Cardiotrophin-1 (CT-1) is known to promote survival but also to induce an elongated morphology of isolated cardiomyocytes. The latter observation led to the hypothesis that CT-1, which is chronically augmented in human heart failure, might induce eccentric cardiac hypertrophy and contractile failure. To address this question, heart tissues reconstituted from neonatal rat cardiomyocytes (engineered heart tissue, EHT) were used as multicellular in vitro test systems. CT-1 dose-ependently affected contractile function in EHTs. After treatment with 0.1 nM CT-1 (concentration corresponds to plasma levels found in humans) for 10 days (day 1-11), twitch tension significantly decreased to 0.30±0.04 mN (n=15) versus 0.45±0.04 mN (n=16) in controls. Furthermore, the positive inotropic effects of cumulative concentrations of Ca²⁺ and isoprenaline were significantly diminished. Maximum isoprenaline-induced increase in twitch tension amounted to 0.27±0.04 mN (n=15) vs. 0.47±0.06 mN (n=16) in controls (p<0.001). When EHTs were treated for only 5 days (day 6-11) qualitatively similar results were obtained but changes were less pronounced. Immunostaining of whole mount EHT preparations revealed that after CT-1 treatment (1) the number of non-myocyte cells significantly increased by 98% (1 nM CT-1, 10 d), and (2) myocytes did not form compact longitudinally oriented muscle bundles. Interestingly, expression of the Ca²⁺-handling protein calsequestrin was markedly reduced (69±7% of Ctr) by treatment with CT-1 (10 d, 0.1 nM). In summary, our results demonstrate that long-term exposure to CT-1 induces contractile dysfunction in EHTs. Structural changes due to impaired differentiation and/or remodeling of the heart tissue may play an important role.