Augmenting Workload Drives T-tubule Assembly in Developing Cardiomyocytes.

Contraction of cardiomyocytes is initiated at subcellular elements called dyads, where L-type Ca 2+ channels in t-tubules are located within close proximity to Ryanodine Receptors in the Sarcoplasmic Reticulum. While evidence from small rodents indicates that dyads are assembled gradually in the developing heart, it is unclear how this process occurs in large mammals. We presently examined dyadic formation in fetal and newborn sheep (Ovis aries), and the regulation of this process by fetal cardiac workload. By employing advanced imaging methods, we demonstrated that t-tubule growth and dyadic assembly proceed gradually during fetal sheep development, from 93 days of gestational age until birth (147 days). This process parallels progressive increases in fetal systolic blood pressure, and includes step-wise colocalization of L-type Ca 2+ channels and the Na + -Ca 2+ exchanger with Ryanodine Receptors. These proteins are upregulated together with the dyadic anchor junctophilin-2 (JPH2) during development, alongside changes in the expression of amphiphysin-2 (BIN1) and its partner proteins myotubularin (MTM1) and dynamin-2 (DNM2). Increasing fetal systolic load by infusing plasma or occluding the post-ductal aorta accelerated t-tubule growth. Conversely, reducing fetal systolic load with infusion of enalaprilat, an angiotensin converting enzyme inhibitor, blunted t-tubule formation. Interestingly, altered t-tubule densities did not relate to changes in dyadic junctions, or marked changes in the expression of dyadic regulatory proteins, indicating that distinct signals are responsible for maturation of the Sarcoplasmic Reticulum. In conclusion, augmenting blood pressure and workload during normal fetal development critically promote t-tubule growth, while additional signals contribute to dyadic assembly. KEY POINTS: T-tubule growth and dyadic assembly proceed gradually in cardiomyocytes during fetal sheep development, from 93 days of gestational age until the post-natal stage. Increasing fetal systolic load by infusing plasma or occluding the post-ductal aorta accelerated t-tubule growth and hypertrophy. In contrast, reducing fetal systolic load by enalaprilat infusion slowed t-tubule development and decreased cardiomyocyte size. Load-dependent modulation of t-tubule maturation was linked to altered expression patterns of the t-tubule regulatory proteins junctophilin-2, amphiphysin-2 (BIN1), and its protein partners. Altered t-tubule densities did not influence dyadic formation, indicating that distinct signals are responsible for maturation of the sarcoplasmic reticulum. Abstract figure legend In the sheep heart, t-tubule growth begins in the fetal stage of development. Even at this early stage, growing t-tubules contain both L-type Ca 2+ channels (LTCCs) and Na + -Ca 2+ exchanger (NCX), which are colocalized with ryanodine receptors (RyRs) in dyadic junctions with the sarcoplasmic reticulum (SR). Progressive dyadic maturation is linked to upregulation of Junctophilin-2 and BIN1, and this structural organization continues after birth. T-tubule development is highly workload sensitive. Indeed, in the healthy developing heart, gradually increasing blood pressure drives t-tubule maturation, and this process can be accelerated or blunted by interventions which increase or reduce fetal systolic load, respectively. This article is protected by copyright. All rights reserved.