Hyperactive ryanodine receptors in human heart failure and ischaemic cardiomyopathy reside outside of couplons

Aims In ventricular myocytes from humans and large mammals, the transverse and axial tubular system (TATS) network is less extensive than in rodents with consequently a greater proportion of ryanodine receptors (RyRs) not coupled to this membrane system. TATS remodelling in heart failure (HF) and after myocardial infarction (MI) increases the fraction of non-coupled RyRs. Here we investigate whether this remodelling alters the activity of coupled and non-coupled RyR sub-populations through changes in local signalling. We study myocytes from patients with end-stage HF, compared with non-failing (non-HF), and myocytes from pigs with MI and reduced left ventricular (LV) function, compared with sham intervention (SHAM).Methods and resultsSingle LV myocytes for functional studies were isolated according to standard protocols. Immunofluorescent staining visualized organization of TATS and RyRs. Ca2+ was measured by confocal imaging (fluo-4 as indicator) and using whole-cell patch-clamp (37°C). Spontaneous Ca2+ release events, Ca2+ sparks, as a readout for RyR activity were recorded during a 15 s period following conditioning stimulation at 2 Hz. Sparks were assigned to cell regions categorized as coupled or non-coupled sites according to a previously developed method. Human HF myocytes had more non-coupled sites and these had more spontaneous activity than in non-HF. Hyperactivity of these non-coupled RyRs was reduced by Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition. Myocytes from MI pigs had similar changes compared with SHAM controls as seen in human HF myocytes. As well as by CaMKII inhibition, in MI, the increased activity of non-coupled sites was inhibited by mitochondrial reactive oxygen species (mito-ROS) scavenging. Under adrenergic stimulation, Ca2+ waves were more frequent and originated at non-coupled sites, generating larger Na+/Ca2+ exchange currents in MI than in SHAM. Inhibition of CaMKII or mito-ROS scavenging reduced spontaneous Ca2+ waves, and improved excitation–contraction coupling.ConclusionsIn HF and after MI, RyR microdomain re-organization enhances spontaneous Ca2+ release at non-coupled sites in a manner dependent on CaMKII activation and mito-ROS production. This specific modulation generates a substrate for arrhythmia that appears to be responsive to selective pharmacologic modulation

Altered adrenergic response in myocytes bordering a chronic myocardial infarction underlies <i>in vivo</i> triggered activity and repolarization instability

Ventricular arrhythmias are a major complication early 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 (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 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 2+ handling at baseline and myocyte hypertrophy were present throughout the LV. Expression of some of the related genes was however different between the regions. In conclusion, altered myocyte adrenergic responses in the peri‐infarct, but not in the remote region, provide a source of triggered activity in vivo and of repolarization instability amplifying the substrate for re‐entry. These findings stimulate further exploration of region‐specific therapies targeting myocytes and autonomic modulation

MSK-Mediated Phosphorylation of Histone H3 Ser28 Couples MAPK Signalling with Early Gene Induction and Cardiac Hypertrophy

Heart failure is a leading cause of death that develops subsequent to deleterious hypertrophic cardiac remodelling. MAPK pathways play a key role in coordinating the induction of gene expression during hypertrophy. Induction of the immediate early gene (IEG) response including activator protein 1 (AP-1) complex factors is a necessary and early event in this process. How MAPK and IEG expression are coupled during cardiac hypertrophy is not resolved. Here, in vitro, in rodent models and in human samples, we demonstrate that MAPK-stimulated IEG induction depends on the mitogen and stress-activated protein kinase (MSK) and its phosphorylation of histone H3 at serine 28 (pH3S28). pH3S28 in IEG promoters in turn recruits Brg1, a BAF60 ATP-dependent chromatin remodelling complex component, initiating gene expression. Without MSK activity and IEG induction, the hypertrophic response is suppressed. These studies provide new mechanistic insights into the role of MAPK pathways in signalling to the epigenome and regulation of gene expression during cardiac hypertrophy

Myofibroblast modulation of cardiac myocyte structure and function

After myocardial infarction, resident fibroblasts (Fb) differentiate towards myofibroblasts (MyoFb), generating the scar tissue and the interstitial fibrosis seen in the adjacent myocardium. Fb and MyoFb have the potential to interact with cardiac myocytes (CMs) but insight into the phenotype-specific role and mode of interaction is still incomplete. Our objectives are to further define the modulation of CMs by MyoFbs compared to Fbs, as well as the role of direct contact through gap junctions vs. soluble mediators, using Fbs and CMs from pig left ventricle. Fbs were treated to maintain an undifferentiated state (SD-208) or to attain full differentiation to MyoFb (TGF-β1). Fbs and MyoFbs were co-cultured with CMs, with the possibility of direct contact or separated by a Thincert membrane. Only in direct co-culture, both Fbs and MyoFbs were able to decrease CM viability after 2 days. Only MyoFbs induced significant distal spreading of CMs in both direct and indirect co-culture. MyoFbs, but not Fbs, readily made connections with CMs in direct co-culture and connexin 43 expression in MyoFb was higher than in Fb. When coupled to CMs, MyoFbs reduced the CM action potential duration and hyperpolarized the CM resting membrane potential. Uncoupling reversed these effects. In conclusion, MyoFbs, but not Fbs, alter the CM structural phenotype. MyoFbs, but not Fbs, are likely to electrically connect to CMs and thereby modulate the CM membrane potential. These data provide further support for an active role of MyoFbs in the arrhythmogenic substrate after cardiac remodelling.status: publishe

Reduced mitochondrial respiration in the ischemic as well as in the remote nonischemic region in postmyocardial infarction remodeling

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.status: publishe

Myofibroblast Phenotype and Reversibility of Fibrosis in Patients With End-Stage Heart Failure

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.status: publishe