlates with increased phosphorylation of the myofilament protein cardiac myosin binding protein-C. These results reveal BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium.

Fig. 1. β-AR-stimulated augmentation of I_Ca and Ca²⁺ transients is diminished in aging.

a Representative whole-cell currents elicited from young and old ventricular myocytes before (control; black) and during application of ISO (blue). b Dot-plots showing the fold-change in peak current with ISO in young and old myocytes. c Dot-plots showing peak I_Ca density before and after ISO. d Plots showing the voltage dependence of I_Ca density for both groups before and after ISO. e Voltage dependence of the normalized conductance (G/G_max) fit with Boltzmann functions and f dot-plots showing the V_1/2 of activation for each group. N-numbers for patch clamp data in (b–f ) is as follows: young (N = 9, n = 13) and old (N = 5, n = 11). g Representative Ca²⁺ transients recorded before (black) and after ISO (blue) from paced young and old myocytes. h Dot-plots showing the fold increase in Ca²⁺ transient amplitude after ISO and i Ca²⁺ transient amplitude before and after ISO. N-numbers for Ca²⁺ transient data in (h, i) is as follows: young (N = 5, n = 15) and old (N = 3, n = 15). Unpaired two-tailed Student’s t-tests were performed on data sets displayed in (b, h). Two-way ANOVAs with multiple comparison post-hoc tests were performed on data displayed in (c, f, i). Young data in (b–f ) is pooled from NIA young and JAX young myocytes, as no significant differences were found in I_Ca when NIA young and JAX young myocytes were compared (see Supplementary Fig. 1a–e). Young data in (h, i) is from NIA young. Data are presented as mean ± SEM. Source data are provided in the Source Data file.

Fig. 2. Basal super-clustering and impaired β-AR responsiveness of Ca_V1.2 and RyR2 in aged myocytes.

a Single-molecule localization microscopy (SMLM) map showing Ca_V1.2 channel localization and distribution in the t-tubules of young and old ventricular myocytes with or without ISO stimulation. Yellow boxes indicate the location of the regions of interest magnified in the top right of each image. b Dot-plots showing mean Ca_V1.2 channel cluster areas in young (control: N = 3, n = 16; ISO: N = 3, n = 16) and old (control: N = 4, n = 11; ISO: N = 4, n = 10) myocytes. c, d show the same for RyR2 in young (control: N = 3, n = 15; ISO: N = 3, n = 15) and old (control: N = 3, n = 12; ISO: N = 3, n = 11) myocytes. Statistical analyses on data summarized in (b, d) were performed using two-way ANOVAs with multiple comparison post-hoc tests. Young data in b and d are from NIA young. Data are presented as mean ± SEM. Source data are provided in the Source Data file.

Fig. 3. Dynamic TIRF imaging reveals age-associated trafficking deficits of Ca_V1.2.

a, b Representative TIRF images of transduced Ca_vβ_2a-paGFP young (a) and old (b) ventricular myocytes before (top) and after ISO (bottom). Channel populations that were inserted (red), endocytosed (blue), or static (white) during the ISO treatment are represented to the right. c, d Dot-plots summarizing the quantification of inserted (c) and endocytosed (d) Ca_vβ_2a-paGFP populations. e is a compilation of the data to show the relative % of the channel pool that is inserted, endocytosed, and static. f is a summary plot showing the net % change of plasma membrane Ca_vβ_2a-paGFP after ISO for each cohort of young and old myocytes. N-numbers for data in (c–f ) are as follows: young (N = 3, n = 9) and old (N = 3, n = 10). Statistical analysis was performed on data in (c–f ) using unpaired two-tailed Student’s t-tests. Young data in (c–f ) are from JAX young. Data are presented as mean ± SEM. Source data are provided in the Source Data file.

Fig. 4. Aging impairs β-AR-stimulated Ca_V1.2 recycling.

a Airyscan super-resolution images of Ca_V1.2 (green) and EEA1 (magenta) immunostained myocytes with and without ISO. Bottom: Binary colocalization maps show pixels in which Ca_V1.2 and EEA1 completely overlapped. b dot-plots summarizing % colocalization between EEA1 and Ca_V1.2 young (control: N = 3, n = 16; ISO: N = 3, n = 16) and old (control: N = 3, n = 14; ISO: N = 3, n = 14) myocytes, and c EEA1-positive endosome areas in young (control: N = 3, n = 15; ISO: N = 3, n = 16) and old (control: N = 3, n = 14; ISO: N = 3, n = 13) myocytes. Data were analyzed using two-way ANOVAs with multiple comparison post-hoc tests. Young data in (b, c) are from JAX young. Note no significant differences in EEA1/Ca_V1.2 colocalization, responsivity to ISO, or endosome size was detected when young JAX and young NIA myocytes were compared (Supplementary Fig. 1h, i). Data are presented as mean ± SEM. Source data are provided in the Source Data file.

Fig. 5. shRNA-mediated BIN1 knockdown restores youthful expression levels and localization patterns.

a Western blot of BIN1 expression in whole heart lysates from young and old mice probed with 2F11 (top) and 99D (bottom). Short and long exposures are displayed. Total ponceau was used for normalization. b Histogram showing normalized BIN1 levels in old relative to young for 2F11 and 99D (N = 6 biological replicates). c Quantitative RT-PCR analysis of pan-bin1, bin1, bin1 + 13, bin1 + 17, and bin1 + 13 + 17 transcripts for young and old myocytes are displayed normalized to gapdh (N = 3 per group, samples from each N were ran in triplicate through three separate PCR runs). d Representative Airyscan images of young (N = 3, n = 20), old (N = 3, n = 15), old shRNA-scrmb (N = 3, n = 16) and old shRNA-mBIN1 (N = 3, n = 19) myocytes immunostained against BIN1. e Western blot of BIN1 expression in whole heart lysates from: young, old, shRNA-mBIN1, and shRNA-scrmb transduced mice probed with 2F11. Short and long exposures are displayed. Total ponceau was used for normalization. f Histogram showing normalized BIN1 levels relative to young (N = 3 biological replicates). Statistical analysis was performed on data in (b) using unpaired two-tailed Student’s t-tests, on data in (c) using unpaired one-tailed Student’s t-tests, and on data in (f ) using one-way ANOVAs with multiple comparison post-hoc tests. Young data in (a, b, e, f ) are from JAX young, and young data in (c, d) are from NIA young myocytes. Note there was no significant difference in BIN1 expression when JAX and NIA young mice were compared (see Supplementary Fig. 1j–l). Data are presented as mean ± SEM. See Supplemental Information for full scans and number of technical replicates for western blots. Source data are provided in the Source Data file.

Fig. 6. BIN1 knockdown restores β-AR augmentation of Ca²⁺ transients and RyR2 clustering dynamics.

a Representative whole-cell currents from shRNA-scrmb and shRNA-mBIN1 myocytes before and during ISO application. b fold change in peak I_Ca with ISO in young, old, shRNA-scrmb (N = 3, n = 7), and shRNA-mBIN1 (N = 3, n = 9) myocytes. c Representative Ca²⁺ transients recorded from old shRNA-scrmb and shRNA-mBIN1 myocytes before and after ISO. d Fold increase after ISO from young, old, shRNA-scrmb (N = 3, n = 14), and shRNA-mBIN1 (N = 5, n = 18) myocytes. e SMLM localization maps showing Ca_V1.2 channel localization on t-tubules of myocytes from old shRNA-scrmb and shRNA-mBIN1, with or without ISO stimulation. Regions of interest are highlighted by yellow boxes. f Fold change in mean Ca_V1.2 channel cluster area with ISO in the young, old, old shRNA-scrmb (control: N = 3, n = 14; ISO: N = 3, n = 13) and shRNA-mBIN1 myocytes (control: N = 3, n = 12; ISO: N = 3, n = 11). g, h show the same layout for RyR2 immunostained old shRNA-scrmb (control: N = 3, n = 12; ISO: N = 3, n = 13) and shRNA-mBIN1 myocytes (control: N = 3, n = 9; ISO: N = 3, n = 8). Old and young data points in (b, d, f, h) are reproduced from data in Figs. 1b, h, and 2b, d respectively. Statistical analysis was performed on data in (b, d, f, h) using one-way ANOVAs with multiple comparison post-hoc tests. Young data in (b) are pooled from NIA young and JAX young myocytes, data in (d, f, h) are from NIA young myocytes. Note there was no significant difference in I_Ca, Ca_V1.2, and RyR2 cluster areas when JAX and NIA young mice were compared (see Supplementary Fig. 1a–g). Data are presented as mean ± SEM. Source data are provided in the Source Data file.

Fig. 7. BIN1 knockdown improves cardiac contractility in old mice.

a Representative M-mode echocardiogram images from conscious young and old mice. b Summary dot-plots showing fractional shortening (FS) in young (N = 10) and old (N = 30) mice are shown. c, d Representative M-mode echocardiogram images from conscious old mice before and two, four, and six weeks after RO-injection of shRNA-scrmb (c) and shRNA-mBIN1 (d). e Summary dot-plots for FS showing paired results before and two weeks after RO-injection for shRNA-scrmb (N = 14) and shRNA-mBIN1 (N = 14). f summary dot-plots for FS showing paired results before and two, four, and six weeks after RO-injection for shRNA-scrmb (N = 5) and shRNA-mBIN1 (N = 5). Statistical analysis was performed on data in (b) using unpaired two-tailed Student’s t-tests, on data in (e) using paired two-tailed Student’s t-tests, and on data in (f ) using one-way ANOVAs with multiple comparison post-hoc tests. Young data are from NIA young mice. Data are presented as mean ± SEM. Source data are provided in the Source Data file.

Fig. 8. BIN1 knockdown does not recover age-related diastolic dysfunction.

a, b Representative pulsed-wave (PW) Doppler images from unconscious young (a) and old (b) mice before and two, four, and six weeks after RO-injection of shRNA-scrmb (left) and shRNA-mBIN1 (right). Summary dot-plots for young (N = 10) and old (N = 19) mice, paired results before and two weeks after RO-injection of old mice with shRNA-scrmb (N = 10) and shRNA-mBIN1 (N = 9), and paired results before and two, four and six weeks after RO-injection of old mice with shRNA-scrmb (N = 4) and shRNA-mBIN1 (N = 4) for the following diastolic measurements are displayed: c–e isovolumetric relaxation time (IVRT) and, f–h mitral valve E/A ratio (MV E/A). Statistical analysis was performed on data in (c, f ) using unpaired two-tailed Student’s t-tests, on data in (d, g) using paired two-tailed Student’s t-tests, and on data in (e, h) using one-way ANOVAs with multiple comparison post-hoc tests. Young data are from NIA young mice. Data are presented as mean ± SEM. Source data are provided in the Source Data file.

Fig. 9. Myofilament Ca²⁺ sensitivity is altered in aging.

a Western blot of total (top) and pS23/24 (bottom) cTnI expression in whole heart lysates from young, old, shRNA-mBIN1 or shRNA-scrmb transduced mice. Ponceau was used for normalization. b Histogram showing normalized total cTnI levels relative to young (N = 3 biological replicates). c Histogram showing cTnI pS23/24 normalized to total cTnI relative to young (N = 3 biological replicates). d Western blot of total (top) and pS273 (bottom) cMyBP-C expression in whole heart lysates from young, old, shRNA-mBIN1 or shRNA-scrmb transduced mice. Ponceau was used for normalization. e Histogram showing normalized total cMyBP-C levels relative to young (N = 3 biological replicates). f Histogram showing cMyBP-C pS273 normalized to total cMyBP-C relative to young (N = 3 biological replicates). Statistical analysis was performed on data in (b, c, e, f ) using one-way ANOVAs with multiple comparisons post-hoc test. Young data are from JAX young mice. Note there was no significant difference in cTnI (total and pS23/24) or cMyBP-C (total and pS273) expression when JAX and NIA-sourced young mice were compared (see Supplementary Fig. 1m–r). Data are presented as mean ± SEM. See Supplemental Information for uncropped scans and number of technical replicates for western blots. Source data are provided in the Source Data file.

Fig. 10. BIN1 knockdown rejuvenates the aging heart.

The main findings of our study graphically illustrated and summarized. Top: In healthy young cells, Ca_V1.2 channels undergo endosomal recycling, where channels on endosomes are either marked for degradation through the late endosome pathway or are recycled to the sarcolemma through the fast and slow recycling pathways. Following β-adrenergic receptor (β-AR) stimulation, a pool of channels localized to endosomes are mobilized to the membrane, resulting in larger Ca_V1.2 clusters along t-tubules. Across the dyad, RyR2 clusters on the sarcoplasmic reticulum also increase following βAR stimulation ensuring efficient Ca²⁺-induced Ca²⁺-release. This increase in cytosolic Ca²⁺, along with increased phosphorylation of cardiac Troponin I (cTnI) and cardiac myosin binding protein-C (cMyBP-C) within the sarcomere, allows for enhanced contractility under acute stress to cope with elevated hemodynamic and metabolic demands. Bottom: In aging, Bridging Integrator 1 (BIN1) protein levels are increased, accompanied by a swelling of endosomes and subsequent dysregulation of endosomal trafficking of Ca_V1.2. Ca_V1.2 and RyR2 channels are basally super-clustered at the dyads and lose β-AR responsivity. Reduced phosphorylation of cTnI and cMyBP-C result in systolic and diastolic dysfunction. BIN1 knockdown in aging recovers RyR2 clustering plasticity and Ca²⁺ transient responsivity to β-AR stimulation. Phosphorylation of cMyBP-C is basally restored, and contractility is recovered to youthful levels. Thus, BIN1 knockdown rejuvenates the aging heart. Created with BioRender.com.