Prelamin A mediates myocardial inflammation in dilated and HIV-associated cardiomyopathies

Cardiomyopathies are complex heart muscle diseases that can be inherited or acquired. Dilated cardiomyopathy can result from mutations in LMNA, encoding the nuclear intermediate filament proteins lamin A/C. Some LMNA mutations lead to accumulation of the lamin A precursor, prelamin A, which is disease causing in a number of tissues, yet its impact upon the heart is unknown. Here, we discovered myocardial prelamin A accumulation occurred in a case of dilated cardiomyopathy, and we show that a potentially novel mouse model of cardiac-specific prelamin A accumulation exhibited a phenotype consistent with inflammatory cardiomyopathy, which we observed to be similar to HIV-associated cardiomyopathy, an acquired disease state. Numerous HIV protease therapies are known to inhibit ZMPSTE24, the enzyme responsible for prelamin A processing, and we confirmed that accumulation of prelamin A occurred in HIV+ patient cardiac biopsies. These findings (a) confirm a unifying pathological role for prelamin A common to genetic and acquired cardiomyopathies; (b) have implications for the management of HIV patients with cardiac disease, suggesting protease inhibitors should be replaced with alternative therapies (i.e., nonnucleoside reverse transcriptase inhibitors); and (c) suggest that targeting inflammation may be a useful treatment strategy for certain forms of inherited cardiomyopathy.


Microscopy 1
Immunohistochemical and general tissue staining was analysed by bright-field light microscopy using 2 an Axioskop 2 microscope (Carl Zeiss MicroImaging, NY). Immunofluorescence was analysed using 3 wide-field fluorescence microscopy (IX81 microscope, Olympus). Where images captured were 4 subjected to quantitative analysis, this was performed by counting by eye, or semi automated with the 5 use of selection and measuring tools made freely available by ImageJ software. All massays were 6 performed at the James Black Centre, King's College London and in each case the investigator 7 performing the analysis was blinded to group assignment. Images for publication were captured using 8 a Leica SP5 confocal microscope (Leica Microsystems, UK). Confocal images were processed in 9 Photoshop CS3 (Adobe) and minimal adjustments to brightness and contrast were made where 10 necessary, e.g. when images were deemed too dark for comfortable viewing. 11 Immunoblotting 12 Cryopreserved heart tissue was homogenised by placement into a pre-liquid nitrogen cooled stainless 13 steel chamber followed by crushing administered by a pre-liquid nitrogen cooled stainless steel rod 14 and rubber mallet. 200 μl lysis/sample buffer (3.7 M urea, 134.6 mM Tris pH 6.8; 5.4% SDS; 2.3% 15 NP-40; 4.45% beta-mercaptoethanol; 4% glycerol and 6 mg/100 ml bromophenolblue) was added. 16 And frozen pellets placed into Eppendorf tubes. The tubes were heated to 85°C for 5 minutes. A 17 chamber was prepared with an SDS gel of 10% acrylamide concentration. 20l was then loaded into 18 each well of the gel. The proteins were separated at 150 mV. Transfer was achieved in wet conditions. 19 The proteins were subject to electrophoretic transfer onto PVDF membranes that had been incubated 20 in methanol for 20 seconds and then washed in distilled water. The transfer conditions were 65 Amps 21 at 4°C ON. The membrane was blocked for 1 hour with 5% Milk in TBS-T. Primary antibodies were 22 added (Table 2.2) with TBS-T/5% milk or TBS-T 5% bovine serum albumin and incubated overnight 23 at 4°C. The membrane was washed 3x5 min with TBS-T. The blots were then incubated in secondary 24 antibody coupled to horseradish peroxidise for 1 hour at room temperature or secondary antibody 25 conjugated to a fluorophore or horse radish peroxidase for 1 hour. The blots were again washed with rehydration, sections were washed under running tap water for 5 min, and then placed in Harris' 1 haematoxylin solution for 5 min. The sections were then washed under running tap water until the 2 water ran clear followed by differentiation in acid alcohol. The sections were again placed under 3 running tap water for 5 min and then stained with eosin solution for 3 min. The sections were dunked 4 5 times in tap water and then subjected to dehydration by sequential ethanol treatment: 25%, 50%, 5 75%, 96% (1 min each) 100%, (2x3 min) xylene, (2x5 min). Coverslips were mounted using DPX and 6 allowed to dry overnight. 7

Picro-sirius red staining 8
Following rehydration, slides were left in ddH 2 O for 5 min followed by a 30 second incubation in 9 0.2% phosphomolybdic acid. Slides were rinsed in ddH 2 O and then left in 1% Sirius red solution for 10 90 min. Slides were washed 2x2 min in acidified ddH 2 O (0.05% acetic acid) followed by a 15 min 11 incubation in Picric acid. Slides were rinsed several times in ddH 2 O followed by dehydration by 12 sequential ethanol treatment: Glass coverslips were mounted with DPX and allowed to dry overnight. 13

Senescence associated β-Galactosidase assay 14
Hearts were resected from mice and immediately frozen and cryosectioned into 5mm thick slices and 15 mounted onto superfrost slides. Sections were allowed to dry for 1-2 hrs. Samples were fixed in 1% 16 formalin solution for 10 min RT. Senescence associated β-Galactosidase (SA-β-Gal) staining solution 17 was freshly made and applied to each heart section so that they were generously submerged (approx. 18 50 μl per heart section). The slides were incubated in a humidified chamber overnight (approx. 16h) at 19 37°C. The slides were washed 2x5 min in PBS and once in 100% methanol for 2 min, the heart 20 sections were then viewed and photographed under bright-field microscopy. 21

Cardiac troponin T (cTnT) Enzyme Linked Immuno-Sorbent Assay (ELISA) 22
Cardiac puncture under terminal anaesthesia was performed and blood was collected in an EDTA 23 coated Eppendorf and spun at 13,000 g 4⁰C for 15 minutes to sediment red blood cells. Plasma was collected and subject to ELISA for cTnT (Elabscience, USA) according to manufacturer's 1 instructions. 2

TUNEL staining 3
Deparaffinised sections were digested in 20 μg/ml proteinase K solution at 37°C for 10 min and 4 washed 3x5 min in PBS. Sections were then incubated in 3% H 2 O 2 for 10 min RT to block 5 endogenous peroxidase activity and washed 3x5 min in PBS. Sections were then pre-incubated in TdT 6 reaction buffer for 10 min RT and then incubated in TdT reaction mixture for 1 hour at 37°C in a 7 humidified chamber. Sections were then washed in stop wash buffer for 10 min RT and rinsed in 8 PBS-T 3x5 min. Sections were incubated with streptavidin-HRP for 20 min RT. Sections were 9 incubated in DAB chromagen solution for 1-10 min (until brown staining appeared) and left under 10 running tap water for 5 min. Coverslips were mounted with DPX and allowed to dry for 2 hour or 11 overnight (ON). 12