Sarcoplasmic reticulum (SR) calcium pump function requires a high local ATP/ADP ratio, which can be maintained by direct nucleotide channeling from mitochondria, and by SR-bound creatine kinase (CK)-catalyzed phosphor-transfer from phosphocreatine. We hypothesized that SR calcium uptake supported by mitochondrial direct nucleotide channeling, but not bound CK, depends on the juxtaposition of these organelles. To test this, we studied a well-described model of cytoarchitectural disorganization, the muscle LIM protein (MLP)-null mouse heart. Subcellular organization was characterized using electron microscopy, and mitochondrial, SR and myofibrillar function were assessed in saponin-permeabilized fibers by measuring respiration rates and caffeine-induced tension transients. MLP-null hearts had fewer, less tightly-packed intermyofibrillar mitochondria, and more subsarcolemmal mitochondria. The apparent mitochondrial Km for ADP was significantly lower in MLP-null heart than control (175±15 versus 270±33 µM), indicating greater ADP accessibility, although maximal respiration rate, mitochondrial content and total CK activity were unaltered. MLP-null myofiber active tension was 54% lower than control (39°3 versus 18°1 mN/mm2), consistent with cytoarchitectural disorganization. MLP-null fiber SR calcium loading was similar to control with bound-CK support, but ~36% lower than control with mitochondrial support (p<0.05). Mitochondrial support for SR calcium uptake was also specifically decreased in desmin-null hearts, another model of cytoarchitectural perturbation. Thus, despite normal oxidative capacity, direct nucleotide channeling to the SR was impaired in MLP deficiency, concomitant with looser mitochondrial packing and increased nucleotide accessibility to this organelle. Changes in cytoarchitecture may therefore impair subcellular energy transfer and contribute to energetic and contractile dysfunction.