Protein Inhibitor of NOS1 Plays a Central Role in the Regulation of NOS1 Activity in Human Dilated Hearts

An essential factor for the production of nitric oxide by nitric oxide synthase 1 (NOS1), major modulator of cardiac function, is the cofactor tetrahydrobiopterin (BH4). BH4 is regulated by GTP cyclohydrolase 1, the rate-limiting enzyme in BH4 biosynthesis which catalyses the formation of dihydroneopterin 3′triphosfate from GTP, producing BH4 after two further steps catalyzed by 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase. However, there are other essential factors involved in the regulation of NOS1 activity, such as protein inhibitor of NOS1 (PIN), calmodulin, heat shock protein 90, and NOS interacting protein. All these molecules have never been analysed in human non-ischemic dilated hearts (DCM). In this study we demonstrated that the upregulation of cardiac NOS1 is not accompanied by increased NOS1 activity in DCM, partly due to the elevated PIN levels and not because of alterations in biopterin biosynthesis. Notably, the PIN concentration was significantly associated with impaired ventricular function, highlighting the importance of this NOS1 activity inhibitor in Ca2+ homeostasis. These results take a central role in the current list of targets for future studies focused on the complex cardiac dysfunction processes through more efficient harnessing of NOS1 signalling.


RNA extraction
Heart samples were homogenised in TRIzol ® reagent in a TissueLysser LT (Qiagen, UK). All RNA extractions were performed using a PureLink™ Kit according to the manufacturer's instructions (Ambion Life Technologies, CA, USA). RNA was quantified using a NanoDrop1000 spectrophotometer (Thermo Fisher Scientific, UK), and the purity and integrity of the RNA samples were measured using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano LabChip kit (Agilent Technologies, Spain). All samples showed a 260/280 ratio >2.0 and an RNA integrity number ≥9.

RNA-seq
The RNA samples were isolated using a MicroPoly(A) Purist Kit™ (Ambion, USA). The total polyA-RNA samples were used to generate whole transcriptome libraries that were sequenced on a SOLiD 5500XL platform as per the manufacturer's recommendations (Life Technologies, CA). The amplified cDNA quality was analysed using the Bioanalyzer 2100 DNA 1000 kit (Agilent Technologies, Spain), and the cDNA was quantified using the Qubit 2.0 Fluorometer (Invitrogen, UK). Whole transcriptome libraries were used to generate SOLiD templated beads by following the SOLiD Templated Bead Preparation guide. Bead quality was estimated based on WFA (workflow analysis) parameters. The samples were sequenced using the 50625 paired-end protocol, which generated 75 nt + 35 nt (Paired-End) + 5 nt (Barcode) sequences. Quality data were measured using the SETS software parameters (SOLiD Experimental Tracking System).

Computational analysis of RNA-seq data
The initial whole transcriptome paired-end reads obtained from the sequencing were mapped against the latest version of the human genome (Version GRchr37/hg19) by using the Life Technologies mapping algorithm (http://www.lifetechnologies.com/). The aligned records were reported in the BAM/SAM format 1 . Bad quality reads (Phred score <10) were eliminated using the Picard Tools software 2 . The isoform and gene predictions were subsequently estimated using the cufflinks method 3 , and the expression levels were calculated using the HTSeq software 4 . The Edge method was applied to analyse the differential expression between conditions 5 . This method relies on a Poisson model to estimate the RNAseq data variance for differential expression. We selected genes and isoforms that were calculated to exhibit P<0.05 and fold-change >1.5.

RT-qPCR analysis
Reverse transcription was carried out using 1 μg total RNA and M-MLV enzyme (Invitrogen were added for 3 hours at 4 °C, centrifugated, and washed three times. This was followed by resuspending the pellet with 20 μL SDS sample buffer and heating the samples to 95 °C for 5 minutes. Thereafter, samples were separated by electrophoresis and immunoblotted.

NOS activity
NOS activity was measured using radiochemical detection of L-arginine to L-citrulline conversion, as described previously. 7 Briefly, separation of the products of L-arginine metabolism was obtained by ion exchange chromatography (Jasco Ltd.) and on-line radiochemical scintillation detection (Lablogic Systems Ltd). Recorded data were analysed using Azur software (Datalys, France). LV was homogenized ice-cold Krebs' HEPES Buffer containing 5 μmol/L or-NOHA (to inhibit arginase activity). After centrifugation (13,000 rpm for 10 mins at 4ºC), the supernatant was then incubated for 30 mins on ice with added NOS cofactors except BH4 (i.e., 10 μmol/L FAD, 10 μmol/L FMN, 1 mmol/L NADPH), in the presence or absence of either the non-specific NOS inhibitor, L-NAME (1 mmol/L), or the NOS1-selective inhibitor SMTC (100 nmol/L), followed by 4 hours incubation at 37ºC with 3 μL of labelled 14 C L-arginine (Amersham Biosciences UK Ltd.). Trichloroacetic acid (10%) was then added to de-proteinate the samples, prior to centrifugation. The supernatant was placed into the auto-sampler cooled to 4ºC for chromatographic analysis. Standards of 14 Clabelled Larginine (1 μmol/L), L-citrulline (0.1 μmol/L), and L-ornithine (0.2 μmol/L, all from Amersham Bioscience UK Ltd.) were used to determine elution time. Chromatographic peaks were integrated and expressed as a proportion of total 14 C counts for each sample.
Results were expressed as the L-NAME-or SMTC-inhibitable fraction. NOS activity was normalised (500 μg of protein per reaction).

Biopterins determinations
The detection of the different biopterins BH4, BH2 and B was performed by separation and quantification by reverse phase HPLC. 8 Tissues were prepared from frozen (in -80°C storage); 20-30 mg of LV samples were homogenised as previously described in 500 μl ice cold re-suspension buffer, followed by 15 min centrifugation at 13000 rpm and 4ºC. A volume of 180 μl of the supernatant was taken from samples and standards and added to Eppendorf tubes containing 20 μl of ice-cold 10x acid precipitation buffer. These were mixed well by vortexing and centrifuged for 5 min at 13000 rpm (4ºC). Supernatants were injected into the isocratic HPLC system; biopterins were separated using a Carbon-18 column (Hichrom) with a flow of mobile phase at the rate of 1.3 ml/min. Biopterins were quantified by sequential electrochemical (Coulochem III, ESA Inc, USA) and fluorescence (Jasco Ltd) detection. Biopterin concentrations were quantified by comparison with BH4, BH2 and B reference standards and normalised to protein content.