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The fast transient outward potassium current (Ito,f) plays a key role in phase 1 repolarization of the human cardiac action potential (AP) and its reduction in heart failure (HF) contributes to the loss of contractility. Therefore, restoring Ito,f might be beneficial for treating HF. The coding sequence of a P2A peptide was cloned, in frame, between Kv4.3 and KChIP2.1 genes and ribosomal skipping was confirmed by Western blotting. Typical Ito,f properties with slowed inactivation and accelerated recovery from inactivation due to the association of KChIP2.1 with Kv4.3 was seen in transfected HEK293 cells. Both bicistronic components trafficked to the plasmamembrane and in adenovirus transduced rabbit cardiomyocytes both t-tubular and sarcolemmal construct labelling appeared. The resulting current was similar to Ito,f seen in human ventricular cardiomyocytes and was 50% blocked at ~0.8 mmol/l 4-aminopyridine and increased ~30% by 5 μmol/l NS5806 (an Ito,f agonist). Variation in the density of the expressed Ito,f, in rabbit cardiomyocytes recapitulated typical species-dependent variations in AP morphology. Simultaneous voltage recording and intracellular Ca2+ imaging showed that modification of phase 1 to a non-failing human phenotype improved the rate of rise and magnitude of the Ca2+ transient. Ito,f expression also reduced AP triangulation but did not affect ICa,L and INa magnitudes. This raises the possibility for a new gene-based therapeutic approach to HF based on selective phase 1 modification. ispartof: JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY vol:164 pages:29-41 ispartof: location:England status: published

Ischemic heart disease is the most common cause of lethal ventricular arrhythmias and sudden cardiac death (SCD). In patients who are at high risk after myocardial infarction, implantable cardioverter defibrillators are the most effective treatment to reduce incidence of SCD and ablation therapy can be effective for ventricular arrhythmias with identifiable culprit lesions. Yet, these approaches are not always successful and come with a considerable cost, while pharmacological management is often poor and ineffective, and occasionally proarrhythmic. Advances in mechanistic insights of arrhythmias and technological innovation have led to improved interventional approaches that are being evaluated clinically, yet pharmacological advancement has remained behind. We review the mechanistic basis for current management and provide a perspective for gaining new insights that centre on the complex tissue architecture of the arrhythmogenic infarct and border zone with surviving cardiac myocytes as the source of triggers and central players in re-entry circuits. Identification of the arrhythmia critical sites and characterisation of the molecular signature unique to these sites can open avenues for targeted therapy and reduce off-target effects that have hampered systemic pharmacotherapy. Such advances are in line with precision medicine and a patient-tailored therapy. ispartof: CELLS vol:10 issue:10 ispartof: location:Switzerland status: published

Aims Takotsubo syndrome (TTS) is an acute heart failure, typically triggered by high adrenaline during physical or emotional stress. It is distinguished from myocardial infarction (MI) by a characteristic pattern of ventricular basal hypercontractility with hypokinesis of apical segments, and absence of coronary occlusion. We aimed to understand whether recently discovered circulating biomarkers miR-16 and miR-26a, which differentiate TTS from MI at presentation, were mechanistically involved in the pathophysiology of TTS. Methods and results miR-16 and miR-26a were co-overexpressed in rats with AAV and TTS induced with an adrenaline bolus. Untreated isolated rat cardiomyocytes were transfected with pre-/anti-miRs and functionally assessed. Ventricular basal hypercontraction and apical depression were accentuated in miR-transfected animals after induction of TTS. In vitro miR-16 and/or miR-26a overexpression in isolated apical (but not basal) cardiomyocytes produced strong depression of contraction, with loss of adrenaline sensitivity. They also enhanced the initial positive inotropic effect of adrenaline in basal cells. Decreased contractility after TTS-miRs was reproduced in non-failing human apical cardiomyocytes. Bioinformatic profiling of miR targets, followed by expression assays and functional experiments, identified reductions of CACNB1 (L-type calcium channel Cavβ subunit), RGS4 (regulator of G-protein signalling 4) and G-protein subunit Gβ (GNB1) as underlying these effects. Conclusion miR-16 and miR-26a sensitise the heart to TTS-like changes produced by adrenaline. Since these miRs have been associated with anxiety and depression, they could provide a mechanism whereby priming of the heart by previous stress causes an increased likelihood of TTS in the future. Translational perspective TTS-associated miRs have the potential to be active players predisposing to TTS. Feasibly, their measurement in recovered TTS patients during subsequent periods of stress could be used to predict likelihood of recurrence, a significant risk in this population, and allow preventative action. Since they have been reported as raised in anxiety and depression, they could be part of a priming mechanism where chronic stress predisposes to an acute episode. Understanding the mechanistic basis for the sensitisation may also allow design of other prophylactic pharmacological therapies, including the pre/anti-miR constructs which are now starting to reach the clinic.

BACKGROUND: Interstitial fibrosis is an important component of diastolic, and systolic, dysfunction in heart failure (HF) and depends on activation and differentiation of fibroblasts into myofibroblasts (MyoFb). Recent clinical evidence suggests that in late-stage HF, fibrosis is not reversible. OBJECTIVES: The study aims to examine the degree of differentiation of cardiac MyoFb in end-stage HF and the potential for their phenotypic reversibility. METHODS: Fibroblasts were isolated from the left ventricle of the explanted hearts of transplant recipients (ischemic and dilated cardiomyopathy), and from nonused donor hearts. Fibroblasts were maintained in culture without passaging for 4 or 8 days (treatment studies). Phenotyping included functional testing, immunostaining, and expression studies for markers of differentiation. These data were complemented with immunohistology and expression studies in tissue samples. RESULTS: Interstitial fibrosis with cross-linked collagen is prominent in HF hearts, with presence of activated MyoFbs. Tissue levels of transforming growth factor (TGF)-β1, lysyl oxidase, periostin, and osteopontin are elevated. Fibroblastic cells isolated from HF hearts are predominantly MyoFb, proliferative or nonproliferative, with mature α-smooth muscle actin stress fibers. HF MyoFb express high levels of profibrotic cytokines and the TGF-β1 pathway is activated. Inhibition of TGF-β1 receptor kinase in HF MyoFb promotes dedifferentiation of MyoFb with loss of α-smooth muscle actin and depolymerization of stress fibers, and reduces the expression of profibrotic genes and cytokines levels to non-HF levels. CONCLUSION: MyoFb in end-stage HF have a variable degree of differentiation and retain the capacity to return to a less activated state, validating the potential for developing antifibrotic therapy targeting MyoFb. ispartof: JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY vol:73 issue:18 pages:2267-2282 ispartof: location:United States status: published

Fibroblast (Fb) differentiation and interstitial fibrosis contribute to cardiac remodeling and loss of function after myocardial infarction (MI). We investigated regional presence and regulation of fibrosis in a pig MI model. In vivo analysis of regional function and perfusion defined three regions: the scar, the myocardium adjacent to the scar (MIadjacent, reduced function, reduced perfusion reserve), and the remote myocardium (MIremote, minimal functional deficit, maintained perfusion). Interstitial and perivascular fibrosis, and increase of collagen type I, was only observed in the MIadjacent. Fb activated protein-alpha (FAP-α) was enriched in MIadjacent compared to MIremote. TGF-β1, which triggers Fb differentiation, was upregulated in both MIadjacent and MIremote, whereas lysyl oxidase, a regulator of collagen cross-linking, and the proteoglycans decorin and biglycan were only increased in the MIadjacent. Fb isolated and cultured for 4 days had myoFb characteristics with little difference between MIremote and MIadjacent, although RNA sequencing revealed differences in gene expression profiles. Fbs from all regions maintained proliferative capacity, and induced contraction of 3-D collagen matrices but scar myoFb was more effective. These data suggest that after MI, signaling through TGF-β1, possibly related to increased mechanical load, drives Fb activation throughout the left ventricle while regional signaling determines further maturation and extracellular matrix remodeling after MI.

Beat-to-beat variability of ventricular repolarization (BVR) has been proposed as a strong predictor of Torsades de Pointes (TdP). BVR is also observed at the myocyte level, and a number of studies have shown the importance of calcium handling in influencing this parameter. The chronic AV block (CAVB) dog is a model of TdP arrhythmia in cardiac hypertrophy, and myocytes from these animals show extensive remodeling, including of Ca2 + handling. This remodeling process also leads to increased BVR. We aimed to determine the role that (local) Ca2 + handling plays in BVR.\ud \ud In isolated LV myocytes an exponential relationship was observed between BVR magnitude and action potential duration (APD) at baseline. Inhibition of Ca2 + release from sarcoplasmic reticulum (SR) with thapsigargin resulted in a reduction of [Ca2 +]i, and of both BVR and APD. Increasing ICaL in the presence of thapsigargin restored APD but BVR remained low. In contrast, increasing ICaL with preserved Ca2 + release increased both APD and BVR. Inhibition of Ca2 + release with caffeine, as with thapsigargin, reduced BVR despite maintained APD. Simultaneous inhibition of Na+/Ca2 + exchange and ICaL decreased APD and BVR to similar degrees, whilst increasing diastolic Ca2 +. Buffering of Ca2 + transients with BAPTA reduced BVR for a given APD to a greater extent than buffering with EGTA, suggesting subsarcolemmal Ca2 + transients modulated BVR to a larger extent than the cytosolic Ca2 + transient.\ud \ud In conclusion, BVR in hypertrophied dog myocytes, at any APD, is strongly dependent on SR Ca2 + release, which may act through modulation of the l-type Ca2 + current in a subsarcolemmal microdomain.

In large mammals, many SR Ca2+ channels (RyRs) are uncoupled from sarcolemma or T-tubules (TTs). Tools for exploring RyR regulation in relation to T-tubule proximity are limited. Here we present a method to measure TT distance and investigate release from coupled and uncoupled RyRs. In isolated pig ventricular myocytes consecutive vertical confocal images were taken after labeling with wheat germ agglutinin-Alexa594. After image processing, the 3-D TT geometry was assessed using Euclidean distance mapping. Whole-cell voltage clamp was subsequently used to elicit Ca2+ release at 0.5, 1 and 2 Hz and Ca2+ was recorded in a confocal line scan (0.3 micron pixel-1, 649 Hz). The temporal mid-point of the local Ca2+ transient upstroke (TF50) was used to assess latency of release. Correlation of the TF50 for each site with distance to TT in 3-D yielded a linear relationship for distance between 0.5 and 3 μm from T-tubules. This allowed the use of TF50 to map subcellular Ca2+ release regions as coupled ( 2 μm). In coupled regions, TF50 occurred within 18 ms, 17 ms and 12 ms, respectively for 0.5, 1 and 2 Hz. This resulted in 56% (0.5 Hz), 57% (1 Hz) and 49% (2 Hz) of coupled regions. Spark frequency increased with stimulation frequency in coupled release sites but significantly less so in uncoupled ones. In the presence of the F-actin disrupter Cytochalasin D, this increase in spark frequency was abolished and there was no difference between coupled and uncoupled sites. Therefore, the regional discrimination of RyR reveals a preferential activation of coupled RyRs with frequency, which is dependent on a cytoskeletal interaction.

Purpose: In ventricular myocytes of rodents, ryanodine receptors (RyRs) are typically organized at Z-lines where the sarcoplasmic reticulum forms dyads with T-tubules (TTs). In large mammals, TT density is lower and not all RyRs are coupled to the TTs. Recently, we have shown that non-coupled RyRs lack a CaMKII and ROS-dependent microdomain modulation during rate adaptation. Here we examine how this microdomain modulation may be altered in ischemic cardiomyopathy where the fraction of non-coupled RyRs is increased. Methods: Using an established pig model of chronic ischemia and myocardial infarction (MI, N=7), we studied myocytes adjacent to the MI and compared these to myocytes from SHAM pigs (N=5) using whole-cell voltage clamp with Fluo-4 as a [Ca2+]i indicator and confocal line scan imaging. Spontaneous Ca2+ sparks were recorded during a 15s period following stimulation and assigned to different subcellular regions categorized as coupled or non-coupled RyR using a specific algorithm. All data were obtained in at least 3 different animals. Results: In myocytes from SHAM, we confirmed the specific modulation of coupled, but not of non-coupled, RyR as seen in control hearts, i.e. an increase of spark frequency at 2 Hz stimulation that is dependent on CaMKII and NOX2-generated ROS (#sparks/100 μm/s in SHAM: 0.8 ± 0.1 at 0.5 Hz to 2.3 ± 0.2 at 2 Hz, vs. 1.5 ± 0.2 with AIP at 2 Hz, and 1.1 ± 0.2 with gp91 ds-tat peptide at 2 Hz, p-value < 0.05 vs. control, n=7-14 in each group). In MI myocytes this modulation of coupled RyR was absent (#sparks/100 μm/s at 2 Hz not different from at 0.5 Hz and no effect of AIP and gp-91 ds-tat peptide). The fraction of non-coupled RyR was larger in MI (orphaned RyR) and their spark frequency at 2 Hz was significantly higher compared to SHAM. In contrast to SHAM, the response of these orphaned RyR was sensitive to CaMKII inhibition (AIP) (at 2 Hz #sparks/100 μm/s was 5.3 ± 0.5 at baseline vs. 3.1 ± 0.6 with AIP, p-value < 0.05, n=8-10 in each group). A significant reduction in spark frequency was observed in these orphaned RyRs after global ROS scavenging (NAC) (2 Hz #sparks/100 μm/s was 5.2 ± 0.5 at baseline vs. 2.9 ± 0.6 with NAC, p-value < 0.05, n=9-10 in each group) and after mitochondrial ROS inhibition using mitoTEMPO (at 2 Hz #sparks/100 μm/s was 2.9 ± 0.4 at baseline vs. 1.1 ± 0.3 with mitoTEMPO, p-value < 0.001, n=8 in each group) while NOX2 inhibition had no effect (gp91 ds-tat peptide, n=10–14). Conclusion: After MI there is a novel RyR microdomain organization favoring sparks in orphaned RyR, possibly related to mitochondrial ROS production.