Protein Kinase C Alpha Cellular Distribution, Activity, and Proximity with Lamin A/C in Striated Muscle Laminopathies

Abstract

Striated muscle laminopathies are cardiac and skeletal muscle conditions caused by mutations in the lamin A/C gene (LMNA). LMNA codes for the A-type lamins, which are nuclear intermediate filaments that maintain the nuclear structure and nuclear processes such as gene expression. Protein kinase C alpha (PKC-α) interacts with lamin A/C and with several lamin A/C partners involved in striated muscle laminopathies. To determine PKC-α's involvement in muscular laminopathies, PKC-α's localization, activation, and interactions with the A-type lamins were examined in various cell types expressing pathogenic lamin A/C mutations. The results showed aberrant nuclear PKC-α cellular distribution in mutant cells compared to WT. PKC-α activation (phos-PKC-α) was decreased or unchanged in the studied cells expressing LMNA mutations, and the activation of its downstream targets, ERK 1/2, paralleled PKC-α activation alteration. Furthermore, the phos-PKC-α-lamin A/C proximity was altered. Overall, the data showed that PKC-α localization, activation, and proximity with lamin A/C were affected by certain pathogenic LMNA mutations, suggesting PKC-α involvement in striated muscle laminopathies.

Keywords: DCM; EDMD; L-CMD; lamin A/C; protein kinase C alpha; striated muscle laminopathies.

Figure 1

Lamin A/C protein schematic showing the locations of the pathogenic amino acid changes studied, their corresponding phenotypes and the PKC-α binding site (indicated by the black dotted line box). DCM—dilated cardiomyopathy; EDMD—Emery–Dreifuss muscular dystrophy; L-CMD—LMNA related congenital muscular dystrophy; and DCM-CD—dilated cardiomyopathy with conduction defects.

Figure 2

Cellular localization of total PKC-α in transfected H9C2 cells expressing either WT or mutant human lamins A and C. (A) Exogenous eCFP-human lamin A and eYFP-human lamin C are shown with the immunostaining for endogenous PKC-α (red) in WT and mutant LMNA expressing H9C2 cells. The composite image showing total PKC-α, eCFP-human lamin A and eYFP-human lamin C for each group is shown in the leftmost panel. A representative untransfected cell (indicated by a white asterisk) shows cytoplasmic PKC-α localization comparable to PKC-α localization in WT LMNA transfected cells. The white line across a cell in the lamin C and total PKC-α panels indicates the measurement path used to generate the fluorescence intensity profiles for lamin C and PKC-α shown in the rightmost panel. Since lamins A and C colocalize, the plot profile for eYFP-human lamin C only is included to show nuclear demarcation of fluorescence signal. Scale bar: 15 μm for all immunofluorescence images. (B) Mean (±SEM) net nuclear PKC-α fluorescence intensity of each group: WT (grey circles), D192G (blue diamonds), H222P (green triangle) and Y481X (orange diamonds). The net nuclear PKC-α fluorescence intensity of each cell = adjusted nuclear PKC-α fluorescence intensity − mean of the adjusted cytoplasmic PKC-α fluorescence intensities. n ≥ 3 independent sets were analyzed (≥14 cells/group are analyzed). Significance (*) was set at p < 0.05.

Figure 3

Cellular localization of phosphorylated PKC-α (phos-PKC-α) in control and patient myoblasts. (A) Immunostaining for DAPI, lamin A/C and phos-PKC-α in control and patient myoblasts. The composite image showing DAPI, lamin A/C and phos-PKC-α for each group is shown in the leftmost panel. The white line across a cell in the DAPI and phos-PKC-α panels indicates the measurement path used to generate the fluorescence intensity profiles for DAPI and phos-PKC-α shown in the rightmost panel. The plot profile for DAPI is included to show nuclear demarcation of fluorescence signal. Scale bar: 15 μm for all immunofluorescence images. (B) Mean (±SEM) net nuclear phos-PKC-α fluorescence intensity of each group: control (grey circles), ΔK32 (blue diamonds) and R249W (pink triangles). The net nuclear phos-PKC-α fluorescence intensity of each cell = adjusted nuclear phos-PKC-α fluorescence intensity − mean of the adjusted cytoplasmic phos-PKC-α fluorescence intensities. n = 2 technical replicates (one patient/group; ≥ 66 cells/group were analyzed). Significance **** means p < 0.0001.

Figure 4

Western blot analyses for PKC-α in whole cell protein extracts from patient and mouse model myoblasts. Representative blots for human myoblasts (A) and mouse model myoblasts (C). Mean (± SEM) protein level relative to control (or WT) is shown for human myoblasts (B) and mouse model myoblasts (D). n ≥ 3 technical replicates (one patient or mouse model/group). Significance (*) was set at p < 0.05.

Figure 5

Proximity ligation assay (PLA) between lamin A/C and phos-PKC-α in human (A) and mouse model (C) myoblasts. Mean (±SEM) PLA signal/nucleus in human (B) and mouse model (D) myoblasts. Each nucleus was stained with DAPI (blue) and the PLA signal representing the proximity of lamin A/C and phos-PKC-α is in white. n = 2 technical replicates (one patient/group; ≥92 nuclei/group were analyzed) for human myoblasts and n = 1 experimental set (≥46 nuclei/group were analyzed) for mouse models’ myoblasts. Scale bar: 15 μm for all PLA images. Significance (*) was set at p < 0.05; ** means p < 0.01 and *** means p < 0.001.

Figure 6

Western blot analyses for ERK 1/2 in whole cell protein extracts from patient and mouse model myoblasts. Representative blots for human myoblasts (A) and mouse model myoblasts (C). Mean (±SEM) protein level relative to control (or WT) is shown for human myoblasts (B) and mouse model myoblasts (D). n ≥ 2 technical replicates (one patient or mouse model/group).