Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are caused by mutations in thin filament regulatory proteins that confer distinct primary alterations of cardiac contractility. We believe that altered Ca2+-buffering by mutant thin filaments leads to altered Ca2+ handling and, via Ca2+-dependent signalling pathways, contributes to disease pathogenisis. We are studying the in situ effect on Ca2+ flux of a HCM and a DCM causing mutation in human cardiac troponin T (TnT) (R92Q and R131W), troponin I (TnI) (R145G and K36Q) and α-tropomyosin (αTM) (D175N and E40K) by adenoviral mediated expression in adult guinea pig left ventricular cardiomyocytes. Western blot analysis of infected cells showed that recombinant FLAG tagged protein comprised ∼50% of the total TnT, TnI or αTM in these cells 48 hours after infection. Localisation of the FLAG tag epitope by immunofluorescence indicated that the expressed proteins were present at the I-band of the sarcomere in all cases. Analysis of unloaded sarcomere shortening showed that cells infected with HCM mutations increased the contractile velocity and gave a significant reduction in basal sarcomere length. By contrast DCM causing mutant proteins prolonged relaxation and reduced the magnitude of contraction. Analysis of Ca2+ transients of the same cells using fura-2 loading, indicated that the HCM mutations caused a significant increase the diastolic Ca2+ concentration. The DCM mutations increased the time to complete Ca2+ reuptake, with no change to the transient amplitude, despite the observed decrease in contraction. Caffeine challenging indicated that the presence of a HCM mutation reduced SR load and may predispose the sodium calcium exchanger (NCX) to contribute more strongly to the Ca2+ transient, whilst DCM mutations increase the rate of Ca2+ extrusion by the NCX. In order investigate whether acute alterations to calcium flux alter Ca2+-dependent signalling cascades, the nuclear translocation of the Transcription Factor of Activated T-cells-3 (NFAT-c3) was assessed. Immunofluorescent staining of chronically paced cardiomyocytes expressing HCM mutant cTnI (R145G) showed a 2.5 fold increase in nuclear NFAT compared to unpaced or wild type infected cells. Cardiomyocytes expressing DCM mutant cTnI (K36Q) gave a 1.8 fold NFAT nuclear translocation compared to cells expressing the wild type proteins which was independent of pacing. These data suggest that the acute effects of thin filament mutations may act, at least in part, via calcium signalling to drive the macroscopic remodelling observed in the pathological disease states of HCM and DCM.