Cardiomyocyte-Specific Expression of Lamin A Improves Cardiac Function in <em>Lmna</em>^−/− Mice

<div><p><em>Lmna</em>^−/− mice display multiple tissue defects and die by 6–8 weeks of age reportedly from dilated cardiomyopathy with associated conduction defects. We sought to determine whether restoration of lamin A in cardiomyocytes improves cardiac function and extends the survival of <em>Lmna</em>^−/− mice. We observed increased total desmin protein levels and disorganization of the cytoplasmic desmin network in ∼20% of <em>Lmna</em>^−/− ventricular myocytes, rescued in a cell-autonomous manner in <em>Lmna</em>^−/− mice expressing a cardiac-specific lamin <em>A</em> transgene (<em>Lmna</em>^−/−; Tg). <em>Lmna</em>^−/−; Tg mice displayed significantly increased contractility and preservation of myocardial performance compared to <em>Lmna</em>^−/− mice. <em>Lmna</em>^−/−; Tg mice attenuated ERK1/2 phosphorylation relative to <em>Lmna</em>^−/− mice, potentially underlying the improved localization of connexin43 to the intercalated disc. Electrocardiographic recordings from <em>Lmna</em>^−/− mice revealed arrhythmic events and increased frequency of PR interval prolongation, which is partially rescued in <em>Lmna</em>^−/−; Tg mice. These findings support our observation that <em>Lmna</em>^−/−; Tg mice have a 12% median extension in lifespan compared to <em>Lmna</em>^−/− mice. While significant, <em>Lmna</em>^−/−; Tg mice only have modest improvement in cardiac function and survival likely stemming from the observation that only 40% of <em>Lmna</em>^−/−; Tg cardiomyocytes have detectable lamin A expression. Cardiomyocyte-specific restoration of lamin A in <em>Lmna</em>^−/− mice improves heart-specific pathology and extends lifespan, demonstrating that the cardiac pathology of <em>Lmna</em>^−/− mice limits survival. The expression of lamin A is sufficient to rescue certain cellular defects associated with loss of A-type lamins in cardiomyocytes in a cell-autonomous fashion.</p> </div

FLAG-lamin A is highly expressed, tissue-specific, and mosaic.

<p>(A) Western blot analysis of <i>Lmna</i>^+/+, <i>Lmna</i>^−/−, and <i>Lmna</i>^−/−; Tg heart lysates that were probed with lamin A/C antibody. (B) Western blot analysis of heart, gastrocnemius, quadriceps, thymus, spleen, kidney, liver, and lung from <i>Lmna</i>^+/+; Tg (upper panel) and <i>Lmna</i>^−/−; Tg (lower panel) mice probing with lamin A/C antibody. The asterisk indicates a cross-reactive band present in all lanes. (C) Indirect immunofluorescence micrograph of <i>Lmna</i>^−/−; Tg heart cross-section stained for MF-20 (cardiomyocytes; green), FLAG (transgene; red), and DAPI (nuclei, blue). Note that the mosaic expression of the transgene is limited to only cardiomyocytes. Scale bar denotes 100 µm. Inset is zoomed in view from the same image. (D) Quantitation of the percent of cardiomyocytes expressing FLAG-lamin A (N = 3 each; ∼500 cardiomyocytes were scored for each group) (E) Body weights of <i>Lmna</i>^+/+, <i>Lmna</i>^−/−, and <i>Lmna</i>^−/−; Tg mice (N = 10, 11, and 8 at 4–6 weeks respectively and N = 10, 7, and 5 at 6–8 weeks respectively).</p

<i>Lmna</i>^−/−; Tg mice display improved contractile function compared to <i>Lmna</i>^−/− littermates.

<p>(A) Representative echocardiograms of <i>Lmna</i>^−/−, <i>Lmna</i>^−/−; Tg, and control littermates. (B) Normalized left ventricular end-systolic diameter (LVESD) measurements are increased in <i>Lmna</i>^−/− hearts compared to control hearts and are improved in <i>Lmna</i>^−/−; Tg hearts. (C) Normalized left ventricular end-diastolic diameter (LVEDD) measurements are increased in <i>Lmna</i>^−/− hearts and are not improved in <i>Lmna</i>^−/−; Tg hearts. (D) Fractional shortening is decreased in <i>Lmna</i>^−/− hearts compared to control hearts and is improved in <i>Lmna</i>^−/−; Tg hearts. (E) Myocardial performance index (MPI) is increased (worse) in <i>Lmna</i>^−/− mice, but is improved in <i>Lmna</i>^−/−; Tg mice. For all of the above experiments: Control, N = 13; <i>Lmna</i>^−/−, N = 15; <i>Lmna</i>^−/−; Tg, N = 12 for 4–8 weeks of age. One-way ANOVA analyses were performed (B–E) and significant genotype differences are listed above for each panel. Post-tests were performed between genotypes and significance is listed as follows: * P<0.05; ** P<0.01; *** P<0.001; n.s. not significant.</p

Transgenic expression of lamin A in <i>Lmna</i>^−/− cardiomyocytes partially restores ERK1/2 activation and gap junction protein localization.

<p>(A) Western blot analysis of <i>Lmna</i>^+/+, <i>Lmna</i>^−/−, and <i>Lmna</i>^−/−; Tg mouse heart lysates. Phosphorylated-ERK1/2 (pERK1/2) levels are increased in <i>Lmna</i>^−/− compared to <i>Lmna</i>^+/+ and are decreased in <i>Lmna</i>^−/−; Tg hearts while total ERK1/2 levels are unchanged. Total Cx43 protein levels detected by NT1 are unchanged, while cytoplasmic levels of Cx43 as detected by CT1 are increased. α-tubulin is shown as a loading control. (B) Quantification of pERK1/2 levels normalized to total ERK1/2 levels in 5–7 week old <i>Lmna</i>^+/+ (N = 13), <i>Lmna</i>^−/− (N = 16), and <i>Lmna</i>^−/−; Tg (N = 11) mouse heart lysates. One-way ANOVA analyses were performed for significance with post-test significance values as follows: ** P<0.01; n.s. not significant. (C–E) Indirect immunofluorescence micrographs of pan-cadherin (red) and Cx43 (green) in <i>Lmna</i>^+/+ (C), <i>Lmna</i>^−/− (D), and (E) <i>Lmna</i>^−/−; Tg heart ventricle sections taken at equivalent exposure times from 6-week old mice. Asterisks mark intercalated discs with qualitative decreases in Cx43 relative to pan-cadherin staining. White scale bar denotes 50 µm. (F) Distribution of individual intercalated disks summarizing relative frequency of Cx43 co-association index in 5–7-week old <i>Lmna</i>^+/+ (N = 3; 350 intercalated discs analyzed total), <i>Lmna</i>^−/− (N = 3; 349 intercalated discs analyzed total), and <i>Lmna</i>^−/−; Tg (N = 3; 367 intercalated discs analyzed total) mice. A non-linear regression for Gaussian distribution was fitted for each group tested (<i>Lmna</i>^+/+ R² = 0.91; <i>Lmna</i>^−/− R² = 0.96; <i>Lmna</i>^−/−; Tg R² = 0.80). (G) Combined Cx43 co-association indices from multiple experiments normalized against their respective <i>Lmna</i>^+/+ control (N = 3). One-way ANOVA analyses were performed for significance with post-test significance values as follows: ** P<0.01; n.s. not significant.</p

Extension of median and maximal lifespan in <i>Lmna</i>^−/− mice expressing cardiac lamin A.

<p><i>Lmna</i>^−/−; Tg mice display a 12% and 15% increase in median and maximal lifespans, respectively, compared to <i>Lmna</i>^−/− littermates (<i>Lmna</i>^+/+; Tg N = 10; <i>Lmna</i>^−/− N = 24; <i>Lmna</i>^−/−; Tg N = 28). The Log-Rank test, which measures significance evenly across all time points, reports a significance probability of P<0.0114</p

Transgenic expression of lamin A in <i>Lmna</i>^−/− cardiomyocytes results in partial restoration of desmin phenotype but not cardiac remodeling markers.

<p>(A) Western blot analysis of <i>Lmna</i>^+/+, <i>Lmna</i>^−/−, and <i>Lmna</i>^−/−; Tg whole heart lysates. Desmin is increased in <i>Lmna</i>^−/− heart lysates compared to <i>Lmna</i>^+/+ and is partially attenuated in <i>Lmna</i>^−/−; Tg mice (middle blot). α-tubulin is shown as a loading control (bottom blot). (B–D) Indirect immunofluorescence micrographs showing desmin (red), FLAG (green), and DAPI (blue) in <i>Lmna</i>^+/+ (B), <i>Lmna</i>^−/− (C), and <i>Lmna</i>^−/−; Tg (D) heart ventricle sections. Asterisks mark intercalated discs, which co-localize with a pan-cadherin antibody (not shown) and arrows indicate ventricular myocytes with accumulated desmin in the cytoplasm. Scale bar denotes 50 µm. (D) Note the lack of ventricular myocytes positive for both desmin cytoplasmic accumulation and transgene expression. (E) Graph summarizing the desmin cytoplasmic accumulation phenotype observed in <i>Lmna</i>^−/− ventricular myocytes across different mouse backgrounds aged 5–7 weeks (N = 3 for <i>Lmna</i>^+/+, <i>Lmna</i>^−/−, and <i>Lmna</i>^−/−; Tg; ** P<0.01; *** P<0.001).</p

Lmna^−/−; Tg mice show improved conduction parameters compared to Lmna^−/− mice by ECG analysis.

<p>(A) Box-and-whiskers plot of pooled <i>Lmna</i>^+/+ PR intervals (N = 5) compared with single animal PR intervals for each of the following genetic backgrounds at 5-7 weeks of age: <i>Lmna</i>^+/+; Tg, <i>Lmna</i>^−/− and <i>Lmna</i>^−/−; Tg. The whiskers represent at 5–95% confidence interval and individual outliers are represented by their respective dots. (B) Fraction of mice which display >30% abnormally prolonged PR interval. <i>Lmna</i>^+/+ (N = 5), <i>Lmna</i>^+/+; Tg (N = 5), <i>Lmna</i>^−/− (N = 6), and <i>Lmna</i>^−/−; Tg (N = 5). (C–E) ECG traces from a <i>Lmna</i>^+/+ mouse (C), a <i>Lmna</i>^−/− mouse exhibiting an atrial premature beat (D) and a <i>Lmna</i>^−/− mouse displaying multifocal atrial rhythm (E). Note the changes in P-wave morphology and cycle length. Arrows denote ectopic P-waves. RR intervals are shown above.</p