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Background: The initial derivation of cardiac power output (CPO) included the difference between mean arterial pressure (MAP) and right atrial pressure (RAP) in the numerator, before multiplying by cardiac output (CO). We hypothesized that the inclusion of RAP (CPO-RAP) would enhance the prognostic performance of this parameter in those with an elevated RAP., Methods and Results: We obtained patient-level data from the ESCAPE trial via the Biolincc database. Participants with full final hemodynamics were included in the analysis. The CPO-RAP was calculated as [(MAP - RAP) × CO)]/451 Watts (W), and the CPO was calculated as (MAP × CO)/451. The primary outcome was freedom from left ventricular assist device, heart transplant, or death at 6 months. Included participants (n = 157) were a median of 58 years of age (interquartile range [IQR] 49-67 years), 27% were women, and 59% had ischemic cardiomyopathy. The median CPO was 0.70 W (IQR 0.50-0.90 W), and the median CPO-RAP was 0.62 W (IQR 0.47-0.79 W). In univariable logistic regressions, the CPO was not associated with the primary outcome (odds ratio 0.32, 95% confidence interval 0.08-1.29, P = .11), but the CPO-RAP was (odds ratio 0.10, 95% confidence interval 0.02-0.54, P < .01). In Kaplan-Meier analyses, there were no significant difference in outcomes with CPO (76% vs 64%, P = .08), but for CPO-RAP, there were significant differences in outcomes (81% vs 63%, P = .01). When further delineating CPO-RAP by RAP above or below the median, there was no significant difference in the outcome for participants with a RAP 8 or less (94% vs 79%, P = .07), but a significant difference in participants with a RAP of more than 8 mm Hg (66% vs 45%, P < .05)., Conclusions: The inclusion of RAP resulted in a significant association with the primary outcome; CPO alone was not., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Atherosclerosis is the predominant cause of coronary artery disease. The last several decades have witnessed significant advances in lipid-lowering therapies, which comprise a central component of atherosclerotic cardiovascular disease prevention. In addition to cardiovascular risk reduction with dyslipidemia management, some lipid-based therapies show promise at the level of the atherosclerotic plaque itself through mechanisms governing lipid accumulation, plaque stability, local inflammation, endothelial dysfunction, and thrombogenicity. The capacity of lipid-lowering therapies to modify atherosclerotic plaque burden, size, composition, and vulnerability should correlate with their ability to reduce disease progression. This review discusses plaque characteristics, diagnostic modalities to evaluate these characteristics, and how they are altered by current and emerging lipid-lowering therapies, all in human coronary artery disease., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V.)

Background: Tetralogy of Fallot is a congenital heart defect diagnosed in infancy. Assessment of right ventricular size and function is important for evaluation of patients with tetralogy of Fallot, but these quantitative measures are challenging by echocardiography. This study evaluates a semi-automated software (EchoInsight®, Epsilon Imaging) by comparing its measures to manual measures in children with tetralogy of Fallot., Methods: Echocardiographic measurements were performed using manual techniques and semi-automated software. Right ventricular measurements included end-diastolic and end-systolic area, fractional area change, chamber dimensions, and tricuspid annular plane systolic excursion. Reliability, correlation, and agreement between manual and semi-automated measures were assessed., Results: Echocardiograms for 46 patients were analysed. Intra- and inter-observer reliabilities for semi-automated measures were good with intraclass correlation coefficients all over 0.95 and 0.85, respectively. There was high correlation between manual and semi-automated methods for areas and dimensions (r = 0.91-0.98). Tricuspid annular plane systolic excursion measures and fractional area change also correlated, albeit less strongly. The semi-automated measurements of end-systolic and end-diastolic area were a 20 and 47% higher than manual methods, respectively.The semi-automated method yielded a relative 52% lower fractional area change compared to the manual method., Conclusions: The semi-automated software generates quantitative right ventricular measures in children with tetralogy of Fallot with good reliability and good correlation with manual methods for all measures, but with significant difference between manual and semi-automated techniques for area and functional measures. The specific right ventricular geometry in tetralogy of Fallot children may be why, compared to normal anatomy, greater differences were observed between the two techniques.

Competing Interests: Dr Sarswat has been a consultant and has served on advisory boards for Alnylam, Akcea, and Pfizer; and has received grant funding from Pfizer. Dr Derman has served on an advisory board for Sanofi. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Aims: The TOPCAT trial showed no benefit for spironolactone in heart failure patients with preserved ejection fraction (HFpEF). Post-hoc, spironolactone helped participants from the Americas, but not Eastern Europe. Determining which patients with HFpEF could respond like TOPCAT's responders should help guide their care. We aimed to develop a TOPCAT Trial Score (TS) as a composite metric to identify such patients., Methods and Results: From the TOPCAT individual-level data, we calculated a TS of age, body mass index, systolic blood pressure, heart rate, creatinine, potassium, glucose, left ventricular ejection fraction, and left atrial volume for each participant as a weighted distance in multidimensional space from the theoretical perfectly average Americas participant. Logistic regression was used to measure TS and spironolactone as predictors of TOPCAT's primary outcome. The relationship between TS and the H 2 FPEF score was also determined in TOPCAT and a registry cohort of real-world patients in the U.S. with HFpEF. A bimodal distribution of TS separated American (n = 1766) and Eastern European (n = 1,677) participants. Those with lower TS showed no significant response to spironolactone. Spironolactone's benefit rose with rising TS [β interaction  = -0.28 (P < 0.01)]. Significantly more American participants had benefit from spironolactone based on higher TS (> 1.14), in addition to higher likelihood of HFpEF based on higher H 2 FPEF scores (≥3). The cohort of real-world patients with HFpEF had even higher TS than American TOPCAT participants., Conclusions: Patients with HFpEF can be quantified by the TS to capture the likelihood of benefit from spironolactone., (© 2021 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Objective: To rapidly exclude severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using artificial intelligence applied to the electrocardiogram (ECG)., Methods: A global, volunteer consortium from 4 continents identified patients with ECGs obtained around the time of polymerase chain reaction-confirmed COVID-19 diagnosis and age- and sex-matched controls from the same sites. Clinical characteristics, polymerase chain reaction results, and raw electrocardiographic data were collected. A convolutional neural network was trained using 26,153 ECGs (33.2% COVID positive), validated with 3826 ECGs (33.3% positive), and tested on 7870 ECGs not included in other sets (32.7% positive). Performance under different prevalence values was tested by adding control ECGs from a single high-volume site., Results: The area under the curve for detection of acute COVID-19 infection in the test group was 0.767 (95% CI, 0.756 to 0.778; sensitivity, 98%; specificity, 10%; positive predictive value, 37%; negative predictive value, 91%). To more accurately reflect a real-world population, 50,905 normal controls were added to adjust the COVID prevalence to approximately 5% (2657/58,555), resulting in an area under the curve of 0.780 (95% CI, 0.771 to 0.790) with a specificity of 12.1% and a negative predictive value of 99.2%., Conclusion: Infection with SARS-CoV-2 results in electrocardiographic changes that permit the artificial intelligence-enhanced ECG to be used as a rapid screening test with a high negative predictive value (99.2%). This may permit the development of electrocardiography-based tools to rapidly screen individuals for pandemic control., (Copyright © 2021. Published by Elsevier Inc.)

Aims: Aortic pulsatility index (API), calculated as (systolic-diastolic blood pressure)/pulmonary capillary wedge pressure (PCWP), is a novel haemodynamic measurement representing both cardiac filling pressures and contractility. We hypothesized that API would better predict clinical outcomes than traditional haemodynamic metrics of cardiac function., Methods and Results: The Evaluation Study of Congestive Heart Failure and Pulmonary Artery Catheterization Effectiveness (ESCAPE) trial individual-level data were used. Routine haemodynamic measurements, including Fick cardiac index (CI), and the advanced haemodynamic metrics of API, cardiac power output (CPO), and pulmonary artery pulsatility index (PAPI) were calculated after final haemodynamic-monitored optimization. The primary outcome was a composite endpoint of death or need for orthotopic heart transplant (OHT) or left ventricular assist device (LVAD) at 6 months. A total of 433 participants were enrolled in the ESCAPE trial of which 145 had final haemodynamic data. Final API measurements predicted the primary outcome, OR 0.47 (95% CI 0.32-0.70, P < 0.001), while CI, CPO, and PAPI did not. Receiver operator characteristic analyses of final advanced haemodynamic measurements indicated API best predicted the primary outcome with a cutoff of 2.9 (sensitivity 76.2%, specificity 55.3%, correctly classified 61.4%, area-under-the-curve 0.71), compared with CPO, CI, and PAPI. Kaplan-Meier analyses indicated API ≥ 2.9 was associated with greater freedom from the primary outcome (83.5%), compared with API < 2.9 (58.4%), P = 0.001. While PAPI was also significantly associated, CI and CPO were not., Conclusions: The novel haemodynamic measurement API better predicted clinical outcomes in the ESCAPE trial when compared with traditional invasive haemodynamic metrics of cardiac function., (© 2021 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Background: The role of donor-derived cell-free DNA (dd-cfDNA) in screening for cardiac allograft vasculopathy (CAV) is unknown. We hypothesized that dd-cfDNA correlates with CAV, markers of inflammation, and angiogenesis in stable heart transplant (HT) recipients., Methods: Sixty-five HT recipients ≥2 years post-transplant, without recent rejection, were stratified by high (≥0.12%) versus low levels (<0.12%) of dd-cfDNA. A targeted amplification, next-generation sequencing assay (AlloSure ® ; CareDx, Inc.) was used to detect dd-cfDNA. Peripheral blood inflammatory and angiogenesis markers were assessed using a multiplex immunoassay system (Bioplex ® )., Results: Of 65 patients, 58 patients had a known CAV status and were included. Thirty had high levels of dd-cfDNA (≥0.12%), and 28 had low levels (<0.12%). CAV was present in 63% of patients with high dd-cfDNA vs. 35% with low dd-cfDNA (p = .047). Donor-specific antibodies were present in 25% of patients with high dd-cfDNA vs. 3.8% in those with low dd-cfDNA (p = .03). There were no differences in rejection episodes, inflammatory, or angiogenesis markers. Importantly, dd-cfDNA levels were not different when stratified by time post-transplant., Conclusions: Higher dd-cfDNA levels were associated with CAV in stable chronic HT recipients. Further studies are warranted to investigate a possible association between dd-cfDNA levels and CAV severity and whether dd-cfDNA can predict CAV progression., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. We previously showed that macrophages in the atherogenic plaque undergo RIPK3 (receptor-interacting serine/threonine-protein kinase 3)-MLKL (mixed lineage kinase domain-like protein)-dependent programmed necroptosis in response to sterile ligands such as oxidized low-density lipoprotein and damage-associated molecular patterns and that necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which acts as a master switch that controls whether the cell undergoes NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells)-dependent inflammation, caspase-dependent apoptosis, or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is driven largely by NF-κB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-κB-dependent inflammation in early atherogenic lesions, and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis., Methods: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and used loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 antisense oligonucleotides to Apoe -/- mice fed a cholesterol-rich (Western) diet for 8 weeks., Results: We find that RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 antisense oligonucleotides led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, P <0.01) and plasma inflammatory cytokines (IL-1α [interleukin 1α], IL-17A [interleukin 17A], P <0.05) in comparison with controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NF-κB, TNFα [tumor necrosis factor α], IL-1α) and in vivo lipopolysaccharide- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B , E-selectin , and monocyte attachment., Conclusions: We identify RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

Background: High sensitivity cardiac troponin-T (hs-TnT) has been associated with mortality in patients hospitalized with COVID-19. We aimed to determine if hs-TnT levels and their timing are independent predictors of adverse events in these patients., Design: Retrospective chart review was performed for all patients hospitalized at our institution between 23 March 2020 and 13 April 2020 who were found to be COVID-19-positive. Clinical, demographic, and laboratory variables including initial and peak hs-TnT were recorded. Univariable and multivariable analyses were completed for a primary composite endpoint of in-hospital death, intubation, need for critical care, or cardiac arrest., Results: In the 276 patients analysed, initial hs-TnT above the median (≥17 ng/L) was associated with increased length of stay, need for vasoactive medications, and death, along with the composite endpoint (OR 3.92, p  < 0.001). Multivariable analysis demonstrated that elevated initial hs-TnT was independently associated with the primary endpoint (OR 2.92, p  = 0.01). Late-peaking hs-TnT (OR 2.19 for each additional day until peak, p  < 0.001) was also independently associated with the composite endpoint., Conclusions: In patients hospitalized with COVID-19, hs-TnT identifies patients at high risk for adverse in-hospital events, and trends of hs-TnT over time, particularly during the first day, provide additional prognostic information.

New Findings: What is the central question of this study? Are circulating monocyte markers correlated with their derived macrophage polarization patterns and coronary artery disease severity? What is the main finding and its importance? There was an inverse relationship between circulating CD16 + monocytes (high) and M2 macrophages (low) that marked coronary disease severity, and the differences in polarization of macrophages were seen despite a week of cell culture ex vivo. This study highlights the importance, and potential prognostic implications, of circulating monocyte and descendant macrophage phenotypes in coronary artery disease., Abstract: Monocytes and macrophages are central to atherosclerosis, but how they combine to mark progression of human coronary artery disease (CAD) is unclear. We tested whether patients' monocyte subtypes paired with their derived macrophage profiles were correlated with extent of CAD. Peripheral blood was collected from 40 patients undergoing cardiac catheterization, and patients were categorized as having no significant CAD, single vessel disease or multivessel disease according to the number of affected coronary arteries. Mononuclear cells were measured for the monocyte markers CD14 and CD16 by flow cytometry, and separate monocytes were cultured into macrophages over 7 days and measured for the polarization markers CD86 and CD206. At baseline, patients with a greater CAD burden were older, with higher rates of statin, β-blocker and antiplatelet drug use, whereas other characteristics were similar across the spectrum of coronary disease. CD16 + (both intermediate and non-classical) monocytes were elevated in patients with single vessel and multivessel disease compared with those without significant CAD (P < 0.05), whereas regulatory M2 macrophages (CD206 + ) were decreased in patients with single vessel and multivessel disease (P < 0.001). An inverse relationship between paired CD16 + monocytes and M2 macrophages marked CAD severity. On multivariable linear regression, CAD severity was associated, along with age and traditional cardiovascular risk factors, with CD16 + monocytes (directly) and M2 macrophages (inversely). Circulating monocytes may influence downstream polarization of lesional macrophages, and these measures of monocyte and macrophage subtypes hold potential as biomarkers in CAD., (© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society.)

Introduction: Evaluating the applicability of a clinical trial to a specific patient is difficult. A novel framework, the Trial Score, was created to quantify the generalizability of a trial's result based on participants' baseline characteristics and not on the trial's inclusion and exclusion criteria., Methods: For each Systolic Blood Pressure Intervention Trial (SPRINT) participant, the Euclidean distance in six-dimensional space from the theoretical "average" participant was calculated to produce an individual Trial Score that incorporates multiple distinct continuous-variable baseline characteristics. We prospectively defined the "data-rich," "data-limited," and "data-free" zones as Trial Scores < 90th percentile, the 90th-97.5th percentile, and >97.5th percentile, respectively. Trial Scores were then calculated for National Health and Nutrition Examination Survey participants to map data zones of the general population. Individual participant data from the Action to Control Cardiovascular Risk in Diabetes blood pressure trial (ACCORD-BP) was used to test if participants further from the average SPRINT participant behave differently than the overall SPRINT results., Results: The National Health and Nutrition Examination Survey cohort and the ACCORD-BP trial demonstrate large percentages of participants in SPRINT's data-free and data-limited zones. Time-to-event rates seen with intensive and standard blood pressure control in SPRINT were the same as ACCORD-BP participants within SPRINT's data-rich zone (hazard ratio 0.97, p = 0.84 and hazard ratio 0.95, p = 0.70). However, these rates were significantly different than those of ACCORD-BP participants outside SPRINT's data-rich zone (hazard ratio 0.64, p < 0.01 and hazard ratio 0.77, p < 0.01)., Conclusions: ACCORD-BP participants with SPRINT Trial Scores in the 90th percentile or below have similar event rates to SPRINT participants in both the intensive and standard blood pressure groups. Quantifying the difference between an individual patient and the average clinical trial participant holds promise as a tool to more precisely determine applicability of a specific trial to individual patients.

Monocyte chemoattractant protein-1 (MCP-1) stimulates the migration of monocytes to inflammatory sites, leading to the progression of many diseases. Recently, we described a monocyte-targeting peptide amphiphile micelle (MCP-1 PAM) incorporated with the chemokine receptor CCR2 binding motif of MCP-1, which has a high affinity for monocytes in atherosclerotic plaques. We further report here the biomimetic components of MCP-1 PAMs and the influence of the nanoparticle upon binding to monocytes. We report that MCP-1 PAMs have enhanced secondary structure compared to the MCP-1 peptide. As a result, MCP-1 PAMs displayed improved binding and chemoattractant properties to monocytes, which upregulated the inflammatory signaling pathways responsible for monocyte migration. Interestingly, when MCP-1 PAMs were incubated in the presence of prostate cancer cells in vitro, the particle displayed anticancer efficacy by reducing CCR2 expression. Given that monocytes play an important role in tumor cell migration and invasion, our results demonstrate that PAMs can improve the native biofunctional properties of the peptide and may be used as an effective inhibitor to prevent chemokine-receptor interactions that promote disease progression., Competing Interests: Notes The authors declare no competing financial interest.

Systemic inflammation promotes cardiovascular disease. Inflammatory connective tissue diseases (CTD) like lupus and rheumatoid arthritis associate with cardiovascular risk, but it is unknown whether particular groups of patients have enhanced propensity for atherosclerotic cardiovascular disease (ASCVD) associated with their CTD. Analysis of aggregate health record data at a large U.S. academic center identified CTD and ASCVD status for 287,467 African American and white adults. ASCVD prevalence in those with CTD was 29.7% for African Americans and 14.7% for white patients with prevalence ratios, compared to those without CTD, of 3.1 and 1.8, respectively. When different types of CTD were analyzed individually (rheumatoid arthritis; lupus; scleroderma; Sjögren Syndrome; dermatomyositis/polymyositis; unspecified/mixed CTD; other inflammatory arthropathy), increased ASCVD rates were found in nearly all subsets, always with higher prevalence ratios in African Americans. The prevalence ratio of ASCVD was particularly high in young African Americans. Furthermore, individuals lacking traditional cardiovascular risk factors had more ASCVD if they had CTD (prevalence ratio 2.9). Multivariate analysis confirmed a positive interaction between CTD and African-American race and a negative interaction between CTD and age. The factors driving the observed disproportionate CTD-associated ASCVD in African Americans, young adults, and those without traditional risk factors warrant further study.

Atherosclerosis is a multifactorial inflammatory disease that can progress silently for decades and result in myocardial infarction, stroke, and death. Diagnostic imaging technologies have made great strides to define the degree of atherosclerotic plaque burden through the severity of arterial stenosis. However, current technologies cannot differentiate more lethal "vulnerable plaques," and are not sensitive enough for preventive medicine. Imaging early molecular markers and quantifying the extent of disease progression continues to be a major challenge in the field. To this end, monocyte-targeting, peptide amphiphile micelles (PAMs) are engineered through the incorporation of the chemokine receptor CCR2-binding motif of monocyte chemoattractant protein-1 (MCP-1) and MCP-1 PAMs are evaluated preclinically as diagnostic tools for atherosclerosis. Monocyte-targeting is desirable as the influx of monocytes is a marker of early lesions, accumulation of monocytes is linked to atherosclerosis progression, and rupture-prone plaques have higher numbers of monocytes. MCP-1 PAMs bind to monocytes in vitro, and MCP-1 PAMs detect and discriminate between early- and late-stage atherosclerotic aortas. Moreover, MCP-1 PAMs are found to be eliminated via renal clearance and the mononuclear phagocyte system (MPS) without adverse side effects. Thus, MCP-1 PAMs are a promising new class of diagnostic agents capable of monitoring the progression of atherosclerosis., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The organization of the plasma membrane is both highly complex and highly dynamic. One manifestation of this dynamic complexity is the lateral mobility of proteins within the plane of the membrane, which is often an important determinant of intermolecular protein-binding interactions, downstream signal transduction, and local membrane mechanics. The mode of membrane protein mobility can range from random Brownian motion to immobility and from confined or restricted motion to actively directed motion. Several methods can be used to distinguish among the various modes of protein mobility, including fluorescence recovery after photobleaching, single-particle tracking, fluorescence correlation spectroscopy, and variations of these techniques. Here, we present both a brief overview of these methods and examples of their use to elucidate the dynamics of membrane proteins in mammalian cells-first in erythrocytes, then in erythroblasts and other cells in the hematopoietic lineage, and finally in non-hematopoietic cells. This multisystem analysis shows that the cytoskeleton frequently governs modes of membrane protein motion by stably anchoring the proteins through direct-binding interactions, by restricting protein diffusion through steric interactions, or by facilitating directed protein motion. Together, these studies have begun to delineate mechanisms by which membrane protein dynamics influence signaling sequelae and membrane mechanical properties, which, in turn, govern cell function., (© 2013 Elsevier Inc. All rights reserved.)

Macrophages migrate to sites of insult during normal inflammatory responses. Integrins guide such migration, but the transmission of signals from integrins into the requisite cytoskeletal changes is poorly understood. We have discovered that the hematopoietic adaptor protein Skap2 is necessary for macrophage migration, chemotaxis, global actin reorganization and local actin reorganization upon integrin engagement. Binding of phosphatidylinositol [3,4,5]-triphosphate to the Skap2 pleckstrin-homology (PH) domain, which relieves its conformational auto-inhibition, is critical for this integrin-driven cytoskeletal response. Skap2 enables integrin-induced tyrosyl phosphorylation of Src-family kinases (SFKs), Adap, and Sirpα, establishing their roles as signaling partners in this process. Furthermore, macrophages lacking functional Sirpα unexpectedly have impaired local integrin-induced responses identical to those of Skap2(-/-) macrophages, and Skap2 requires Sirpα for its recruitment to engaged integrins and for coordinating downstream actin rearrangement. By revealing the positive-regulatory role of Sirpα in a Skap2-mediated mechanism connecting integrin engagement with cytoskeletal rearrangement, these data demonstrate that Sirpα is not exclusively immunoinhibitory, and illuminate previously unexplained observations implicating Skap2 and Sirpα in mouse models of inflammatory disease.

Mechanical stresses that are preferentially transmitted across the cell surface via transmembrane integrin receptors activate gene transcription by triggering production of intracellular chemical second messengers, such as cAMP. Here we show that the sensitivity of the cAMP signaling pathway to mechanical stresses transferred across beta1 integrins is mediated by force-dependent activation of the heterotrimeric G protein subunit Galphas within focal adhesions at the site of stress application. Galphas is recruited to focal adhesions that form within minutes following clustering of beta1 integrins induced by cell binding to magnetic microbeads coated with activating integrin ligands, and beta1 integrin and Galphas co-precipitate when analyzed biochemically. Stress application to activated beta1 integrins using magnetic twisting cytometry increases Galphas recruitment and activates these large G proteins within focal adhesions, as measured by binding of biotinylated azido-anilido-GTP, whereas application of similar stresses to inactivated integrins or control histocompatibility antigens has little effect. This response is relevant physiologically as application of mechanical strain to cells bound to flexible extracellular matrix-coated substrates induce translocation of phospho-CREB to the nucleus, which can be attenuated by inhibiting Galphas activity, either using the inhibitor melittin or suppressing its expression using siRNA. Although integrins are not typical G protein-coupled receptors, these results show that integrins focus mechanical stresses locally on heterotrimeric G proteins within focal adhesions at the site of force application, and transduce mechanical stimuli into an intracellular cAMP signaling response by activating Galphas at these membrane signaling sites.

A "two-hit" hypothesis predicts a second somatic hit, in addition to the germline mutation, as a prerequisite to cystogenesis and has been proposed to explain the focal nature for renal cyst formation in autosomal dominant polycystic kidney disease (ADPKD). It was reported previously that Pkd1(null/null) mouse kidney epithelial cells are unresponsive to flow stimulation. This report shows that Pkd1(+/null) cells are capable of responding to mechanical flow stimulation by changing their intracellular calcium concentration in a manner similar to that of wild-type cells. This paper reports that human renal epithelia require a higher level of shear stress to evoke a cytosolic calcium increase than do mouse renal epithelia. Both immortalized and primary cultured renal epithelial cells that originate from normal and nondilated ADPKD human kidney tubules display normal ciliary expression of the polycystins and respond to fluid-flow shear stress with the typical change in cytosolic calcium. In contrast, immortalized and primary cultured cyst-lining epithelial cells from ADPKD patients with mutations in PKD1 or with abnormal ciliary expression of polycystin-1 or -2 were not responsive to fluid shear stress. These data support a two-hit hypothesis as a mechanism of cystogenesis. This report proposes that calcium response to fluid-flow shear stress can be used as a readout of polycystin function and that loss of mechanosensation in the renal tubular epithelia is a feature of PKD cysts.

Studies of mechanotransduction mediated by stress-sensitive ion channels generally focus on the site of force application to the cell. Here we show that global, cell-wide changes in cytoskeletal structure and mechanics can regulate mechanotransduction previously shown to be triggered by activation of the mechanosensitive calcium channel, polycystin-2, in the apical primary cilium of renal epithelial cells [S.M. Nauli, F.J. Alenghat, Y. Luo, E. Williams, P. Vassilev, X. Li, A.E. Elia, W. Lu, E.M. Brown, S.J. Quinn, D.E. Ingber, J. Zhou, Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat. Genet. 33 (2003) 129-37]. Disrupting cytoplasmic microfilaments or microtubules in these cells eliminated fluid shear stress-induced increase of intracellular calcium. Altering the cytoskeletal force balance by inhibiting actomyosin-based tension generation (using 2,3-butanedione monoxime), interfering with microtubule polymerization (using nocodazole, cochicine, or taxol), or disrupting basal integrin-dependent extracellular matrix adhesions (using soluble GRGDSP peptide or anti-beta1 integrin antibody), also inhibited the calcium spike in response to fluid stress. These data indicate that although fluid stress-induced displacement of the primary cilium may be transduced into a calcium spike through activation of polycystin-2 and associated calcium-induced calcium release from intracellular stores, this mechanotransduction response is governed by global mechanical cues, including isometric tension (prestress) within the entire cytoskeleton and intact adhesions to extracellular matrix.

A permanent magnetic microneedle was developed to apply tensional forces to integrin receptors via ligand-coated magnetic microbeads while optically analyzing the mechanical properties of individual focal adhesions. Force application (130 pN for 3 s) through activated beta1 integrins produced less bead displacement than when unligated integrins were stressed. This strengthening response differed markedly on a bead-by-bead basis, correlated directly with local focal adhesion assembly, and was similar when analyzed at 4 degrees C, indicating that it was due to passive material properties of the cell. Viscoelastic analysis clarified that recruitment of focal adhesion proteins increased the local elastic stiffness of the adhesion complex without changing its viscous behavior. These data indicate that individual focal adhesions exhibit distinct mechanical properties that depend upon local focal adhesion assembly, and that these local variations in micromechanics can be detected and analyzed within living cells using the permanent magnetic microneedle technique.

This paper focuses on the development of magnetic cellular switches to enable magnetic control of intracellular functions in living mammalian cells, including receptor signal transduction and gene transcription. Our approach takes advantage of the mechanosensitivity of adenosine 3',5'-monophosphate (cAMP) induction and downstream transcription controlled by the cAMP regulatory element (CRE) to engineer gene constructs that optically report gene expression in living cells. We activate transcription of these gene reporters by applying magnetic (mechanical) stress to magnetic microbeads bound to cell surface integrin receptors. In these gene reporter constructs, CRE motifs drive the expression of fluorescent proteins or enzymes that produce fluorescent products, such as DsRed and β-lactamase (BLA), respectively. We demonstrate that a chemical inducer of cAMP (forskolin) increases expression of CRE-DsRed in living cells. More importantly, a threefold increase in CRE-BLA expression is induced by application of mechanical stress to magnetic microbeads (4.5 µm) bound to cell surface integrin receptors. Induction of cAMP could be detected within 5 min using a protein fragment complementation assay involving interactions between the KID and KIX domains of the CRE binding protein linked to complementary halves of the BLA enzyme. These studies confirm that application of magnetic stress to integrins induces gene transcription by activating the cAMP-dependent transcription factor CREB. Ongoing studies focus on optimizing sensitivity and reducing signal-to-noise by establishing stable cell lines that express these gene reporters. These studies collectively demonstrate the feasibility of using magnetic technologies to control function in living mammalian cells and, hence, support the possibility of developing magnetically-actuated cellular components for use in future micro- and nanotechnologies.

Several proteins implicated in the pathogenesis of polycystic kidney disease (PKD) localize to cilia. Furthermore, cilia are malformed in mice with PKD with mutations in TgN737Rpw (encoding polaris). It is not known, however, whether ciliary dysfunction occurs or is relevant to cyst formation in PKD. Here, we show that polycystin-1 (PC1) and polycystin-2 (PC2), proteins respectively encoded by Pkd1 and Pkd2, mouse orthologs of genes mutated in human autosomal dominant PKD, co-distribute in the primary cilia of kidney epithelium. Cells isolated from transgenic mice that lack functional PC1 formed cilia but did not increase Ca(2+) influx in response to physiological fluid flow. Blocking antibodies directed against PC2 similarly abolished the flow response in wild-type cells as did inhibitors of the ryanodine receptor, whereas inhibitors of G-proteins, phospholipase C and InsP(3) receptors had no effect. These data suggest that PC1 and PC2 contribute to fluid-flow sensation by the primary cilium in renal epithelium and that they both function in the same mechanotransduction pathway. Loss or dysfunction of PC1 or PC2 may therefore lead to PKD owing to the inability of cells to sense mechanical cues that normally regulate tissue morphogenesis.

Mechanical stresses modulate cell function by either activating or tuning signal transduction pathways. Mechanotransduction, the process by which cells convert mechanical stimuli into a chemical response, occurs both in cells specialized for sensing mechanical cues and in parenchymal cells whose primary function is not mechanosensory. However, common among the various responses to mechanical stress is the importance of direct or indirect connections between the internal cytoskeleton, the extracellular matrix (ECM), and traditional signal transducing molecules. In many instances, these elements converge at focal adhesions, sites of structural attachment between the cytoskeleton and ECM that are anchored by cell surface integrin receptors. Alenghat and Ingber discuss the accumulating evidence for the central role of cytoskeleton, ECM, and integrin-anchored focal adhesions in several mechanotransduction pathways.

A magnetic tweezer was constructed to apply controlled tensional forces (10 pN to greater than 1 nN) to transmembrane receptors via bound ligand-coated microbeadswhile optically measuring lateral bead displacements within individual cells. Use of this system with wild-type F9 embryonic carcinoma cells and cells from a vinculin knockout mouse F9 Vin (-/-) revealed much larger differences in the stiffness of the transmembrane integrin linkages to the cytoskeleton than previously reported using related techniques that measured average mechanical properties of large cell populations. The mechanical properties measured varied widely among cells, exhibiting an approximately log-normal distribution. The median lateral bead displacement was 2-fold larger in F9 Vin (-/-) cells compared to wild-type cells whereas the arithmetic mean displacement only increased by 37%. We conclude that vinculin serves a greater mechanical role in cells than previously reported and that this magnetic tweezer device may be useful for probing the molecular basis of cell mechanics within single cells., (Copyright 2000 Academic Press.)

This study was carried out to discriminate between two alternative hypotheses as to how cells sense mechanical forces and transduce them into changes in gene transcription. Do cells sense mechanical signals through generalized membrane distortion or through specific transmembrane receptors, such as integrins? Here we show that mechanical stresses applied to the cell surface alter the cyclic AMP signalling cascade and downstream gene transcription by modulating local release of signals generated by activated integrin receptors in a G-protein-dependent manner, whereas distortion of integrins in the absence of receptor occupancy has no effect.