Structural and functional changes ensue in cardiac cell networks when cells are guided by three-dimensional scaffold topography. We report enhanced synchronous pacemaking activity in association with slow diastolic rise in intracellular Ca2+ concentration ([Ca2+]i) in cell networks grown on microgrooved scaffolds. Topography-driven changes in cardiac electromechanics were characterized by the frequency dependence of [Ca2+]i in syncytial structures formed of ventricular myocytes cultured on microgrooved elastic scaffolds (G). Cells were electrically paced at 0.5-5 Hz, and [Ca2+]i was determined using microscale ratiometric (fura 2) fluorescence. Compared with flat (F) controls, the G networks exhibited elevated diastolic [Ca2+]i at higher frequencies, increased systolic [Ca2+]i across the entire frequency range, and steeper restitution of Ca2+ transient half-width (n = 15 and 7 for G and F, respectively, P < 0.02). Significant differences in the frequency response of force-related parameters were also found, e.g., overall larger total area under the Ca2+ transients and faster adaptation of relaxation time to pacing rate (P < 0.02). Altered [Ca2+]i dynamics were paralleled by higher occurrence of spontaneous Ca2+ release and increased sarcoplasmic reticulum load (P < 0.02), indirectly assessed by caffeine-triggered release. Electromechanical instabilities, i.e., Ca2+ and voltage alternans, were more often observed in G samples. Taken together, these findings 1) represent some of the first functional electromechanical data for this in vitro system and 2) demonstrate direct influence of the microstructure on cardiac function and susceptibility to arrhythmias via Ca(2+)-dependent mechanisms. Overall, our results substantiate the idea of guiding cellular phenotype by cellular microenvironment, e.g., scaffold design in the context of tissue engineering.