(559.1) Developmental Regulation of Sarcomere Branching in Cardiomyocytes

Poster Board Number: E155

Peter Ajayi (Muscle Energetics Laboratory, NHLBI), Yuho Kim (Muscle Energetics Laboratory, NHLBI, University of Massachusetts Lowell), Christopher Bleck (Electron Microscopy Core, NHLBI), Brian Glancy (Muscle Energetics Laboratory, NHLBI, National Institute of Arthritis and Musculoskeletal and Skin Diseases)

Cardiac muscles undergo constant contractions to continuously supply oxygenated blood to both central and peripheral organs. To sustain such large demand, cardiac muscles must convert biological fuel to functional force via the contraction of their sarcomeres. Using nanoscale 3D imaging analysis, our group demonstrated that sarcomeres within the cardiac muscle of mature mice (≥ 2 months) are frequently branched to form a unified myofibrillar matrix across the entire cell, thereby providing the architecture needed for both longitudinal and lateral transmission of active force. How these contractile networks are formed during postnatal development remains unclear. Here we used 3D focused-ion beam scanning electron microscopy (FIB-SEM) to evaluate myofibrillar ultrastructure within cardiac muscle cells in mice at postnatal (P) days 1, 7, 14, and 42, respectively. 3D reconstruction of myofibrillar structures showed highly connected networks at all time points. Quantitative assessment of sarcomere branch-points revealed that branching frequency is varied by developmental process. The percentages of sarcomeres with at least one branch-point are as follows: 57 ± 2.4% (mean ± SE, 14 cells, 129 myofibrils, 802 sarcomeres) in P1, 64 ± 2.1% (17 cells, 161 myofibrils, 795 sarcomeres) in P7, 49 ± 3.9% (4 cells, 50 myofibrils, 250 sarcomeres) in P14, and 27 ± 1.1 % (7 cells, 236 myofibrils, 2320 sarcomeres) in P42. Data was further separated based on whether a single sarcomere is branched into two (single-branch, Fig 1E, F) or more (multi-branch, Fig 1G, H) structures to delineate the nature and overall magnitude of branching within cardiomyocytes (Fig 2B- D). Across the postnatal development (P1 to P42), there was a 0.4-fold change in the magnitude of branching within ten sarcomeres (Fig 2D; 7.9±0.41, 11±0.45, 6.5±0.58, and 3.3±0.16 for P1, P7, P14, and P42, respectively). All changes were statistically significant at Plt;0.05. In conclusion, the frequency of sarcomere branching was highest at P7 and progressively declined until maturation. Findings indicate that while the architecture needed for active force transmission across the length and width of the cell is already formed at birth, the connectivity of cardiac contractile networks is dynamic throughout postnatal development. These results suggest that sarcomere branching dynamics may provide a target for modulating the contractility of cardiac muscle cells.

Support or Funding Information

This work was supported by the Division of Intramural Research of the National Heart Lung and Blood Institute and the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases.

lt;pgt;This work was supported by the Division of Intramural Research of the National Heart Lung and Blood Institute and the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases.lt;/pgt;





Figure 1: Connectivity of the myofibrillar matrix during postnatal cardiac development. A-H) Representative 3D renderings of the myofibrillar networks in early postnatal (P1) (A, C, D), one week old (P7) (H), and mature (P42) (B, G, E, F) cardiomyocytes within a mouse heart. Various colors represent individual myofibrillar segments within the network. An example of a single sarcomere branch is shown in E and F. An example of a multi-branch is shown in G and H. Scale bars - 2 µm.; Figure 2: Sarcomere branching is variable across postnatal development. A) Percentage of sarcomeres with at least one branch. B) Percentage of sarcomere with one branching event. C) Percentage of sarcomeres with more than one branching event. D) Overall frequency of sarcomere branching. * indicates significantly different from P1. # indicates significantly different from P7. + indicates significantly different from P14. One-way ANOVA was used (p < 0.05).