TingTing Hong

Cardiac Fibroblasts Regulate Myocardial Proliferation through β1 Integrin Signaling

Growth and expansion of ventricular chambers is essential during heart development and is achieved by proliferation of cardiac progenitors. Adult cardiomyocytes, by contrast, achieve growth through hypertrophy rather than hyperplasia. Although epicardial-derived signals may contribute to the proliferative process in myocytes, the factors and cell types responsible for development of the ventricular myocardial thickness are unclear. Using a coculture system, we found that embryonic cardiac fibroblasts induced proliferation of cardiomyocytes, in contrast to adult cardiac fibroblasts that promoted myocyte hypertrophy. We identified fibronectin, collagen, and heparin-binding EGF-like growth factor as embryonic cardiac fibroblast-specific signals that collaboratively promoted cardiomyocyte proliferation in a paracrine fashion. Myocardial beta1-integrin was required for this proliferative response, and ventricular cardiomyocyte-specific deletion of beta1-integrin in mice resulted in reduced myocardial proliferation and impaired ventricular compaction. These findings reveal a previously unrecognized paracrine function of embryonic cardiac fibroblasts in regulating cardiomyocyte proliferation.

Cardiomyocyte-Specific Deletion of the Vitamin D Receptor Gene Results in Cardiac Hypertrophy

Background— A variety of studies carried out using either human subjects or laboratory animals suggest that vitamin D and its analogues possess important beneficial activity in the cardiovascular system. Using Cre-Lox technology we have selectively deleted the vitamin D receptor (VDR) gene in the cardiac myocyte in an effort to better understand the role of vitamin D in regulating myocyte structure and function. Methods and Results— Targeted deletion of the exon 4 coding sequence in the VDR gene resulted in an increase in myocyte size and left ventricular weight/body weight versus controls both at baseline and following a 7-day infusion of isoproterenol. There was no increase in interstitial fibrosis. These knockout mice demonstrated a reduction in end-diastolic and end-systolic volume by echocardiography, activation of the fetal gene program (ie, increased atrial natriuretic peptide and alpha skeletal actin gene expression), and increased expression of modulatory calcineurin inhibitory protein 1 (MCIP1), a direct downstream target of calcineurin/nuclear factor of activated T cell signaling. Treatment of neonatal cardiomyocytes with 1,25-dihydroxyvitamin D partially reduced isoproterenol-induced MCIP1 mRNA and protein levels and MCIP1 gene promoter activity. Conclusions— Collectively, these studies demonstrate that the vitamin D-VDR signaling system possesses direct, antihypertrophic activity in the heart. This appears to involve, at least in part, suppression of the prohypertrophic calcineurin/NFAT/MCIP1 pathway. These studies identify a potential mechanism to account for the reported beneficial effects of vitamin D in the cardiovascular system.

Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium

Gap junctions form electrical conduits between adjacent myocardial cells, permitting rapid spatial passage of the excitation current essential to each heartbeat. Arrhythmogenic decreases in gap junction coupling are a characteristic of stressed, failing, and aging myocardium, but the mechanisms of decreased coupling are poorly understood. We previously found that microtubules bearing gap junction hemichannels (connexons) can deliver their cargo directly to adherens junctions. The specificity of this delivery requires the microtubule plus-end tracking protein EB1. We performed this study to investigate the hypothesis that the oxidative stress that accompanies acute and chronic ischemic disease perturbs connexon forward trafficking. We found that EB1 was displaced in ischemic human hearts, stressed mouse hearts, and isolated cells subjected to oxidative stress. As a result, we observed limited microtubule interaction with adherens junctions at intercalated discs and reduced connexon delivery and gap junction coupling. A point mutation within the tubulin-binding domain of EB1 reproduced EB1 displacement and diminished connexon delivery, confirming that EB1 displacement can limit gap junction coupling. In zebrafish hearts, oxidative stress also reduced the membrane localization of connexin and slowed the spatial spread of excitation. We anticipate that protecting the microtubule-based forward delivery apparatus of connexons could improve cell-cell coupling and reduce ischemia-related cardiac arrhythmias.

BIN1 localizes the L-type calcium channel to cardiac T-tubules.

Cardiac tubular-like membrane invaginations contain the membrane scaffolding protein BIN1, which tethers dynamic microtubules that deliver calcium channels directly to T-tubule membrane.

Actin Cytoskeleton Rest Stops Regulate Anterograde Traffic of Connexin 43 Vesicles to the Plasma Membrane

Rationale: The intracellular trafficking of connexin 43 (Cx43) hemichannels presents opportunities to regulate cardiomyocyte gap junction coupling. Although it is known that Cx43 hemichannels are transported along microtubules to the plasma membrane, the role of actin in Cx43 forward trafficking is unknown. Objective: We explored whether the actin cytoskeleton is involved in Cx43 forward trafficking. Methods and Results: High-resolution imaging reveals that Cx43 vesicles colocalize with nonsarcomeric actin in adult cardiomyocytes. Live-cell fluorescence imaging reveals Cx43 vesicles as stationary or traveling slowly (average speed 0.09 &mgr;m/s) when associated with actin. At any time, the majority (81.7%) of vesicles travel at subkinesin rates, suggesting that actin is important for Cx43 transport. Using Cx43 containing a hemagglutinin tag in the second extracellular loop, we developed an assay to detect transport of de novo Cx43 hemichannels to the plasma membrane after release from Brefeldin A-induced endoplasmic reticulum/Golgi vesicular transport block. Latrunculin A (for specific interference of actin) was used as an intervention after reinitiation of vesicular transport. Disruption of actin inhibits delivery of Cx43 to the cell surface. Moreover, using the assay in primary cardiomyocytes, actin inhibition causes an 82% decrease (P<0.01) in de novo endogenous Cx43 delivery to cell–cell borders. In Langendorff-perfused mouse heart preparations, Cx43/&bgr;-actin complexing is disrupted during acute ischemia, and inhibition of actin polymerization is sufficient to reduce levels of Cx43 gap junctions at intercalated discs. Conclusions: Actin is a necessary component of the cytoskeleton-based forward trafficking apparatus for Cx43. In cardiomyocytes, Cx43 vesicles spend a majority of their time pausing at nonsarcomeric actin rest stops when not undergoing microtubule-based transport to the plasma membrane. Deleterious effects on this interaction between Cx43 and the actin cytoskeleton during acute ischemia contribute to losses in Cx43 localization at intercalated discs.

BIN1 is reduced and Cav1.2 trafficking is impaired in human failing cardiomyocytes

BACKGROUND Heart failure is a growing epidemic, and a typical aspect of heart failure pathophysiology is altered calcium transients. Normal cardiac calcium transients are initiated by Cav1.2 channels at cardiac T tubules. Bridging integrator 1 (BIN1) is a membrane scaffolding protein that causes Cav1.2 to traffic to T tubules in healthy hearts. The mechanisms of Cav1.2 trafficking in heart failure are not known. OBJECTIVE To study BIN1 expression and its effect on Cav1.2 trafficking in failing hearts. METHODS Intact myocardium and freshly isolated cardiomyocytes from nonfailing and end-stage failing human hearts were used to study BIN1 expression and Cav1.2 localization. To confirm Cav1.2 surface expression dependence on BIN1, patch-clamp recordings were performed of Cav1.2 current in cell lines with and without trafficking-competent BIN1. Also, in adult mouse cardiomyocytes, surface Cav1.2 and calcium transients were studied after small hairpin RNA-mediated knockdown of BIN1. For a functional readout in intact heart, calcium transients and cardiac contractility were analyzed in a zebrafish model with morpholino-mediated knockdown of BIN1. RESULTS BIN1 expression is significantly decreased in failing cardiomyocytes at both mRNA (30% down) and protein (36% down) levels. Peripheral Cav1.2 is reduced to 42% by imaging, and a biochemical T-tubule fraction of Cav1.2 is reduced to 68%. The total calcium current is reduced to 41% in a cell line expressing a nontrafficking BIN1 mutant. In mouse cardiomyocytes, BIN1 knockdown decreases surface Cav1.2 and impairs calcium transients. In zebrafish hearts, BIN1 knockdown causes a 75% reduction in calcium transients and severe ventricular contractile dysfunction. CONCLUSIONS The data indicate that BIN1 is significantly reduced in human heart failure, and this reduction impairs Cav1.2 trafficking, calcium transients, and contractility.

A 14-3-3 Mode-1 Binding Motif Initiates Gap Junction Internalization During Acute Cardiac Ischemia

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14‐3‐3 proteins can regulate protein trafficking, and a 14‐3‐3 mode‐1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C‐terminus. We hypothesized that Cx43Ser373 phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14‐3‐3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14‐3‐3τ in cell lines increases gap junction plaque size at cell–cell borders. Cx43S373A mutation prevents Cx43/14‐3‐3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff‐perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no‐flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen‐activated protein kinase has previously been implicated in Cx43 internalization. The Cx43S373A mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43Ser373 phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.

Dynasore Protects Mitochondria and Improves Cardiac Lusitropy in Langendorff Perfused Mouse Heart

Background Heart failure due to diastolic dysfunction exacts a major economic, morbidity and mortality burden in the United States. Therapeutic agents to improve diastolic dysfunction are limited. It was recently found that Dynamin related protein 1 (Drp1) mediates mitochondrial fission during ischemia/reperfusion (I/R) injury, whereas inhibition of Drp1 decreases myocardial infarct size. We hypothesized that Dynasore, a small noncompetitive dynamin GTPase inhibitor, could have beneficial effects on cardiac physiology during I/R injury. Methods and Results In Langendorff perfused mouse hearts subjected to I/R (30 minutes of global ischemia followed by 1 hour of reperfusion), pretreatment with 1 µM Dynasore prevented I/R induced elevation of left ventricular end diastolic pressure (LVEDP), indicating a significant and specific lusitropic effect. Dynasore also decreased cardiac troponin I efflux during reperfusion and reduced infarct size. In cultured adult mouse cardiomyocytes subjected to oxidative stress, Dynasore increased cardiomyocyte survival and viability identified by trypan blue exclusion assay and reduced cellular Adenosine triphosphate(ATP) depletion. Moreover, in cultured cells, Dynasore pretreatment protected mitochondrial fragmentation induced by oxidative stress. Conclusion Dynasore protects cardiac lusitropy and limits cell damage through a mechanism that maintains mitochondrial morphology and intracellular ATP in stressed cells. Mitochondrial protection through an agent such as Dynasore can have clinical benefit by positively influencing the energetics of diastolic dysfunction.

Plasma BIN1 correlates with heart failure and predicts arrhythmia in patients with arrhythmogenic right ventricular cardiomyopathy

BACKGROUND Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disorder involving diseased cardiac muscle. Bridging integrator 1 (BIN1) is a membrane-associated protein important to cardiomyocyte homeostasis and is downregulated in cardiomyopathy. We hypothesized that BIN1 could be released into the circulation and that blood-available BIN1 can provide useful data on the cardiac status of patients whose hearts are failing secondary to ARVC. OBJECTIVE To determine whether plasma BIN1 levels can be used to measure disease severity in patients with ARVC. METHODS We performed a retrospective cohort study of 24 patients with ARVC. Plasma BIN1 levels were assessed for their ability to correlate with cardiac functional status and predict ventricular arrhythmias. RESULTS Mean plasma BIN1 levels were decreased in patients with ARVC with heart failure (15 ± 7 vs 60 ± 17 in patients without heart failure, P <.05; the plasma BIN1 level was 60 ± 10 in non-ARVC normal controls). BIN1 levels correlated inversely with number of previous ventricular arrhythmia (R = -.47; P <.05), and low BIN1 levels correctly classified patients with advanced heart failure or ventricular arrhythmia (receiver operator curve area under the curve of 0.88 ± 0.07). Low BIN1 levels also predicted future ventricular arrhythmias (receiver operator curve area under the curve of 0.89 ± 0.09). In a stratified analysis, BIN1 levels could predict future arrhythmias in patients without severe heart failure (n = 20) with an accuracy of 82%. In the 7 patients with ARVC with serial blood samples, all of whom had evidence of disease progression during follow-up, plasma BIN1 levels decreased significantly (a decrease of 63%; P <.05). CONCLUSIONS Plasma BIN1 level seems to correlate with cardiac functional status and the presence or absence of sustained ventricular arrhythmias in a small cohort of patients with ARVC and can predict future ventricular arrhythmias.

Isoproterenol Promotes Rapid Ryanodine Receptor Movement to Bridging Integrator 1 (BIN1)–Organized Dyads

Background— The key pathophysiology of human acquired heart failure is impaired calcium transient, which is initiated at dyads consisting of ryanodine receptors (RyRs) at sarcoplasmic reticulum apposing CaV1.2 channels at t-tubules. Sympathetic tone regulates myocardial calcium transients through &bgr;-adrenergic receptor (&bgr;-AR)–mediated phosphorylation of dyadic proteins. Phosphorylated RyRs (P-RyR) have increased calcium sensitivity and open probability, amplifying calcium transient at a cost of receptor instability. Given that bridging integrator 1 (BIN1) organizes t-tubule microfolds and facilitates CaV1.2 delivery, we explored whether &bgr;-AR–regulated RyRs are also affected by BIN1. Methods and Results— Isolated adult mouse hearts or cardiomyocytes were perfused for 5 minutes with the &bgr;-AR agonist isoproterenol (1 µmol/L) or the blockers CGP+ICI (baseline). Using biochemistry and superresolution fluorescent imaging, we identified that BIN1 clusters P-RyR and CaV1.2. Acute &bgr;-AR activation increases coimmunoprecipitation between P-RyR and cardiac spliced BIN1+13+17 (with exons 13 and 17). Isoproterenol redistributes BIN1 to t-tubules, recruiting P-RyRs and improving the calcium transient. In cardiac-specific Bin1 heterozygote mice, isoproterenol fails to concentrate BIN1 to t-tubules, impairing P-RyR recruitment. The resultant accumulation of uncoupled P-RyRs increases the incidence of spontaneous calcium release. In human hearts with end-stage ischemic cardiomyopathy, we find that BIN1 is also 50% reduced, with diminished P-RyR association with BIN1. Conclusions— On &bgr;-AR activation, reorganization of BIN1-induced microdomains recruits P-RyR into dyads, increasing the calcium transient while preserving electric stability. When BIN1 is reduced as in human acquired heart failure, acute stress impairs microdomain formation, limiting contractility and promoting arrhythmias.