Abnormal splice-out of the exon 7-encoded segment in the N-terminal variable region of cardiac troponin T (cTnT-ΔE7) was found in turkeys and, together with the inclusion of embryonic exon (eTnT), in adult dogs with a correlation with dilated cardiomyopathy. Overexpression of these cTnT variants in transgenic mouse hearts significantly decreased cardiac function. To further investigate the functional effect of cTnT-ΔE7 or ΔE7+eTnT in vivo under systemic regulation, echocardiography was carried out in single and double-transgenic mice. No atrial enlargement, ventricular hypertrophy or dilation was detected in the hearts of 2-month-old cTnT-ΔE7 and ΔE7+eTnT mice in comparison to wild-type controls, indicating a compensated state. However, left ventricular fractional shortening and ejection fraction were decreased in ΔE7 and ΔE7+eTnT mice, and the response to isoproterenol was lower in ΔE7+eTnT mice. Left ventricular outflow tract velocity and gradient were decreased in the transgenic mouse hearts, indicating decreased systolic function. Ex vivo working heart function showed that high afterload or low preload resulted in more severe decreases in the systolic function and energetic efficiency of cTnT-ΔE7 and ΔE7+eTnT hearts. On the other hand, increases in preload demonstrated preserved Frank-Starling responses and minimized the loss of cardiac function and efficiency. The data demonstrate that the N-terminal variable region of cardiac TnT regulates systolic function of the heart.