Doxorubicin (DOX) is an efficacious chemotherapy compound used to treat various cancers which elicits severe side effects, including heart failure. Uptake of DOX by cardiomyocytes causes metabolic dysfunction and cell death but causal mechanisms remain largely undefined. We applied genome-wide CRISPR/Cas9 knockout screens to discover genetic modifiers of DOX-induced cardiomyocyte cell death, and independently, DOX uptake and clearance. Both screens discovered known and novel factors. In cell death screens and validation studies, loss of retinoic acid receptor- α (RARA) predisposed cardiomyocytes to DOX-mediated cell death. Conversely, RARA activation reduced DOX cytotoxicity in wild type cardiomyocytes. RNA-Seq analysis revealed that whilst DOX caused large-scale suppression of metabolic and mitochondrial gene expression, RARA activation mitigated this effect. In DOX accumulation screens, an essential role for lysosomes in DOX clearance was observed. Loss of Sphingolipid Transporter 1 (SPNS1) led to DOX hyperaccumulation, suppression of autophagy, increased DNA damage, and increased cell death. Hence, SPNS1 plays a key role in buffering against DOX accumulation and toxicity. Collectively, our study nominated hundreds of drug-gene interactions, providing a springboard for exploration of causal mechanisms, and a technical framework for future screening campaigns.

Objective Animal models suggest that BRCA1/2 mutations increase doxorubicin-induced cardiotoxicity risk but data in humans are limited. We aimed to determine whether germline BRCA1/2 mutations are associated with cardiac dysfunction in breast cancer survivors. Methods In a single-center cross-sectional study, stage I-III breast cancer survivors were enrolled according to three groups: (1) BRCA1/2 mutation carriers treated with doxorubicin; (2) BRCA1/2 mutation non-carriers treated with doxorubicin; and (3) BRCA1/2 mutation carriers treated with non-doxorubicin cancer therapy. In age-adjusted analysis, core-lab quantitated measures of echocardiography-derived cardiac function and cardiopulmonary exercise testing (CPET) were compared across the groups. A complementary in vitro study was performed to assess the impact of BRCA1 loss of function on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) survival following doxorubicin exposure. Results Sixty-seven women with mean (standard deviation) age of 50 (11) years were included. Age-adjusted left ventricular ejection fraction (LVEF) was lower in participants receiving doxorubicin regardless of BRCA1/2 mutation status (p = 0.03). In doxorubicin-treated BRCA1/2 mutation carriers and non-carriers, LVEF was lower by 5.4% (95% CI; −9.3, −1.5) and 4.8% (95% CI; −9.1, −0.5), respectively compared to carriers without doxorubicin exposure. No significant differences in VO2max were observed across the three groups (poverall = 0.07). Doxorubicin caused a dose-dependent reduction in viability of iPSC-CMs in vitro without differences between BRCA1 mutant and wild type controls (p > 0.05). Conclusions BRCA1/2 mutation status was not associated with differences in measures of cardiovascular function or fitness. Our findings do not support a role for increased cardiotoxicity risk with BRCA1/2 mutations in women with breast cancer.

In the heart, alternative splicing of the igf-I gene produces two isoforms: IGF-IEa and IGF-IEc, (Mechano-growth factor, MGF). The sequence divergence between their E-domain regions suggests differential isoform function. To define the biological actions of MGF’s E-domain, we performed in silico analysis of the unique C-terminal sequence and identified a phosphorylation consensus site residing within a putative 14-3-3 binding motif. To test the functional significance of Ser 18 phosphorylation, phospho-mimetic (S/E18) and phospho-null (S/A18) peptides were delivered to mice at different doses for 2 weeks. Cardiovascular function was measured using echocardiography and a pressure-volume catheter. At the lowest (2.25 mg/kg/day) and highest (9 mg/kg/day) doses, the peptides produced a depression in systolic and diastolic parameters. However, at 4.5 mg/kg/day the peptides produced opposing effects on cardiac function. Fractional shortening analysis also showed a similar trend, but with no significant change in cardiac geometry. Microarray analysis discovered 21 genes (FDR p < 0.01), that were expressed accordant with the opposing effects on contractile function at 4.5 mg/kg/day, with the nuclear receptor subfamily 4 group A member 2 (Nr4a2) identified as a potential target of peptide regulation. Testing the regulation of the Nr4a family, showed the E-domain peptides modulate Nr4a gene expression following membrane depolarization with KCl in vitro. To determine the potential role of 14-3-3 proteins, we examined 14-3-3 isoform expression and distribution. 14-3-3γ localized to the myofilaments in neonatal cardiac myocytes, the cardiac myocytes and myofilament extracts from the adult heart. Thermal shift analysis of recombinant 14-3-3γ protein showed the S/A18 peptide destabilized 14-3-3γ folding. Also, the S/A18 peptide significantly inhibited 14-3-3γ’s ability to interact with myosin binding protein C (MYPC3) and phospholamban (PLN) in heart lysates from dobutamine injected mice. Conversely, the S/E18 peptide showed no effect on 14-3-3γ stability, did not inhibit 14-3-3γ’s interaction with PLN but did inhibit the interaction with MYPC3. Replacing the glutamic acid with a phosphate group on Ser 18 (pSer18), significantly increased 14-3-3γ protein stability. We conclude that the state of Ser 18 phosphorylation within the 14-3-3 binding motif of MGF’s E-domain, modulates protein-protein interactions within the 14-3-3γ interactome, which includes proteins involved in the regulation of contractile function.

amino-cytoplasmic domain is not required for the internalisation of low density uptake ABSTRACT The uptake of oxidised low density lipoproteins (OxLDL) by CD36-expressing macrophages in the arterial intima and the ensuing ‘foam cell’ formation represents a crucial step in the initiation and development of atherosclerotic plaques. This study has addressed the function of the CD36 amino-cytoplasmic domain in the binding and internalisation of OxLDL. A selection of CD36 amino-cytoplasmic domain mutants were generated and stably expressed in HEK293 cells. The capacity of three mutants (CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD-) to bind and endocytose OxLDL was then studied using immunofluorescence microscopy and quantitative fluoremetry. Each of the CD36 constructs was expressed at differing levels at the cell surface as measured by flow cytometry and western blotting. Following incubation with DiI-OxLDL, cells bearing the CD36_wild-type, CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD - construct all internalized DiI-OxLDL into endosomal structures whereas empty vector-transfected cells failed to do so indicating that unlike the carboxy-cytoplasmic domain, the amino-cytoplasmic domain is not essential for the endocytosis of OxLDL. To conclude, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 amino-cytoplasmic domain. However, the amino-cytoplasmic domain may conceivably be implicated in the maturation of CD36. the uptake of DiI-OxLDL by cells expressing each of the CD36 constructs. Consistent with the immunofluorescence work, cells bearing the different mutants were capable of binding and endocytosing OxLDL. In such analyses, there was a notable difference in the quantity of DiI-OxLDL taken up by each cell type. Cells expressing the wild-type and C3/7-A constructs both internalised approximately two-fold more DiI-OxLDL than ev-transfected cells, whilst cells expressing the D4/R5-A and nCPD - constructs internalised approximately four-fold and five-fold more DiI-OxLDL, respectively than the ev-transfected cells. These differences were felt to be due at least partially to the fact that the different constructs were expressed at different levels at the cell surface of transfectants. As a means of compensating for the disparity in construct expression, all DiI-OxLDL uptake values were normalised against the amount of CD36 expressed by each cell line. After doing this, there was a negligible difference in the efficacy of each cell-type to take up DiI-OxLDL, again suggesting that the CD36 amino-cytoplasmic domain is not involved in the endocytotic mechanism. To conclude, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 amino cytoplasmic domain. However, this region may somehow be implicated in the maturation of CD36. Specifically, it could be involved in retarding the movement of the CD36 polypeptide – following translation - from the cytoplasm to the plasma membrane in order for it to attain its final, native state structure and receive additional moieties such as sugar chains and palmitoyl groups.

Mutations in B cell lymphoma 2-associated athanogene 3 (BAG3) are recurrently associated with dilated cardiomyopathy (DCM) and muscular dystrophy. Using isogenic genome-edited human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we examined how a DCM-causing BAG3 mutation (R477H), as well as complete loss of BAG3 (KO), impacts myofibrillar organization and chaperone networks. Although unchanged at baseline, fiber length and alignment declined markedly in R477H and KO iPSC-CMs following proteasome inhibition. RNA sequencing revealed extensive baseline changes in chaperone- and stress response protein-encoding genes, and protein levels of key BAG3 binding partners were perturbed. Molecular dynamics simulations of the BAG3-HSC70 complex predicted a partial disengagement by the R477H mutation. In line with this, BAG3-R477H bound less HSC70 than BAG3-WT in coimmunoprecipitation assays. Finally, myofibrillar disarray triggered by proteasome inhibition in R477H cells was mitigated by overexpression of the stress response protein heat shock factor 1 (HSF1). These studies reveal the importance of BAG3 in coordinating protein quality control subsystem usage within the cardiomyocyte and suggest that augmenting HSF1 activity might be beneficial as a means to mitigate proteostatic stress in the context of BAG3-associated DCM.

Angiopoietin-like 3 ( ANGPTL3 ) associates strongly with blood lipid phenotypes in human genetics studies and has thus emerged as a promising therapeutic target for plasma lipids. Our previous work showed that there was a 34% lower risk of coronary heart disease (CHD) among carriers of an ANGPTL3 loss-of-function (LOF) mutation compared with non-carriers in a meta-analysis of 21,980 patients with CHD and 158,200 control individuals. The role of ANGPTL3 in lipid metabolism is unequivocal, but the link between ANGPTL3 and glucose metabolism remains unclear. To determine the effects of mouse Angptl3 on insulin resistance, we used CRISPR-Cas9 to generate Angptl3 knockout mice, which exhibited decreased TG (61%, P < 0.001), decreased cholesterol (31%, P < 0.002) and no detectable ANGPTL3 protein in both liver lysis and circulation. The body weight and glucose level in plasma are similar between Angptl3 knockout and wild-type mice at normal chow. However, when fed on high-fat-diet for 5 months, the glucose level was significant lower in Angptl3 knockout mice compared to wild-type in both genders after 4 hours fasting (11.7% lower in male and 10.6% lower in female). More importantly, insulin sensitivity, measured by intraperitoneal glucose tolerance was significantly higher among Angptl3 knockout mice compared to wild-type. Together, these data suggests that LOF of ANGPTL3 might have a favorable impact on glucose homeostasis, which marks out ANGPTL3 as a unique therapeutic target for both CHD and T2DM.

Mitochondrial homeostasis depends on mitophagy, the programmed degradation of mitochondria. Only a few proteins are known to participate in mitophagy. Here we develop a multidimensional CRISPR-Cas9 genetic screen, using multiple mitophagy reporter systems and pro-mitophagy triggers, and identify numerous components of parkin-dependent mitophagy(1). Unexpectedly, we find that the adenine nucleotide translocator (ANT) complex is required for mitophagy in several cell types. Whereas pharmacological inhibition of ANT-mediated ADP/ATP exchange promotes mitophagy, genetic ablation of ANT paradoxically suppresses mitophagy. Notably, ANT promotes mitophagy independently of its nucleotide translocase catalytic activity. Instead, the ANT complex is required for inhibition of the presequence translocase TIM23, which leads to stabilization of PINK1, in response to bioenergetic collapse. ANT modulates TIM23 indirectly via interaction with TIM44, which regulates peptide import through TIM23(2). Mice that lack ANT1 show blunted mitophagy and consequent profound accumulation of aberrant mitochondria. Disease-causing human mutations in ANT1 abrogate binding to TIM44 and TIM23 and inhibit mitophagy. Together, our findings show that ANT is an essential and fundamental mediator of mitophagy in health and disease.

Background: Truncating variants in the Titin gene (TTNtvs) are common in individuals with idiopathic dilated cardiomyopathy (DCM). However, a comprehensive genomics-first evaluation of the impact of TTNtvs in different clinical contexts, and the evaluation of modifiers such as genetic ancestry, has not been performed. Methods: We reviewed whole exome sequence data for >71 000 individuals (61 040 from the Geisinger MyCode Community Health Initiative (2007 to present) and 10 273 from the PennMedicine BioBank (2013 to present) to identify anyone with TTNtvs. We further selected individuals with TTNtvs in exons highly expressed in the heart (proportion spliced in [PSI] >0.9). Using linked electronic health records, we evaluated associations of TTNtvs with diagnoses and quantitative echocardiographic measures, including subanalyses for individuals with and without DCM diagnoses. We also reviewed data from the Jackson Heart Study to validate specific analyses for individuals of African ancestry. Results: Identified with a TTNtv in a highly expressed exon (hiPSI) were 1.2% individuals in PennMedicine BioBank and 0.6% at Geisinger. The presence of a hiPSI TTNtv was associated with increased odds of DCM in individuals of European ancestry (odds ratio [95% CI]: 18.7 [9.1–39.4] {PennMedicine BioBank} and 10.8 [7.0–16.0] {Geisinger}). hiPSI TTNtvs were not associated with DCM in individuals of African ancestry, despite a high DCM prevalence (odds ratio, 1.8 [0.2–13.7]; P =0.57). Among 244 individuals of European ancestry with DCM in PennMedicine BioBank, hiPSI TTNtv carriers had lower left ventricular ejection fraction (β=–12%, P =3×10 –7 ), and increased left ventricular diameter (β=0.65 cm, P =9×10 –3 ). In the Geisinger cohort, hiPSI TTNtv carriers without a cardiomyopathy diagnosis had more atrial fibrillation (odds ratio, 2.4 [1.6–3.6]) and heart failure (odds ratio, 3.8 [2.4–6.0]), and lower left ventricular ejection fraction (β=–3.4%, P =1×10 –7 ). Conclusions: Individuals of European ancestry with hiPSI TTNtv have an abnormal cardiac phenotype characterized by lower left ventricular ejection fraction, irrespective of the clinical manifestation of cardiomyopathy. Associations with arrhythmias, including atrial fibrillation, were observed even when controlling for cardiomyopathy diagnosis. In contrast, no association between hiPSI TTNtvs and DCM was discerned among individuals of African ancestry. Given these findings, clinical identification of hiPSI TTNtv carriers may alter clinical management strategies.

The introduction of clinical sequencing is dramatically increasing the discovery of variants of uncertain significance (VUSs) in genes linked to inherited cardiomyopathies. We have established a platform for rapid and efficient insertion of TNNT2 gene variants into an induced pluripotent stem cell (iPSC) line to generate an allelic series of isogenic clones for differentiation into cardiomyocytes (iPSC-CMs) for functional annotation of the variants. We first used CRISPR-Cas9 to introduce known pathogenic variants into iPSCs from a healthy person or to correct pathogenic variants in iPSCs from patients with severe cardiomyopathy. Whereas normal/corrected iPSC-CMs responded to isoproterenol treatment with a 50%-70% increase in spontaneous beating rate as assessed by patch-clamp studies, iPSC-CMs with pathogenic variants had minimal responses (close to 0%). Due to the inefficiency of CRISPR-Cas9 in introducing/correcting variants in iPSCs, we next used dual integrase cassette exchange (DICE) to allow for the introduction of a large number of variants in parallel into a pool of cells. In a single pilot use of the DICE platform, we isolated heterozygous clones with 14 unique variants, >10% of all TNNT2 coding variants cataloged in ClinVar. We found that iPSC-CMs with any of 7 VUSs or likely pathogenic variants were impaired in their response to isoproterenol, in contrast to control DICE-treated iPSC-CMs. Finally, we sought to apply the DICE platform to a patient case in real time. A 65-year-old woman with severe hypertrophic cardiomyopathy underwent gene panel testing that identified a single VUS, TNNT2 E251D. Between the first and second clinic visits (~10 weeks), we were able to use DICE to rapidly and efficiently generate iPSC-CMs with the E251D variant and determine they had normal responses to isoproterenol, suggesting that the variant is not pathogenic. Guided by this finding, we recommended that the patient’s children and grandchildren not undergo cascade genetic screening for the E251D variant. In conclusion, this work establishes the feasibility of rapid functional annotation of cardiomyopathy gene variants, which after further validation could be incorporated into clinical practice as a line of evidence to support variant classification.

Circulation. 2018;138:2852–2854. DOI: 10.1161/CIRCULATIONAHA.118.035028 2852

Mitochondrial Transcription Termination Factor 4 ( MTERF4 ) is a transcription factor involved in mitochondrial ribosomal biogenesis and was identified as a gene of interest via whole exome sequencing in a pediatric patient with hypertrophic cardiomyopathy (HCM) at Children’s Hospital of Wisconsin. The variants of interest identified in MTERF4 have not been previously reported in the literature or associated with HCM. Here, we combined precision genome editing via CRISPR/Cas9 system with human iPSC-derived cardiomyocytes (hiPSC-derived CMs) to model the HCM patient-specific MTERF4 variants. We hypothesize that MTERF4 variants are contributing to cardiomyocyte impairment, leading to the development of the hypertrophic phenotype. To improve our efficiency of CRISPR/Cas9 precision genome editing in hiPSCs, we are using a co-targeting with selection method. MTERF4 mutant clones and control hiPSCs (obtained from co-targeted but unmodified by CRISPR/Cas9) were identified by Sanger sequencing and assessed for pluripotency using immunostaining and gene expression. MTERF4 clones and controls were subjected to our modified Palecek matrix-overlay method for CM differentiation and evaluated at 3-4 weeks for cell size, mitochondrial function, and gene expression. The hiPSC-derived CMs cell size analysis by average pixel area of the MTERF4 mutant indicated that they are significantly (p=0.0012) larger compared to MTERF4 control. The assessment of mitochondrial function demonstrated that the maximal mitochondrial respiration may be reduced in MTERF4 mutant hiPSC-derived CMs compared to control (p=0.056). In known cardiovascular disease genes (NPPA, NPPB, GATA4, TNNT, MYL7, MYH7) associated with HCM, gene expression (qRT-PCR) were elevated. Preliminary data support our hypothesis through changes in CMs size, mitochondrial function, and transcriptional expression for one MTERF4 mutant clone and control. This approach has generated an in vitro tool to evaluate aspects diseases such as cardiomyopathy that can be used for diagnostic screening and therapy. Repeating our initial studies, and adding mechanistic studies, with additional MTERF4 mutant and control clones will further validate our hypothesis.

High throughput sequencing continues to uncover novel potentially pathogenic variants in cases of familial cardiomyopathy. Clinical interpretation of such variants is challenging, and hence definitive assignment of causality frequently remains unestablished. A case in point is provided by BAG3, a critical regulator of protein quality control in cardiomyocytes, which has been recurrently linked to dilated cardiomyopathy (DCM). Using theoretical and experimental approaches, we assessed the impact of a functionally-uncharacterized DCM-associated variant in BAG3 (c.1430G>A; p.Arg477His). Molecular dynamics simulations of wildtype and mutant forms of BAG3 suggested the residue substitution perturbs a crucial interaction between BAG3 and HSC70 by altering electrostatic field distribution, conformational rigidity and side-chain orientation. Acute and stable expression of the mutant BAG3 protein led to reduced HSC70 engagement and protein aggregation in HEK293 and HL1 cells. Using the CRISPR-Cas9 system, we then introduced the variant into a healthy induced pluripotent stem cell line line and upon differentiation into cardiomyocytes, observed protein aggregation and a sensitivity to pro-apoptotic stressors. In conclusion, disease-in-a-dish modeling was used to study the molecular and cellular consequences of a potentially pathogenic BAG3 variant. We define what we believe is the primary molecular lesion and propose ectopic protein quality control and potentiated stress sensitivity as possible pathogenic drivers and potential therapeutic entry points.

Background Interpreting genetic variants is one of the greatest challenges impeding analysis of rapidly increasing volumes of genomic data from patients. For example, SHROOM3 is an associated risk gene for CKD, yet causative mechanism(s) of SHROOM3 allele(s) are unknown.Methods We used our analytic pipeline that integrates genetic, computational, biochemical, CRISPR/Cas9 editing, molecular, and physiologic data to characterize coding and noncoding variants to study the human SHROOM3 risk locus for CKD.Results We identified a novel SHROOM3 transcriptional start site, which results in a shorter isoform lacking the PDZ domain and is regulated by a common noncoding sequence variant associated with CKD (rs17319721, allele frequency: 0.35). This variant disrupted allele binding to the transcription factor TCF7L2 in podocyte cell nuclear extracts and altered transcription levels of SHROOM3 in cultured cells, potentially through the loss of repressive looping between rs17319721 and the novel start site. Although common variant mechanisms are of high utility, sequencing is beginning to identify rare variants involved in disease; therefore, we used our biophysical tools to analyze an average of 112,849 individual human genome sequences for rare SHROOM3 missense variants, revealing 35 high-effect variants. The high-effect alleles include a coding variant (P1244L) previously associated with CKD (P=0.01, odds ratio=7.95; 95% CI, 1.53 to 41.46) that we find to be present in East Asian individuals at an allele frequency of 0.0027. We determined that P1244L attenuates the interaction of SHROOM3 with 14-3-3, suggesting alterations to the Hippo pathway, a known mediator of CKD.Conclusions These data demonstrate multiple new SHROOM3-dependent genetic/molecular mechanisms that likely affect CKD.

Hypertension is one of the most prevalent diseases worldwide and a major risk factor for renal failure and cardiovascular disease. The role of albuminuria, a common feature of hypertension and robust predictor of cardiorenal disorders, remains incompletely understood. The goal of this study was to investigate the mechanisms leading to albuminuria in the kidney of a rat model of hypertension, the Dahl salt-sensitive (SS) rat. To determine the relative contributions of the glomerulus and proximal tubule (PT) to albuminuria, we applied intravital two-photon-based imaging to investigate the complex renal physiological changes that occur during salt-induced hypertension. Following a high-salt diet, SS rats exhibited elevated blood pressure, increased glomerular sieving of albumin (GSCalb = 0.0686), relative permeability to albumin (+Δ16%), and impaired volume hemodynamics (-Δ14%). Serum albumin but not serum globulins or creatinine concentration was decreased (-0.54 g/dl), which was concomitant with increased filtration of albumin (3.7 vs. 0.8 g/day normal diet). Pathologically, hypertensive animals had significant tubular damage, as indicated by increased prevalence of granular casts, expansion and necrosis of PT epithelial cells (+Δ2.20 score/image), progressive augmentation of red blood cell velocity (+Δ269 µm/s) and micro vessel diameter (+Δ4.3 µm), and increased vascular injury (+Δ0.61 leakage/image). Therefore, development of salt-induced hypertension can be triggered by fast and progressive pathogenic remodeling of PT epithelia, which can be associated with changes in albumin handling. Collectively, these results indicate that both the glomerulus and the PT contribute to albuminuria, and dual treatment of glomerular filtration and albumin reabsorption may represent an effective treatment of salt-sensitive hypertension.

Genome editing in induced pluripotent stem cells is currently hampered by the laborious and expensive nature of identifying homology-directed repair (HDR)-modified cells. We present an approach where isolation of cells bearing a selectable, HDR-mediated editing event at one locus enriches for HDR-mediated edits at additional loci. This strategy, called co-targeting with selection, improves the probability of isolating cells bearing HDR-mediated variants and accelerates the production of disease models.

Alternative splicing contributes to gene expression dynamics in many tissues, yet its role in auditory development remains unclear. We performed whole-exome sequencing in individuals with sensorineural hearing loss (SNHL) and identified pathogenic mutations in Epithelial Splicing-Regulatory Protein 1 (ESRP1). Patient-derived induced pluripotent stem cells showed alternative splicing defects that were restored upon repair of an ESRP1 mutant allele. To determine how ESRP1 mutations cause hearing loss, we evaluated Esrp1-/- mouse embryos and uncovered alterations in cochlear morphogenesis, auditory hair cell differentiation, and cell fate specification. Transcriptome analysis revealed impaired expression and splicing of genes with essential roles in cochlea development and auditory function. Aberrant splicing of Fgfr2 blocked stria vascularis formation due to erroneous ligand usage, which was corrected by reducing Fgf9 gene dosage. These findings implicate mutations in ESRP1 as a cause of SNHL and demonstrate the complex interplay between alternative splicing, inner ear development, and auditory function.

Mitochondrial dysfunction contributes to myriad monogenic and complex pathologies. To understand the underlying mechanisms, it is essential to define the full complement of proteins that modulate mitochondrial function. To identify such proteins, we performed a meta-analysis of publicly available gene expression data. Gene co-expression analysis of a large and heterogeneous compendium of microarray data nominated a sub-population of transcripts that whilst highly correlated with known mitochondrial protein-encoding transcripts (MPETs), are not themselves recognized as generating proteins either localized to the mitochondrion or pertinent to functions therein. To focus the analysis on a medically-important condition with a strong yet incompletely understood mitochondrial component, candidates were cross-referenced with an MPET-enriched module independently generated via genome-wide co-expression network analysis of a human heart failure gene expression dataset. The strongest uncharacterized candidate in the analysis was Leucine Rich Repeat Containing 2 (LRRC2). LRRC2 was found to be localized to the mitochondria in human cells and transcriptionally-regulated by the mitochondrial master regulator Pgc-1α. We report that Lrrc2 transcript abundance correlates with that of β-MHC, a canonical marker of cardiac hypertrophy in humans and experimentally demonstrated an elevation in Lrrc2 transcript in in vitro and in vivo rodent models of cardiac hypertrophy as well as in patients with dilated cardiomyopathy. RNAi-mediated Lrrc2 knockdown in a rat-derived cardiomyocyte cell line resulted in enhanced expression of canonical hypertrophic biomarkers as well as increased mitochondrial mass in the context of increased Pgc-1α expression. In conclusion, our meta-analysis represents a simple yet powerful springboard for the nomination of putative mitochondrially-pertinent proteins relevant to cardiac function and enabled the identification of LRRC2 as a novel mitochondrially-relevant protein and regulator of the hypertrophic response.

Shortly after the discovery of endothelial progenitor cells (EPCs) in 1997, many clinical trials were conducted using EPCs as a cellular based therapy with the goal of restoring damaged organ function by inducing growth of new blood vessels (angiogenesis). Results were disappointing, largely because the cellular and molecular mechanisms of EPC-induced angiogenesis were not clearly understood. Following injection, EPCs must migrate to the target tissue and engraft prior to induction of angiogenesis. In this study EPC migration was investigated in response to tumor necrosis factor α (TNFα), a pro-inflammatory cytokine, to test the hypothesis that organ damage observed in ischemic diseases induces an inflammatory signal that is important for EPC homing. In this study, EPC migration and incorporation were modeled in vitro using a coculture assay where TNFα treated EPCs were tracked while migrating toward vessel-like structures. It was found that TNFα treatment of EPCs increased migration and incorporation into vessel-like structures. Using a combination of genomic and proteomic approaches, NF-kB mediated upregulation of CADM1 was identified as a mechanism of TNFα induced migration. Inhibition of NF-kB or CADM1 significantly decreased migration of EPCs in vitro suggesting a role for TNFα signaling in EPC homing during tissue repair. Stem Cells 2016;34:1922-1933.

Hypertension is one of the most prevalent diseases worldwide, and is a major risk factor for developing albuminuria, renal failure and cardiovascular diseases. The goal of this study was to investigate the mechanisms leading to albuminuria in the kidney of a canonical rat model of hypertension, the Dahl salt-sensitive (SS) rat. To determine the relative contributions of the glomerulus and proximal tubule (PT) to albuminuria, we applied intravital two-photon-based imaging to investigate complex changes in renal function that occur during salt-induced hypertension. Following a high salt (HS) diet, SS rats exhibited elevated blood pressure, accompanied by increased glomerular sieving of albumin (GSC alb =0.0686) and decreased serum albumin (-Δ0.54 g/dL), which corresponded to increased daily filtered albumin up to 3.7 vs 0.8 g at normal diet. Pathologically, hypertensive animals had significant tubular damage as indicated by increased prevalence of granular casts (+Δ2.20 casts/image), dilation/expansion and necrosis of PT epithelial cells, increased vascular injury (+Δ0.61 leakage/image), and progressive inflammation. HS diet significantly reduced total PT uptake of albumin, which also coincided with reduced transcellular transport of albumin back into circulation. Collectively, these results indicate that both the glomerulus and the proximal tubule contribute to albuminuria and dual treatment of glomerular filtration and albumin reabsorption may represent an effective treatment of salt-sensitive hypertension.