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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.