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Abstract
Pathological cardiac hypertrophy is a debilitating condition characterized by deleterious thickening of the myocardium, dysregulated Ca2+ signaling within cardiomyocytes, and contractile dysfunction. Importantly, the nanoscale organization, localization, and patterns of expression of critical Ca2+ handling regulators including dihydropyridine receptor (DHPR), ryanodine receptor 2 (RyR2), phospholamban (PLN), and sarco/endoplasmic reticulum Ca2+-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.
Highlights
The sarcoplasmic reticulum (SR) is a multi-functional organelle that is essential in the proper functioning of cardiomyocytes
Traditional diffraction-limited microscopy can only achieve a lateral resolution of 150 nm while super resolution imaging via direct stochastic optical reconstruction microscopy (dSTORM) can achieve a lateral resolution of 30 nm, enabling more accurate visualization of proteins and provide better localization of individual fluorescent reporters
In our study we have shown quantitative differences in the 2-D nanoscale spatial distribution of critical sarcoplasmic reticulum proteins with transverse aortic constriction (TAC), which we feel provides a comprehensive understanding of disease progression alongside usual biochemical analyses
Summary
The sarcoplasmic reticulum (SR) is a multi-functional organelle that is essential in the proper functioning of cardiomyocytes. Changes in expression levels and biochemical properties in many of the SR proteins, including DHPR, RyR2, SERCA2A, and PLN are well established[4,5,6,7] In addition to these widescale biochemical changes, spatial changes in L-type Ca2+ channels and changes of microdomain interactions between potassium channels and caveolin-3 have been shown to trigger arrhythmias in heart failure[8,9]. Using direct stochastic optical reconstruction microscopy (dSTORM), we visualized the changes in localization, clustering, and patterns of expression of several proteins critical in SR Ca2+ cycling in hypertrophic hearts. These approaches provide novel characterization of the physiology underlying the pressure-overload cardiac hypertrophy phenotype and provides new mechanistic insights into this disease. Our findings reveal a robust reorganization in SR protein clusters in nanoscale in response to pathological cardiac hypertrophy
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