Abstract Body: Background: Mutations in desmoplakin often lead to dilated cardiomyopathy. Here, we investigate the pathogenesis of DSP-R451G, a missense mutation that results in complete degradation of desmoplakin protein. We hypothesize that a Src-dependent shortening of resting sarcomere length contributes, at least in part, to contractile dysfunction. Methods: We use iPSC-derived engineered heart tissues (EHTs) bearing heterozygous and homozygous DSP-R451G and a heterozygous Dsp^WT/R451G knock-in mouse to investigate mechanisms of hypocontractility. To expand the relevance of these findings, we replicate key signaling and sarcomeric features in left-ventricular specimens from 3 patients with DSP-linked cardiomyopathy. Results: DSP-R451G EHTs exhibit contractile dysfunction (WT peak force 149 µN, R451G 90 µN, p < 0.0001, Fig. A, N = 16), a result that is recapitulated in isolated ventricular cardiomyocytes from Dsp^WT/R451G mice (fractional shortening, 9.4% vs 7.9%, p = 0.037, N = 3). R451G EHTs also have shorter resting sarcomere lengths (1.98 µm vs 1.79 µm, p = 0.0007, N = 3), also replicated in murine and human left-ventricular samples (Fig. B, N = 3 patients/group). Importantly, phosphorylation of PKC, which regulates sarcomere length, and Src, a known PKC interactor, are increased in DSP-R451G EHTs and human samples. Blockade of Src with low-dose dasatinib not only rescues sarcomere length to the control value (1.91 µm vs 2.07 µm, p = 0.0014, Fig. C), but reverses the contractile deficit seen in DSP-R451G tissues (peak force, 144 µN [WT vehicle], 101 µN [DSP vehicle], 147 µN [DSP-dasatinib], p = 0.0104, Fig. D). Conclusion: We demonstrate that loss of desmoplakin results in activation of PKC and Src signaling, which underly shorter resting sarcomere length and contractile dysfunction. Blockade of Src signaling with an FDA-approved drug successfully restores sarcomere length and rescues contractility, suggesting a novel targetable pathway in desmoplakin cardiomyopathy.