Abstract Body (Do not enter title and authors here): Mutations in TNNC1, encoding cardiac troponin C (cTnC), are associated with dilated (DCM) and hypertrophic (HCM) cardiomyopathy. The N-terminal domain of cTnC is essential for Ca²⁺ mediated thin filament activation. Interestingly, two rare human variants—I4M and A8V—located just four residues apart in the N-terminal helix, lead to opposite cardiomyopathy phenotypes. The biophysical mechanisms underlying this divergence remain unclear. We generated knock-in mouse models harboring human cTnC-I4M^+/− or cTnC-A8V^-/-. LC-MS previously showed ~21% incorporation of the mutant cTnC protein into the myofilament in cTnC-A8V^-/- mice. In contrast, our analysis revealed higher incorporation (~61%) of the I4M mutant. At 4-weeks of age, echocardiography of cTnC-I4M^+/− mice demonstrated DCM remodeling, characterized by significant increases in left ventricular internal diameter, end-diastolic and end-systolic volume, and wall thinning. Fractional shortening (FS%) and ejection fraction (EF%) declined significantly relative to controls (~28% vs ~22%, ~55% vs 45%, respectively). Notably, dobutamine (0.75 mg/kg i.p.) acutely restore contractile performance, likely due to enhanced β-adrenergic responsiveness. Small-angle X-ray diffraction revealed an increased I_1,1/I_1,o intensity ratio (0.38 vs 0.27), consistent with abnormal myosin head disorganization independent of Ca²⁺ activation. Conversely, cTnC-A8V^-/− mice displayed hypertrophic and restrictive features, including preserved or enhanced FS% and EF% with concentric wall thickening. Pressure–volume loop analysis showed elevated end-diastolic pressure-volume relationship (0.10 vs 0.17) and prolonged relaxation constant (τ ~12 ms vs. ~5 ms controls). Although direct measures of Ca²⁺ handling and sarcomere length is forthcoming, published work by our lab showed a leftward shift in the force–pCa curve for cTnC-A8V^-/- papillary muscles, while cTnC- I4M^+/− fibers showed Ca²⁺desensitization. Ongoing studies aim to quantify Ca²⁺ transients and ATP energetics to further delineate these mechanisms. These findings highlight that N-terminal cTnC mutations drive disease through a network of alterations: (1) allosteric perturbations of thin-filament activation (I4M desensitizes; A8V sensitizes), (2) kinetic modifications of Ca²⁺ binding/release that skew cross-bridge cycling timing and duty cycle. Our data underscore how adjacent residues within the N-helix of cTnC can elicit divergent structural and functional consequences.