William Kutschke

A Dynamic Pathway for Calcium-Independent Activation of CaMKII by Methionine Oxidation

Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA-/- mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.

T-Tubule Remodeling During Transition From Hypertrophy to Heart Failure

Rationale: The transverse tubule (T-tubule) system is the ultrastructural substrate for excitation–contraction coupling in ventricular myocytes; T-tubule disorganization and loss are linked to decreased contractility in end stage heart failure (HF). Objective: We sought to examine (1) whether pathological T-tubule remodeling occurs early in compensated hypertrophy and, if so, how it evolves during the transition from hypertrophy to HF; and (2) the role of junctophilin-2 in T-tubule remodeling. Methods and Results: We investigated T-tubule remodeling in relation to ventricular function during HF progression using state-of-the-art confocal imaging of T-tubules in intact hearts, using a thoracic aortic banding rat HF model. We developed a quantitative T-tubule power (TTpower) index to represent the integrity of T-tubule structure. We found that discrete local loss and global reorganization of the T-tubule system (leftward shift of TTpower histogram) started early in compensated hypertrophy in left ventricular (LV) myocytes, before LV dysfunction, as detected by echocardiography. With progression from compensated hypertrophy to early and late HF, T-tubule remodeling spread from the LV to the right ventricle, and TTpower histograms of both ventricles gradually shifted leftward. The mean LV TTpower showed a strong correlation with ejection fraction and heart weight to body weight ratio. Over the progression to HF, we observed a gradual reduction in the expression of a junctophilin protein (JP-2) implicated in the formation of T-tubule/sarcoplasmic reticulum junctions. Furthermore, we found that JP-2 knockdown by gene silencing reduced T-tubule structure integrity in cultured adult ventricular myocytes. Conclusions: T-tubule remodeling in response to thoracic aortic banding stress begins before echocardiographically detectable LV dysfunction and progresses over the development of overt structural heart disease. LV T-tubule remodeling is closely associated with the severity of cardiac hypertrophy and predicts LV function. Thus, T-tubule remodeling may constitute a key mechanism underlying the transition from compensated hypertrophy to HF.

MyD88 mediated inflammatory signaling leads to CaMKII oxidation, cardiac hypertrophy and death after myocardial infarction

The toll-like receptors (TLR) and myocardial infarction (MI) promote NF-κB-dependent inflammatory transcription and oxidative injury in myocardium. The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is activated by oxidation and contributes to NF-κB-dependent transcription, myocardial hypertrophy and post-MI death. The myeloid differentiation protein 88 (MyD88) is an adapter protein critical for many TLR functions, but downstream targets for TLR/MyD88 signaling in MI are not well understood. We asked if CaMKII and TLR/MyD88 pathways are interconnected and if TLR/MyD88 contributes to adverse outcomes after MI. Here we show that TLR-4 activation by lipopolysaccharide (LPS) induces CaMKII oxidation (ox-CaMKII) in cardiomyocytes. MI enhances ox-CaMKII in wild type (WT) hearts but not in MyD88(-/-) hearts that are defective in MyD88-dependent TLR signaling. In post-MI WT hearts expression of pro-inflammatory genes TNF-α (Tnfa), complement factor B (Cfb), myocyte death and fibrosis were significantly increased, but increases were significantly less in MyD88(-/-) hearts after MI. MyD88(-/-) cardiomyocytes were defective in NF-κB activation by LPS but not by the MyD88-independent TLR agonist poly(I:C). In contrast, TNF-α induced Cfb gene expression was not deficient in MyD88(-/-) cardiomyocytes. Several hypertrophy marker genes were upregulated in both WT and MyD88(-/-) hearts after MI, but Acta1 was significantly attenuated in MyD88(-/-) hearts, suggesting that MyD88 selectively affects expression of hypertrophic genes. Post-MI cardiac hypertrophy, inflammation, apoptosis, ox-CaMKII expression and mortality were significantly reduced in MyD88(-/-) compared to WT littermates. These data suggest that MyD88 contributes to CaMKII oxidation and is important for adverse hypertrophic and inflammatory responses to LPS and MI.

Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart

Significance Mitochondrial Ca2+ is a fundamental signal that allows for adaptation to physiological stress but a liability during ischemia-reperfusion injury in heart. On one hand, mitochondrial Ca2+ entry coordinates energy supply and demand in myocardium by increasing the activity of matrix dehydrogenases to augment ATP production by oxidative phosphorylation. On the other hand, inhibiting mitochondrial Ca2+ overload is promulgated as a therapeutic approach to preserve myocardial tissue following ischemia-reperfusion injury. We developed a new mouse model of myocardial-targeted transgenic dominant-negative mitochondrial Ca2+ uniporter (MCU) expression to test consequences of chronic loss of MCU-mediated Ca2+ entry in heart. Here we show that MCU inhibition has unanticipated consequences on extramitochondrial pathways affecting oxygen utilization, cytoplasmic Ca2+ homeostasis, physiologic responses to stress, and pathologic responses to ischemia-reperfusion injury. Myocardial mitochondrial Ca2+ entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca2+ are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca2+ uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca2+ entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU–permeabilized myocardial fibers or isolated mitochondria in low Ca2+ were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca2+ homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca2+] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca2+ homeostasis. Mitochondrial Ca2+ overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca2+ homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.

Overexpression of junctophilin-2 does not enhance baseline function but attenuates heart failure development after cardiac stress

Significance Normal cardiac function requires coordinated contraction of working myocytes, which is initiated by a specific communication between Ca2+ channels on the transverse (T)-tubule membrane and ryanodine receptors on the sarcoplasmic reticulum (SR) membrane. Junctophilin-2 (JP2) is a structural protein that induces docking of SR to T-tubules to form dyads and that indirectly stabilizes the T-tubule network in ventricular cardiomyocytes. JP2 is frequently down-regulated in heart failure, in parallel with a disruption of the T-tubule network and loss of normal excitation-contraction coupling. Here we show that overexpression of JP2 stabilizes the T-tubule network and attenuates heart failure after cardiac stress. These data suggest that future treatment of heart disease may include strategies to stabilize the architecture of T-tubules and cardiac dyads. Heart failure is accompanied by a loss of the orderly disposition of transverse (T)-tubules and a decrease of their associations with the junctional sarcoplasmic reticulum (jSR). Junctophilin-2 (JP2) is a structural protein responsible for jSR/T-tubule docking. Animal models of cardiac stresses demonstrate that down-regulation of JP2 contributes to T-tubule disorganization, loss of excitation-contraction coupling, and heart failure development. Our objective was to determine whether JP2 overexpression attenuates stress-induced T-tubule disorganization and protects against heart failure progression. We therefore generated transgenic mice with cardiac-specific JP2 overexpression (JP2-OE). Baseline cardiac function and Ca2+ handling properties were similar between JP2-OE and control mice. However, JP2-OE mice displayed a significant increase in the junctional coupling area between T-tubules and the SR and an elevated expression of the Na+/Ca2+ exchanger, although other excitation-contraction coupling protein levels were not significantly changed. Despite similar cardiac function at baseline, overexpression of JP2 provided significantly protective benefits after pressure overload. This was accompanied by a decreased percentage of surviving mice that developed heart failure, as well as preservation of T-tubule network integrity in both the left and right ventricles. Taken together, these data suggest that strategies to maintain JP2 levels can prevent the progression from hypertrophy to heart failure.

β-Adrenergic receptor antagonists ameliorate myocyte T-tubule remodeling following myocardial infarction

β‐Adrenergic receptor (AR) blockers provide substantial clinical benefits, including improving overall survival and left ventricular (LV) function following myocardial infarction (MI), though the mechanisms remain incompletely defined. The transverse‐tubule (T‐tubule) system of ventricular myocytes is an important determinant of cardiac excitation‐contraction function. T‐tubule remodeling occurs early during LV failure. We hypothesized that β‐AR blockers prevent T‐tubule remodeling and thereby provide therapeutic benefits. A murine model of MI was utilized to examine the effect of β‐AR blockers on T‐tubule remodeling following LV MI. We applied the in situ imaging of T‐tubule structure from Langendorff‐perfused intact hearts with laser scanning confocal microscopy. We found that MI caused remarkable T‐tubule remodeling near the infarction border zone and moderate LV remodeling remote from the MI. Metoprolol and carvedilol administered 6 d after MI for 4 wk each increased the T‐tubule integrity at the remote and border zones. At the molecular level, both β‐AR blockers restored border and remote zone expression of junctophilin‐2 (JP‐2), which is involved in T‐tubule organization and formation of the T‐tubule/sarcoplasmic reticulum junctions. In contrast, β‐AR blockers had no significant effects on caveolin‐3 expression. In summary, our data show that β‐AR antagonists can protect against T‐tubule remodeling after MI, suggesting a novel therapeutic mechanism of action for this drug class. Preservation of JP‐2 expression may contribute to the beneficial effects of metoprolol and carvedilol on T‐tubule remodeling.— Chen, B., Li, Y., Jiang, S., Xie, Y.‐P., Guo, A., Kutschke, W., Zimmerman, K., Weiss, R. M., Miller, F. J., Anderson, M. E., Song, L.‐S. β‐Adrenergic receptor antagonists ameliorate myocyte T‐tubule remodeling following myocardial infarction. FASEB J. 26, 2531‐2537 (2012). www.fasebj.org

Critical roles of junctophilin-2 in T-tubule and excitation–contraction coupling maturation during postnatal development

AIMS Emerging evidence indicates a critical role for junctophilin-2 (JP2) in T-tubule integrity and assembly of cardiac dyads in adult ventricular myocytes. In the postnatal stage, one of the critical features of myocyte maturation is development of the T-tubule system, though the mechanisms remain poorly understood. In this study, we aim to determine whether JP2 is required for normal cardiac T-tubule maturation. METHODS AND RESULTS Using in situ confocal imaging of intact murine hearts, we found T-tubules were absent in both left- and right-ventricular myocytes at postnatal Day 8 and did not appear until Day 10. Quantification of T-tubule structural integrity using the T-tubule power (TT(power)) index revealed a progressive increase in TT(power) between postnatal Days 10 and 19. By postnatal Day 19, TT(power) was similar to that in adult murine cardiomyocytes, indicative of a nearly matured T-tubule network. JP2 levels increased dramatically during development, reaching levels observed in adult hearts by postnatal Day 14. Deficiency of JP2, using a mouse model in which a JP2-specific shRNA is expressed during embryonic development, severely impaired T-tubule maturation, with equivalent decreases in the left- and right-ventricular TT(power). We also detected a gradual increase in the density of transverse but not longitudinal tubules during development, and JP2 deficiency abolished the increase in the density of transverse elements. Alterations in T-tubules caused significant reduction in Ca(2+) transient amplitude and marked increase in Ca(2+) release dyssynchrony, Ca(2+) alternans, and spontaneous Ca(2+) waves, leading to contractile failure. CONCLUSION Our data identify a critical role for JP2 in T-tubule and excitation-contraction coupling maturation during development.

Effects of deep sedation or general anesthesia on cardiac function in mice undergoing cardiovascular magnetic resonance

BackgroundGenetically engineered mouse models of human cardiovascular disease provide an opportunity to understand critical pathophysiological mechanisms. Cardiovascular magnetic resonance (CMR) provides precise reproducible assessment of cardiac structure and function, but, in contrast to echocardiography, requires that the animal be immobilized during image acquisition. General anesthetic regimens yield satisfactory images, but have the potential to significantly perturb cardiac function. The purpose of this study was to assess the effects of general anesthesia and a new deep sedation regimen, respectively, on cardiac function in mice as determined by CMR, and to compare them to results obtained in mildly sedated conscious mice by echocardiography.ResultsIn 6 mildly sedated normal conscious mice assessed by echo, heart rate was 615 ± 25 min-1 (mean ± SE) and left ventricular ejection fraction (LVEF) was 0.94 ± 0.01. In the CMR studies of normal mice, heart rate was slightly lower during deep sedation with morphine/midazolam (583 ± 30 min-1), but the difference was not statistically significant. General anesthesia with 1% inhaled isoflurane significantly depressed heart rate (468 ± 7 min-1, p < 0.05 vs. conscious sedation). In 6 additional mice with ischemic LV failure, trends in heart rate were similar, but not statistically significant. In normal mice, deep sedation depressed LVEF (0.79 ± 0.04, p < 0.05 compared to light sedation), but to a significantly lesser extent than general anesthesia (0.60 ± 0.04, p < 0.05 vs. deep sedation).In mice with ischemic LV failure, ejection fraction measurements were comparable when performed during light sedation, deep sedation, and general anesthesia, respectively. Contrast-to-noise ratios were similar during deep sedation and during general anesthesia, indicating comparable image quality. Left ventricular mass measurements made by CMR during deep sedation were nearly identical to those made during general anesthesia (r2 = 0.99, mean absolute difference < 4%), indicating equivalent quantitative accuracy obtained with the two methods. The imaging procedures were well-tolerated in all mice.ConclusionIn mice with normal cardiac function, CMR during deep sedation causes significantly less depression of heart rate and ejection fraction than imaging during general anesthesia with isoflurane. In mice with heart failure, the sedation/anesthesia regimen had no clear impact on cardiac function. Deep sedation and general anesthesia produced CMR with comparable image quality and quantitative accuracy.

Aortic valve sclerosis in mice deficient in endothelial nitric oxide synthase

Risk factors for fibrocalcific aortic valve disease (FCAVD) are associated with systemic decreases in bioavailability of endothelium-derived nitric oxide (EDNO). In patients with bicuspid aortic valve (BAV), vascular expression of endothelial nitric oxide synthase (eNOS) is decreased, and eNOS(-/-) mice have increased prevalence of BAV. The goal of this study was to test the hypotheses that EDNO attenuates profibrotic actions of valve interstitial cells (VICs) in vitro and that EDNO deficiency accelerates development of FCAVD in vivo. As a result of the study, coculture of VICs with aortic valve endothelial cells (vlvECs) significantly decreased VIC activation, a critical early phase of FCAVD. Inhibition of VIC activation by vlvECs was attenuated by N(G)-nitro-l-arginine methyl ester or indomethacin. Coculture with vlvECs attenuated VIC expression of matrix metalloproteinase-9, which depended on stiffness of the culture matrix. Coculture with vlvECs preferentially inhibited collagen-3, compared with collagen-1, gene expression. BAV occurred in 30% of eNOS(-/-) mice. At age 6 mo, collagen was increased in both bicuspid and trileaflet eNOS(-/-) aortic valves, compared with wild-type valves. At 18 mo, total collagen was similar in eNOS(-/-) and wild-type mice, but collagen-3 was preferentially increased in eNOS(-/-) mice. Calcification and apoptosis were significantly increased in BAV of eNOS(-/-) mice at ages 6 and 18 mo. Remarkably, these histological changes were not accompanied by physiologically significant valve stenosis or regurgitation. In conclusion, coculture with vlvECs inhibits specific profibrotic VIC processes. In vivo, eNOS deficiency produces fibrosis in both trileaflet and BAVs but produces calcification only in BAVs.

Calcium/Calmodulin‐Dependent Kinase II Inhibition in Smooth Muscle Reduces Angiotensin II–Induced Hypertension by Controlling Aortic Remodeling and Baroreceptor Function

Background Multifunctional calcium/calmodulin-dependent kinase II (CaMKII) is activated by angiotensin II (Ang II) in cultured vascular smooth muscle cells (VSMCs), but its function in experimental hypertension has not been explored. The aim of this study was to determine the impact of CaMKII inhibition selectively in VSMCs on Ang II hypertension. Methods and Results Transgenic expression of a CaMKII peptide inhibitor in VSMCs (TG SM-CaMKIIN model) reduced the blood pressure response to chronic Ang II infusion. The aortic depressor nerve activity was reset in hypertensive versus normotensive wild-type animals but not in TG SM-CaMKIIN mice, suggesting that changes in baroreceptor activity account for the blood pressure difference between genotypes. Accordingly, aortic pulse wave velocity, a measure of arterial wall stiffness and a determinant of baroreceptor activity, increased in hypertensive versus normotensive wild-type animals but did not change in TG SM-CaMKIIN mice. Moreover, examination of blood pressure and heart rate under ganglionic blockade revealed that VSMC CaMKII inhibition abolished the augmented efferent sympathetic outflow and renal and splanchnic nerve activity in Ang II hypertension. Consequently, we hypothesized that VSMC CaMKII controls baroreceptor activity by modifying arterial wall remodeling in Ang II hypertension. Gene expression analysis in aortas from normotensive and Ang II–infused mice revealed that TG SM-CaMKIIN aortas were protected from Ang II–induced upregulation of genes that control extracellular matrix production, including collagen. VSMC CaMKII inhibition also strongly altered the expression of muscle contractile genes under Ang II. Conclusions CaMKII in VSMCs regulates blood pressure under Ang II hypertension by controlling structural gene expression, wall stiffness, and baroreceptor activity.