A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria.

Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.

Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds.

Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.

Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation.

While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.

CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.

The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

Estimation of Stressed Blood Volume in Patients With Cardiogenic Shock From Acute Myocardial Infarction and Decompensated Heart Failure.

BACKGROUND: Sympathetically mediated redistribution of blood from the unstressed venous reservoir to the hemodynamically active stressed compartment is thought to contribute to congestion in cardiogenic shock (CS). We used a novel computational method to estimate stressed blood volume (SBV) in CS and assess its relationship with clinical outcomes. METHODS AND RESULTS: Hemodynamic parameters including estimated SBV (eSBV) were compared among patients from the Cardiogenic Shock Working Group registry with a complete set of hemodynamic data. eSBV was compared across shock etiologies (acute myocardial infarction and CS (AMI-CS) vs heart failure with CS (HF-CS), Society for Cardiovascular Angiography and Interventions stage, and between survivors and nonsurvivors. Among 528 patients with patients analyzed, the mean eSBV was 2423 mL/70 kg and increased with increasing Society for Cardiovascular Angiography and Interventions stage (B, 2029 mL/70 kg; C, 2305 mL/70 kg; D, 2496 mL/70 kg; E, 2707 mL/70 kg; P < .001). The eSBV was significantly greater among patients with HF-CS who died compared with survivors (2733 vs 2357 mL/70 kg; P < .001), whereas no significant difference was observed between outcome groups in AMI-CS (2501 mL/70 kg vs 2384 mL/70 kg; P = .19). CONCLUSIONS: eSBV is a novel integrated index of congestion which correlates with shock severity. eSBV was higher in patients with HF-CS who died; no difference was observed in patients with AMI-CS, suggesting that congestion may play a more significant role in the deterioration of patients with HF-CS.

Right Ventricular Dysfunction Is Common and Identifies Patients at Risk of Dying in Cardiogenic Shock.

BACKGROUND: Understanding the prognostic impact of right ventricular dysfunction (RVD) in cardiogenic shock (CS) is a key step toward rational diagnostic and treatment algorithms and improved outcomes. Using a large multicenter registry, we assessed (1) the association between hemodynamic markers of RVD and in-hospital mortality, (2) the predictive value of invasive hemodynamic assessment incorporating RV evaluation, and (3) the impact of RVD severity on survival in CS. METHODS AND RESULTS: Inpatients with CS owing to acute myocardial infarction (AMI) or heart failure (HF) between 2016 and 2019 were included. RV parameters (right atrial pressure, right atrial/pulmonary capillary wedge pressure [RA/PCWP], pulmonary artery pulsatility index [PAPI], and right ventricular stroke work index [RVSWI]) were assessed between survivors and nonsurvivors, and between etiology and SCAI stage subcohorts. Multivariable logistic regression analysis determined hemodynamic predictors of in-hospital mortality; the resulting models were compared with SCAI staging alone. Nonsurvivors had a significantly higher right atrial pressure and RA/PCWP and lower PAPI and RVSWI than survivors, consistent with more severe RVD. Compared with AMI, patients with HF had a significantly lower RA/PCWP (0.58 vs 0.66, P = .001) and a higher PAPI (2.71 vs 1.78, P < .001) and RVSWI (5.70 g-m/m2 vs 4.66 g-m/m2, P < .001), reflecting relatively preserved RV function. Paradoxically, multiple RVD parameters (PAPI, RVSWI) were associated with mortality in the HF but not the AMI cohort. RVD was more severe with advanced SCAI stage, although its prognostic value was progressively diluted in stages D and E. Multivariable modelling incorporating the RA/PCWP improved the predictive value of SCAI staging (area under the curve [AUC] 0.78 vs 0.73, P < .001), largely driven by patients with HF (AUC 0.82 vs 0.71, P < .001). CONCLUSIONS: RVD is associated with poor outcomes in CS, with key differences across etiology and shock severity. Further studies are needed to assess the usefulness of RVD assessment in guiding therapy.

Clinical Course of Patients in Cardiogenic Shock Stratified by Phenotype.

BACKGROUND: Cardiogenic shock (CS) patients remain at 30% to 60% in-hospital mortality despite therapeutic innovations. Heterogeneity of CS has complicated clinical trial design. Recently, 3 distinct CS phenotypes were identified in the CSWG (Cardiogenic Shock Working Group) registry version 1 (V1) and external cohorts: I, "noncongested;" II, "cardiorenal;" and III, "cardiometabolic" shock. OBJECTIVES: The aim was to confirm the external reproducibility of machine learning-based CS phenotypes and to define their clinical course. METHODS: The authors included 1,890 all-cause CS patients from the CSWG registry version 2. CS phenotypes were identified using the nearest centroids of the initially reported clusters. RESULTS: Phenotypes were retrospectively identified in 796 patients in version 2. In-hospital mortality rates in phenotypes I, II, III were 23%, 41%, 52%, respectively, comparable to the initially reported 21%, 45%, and 55% in V1. Phenotype-related demographic, hemodynamic, and metabolic features resembled those in V1. In addition, 58.8%, 45.7%, and 51.9% of patients in phenotypes I, II, and III received mechanical circulatory support, respectively (P = 0.013). Receiving mechanical circulatory support was associated with increased mortality in cardiorenal (OR: 1.82 [95% CI: 1.16-2.84]; P = 0.008) but not in noncongested or cardiometabolic CS (OR: 1.26 [95% CI: 0.64-2.47]; P = 0.51 and OR: 1.39 [95% CI: 0.86-2.25]; P = 0.18, respectively). Admission phenotypes II and III and admission Society for Cardiovascular Angiography and Interventions stage E were independently associated with increased mortality in multivariable logistic regression compared to noncongested "stage C" CS (P < 0.001). CONCLUSIONS: The findings support the universal applicability of these phenotypes using supervised machine learning. CS phenotypes may inform the design of future clinical trials and enable management algorithms tailored to a specific CS phenotype.

Clinical Presentation and In-Hospital Trajectory of Heart Failure and Cardiogenic Shock.

BACKGROUND: Heart failure-related cardiogenic shock (HF-CS) remains an understudied distinct clinical entity. OBJECTIVES: The authors sought to profile a large cohort of patients with HF-CS focused on practical application of the SCAI (Society for Cardiovascular Angiography and Interventions) staging system to define baseline and maximal shock severity, in-hospital management with acute mechanical circulatory support (AMCS), and clinical outcomes. METHODS: The Cardiogenic Shock Working Group registry includes patients with CS, regardless of etiology, from 17 clinical sites enrolled between 2016 and 2020. Patients with HF-CS (non-acute myocardial infarction) were analyzed and classified based on clinical presentation, outcomes at discharge, and shock severity defined by SCAI stages. RESULTS: A total of 1,767 patients with HF-CS were included, of whom 349 (19.8%) had de novo HF-CS (DNHF-CS). Patients were more likely to present in SCAI stage C or D and achieve maximum SCAI stage D. Patients with DNHF-CS were more likely to experience in-hospital death and in- and out-of-hospital cardiac arrest, and they escalated more rapidly to a maximum achieved SCAI stage, compared to patients with acute-on-chronic HF-CS. In-hospital cardiac arrest was associated with greater in-hospital death regardless of clinical presentation (de novo: 63% vs 21%; acute-on-chronic HF-CS: 65% vs 17%; both P < 0.001). Forty-five percent of HF-CS patients were exposed to at least 1 AMCS device throughout hospitalization. CONCLUSIONS: In a large contemporary HF-CS cohort, we identified a greater incidence of in-hospital death and cardiac arrest as well as a more rapid escalation to maximum SCAI stage severity among DNHF-CS. AMCS use in HF-CS was common, with significant heterogeneity among device types. (Cardiogenic Shock Working Group Registry [CSWG]; NCT04682483).

Device profile of the Impella 5.0 and 5.5 system for mechanical circulatory support for patients with cardiogenic shock: overview of its safety and efficacy.

INTRODUCTION: Trans-valvular micro-axial flow pumps such as Impella are increasingly utilized in patients with cardiogenic shock [CS]. A number of different Impella devices are now available providing a wide range of cardiac output. Among these, the Impella 5.0 and recently introduced Impella 5.5 pumps can provides 5.55 L/min of flow, enabling complete left ventricular support with more favorable hemodynamic effects on myocardial oxygen consumption and left ventricular unloading. These devices require placement of a surgical conduit graft for endovascular delivery, but are increasingly being used in patients with CS due to acutely decompensated heart failure [ADHF], acute myocardial infarction [AMI] and after cardiac surgery as a bridge to transplant or durable ventricular assist device surgery or myocardial recovery. AREAS COVERED: This review focuses on the device profile and use of the Impella 5.0 and 5.5 systems in patients with CS. Specifically; we reviewed the published literature for Impella 5.0 device to summarize data regarding safety and efficacy. EXPERT OPINION: The Impella 5.0 and 5.5 are trans-valvular micro-axial flow pumps for which the current data suggest excellent safety and efficacy profiles as approaches to provide circulatory support, myocardial unloading, and axillary placement enabling patient mobilization and rehabilitation. ABBREVIATIONS: pMCS, Percutaneous mechanical circulatory support devices; CS, Cardiogenic shock; ADHF, Acute decompensated heart failure; AMI, Acute myocardial infarction; LVAD, Left ventricular assist deviceI; ABP, Intra-aortic balloon pump; VA-ECLS, Veno-arterial extracorporeal life support.

Criteria for Defining Stages of Cardiogenic Shock Severity.

BACKGROUND: Risk-stratifying patients with cardiogenic shock (CS) is a major unmet need. The recently proposed Society for Cardiovascular Angiography and Interventions (SCAI) staging system for CS severity lacks uniform criteria defining each stage. OBJECTIVES: The purpose of this study was to test parameters that define SCAI stages and explore their utility as predictors of in-hospital mortality in CS. METHODS: The CS Working Group registry includes patients from 17 hospitals enrolled between 2016 and 2021 and was used to define clinical profiles for CS. We selected parameters of hypotension and hypoperfusion and treatment intensity, confirmed their association with mortality, then defined formal criteria for each stage and tested the association between both baseline and maximum Stage and mortality. RESULTS: Of 3,455 patients, CS was caused by heart failure (52%) or myocardial infarction (32%). Mortality was 35% for the total cohort and higher among patients with myocardial infarction, out-of-hospital cardiac arrest, and treatment with increasing numbers of drugs and devices. Systolic blood pressure, lactate level, alanine transaminase level, and systemic pH were significantly associated with mortality and used to define each stage. Using these criteria, baseline and maximum stages were significantly associated with mortality (n = 1,890). Lower baseline stage was associated with a higher incidence of stage escalation and a shorter duration of time to reach maximum stage. CONCLUSIONS: We report a novel approach to define SCAI stages and identify a significant association between baseline and maximum stage and mortality. This approach may improve clinical application of the staging system and provides new insight into the trajectory of hospitalized CS patients. (Cardiogenic Shock Working Group Registry [CSWG]; NCT04682483).

Clinical Outcomes Associated With Acute Mechanical Circulatory Support Utilization in Heart Failure Related Cardiogenic Shock.

BACKGROUND: Cardiogenic shock occurring in the setting of advanced heart failure (HF-CS) is increasingly common. However, recent studies have focused almost exclusively on acute myocardial infarction-related CS. We sought to define clinical, hemodynamic, metabolic, and treatment parameters associated with clinical outcomes among patients with HF-CS, using data from the Cardiogenic Shock Working Group registry. METHODS: Patients with HF-CS were identified from the multicenter Cardiogenic Shock Working Group registry and divided into 3 outcome categories assessed at hospital discharge: mortality, heart replacement therapy (HRT: durable ventricular assist device or orthotopic heart transplant), or native heart survival. Clinical characteristics, hemodynamic, laboratory parameters, drug therapies, acute mechanical circulatory support device (AMCS) utilization, and Society of Cardiovascular Angiography and Intervention stages were compared across the 3 outcome cohorts. RESULTS: Of the 712 patients with HF-CS identified, 180 (25.3%) died during their index admission, 277 (38.9%) underwent HRT (durable ventricular assist device or orthotopic heart transplant), and 255 (35.8%) experienced native heart survival without HRT. Patients who died had the highest right atrial pressure and heart rate and the lowest mean arterial pressure of the 3 outcome groups (P<0.01 for all). Biventricular and isolated left ventricular congestion were common among patients who died or underwent HRT, respectively. Lactate, blood urea nitrogen, serum creatinine, and aspartate aminotransferase were highest in patients with HF-CS experiencing in-hospital death. Intraaortic balloon pump was the most commonly used AMCS device in the overall cohort and among patients receiving HRT. Patients receiving >1 AMCS device had the highest in-hospital mortality rate irrespective of the number of vasoactive drugs used. Mortality increased with deteriorating Society of Cardiovascular Angiography and Intervention stages (stage B: 0%, stage C: 10.7%, stage D: 29.4%, stage E: 54.5%, 1-way ANOVA=<0.001). CONCLUSIONS: Patients with HF-CS experiencing in-hospital mortality had a high prevalence of biventricular congestion and markers of end-organ hypoperfusion. Substantial heterogeneity exists with use of AMCS in HF-CS with intraaortic balloon pump being the most common device used and high rates of in-hospital mortality after exposure to >1 AMCS device.

The science of safety: complications associated with the use of mechanical circulatory support in cardiogenic shock and best practices to maximize safety.

Acute mechanical circulatory support (MCS) devices are widely used in cardiogenic shock (CS) despite a lack of high-quality clinical evidence to guide their use. Multiple devices exist across a spectrum from modest to complete support, and each is associated with unique risks. In this review, we summarize existing data on complications associated with the three most widely used acute MCS platforms: the intra-aortic balloon pump (IABP), Impella systems, and veno-arterial extracorporeal membrane oxygenation (VA-ECMO). We review evidence from available randomized trials and highlight challenges comparing complication rates from case series and comparative observational studies where a lack of granular data precludes appropriate matching of patients by CS severity. We further offer a series of best practices to help shock practitioners minimize the risk of MCS-associated complications and ensure the best possible outcomes for patients.

Central Venous Pressure and Clinical Outcomes During Left-Sided Mechanical Support for Acute Myocardial Infarction and Cardiogenic Shock.

Background: Right ventricular failure (RVF) is associated with increased mortality among patients receiving left ventricular mechanical circulatory support (LV-MCS) for cardiogenic shock and requires prompt recognition and management. Increased central venous pressure (CVP) is an indicator of potential RVF. Objectives: We studied whether elevated CVP during LV-MCS for acute myocardial infarction complicated by cardiogenic shock is associated with higher mortality. Methods: Between January 2014 and June 2019, we analyzed hemodynamic parameters during Impella LV-MCS from 28 centers in the United States participating in the global, prospective catheter-based ventricular assist device (cVAD) study. A total of 132 patients with a documented CVP measurement while on Impella left-sided support for cardiogenic shock were identified. Results: CVP was significantly higher among patients who died in the hospital (14.0 vs. 11.7 mmHg, p = 0.014), and a CVP >12 identified patients at significantly higher risk for in-hospital mortality (65 vs. 45%, p = 0.02). CVP remained significantly associated with in-hospital mortality even after adjustment in a multivariable model (adjusted OR 1.10 [95% CI 1.02-1.19] per 1 mmHg increase). LV-MCS suction events were non-significantly more frequent among patients with high vs. low CVP (62.11 vs. 7.14 events, p = 0.067). Conclusion: CVP is a single, readily accessible hemodynamic parameter which predicts a higher rate of short-term mortality and may identify subclinical RVF in patients receiving LV-MCS for cardiogenic shock.