AIMS: This study explored the lateral crest structures of adult cardiomyocytes (CMs) within healthy and diseased cardiac tissue. METHODS AND RESULTS: Using high-resolution electron and atomic force microscopy, we performed an exhaustive quantitative analysis of the three-dimensional (3D) structure of the CM lateral surface in different cardiac compartments from various mammalian species (mouse, rat, cow, and human) and determined the technical pitfalls that limit its observation. Although crests were observed in nearly all CMs from all heart compartments in all species, we showed that their heights, dictated by the subsarcolemmal mitochondria number, substantially differ between compartments from one species to another and tightly correlate with the sarcomere length. Differences in crest heights also exist between species; for example, the similar cardiac compartments in cows and humans exhibit higher crests than rodents. Unexpectedly, we found that lateral surface crests establish tight junctional contacts with crests from neighbouring CMs. Consistently, super-resolution SIM or STED-based immunofluorescence imaging of the cardiac tissue revealed intermittent claudin-5-claudin-5 interactions in trans via their extracellular part and crossing the basement membrane. Finally, we found a loss of crest structures and crest-crest contacts in diseased human CMs and in an experimental mouse model of left ventricle barometric overload. CONCLUSION: Overall, these results provide the first evidence for the existence of differential CM surface crests in the cardiac tissue as well as the existence of CM-CM direct physical contacts at their lateral face through crest-crest interactions. We propose a model in which this specific 3D organization of the CM lateral membrane ensures the myofibril/myofiber alignment and the overall cardiac tissue cohesion. A potential role in the control of sarcomere relaxation and of diastolic ventricular dysfunction is also discussed. Whether the loss of CM surface crests constitutes an initial and common event leading to the CM degeneration and the setting of heart failure will need further investigation.