Rationale Sarcoplasmic reticulum (SR) Ca2+ cycling is key to normal excitation-contraction coupling but may also contribute to pathological cardiac alternans and arrhythmia. Objective To measure intra-SR free [Ca2+] ([Ca2+]SR) changes in intact hearts during alternans and ventricular fibrillation (VF). Methods and Results Simultaneous optical mapping of Vm (with RH237) and [Ca2+]SR (with Fluo-5N AM) was performed in Langendorff-perfused rabbit hearts. Alternans and VF were induced by rapid pacing. SR Ca2+ and action potential duration (APD) alternans occurred in-phase, but SR Ca2+ alternans emerged first as cycle length was progressively reduced (217±10ms vs. 190±13ms, p<0.05). Ryanodine receptor (RyR) refractoriness played a key role in the onset of SR Ca2+ alternans, with SR Ca2+ release alternans routinely occurring without changes in diastolic [Ca2+]SR. Sensitizing RyR with caffeine (200μM) significantly reduced the pacing threshold for both SR Ca2+ and APD alternans (188±15ms and 173±12ms, p<0.05 vs. baseline). Caffeine also reduced the magnitude of spatially discordant SR Ca2+ alternans, but not APD alternans, the pacing threshold for discordance, or threshold for VF. During VF, [Ca2+]SR was high, but RyR remained nearly continuously refractory, resulting in minimal SR Ca2+ release throughout VF. Conclusions In intact hearts RyR refractoriness initiates SR Ca2+ release alternans, that can be amplified by diastolic [Ca2+]SR alternans and lead to APD alternans. Sensitizing RyR suppresses spatially concordant, but not discordant SR Ca2+ and APD alternans. Despite increased [Ca2+]SR during VF, SR Ca2+ release was nearly continuously refractory. This novel method provides insight into SR Ca2+ handling during cardiac alternans and arrhythmia.

Optimal healing of damaged tissue following myocardial infarction (MI) requires a coordinated cellular response that can be divided into three phases: inflammatory, proliferative/reparative, and maturation. The inflammatory phase, characterized by rapid influx of cytokines, chemokines, and immune cells, is critical to the removal of damaged tissue. The onset of the proliferative/reparative phase is marked by increased proliferation of myofibroblasts and secretion of collagen to replace dead tissue. Lastly, crosslinking of collagen fibers and apoptosis of immune cells marks the maturation phase. Excessive inflammation or fibrosis has been linked to increased incidence of arrhythmia and other MI-related pathologies. This review describes the roles of inflammation and fibrosis in arrhythmogenesis and prospective therapies for anti-arrhythmic treatment.

Background Atherosclerotic animal models show increased recruitment of inflammatory cells to the heart following myocardial infarction (MI), which impacts ventricular function and remodeling. Objective To determine whether increased myocardial inflammation following MI also contributes to arrhythmias. Methods MI was created in 3 mouse models: 1) atherosclerotic (ApoE−/− on atherogenic diet; n=12), 2) acute inflammation (wild-type [WT] given daily lipopolysaccharide [LPS], 10µg/day; n=7), and 3) WT (n=14). Sham-operated (n=4) mice were also studied. Four days post-MI, an inflammatory protease-activatable fluorescent probe (Prosense680) was injected intravenously to quantify myocardial inflammation on day 5. Optical mapping with voltage-sensitive dye was performed on day 5 to assess electrophysiology and arrhythmia susceptibility. Results Inflammatory activity (Prosense680 fluorescence) was increased approximately 2-fold in ApoE+MI and LPS+MI hearts versus WT+MI (p<0.05) and 3-fold versus Sham (p<0.05). ApoE+MI and LPS+MI hearts also had prolonged action potential duration, slowed conduction velocity, and increased susceptibility to pacing-induced arrhythmias (56% and 71%; vs. 13% for WT+MI and 0% for Sham, respectively, p<0.05 for ApoE+MI and LPS+MI groups versus both WT+MI and Sham). Increased macrophage accumulation in ApoE+MI and LPS+MI hearts was confirmed with immunofluorescence. Macrophages were associated with areas of connexin-43 (Cx43) degradation and a 2-fold decrease in Cx43 expression was found in ApoE+MI versus WT+MI hearts (p<0.05). ApoE+MI hearts also had a 3-fold increase in interleukin-1β expression, an inflammatory cytokine known to degrade Cx43. Conclusions Underlying atherosclerosis exacerbates post-MI electrophysiological remodeling and arrhythmias. LPS+MI hearts fully recapitulate the atherosclerotic phenotype, suggesting myocardial inflammation as a key contributor to post-MI arrhythmia.

Background Interleukin-1 beta (IL-1β) is a key regulator of the inflammatory response following myocardial infarction (MI), by modulating immune cell recruitment, cytokine production, and extracellular matrix turnover. Elevated levels of IL-1β are associated with adverse remodeling, and inhibition of IL-1 signaling following MI results in improved contractile function. Objective The goal of this study was to determine if IL-1 signaling also contributes to post-MI arrhythmogenesis. Methods MI was created in two murine models of elevated inflammation: atherosclerotic on Western diet or wild-type with a sub-septic dose of lipopolysaccharide. The role of IL-1β was assessed with the IL-1 receptor antagonist, anakinra (10mg/kg/day, starting 24h post-MI). Results In vivo and ex vivo molecular imaging showed reduced myocardial inflammation following a 4-day course of anakinra treatment, despite no change in infarct size. At day 5 post-MI, high-speed optical mapping of transmembrane potential (Vm) and intracellular Ca2+ in isolated hearts revealed that IL-1β inhibition improved conduction velocity, reduced action potential duration dispersion, improved intracellular Ca2+ handling, decreased Vm and Ca2+ alternans magnitude, and reduced spontaneous and inducible ventricular arrhythmias. These functional improvements were linked to increased expression of connexin43 and sarcoplasmic reticulum Ca2+-ATPase (SERCA). Conclusions This study revealed a novel mechanism for IL-1β in contributing to defective excitation-contraction coupling and arrhythmogenesis in the post-MI heart. Our results suggest that inhibition of IL-1 signaling post-MI may represent a novel anti-arrhythmic therapy.

Objective Traumatic joint injuries initiate a surge of inflammatory cytokines and proteases that may contribute to cartilage and subchondral bone degeneration. Detecting these early biological processes in animal models of post-traumatic osteoarthritis (PTOA) typically involves ex vivo analysis of blood serum or synovial fluid biomarkers, or destructive histological analysis of the joint. In this study, we used in vivo fluorescence reflectance imaging (FRI) to quantify protease activity, matrix metalloproteinase (MMP) activity, and Cathepsin K activity in mice following ACL rupture. We hypothesized that these processes would be elevated at early time points following traumatic joint injury (1–14 days), but would return to control levels at later time points (4–8 weeks). Design Mice were injured via tibial compression overload, and FRI imaging was performed at multiple time points from 1–56 days after injury using commercially available activatable fluorescent tracers to quantify protease activity, MMP activity, and cathepsin K activity in injured vs. uninjured knees. PTOA was assessed at 56 days post-injury using micro-computed tomography and whole-joint histology. Results Protease activity, MMP activity, and cathepsin K activity were all significantly increased in injured knees relative to uninjured knees at all time points, peaking at 1–7 days post-injury, then decreasing at later time points while still remaining elevated relative to controls. Conclusions This study establishes FRI imaging as a reliable method for in vivo quantification of early biological processes in a translatable mouse model of PTOA, and provides crucial information about the time course of inflammation and biological activity following joint injury. These data may inform future studies aimed at targeting early inflammation to reduce the development of PTOA.