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.

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.

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.

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.

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.

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.

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.

Localization and functional consequences of a direct interaction between TRIOBP-1 and hERG proteins in the heart.

Reduced levels of the cardiac human (h)ERG ion channel protein and the corresponding repolarizing current IKr can cause arrhythmia and sudden cardiac death, but the underlying cellular mechanisms controlling hERG surface expression are not well understood. Here, we identified TRIOBP-1, an F-actin-binding protein previously associated with actin polymerization, as a putative hERG-interacting protein in a yeast-two hybrid screen of a cardiac library. We corroborated this interaction by performing Förster resonance energy transfer (FRET) in HEK293 cells and co-immunoprecipitation in HEK293 cells and native cardiac tissue. TRIOBP-1 overexpression reduced hERG surface expression and current density, whereas reducing TRIOBP-1 expression via shRNA knockdown resulted in increased hERG protein levels. Immunolabeling in rat cardiomyocytes showed that native TRIOBP-1 colocalized predominantly with myosin-binding protein C and secondarily with rat ERG. In human stem cell-derived cardiomyocytes, TRIOBP-1 overexpression caused intracellular co-sequestration of hERG signal, reduced native IKr and disrupted action potential repolarization. Ca2+ currents were also somewhat reduced and cell capacitance was increased. These findings establish that TRIOBP-1 interacts directly with hERG and can affect protein levels, IKr magnitude and cardiac membrane excitability.

Health Policy Perspective: Medicaid and State Politics Beyond COVID.

The COVID-19 pandemic is poised to drastically alter the Medicaid program. While state Medicaid programs are currently expanding coverage policies and enrollment to address acute public health needs, states will soon face significant budget shortfalls. These impending changes may renew partisan debates about restrictive policies like work requirements, which generally require beneficiaries to verify their participation in certain activities-such as employment, job search, or training programs-in order to receive or retain coverage. We argue that restrictive Medicaid policies are driven, to a great extent, by political party affiliation, highlighting the outsized role of partisanship in Medicaid policy adoption. To combat these dynamics, additional efforts are needed to improve community-informed decision-making, strengthen evaluation approaches to tie evidence to policymaking, and boost participation in and understanding of the political processes that affect policy change.

Federalism Complicates the Response to the COVID-19 Health and Economic Crisis: What Can Be Done?

Federalism has complicated the US response to the novel coronavirus. States' actions to address the pandemic have varied widely, and federal and state officials have provided conflicting messages. This fragmented approach has surely cost time and lives. Federalism will shape the long-term health and economic impacts of COVID-19, including plans for the future, for at least two reasons: First, federalism exacerbates inequities, as some states have a history of underinvesting in social programs, especially in certain communities. Second, many of the states with the deepest needs are poorly equipped to respond to emergencies due to low taxes and distrust of government, leading to inadequate infrastructure. These dynamics are not new, but they have been laid bare by this crisis. What can policy makers do to address the inequities in health and economic outcomes that federalism intensifies? The first section of this article offers a case study of the Mississippi Delta to illustrate the role of federalism in perpetuating the connection between place, health, and economics. The second section examines challenges that safety net programs will face when moving beyond the acute phase of COVID-19. The final section explores near-, middle-, and long-term policy options to mitigate federalism's harmful side effects.

Have the ACA's Exchanges Succeeded? It's Complicated.

The fight over health insurance exchanges epitomizes the rapid evolution of health reform politics in the decade since the passage of the Affordable Care Act (ACA). The ACA's drafters did not expect the exchanges to be contentious because they would expand private insurance coverage to low- and middle-income individuals who were increasingly unable to obtain employer-sponsored health insurance. Instead, exchanges became one of the primary fronts in the war over Obamacare. Have the exchanges been successful? The answer is not straightforward and requires a historical perspective through a federalism lens. What the ACA has accomplished has depended largely on whether states were invested in or resistant to implementation, as well as individual decisions by state leaders working with federal officials. Our account demonstrates that the states that have engaged with the ACA most consistently appear to have experienced greater exchange-related success. But each aspect of states' engagement with or resistance to the ACA must be counted to fully paint this picture, with significant variation among states. This variation should give pause to those considering next steps in health reform, because state variation can mean innovation and improvement but also lack of coverage, disparities, and diminished access to care.

Cotranslational association of mRNA encoding subunits of heteromeric ion channels.

Oligomers of homomeric voltage-gated potassium channels associate early in biogenesis as the nascent proteins emerge from the polysome. Less is known about how proteins emerging from different polysomes associate to form hetero-oligomeric channels. Here, we report that alternate mRNA transcripts encoding human ether-à-go-go-related gene (hERG) 1a and 1b subunits, which assemble to produce ion channels mediating cardiac repolarization, are physically associated during translation. We show that shRNA specifically targeting either hERG 1a or 1b transcripts reduced levels of both transcripts, but only when they were coexpressed heterologously. Both transcripts could be copurified with an Ab against the nascent hERG 1a N terminus. This interaction occurred even when translation of 1b was prevented, indicating the transcripts associate independent of their encoded proteins. The association was also demonstrated in cardiomyocytes, where levels of both hERG transcripts were reduced by either 1a or 1b shRNA, but native KCNE1 and ryanodine receptor 2 (RYR2) transcripts were unaffected. Changes in protein levels and membrane currents mirrored changes in transcript levels, indicating the targeted transcripts were undergoing translation. The physical association of transcripts encoding different subunits provides the spatial proximity required for nascent proteins to interact during biogenesis, and may represent a general mechanism facilitating assembly of heteromeric protein complexes involved in a range of biological processes.

Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain.

The human human ether-à-go-go-related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained.

Expanding Medicaid Access without Expanding Medicaid: Why Did Some Nonexpansion States Continue the Primary Care Fee Bump?

Six states that have rejected the Patient Protection and Affordable Care Act's (ACA) Medicaid expansion nonetheless extended the primary care "fee bump," by which the federal government increased Medicaid fees for primary care services up to 100 percent of Medicare fees during 2013-14. We conducted semistructured interviews with leaders in five of these states, as well as in three comparison states, to examine why they would continue a provision of the ACA that moderately expands access at significant state expense while rejecting the expansion and its large federal match, focusing on relevant economic, political, and procedural factors. We found that fee bump extension proposals were more successful where they were dissociated from major national policy debates, actionable with the input of relatively few stakeholder entities, and well aligned with preexisting policy-making structures and decision trends. Republican proposals to cap or reduce federal funding for Medicaid, if enacted, would compel states to contain program costs. In this context, states' established decision-making processes for updating Medicaid fee schedules, which we elucidate in this study, may shape the future of the Medicaid program.

Undermining the ACA through the executive branch and federalism: what the Trump administration's approach to health reform means for older Americans.

The ACA has survived multiple existential threats in the legislative and judicial branches, including dozens of congressional attempts at repeal and two major Supreme Court cases. Even as it seems that the ACA is here to stay, what the law accomplishes is far from settled. The Trump administration is using executive powers to weaken the law, in many cases using the same powers that President Obama used to strengthen the effects of the reform. States have responded by seeking flexibility to pursue reforms, such as work requirements, that could not pass Congress and that were not allowed by the Obama administration. There is no indication that the ACA is imploding as President Trump has predicted and seems to desire, although these changes have a real and substantial impact on the lives of many Americans, including the near-elderly in unique ways.

A series of unfortunate events: implications of Republican efforts to repeal and replace the Affordable Care Act For older adults.

This paper discusses Republican efforts to repeal the 2010 Patient Protection and Affordable Care Act (ACA) over President Trump's first year in office (2017) and their impact on near-elderly Americans (50-64 years old). We describe how the ACA's provisions for strengthening health care coverage were particularly advantageous for near-elderly Americans: The law shored up employer-sponsored health care, expanded Medicaid, and-most important-created conditions for a strong individual health insurance market. We then describe Republican efforts to undermine the ACA in the years immediately following its passage, followed by detailed discussion of Republican proposals to repeal and replace the ACA during 2017. We conclude by discussing factors informing the fate of Republican legislation in this area, the potential consequences of the legislation that ultimately passed, and the prospects for future attempts to repeal and replace the ACA through the legislative process.

Social Work's Role in Medicaid Reform: A Qualitative Study.

OBJECTIVES: To critically analyze social work's role in Medicaid reform. METHODS: We conducted semistructured interviews with 46 stakeholders from 10 US states that use a range of Medicaid reform approaches. We identified participants using snowball and purposive sampling. We gathered data in 2016 and analyzed them using qualitative methods. RESULTS: Multiple themes emerged: (1) social work participates in Medicaid reform through clinical practice, including care coordination and case management; (2) there is a gap between social work's practice-level and systems-level involvement in Medicaid innovations; (3) factors hindering social work's involvement in systems-level practice include lack of visibility, insufficient clarity on social work's role and impact, and too few resources within professional organizations; and (4) social workers need more training in health transformation payment models and policy. CONCLUSIONS: Social workers have unique skills that are valuable to building health systems that promote population health and reduce health inequities. Although there is considerable opportunity for social work to increase its role in Medicaid reform, there is little social work involvement at the systems level.

hERG 1b is critical for human cardiac repolarization.

The human ether-à-go-go-related gene (hERG; or KCNH2) encodes the voltage-gated potassium channel underlying IKr, a repolarizing current in the heart. Mutations in KCNH2 or pharmacological agents that reduce IKr slow action potential (AP) repolarization and can trigger cardiac arrhythmias associated with long QT syndrome. Two channel-forming subunits encoded by KCNH2 (hERG 1a and 1b) are expressed in cardiac tissue. In heterologous expression systems, these subunits avidly coassemble and exhibit biophysical and pharmacological properties distinct from those of homomeric hERG 1a channels. Despite these findings, adoption of hERG 1a/1b heteromeric channels as a model for cardiac IKr has been hampered by the lack of evidence for a direct functional role for the 1b subunit in native tissue. In this study, we measured IKr and APs at physiological temperature in cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs). We found that specific knockdown of the 1b subunit using shRNA caused reductions in 1b mRNA, 1b protein levels, and IKr magnitude by roughly one-half. AP duration was increased and AP variability was enhanced relative to controls. Early afterdepolarizations, considered cellular substrates for arrhythmia, were also observed in cells with reduced 1b expression. Similar behavior was elicited when channels were effectively converted from heteromers to 1a homomers by expressing a fragment corresponding to the 1a-specific N-terminal Per-Arnt-Sim domain, which is omitted from hERG 1b by alternate transcription. These findings establish that hERG 1b is critical for normal repolarization and that loss of 1b is proarrhythmic in human cardiac cells.

Policy Diffusion across Disparate Disciplines: Private- and Public-Sector Dynamics Affecting State-Level Adoption of the ACA.

The ACA entails a number of provisions that are profoundly changing the way the states ensure access to medical care, including the expansion of Medicaid and the maintenance of health insurance exchanges. Here, we argue that while federal policy is the originating force of whether these provisions are adopted, individual state decisions are made within a larger ecosystem. This ecosystem has two main components: (1) complementary and competing state and federal policies; and (2) medical provision by a variety of suppliers. Specifically, the merits, costs, and uncertainties associated with adopting these provisions cannot be considered by the states in a vacuum-they may interact with a large set of simultaneously launched or existing local, state, and federal policies aimed at ensuring access to medical care. They may also interact with specific state and federal reimbursement policies and other requirements facing local hospitals and medical providers. We illustrate by example how these interactions may have important implications for the diffusion of ACA provisions. One implication of this perspective is that future empirical work on the rate, determinants, and impacts of ACA coverage expansions on individual and aggregate well-being must incorporate systematic study of this complex public-private sector ecosystem.