Connexin 43 as a novel Ca2+ transport pathway in the nuclear envelope of human induced pluripotent stem cell derived cardiomyocytes

Abstract

Nuclear envelope (NE) is a double lipid bilayer separating the nucleus from the cytosol. While cytoplasmic and sarcoplasmic reticulum (SR) Ca²⁺ signaling is extensively studied, the role of NE in cellular Ca²⁺ dynamics and the identity and regulation of nuclear Ca²⁺ transporters remain less explored.

NE-associated Ca²⁺ activity was examined using a genetically engineered fluorescent Ca²⁺ probe targeting nuclear LaminB1 at the inner NE membrane in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). Confocal imaging of NE revealed larger and delayed but slower rise of Ca²⁺ in the nuclear lamina compared to I_Ca triggered cytosolic rise in Ca²⁺. Caffeine induced ∼4X larger Ca²⁺rise in NE lamina compared to I_Ca. To determine whether NE Ca²⁺ signaling depended on SR Ca²⁺ release, we measured the nuclear Ca²⁺signals of cells expressing genetically CICR-impaired RyR2 mutations (E3848A, Q3925E), where SR Ca²⁺ release was fully suppressed. In these CICR-deleted cells, although caffeine failed to activate robust NE Ca²⁺ transients, spontaneous beating persisted activating NE Ca²⁺ transients, suggesting Ca²⁺ signaling pathway remodeling and activation of an alternate Ca²⁺ pathway. Confocal imaging of hiPSCCMs infected with antibodies to Cx43 identified robust Cx43 expression in the NE, the inhibition of which by Gap19 protein blocked the rise of nuclear lamina Ca²⁺-transients. We conclude, that while SR Ca²⁺ release is essential in replenishing the NE Ca²⁺ content, RyR2 mutations that delete CICR induce remodeling of Ca²⁺signaling pathway that may include Cx43 to maintain Ca²⁺ fluxes critical for spontaneous beating and triggering of Ca²⁺transients.