Dilated cardiomyopathy mutations in thin-filament regulatory proteins reduce contractility, suppress systolic Ca²⁺, and activate NFAT and Akt signaling

Fig. 1.. Expression and localization of adenovirally expressed FLAG tagged dilated cardiomyopathy (DCM) mutant protein in guinea pig left ventricular cardiomyocytes. WT, wild type; TnT, cardiac troponin T (R131W); TnI, cardiac troponin I (K36Q); α-TM, α-tropomyosin (E40K). A: relative protein expression of FLAG tagged human recombinant WT/R131W TnT, WT/K36Q TnI, and WT/E40K α-TM. The presence of an NH2- or COOH-terminal FLAG tag created a band shift in expressed protein to allow for direct comparison to endogenous guinea pig subunits. Blots were probed using anti-TnT, anti-TnI and anti-α-TM primary antibodies, respectively. B: same membrane probed using anti-FLAG tag primary antibody. The relative expression level of recombinant protein as a percentage of the total is plotted in C (n = 4). Box and whisker plot give the median (line), standard deviation (box) and maximum and minimum data spread (whiskers). Localization of adenovirally expressed FLAG tagged DCM mutant protein in guinea pig left ventricular cardiomyocytes is shown in D. Adenovirally expressed FLAG tagged protein was localized to the I band in laminin immobilized cardiomyocytes using an anti-FLAG tag primary antibody (with…

Fig. 2.. Unloaded sarcomere shortening measurements in adenovirally infected guinea pig left ventricular cardiomyocytes containing dilated cardiomyopathy (DCM)-causing mutations. WT, wild type; TnT, cardiac troponin T (R131W); TnI, cardiac troponin I (K36Q); α-TM, α-tropomyosin (E40K). A–C: unloaded sarcomere shortening measurements at 1 Hz pacing for TnT R131W, TnI K36Q, and α-TM E40K, respectively. D–G: basal sarcomere length, fractional shortening, time to 50% contraction, and time to 50% relaxation, respectively. Each curve was averaged from multiple cells (n) taken from at least 4 separate cell preparations; total n numbers are given in the legends of each plot. Box and whisker plots compare WT with DCM mutant for each gene tested and give the median (line), standard deviation (box), and maximum and minimum data spread (whiskers). All data were tested for normality (D’Agostino-Pearson), and significance values calculated using either Mann-Whitney or standard t-tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.. Ca2+-transient measurements in adenovirally infected guinea pig left ventricular cardiomyocytes containing dilated cardiomyopathy (DCM)-causing mutations. WT, wild type; TnT, cardiac troponin T (R131W); TnI, cardiac troponin I (K36Q); α-TM, α-tropomyosin (E40K). A–C: Ca2+-transient measurements at 0.5 Hz pacing, comparing WT to TnT R131W, TnI K36Q, and α-TM E40K, respectively. D–H: diastolic intracellular Ca2+ concentration ([Ca2+]i) systolic [Ca2+]i, Ca2+-transient amplitude, time to 50% (T50) sarcoplasmic reticulum (SR) Ca2+ release, and time to 50% SR Ca2+ reuptake, respectively. Each curve was averaged from multiple cells (n) taken from at least 4 separate cell preparations; total n numbers are given in the legends of each plot. Box and whisker plots compare WT with DCM mutant for each gene tested and give the median (line), standard deviation (box) and maximum and minimum data spread (whiskers). All data were tested for normality (D’Agostino-Pearson), and significance values calculated using either Mann-Whitney or standard t-tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4.. Dilated cardiomyopathy (DCM) mutant-infected cardiomyocytes have increased sarcoplasmic reticulum (SR) load and sodium-calcium exchanger (NCX) activity with reduced fractional SR Ca2+ release and sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2) activity. WT, wild type; TnT, cardiac troponin T (R131W); TnI, cardiac troponin I (K36Q); α-TM, α-tropomyosin (E40K). A: representative caffeine-induced intracellular Ca2+ concentration ([Ca2+]i) transients generated by the direct perfusion of 10 mM caffeine after a 5-s pause of pacing for WT, TnT, TnT R131W, WT TnI, TnI K36Q, WT α-TM, and α-TM E40K-transduced cardiomyocytes, respectively. Box and whisker plots show the SR load (B), fractional release of Ca2+ (calculated from the caffeine-transient amplitudes in B subtracted from the preceding Ca2+-transient amplitude shown in Fig. 2F (C). NCX activity was measured from the τ-decay rates of caffeine induced Ca2+ transients (D). SERCA2a activity was calculated by the subtraction of τ-decay constants derived from Fig. 2H and caffeine-transient (NCX) τ-decay rates from D (E). Box and whisker plots compare WT with DCM mutant for each gene tested and give the median (line), standard deviation…

Fig. 5.. The ratio of monomeric vs. pentameric phospholamban (PLN) is increased by α-tropomyosin (α-TM; E40K)-dilated cardiomyopathy (DCM)-mutant protein expression following chronic pacing. WT, wild type; TnT, cardiac troponin T (R131W); TnI, cardiac troponin I (K36Q). Left ventricular cardiomyocytes were paced for 8 h at 0.5 Hz and 10 V at 37°C. Western blotting of PLN under nondenaturing conditions indicates that the total PLN levels are unchanged (A and D). When normalized to GAPDH loading controls (B), however, presence of the DCM mutation E40K in α-TM causes significant increase of the monomeric protein levels (C), driven by a reduction in PKA-dependent phosphorylation of serine-16 (E and G) and a profound reduction in CAMKII-dependent phosphorylation of threonine-17 when normalized to total PLN levels (F and H). Box and whisker plots give the median (line), standard deviation (box), and maximum and minimum data spread (whiskers) to compare DCM mutant to WT for each gene tested, and significance value was calculated using a Mann-Whitney test. *P < 0.05; **P < 0.01; ****P < 0.0001; n = 4 cell isolations for each group.

Fig. 6.. Dilated cardiomyopathy (DCM) mutant-infected cardiomyocytes have pacing-independent, dephosphorylation with concordant increased nuclear factor of activated T cells (NFAT) nuclear localization. WT, wild type; TnT, cardiac troponin T (R131W); TnI, cardiac troponin I (K36Q); α-TM, α-tropomyosin (E40K). A: representative cropped immunofluorescence images of single cardiomyocytes using anti-NFAT-C3 antibody. Individual panels compare DCM mutant to WT infected cardiomyocytes either unpaced or paced at 0.5 Hz for 4 h. The relative nuclear intensity of NFAT (colocalized with DAPI) (vs. cytoplasmic) is calculated using ImageJ, to compare WT to mutant cardiomyocytes for unpaced (B) and paced (C) cells, respectively. Concordant NFAT dephosphorylation is seen in Western blots comparing unpaced and paced, DCM and WT-transduced cardiomyocyte lysates, were developed with anti-NFAT, Anti-phospho-Ser-165 NFAT or anti-GAPDH antibody. Representative blots are shown in D. Box and whisker plots (E and F) compare DCM mutant to WT for each gene tested and for unpaced or paced cardiomyocytes, respectively. All box and whisker plots give the median (line), standard deviation (box), and maximum and…

Fig. 7.. Dilated cardiomyopathy (DCM) mutant cardiomyocytes increase pacing independent Akt phosphorylation with no change to ERK phosphorylation compared with wild type (WT). TnT, cardiac troponin T (R131W); TnI, cardiac troponin I (K36Q); α-TM, α-tropomyosin (E40K). Western blots comparing unpaced and paced (at 0.5 Hz for 8 h), DCM and WT-transduced cardiomyocyte lysates, were developed following incubation with anti-Akt, anti-phospho-Ser-473 Akt, anti-ERK, anti-Tyr-204 ERK or anti-GAPDH antibody. Representative blots for Akt and ERK are shown in (A and D), respectively. Box and whisker plots show the relative densitometry analysis of phosphorylated vs. total protein, give the median (line), standard deviation (box), and maximum and minimum data spread (whiskers) to compare DCM mutant to WT for each gene tested and for either unpaced (B and E) or paced (C and F) cardiomyocytes. The dot plot (G) shows the average phosphorylation level of ERK in each group to compare unpaced to paced cardiomyocytes; black line gives the mean average. Significance values were calculated using a Mann-Whitney test. *P < 0.05; **P < 0.01; ***P < 0.001; n = 5–8 blots for each group.