Dynamic Myofibrillar Remodeling in Live Cardiomyocytes under Static Stretch.

An increase in mechanical load in the heart causes cardiac hypertrophy, either physiologically (heart development, exercise and pregnancy) or pathologically (high blood pressure and heart-valve regurgitation). Understanding cardiac hypertrophy is critical to comprehending the mechanisms of heart development and treatment of heart disease. However, the major molecular event that occurs during physiological or pathological hypertrophy is the dynamic process of sarcomeric addition, and it has not been observed. In this study, a custom-built second harmonic generation (SHG) confocal microscope was used to study dynamic sarcomeric addition in single neonatal CMs in a 3D culture system under acute, uniaxial, static, sustained stretch. Here we report, for the first time, live-cell observations of various modes of dynamic sarcomeric addition (and how these real-time images compare to static images from hypertrophic hearts reported in the literature): 1) Insertion in the mid-region or addition at the end of a myofibril; 2) Sequential addition with an existing myofibril as a template; and 3) Longitudinal splitting of an existing myofibril. The 3D cell culture system developed on a deformable substrate affixed to a stretcher and the SHG live-cell imaging technique are unique tools for real-time analysis of cultured models of hypertrophy.

Laser cell-micropatterned pair of cardiomyocytes: the relationship between basement membrane development and gap junction maturation.

The basement membrane (BM), a network of laminin and collagen IV, mechanically supports individual cells and directly mediates cell-cell and cell-extracellular matrix (ECM) interactions. For example, the BM network that tightly encloses each cardiomyocyte (CM) mediates the alignment of CMs with collagen I in the ECM. Additionally, the BM-laminin is involved in the formation of gap junctions (GJs), which regulate electrical coupling between two CMs in the myocardium. The role of BM in GJ maturation remains unclear because of the complicated in vivo structures and lack of an ideal in vitro culturing mode. In this study, our laser cell-micropatterning system was used to place two neonatal CMs (NCMs) in contact on an aligned collagen gel (ACG) to study the relationship between GJ maturation and BM development. The results of double immunofluorescence staining and confocal imaging showed that BM-laminin was deposited earlier than the formation of GJs in the intercellular space and that newly expressed connexin 43 clusters were preferentially assembled near the deposited BM structures. Eventually the BM network surrounded the GJs.