Regional heterogeneity in mitochondrial volume and ATP5A1 expression in SA node myocytes. (A) Representative RNAscope in situ hybridization image of a whole-mount SA node labeled for ATP5A1 mRNA (white). Insets show higher magnification views of ATP5A1 puncta in superior (blue outlines) and inferior (orange outlines) regions. Scale bars: 100 µm (whole mount), 10 µm (insets). (B) Quantification of ATP5A1 punctate density in superior and inferior SA node myocytes. (C) Representative three-dimensional surface renderings of the mitochondrial reticulum (MitoTracker Green) in myocytes isolated from superior (top) and inferior (bottom) regions. (D) Quantification of total mitochondrial volume per myocyte (N = 4 mice per group). (E and F) Frequency distributions of mitochondrial volume (E) and integrated Ca²⁺ signal mass (F) pooled across all analyzed myocytes. Red curves indicate Gaussian fits, resolving two populations. The green dashed line (≈2.2 × 10³ µm³) denotes the intersection used to classify low- and high-volume/mass phenotypes. (G) Relationship between mitochondrial volume and Ca²⁺ signal mass for individual myocytes. The red curve represents an exponential fit (R² = 0.73). Insets show maximum-intensity projections of representative Ca²⁺ transients from low- and high-volume groups. Scale bars, 5 µm. P values are shown above comparisons. Large circles denote per-animal means; small circles indicate individual biological replicates. N represents the number of independent mice.