Temporal expression and spatial distribution of the proteoglycan versican during cardiac fibrosis development.

Cardiac fibrosis is a central pathological feature in several cardiac diseases, but the underlying molecular players are insufficiently understood. The extracellular matrix proteoglycan versican is elevated in heart failure and suggested to be a target for treatment. However, the temporal expression and spatial distribution of versican and the versican cleavage fragment containing the neoepitope DPEAAE in cardiac fibrosis remains to be elucidated. In this study, we have examined versican during cardiac fibrosis development in a murine pressure overload model and in patients with cardiomyopathies. We found that versican, mainly the V1 isoform, was expressed immediately after induction of pressure overload, preceding collagen accumulation, and versican protein levels extended from the perivascular region into the cardiac interstitium. In addition, we found increased production of versican by collagen expressing fibroblasts, and that it was deposited extensively in the fibrotic extracellular matrix during pressure overload. In cardiac cell cultures, the expression of versican was induced by the pro-fibrotic transforming growth factor beta and mechanical stretch. Furthermore, we observed that the proteolytic cleavage of versican (DPEAAE fragment) increased in the late phase of fibrosis development during pressure overload. In patients with hypertrophic and dilated cardiomyopathies, we found elevated levels of versican and a positive correlation between versican and collagen mRNA in the heart, as well as increased cleavage of full-length protein. Taken together, the temporal expression profile and the spatial distribution of both the full-length versican and the DPEAAE fragment observed in this study indicates a role for versican in development of cardiac fibrosis.

InsP3R-RyR channel crosstalk augments sarcoplasmic reticulum Ca2+ release and arrhythmogenic activity in post-MI pig cardiomyocytes.

Ca2+ transients (CaT) underlying cardiomyocyte (CM) contraction require efficient Ca2+ coupling between sarcolemmal Ca2+ channels and sarcoplasmic reticulum (SR) ryanodine receptor Ca2+ channels (RyR) for their generation; reduced coupling in disease contributes to diminished CaT and arrhythmogenic Ca2+ events. SR Ca2+ release also occurs via inositol 1,4,5-trisphosphate receptors (InsP3R) in CM. While this pathway contributes negligeably to Ca2+ handling in healthy CM, rodent studies support a role in altered Ca2+ dynamics and arrhythmogenic Ca2+ release involving InsP3R crosstalk with RyRs in disease. Whether this mechanism persists in larger mammals with lower T-tubular density and coupling of RyRs is not fully resolved. We have recently shown an arrhythmogenic action of InsP3-induced Ca2+ release (IICR) in end stage human heart failure (HF), often associated with underlying ischemic heart disease (IHD). How IICR contributes to early stages of disease is however not determined but highly relevant. To access this stage, we chose a porcine model of IHD, which shows substantial remodelling of the area adjacent to the infarct. In cells from this region, IICR preferentially augmented Ca2+ release from non-coupled RyR clusters that otherwise showed delayed activation during the CaT. IICR in turn synchronised Ca2+ release during the CaT but also induced arrhythmogenic delayed afterdepolarizations and action potentials. Nanoscale imaging identified co-clustering of InsP3Rs and RyRs, thereby allowing Ca2+-mediated channel crosstalk. Mathematical modelling supported and further delineated this mechanism of enhanced InsP3R-RyRs coupling in MI. Our findings highlight the role of InsP3R-RyR channel crosstalk in Ca2+ release and arrhythmia during post-MI remodelling.

Identification of Cx43 variants predisposing to ventricular fibrillation in the acute phase of ST-elevation myocardial infarction.

AIMS: Ventricular fibrillation (VF) occurring in the acute phase of ST-elevation myocardial infarction (STEMI) is the leading cause of sudden cardiac death worldwide. Several studies showed that reduced connexin 43 (Cx43) expression and reduced conduction velocity increase the risk of VF in acute myocardial infarction (MI). Furthermore, genetic background might predispose individuals to primary VF (PVF). The primary objective was to evaluate the presence of GJA1 variants in STEMI patients. The secondary objective was to evaluate the arrhythmogenic impact of GJA1 variants in STEMI patients with VF. METHODS AND RESULTS: The MAP-IDM prospective cohort study included 966 STEMI patients and was designed to identify genetic predisposition to VF. A total of 483 (50.0%) STEMI patients with PVF were included. The presence of GJA1 variants increased the risk of VF in STEMI patients [from 49.1 to 70.8%, P = 0.0423; odds ratio (OR): 0.40; 95% confidence interval: 0.16-0.97; P = 0.04]. The risk of PVF decreased with beta-blocker intake (from 53.5 to 44.8%, P = 0.0085), atrial fibrillation (from 50.7 to 26.4%, P = 0.0022), and with left ventricular ejection fraction >50% (from 60.2 to 41.4%, P < 0.0001). Among 16 GJA1 variants, three novel heterozygous missense variants were identified in three patients: V236I, H248R, and I327M. In vitro studies of these variants showed altered Cx43 localization and decreased cellular communication, mainly during acidosis. CONCLUSION: Connexin 43 variants are associated with increased VF susceptibility in STEMI patients. Restoring Cx43 function may be a potential therapeutic target to prevent PVF in patients with acute MI. CLINICAL TRIAL REGISTRATION: Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT00859300.

Altered adrenergic response in myocytes bordering a chronic myocardial infarction underlies in vivo triggered activity and repolarization instability.

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 < 0.001), correlating with DAD activity. In contrast to these regional distinctions under ISO, alterations in Ca2+ handling at baseline and myocyte hypertrophy were present throughout the left ventricle (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 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.

Calcium Signaling in Cardiomyocyte Function.

Rhythmic increases in intracellular Ca2+ concentration underlie the contractile function of the heart. These heart muscle-wide changes in intracellular Ca2+ 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. Ca2+ signaling pathways also regulate gene expression and cardiomyocyte growth during development and in pathology. These fundamental roles of Ca2+ in the heart are illustrated by the prevalence of altered Ca2+ 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 Ca2+ 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 Ca2+ handling and dysregulation in cardiomyocytes of great importance.

Age-dependent ventricular arrhythmias risk, structural and molecular remodeling in systemic arterial hypertension.

INTRODUCTION: The left ventricular hypertrophy (LVH)-ventricular arrhythmias relationship associated with arterial hypertension and aging remains controversial. We aimed to assess the age-dependency of ventricular arrhythmias in spontaneously hypertensive rats (SHRs) and the corresponding ventricular structural and molecular remodeling. MATERIALS AND METHODS: Ventricular arrhythmias were quantified using 24-h radiotelemetry ECG monitoring in eight SHRs and four Wistar-Kyoto (WKY) rats at 14 (young), 24 (adult), and 48 (aging) weeks of age. Left ventricular histology and mRNA expressions of 89 proarrhythmogenic genes were assessed in six additional groups (n=4 each) of young, adult, and aging SHRs and WKYs. RESULTS: Regardless of their age, SHRs presented more premature ventricular contractions (PVCs) than age-matched WKYs (p<0.01). The arrhythmogenicity peak occurred in adult SHRs; ventricular tachycardias only occurred in adult SHRs. Among the SHRs, LV thickness, interstitial fibrosis, and the number of deregulated genes increased with age. Kcnj11 expression was deregulated in adult, but not in young or aging SHRs. DISCUSSION: This study confirms the presence of higher ventricular ectopy in SHRs than in age-matched WKYs. LVH appeared to be an adaptive, antiarrhythmic process. Myocardial energetic changes with advancing age, as reflected by Kcnj11 expression changes, could underlie this age-dependency of ventricular arrhythmias.