Bradley N. Plummer

Spontaneous calcium release in tissue from the failing canine heart

Abnormalities in calcium handling have been implicated as a significant source of electrical instability in heart failure (HF). While these abnormalities have been investigated extensively in isolated myocytes, how they manifest at the tissue level and trigger arrhythmias is not clear. We hypothesize that in HF, triggered activity (TA) is due to spontaneous calcium release from the sarcoplasmic reticulum that occurs in an aggregate of myocardial cells (an SRC) and that peak SCR amplitude is what determines whether TA will occur. Calcium and voltage optical mapping was performed in ventricular wedge preparations from canines with and without tachycardia-induced HF. In HF, steady-state calcium transients have reduced amplitude [135 vs. 170 ratiometric units (RU), P < 0.05] and increased duration (252 vs. 229 s, P < 0.05) compared with those of normal. Under control conditions and during beta-adrenergic stimulation, TA was more frequent in HF (53% and 93%, respectively) compared with normal (0% and 55%, respectively, P < 0.025). The mechanism of arrhythmias was SCRs, leading to delayed afterdepolarization-mediated triggered beats. Interestingly, the rate of SCR rise was greater for events that triggered a beat (0.41 RU/ms) compared with those that did not (0.18 RU/ms, P < 0.001). In contrast, there was no difference in SCR amplitude between the two groups. In conclusion, TA in HF tissue is associated with abnormal calcium regulation and mediated by the spontaneous release of calcium from the sarcoplasmic reticulum in aggregates of myocardial cells (i.e., an SCR), but importantly, it is the rate of SCR rise rather than amplitude that was associated with TA.

Aberrant S-nitrosylation mediates calcium-triggered ventricular arrhythmia in the intact heart

Nitric oxide (NO) derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca2+ handling proteins. Deficient S-nitrosylation of the cardiac ryanodine receptor (RyR2) has a variable effect on SR Ca2+ leak/sparks in isolated myocytes, likely dependent on the underlying physiological state. It remains unknown, however, whether such molecular aberrancies are causally related to arrhythmogenesis in the intact heart. Here we show in the intact heart, reduced NOS1 activity increased Ca2+-mediated ventricular arrhythmias only in the setting of elevated myocardial [Ca2+]i. These arrhythmias arose from increased spontaneous SR Ca2+ release, resulting from a combination of decreased RyR2 S-nitrosylation (RyR2-SNO) and increased RyR2 oxidation (RyR-SOx) (i.e., increased reactive oxygen species (ROS) from xanthine oxidoreductase activity) and could be suppressed with xanthine oxidoreductase (XOR) inhibition (i.e., allopurinol) or nitric oxide donors (i.e., S-nitrosoglutathione, GSNO). Surprisingly, we found evidence of NOS1 down-regulation of RyR2 phosphorylation at the Ca2+/calmodulin-dependent protein kinase (CaMKII) site (S2814), suggesting molecular cross-talk between nitrosylation and phosphorylation of RyR2. Finally, we show that nitroso–redox imbalance due to decreased NOS1 activity sensitizes RyR2 to a severe arrhythmic phenotype by oxidative stress. Our findings suggest that nitroso–redox imbalance is an important mechanism of ventricular arrhythmias in the intact heart under disease conditions (i.e., elevated [Ca2+]i and oxidative stress), and that therapies restoring nitroso–redox balance in the heart could prevent sudden arrhythmic death.

Spontaneous calcium oscillations during diastole in the whole heart: the influence of ryanodine reception function and gap junction coupling

Triggered arrhythmias due to spontaneous cytoplasmic calcium oscillations occur in a variety of disease conditions; however, their cellular mechanisms in tissue are not clear. We hypothesize that spontaneous calcium oscillations in the whole heart are due to calcium release from the sarcoplasmic reticulum and are facilitated by calcium diffusion through gap junctions. Optical mapping of cytoplasmic calcium from Langendorff perfused guinea pig hearts (n = 10) was performed using oxygenated Tyrodes solution (in mM): 140 NaCl, 0.7 MgCl, 4.5 KCl, 5.5 dextrose, 5 HEPES, and 5.5 CaCl₂ (pH 7.45, 34°C). Rapid pacing was used to induce diastolic calcium oscillations. In all preparations, pacing-induced multicellular diastolic calcium oscillations (m-SCR) occurred across most of the mapping field, at all pacing rates tested. Ryanodine (1 μM) eliminated all m-SCR activity. Low-dose caffeine (1 mM) increased m-SCR amplitude (+10.4 ± 4.4%, P < 0.05) and decreased m-SCR time-to-peak (-17.4 ± 6.7%, P < 0.05) and its temporal synchronization (i.e., range) across the mapping field (-26.9 ± 17.1%, P < 0.05). Surprisingly, carbenoxolone increased the amplitude of m-SCR activity (+14.8 ± 4.1%, P < 0.05) and decreased m-SCR time-to-peak (-11.3 ± 9.6%, P < 0.01) and its synchronization (-37.0 ± 19.1%, P < 0.05), similar to caffeine. In isolated myocytes, carbenoxolone (50 μM) had no effect on the frequency of aftercontractions, suggesting the effect of cell-to-cell uncoupling on m-SCR activity is tissue specific. Therefore, in the whole heart, overt m-SCR activity caused by calcium release from the SR can be induced over a broad range of pacing rates. Enhanced ryanodine receptor open probability and, surprisingly, decreased cell-to-cell coupling increased the amplitude and temporal synchronization of spontaneous calcium release in tissue.

Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+-release Channel RyR1 by S-Palmitoylation

Background: Excitation-contraction coupling in striated muscle requires intracellular Ca2+ release through ryanodine receptor/Ca2+-release channels (RyRs). Results: S-Palmitoylation is a previously unidentified post-translational modification of skeletal muscle RyR1. Diminishing S-palmitoylation significantly diminishes RyR1 activity including stimulus-dependent Ca2+ release. Conclusion: S-Palmitoylation provides a previously unidentified mechanism to regulate Ca2+ flux in skeletal muscle. Significance: S-Palmitoylation is likely to regulate Ca2+ flux in many cell types. The ryanodine receptor/Ca2+-release channels (RyRs) of skeletal and cardiac muscle are essential for Ca2+ release from the sarcoplasmic reticulum that mediates excitation-contraction coupling. It has been shown that RyR activity is regulated by dynamic post-translational modifications of Cys residues, in particular S-nitrosylation and S-oxidation. Here we show that the predominant form of RyR in skeletal muscle, RyR1, is subject to Cys-directed modification by S-palmitoylation. S-Palmitoylation targets 18 Cys within the N-terminal, cytoplasmic region of RyR1, which are clustered in multiple functional domains including those implicated in the activity-governing protein-protein interactions of RyR1 with the L-type Ca2+ channel CaV1.1, calmodulin, and the FK506-binding protein FKBP12, as well as in “hot spot” regions containing sites of mutations implicated in malignant hyperthermia and central core disease. Eight of these Cys have been identified previously as subject to physiological S-nitrosylation or S-oxidation. Diminishing S-palmitoylation directly suppresses RyR1 activity as well as stimulus-coupled Ca2+ release through RyR1. These findings demonstrate functional regulation of RyR1 by a previously unreported post-translational modification and indicate the potential for extensive Cys-based signaling cross-talk. In addition, we identify the sarco/endoplasmic reticular Ca2+-ATPase 1A and the α1S subunit of the L-type Ca2+ channel CaV1.1 as S-palmitoylated proteins, indicating that S-palmitoylation may regulate all principal governors of Ca2+ flux in skeletal muscle that mediates excitation-contraction coupling.

Targeted Antioxidant Treatment Decreases Cardiac Alternans Associated With Chronic Myocardial Infarction

Background—In myocardial infarction (MI), repolarization alternans is a potent arrhythmia substrate that has been linked to Ca2+ cycling proteins, such as sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), located in the sarcoplasmic reticulum. MI is also associated with oxidative stress and increased xanthine oxidase (XO) activity, an important source of reactive oxygen species (ROS) in the sarcoplasmic reticulum that may reduce SERCA2a function. We hypothesize that in chronic MI, XO-mediated oxidation of SERCA2a is a mechanism of cardiac alternans. Methods and Results—Male Lewis rats underwent ligation of the left anterior descending coronary artery (n=54) or sham procedure (n=24). At 4 weeks, optical mapping of intracellular Ca2+ and ROS was performed. ECG T-wave alternans (ECG ALT) and Ca2+ transient alternans (Ca2+ALT) were induced by rapid pacing (300–120 ms) before and after the XO inhibitor allopurinol (ALLO, 50 µmol/L). In MI, ECG ALT (2.32±0.41%) and Ca2+ ALT (22.3±4.5%) were significantly greater compared with sham (0.18±0.08%, P<0.001; 0.79±0.32%, P<0.01). Additionally, ROS was increased by 137% (P<0.01) and oxidation of SERCA2a by 30% (P<0.05) in MI compared with sham. Treatment with ALLO significantly decreased ECG ALT (−77±9%, P<0.05) and Ca2+ ALT (−56±7%, P<0.05) and, importantly, reduced ROS (−65%, P<0.01) and oxidation of SERCA2a (−38%, P<0.05). CaMKII inhibition and general antioxidant treatment had no effect on ECG ALT and Ca2+ ALT. Conclusions—These results demonstrate, for the first time, that in MI, increased ROS from XO is a significant cause of repolarization alternans. This suggests that targeting XO ROS production may be effective at preventing arrhythmia substrates in chronic MI.

Targeted SERCA2a Gene Delivery to Restore Electrical Stability in the Failing Heart

Background— Recently, we reported that sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), the pump responsible for reuptake of cytosolic calcium during diastole, plays a central role in the molecular mechanism of cardiac alternans. Heart failure (HF) is associated with impaired myocardial calcium handling, deficient SERCA2a, and increased susceptibility to cardiac alternans. Therefore, we hypothesized that restoring deficient SERCA2a by gene transfer will significantly reduce arrhythmogenic cardiac alternans in the failing heart. Methods and Results— Adult guinea pigs were divided into 3 groups: control, HF, and HF+AAV9.SERCA2a gene transfer. HF resulted in a decrease in left ventricular fractional shortening compared with controls (P<0.001). As expected, isolated HF myocytes demonstrated slower sarcoplasmic reticulum calcium uptake, decreased Ca2+ release, and increased diastolic Ca2+ (P<0.05) compared with controls. Moreover, SERCA2a, cardiac ryanodine receptor 2, and sodium-calcium exchanger protein expression was decreased in HF compared with control (P<0.05). As predicted, HF increased susceptibility to cardiac alternans, as evidenced by decreased heart rate thresholds for both Vm alternans and Ca alternans compared with controls (P<0.01). Interestingly, in vivo gene transfer of AAV9.SERCA2a in the failing heart improved left ventricular contractile function (P<0.01), suppressed cardiac alternans (P<0.01), and reduced ryanodine receptor 2 Po secondary to reduction of ryanodine receptor 2–PS2814 (P<0.01). This ultimately resulted in a decreased incidence of inducible ventricular arrhythmias (P=0.05). Conclusions— These data show that SERCA2a gene transfer in the failing heart not only improves contractile function but also directly restores electric stability through the amelioration of key arrhythmogenic substrate (ie, cardiac alternans) and triggers (ie, sarcoplasmic reticulum Ca2+ leak).

Arrhythmogenic Cardiac Alternans in Heart Failure is Suppressed by Late Sodium Current Blockade by Ranolazine

BACKGROUND Cardiac alternans is promoted by heart failure (HF)-induced calcium (Ca2+) cycling abnormalities. Late sodium current (INa,L) is enhanced in HF and promotes Ca2+ overload; however, mechanisms underlying an antiarrhythmic effect of INa,L blockade in HF remain unclear. OBJECTIVE The purpose of this study was to determine whether ranolazine suppresses cardiac alternans in HF by normalizing Ca2+ cycling. METHODS Transmural dual optical mapping of Ca2+ transients and action potentials was performed in wedge preparations from 8 HF and 8 control (normal) dogs. Susceptibility to action potential duration alternans (APD-ALT) and Ca2+ transient alternans (Ca-ALT) was compared at baseline and with ranolazine (5-10 μM). RESULTS HF increased APD- and Ca-ALT compared to normal (both P <.05), and ranolazine suppressed APD- and Ca-ALT in both groups (P <.05). The incidence of spatially discordant alternans (DIS-ALT) was increased by HF (8/8) compared to normal (4/8; P <.05), and ranolazine decreased DIS-ALT in HF (4/8; P <.05).Not only did ranolazine mitigate HF-induced Ca2+ overload, it also attenuated APD-ALT to Ca-ALT gain (amount of APD-ALT produced by Ca-ALT). In HF, APD-ALT to Ca-ALT gain was significantly increased (0.55 ± 0.02) compared to normal (0.44 ± 0.02; P <.05) and was normalized by ranolazine (0.36 ± 0.05; P <.05), representing a complementary mechanism by which INa,L blockade suppressed cardiac alternans. CONCLUSION Ranolazine attenuated arrhythmogenic cardiac alternans in HF, both by suppressing Ca-ALT and decreasing the coupling gain of APD-ALT to Ca-ALT. Blockade of INa,L may reverse impaired Ca2+ cycling to mitigate cardiac alternans, representing a mechanism underlying the antiarrhythmic benefit of INa,L blockade in HF.

Arrhythmogenic Delayed Afterdepolarizations Are Promoted by Severe Hypothermia But Not Therapeutic Hypothermia

BACKGROUND Severe hypothermia (SH) is known to be arrhythmogenic, but the effect of therapeutic hypothermia (TH) on arrhythmias is unclear. It is hypothesized that susceptibility to Ca-mediated arrhythmia triggers would be increased only by SH.Methods and Results:Spontaneous Ca release (SCR) and resultant delayed afterdepolarizations (DADs) were evaluated by optical mapping in canine wedge preparations during normothermia (N, 36℃), TH (32℃) or SH (28℃; n=8 each). The slope (amplitude/rise time) of multicellular SCR (mSCR) events, a determinant of triggered activity, was suppressed in TH (24.4±3.4%/s vs. N: 41.5±6.0%/s), but significantly higher in SH (96.3±8.1%/s) producing higher amplitude DADs in SH (35.7±1.6%) and smaller in TH (5.3±1.0% vs. N: 10.0±1.1%, all P<0.05). Triggered activity was only observed in SH. In isolated myocytes, sarcoplasmic reticulum (SR) Ca release kinetics slowed in a temperature-dependent manner, prolonging Ca transient rise time [33±3 (N) vs. 50±6 (TH) vs. 88±12 ms (SH), P<0.05], which can explain the decreased mSCR slope and DAD amplitude in TH. Although the SR Ca content was similar in TH and SH, Ca spark frequency was markedly increased only in SH, suggesting that increased ryanodine receptor open probability could explain the increased triggered activity during SH. CONCLUSIONS Temperature dependence of Ca release can explain susceptibility to Ca-mediated arrhythmia triggers in SH. This may therefore explain the increased risk of lethal arrhythmia in SH, but not during TH.

Calcium-Mediated Arrhythmia Substrates Associated with Oxidative Stress during Myocardial Infarction

Following myocardial infarction (MI), ventricular arrhythmias commonly originate from the MI border zone (BZ) but the cause for this is not clear. Increased oxidative stress is a hallmark of MI and an important mechanism of intracellular calcium dysregulation. We hypothesize that following MI, increased oxidative stress in the infarct BZ creates a substrate for calcium-mediated arrhythmias. Methods: Male Lewis rats (n=12) underwent ligation of the left-anterior descending artery. At 4 weeks, hearts were isolated and high-resolution optical mapping of intracellular calcium (Indo-1AM) and reactive oxygen species (ROS, dichlorofluorescein diacetate) were performed. Blebbistatin (7μM) was used to eliminate motion artifact. Calcium transient alternans (CaT_Alt) and multicellular-spontaneous calcium release (mSCR) were induced by rapid pacing (200–670 bpm) before and after the CaMKII blocker KN-93. Sites within 2mm of the anatomical scar border where designated as BZ. All other sites in the mapping field outside the BZ and scar were considered remote. Results: CaT_Alt and mSCR activity were significantly greater in the MI BZ (15±3%, 20±2%) as compared to remote sites (3.3±2%, p<0.01; 9.0±2%, p<0.001), respectively. Additionally, ROS density was increased by 283±53% (p<0.001) in the BZ compared to remote regions, which was confirmed offline by tissue sample analysis. Interestingly, treatment with KN-93 significantly decreased CaT_Alt in the BZ by 50±10% (p<0.05) but did not decrease mSCR activity. Conclusions: These results demonstrate that increased ROS in the infarct BZ is associated with a significant increase in calcium-mediated arrhythmia substrates (CaT_Alt and mSCR). In addition, CaMKII activation may be a mechanism of CaT_Alt but not spontaneous calcium release in the BZ, suggesting multiple calcium regulatory targets of oxidative stress associated with MI.