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Pulmonary hypertension is a complex disorder characterized by pulmonary vascular remodeling and right ventricular hypertrophy, leading to right heart failure. The mechanisms underlying this process are not well understood. We hypothesize that the structural remodeling occurring in the cardiomyocytes of the right ventricle affects the cytosolic Ca 2+ handling leading to arrhythmias. After 12 days of monocrotaline-induced pulmonary hypertension in rats, epicardial mapping showed electrical remodeling in both ventricles. In myocytes isolated from the hypertensive rats, a combination of high-speed camera and confocal line-scan documented a prolongation of Ca 2+ transients along with a higher local Ca 2+ -release activity. These Ca 2+ transients were less synchronous than in controls, likely due to disorganized transverse-axial tubular system. In fact, following pulmonary hypertension, hypertrophied right ventricular myocytes showed significantly reduced number of transverse tubules and increased number of axial tubules; however, Stimulation Emission Depletion microscopy demonstrated that the colocalization of L-type Ca 2+ channels and RyR2 (ryanodine receptor 2) remained unchanged. Finally, Stimulation Emission Depletion microscopy and super-resolution scanning patch-clamp analysis uncovered a decrease in the density of active L-type Ca 2+ channels in right ventricular myocytes with an elevated open probability of the T-tubule anchored channels. This may represent a general mechanism of how nanoscale structural changes at the early stage of pulmonary hypertension impact on the development of the end stage failing phenotype in the right ventricle.

Dysregulated intracellular Ca

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: Sympathetic activation in ischemic heart disease can cause lethal arrhythmias. These often are preceded by premature ventricular complexes (PVCs), which at the cellular level could result from delayed afterdepolarizations. OBJECTIVE: The purpose of this study was to identify and map vulnerable areas for arrhythmia initiation after myocardial infarction (MI) and to explore the link between PVCs and cellular events. METHODS: Anterior-septal wall MI was induced by 120 minutes of coronary occlusion followed by reperfusion (27 MI and 16 sham pigs). After 4 weeks, EnSite™ electroanatomic mapping combined with imaging was performed to precisely locate PVC sites of origin and subsequently record monophasic action potentials. Cardiomyocytes were isolated from different regions to study regional cellular remodeling. Isoproterenol was used as a surrogate for adrenergic stimulation both in vivo and in cardiomyocytes. RESULTS: PVCs originated from the MI border zone (BZ) and occurred at discrete areas with clusters of PVCs within the BZ. At these sites, frequent delayed afterdepolarizations and occasional associated spontaneous action potentials translating to a PVC were present. Cardiomyocytes isolated from the MI BZ exhibited more spontaneous action potentials than cardiomyocytes from remote regions. Sensitivity to adrenergic stimulation was increased in MI, in vivo and in cardiomyocytes. In awake, freely moving MI animals, frequent PVCs, ventricular arrhythmia, and sudden cardiac death occurred spontaneously at moderately elevated heart rates. CONCLUSION: Post-MI, arrhythmias initiate from discrete vulnerable areas within the BZ, where delayed afterdepolarizations, related to increased adrenergic response of BZ cardiomyocytes, can generate PVCs. ispartof: HEART RHYTHM vol:18 issue:11 pages:1976-1987 ispartof: location:United States status: published

Dries et al. show that injured myocardial slices from the subendocardium are more susceptible to spontaneous Ca2+ release events and whole-slice contractions than those from the subepicardium, and that this is reduced by CaMKII inhibition., Spontaneous Ca2+ release (SCR) can cause triggered activity and initiate arrhythmias. Intrinsic transmural heterogeneities in Ca2+ handling and their propensity to disease remodeling may differentially modulate SCR throughout the left ventricular (LV) wall and cause transmural differences in arrhythmia susceptibility. Here, we aimed to dissect the effect of cardiac injury on SCR in different regions in the intact LV myocardium using cryoinjury on rat living myocardial slices (LMS). We studied SCR under proarrhythmic conditions using a fluorescent Ca2+ indicator and high-resolution imaging in LMS from the subendocardium (ENDO) and subepicardium (EPI). Cryoinjury caused structural remodeling, with loss in T-tubule density and an increased time of Ca2+ transients to peak after injury. In ENDO LMS, the Ca2+ transient amplitude and decay phase were reduced, while these were not affected in EPI LMS after cryoinjury. The frequency of spontaneous whole-slice contractions increased in ENDO LMS without affecting EPI LMS after injury. Cryoinjury caused an increase in foci that generates SCR in both ENDO and EPI LMS. In ENDO LMS, SCRs were more closely distributed and had reduced latencies after cryoinjury, whereas this was not affected in EPI LMS. Inhibition of CaMKII reduced the number, distribution, and latencies of SCR, as well as whole-slice contractions in ENDO LMS, but not in EPI LMS after cryoinjury. Furthermore, CaMKII inhibition did not affect the excitation–contraction coupling in cryoinjured ENDO or EPI LMS. In conclusion, we demonstrate increased arrhythmogenic susceptibility in the injured ENDO. Our findings show involvement of CaMKII and highlight the need for region-specific targeting in cardiac therapies.

Cx43, a major cardiac connexin, forms precursor hemichannels that accrue at the intercalated disc to assemble as gap junctions. While gap junctions are crucial for electrical conduction in the heart, little is known about the potential roles of hemichannels. Recent evidence suggests that inhibiting Cx43 hemichannel opening with Gap19 has antiarrhythmic effects. Here, we used multiple electrophysiology, imaging, and super-resolution techniques to understand and define the conditions underlying Cx43 hemichannel activation in ventricular cardiomyocytes, their contribution to diastolic Ca2+ release from the sarcoplasmic reticulum, and their impact on electrical stability. We showed that Cx43 hemichannels were activated during diastolic Ca2+ release in single ventricular cardiomyocytes and cardiomyocyte cell pairs from mice and pigs. This activation involved Cx43 hemichannel Ca2+ entry and coupling to Ca2+ release microdomains at the intercalated disc, resulting in enhanced Ca2+ dynamics. Hemichannel opening furthermore contributed to delayed afterdepolarizations and triggered action potentials. In single cardiomyocytes, cardiomyocyte cell pairs, and arterially perfused tissue wedges from failing human hearts, increased hemichannel activity contributed to electrical instability compared with nonfailing rejected donor hearts. We conclude that microdomain coupling between Cx43 hemichannels and Ca2+ release is a potentially novel, targetable mechanism of cardiac arrhythmogenesis in heart failure. Copyright: © 2021, American Society for Clinical Investigation. We sincerely thank Ellen Cocquyt, Diego De Baere, Vicky Pauwelyn, Annemie Biesemans, Roxane Menten, and Mingliang Zhang for superb technical support. We would also like to thank the heart failure unit, the transplant surgical team, and the transplant coordinating team of UZ Leuven for help in providing the human explant hearts. This work was supported by the Fund for Scientific Research Flanders (project grants to LL, KRS, and GB; a postdoctoral fellowship to ED; and PhD fellowships to MDS and MA); Ghent University (a postdoctoral fellowship to KW and PhD fellowships to AL and TN); the Interuniversity Attraction Poles P7/10 to KRS and LL; NIH (project grants to ER and MD); the Fondation Leducq (transatlantic network award to MD); and a grant from the Ministry of Science and Higher Education of the Russian Federation, agreement 075-15-2020-800, to AVP.

Key points Ventricular arrhythmias are a major complication after myocardial infarction (MI), associated with sympathetic activation. The structurally heterogeneous peri‐infarct zone is a known substrate, but the functional role of the myocytes is less well known.Recordings of monophasic action potentials in vivo reveal that the peri‐infarct zone is a source of delayed afterdepolarizations (DADs) and has a high beat‐to‐beat variability of repolarization (BVR) during adrenergic stimulation (isoproterenol, ISO).Myocytes isolated from the peri‐infarct region have more DADs and spontaneous action potentials, with spontaneous Ca2+ release, under ISO. These myocytes also have reduced repolarization reserve and increased BVR. Other properties of post‐MI remodelling are present in both peri‐infarct and remote myocytes.These data highlight the importance of altered myocyte adrenergic responses in the peri‐infarct region as source and substrate of post‐MI arrhythmias. Abstract Ventricular arrhythmias are a major early complication after myocardial infarction (MI). The heterogeneous peri‐infarct zone forms a substrate for re‐entry while arrhythmia initiation is often associated with sympathetic activation. We studied the mechanisms triggering these post‐MI arrhythmias in vivo and their relation to regional myocyte remodelling. In pigs with chronic MI (6 weeks), in vivo monophasic action potentials were simultaneously recorded in the peri‐infarct and remote regions during adrenergic stimulation with isoproterenol (isoprenaline; ISO). Sham animals served as controls. During infusion of ISO in vivo, the incidence of delayed afterdepolarizations (DADs) and beat‐to‐beat variability of repolarization (BVR) was higher in the peri‐infarct than in the remote region. Myocytes isolated from the peri‐infarct region, in comparison to myocytes from the remote region, had more DADs, associated with spontaneous Ca2+ release, and a higher incidence of spontaneous action potentials (APs) when exposed to ISO (9.99 ± 4.2 vs. 0.16 ± 0.05 APs/min, p = 0.004); these were suppressed by CaMKII inhibition. Peri‐infarct myocytes also had reduced repolarization reserve and increased BVR (26 ± 10 ms vs. 9 ± 7 ms, P, Key points Ventricular arrhythmias are a major complication after myocardial infarction (MI), associated with sympathetic activation. The structurally heterogeneous peri‐infarct zone is a known substrate, but the functional role of the myocytes is less well known.Recordings of monophasic action potentials in vivo reveal that the peri‐infarct zone is a source of delayed afterdepolarizations (DADs) and has a high beat‐to‐beat variability of repolarization (BVR) during adrenergic stimulation (isoproterenol, ISO).Myocytes isolated from the peri‐infarct region have more DADs and spontaneous action potentials, with spontaneous Ca2+ release, under ISO. These myocytes also have reduced repolarization reserve and increased BVR. Other properties of post‐MI remodelling are present in both peri‐infarct and remote myocytes.These data highlight the importance of altered myocyte adrenergic responses in the peri‐infarct region as source and substrate of post‐MI arrhythmias.

Rhythmic increases in intracellular Ca(2+) concentration underlie the contractile function of the heart. These heart muscle-wide changes in intracellular Ca(2+) are induced and coordinated by electrical depolarization of the cardiomyocyte sarcolemma by the action potential. Originating at the sinoatrial node, conduction of this electrical signal throughout the heart ensures synchronization of individual myocytes into an effective cardiac pump. Ca(2+) signaling pathways also regulate gene expression and cardiomyocyte growth during development and in pathology. These fundamental roles of Ca(2+) in the heart are illustrated by the prevalence of altered Ca(2+) homeostasis in cardiovascular diseases. Indeed, heart failure (an inability of the heart to support hemodynamic needs), rhythmic disturbances, and inappropriate cardiac growth all share an involvement of altered Ca(2+) handling. The prevalence of these pathologies, contributing to a third of all deaths in the developed world as well as to substantial morbidity makes understanding the mechanisms of Ca(2+) handling and dysregulation in cardiomyocytes of great importance.

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

Key points The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca2+/calmodulin-dependent kinase II (CaMKII) activation during high-frequency stimulation. Sympathetic stimulation through β-adrenergic receptors activates an integrated signalling cascade, enhancing Ca2+ cycling and is at least partially mediated through CaMKII. Here we report that CaMKII activation during β-adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs. In contrast, the increase in the Ca2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca2+ transient are primarily protein kinase A-dependent. The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. Abstract In cardiac myocytes, β-adrenergic stimulation enhances Ca2+ cycling through an integrated signalling cascade modulating L-type Ca2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca2+/calmodulin-dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole-cell voltage-clamp and confocal line-scan imaging with Fluo-4 as a [Ca2+]i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca2+ sparks, which are assigned to coupled and non-coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces spark frequency in coupled RyRs only; NOS1 inhibition mimics the effect of CaMKII inhibition. Moreover, ISO induces the repetitive activation of coupled RyR clusters through CaMKII activation. Immunostaining shows high levels of CaMKII phosphorylation at the dyadic cleft. CaMKII inhibition reduces ICaL and local Ca2+ transients during depolarizing steps but has only modest effects on amplitude or relaxation of the global Ca2+ transient. In contrast, protein kinase A (PKA) inhibition reduces spark frequency in all RyRs concurrently with a reduction of sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude and relaxation. In conclusion, CaMKII activation during β-adrenergic stimulation is restricted to the dyadic cleft microdomain, enhancing LTCC-triggered local Ca2+ release as well as spontaneous diastolic Ca2+ release whilst PKA is the major pathway increasing global Ca2+ cycling. Selective CaMKII inhibition may reduce potentially arrhythmogenic release without negative inotropy.

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.

Rationale: In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs. Objective: Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation. Methods and Results: We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca 2+ transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K 5 fluo-4 as Ca 2+ indicator). Ca 2+ sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca 2+ /calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca 2+ /calmodulin-dependent kinase II blockers autocamtide-2–related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca 2+ /calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca 2+ transients, frequency-dependent increase of the rate of Ca 2+ rise was seen in coupled RyR regions only and abolished by autocamtide-2–related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost. Conclusions: Coupled RyRs have a distinct modulation by Ca 2+ /calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca 2+ /reactive oxygen species microdomain, and subject to modification with disease.

Scarring and remodeling of the left ventricle (LV) after myocardial infarction (MI) results in ischemic cardiomyopathy with reduced contractile function. Regional differences related to persisting ischemia may exist. We investigated the hypothesis that mitochondrial function and structure is altered in the myocardium adjacent to MI with reduced perfusion (MIadjacent) and less so in the remote, nonischemic myocardium (MIremote). We used a pig model of chronic coronary stenosis and MI ( n = 13). Functional and perfusion MR imaging 6 wk after intervention showed reduced ejection fraction and increased global wall stress compared with sham-operated animals (Sham; n = 14). Regional strain in MIadjacent was reduced with reduced contractile reserve; in MIremote strain was also reduced but responsive to dobutamine and perfusion was normal compared with Sham. Capillary density was unchanged. Cardiac myocytes isolated from both regions had reduced basal and maximal oxygen consumption rate, as well as through complex I and II, but complex IV activity was unchanged. Reduced respiration was not associated with detectable reduction of mitochondrial density. There was no significant change in AMPK or glucose transporter expression levels, but glycogen content was significantly increased in both MIadjacent and MIremote. Glycogen accumulation was predominantly perinuclear; mitochondria in this area were smaller but only in MIadjacent where also subsarcolemmal mitochondria were smaller. In conclusion, after MI reduction of mitochondrial respiration and glycogen accumulation occur in all LV regions suggesting that reduced perfusion does not lead to additional specific changes and that increased hemodynamic load is the major driver for changes in mitochondrial function.

Key points The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca2+/calmodulin‐dependent kinase II (CaMKII) activation during high‐frequency stimulation.Sympathetic stimulation through β‐adrenergic receptors activates an integrated signalling cascade, enhancing Ca2+ cycling and is at least partially mediated through CaMKII.Here we report that CaMKII activation during β‐adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs.In contrast, the increase in the Ca2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca2+ transient are primarily protein kinase A‐dependent.The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. Abstract In cardiac myocytes, β‐adrenergic stimulation enhances Ca2+ cycling through an integrated signalling cascade modulating L‐type Ca2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca2+/calmodulin‐dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole‐cell voltage‐clamp and confocal line‐scan imaging with Fluo‐4 as a [Ca2+]i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca2+ sparks, which are assigned to coupled and non‐coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces spark frequency in coupled RyRs only; NOS1 inhibition mimics the effect of CaMKII inhibition. Moreover, ISO induces the repetitive activation of coupled RyR clusters through CaMKII activation. Immunostaining shows high levels of CaMKII phosphorylation at the dyadic cleft. CaMKII inhibition reduces I CaL and local Ca2+ transients during depolarizing steps but has only modest effects on amplitude or relaxation of the global Ca2+ transient. In contrast, protein kinase A (PKA) inhibition reduces spark frequency in all RyRs concurrently with a reduction of sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude and relaxation. In conclusion, CaMKII activation during β‐adrenergic stimulation is restricted to the dyadic cleft microdomain, enhancing LTCC‐triggered local Ca2+ release as well as spontaneous diastolic Ca2+ release whilst PKA is the major pathway increasing global Ca2+ cycling. Selective CaMKII inhibition may reduce potentially arrhythmogenic release without negative inotropy., Key points The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca2+/calmodulin‐dependent kinase II (CaMKII) activation during high‐frequency stimulation.Sympathetic stimulation through β‐adrenergic receptors activates an integrated signalling cascade, enhancing Ca2+ cycling and is at least partially mediated through CaMKII.Here we report that CaMKII activation during β‐adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs.In contrast, the increase in the Ca2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca2+ transient are primarily protein kinase A‐dependent.The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events.

Ventricular myocytes remodel after myocardial infarction (MI). Myocytes hypertrophy, mitochondria are affected and the density of T-tubules reduces, while their distance to ryanodine receptors (RyRs) and mitochondria increases. Reactive oxygen species (ROS) production may increase and modulate RyR function within microdomains. We investigated the hypothesis that distance to mitochondria may be an independent determinant of RyR activity, in particular after MI.We studied permeabilized myocytes, where the influences of sarcolemma, bulk [Ca] and [ROS] in sham vs. MI are minimized. Cells were isolated from 6-weeks post-MI pig hearts, from the area adjacent to the MI and matching tissue in sham. xyt scanning served to characterize Ca sparks at the focal plane, relative to the xyz distribution of mitochondria. Mitochondrial z-stacks were processed using standard procedures, while a xyt spark detector sharing features with published algorithms [Kong et al (2008) Biophys J. 95(12):6016-24; Banyasz et al. (2007) Biophys J. 92(12):4458-65] was created. Spark frequency was plotted as a function of the distance between each spark's center of mass (at first detection) and its closest outer mitochondrial membrane (OMM).Control experiments in healthy permeabilized cells showed that, in our conditions, ROS were produced by mitochondria. In healthy and diseased myocytes, most sparks occurred within 0.5 microns of an OMM. However, it was found that the relationship between spark frequency and spark center-OMM distance did not change with MI. Yet in experiments in intact cells, scavenging of mitochondrial ROS affected spark production in MI predominantly in RyRs at a distance from the T-tubules. These results might indicate that ROS production by mitochondria likely affects all RyRs due to their ubiquitous nature but that further regulatory factors of RyR activity may be related to the presence of an intact sarcolemma.

55 Establishing a model for intravital imaging of the beating murine heart {#article-title-2} Introduction: While stem cell (SC)-based therapy following heart injury holds promise, clinical success is compromised by poor SC recruitment to the heart following infusion. One solution is to identify strategies that may increase SC capture in cardiac microvessels. We have routinely used intravital microscopy (IVM), a method of live in vivo imaging of the microcirculation, to monitor cell trafficking. To do so in the heart would help facilitate research aimed at improving SC delivery. However, IVM of the beating mouse heart remains challenging due to difficulties with organ access and impractical degrees of tissue motion. The study presents a method that allows for cardiac IVM using a 3D-printed stabilizing device. Methods: Anaesthetized mice were artificially ventilated using a small rodent ventilator which delivered medical oxygen. PEEP was applied by immersing the ventilator outlet in 4cm of ddH2O. A chest wall incision, expanded with retractors, facilitated the adhesive positioning of a specially designed tissue stabilizer onto the heart surface. This limited respiratory and cardiac contractile motion in a small area without compromising overall heart function. Imaging was performed through a small hole in the centre of the stabilizer. Preliminary studies were performed in the myocardial ischemia-reperfusion injury model. This injury was established by occlusion of the left anterior descending artery (LAD). The vessel was occluded for 45 minutes using a small piece of plastic tubing within a loop which was placed under the LAD. Identification of vascular dynamics (e.g. cell recruitment, monitoring of vascular no-reflow) took place at various points during reperfusion. Administration of FITC-BSA allowed for visualisation of the microvasculature. Endogenous neutrophils were labelled by intra-arterial administration of 6µg eFluor-660 labelled anti-Gr-1 antibody. Results: Infusion of FITC-BSA permitted visualization of the coronary microcirculation. Vascular integrity was not disturbed and flow was visible as a result of application of the stabiliser. Endogenous (labelled) neutrophils were easily identified circulating in the cardiac microcirculation. Following cardiac IR, we identified significantly enhanced neutrophil recruitment as early as 60 minutes post-reperfusion, when compared to sham-operated control animals. Furthermore, using FITC-BSA visualization of the coronary microcirculation, we identified areas of vascular no-reflow following cardiac IR. Finally, systemically administered SCs (fluorescently labelled HSCs) could be identified trafficking through the cardiac microvasculature following cardiac IR injury. Conclusion: We present a model for intravital imaging of the mouse beating heart in vivo. This method may be useful in monitoring cell dynamics and microvascular disturbances in the heart at the single cell level. # 56 Altered RyR microdomains lead to more Ca2+ waves in non-coupled RyRs after myocardial infarction {#article-title-3} Purpose: In ventricular myocytes of large mammals, a significant number of ryanodine receptors (RyRs) are not in couplons (i.e. non-coupled RyRs). Previous research from our lab has shown that these non-coupled receptors lack CaMKII and NOX2 microdomain modulation. Furthermore, after MI TTs are lost and hence the fraction of non-coupled RyRs is increased. We investigated whether this affected microdomain signaling and diastolic Ca2+ waves which are potentially arrhythmogenic. Methods: Using an established pig model of chronic ischemia and myocardial infarction (MI, N=16), we studied myocytes from the area adjacent to the MI and compared these to myocytes from SHAM pigs (N=13) using whole-cell voltage clamp with Fluo-4 as a [Ca2+]i indicator together with confocal line scan imaging. Myocytes were stimulated at a high frequency (2 Hz) in the presence of isoproterenol (10 nM; ISO). Ca2+ waves were recorded during a 15 s rest period following stimulation and assigned to different subcellular regions categorized as coupled or non-coupled RyRs using a specific algorithm. Results: Conditioning myocytes at high frequency in the presence of ISO induced more Ca2+ waves in MI myocytes (0.21 ± 0.1 #waves/100µm/s; n=62) compared to SHAM myocytes (0.15 ± 0.1 #waves/100µm/s; n=59). No differences were found in wave foci, but a significant reduction in foci recruitment occurred after MI (57 % in MI vs. 70 % in SHAM). Moreover, in healthy myocytes more Ca2+ waves occurred in coupled compared to non-coupled in a 2:1 ratio. In MI myocytes, however, the subcellular site of wave occurrence was reversed to non-coupled sites resulting in a 1:2 ratio for coupled:non-coupled RyRs. Specific blockers directly (S107) or indirectly (AIP inhibiting CaMKII, mitoTEMPO scavenging mitochondrial ROS) targeting RyRs were investigated. In healthy myocytes, none of the blockers significantly affected wave frequency, neither at the global nor in the coupled versus non-coupled RyRs. However, in myocytes from MI animals, S107, mitoTEMPO and AIP all reduced the global increase in wave frequency. Subcellular analysis of different RyR subsets showed that in MI S107 (0.06 ± 0.1 #waves/100µm/s; n=27), AIP (0.06 ± 0.1 #waves/100µm/s; n=25) and mitoTEMPO (0.08 ± 0.1 #waves/100µm/s; n=22) reduced the wave frequency in non-coupled RyRs without significantly affecting coupled RyRs. Conclusion: After MI, Ca2+ waves arise predominantly in non-coupled RyRs. The non-coupled RyRs after MI are modulated via local microdomains of CaMKII and mitochondrial ROS. These data highlight the arrhythmogenic nature of the non-coupled RyR clusters, which are at a higher density after MI, and suggest there may be a potential for selective pharmacological targeting. # 57 Intravital microscopy (IVM), a new method for in vivo imaging of monocyte homing in a mouse hind limb arteriogenesis model {#article-title-4} Therapeutic augmentation of collateral vessel growth (arteriogenesis) is of particular clinical interest. Monocytes are key players within this process. Quantification of collateralization in small animal models is, however, difficult and the commonly used techniques are post-mortem or histological assays. Live imaging of monocyte homing to sites of arteriogenesis in vivo has not been demonstrated until now. Therefore, an intravital microscopy (IVM) technique was established to visualize this process. The IVM is a non-invasive optical imaging technique offering high resolution, deep tissue penetration and video capturing. Nowadays only already located (homed) cells could be detected by post mortem examination of the tissue. Real time transmigration and interaction of cells had never been visualized under these circumstances. By the use of this innovative and unique method we were able to examine the kinetics of monocyte homing in vivo and endogenous development of collateral arteries within the hind limb of C57BL6 mice after ligature of one femoral artery. The morphology of the collateral vessels in the adductor muscle was determined by 3D imaging and blood flow was visualized within the growing arteries. Our results were validated by histological workup and laser Doppler perfusion imaging (LDPI). For the in vivo experiments, mice were anesthetized intraperitoneally and placed on a heating plate (37°C). Then, the leg was fixed between two adjustable stamps and care was taken to keep the region of interest moist. A cover glass was positioned on top of the stamps and adjusted before imaging was started. A Zeiss LSM 710 NLO with a 20x water immersion lens (NA 1.0) or a Leica SP 5 LSM microscope with a 10x dry lens (NA 0.4) were used in either 2 Photon or LSM mode, respectively. With both systems we were able to achieve deep tissue penetration up to 1 mm, thereby capturing multi-colour videos of living cells. Histological workup of hind limb tissue sections of C57BL6 mice approved the increase of collateralization. The ratio of collateral arteries (ligated/unligated leg) was calculated (each group n=4). The intra individual ratio increased from 1.0±0.07 (prior to ligation) to 1.32±0.17 two weeks and 1.44±0.11 three weeks after ligation (P

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.