BPS2026 – Sub-sarcomeric spatial multiomics reveals distinct organization of excitation-contraction coupling mRNA translation

The spatial organization of protein synthesis is emerging as a key regulator of cell function, but its role within the heart remains unclear. In cardiomyocytes, excitation-contraction coupling depends on the precise alignment of calcium-handling complexes whose maintenance by localized mRNA translation is poorly understood. We developed a 3D spatial multiomics platform that simultaneously maps multiple mRNAs and structural proteins at a ∼120-nm resolution, enabling the sub-sarcomeric analysis of cardiac mRNA organization. Single-cell analysis of adult ventricular myocytes revealed transcript-specific spatial patterns among ECC-related mRNAs. Ryr2, Calm1, and Jph2 mRNAs showed pronounced Z-line enrichment (∼80% within 200 nm of α-actinin), whereas Atp2a2 (SERCA2a), Calm2, Calm3, Cacna1c (LTCC), and Camk2d were more broadly distributed. Colocalization with RN18S rRNA revealed most ECC mRNAs (70%–90%) overlapped with ribosomes. These mRNAs were distributed across two distinct translation-associated microdomains by either aligning with, or longitudinally between, Z-lines. Specifically, Ryr2, Calm1–3, Jph2, and Cacna1c were enriched within Z-line-associated ribosomes, whereas Atp2a2, Atp1a1, and Camk2d predominately populated longitudinal ribosomes. Across cells, ∼40% of all transcript localizations occurred within multi-transcript clusters (≤200 nm), ∼60% of which resided at Z-line ribosomes. These Z-line clusters, often including Ryr2-Jph2-Cacna1c or Ryr2-Calm3-Cacna1c, reached groupings of up to six distinct mRNAs, indicating a high local density of translation-associated assemblies. By contrast, inter-Z-line ribosomes formed smaller, pair-dominated clusters—typically involving Atp2a2 and Camk2d—consistent with distributed translation of calcium-reuptake and signaling modules. These findings reveal that ECC mRNAs are partitioned and cooperatively clustered within two spatially distinct translation-associated microdomains—Z-line-aligned and inter-Z-line—representing a previously unrecognized organizational principle of sub-sarcomeric protein synthesis that may underlie local renewal of the heartbeat machinery.