The high prevalence and poor prognosis of heart failure are two key drivers for research into cardiac electrophysiology and regeneration. Dyssynchrony in calcium release and loss of structural organization within individual cardiomyocytes (CM) has been linked to reduced contractile strength and arrhythmia. Correlating calcium dynamics and cell microstructure requires multidimensional imaging with high spatiotemporal resolution. In light-sheet fluorescence microscopy (LSFM), selective plane illumination enables fast optically sectioned imaging with lower phototoxicity, making it suitable for imaging subcellular dynamics. In this work, a custom remote refocusing LSFM system is applied to studying calcium dynamics in isolated CM, cardiac cell cultures and tissue slices. The spatial resolution of the LSFM system was modelled and experimentally characterized. Simulation of the illumination path in Zemax was used to estimate the light-sheet beam waist and confocal parameter. Automated MATLAB-based image analysis was used to quantify the optical sectioning and the 3D point spread function using Gaussian fitting of bead image intensity distributions. The results demonstrated improved and more uniform axial resolution and optical sectioning with the tighter focused beam used for axially swept light-sheet microscopy. High-speed dual-channel LSFM was used for 2D imaging of calcium dynamics in correlation with the t-tubule structure in left and right ventricle cardiomyocytes at 395 fps. The high spatio-temporal resolution enabled the characterization of calcium sparks. The use of para-nitro-blebbistatin (NBleb), a non-phototoxic, low fluorescence contraction uncoupler, allowed 2D-mapping of the spatial dyssynchrony of calcium transient development across the cell. Finally, aberration-free remote refocusing was used for high-speed volumetric imaging of calcium dynamics in human induced pluripotent stem-cell derived cardiomyocytes (hiPSC-CM) and their co-culture with adult-CM. 3D-imaging at up to 8 Hz demonstrated the synchronization of calcium transients in co-culture, with increased coupling with longer co-culture duration, uninhibited by motion uncoupling with NBleb.