T-tubule remodeling during cardiac pathologies such as heart failure leads to impaired contractility of the heart. Increased cardiac load has been suggested to play a role, and we previously identified wall stress as an important trigger of these structural alterations. Specifically, we showed that elevated diastolic wall stress induces t-tubule loss and re-organization in areas surrounding a myocardial infarction (MI) in an experimental rat heart failure model (Frisk et al., Cardiovasc Res, 2016). The precise mechanical stimuli and effectors involved, however, still remain to be elucidated. We therefore presently investigated how preload affects t-tubule structure during compensatory and decompensatory remodeling and hypothesized that preload acts as specific and dynamic regulator of the t-tubule network. This hypothesis was examined using explanted hearts from mouse models of cardiac pathology and patients. The effects of increased preload were first investigated in mice following aortic shunt. At one week post-surgery, t-tubule density was increased while after 20 weeks (where preload was more markedly elevated) t-tubule density was decreased. Similarly, stretching isolated papillary muscles to reproduce a modest increase in diastolic stress increased t-tubule density, whereas excessive stretch beyond the optimal muscle length for force generation resulted in loss. In mice with dilated hearts and highly increased diastolic wall stress due to MI or aortic banding, t-tubules were also degraded, whereas compensatory increases in t-tubule density were observed at lower loads. A similar pattern of t-tubule loss was observed in explanted hearts; patients with dilated cardiomyopathy exhibited reduced t-tubule density while t-tubule proliferation was observed in patients with hypertrophic cardiomyopathy and aortic stenosis where diastolic wall stress was only slightly increased. Taken together, these results indicate that there is a bell-shaped relationship between t-tubule density and preload, with the healthy heart positioned on the rising phase of the curve. Moderately increased preload is associated with a compensatory increase in t-tubule density, likely aimed at adapting to increased contractile demand. However, excessive preload, such as during heart failure development, is detrimental as triggered t-tubule loss contributes to declining cardiac performance.