ATAC-Seq Reveals an Isl1 Enhancer That Regulates Sinoatrial Node Development and Function.

RATIONALE: Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate the heartbeat. Although critical SAN transcription factors, including Isl1 (Islet-1), Tbx3 (T-box transcription factor 3), and Shox2 (short-stature homeobox protein 2), have been identified, the cis-regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified. OBJECTIVE: To define the epigenetic profile of PCs using comparative ATAC-seq (assay for transposase-accessible chromatin with sequencing) and to identify novel enhancers involved in SAN gene regulation, development, and function. METHODS AND RESULTS: We used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched assay for transposase-accessible chromatin peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF (transcription factor) binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at the Isl1 locus that was active specifically in the cardiac inflow at embryonic day 8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations. CONCLUSIONS: (1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) cis-regulation of Isl1 specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding human ISL1 enhancer may regulate human SAN function.

Coronary reperfusion and clinical outcomes after thrombus aspiration during primary percutaneous coronary intervention: findings from the HORIZONS-AMI trial.

OBJECTIVES: To assess the quality of coronary reperfusion and long-term clinical outcomes of patients enrolled in the HORIZONS-AMI trial according to the use of thrombus aspiration (TA). BACKGROUND: The impact of manual TA on microvascular perfusion and clinical outcomes in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (pPCI) is unsettled. METHODS: In this retrospective, nonrandomized, subgroup analysis, the authors evaluated thrombolysis in myocardial infarction (TIMI) flow, tissue myocardial perfusion grade (TMPG), ST-segment resolution (STR), net adverse clinical events (NACE), and major adverse cardiac events (MACE) in patients undergoing pPCI with or without manual TA. RESULTS: A total of 318 patients had pPCI with upfront TA, and 2,917 patients had pPCI without TA. Patients who had TA were more likely to have TIMI 0/1 flow at baseline (75.1% vs. 63.7%, P < 0.0001). There was no difference in the rates of final TIMI 3 flow (90.2% vs. 92.3%, P = 0.19) or dynamic TMPG 2-3 (77.4% vs. 76.4%, P = 0.68). STR ≥70% was similar in both groups at 60 minutes but higher in the TA group at discharge (71.8% vs. 64.6%, P = 0.02). After multivariable adjustment, TA did not predict MACE at 30 days (HR 0.96 [0.51-1.80], P = 0.90), 1 year (HR 1.03 [0.68-1.55], P = 0.89), or 3 years (HR 1.13 [0.86-1.48], P = 0.39). Stent thrombosis did not differ at 1 year or 3 years. CONCLUSIONS: In STEMI patients undergoing pPCI, the use of manual TA was associated with improved STR at discharge, but not with any difference in final TIMI flow, TMPG, or MACE.

An injury-responsive mmp14b enhancer is required for heart regeneration.

Mammals have limited capacity for heart regeneration, whereas zebrafish have extraordinary regeneration abilities. During zebrafish heart regeneration, endothelial cells promote cardiomyocyte cell cycle reentry and myocardial repair, but the mechanisms responsible for promoting an injury microenvironment conducive to regeneration remain incompletely defined. Here, we identify the matrix metalloproteinase Mmp14b as an essential regulator of heart regeneration. We identify a TEAD-dependent mmp14b endothelial enhancer induced by heart injury in zebrafish and mice, and we show that the enhancer is required for regeneration, supporting a role for Hippo signaling upstream of mmp14b. Last, we show that MMP-14 function in mice is important for the accumulation of Agrin, an essential regulator of neonatal mouse heart regeneration. These findings reveal mechanisms for extracellular matrix remodeling that promote heart regeneration.

Single Cell Multi-Omics of an iPSC Model of Human Sinoatrial Node Development Reveals Genetic Determinants of Heart Rate and Arrhythmia Susceptibility.

Cellular heterogeneity within the sinoatrial node (SAN) is functionally important but has been difficult to model in vitro , presenting a major obstacle to studies of heart rate regulation and arrhythmias. Here we describe a scalable method to derive sinoatrial node pacemaker cardiomyocytes (PCs) from human induced pluripotent stem cells that recapitulates differentiation into distinct PC subtypes, including SAN Head, SAN Tail, transitional zone cells, and sinus venosus myocardium. Single cell (sc) RNA-sequencing, sc-ATAC-sequencing, and trajectory analyses were used to define epigenetic and transcriptomic signatures of each cell type, and to identify novel transcriptional pathways important for PC subtype differentiation. Integration of our multi-omics datasets with genome wide association studies uncovered cell type-specific regulatory elements that associated with heart rate regulation and susceptibility to atrial fibrillation. Taken together, these datasets validate a novel, robust, and realistic in vitro platform that will enable deeper mechanistic exploration of human cardiac automaticity and arrhythmia.

Cold shock domain-containing protein E1 is a posttranscriptional regulator of the LDL receptor.

The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current therapies and known causes of familial hypercholesterolemia. We found that cold shock domain-containing protein E1 (CSDE1) enhanced hepatic LDLR messenger RNA (mRNA) decay via its 3' untranslated region and regulated atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens in a tissue culture model, we identified 40 specific regulators of the LDLR that were not previously identified by observational human genetic studies. Among these, we demonstrated that, in HepG2 cells, CSDE1 regulated the LDLR at least as strongly as statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. In addition, we showed that hepatic gene silencing of Csde1 treated diet-induced dyslipidemia in mice to a similar degree as Pcsk9 silencing. These results suggest the therapeutic potential of targeting CSDE1 to manipulate the posttranscriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. Our approach of modeling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, may serve as a generalizable template for the identification of therapeutic targets in other human disease states.

Locations of the beta1 transmembrane helices in the BK potassium channel.

BK channels are composed of alpha-subunits, which form a voltage- and Ca(2+)-gated potassium channel, and of modulatory beta-subunits. The beta1-subunit is expressed in smooth muscle, where it renders the BK channel sensitive to [Ca(2+)](i) in a voltage range near the smooth-muscle resting potential and slows activation and deactivation. BK channel acts thereby as a damped feedback regulator of voltage-dependent Ca(2+) channels and of smooth muscle tone. We explored the contacts between alpha and beta1 by determining the extent of endogenous disulfide bond formation between cysteines substituted just extracellular to the two beta1 transmembrane (TM) helices, TM1 and TM2, and to the seven alpha TM helices, consisting of S1-S6, conserved in all voltage-dependent potassium channels, and the unique S0 helix, which we previously concluded was partly surrounded by S1-S4. We now find that the extracellular ends of beta1 TM2 and alpha S0 are in contact and that beta1 TM1 is close to both S1 and S2. The extracellular ends of TM1 and TM2 are not close to S3-S6. In almost all cases, cross-linking of TM2 to S0 or of TM1 to S1 or S2 shifted the conductance-voltage curves toward more positive potentials, slowed activation, and speeded deactivation, and in general favored the closed state. TM1 and TM2 are in position to contribute, in concert with the extracellular loop and the intracellular N- and C-terminal tails of beta1, to the modulation of BK channel function.

Location of the beta 4 transmembrane helices in the BK potassium channel.

Large-conductance, voltage- and Ca(2+)-gated potassium (BK) channels control excitability in a number of cell types. BK channels are composed of alpha subunits, which contain the voltage-sensor domains and the Ca(2+)- sensor domains and form the pore, and often one of four types of beta subunits, which modulate the channel in a cell-specific manner. beta 4 is expressed in neurons throughout the brain. Deletion of beta 4 in mice causes temporal lobe epilepsy. Compared with channels composed of alpha alone, channels composed of alpha and beta 4 activate and deactivate more slowly. We inferred the locations of the two beta 4 transmembrane (TM) helices TM1 and TM2 relative to the seven alpha TM helices, S0-S6, from the extent of disulfide bond formation between cysteines substituted in the extracellular flanks of these TM helices. We found that beta 4 TM2 is close to alpha S0 and that beta 4 TM1 is close to both alpha S1 and S2. At least at their extracellular ends, TM1 and TM2 are not close to S3-S6. In six of eight of the most highly crosslinked cysteine pairs, four crosslinks from TM2 to S0 and one each from TM1 to S1 and S2 had small effects on the V(50) and on the rates of activation and deactivation. That disulfide crosslinking caused only small functional perturbations is consistent with the proximity of the extracellular ends of TM2 to S0 and of TM1 to S1 and to S2, in both the open and closed states.

The BK potassium channel in the vascular smooth muscle and kidney: α- and ß-subunits.

The large-conductance voltage and calcium-sensitive BK channel is important in many electrically active cells. Its unique sensitivity to both intracellular calcium levels and membrane potential makes it a key regulator of intracellular calcium, a critical second messenger in cells. The BK channel is expressed ubiquitously in the body and has particularly significant roles in the neuronal and smooth muscle cells. More recently, the importance of BK channel in the pathophysiology of hypertension has been demonstrated. In this review, we will focus on function, regulation, and clinical significance of the BK channel. We will also specifically concentrate on one of its ß-subunits, ß1, which is important in vascular and renal physiology.

A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish.

Zebrafish is a powerful model for forward genetics. Reverse genetic approaches are limited by the time required to generate stable mutant lines. We describe a system for gene knockout that consistently produces null phenotypes in G0 zebrafish. Yolk injection of sets of four CRISPR/Cas9 ribonucleoprotein complexes redundantly targeting a single gene recapitulated germline-transmitted knockout phenotypes in >90% of G0 embryos for each of 8 test genes. Early embryonic (6 hpf) and stable adult phenotypes were produced. Simultaneous multi-gene knockout was feasible but associated with toxicity in some cases. To facilitate use, we generated a lookup table of four-guide sets for 21,386 zebrafish genes and validated several. Using this resource, we targeted 50 cardiomyocyte transcriptional regulators and uncovered a role of zbtb16a in cardiac development. This system provides a platform for rapid screening of genes of interest in development, physiology, and disease models in zebrafish.

Orientations and proximities of the extracellular ends of transmembrane helices S0 and S4 in open and closed BK potassium channels.

The large-conductance potassium channel (BK) α subunit contains a transmembrane (TM) helix S0 preceding the canonical TM helices S1 through S6. S0 lies between S4 and the TM2 helix of the regulatory ß1 subunit. Pairs of Cys were substituted in the first helical turns in the membrane of BK α S0 and S4 and in ß1 TM2. One such pair, W22C in S0 and W203C in S4, was 95% crosslinked endogenously. Under voltage-clamp conditions in outside-out patches, this crosslink was reduced by DTT and reoxidized by a membrane-impermeant bis-quaternary ammonium derivative of diamide. The rate constants for this reoxidation were not significantly different in the open and closed states of the channel. Thus, these two residues are approximately equally close in the two states. In addition, 90% crosslinking of a second pair, R20C in S0 and W203C in S4, had no effect on the V50 for opening. Taken together, these findings indicate that separation between residues at the extracellular ends of S0 and S4 is not required for voltage-sensor activation. On the contrary, even though W22C and W203C were equally likely to form a disulfide in the activated and deactivated states, relative immobilization by crosslinking of these two residues favored the activated state. Furthermore, the efficiency of recrosslinking of W22C and W203C on the cell surface was greater in the presence of the ß1 subunit than in its absence, consistent with ß1 acting through S0 to stabilize its immobilization relative to α S4.

Positions of ß2 and ß3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel.

Large-conductance voltage- and Ca(2+)-gated K(+) channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory ß subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in ß2 and ß3, as in ß1 and ß4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse ß3 (mß3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0-αS6. We identified by elimination mß3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in ß1. These are conserved in ß2-ß4, which have four additional Cys (eight in total), except that mß3 has one fewer. In ß1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mß3, the free Cys is at position 7; position 2 lacks a Cys present in all other ß2-ß4; and the disulfide pattern is 1-8, 3-4, and 5-6. Presumably, Cys 2 cross-links to Cys 7 in all other ß2-ß4. Cross-linking of mß3 Cys152 to Cys substituted in the flanks of αS0-S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mß3 perturbed. Although the mß3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.

Location of modulatory beta subunits in BK potassium channels.

Large-conductance voltage- and calcium-activated potassium (BK) channels contain four pore-forming alpha subunits and four modulatory beta subunits. From the extents of disulfide cross-linking in channels on the cell surface between cysteine (Cys) substituted for residues in the first turns in the membrane of the S0 transmembrane (TM) helix, unique to BK alpha, and of the voltage-sensing domain TM helices S1-S4, we infer that S0 is next to S3 and S4, but not to S1 and S2. Furthermore, of the two beta1 TM helices, TM2 is next to S0, and TM1 is next to TM2. Coexpression of alpha with two substituted Cys's, one in S0 and one in S2, and beta1 also with two substituted Cys's, one in TM1 and one in TM2, resulted in two alphas cross-linked by one beta. Thus, each beta lies between and can interact with the voltage-sensing domains of two adjacent alpha subunits.

Clinical outcomes after cardiac transplantation in muscular dystrophy patients.

BACKGROUND: Patients with muscular dystrophy are at risk of developing a dilated cardiomyopathy and can progress to advanced heart failure. At present, it is not known whether such patients can safely undergo cardiac transplantation. METHODS: This was a retrospective review of the Cardiac Transplant Research Database, a multi-institutional registry of 29 transplant centers in the United States, from the years 1990 to 2005. The post-cardiac transplant outcomes of 29 patients with muscular dystrophy were compared with 275 non-muscular dystrophy patients with non-ischemic cardiomyopathy, matched for age, body mass index, gender, and race. RESULTS: Becker's muscular dystrophy was present in 52% of the patients. Survival in the muscular dystrophy patients was similar to the controls at 1 year (89% vs 91%; p = 0.5) and at 5 years (83% vs 78%; p = 0.5). The differences in rates of cumulative infection, rejection, or allograft vasculopathy between the 2 groups were not significant (p > 0.5 for all comparisons). CONCLUSIONS: Recognizing the limitations of the present investigation (ie, selection bias and data lacking in the functional capacity of the muscular dystrophy patients), the current study suggests that the clinical outcomes after cardiac transplantation in selected patients with muscular dystrophy are similar to those seen in age-matched patients with non-ischemic cardiomyopathy.