Heart failure is an important, and growing, cause of morbidity and mortality. Half of patients with heart failure have preserved ejection fraction, for whom therapeutic options are limited. Here we report that cardiac bridging integrator 1 gene therapy to maintain subcellular membrane compartments within cardiomyocytes can stabilize intracellular distribution of calcium-handling machinery, preserving diastolic function in hearts stressed by chronic beta agonist stimulation and pressure overload. This study identifies that maintenance of intracellular architecture and, in particular, membrane microdomains at t-tubules, is important in the setting of sympathetic stress. Stabilization of membrane microdomains may be a pathway for future therapeutic development.