CRISPLD1: a novel conserved target in the transition to human heart failure

Heart failure is a major health problem worldwide with a significant morbidity and mortality rate. Although studied extensively in animal models, data from patients at the compensated disease stage are lacking. We sampled myocardium biopsies from aortic stenosis patients with compensated hypertrophy and moderate heart failure and used transcriptomics to study the transition to failure. Sequencing and comparative analysis of analogous samples of mice with transverse aortic constriction identified 25 candidate genes with similar regulation in response to pressure overload, reflecting highly conserved molecular processes. The gene cysteine-rich secretory protein LCCL domain containing 1 (CRISPLD1) is upregulated in the transition to failure in human and mouse and its function is unknown. Homology to ion channel regulatory toxins suggests a role in Ca2+ cycling. CRISPR/Cas9-mediated loss-of-function leads to dysregulated Ca2+ handling in human-induced pluripotent stem cell-derived cardiomyocytes. The downregulation of prohypertrophic, proapoptotic and Ca2+-signaling pathways upon CRISPLD1-KO and its upregulation in the transition to failure implicates a contribution to adverse remodeling. These findings provide new pathophysiological data on Ca2+ regulation in the transition to failure and novel candidate genes with promising potential for therapeutic interventions. Electronic supplementary material The online version of this article (10.1007/s00395-020-0784-4) contains supplementary material, which is available to authorized users.


Supplementary methods:
Expression profiling and identification of conserved candidates.
Generation, quantification and analysis of proteome data.       Information. S8: Sequence information of CRISPLD1-iPSC-KO.

Supplementary methods
Expression profiling and identification of conserved candidates. Differential expression (DE) was assessed for the CH/1wTAC and HF group compared to the control/sham group.
Differential expression analysis was done using DESeq2. Initial DE analysis of the control transcriptomes vs pathology (cut offs: minimal counts > 10, log2FC >+/-1, adjusted p value < 0.05) resulted in a list of significant DEGs for each of the conditions vs. the control. Each gene that were significantly differentially expressed in at least one DE analysis was analyzed for expression in the other condition and the control comparing the normalized counts (see data file S2 and S3) using the following criteria: If the sum of two pathology groups were higher than 20 counts the medians were calculated.
Fold changes higher than 1.5 received negative or positive labels depending on the expression between the 3 conditions. Fold changes below 1.2 indicate no alteration of expression between conditions. Exclusive expression is given by expression higher than 50 counts in one case and less than 10 counts in the other two. Data sets of human and mouse genes were compared under consideration of the expression profiles and 25 genes were identified showing conserved changes in expression during disease progression in human and mouse.
Generation, quantification and analysis of proteome data. For generation of a peptide library, equal amount aliquots from each sample were pooled to a total amount of 80 µg, and separated into eight fractions using a reversed phase spin column (Pierce High pH Reversed-Phase Peptide Fractionation Kit, Thermo Fisher Scientific). All samples were spiked with a synthetic peptide standard used for retention time alignment (iRT Standard, Schlieren, Schweiz).
Qualitative LC/MS/MS analysis was performed using a Top20 data-dependent acquisition method with an MS survey scan of m/z 350-1250 accumulated for 350 ms at a resolution of 30,000 full width at half maximum (FWHM). MS/MS scans of m/z 180-1600 were accumulated for 100 ms at a resolution of 17,500 FWHM and a precursor isolation width of 0.7 FWHM, resulting in a total cycle time of 2.9 s. Precursors above a threshold MS intensity of 125 cps with charge states 2+, 3+, and 4+ were selected for MS/MS, the dynamic exclusion time was set to 30 s. MS/MS activation was achieved by CID using nitrogen as a collision gas and the manufacturer's default rolling collision energy settings. Two technical replicates per reversed phase fraction were analyzed to construct a spectral library.
For quantitative SWATH analysis, MS/MS data were acquired using 65 variable size windows [28] across the 400-1,050 m/z range. Fragments were produced using rolling