Diversity of cells and signals in the cardiovascular system.

This white paper is the outcome of the seventh UC Davis Cardiovascular Research Symposium on Systems Approach to Understanding Cardiovascular Disease and Arrhythmia. This biannual meeting aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2022 Symposium was 'Cell Diversity in the Cardiovascular System, cell-autonomous and cell-cell signalling'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies, and challenges in examining cell and signal diversity, co-ordination and interrelationships involved in cardiovascular function. This paper originates from the topics of formal presentations and informal discussions from the Symposium, which aimed to develop a holistic view of how the multiple cell types in the cardiovascular system integrate to influence cardiovascular function, disease progression and therapeutic strategies. The first section describes the major cell types (e.g. cardiomyocytes, vascular smooth muscle and endothelial cells, fibroblasts, neurons, immune cells, etc.) and the signals involved in cardiovascular function. The second section emphasizes the complexity at the subcellular, cellular and system levels in the context of cardiovascular development, ageing and disease. Finally, the third section surveys the technological innovations that allow the interrogation of this diversity and advancing our understanding of the integrated cardiovascular function and dysfunction.

KCNH2 encodes a nuclear-targeted polypeptide that mediates hERG1 channel gating and expression.

KCNH2 encodes hERG1, the voltage-gated potassium channel that conducts the rapid delayed rectifier potassium current (IKr) in human cardiac tissue. hERG1 is one of the first channels expressed during early cardiac development, and its dysfunction is associated with intrauterine fetal death, sudden infant death syndrome, cardiac arrhythmia, and sudden cardiac death. Here, we identified a hERG1 polypeptide (hERG1NP) that is targeted to the nuclei of immature cardiac cells, including human stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat cardiomyocytes. The nuclear hERG1NP immunofluorescent signal is diminished in matured hiPSC-CMs and absent from adult rat cardiomyocytes. Antibodies targeting distinct hERG1 channel epitopes demonstrated that the hERG1NP signal maps to the hERG1 distal C-terminal domain. KCNH2 deletion using CRISPR simultaneously abolished IKr and the hERG1NP signal in hiPSC-CMs. We then identified a putative nuclear localization sequence (NLS) within the distal hERG1 C-terminus, 883-RQRKRKLSFR-892. Interestingly, the distal C-terminal domain was targeted almost exclusively to the nuclei when overexpressed HEK293 cells. Conversely, deleting the NLS from the distal peptide abolished nuclear targeting. Similarly, blocking α or ß1 karyopherin activity diminished nuclear targeting. Finally, overexpressing the putative hERG1NP peptide in the nuclei of HEK cells significantly reduced hERG1a current density, compared to cells expressing the NLS-deficient hERG1NP or GFP. These data identify a developmentally regulated polypeptide encoded by KCNH2, hERG1NP, whose presence in the nucleus indirectly modulates hERG1 current magnitude and kinetics.

International Alliance of Urolithiasis Guideline on Shockwave Lithotripsy.

Different international associations have proposed their own guidelines on urolithiasis. However, the focus is primarily on an overview of the principles of urolithiasis management rather than step-by-step technical details for the procedure. The International Alliance of Urolithiasis (IAU) is releasing a series of guidelines on the management of urolithiasis. The current guideline on shockwave lithotripsy (SWL) is the third in the IAU guidelines series and provides a clinical framework for urologists and technicians performing SWL. A total of 49 recommendations are summarized and graded, covering the following aspects: indications and contraindications; preoperative patient evaluation; preoperative medication; prestenting; intraoperative analgesia or anesthesia; intraoperative position; stone localization and monitoring; machine and energy settings; intraoperative lithotripsy strategies; auxiliary therapy following SWL; evaluation of stone clearance; complications; and quality of life. The recommendations, tips, and tricks regarding SWL procedures summarized here provide important and necessary guidance for urologists along with technicians performing SWL. PATIENT SUMMARY: For kidney and urinary stones of less than 20 mm in size, shockwave lithotripsy (SWL) is an approach in which the stone is treated with shockwaves applied to the skin, without the need for surgery. Our recommendations on technical aspects of the procedure provide guidance for urologists and technicians performing SWL.

hERG1 channel subunit composition mediates proton inhibition of rapid delayed rectifier potassium current (IKr) in cardiomyocytes derived from hiPSCs.

The voltage-gated channel, hERG1, conducts the rapid delayed rectifier potassium current (IKr) and is critical for human cardiac repolarization. Reduced IKr causes long QT syndrome and increases the risk for cardiac arrhythmia and sudden death. At least two subunits form functional hERG1 channels, hERG1a and hERG1b. Changes in hERG1a/1b abundance modulate IKr kinetics, magnitude, and drug sensitivity. Studies from native cardiac tissue suggest that hERG1 subunit abundance is dynamically regulated, but the impact of altered subunit abundance on IKr and its response to external stressors is not well understood. Here, we used a substrate-driven human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturation model to investigate how changes in relative hERG1a/1b subunit abundance impact the response of native IKr to extracellular acidosis, a known component of ischemic heart disease and sudden infant death syndrome. IKr recorded from immatured hiPSC-CMs displays a 2-fold greater inhibition by extracellular acidosis (pH 6.3) compared with matured hiPSC-CMs. Quantitative RT-PCR and immunocytochemistry demonstrated that hERG1a subunit mRNA and protein were upregulated and hERG1b subunit mRNA and protein were downregulated in matured hiPSC-CMs compared with immatured hiPSC-CMs. The shift in subunit abundance in matured hiPSC-CMs was accompanied by increased IKr. Silencing hERG1b's impact on native IKr kinetics by overexpressing a polypeptide identical to the hERG1a N-terminal Per-Arnt-Sim domain reduced the magnitude of IKr proton inhibition in immatured hiPSC-CMs to levels comparable to those observed in matured hiPSC-CMs. These data demonstrate that hERG1 subunit abundance is dynamically regulated and determines IKr proton sensitivity in hiPSC-CMs.

Reduced health care utilization among young children of immigrants after Donald Trump's election and proposed public charge rule.

Widespread fear among immigrants from hostile 2016 presidential campaign rhetoric decreased social and health care service enrollment (chilling effect). Health care utilization effects among immigrant families with young children are unknown. We examined whether former President Trump's election had chilling effects on well-child visit (WCV) schedule adherence, hospitalizations, and emergency department (ED) visits among children of immigrant vs US-born mothers in 3 US cities. Cross-sectional surveys of children <4 years receiving care in hospitals were linked to 2015-2018 electronic health records. We applied difference-in-difference analysis with a 12-month pre/post-election study period. Trump's election was associated with a 5-percentage-point decrease (-0.05; 95% CI: -0.08, -0.02) in WCV adherence for children of immigrant vs US-born mothers with no difference in hospitalizations or ED visits. Secondary analyses extending the treatment period to a leaked draft of proposed changes to public charge rules also showed significantly decreased WCV adherence among children of immigrant vs US-born mothers. Findings indicate likely missed opportunities for American Academy of Pediatrics-recommended early childhood vaccinations, health and developmental screenings, and family support. Policies and rhetoric promoting immigrant inclusion create a more just and equitable society for all US children.

Enhancing iPSC-CM Maturation Using a Matrigel-Coated Micropatterned PDMS Substrate.

Cardiac myocytes isolated from adult heart tissue have a rod-like shape with highly organized intracellular structures. Cardiomyocytes derived from human pluripotent stem cells (iPSC-CMs), on the other hand, exhibit disorganized structure and contractile mechanics, reflecting their pronounced immaturity. These characteristics hamper research using iPSC-CMs. The protocol described here enhances iPSC-CM maturity and function by controlling the cellular shape and environment of the cultured cells. Microstructured silicone membranes function as a cell culture substrate that promotes cellular alignment. iPSC-CMs cultured on micropatterned membranes display an in-vivo-like rod-shaped morphology. This physiological cellular morphology along with the soft biocompatible silicone substrate, which has similar stiffness to the native cardiac matrix, promotes maturation of contractile function, calcium handling, and electrophysiology. Incorporating this technique for enhanced iPSC-CM maturation will help bridge the gap between animal models and clinical care, and ultimately improve personalized medicine for cardiovascular diseases. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cardiomyocyte differentiation of iPSCs Basic Protocol 2: Purification of differentiated iPSC-CMs using MACS negative selection Basic Protocol 3: Micropatterning on PDMS.

Improving health across sectors: Best practices for the implementation of health in all policies approaches.

Health is influenced by a broad range of factors beyond the typical remit of public health. It is therefore increasingly recognized that multiple sectors need to be engaged to improve population health. Health in All Policies (HiAP) is an approach to systematically consider health across policies and programs. This study assessed best practices and gaps in HiAP operationalization to inform practitioners aiming to incorporate HiAP in their work. We used Delaware as a model state to examine operationalization factors in a jurisdiction planning to implement HiAP. Methods included document review, key informant interviews, focus groups, and a questionnaire conducted in Delaware and virtually. Thematic analysis was used to analyze qualitative data to provide information on best practices and gaps in existing HiAP programs and context in Delaware. Descriptive statistics were used to examine collaboration in Delaware and to support or refute qualitative findings. We identified two gaps that can hinder HiAP implementation: 1) HiAP practitioners do not adequately use strategic communications to increase buy-in across sectors; 2) practitioners do not fully recognize the importance of being adaptable throughout HiAP implementation, which hinders sustainability. Qualitative findings from Delaware offer insight to these gaps and opportunities to address them. Refining the essential elements of HiAP to add: 1) strategic communications across sectors and 2) flexibility throughout HiAP implementation may point the way to more successful adoption of HiAP approaches across jurisdictions. This research demonstrated the importance of examining local perspectives on HiAP before implementation based on a jurisdiction's context.

A database of US state policies to mitigate COVID-19 and its economic consequences.

BACKGROUND: Since COVID-19 first appeared in the United States (US) in January 2020, US states have pursued a wide range of policies to mitigate the spread of the virus and its economic ramifications. Without unified federal guidance, states have been the front lines of the policy response. MAIN TEXT: We created the COVID-19 US State Policy (CUSP) database ( https://statepolicies.com/ ) to document the dates and components of economic relief and public health measures issued at the state level in response to the COVID-19 pandemic. Documented interventions included school and business closures, face mask mandates, directives on vaccine eligibility, eviction moratoria, and expanded unemployment insurance benefits. By providing continually updated information, CUSP was designed to inform rapid-response, policy-relevant research in the context of the COVID-19 pandemic and has been widely used to investigate the impact of state policies on population health and health equity. This paper introduces the CUSP database and highlights how it is already informing the COVID-19 pandemic response in the US. CONCLUSION: CUSP is the most comprehensive publicly available policy database of health, social, and economic policies in response to the COVID-19 pandemic in the US. CUSP documents widespread variation in state policy decisions and implementation dates across the US and serves as a freely available and valuable resource to policymakers and researchers.

The ERG1 K+ Channel and Its Role in Neuronal Health and Disease.

The ERG1 potassium channel, encoded by KCNH2, has long been associated with cardiac electrical excitability. Yet, a growing body of work suggests that ERG1 mediates physiology throughout the human body, including the brain. ERG1 is a regulator of neuronal excitability, ERG1 variants are associated with neuronal diseases (e.g., epilepsy and schizophrenia), and ERG1 serves as a potential therapeutic target for neuronal pathophysiology. This review summarizes the current state-of-the-field regarding the ERG1 channel structure and function, ERG1's relationship to the mammalian brain and highlights key questions that have yet to be answered.

Hysteretic hERG channel gating current recorded at physiological temperature.

Cardiac hERG channels comprise at least two subunits, hERG 1a and hERG 1b, and drive cardiac action potential repolarization. hERG 1a subunits contain a cytoplasmic PAS domain that is absent in hERG 1b. The hERG 1a PAS domain regulates voltage sensor domain (VSD) movement, but hERG VSD behavior and its regulation by the hERG 1a PAS domain have not been studied at physiological temperatures. We recorded gating charge from homomeric hERG 1a and heteromeric hERG 1a/1b channels at near physiological temperatures (36 ± 1 °C) using pulse durations comparable in length to the human ventricular action potential. The voltage dependence of deactivation was hyperpolarized relative to activation, reflecting VSD relaxation at positive potentials. These data suggest that relaxation (hysteresis) works to delay pore closure during repolarization. Interestingly, hERG 1a VSD deactivation displayed a double Boltzmann distribution, but hERG 1a/1b deactivation displayed a single Boltzmann. Disabling the hERG 1a PAS domain using a PAS-targeting antibody similarly transformed hERG 1a deactivation from a double to a single Boltzmann, highlighting the contribution of the PAS in regulating VSD movement. These data represent, to our knowledge, the first recordings of hERG gating charge at physiological temperature and demonstrate that VSD relaxation (hysteresis) is present in hERG channels at physiological temperature.

How Do Medicaid Agencies Improve Substance Use Treatment Benefits? Lessons from Three States' 1115 Waiver Experiences.

CONTEXT: In 2015, the Centers for Medicare and Medicaid Services (CMS) urged state Medicaid programs to use 1115 waiver demonstrations to expand substance use treatment benefits. We analyzed four critical points in states' decision-making processes before expanding benefits. METHODS: We conducted qualitative cross-case comparison of three states that were early adopters of the 1115 waiver request. We conducted 44 interviews with key informants from CMS, Medicaid, and other state agencies, providers, and managed care organizations. FINDINGS: Policy makers expanded substance use treatment in response to "fragmented" care systems and unsustainable funding streams. Medicaid staff had mixed preferences for implementing new benefits via 1115 waivers or state plan amendments. The 1115 waiver process enabled states to provide coverage for residential benefits, but state plan amendments made other services permanent parts of the benefit. Medicaid agencies relied on interorganizational networks to identify evidence-based practices. Medicaid staff secured legislative support for reform by focusing on program integrity concerns and downstream effects of substance use rather than Medicaid beneficiaries' needs. CONCLUSIONS: Decision-making processes were influenced by Medicaid agency characteristics and interorganizational partnerships, not federal executive branch influence. Lessons from early-adopter states provide a road map for other state Medicaid agencies considering similar reform.

Translating Medicaid policy into practice: policy implementation strategies from three US states' experiences enhancing substance use disorder treatment.

BACKGROUND: Despite the important upstream impact policy has on population health outcomes, few studies in implementation science in health have examined implementation processes and strategies used to translate state and federal policies into accessible services in the community. This study examines the policy implementation strategies and experiences of Medicaid programs in three US states that responded to a federal prompt to improve access to evidence-based practice (EBP) substance use disorder (SUD) treatment. METHODS: Three US state Medicaid programs implementing American Society of Addiction Medicine (ASAM) Criteria-driven SUD services under Section 1115 waiver authority were used as cases. We conducted 44 semi-structured interviews with Medicaid staff, providers and health systems partners in California, Virginia, and West Virginia. Interviews were triangulated with document review of state readiness and implementation plans. The Exploration, Preparation, Implementation, Sustainment Framework (EPIS) guided qualitative theme analysis. The Expert Recommendations for Implementing Change and Specify It criteria were used to create a taxonomy of policy implementation strategies used by policymakers to promote providers' uptake of statewide EBP SUD care continuums. RESULTS: Four themes describe states' experiences and outcomes implementing a complex EBP SUD treatment policy directive: (1) Medicaid agencies adapted their inner/outer contexts to align with EBPs and adapted EBPs to fit their local context; (2) enhanced financial reimbursement arrangements were inadequate bridging factors to achieve statewide adoption of new SUD services; (3) despite trainings, service providers and managed care organizations demonstrated poor fidelity to the ASAM Criteria; and (4) successful policy adoption at the state level did not guarantee service providers' uptake of EBPs. States used 29 implementation strategies to implement EBP SUD care continuums. Implementation strategies were used in the Exploration (n=6), Preparation (n=10), Implementation (n=19), and Sustainment (n=6) phases, and primarily focused on developing stakeholder interrelationships, evaluative and iterative approaches, and financing. CONCLUSIONS: This study enhances our understanding of statewide policy implementation outcomes in low-resource, public healthcare settings. Themes highlight the need for additional pre-implementation and sustainment focused implementation strategies. The taxonomy of detailed policy implementation strategies employed by policymakers across states should be tested in future policy implementation research.

Predicting longitudinal service use for individuals with substance use disorders: A latent profile analysis.

INTRODUCTION: Substance use disorders (SUD) are chronic conditions that often warrant coordinated medical care throughout a relapsing and remitting course. However, SUD treatment is frequently measured as a binary outcome, where individuals either receive or do not receive care following the immediate treatment seeking event. This study aimed to describe longitudinal treatment seeking behaviors by assessing service use patterns among individuals with diagnosed SUDs in a safety net hospital that offers a "no wrong door" care model. This study also examined whether certain patient subgroups were more likely to transition to service use patterns that support recovery or treatment disengagement over time. METHODS: The team conducted a retrospective cohort study using electronic health record data from adult patients diagnosed with SUDs (n = 1157) who regularly accessed services at a safety net hospital over a five-year period. The study used latent class analysis (LCA) and latent profile analysis to empirically identify distinct treatment utilization profiles of individuals with SUDs. We used multinomial logistic regression to evaluate predictors of class membership and transitions over a five-year period. RESULTS: The research team identified five distinct service use classes, including patients who disengaged from services (42.4%), or those who predominantly used outpatient substance use services (7.0%), mental health services (13.0%), primary care services (24.7%), or other specialty care services (13.1%). Being female and an older adult were statistically significant predictors for membership in any service use-driven status. Black patients had increased odds for "substance use service" and "primary care" service statuses over time. CONCLUSION: LCA and latent transition analysis (LTA) methods are novel approaches for identifying profiles of patients with higher risk for health services disengagement. SUD treatment engagement strategies are needed to reach males, young adults, and individuals with non-opioid SUDs.

Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating.

The human ERG (hERG) K+ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS-CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.

Examining the implementation of police-assisted referral programs for substance use disorder services in Massachusetts.

BACKGROUND: In response to the dramatic increase in opioid overdose deaths in recent years, police departments and community partners across the United States have begun to implement programs focused on connecting individuals to substance use disorder services. We examined the implementation of police-assisted referral programs from the perspectives of different team members to understand the key components of these programs and strategies used to implement them. METHODS: Qualitative research methods were used to examine the implementation of police-assisted referral programs in five Massachusetts communities between June 2019 and March 2020. Focus groups and interviews were conducted with 33 individuals, including 5 police chiefs, 12 police officers, 6 outreach workers, 4 community-based organization (CBO) directors, 2 interns, 1 clinician, 1 program manager, 1 religious representative, and 1 prevention specialist. RESULTS: Five key themes emerged regarding the implementation of police-assisted referral programs across the communities: 1) program development was an ongoing process; 2) partnerships between police departments and community stakeholders were essential for starting and sustaining a program; 3) high-level leadership influenced program priorities and facilitated implementation; 4) program success was defined in multiple ways; and 5) programs contributed to shifts in beliefs about substance use and addiction among police officers. CONCLUSIONS: Police-assisted referral programs in Massachusetts have adopted a variety of models of service delivery, evolving from post-overdose outreach and walk-in models to more complex hybrid forms. Implementation was facilitated by the support of departmental leadership, particularly the police chief, and the development of key partnerships across institutional boundaries. Communities continue to develop their programs to incorporate additional components, such as new mechanisms of outreach, harm reduction services, and long-term engagement activities. Further evaluation of these programs is needed to understand how each of these unique components may influence a program's impact on future overdoses, entry to treatment, and long-term recovery.

Pursuing Medicaid Block Grants with the Healthy Adult Opportunity Initiative: Dressing Up Old Ideas in New Clothes.

The Trump administration's Healthy Adult Opportunity waiver follows a long history of Republican attempts to retrench the Medicaid program through block grants and to markedly reduce federal spending while providing states with substantially greater flexibility over program structure. Previous block grant proposals were promulgated during the presidential administrations of Ronald Reagan and George W. Bush and majorities in Congress led by House Speaker Newt Gingrich and House Budget Committee Chair and then Speaker Paul Ryan. Most recently, Medicaid block grants featured prominently in Republican efforts to repeal and replace the Affordable Care Act. This essay traces the history of Republican Medicaid block grant proposals, culminating in the Trump administration's Healthy Adult Opportunity initiative. It concludes that the Trump administration's attempt to convert Medicaid into a block grant program through the waiver process is illegal and, if implemented, would leave thousands of people without necessary medical care. This fact, combined with failed legislative efforts to block grant Medicaid during the last forty years, highlights the substantial roadblocks to radically restructuring a popular program that helps millions of Americans.

Neighbourhood income and physical distancing during the COVID-19 pandemic in the United States.

Physical distancing has been the primary strategy to control COVID-19 in the United States. We used mobility data from a large, anonymized sample of smartphone users to assess the relationship between neighbourhood income and physical distancing during the pandemic. We found a strong gradient between neighbourhood income and physical distancing. Individuals in high-income neighbourhoods increased their days at home substantially more than individuals in low-income neighbourhoods did. Residents of low-income neighbourhoods were more likely to work outside the home, compared to residents in higher-income neighbourhoods, but were not more likely to visit locations such as supermarkets, parks and hospitals. Finally, we found that state orders were only associated with small increases in staying home in low-income neighbourhoods. Our findings indicate that people in lower-income neighbourhoods have faced barriers to physical distancing, particularly needing to work outside the home, and that state physical distancing policies have not mitigated these disparities.