Nanoscale reorganization of sarcoplasmic reticulum in pressure-overload cardiac hypertrophy visualized by dSTORM

Abstract

Pathological cardiac hypertrophy is a debilitating condition characterized by deleterious thickening of the myocardium, dysregulated Ca 2+ signaling within cardiomyocytes, and contractile dysfunction. Importantly, the nanoscale organization, localization, and patterns of expression of critical Ca 2+ handling regulators including dihydropyridine receptor (DHPR), ryanodine receptor 2 (RyR2), phospholamban (PLN), and sarco/endoplasmic reticulum Ca 2+ -ATPase 2A (SERCA2A) remain poorly understood, especially during pathological hypertrophy disease progression. In the current study, we induced cardiac pathological hypertrophy via transverse aortic constriction (TAC) on 8-week-old CD1 mice, followed by isolation of cardiac ventricular myocytes. dSTORM super-resolution imaging was then used to visualize proteins at nanoscale resolution at two time points and we quantified changes in protein cluster properties using Voronoi tessellation and 2D Fast Fourier Transform analyses. We showed a decrease in the density of DHPR and RyR2 clusters with pressure-overload cardiac hypertrophy and an increase in the density of SERCA2A protein clusters. PLN protein clusters decreased in density in 2-week TAC but returned to sham levels by 4-week TAC. Furthermore, 2D-FFT analysis revealed changes in molecular organization during pathological hypertrophy, with DHPR and RyR2 becoming dispersed while both SERCA2A and PLN sequestered into dense clusters. Our work reveals molecular adaptations that occur in critical SR proteins at a single molecule during pressure overload-induced cardiomyopathy. Nanoscale alterations in protein localization and patterns of expression of crucial SR proteins within the cardiomyocyte provided insights into the pathogenesis of cardiac hypertrophy, and specific evidence that cardiomyocytes undergo significant structural remodeling during the progression of pathological hypertrophy.

Functional culture and in vitro genetic and small-molecule manipulation of adult mouse cardiomyocytes

“…dSTORM super-resolution imaging. Direct stochastic optical reconstruction microscopy was carried out as described previously 28 . Briefly, stochastic photoswitching of immunostained samples was initiated with a buffer containing 50 mmol L −1 2-mercaptoethylamine (M9768, Sigma-Aldrich), 40 µg mL −1 catalase (C3155, Sigma-Aldrich), 500 µg mL −1 glucose oxidase (G7141, Sigma-Aldrich), 50% (w/v) D-glucose (Sigma-Aldrich), in PBS pH 7.4.…”

Assessing Cardiomyocyte Excitation-Contraction Coupling Site Detection From Live Cell Imaging Using a Structurally-Realistic Computational Model of Calcium Release

“…Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) re-uptakes Ca 2+ from the cytosol into intracellular stores and has been shown to colocalize with RyRs at z-disks (Drago et al, 1998; Eisner et al, 2017; Hadipour-Lakmehsari et al, 2019). SERCA (or effluxing NCX) located near a releasing RyR cluster might alter Ca 2+ diffusion into the imaging plane by reducing signal amplitude or symmetry (depending on the orientation of these channels in relation to the imaging plane).…”

Nanoscale Organization, Regulation, and Dynamic Reorganization of Cardiac Calcium Channels

“…There is little information on alterations in jSR motility during HF although disrupted SR networks have been observed in HF models ( Pinali et al, 2013 ), and altered distributions of SR proteins are observed in hypertrophic myocytes ( Hadipour-Lakmehsari et al, 2019 ). Microtubule growth and stability is decreased by the oxidative stress of MI, resulting in enhanced rates of microtubule catastrophe and impaired delivery of K V 4.2 and K V 4.3 potassium channels in mouse ventricular myocytes ( Drum et al, 2016 ).…”