Table 1.
Characteristics of hearts.
Fig 1.
PCR strategy for the detection of progerin expression in heart and blood.
A Schematic overview of PCR based strategy to analyze progerin mRNA expression in human heart and the blood samples. Shown are the consensus donor splice sequence at the end of exon 11, the sequence of the normal LMNA cryptic splice site, and two common known mutated cryptic splice sites in HGPS patients. B First row: PCR gel showing total lamin A (first arrow) and small amounts of progerin expression (arrow showing Δ150bp band). Second row: Specific progerin expression utilizing primers spanning exon 11 to 12. Third row: RPL32 expression was used for relative quantification of progerin expression. G: Genomic DNA (G) did not reveal any significant LMNA bands in the gel verifying specific mRNA expression.
Fig 2.
Alignment of progerin PCR product cDNA confirms alternative splicing in the heart.
The sequence of purified progerin PCR cDNA (red letters) was aligned to the genomic sequence of LMNA exon 11 (green) to exon 12 (yellow) with the 3´UTR sequence (blue) showing the expected gap of 150bp between exon 11 to exon 12 confirming alternative splicing of progerin.
Fig 3.
Progerin mRNA is upregulated in DCM hearts and is significantly correlated with measures of heart failure but not with age.
A Relative amount of progerin mRNA levels related to the reference gene RPL32 in heart biopsies of patients with DCM (n = 15) and non-failing control hearts (n = 10). Shown are all individual data points, lines show mean ± SD. ** P ≤ 0.01 DCM vs. Control. B Relative amount of progerin mRNA levels related to the reference gene RPL32 in whole blood cells derived from patients with DCM (n = 56) and healthy controls (n = 10). Shown are all individual data points, lines show mean ± SD. P-value was derived from unpaired two-sided T-Test between the groups. C Scatter plot showing the negative correlation between the relative amount of progerin mRNA related to RPL32 with ejection fraction in human heart biopsies (n = 25). ** P ≤ 0.01. D Scatter plot showing the positive correlation between the relative amount of progerin mRNA related to RPL32 with left ventricular enddiastolic diameter (LVEDD) and E Scatter plot showing the positive correlation between progerin mRNA related to RPL32 with the age of the hearts in human heart biopsies (n = 25). N.s. not significant. ** P ≤ 0.01.
Table 2.
Progerin and inflammation in the heart.
Fig 4.
Increased progerin expression and TUNEL+ cells in DCM hearts.
A Immunohistochemistry showing increased numbers of progerin expressing cells (arrows, red nuclei) in DCM hearts compared to non-failing controls. Scale bar represents 100μM (rows 1–2) and 25μM (rows 3–4). B Immunofluorescence stainings revealed increased expression of progerin (green, arrows) in nuclei (blue) of DCM hearts (merged images last row, arrows) compared to non-failing controls. Red: WGA membrane staining, Green: Progerin staining, Blue: DAPI+ nuclei. Scale bar represents 20μM. C TUNEL staining of apoptotic cell death revealed increased numbers of TUNEL+ cells in DCM hearts compared to non-failing controls. Scale bar represents 200μM (rows 1–3) and 20μM (last row). D Quantification of progerin+ nuclei to total nuclei per high power field (HPF) in DCM hearts and Controls (n = 3). ** P ≤ 0.01 DCM vs. Control. E Quantification of apoptotic index (TUNEL+ nuclei to total nuclei) in DCM hearts compared to controls (n = 3). ** P ≤ 0.01 DCM vs. Control.
Fig 5.
Co-expression of apoptotic marker caspase-3 and progerin in DCM.
Immunofluorescence stainings revealed expression of the apoptotic cell death marker cleaved caspase-3 (red, arrow) and progerin (green, arrow) in the nucleus (blue) of a cardiomyocyte delineated by red cell membrane staining with WGA (merged image last row, arrow) compared to non-failing controls. Scale bar represents 20μM.