Increased signaling by Gi-coupled receptors has been implicated in dilated cardiomyopathy. To investigate the mechanisms, we used transgenic mice that develop dilated cardiomyopathy after conditional expression of a cardiac-targeted Gi-coupled receptor (Ro1). Activation of Gi signaling by the Ro1 agonist spiradoline caused decreased cellular cAMP levels and bradycardia in Langendorff perfused hearts. However, acutely terminating Ro1 signaling using the antagonist nor-Binaltorphimine did not reverse the Ro1-induced contractile dysfunction, indicating that the Ro1 cardiomyopathy was not due to acute effects of receptor signaling. Early after initiating Ro1 expression, there was a 40% reduction in the abundance of the SR Ca²⁺ ATPase (P<0.05), and thereafter, progressive impairment of both Ca²⁺ handling and force development assessed using ventricular trabeculae. Six weeks after initiating Ro1 expression, systolic [Ca²⁺] was reduced to 0.61±0.08 µM vs. 0.91±0.07 µM, for control (n=6-8, P<0.05); diastolic [Ca²⁺] was elevated to 0.41±0.07 µM vs. 0.23±0.06 µM for control (n=6-8, P<0.01); and the decline phase of the Ca²⁺ transient (time from peak to 50% decline) was slowed to 0.25±0.02 s vs. 0.13±0.02 s for control (n=6-8, P<0.01). Early after initiating Ro1 expression, there was a 9-fold elevation of matrix-metalloproteinase-2 (P<0.01), which is known to cause myofilament injury. Consistent with this, six weeks after initiating Ro1 expression, Ca²⁺-saturated myofilament force in skinned trabeculae was reduced to 21±2 mN/mm² vs. 38±0.1 mN/mm² for controls (n=3, P <0.01). Furthermore, electron micrographs revealed extensive myofilament damage. These findings may have implications for some forms of human heart failure where increased activity of Gi-coupled receptors leads to impaired Ca²⁺ handling and myofilament injury which contribute to impaired ventricular pump function and heart failure.