Endoplasmic reticulum (ER)-mitochondrial (ER-Mito) interface, termed mitochondrial-ER contacts (MERCs), plays significant roles in the maintenance of bioenergetics and basal cell functions via the exchange of lipids, Ca²⁺, and reactive oxygen species (ROS) in various cell types/tissues. Genetic deletion of mitofusin 2 (Mfn2), one of the key components of ER-Mito tethering, in cardiomyocytes (CMs) in vivo revealed the importance of the microdomains between mitochondria and sarcoplasmic reticulum (SR), a differentiated form of ER in muscle cells, for maintaining normal mitochondrial Ca²⁺ (mtCa²⁺) handling and bioenergetics in the adult heart. However, key questions remain to be answered: 1) What tethering proteins sustain SR-Mito contact site structure in SR-Mito contact sites in the adult ventricular CMs (AVCMs), the predominant cell type in the adult heart? 2) Which MERC proteins operate in AVCMs to mediate specific microdomain functions under physiological conditions? and 3) How are the MERC protein expression profile and function altered in cardiac pathophysiology? In this review, we summarize current knowledge regarding the structure, function, and regulation of SR-Mito microdomains in the heart, with particular focus on AVCMs, which display unique membrane organization and Ca²⁺ handling compared with other cell types. We further explore molecular mechanisms underpinning microdomain dysfunction in cardiac diseases and highlight the emerging roles of MERC proteins in the development and progression of cardiac pathology.