Targeting Mitogen-Activated Protein Kinase Signaling in Mouse Models of Cardiomyopathy Caused by Lamin A/C Gene Mutations.

Lamin A and lamin C are two of the protein building blocks of the nuclear lamina, a meshwork of intermediate filaments on the inner aspect of the nuclear envelope inner membrane ( Aebi, Cohn, Buhle, & Gerace, 1986 ; Fisher, Chaudhary, & Blobel, 1986 ; Goldman, Maul, Steinert, Yang, & Goldman, 1986 ; McKeon, Kirschner, & Caput, 1986 ). They are encoded by the lamin A/C gene ( LMNA ) on chromosome 1q21 and expressed in most differentiated somatic cells ( Lin & Worman, 1993 ; Wydner, McNeil, Lin, Worman, & Lawrence, 1996 ). Human lamin A and lamin C are identical for the first 566 amino acids with lamin C having six unique carboxyl-terminal amino acids and prelamin A, the precursor of lamin A, having 98 unique carboxyl-terminal amino acids. Prelamin A contains a cysteine–aliphatic–aliphatic–any amino acid (CAAX) motif at its carboxyl-terminus, which signals the following series of posttranslational enzymatic modifications: (1) farnesylation of the cysteine, (2) cleavage of the −AAX, and (3) methylation of the carboxyl group of the newly exposed farnesylcysteine ( Bergo, Wahlstrom, Fong, & Young, 2008 ). This modified protein is then recognized by the zinc metalloproteinase ZMPSTE24 and cleaved 15 amino acids from the farnesylated cysteine to yield lamin A ( Rusiñol & Sinensky, 2006 ; Young, Meta, Yang, & Fong, 2006 ). Lamins and the nuclear lamina have been implicated in a wide range of functions from providing structural support to the nucleus to regulating transcription and DNA replication. More recent data also demonstrate that lamins are connected to the cytoskeleton via integral proteins of the inner and outer nuclear membranes, functioning in mechanotransduction and nuclear positioning ( Chang, Worman, & Gundersen, 2015 ). Scientific interest in lamin A and lamin C has significantly increased in the past decade and a half since mutations in LMNA have been connected to a broad range of inherited diseases often called laminopathies ( Worman, Fong, Muchir, & Young, 2009 ). Depending upon the mutation, these diseases predominantly affect either (1) striated muscle, (2) adipose tissue, (3) peripheral nerve, or (4) multiple systems generating progeroid phenotypes. The most frequently occurring LMNA mutations lead to striated muscle diseases virtually always involving the heart. In 1999, Bonne et al. (1999) identified LMNA mutations causing autosomal dominant Emery–Dreifuss muscular dystrophy. Progressive muscle weakness and wasting, contractures of the elbows, ankles, and neck; and dilated cardiomyopathy with an early onset atrioventricular conduction block are the classical clinical features. Soon after, mutations in LMNA were shown to cause dilated cardiomyopathy without significant skeletal muscle involvement, limb-girdle muscular dystrophy type 1B, and cardiomyopathy with variable skeletal muscle involvement ( Brodsky et al., 2000 ; Fatkin et al., 1999 ; Muchir et al., 2000 ). Based on the case series and reports published since these initial discoveries, we now know that the same mutations in LMNA can cause any one of these phenotypes, overlaps of these phenotypes and congenital muscular dystrophy, with dilated cardiomyopathy as a common feature ( Lu, Muchir, Nagy, & Worman, 2011 ). Various cellular signaling pathways are perturbed in diseases arising from mutations in genes encoding nuclear envelope proteins including LMNA ( Dauer & Worman, 2009 ). We have used mouse models of cardiomyopathy caused by LMNA mutations to analyze alterations in cell signaling in affected heart. In particular, our research has focused on abnormal mitogen-activated protein (MAP) kinase signaling and AKT-mTOR signaling in the Lmna H222P/H222P mouse model ( Choi et al., 2012 ; Muchir et al., 2007 ; Muchir, Wu, et al., 2012 ). Here, we review methods underlying our discoveries of altered MAP kinase signaling in hearts of mice with Lmna mutations and its role in the pathogenesis of cardiomyopathy.

Several mouse models of human laminopathies, as well as mice with selective deletions of lamin A or lamin C and altered prelamin A processing, have been generated ( Stewart, Kozlov, Fong, & Young, 2007 ; Zhang, Kieckhaefer, & Cao, 2013 ). While the heart is secondarily affected in some models of progeria, several knockout and knockin mice develop a primary dilated cardiomyopathy, sometimes with accompanying skeletal muscle disease resembling muscular dystrophy ( Table 1 ). The original Lmna −/− mice generated by Sullivan et al. develop dilated cardiomyopathy and muscular dystrophy at an early age, dying by 6–8 weeks ( Nikolova et al., 2004 ; Sullivan et al., 1999 ). One report has suggested that these mice may not be complete knockouts but rather express a truncated prelamin A ( Jahn et al., 2012 ). Nonetheless, these mice and embryonic fibroblasts derived from them have been widely used to obtain important insights regarding the functions of lamin A and lamin C. Another gene trap Lmna knockout line has a shorter lifespan and does not develop left ventricular dilatation prior to death ( Kubben et al., 2011 ). Lmna N195K/N195K mice, Lmna H222P/H222P mice, and homozygous mice expressing nonfarnesylated prelamin A without lamin C all develop cardiomyopathy; however, their phenotypes vary. Lmna N195K/N195K have a significantly shorter lifespan than Lmna H222P/H222P mice or homozygous mice expressing nonfarnesylated prelamin A without lamin C ( Arimura et al., 2005 ; Davies et al., 2010 ; Mounkes et al., 2005 ). Lmna H222P/H222P mice also develop skeletal muscle disease. Lmna Δ32/Δ32 mice have a lifespan of less than 3 weeks and skeletal muscle defects but do not develop left ventricular dilatation prior to death ( Bertrand et al., 2012 ). Lmna H222P/H222P mice and homozygous mice expressing nonfarnesylated prelamin A without lamin C have sex differences in disease severity, with male mice more severely affected than female mice. In humans, cardiomyopathy caused by LMNA mutations is virtually always an autosomal dominant disease. In contrast, heterozygous Lmna knockout and knockin mice generally have normal lifespans. An exception is Lmna Δ32/+ mice, which die between 30 and 70 weeks of age and develop dilated cardiomyopathy ( Cattin et al., 2013 ). Although they apparently have a normal lifespan, the Lmna +/− mice generated by Sullivan et al. have been reported to develop cardiac conduction defects and late-onset left ventricular dilation at around 20 weeks ( Chandar et al., 2010 ; Sullivan et al., 1999 ). It is unclear if other heterozygous lines develop late-onset heart abnormalities, as careful phenotyping of older mice has not been reported in the literature. For most of our research, we have used Lmna H222P/H222P mice generated by Arimura et al. (2005) . We have found this to be a useful model, as the mice do not develop heart abnormalities until 8–10 weeks of age and grow to a size, when they are much easier to analyze than those with earlier-onset disease and shorter lifespans. By 16 weeks of age, male Lmna H222P/H222P mice also have significant cardiac fibrosis, which occurs in humans with cardiomyopathy caused by LMNA mutations ( Holmström et al., 2011 ; Raman, Sparks, Baker, McCarthy, & Wooley, 2007 ). In contrast to the mice expressing nonfarnesylated prelamin A without lamin C, Lmna H222P corresponds to a naturally occurring human disease-causing mutation. Because of the sex differences in disease severity, we have mostly utilized male Lmna H222P/H222P mice. The male mice develop clinically detectable cardiomyopathy at an earlier age than female mice, making it less time consuming and more cost-effective to carry out experiments. One drawback of male Lmna H222P/H222P mice is that at 16–20 weeks of age they develop myopathy that involves the diaphragm as well as other muscle groups, which makes it difficult to determine if cardiomyopathy is the only process responsible for their death.

To determine if altered signal transduction occurs in cardiomyopathy caused by LMNA mutation, we carried out a transcriptomic analysis of hearts of Lmna H222P/H222P male mice at 10 weeks of age. This age is concurrent with the onset of left ventricular dysfunction ( Muchir et al., 2007 ; Muchir, Wu, et al., 2012 ). We first examined similarities in transcription profiles between hearts from wild-type ( n =8), Lmna H222P/+ ( n =7), and Lmna H222P/H222P ( n =6) mice using hierarchical cluster analysis. This analysis revealed a strong consistency between replicates and yielded 104 probe sets in hearts from Lmna H222P/+ mice and 114 in hearts from Lmna H222P/H222P mice with statistically significant differences in expressed compared to wild-type mouse hearts. There were 57 similar probe sets between hearts from Lmna H222P/H222P and Lmna H222P/+ mice. We then analyzed Gene Ontology terms applied to genes to identify functional related groups differentially expressed in hearts of mutant mice compared to control mice. This analysis revealed significant differences in expression of genes in several groups, including those encoding proteins in the extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38α kinase branches of the MAP kinase pathway. This can be visualized using the KEGG-based pathway visualization tool ( Arakawa, Kono, Yamada, Mori, & Tomita, 2005 ; Fig. 1 ). The stepwise protocol used for the transcriptomic analysis as follows: Left ventricles from wild-type, Lmna H222P/+ , and Lmna HH222P/H222P male mice were isolated after cervical dislocation at 10 weeks of age. A rotor-stator homogenizer (Omni International) was used to disrupt 20 mg of tissue in 350 μl of buffer RNeasy Lysis Buffer (Qiagen) containing 1% β-mercaptoethanol until the tissue was uniformly homogeneous. We centrifuged the lysate for 3 min at 13,000× g and transferred the supernatant to a microcentrifuge tube. We then added 1 volume of 70% ethanol and mixed immediately by pipetting. We transferred up to 700 μl of the sample to an RNeasy spin column placed in a 2-ml collection tube, which was centrifuged for 15 s at 8000× g . We discarded the flow-through. We add 700 μl of Buffer RNeasy Wash 1 to the RNeasy spin column and centrifuged for 15 s at 8000× g . We discarded the flow-through. We then added 500 μl of Buffer RPE (Qiagen) and centrifuged for 15 s at 8000× g . We discarded the flow-through. We again added 500 μl of Buffer RPE and centrifuged for 2 min at 8000× g . We discarded the flow-through. We placed the RNeasy spin column in a 1.5-ml tube and added 30 μl of RNAse-free water and centrifuged for 1 min at 8000× g . Adequacy and integrity of extracted RNA were determined by gel electrophoresis and concentrations were measured by ultraviolet absorption spectroscopy (OD 260 and 280 nm). To analyze RNA transcripts, we used GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix). Methods for target preparation, hybridization, fluidics station setup, array washing staining, and probe array scanning were those in the GeneChip Expression Analysis Technical Manual ( http://media.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.pdf ). Image files were obtained through Affymetrix GeneChip software and analyzed by robust multichip analysis using Affymetrix microarray “.cel” image files and GeneTraffic 3.0 software (Stratagene). Genes were identified as being differentially expressed if they met a false discovery rate threshold of q <0.05 in a two-tailed Student’s t -test and showed at least a twofold difference in expression independent of absolute signal intensity. Gene expression changes related to functional groups were analyzed using the class score method in ErmineJ (version 2.1.12; http://www.bioinformatics.ubc.ca/ermineJ/ ) ( Lee, Braynen, Keshav, & Pavlidis, 2005 ) and the database for annotation, visualization, and integrated discovery program ( http://david.abcc.ncifcrf.gov/ ) ( Dennis et al., 2003 ). Alterations in expression of genes encoding component of the MAP kinase pathway, specifically the ERK1/2, JNK, and p38α branches, were visualized using the KEGG-based pathway visualization tool ( Arakawa et al., 2005 ). Transcriptomic data suggesting that expression of genes in the MAP kinase signaling pathway are altered in hearts of Lmna H222P/H222P mice at 10 weeks of age, led us to test whether the activities of the proteins were altered in heart tissue. We used immunoblotting analysis to directly demonstrate increased phosphorylation (activation) of ERK1/2, JNK, and p38α using the following protocol. Hearts were excised from Lmna H222P/H222P and Lmna +/+ mice. Left ventricles were dissected and snap-frozen in liquid nitrogen-cooled isopentane and then homogenized using a roto-stator homogenizer (Omni International) in extraction buffer (25 m M Tris–HCl [pH 7.4], 150 m M NaCl, 5 m M ethylenediaminetetraacetic acid, 10 m M sodium pyrophosphate, 1 m M Na 3 VO 4 , 1% SDS, 1 m M dithiothreitol) containing 25 mg/ml aprotinin and 10 mg/ml leupeptin. Proteins in homogenates (20 μg) were separated by SDS–polyacrylamide gel electrophoresis ( Laemmli, 1970 ), transferred to nitrocellulose membranes (0.45 μ M , Invitrogen), and blotted with optimal dilutions of primary antibodies against ERK1/2 (No sc94, Santa Cruz Biotechnology), phosphorylated ERK1/2 (No 9101, Cell Signaling Technology), JNK (No sc474, Santa Cruz Biotechnology), phosphorylated JNK (No 9251, Cell Signaling), p38α (No 9212, Cell Signaling), or phosphorylated p38α (No 9216, Cell Signaling) in Tris-buffered saline containing 0.1% polysorbate 20 and incubated at room temperature overnight. Antibodies against β-tubulin, β-actin, or GADPH were used as internal controls to normalize the amounts of protein between immunoblots. Blots were then washed in Tris-buffered saline containing 0.1% polysorbate 20, labeled with horseradish peroxidase-conjugated secondary antibodies, and washed again. Recognized proteins were visualized by enhanced chemiluminescence (GE Healthcare Life Sciences). Band densities were calculated using Scion Imaging software (version alpha 4.0.3.2), and kinase activity was estimated by calculating the ratio of phosphorylated MAP kinase/total MAP kinase. This analysis showed increased ratios of phosphorylated to total JNK, ERK1/2, and p38α in hearts of male Lmna H222P/H222P mice compared to those from Lmna +/+ mice ( Fig. 2 ). For ERK1/2, this increased activation occurs in mice as young as 4 weeks of age and for p38α in mice as young as 8 weeks ( Choi et al., 2012 ; Muchir, Wu, et al., 2012 ). Activated/phosphorylated MAP kinases translocate to the nucleus where they modulate the activities of target genes ( Plotnikov, Zehorai, Procaccia, & Seger, 2011 ). We therefore examined nuclear translocation of ERK1/2 in mouse hearts by using an antibody that recognized phosphorylated ERK1/2 in tissue sections. We showed that the phosphorylated form of ERK1/2 is predominantly located in the nucleus of cardiomyocytes. Immunofluorescence microscopic analysis of heart sections from Lmna +/+ mice using these antibodies revealed a faint, rather diffuse pattern, whereas in heart sections from Lmna H222P/H222P mice there was intense and predominantly nuclear fluorescence ( Muchir et al., 2007 ). The protocol we used to analyze nuclear localization of ERK1/2 is as follows: Hearts were excised from Lmna +/+ mice and Lmna H222P/H222P mice and snap-frozen in liquid nitrogen-cooled isopentane; portions of these hearts can be stored in liquid nitrogen for other subsequent experiments. Immunofluorescence labeling of phosphorylated ERK1/2 was performed on transverse frozen sections (8 μm) of left ventricular muscle. Sections were fixed in 3.7% formaldehyde in phosphate-buffered saline for 15 min and then blocked in 5% fetal goat serum in phosphate-buffered saline with 0.1% Triton X-100 for 1 h. This fixation was suitable for the anti-ERK1/2 antibodies used. Sections were incubated in blocking solution with antiphosphorylated ERK1/2 monoclonal antibody (Cell Signaling Technology) overnight at 4 °C followed by washing in phosphate-buffered saline and incubation with Texas red-conjugated goat anti-mouse immunoglobulin G secondary antibody (Invitrogen). Sections were counterstained with 0.1 μg/ml 4′6-diamidino-2-phenylindole (Sigma-Aldrich) to label nuclei. Images were analyzed using a planar immunofluorescence microscope and intensity of phosphorylated ERK1/2 labeling of cardiomyocytes was measured using Scion Imaging software (version alpha 4.0.3.2; Scion Corp.).

The discovery that MAP kinase activities were abnormally elevated in hearts of Lmna H222P/H222P mice led us to hypothesize that decreasing their activities would lead to clinical improvement in the living animals. We therefore examined the effects of treatment with mitogen-activated protein kinase kinase 1/2 (MEK1/2), JNK, and p38α inhibitors on Lmna H222P/H222P mice. Allosteric inhibitors of MEK1/2, the MAP kinase kinase that phosphorylates ERK1/2, have been synthesized and tested in humans, primary for oncology indications ( Zhao & Adjei, 2014 ). Glaxo’s trametinib was the first drug in this class to be approved by the U.S. Food and Drug Administration ( Wright & McCormack, 2013 ). We have used several other MEK1/2 inhibitors to examine the effects of blocking ERK1/2 activity in hearts of Lmna H222P/H222P mice. Our initial published studies utilized PD098059 , which was one of the first small molecule inhibitors of MEK1/2 synthesized by scientists at Parke-Davis Pharmaceutical Research Division of Warner-Lambert Co. (now Pfizer) ( Dudley, Pang, Decker, Bridges, & Saltiel, 1995 ). Although PD098059 inhibits MEK1/2 in vitro and in vivo ( Alessi, Cuenda, Cohen, Dudley, & Saltiel, 1995 ), it never advanced in clinical development. In subsequent studies, we therefore switched to selumetinib, a potent and selective MEK1/2 inhibitor discovered by scientists at Array BioPharma and licensed to AstraZeneca for clinical development; it is currently being studied in phase III clinical trials ( Ciombor & Bekaii-Saab, 2015 ; Yeh et al., 2007 ). Pharmacological inhibitors of JNK have been synthesized but their clinical development has been rather limited ( Bogoyevitch & Arthur, 2008 ). They have been hypothesized to be potentially therapeutically useful in a wide range of conditions including neurodegeneration, inflammation, diabetes, viral infections, and fibrosis; however, there are no human clinical data to support these indications. One, PGL5001, has been studied in a phase II clinical trial for inflammatory endometriosis ( Barnes, 2013 ). In our studies of Lmna H222P/H222P mice, we used the reversible ATP-competitive JNK inhibitor SP600125 ( Bennett et al., 2001 ). Several small molecule inhibitors of p38α have been synthesized ( Coulthard, White, Jones, McDermott, & Burchill, 2009 ). Dilmapimod has been used in phase II clinical trials for neuropathic pain and chronic obstructive pulmonary disease ( Anand et al., 2011 ; Betts et al., 2015 ; Singh, Smyth, Borrill, Sweeney, & Tal-Singer, 2010 ). The p38α inhibitor ARRY-371797, discovered at Array BioPharma, has been studied in a phase II trial for acute inflammatory pain ( Coulthard et al., 2009 ). According to the company, its development for this indication was discontinued because the results were similar to other p38 inhibitors evaluated in rheumatoid arthritis ( http://investor.arraybiopharma.com/phoenix.zhtml?c=123810&p=irol-newsArticle&ID=1305781 ). We used ARRY-371797 in our studies of Lmna H222P/H222P mice. Based on its beneficial effects on left ventricular diameters and fractional shortening in these mice ( Muchir, Wu, et al., 2012 ), Array BioPharma has started a phase II trial of ARRY-371797 for patients with cardiomyopathy caused by LMNA mutations ( https://clinicaltrials.gov/ct2/show/NCT02057341 ). A summary of the MAP kinase inhibitors used is provided in Table 2 . For MEK1/2 inhibition, PD098059 (Selleck Chemicals) or selumetinib (Selleck Chemicals) were delivered to male Lmna H222P/H222P mice by intraperitoneal injection using a 27-gauge 5/8-in. needle and syringe. For JNK inhibition, SP600125 (Calbiochem) was similarly delivered. The drugs were dissolved in dimethyl sulfoxide (Sigma-Aldrich) at a concentration of 1.0 or 0.5 mg/ml. Doses were 3 mg/kg/day for PD098059 , 1 mg/kg/day for selumetinib, and 1 mg/kg for SP600125, given 5 days a week. To study the effects of an angiotensin-converting-enzyme inhibitor, benazepril (Sigma-Aldrich) was dissolved in dimethyl sulfoxide and similarly administered by intraperitoneal injection at a dose of 10 mg/kg/day. The placebo control consisted of dimethyl sulfoxide alone and was delivered similarly. During treatment, mice were fed a chow diet and housed in a barrier facility. For p38α inhibition, ARRY-371797 (Array BioPharma) was delivered to male Lmna H222P/H222P mice by oral gavage. It was dissolved in Water for Injection (Gibco) at a concentration of 0.5 mg/ml and dosed at 30 mg/kg twice a day. The placebo consisted of the same volume of Water for Injection. To assess effects on heart, treatment was started either when mice were 8 weeks of age (asymptomatic) and continued until 16 weeks or, alternatively, started at 16 weeks of age (symptomatic) and continued until 20 weeks of age. At 16 or 20 weeks of age, mice were analyzed by echocardiography, then euthanized and hearts were excised for biochemical and histological analyses (see below). To assess the effects of treatment on survival, selumetinib dissolved in dimethyl sulfoxide was diluted in drinking water to a concentration of 0.2 mg/ml; the same volume of dimethyl sulfoxide diluted in drinking water was used as placebo. Treatment was begun at 16 weeks of age and continued until the endpoints were reached (see below).

As summarized in Table 3 , treatment of Lmna H222P/H222P mice with MAP kinase inhibitors has various beneficial effects. Most of our work focused on MEK1/2 inhibitors because, as a class, they are further along in clinical development than JNK and p38α inhibitors. When treatment with a MEK1/2 inhibitor is begun at 8 weeks of age, before the onset of symptoms, male Lmna H222P/H222P mice have significantly smaller left ventricular end systolic and end diastolic diameters and a significantly greater left ventricular ejection fraction, with values similar to wild-type mice, at 16 weeks of age ( Muchir et al., 2009 ). Treatment with the JNK inhibitor SP600125 and analysis at the same ages gives similar results ( Wu et al., 2010 ). Male Lmna H222P/H222P mice treated with MEK1/2 inhibitors or a JNK inhibitor starting at 16 weeks of age, when heart function is already abnormal, have significantly greater left ventricular fractional shortening and ejection fraction, significantly smaller left ventricular diameters and significantly less left ventricular fibrosis than placebo-treated mice at 20 weeks of age ( Muchir, Reilly, et al., 2012 ; Wu et al., 2011 ). MEK1/2 inhibitor treatment from 16 to 20 weeks also decreases serum concentrations of natriuretic peptide A, which is secreted in response to increased cardiac filling pressure ( Muchir, Reilly, et al., 2012 ). Treatment with a MEK1/2 inhibitor also has synergistic benefits when combined with an angiotensin-converting enzyme inhibitor ( Muchir, Wu, Sera, Homma, & Worman, 2014 ). Treatment with a p38α inhibitor from 16 to 20 weeks has similar beneficial effects on left ventricular diameters and fractional shortening but does not decrease the expression of genes encoding collagens responsible for fibrosis ( Muchir, Wu, et al., 2012 ). Finally, treatment with a MEK1/2 inhibitor starting at 16 weeks of age significantly prolonged survival of male Lmna H222P/H222P mice ( Muchir, Reilly, et al., 2012 ). The protocols and methods we used to assess the effects of the MAP kinase inhibitors were as follows. Ideally, preclinical drug studies using such protocols such as these should, at a minimum, report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment and the handling of data, as recommended by the U.S. National Institute of Neurological Disorders and Stroke ( Landis et al., 2012 ). Echocardiography provides a convenient, noninvasive method to assess left ventricular diameters and functions in mice ( Rottman, Ni, & Brown, 2007 ). Potential limitations have been related to the small size and rapid heart rate; however, the development of small probes operating at higher frequencies and faster frame rates has overcome these limitations. Care must be taken not to give too much anesthesia to maintain a physiologically relevant heart rate. Mice were anesthetized with 1.5% isoflurane by inhalation and placed on a heating pad at 37 °C. We aimed at maintaining a heart rate of greater than 500/s. Echocardiography was performed using a Visualsonics Vevo 770 ultrasound with a 30 MHz transducer applied to the chest wall. Cardiac ventricular dimensions were measured in two-dimensional mode and M-mode three times for each animal. A typical echocardiographic examination takes approximately 3–5 min. Fractional shortening was calculated using the following formula: fractional shortening (%)=[(left ventricular end diastolic diameter—left ventricular end systolic diameter)/left ventricular end diastolic diameter] × 100. Left ventricular ejection fraction was calculated using the modified Simpson rule ( Folland et al., 1979 ). The echocardiographer was always blind to mouse genotype and treatment. After treatment with MAP kinase inhibitors, hearts were excised from drug-treated and placebo-treated mice to assess inhibition of ERK1/2 (which is activated by MEK1/2), JNK, and p38α. As described in Section 3.2 above, we used immunoblotting to measure their activities. Treatment with inhibitors gave approximately 30–70% decreases in the ratio of the phosphorylated MAP kinase to the total MAP kinase compared to treatment with placebo ( Muchir, Reilly, et al., 2012 ; Muchir et al., 2009 ; Muchir, Wu, et al., 2012 ; Wu et al., 2011 , 2010 ). Natriuretic peptide A, also known as atrial natriuretic factor and atriopeptin, is secreted from the heart in response to increased intravascular volume and right atrial pressure. Its concentration is elevated in the blood in congestive heart failure ( Tikkanen, Fyhrquist, Metsärinne, & Leidenius, 1985 ). We measured natriuretic peptide A in serum from Lmna H222P/H222P mice treated with selumetinib or placebo using the following methods. Natriuretic peptide A is detected in mouse serum samples using a commercial competitive enzyme immunoassay (RayBiotech). We added 100 μl antinatriuretic peptide A antibody solution to each well of the 96-well plate coated with rabbit secondary antibody and incubate for 1.5 h at room temperature. Serum samples containing biotinylated natriuretic peptide A (10 pg/ml) was added to wells of the microplate and incubated for 2.5 h at room temperature. After washing four times in phosphate-buffered saline, we added 100 ml of horseradish peroxidase–streptavidin solution to each well and incubated for 45 min at room temperature. After washing and incubation for 30 min with 100 μl of RayBiotech 3,3′,5,5′-tetramethylbenzidine One-Step substrate reagent in the dark, absorbance was measured at 450 nm. We assessed left ventricular fibrosis in excised hearts of Lmna H222P/H222P treated with either a MAP kinase inhibitor or a placebo using histological and biochemical methods. Histological assessment was a semiquantitative analysis of collagen staining. Biochemical analysis measured the expression of two genes encoding collagens. Sections of left ventricles from Lmna H222P/H222P mice treated with a MAP kinase inhibitor or placebo were fixed in 4% formaldehyde for 48 h, embedded in paraffin, sectioned at 5 μm, and stained with Gomori trichrome, which stains collagen blue. Representative stained sections were photographed using a Microphot SA (Nikon) light microscope attached to a Spot RT Slide camera (Diagnostic Instruments). To quantify fibrosis, micrographs were processed (JMicroVision software) and blue-stained fibrotic tissue measured (ImageJ64 software). For biochemical analysis of collagen gene expression, primers corresponding to mouse Col1a1 and Co1a2 gene cDNAs ( Col1a1 forward 5′-agacggacagtactggatcg-3′ and reverse 5′-gcttcttttccttggggttc-3′; Col1a2 forward 5′-ccgtgcttctcagaacatca-3′ and reverse 5′-gagcagccatcgactaggac-3′) are used from real-time RT-PCR. Reactions contained HotStart-IT SYBR Green qPCR Master Mix (Affymetrix), 200 n M of each primer, and 0.2 ml of template in a 25-ml reaction volume. Amplification was carried out using the ABI 7300 Real-Time PCR System (Applied Biosystems) with an initial denaturation at 95 °C for 2 min followed by 50 cycles at 95 °C for 30 s and 62 °C for 30 s. Relative levels of mRNA expression were calculated using the ΔΔC T method ( Ponchel et al., 2003 ) and normalized to calculated Gapdh mRNA expression level. We demonstrated a significantly prolonged survival of male Lmna H222/H222P mice treated with the MEK1/2 inhibitor selumetinib. We actually used a combined endpoint of survival and required euthanasia for mice that are in distress. This is a more compassionate protocol to limit pain and distress and was encouraged by our institution’s Institutional Animal Care and Use Committee. Selumetinib dissolved in dimethyl sulfoxide was diluted in drinking water to a concentration of 0.2 mg/ml; the same volume of dimethyl sulfoxide diluted in drinking water was used as placebo. Treatment was started when mice were 16 weeks of age and continued until death or until a veterinarian blind to treatment group recommended euthanasia because of undue distress. Criteria for euthanasia were (1) difficulty with normal ambulation, (2) failure to eat or drink, (3) loss of body mass of >20%, (4) rough or unkempt coat, and (5) respiratory distress. Data were analyzed using the Kaplan–Meier estimator ( Kaplan & Meier, 1958 ) followed by a log-rank test with p <0.05 considered statistically significant; GraphPad Prism software was used for this analysis.

The experiments and methods described in this review have demonstrated the utility of a mouse model of cardiomyopathy caused by LMNA mutation in identifying dysregulated cardiac MAP kinase signaling in the hearts of these animals. Pharmacological inhibitors of kinases in the ERK1/2, JNK, and p38α pathways were used to demonstrate a role for their abnormally increased signaling activity in pathogenesis. Several of these kinase inhibitors are in clinical development for other indications and could potentially be developed to treat human subjects with cardiomyopathy caused by LMNA mutations. Abnormally elevated ERK1/2 and p38α activity has indeed been demonstrated in hearts from affected human subjects ( Muchir, Reilly, et al., 2012 ; Muchir, Wu, et al., 2012 ). A small clinical trial of a p38α inhibitor is already underway based on this research and others could be anticipated.