Characterization of a robust mouse model of heart failure with preserved ejection fraction.

Heart failure (HF) is a leading cause of morbidity and mortality particularly in older adults and patients with multiple metabolic comorbidities. Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome with multisystem organ dysfunction in which patients develop symptoms of HF as a result of high left ventricular (LV) diastolic pressure in the context of normal or near normal LV ejection fraction (LVEF; ≥50%). Challenges to create and reproduce a robust rodent phenotype that recapitulates the multiple comorbidities that exist in this syndrome explain the presence of various animal models that fail to satisfy all the criteria of HFpEF. Using a continuous infusion of angiotensin II and phenylephrine (ANG II/PE), we demonstrate a strong HFpEF phenotype satisfying major clinically relevant manifestations and criteria of this pathology, including exercise intolerance, pulmonary edema, concentric myocardial hypertrophy, diastolic dysfunction, histological signs of microvascular impairment, and fibrosis. Conventional echocardiographic analysis of diastolic dysfunction identified early stages of HFpEF development and speckle tracking echocardiography analysis including the left atrium (LA) identified strain abnormalities indicative of contraction-relaxation cycle impairment. Diastolic dysfunction was validated by retrograde cardiac catheterization and analysis of LV end-diastolic pressure (LVEDP). Among mice that developed HFpEF, two major subgroups were identified with predominantly perivascular fibrosis and interstitial myocardial fibrosis. In addition to major phenotypic criteria of HFpEF that were evident at early stages of this model (3 and 10 days), accompanying RNAseq data demonstrate activation of pathways associated with myocardial metabolic changes, inflammation, activation of extracellular matrix (ECM) deposition, microvascular rarefaction, and pressure- and volume-related myocardial stress.NEW & NOTEWORTHY Heart failure with preserved ejection fraction (HFpEF) is an emerging epidemic affecting up to half of patients with heart failure. Here we used a chronic angiotensin II/phenylephrine (ANG II/PE) infusion model and instituted an updated algorithm for HFpEF assessment. Given the simplicity in generating this model, it may become a useful tool for investigating pathogenic mechanisms, identification of diagnostic markers, and for drug discovery aimed at both prevention and treatment of HFpEF.

Necrotic cardiac myocytes skew macrophage polarization towards a classically activated phenotype.

Necrotic and dying cells release damage-associated molecular patterns (DAMPs) that can initiate sterile inflammatory responses in the heart. Although macrophages are essential for myocardial repair and regeneration, the effect of DAMPs on macrophage activation remains unclear. To address this gap in knowledge we studied the effect of necrotic cardiac myocyte extracts on primary peritoneal macrophage (PPM) cultures in vitro. We first performed unbiased transcriptomic profiling with RNA-sequencing of PPMs cultured for up to 72 hours in the presence and absence of: 1) necrotic cell extracts (NCEs) from necrotic cardiac myocytes in order to mimic the release of DAMPs; 2) lipopolysaccharide (LPS), which is known to polarize macrophages towards a classically activated phenotype and 3) Interleukin-4 (IL-4), which is known to promote polarization of macrophages towards an alternatively activated phenotype. NCEs provoke changes in differential gene expression (DEGs) that had considerable overlap with LPS-induced changes, suggesting that NCEs promote macrophage polarization towards a classically activated phenotype. Treating NCEs with proteinase-K abolished the effects of NCEs on macrophage activation, whereas NCE treatment with DNase and RNase did not affect macrophage activation. Stimulation of macrophage cultures with NCEs and LPS resulted in a significant increase in macrophage phagocytosis and interleukin-1ß secretion, whereas treatment with IL-4 had no significant effect on phagocytosis and interleukin-1ß. Taken together, our findings suggest that proteins released from necrotic cardiac myocytes are sufficient to skew the polarization of macrophages towards a classically activated phenotype.

Development of a long-acting relaxin analogue, LY3540378, for treatment of chronic heart failure.

BACKGROUND AND PURPOSE: Chronic heart failure, a progressive disease with limited treatment options currently available, especially in heart failure with preserved ejection fraction (HFpEF), represents an unmet medical need as well as an economic burden. The development of a novel therapeutic to slow or reverse disease progression would be highly impactful to patients and society. Relaxin-2 (relaxin) is a human hormone regulating cardiovascular, renal, and pulmonary adaptations during pregnancy. A short-acting recombinant relaxin, Serelaxin, demonstrated short-term heart failure symptom relief and biomarker improvement in acute heart failure trials. Here, we present the development of a long-acting relaxin analogue to be tested in the treatment of chronic heart failure. EXPERIMENTAL APPROACH: LY3540378 is a long-acting protein therapeutic composed of a human relaxin analogue and a serum albumin-binding VHH domain. KEY RESULTS: LY3540378 is a potent agonist of the relaxin family peptide receptor 1 (RXFP1) and maintains selectivity against RXFP2/3/4 comparable to native relaxin. The half-life of LY3540378 in preclinical species is extended through high affinity binding of the albumin-binding VHH domain to serum albumin. When tested in a single dose administration, LY3540378 elicited relaxin-mediated pharmacodynamic responses, such as reduced serum osmolality and increased renal blood flow in rats. In an isoproterenol-induced cardiac hypertrophy mouse model, treatment with LY3540378 significantly reduced cardiac hypertrophy and improved isovolumetric relaxation time. In a monkey cardiovascular safety study, there were no adverse observations from administration of LY3540378. CONCLUSION AND IMPLICATIONS: LY3540378 demonstrated to be a suitable clinical development candidate, and is progressing in clinical trials.

Selective sweep for an enhancer involucrin allele identifies skin barrier adaptation out of Africa.

The genetic modules that contribute to human evolution are poorly understood. Here we investigate positive selection in the Epidermal Differentiation Complex locus for skin barrier adaptation in diverse HapMap human populations (CEU, JPT/CHB, and YRI). Using Composite of Multiple Signals and iSAFE, we identify selective sweeps for LCE1A-SMCP and involucrin (IVL) haplotypes associated with human migration out-of-Africa, reaching near fixation in European populations. CEU-IVL is associated with increased IVL expression and a known epidermis-specific enhancer. CRISPR/Cas9 deletion of the orthologous mouse enhancer in vivo reveals a functional requirement for the enhancer to regulate Ivl expression in cis. Reporter assays confirm increased regulatory and additive enhancer effects of CEU-specific polymorphisms identified at predicted IRF1 and NFIC binding sites in the IVL enhancer (rs4845327) and its promoter (rs1854779). Together, our results identify a selective sweep for a cis regulatory module for CEU-IVL, highlighting human skin barrier evolution for increased IVL expression out-of-Africa.

Transcriptomic and Functional Analyses of Mitochondrial Dysfunction in Pressure Overload-Induced Right Ventricular Failure.

Background In complex congenital heart disease patients such as those with tetralogy of Fallot, the right ventricle (RV) is subject to pressure overload, leading to RV hypertrophy and eventually RV failure. The mechanisms that promote the transition from stable RV hypertrophy to RV failure are unknown. We evaluated the role of mitochondrial bioenergetics in the development of RV failure. Methods and Results We created a murine model of RV pressure overload by pulmonary artery banding and compared with sham-operated controls. Gene expression by RNA-sequencing, oxidative stress, mitochondrial respiration, dynamics, and structure were assessed in pressure overload-induced RV failure. RV failure was characterized by decreased expression of electron transport chain genes and mitochondrial antioxidant genes (aldehyde dehydrogenase 2 and superoxide dismutase 2) and increased expression of oxidant stress markers (heme oxygenase, 4-hydroxynonenal). The activities of all electron transport chain complexes decreased with RV hypertrophy and further with RV failure (oxidative phosphorylation: sham 552.3±43.07 versus RV hypertrophy 334.3±30.65 versus RV failure 165.4±36.72 pmol/(s×mL), P<0.0001). Mitochondrial fission protein DRP1 (dynamin 1-like) trended toward an increase, while MFF (mitochondrial fission factor) decreased and fusion protein OPA1 (mitochondrial dynamin like GTPase) decreased. In contrast, transcription of electron transport chain genes increased in the left ventricle of RV failure. Conclusions Pressure overload-induced RV failure is characterized by decreased transcription and activity of electron transport chain complexes and increased oxidative stress which are associated with decreased energy generation. An improved understanding of the complex processes of energy generation could aid in developing novel therapies to mitigate mitochondrial dysfunction and delay the onset of RV failure.

The Mechanism of High-Output Cardiac Hypertrophy Arising From Potassium Channel Gain-of-Function in Cantú Syndrome.

Dramatic cardiomegaly arising from gain-of-function (GoF) mutations in the ATP-sensitive potassium (KATP) channels genes, ABCC9 and KCNJ8, is a characteristic feature of Cantú syndrome (CS). How potassium channel over-activity results in cardiac hypertrophy, as well as the long-term consequences of cardiovascular remodeling in CS, is unknown. Using genome-edited mouse models of CS, we therefore sought to dissect the pathophysiological mechanisms linking KATP channel GoF to cardiac remodeling. We demonstrate that chronic reduction of systemic vascular resistance in CS is accompanied by elevated renin-angiotensin signaling, which drives cardiac enlargement and blood volume expansion. Cardiac enlargement in CS results in elevation of basal cardiac output, which is preserved in aging. However, the cardiac remodeling includes altered gene expression patterns that are associated with pathological hypertrophy and are accompanied by decreased exercise tolerance, suggestive of reduced cardiac reserve. Our results identify a high-output cardiac hypertrophy phenotype in CS which is etiologically and mechanistically distinct from other myocardial hypertrophies, and which exhibits key features of high-output heart failure (HOHF). We propose that CS is a genetically-defined HOHF disorder and that decreased vascular smooth muscle excitability is a novel mechanism for HOHF pathogenesis.

Identification of Genes and Pathways Regulated by Lamin A in Heart.

Background Mutations in the LMNA gene, encoding LMNA (lamin A/C), causes distinct disorders, including dilated cardiomyopathies, collectively referred to as laminopathies. The genes (coding and noncoding) and regulatory pathways controlled by LMNA in the heart are not completely defined. Methods and Results We analyzed cardiac transcriptome from wild-type, loss-of-function (Lmna-/-), and gain-of-function (Lmna-/- injected with adeno-associated virus serotype 9 expressing LMNA) mice with normal cardiac function. Deletion of Lmna (Lmna-/-) led to differential expression of 2193 coding and 629 long noncoding RNA genes in the heart (q<0.05). Re-expression of LMNA in the Lmna-/- mouse heart, completely rescued 501 coding and 208 non-coding and partially rescued 1862 coding and 607 lncRNA genes. Pathway analysis of differentially expressed genes predicted activation of transcriptional regulators lysine-specific demethylase 5A, lysine-specific demethylase 5B, tumor protein 53, and suppression of retinoblastoma 1, paired-like homeodomain 2, and melanocyte-inducing transcription factor, which were completely or partially rescued upon reexpression of LMNA. Furthermore, lysine-specific demethylase 5A and 5B protein levels were increased in the Lmna-/- hearts and were partially rescued upon LMNA reexpression. Analysis of biological function for rescued genes identified activation of tumor necrosis factor-α, epithelial to mesenchymal transition, and suppression of the oxidative phosphorylation pathway upon Lmna deletion and their restoration upon LMNA reintroduction in the heart. Restoration of the gene expression and transcriptional regulators in the heart was associated with improved cardiac function and increased survival of the Lmna-/- mice. Conclusions The findings identify LMNA-regulated cardiac genes and their upstream transcriptional regulators in the heart and implicate lysine-specific demethylase 5A and B as epigenetic regulators of a subset of the dysregulated genes in laminopathies.

BET bromodomain inhibition attenuates cardiac phenotype in myocyte-specific lamin A/C-deficient mice.

Mutation in the LMNA gene, encoding lamin A/C, causes a diverse group of diseases called laminopathies. Cardiac involvement is the major cause of death and manifests as dilated cardiomyopathy, heart failure, arrhythmias, and sudden death. There is no specific therapy for LMNA-associated cardiomyopathy. We report that deletion of Lmna in cardiomyocytes in mice leads to severe cardiac dysfunction, conduction defect, ventricular arrhythmias, fibrosis, apoptosis, and premature death within 4 weeks. The phenotype is similar to LMNA-associated cardiomyopathy in humans. RNA sequencing, performed before the onset of cardiac dysfunction, led to identification of 2338 differentially expressed genes (DEGs) in Lmna-deleted cardiomyocytes. DEGs predicted activation of bromodomain-containing protein 4 (BRD4), a regulator of chromatin-associated proteins and transcription factors, which was confirmed by complementary approaches, including chromatin immunoprecipitation sequencing. Daily injection of JQ1, a specific BET bromodomain inhibitor, partially reversed the DEGs, including those encoding secretome; improved cardiac function; abrogated cardiac arrhythmias, fibrosis, and apoptosis; and prolonged the median survival time 2-fold in the myocyte-specific Lmna-deleted mice. The findings highlight the important role of LMNA in cardiomyocytes and identify BET bromodomain inhibition as a potential therapeutic target in LMNA-associated cardiomyopathy, for which there is no specific effective therapy.

TFEB activation in macrophages attenuates postmyocardial infarction ventricular dysfunction independently of ATG5-mediated autophagy.

Lysosomes are at the epicenter of cellular processes critical for inflammasome activation in macrophages. Inflammasome activation and IL-1ß secretion are implicated in myocardial infarction (MI) and resultant heart failure; however, little is known about how macrophage lysosomes regulate these processes. In mice subjected to cardiac ischemia/reperfusion (IR) injury and humans with ischemic cardiomyopathy, we observed evidence of lysosomal impairment in macrophages. Inducible macrophage-specific overexpression of transcription factor EB (TFEB), a master regulator of lysosome biogenesis (Mϕ-TFEB), attenuated postinfarction remodeling, decreased abundance of proinflammatory macrophages, and reduced levels of myocardial IL-1ß compared with controls. Surprisingly, neither inflammasome suppression nor Mϕ-TFEB-mediated attenuation of postinfarction myocardial dysfunction required intact ATG5-dependent macroautophagy (hereafter termed "autophagy"). RNA-seq of flow-sorted macrophages postinfarction revealed that Mϕ-TFEB upregulated key targets involved in lysosomal lipid metabolism. Specifically, inhibition of the TFEB target, lysosomal acid lipase, in vivo abrogated the beneficial effect of Mϕ-TFEB on postinfarction ventricular function. Thus, TFEB reprograms macrophage lysosomal lipid metabolism to attenuate remodeling after IR, suggesting an alternative paradigm whereby lysosome function affects inflammation.

Immunomodulatory role of non-neuronal cholinergic signaling in myocardial injury.

Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the non-neuronal cholinergic system is not well understood. To address the immunomodulatory role of the non-neuronal cholinergic system in the heart we used a previously validated diphtheria toxin (DT)-induced cardiomyocyte ablation model (Rosa26-DTMlc2v-Cre mice). DT-injected Rosa26-DTMlc2v-Cre mice were treated with diluent or Pyridostigmine Bromide (PYR), a reversible cholinesterase inhibitor. PYR treatment resulted in increased survival and decreased numbers of MHC-IIlowCCR2+ macrophages in DT-injected Rosa26-DTMlc2v-Cre mice compared to diluent treated Rosa26-DTMlc2v-Cre mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DTMlc2v-Cre mice with a transgenic mouse line (Chat-ChR2) that constitutively overexpresses the vesicular acetylcholine transporter (VAChT) resulted in decreased expression of Ccl2/7 mRNA and decreased numbers of CD68+ cells in DT-injured Rosa26-DTMlc2v-Cre/Chat-ChR2 mouse hearts, consistent with the pharmacologic studies with PYR. In vitro studies with cultures of LPS-stimulated peritoneal macrophages revealed a concentration-dependent reduction in CCL2 secretion following stimulation with ACh, nicotine and muscarine. Viewed together, these findings reveal a previously unappreciated immunomodulatory role for the non-neuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury.

Simple nutrients bypass the requirement for HLH-30 in coupling lysosomal nutrient sensing to survival.

Lysosomes are ubiquitous acidified organelles that degrade intracellular and extracellular material trafficked via multiple pathways. Lysosomes also sense cellular nutrient levels to regulate target of rapamycin (TOR) kinase, a signaling enzyme that drives growth and suppresses activity of the MiT/TFE family of transcription factors that control biogenesis of lysosomes. In this study, we subjected worms lacking basic helix-loop-helix transcription factor 30 (hlh-30), the Caenorhabditis elegans MiT/TFE ortholog, to starvation followed by refeeding to understand how this pathway regulates survival with variable nutrient supply. Loss of HLH-30 markedly impaired survival in starved larval worms and recovery upon refeeding bacteria. Remarkably, provision of simple nutrients in a completely defined medium (C. elegans maintenance medium [CeMM]), specifically glucose and linoleic acid, restored lysosomal acidification, TOR activation, and survival with refeeding despite the absence of HLH-30. Worms deficient in lysosomal lipase 2 (lipl-2), a lysosomal enzyme that is transcriptionally up-regulated in starvation in an HLH-30-dependent manner, also demonstrated increased mortality with starvation-refeeding that was partially rescued with glucose, suggesting a critical role for LIPL-2 in lipid metabolism under starvation. CeMM induced transcription of vacuolar proton pump subunits in hlh-30 mutant worms, and knockdown of vacuolar H+-ATPase 12 (vha-12) and its upstream regulator, nuclear hormone receptor 31 (nhr-31), abolished the rescue with CeMM. Loss of Ras-related GTP binding protein C homolog 1 RAGC-1, the ortholog for mammalian RagC/D GTPases, conferred starvation-refeeding lethality, and RAGC-1 overexpression was sufficient to rescue starved hlh-30 mutant worms, demonstrating a critical need for TOR activation with refeeding. These results show that HLH-30 activation is critical for sustaining survival during starvation-refeeding stress via regulating TOR. Glucose and linoleic acid bypass the requirement for HLH-30 in coupling lysosome nutrient sensing to survival.

Multiomic approaches to delineate the pathogenesis of cardiac disease.

PURPOSE OF REVIEW: Comprehensive analyses of the genome, transcriptome, proteome and metabolome are instrumental in identifying biomarkers of disease, to gain insight into mechanisms underlying the development of cardiovascular disease, and show promise for better stratifying patients according to disease subtypes. This review highlights recent 'omics' studies, including integration of multiple 'omics' that have advanced mechanistic understanding and diagnosis in humans and animal models. RECENT FINDINGS: Transcriptome-based discovery continues to be a primary method to obtain data for hypothesis generation and the understanding of disease pathogenesis has been enhanced by single cell-based methods capable of revealing heterogeneity in cellular responses. Advances in proteome coverage and quantitation of individual protein species, together with enhanced methods for detecting posttranslational modifications, have improved discovery of protein-based biomarkers. SUMMARY: High-throughput assays capable of quantitating the vast majority of any particular type of biomolecule within a tissue sample, isolated cells or plasma are now available. In order to make best use of the large amount of data that can be generated on given molecule types, as well as their interrelationships in disease, continued development of pattern-recognition algorithms ('machine learning') will be required and the subclassification of disease that is made possible by such algorithms will be likely to inform clinical practice, and vice versa.

Genomic Reorganization of Lamin-Associated Domains in Cardiac Myocytes Is Associated With Differential Gene Expression and DNA Methylation in Human Dilated Cardiomyopathy.

RATIONALE: LMNA (Lamin A/C), a nuclear membrane protein, interacts with genome through lamin-associated domains (LADs) and regulates gene expression. Mutations in the LMNA gene cause a diverse array of diseases, including dilated cardiomyopathy (DCM). DCM is the leading cause of death in laminopathies. OBJECTIVE: To identify LADs and characterize their associations with CpG methylation and gene expression in human cardiac myocytes in DCM. METHODS AND RESULTS: LMNA chromatin immunoprecipitation-sequencing, reduced representative bisulfite sequencing, and RNA-sequencing were performed in 5 control and 5 LMNA-associated DCM hearts. LADs were identified using enriched domain detector program. Genome-wide 331±77 LADs with an average size of 2.1±1.5 Mbp were identified in control human cardiac myocytes. LADs encompassed ≈20% of the genome and were predominantly located in the heterochromatin and less so in the promoter and actively transcribed regions. LADs were redistributed in DCM as evidenced by a gain of 520 and loss of 149 genomic regions. Approximately, 4500 coding genes and 800 long noncoding RNAs, whose levels correlated with the transcript levels of coding genes in cis, were differentially expressed in DCM. TP53 (tumor protein 53) was the most prominent among the dysregulated pathways. CpG sites were predominantly hypomethylated genome-wide in controls and DCM hearts, but overall CpG methylation was increased in DCM. LADs were associated with increased CpG methylation and suppressed gene expression. Integrated analysis identified genes whose expressions were regulated by LADs or CpG methylation, or by both, the latter pertained to genes involved in cell death, cell cycle, and metabolic regulation. CONCLUSIONS: LADs encompass ≈20% of the genome in human cardiac myocytes comprised several hundred coding and noncoding genes. LADs are redistributed in LMNA-associated DCM in association with markedly altered CpG methylation and gene expression. Thus, LADs through genomic alterations contribute to the pathogenesis of DCM in laminopathies.

Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

Lipin 1 regulates glycerolipid homeostasis by acting as a phosphatidic acid phosphohydrolase (PAP) enzyme in the triglyceride-synthesis pathway and by regulating transcription factor activity. Mutations in human lipin 1 are a common cause of recurrent rhabdomyolysis in children. Mice with constitutive whole-body lipin 1 deficiency have been used to examine mechanisms connecting lipin 1 deficiency to myocyte injury. However, that mouse model is confounded by lipodystrophy not phenocopied in people. Herein, 2 muscle-specific mouse models were studied: 1) Lpin1 exon 3 and 4 deletion, resulting in a hypomorphic protein without PAP activity, but which preserved transcriptional coregulatory function; and 2) Lpin1 exon 7 deletion, resulting in total protein loss. In both models, skeletal muscles exhibited a chronic myopathy with ongoing muscle fiber necrosis and regeneration and accumulation of phosphatidic acid and, paradoxically, diacylglycerol. Additionally, lipin 1-deficient mice had abundant, but abnormal, mitochondria likely because of impaired autophagy. Finally, these mice exhibited increased plasma creatine kinase following exhaustive exercise when unfed. These data suggest that mice lacking lipin 1-mediated PAP activity in skeletal muscle may serve as a model for determining the mechanisms by which lipin 1 deficiency leads to myocyte injury and for testing potential therapeutic approaches.-Schweitzer, G. G., Collier, S. L., Chen, Z., McCommis, K. S., Pittman, S. K., Yoshino, J., Matkovich, S. J., Hsu, F.-F., Chrast, R., Eaton, J. M., Harris, T. E., Weihl, C. C., Finck, B. N. Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

Regional Differences in mRNA and lncRNA Expression Profiles in Non-Failing Human Atria and Ventricles.

The four chambers of the human heart play distinct roles in the maintenance of normal cardiac function, and are differentially affected by inherited/acquired cardiovascular disease. To probe the molecular determinants of these functional differences, we examined mRNA and lncRNA expression profiles in the left (LA) and right (RA) atria, the left (LV) and right (RV) ventricles, and the interventricular septum (IVS) of non-failing human hearts (N = 8). Analysis of paired atrial and ventricular samples (n = 40) identified 5,747 mRNAs and 2,794 lncRNAs that were differentially (>1.5 fold; FDR < 0.05) expressed. The largest differences were observed in comparisons between the atrial (RA/LA) and ventricular (RV/LV/IVS) samples. In every case (e.g., LA vs LV, LA vs RV, etc.), >2,300 mRNAs and >1,200 lncRNAs, corresponding to 17-28% of the total transcripts, were differentially expressed. Heterogeneities in mRNA/lncRNA expression profiles in the LA and RA, as well as in the LV, RV and IVS, were also revealed, although the numbers of differentially expressed transcripts were substantially smaller. Gender differences in mRNA and lncRNA expression profiles were also evident in non-failing human atria and ventricles. Gene ontology classification of differentially expressed gene sets revealed chamber-specific enrichment of numerous signaling pathways.

Cardiovascular consequences of KATP overactivity in Cantu syndrome.

Cantu syndrome (CS) is characterized by multiple vascular and cardiac abnormalities including vascular dilation and tortuosity, systemic hypotension, and cardiomegaly. The disorder is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits. However, there is little understanding of the link between molecular dysfunction and the complex pathophysiology observed, and there is no known treatment, in large part due to the lack of appropriate preclinical disease models in which to test therapies. Notably, expression of Kir6.1 and SUR2 does not fully overlap, and the relative contribution of KATP GOF in various cardiovascular tissues remains to be elucidated. To investigate pathophysiologic mechanisms in CS we have used CRISPR/Cas9 engineering to introduce CS-associated SUR2[A478V] and Kir6.1[V65M] mutations to the equivalent endogenous loci in mice. Mirroring human CS, both of these animals exhibit low systemic blood pressure and dilated, compliant blood vessels, as well dramatic cardiac enlargement, the effects being more severe in V65M animals than in A478V animals. In both animals, whole-cell patch-clamp recordings reveal enhanced basal KATP conductance in vascular smooth muscle, explaining vasodilation and lower blood pressure, and demonstrating a cardinal role for smooth muscle KATP dysfunction in CS etiology. Echocardiography confirms in situ cardiac enlargement and increased cardiac output in both animals. Patch-clamp recordings reveal reduced ATP sensitivity of ventricular myocyte KATP channels in A478V, but normal ATP sensitivity in V65M, suggesting that cardiac remodeling occurs secondary to KATP overactivity outside of the heart. These SUR2[A478V] and Kir6.1[V65M] animals thus reiterate the key cardiovascular features seen in human CS. They establish the molecular basis of the pathophysiological consequences of reduced smooth muscle excitability resulting from SUR2/Kir6.1-dependent KATP GOF, and provide a validated animal model in which to examine potential therapeutic approaches to treating CS.

G-protein receptor kinases 2, 5 and 6 redundantly modulate Smoothened-GATA transcriptional crosstalk in fetal mouse hearts.

G-protein receptor kinases (GRKs) regulate adult hearts by modulating inotropic, chronotropic and hypertrophic signaling of 7-transmembrane spanning neurohormone receptors. GRK-mediated desensitization and downregulation of ß-adrenergic receptors has been implicated in adult heart failure; GRKs are therefore a promising therapeutic target. However, germ-line (but not cardiomyocyte-specific) GRK2 deletion provoked lethal fetal heart defects, suggesting an unexplained role for GRKs in heart development. Here we undertook to better understand the consequences of GRK deficiency on fetal heart development by creating mice and cultured murine embryonic fibroblasts (MEFs) having floxed GRK2 and GRK5 alleles on the GRK6 null background; simultaneous conditional deletion of these 3 GRK genes was achieved using Nkx2-5 Cre or adenoviral Cre, respectively. Phenotypes were related to GRK-modulated gene expression using whole-transcriptome RNA sequencing, RT-qPCR, and luciferase reporter assays. In cultured MEFs the atypical 7-transmembrane spanning protein and GRK2 substrate Smoothened (Smo) stimulated Gli-mediated transcriptional activity, which was interrupted by deleting GRK2/5/6. Mice with Nkx2-5 Cre mediated GRK2/5/6 ablation died between E15.5 and E16.5, whereas mice expressing any one of these 3 GRKs (i.e. GRK2/5, GRK2/6 or GRK5/6 deleted) were developmentally normal. GRK2/5/6 triple null mice at E14.5 exhibited left and right heart blood intermixing through single atrioventricular valves or large membranous ventricular septal defects. Hedgehog and GATA pathway gene expression promoted by Smo/Gli was suppressed in GRK2/5/6 deficient fetal hearts and MEFs. These data indicate that GRK2, GRK5 and GRK6 redundantly modulate Smo-GATA crosstalk in fetal mouse hearts, orchestrating transcriptional pathways previously linked to clinical and experimental atrioventricular canal defects. GRK modulation of Smo reflects convergence of conventional neurohormonal signaling and transcriptional regulation pathways, comprising an unanticipated mechanism for spatiotemporal orchestration of developmental gene expression in the heart.

Modulation of subsets of cardiac B lymphocytes improves cardiac function after acute injury.

Despite the long-standing recognition that the immune response to acute myocardial injury contributes to adverse left ventricular (LV) remodeling, it has not been possible to effectively target this clinically. Using 2 different in vivo models of acute myocardial injury, we show that pirfenidone confers beneficial effects in the murine heart through an unexpected mechanism that depends on cardiac B lymphocytes. Naive hearts contained a large population of CD19+CD11b-CD23-CD21-IgD+IgMlo lymphocytes, and 2 smaller populations of CD19+CD11b+ B1a and B1b cells. In response to tissue injury, there was an increase in neutrophils, monocytes, macrophages, as well as an increase in CD19+ CD11b- B lymphocytes. Treatment with pirfenidone had no effect on the number of neutrophils, monocytes, or macrophages, but decreased CD19+CD11b- lymphocytes. B cell depletion abrogated the beneficial effects of pirfenidone. In vitro studies demonstrated that stimulation with lipopolysaccharide and extracts from necrotic cells activated CD19+ lymphocytes through a TIRAP-dependent pathway. Treatment with pirfenidone attenuated this activation of B cells. These findings reveal a previously unappreciated complexity of myocardial B lymphocytes within the inflammatory infiltrate triggered by cardiac injury and suggest that pirfenidone exerts beneficial effects in the heart through a unique mechanism that involves modulation of cardiac B lymphocytes.

Load-Dependent Changes in Left Ventricular Structure and Function in a Pathophysiologically Relevant Murine Model of Reversible Heart Failure.

BACKGROUND: To better understand reverse left ventricular (LV) remodeling, we developed a murine model wherein mice develop LV remodeling after transverse aortic constriction (TAC) and a small apical myocardial infarct (MI) and undergo reverse LV remodeling after removal of the aortic band. METHODS AND RESULTS: Mice studied were subjected to sham (n=6) surgery or TAC+MI (n=12). Two weeks post-TAC+MI, 1 group underwent debanding (referred to as heart failure debanding [HF-DB] mice; n=6), whereas the aortic band remained in a second group (heart failure [HF] group; n=6). LV remodeling was evaluated by 2D echocardiography at 1 day, 2 weeks and 6 weeks post-TAC+MI. The hearts were analyzed by transcriptional profiling at 4 and 6 weeks and histologically at 6 weeks. Debanding normalized LV volumes, LV mass, and cardiac myocyte hypertrophy at 6 weeks in HF-DB mice, with no difference in myofibrillar collagen in the HF and HF-DB mice. LV ejection fraction and radial strain improved after debanding; however, both remained decreased in the HF-DB mice relative to sham and were not different from HF mice at 6 weeks. Hemodynamic unloading in the HF-DB mice was accompanied by a 35% normalization of the HF genes at 2 weeks and 80% of the HF genes at 4 weeks. CONCLUSIONS: Hemodynamic unloading of a pathophysiologically relevant mouse model of HF results in normalization of LV structure, incomplete recovery of LV function, and incomplete reversal of the HF transcriptional program. The HF-DB mouse model may provide novel insights into mechanisms of reverse LV remodeling.