W. Keith Jones

Inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of Bnip3 restrains postinfarction remodeling in mice

Following myocardial infarction, nonischemic myocyte death results in infarct expansion, myocardial loss, and ventricular dysfunction. Here, we demonstrate that a specific proapoptotic gene, Bnip3, minimizes ventricular remodeling in the mouse, despite having no effect on early or late infarct size. We evaluated the effects of ablating Bnip3 on cardiomyocyte death, infarct size, and ventricular remodeling after surgical ischemia/reperfusion (IR) injury in mice. Immediately following IR, no significant differences were observed between Bnip3(-/-) and WT mice. However, at 2 days after IR, apoptosis was diminished in Bnip3(-/-) periinfarct and remote myocardium, and at 3 weeks after IR, Bnip3(-/-) mice exhibited preserved LV systolic performance, diminished LV dilation, and decreased ventricular sphericalization. These results suggest myocardial salvage by inhibition of apoptosis. Forced cardiac expression of Bnip3 increased cardiomyocyte apoptosis in unstressed mice, causing progressive LV dilation and diminished systolic function. Conditional Bnip3 overexpression prior to coronary ligation increased apoptosis and infarct size. These studies identify postischemic apoptosis by myocardial Bnip3 as a major determinant of ventricular remodeling in the infarcted heart, suggesting that Bnip3 may be an attractive therapeutic target.

Novel Cardioprotective Role of a Small Heat-Shock Protein, Hsp20, Against Ischemia/Reperfusion Injury

Background—Heat-shock proteins (Hsps) have been shown to render cardioprotection from stress-induced injury; however, little is known about the role of another small heat-shock protein, Hsp20, which regulates activities of vasodilation and platelet aggregation, in cardioprotection against ischemia injury. We recently reported that increased expression of Hsp20 in cardiomyocytes was associated with improved contraction and protection against &bgr;-agonist–induced apoptosis. Methods and Results—To investigate whether overexpression of Hsp20 exerts protective effects in both ex vivo and in vivo ischemia/reperfusion (I/R) injury, we generated a transgenic (TG) mouse model with cardiac-specific overexpression of Hsp20 (10-fold). TG and wild-type (WT) hearts were then subjected to global no-flow I/R (45 minutes/120 minutes) using the Langendorff preparation. TG hearts exhibited improved recovery of contractile performance over the whole reperfusion period. This improvement was accompanied by a 2-fold decrease in lactate dehydrogenase released from the TG hearts. The extent of infarction and apoptotic cell death was also significantly decreased, which was associated with increased protein ratio of Bcl-2/Bax and reduced caspase-3 activity in TG hearts. Furthermore, in vivo experiments of 30-minute myocardial ischemia, via coronary artery occlusion, followed by 24-hour reperfusion, showed that the infarct region–to–risk region ratio was 8.1±1.1% in TG hearts (n=7), compared with 19.5±2.1% in WT hearts (n=11, P<0.001). Conclusions—Our data demonstrate that increased Hsp20 expression in the heart protects against I/R injury, resulting in improved recovery of cardiac function and reduced infarction. Thus, Hsp20 may constitute a new therapeutic target for ischemic heart diseases.

Peripheral Nociception Associated With Surgical Incision Elicits Remote Nonischemic Cardioprotection Via Neurogenic Activation of Protein Kinase C Signaling

Background— Although remote ischemic stimuli have been shown to elicit cardioprotection against ischemia/reperfusion injury, there is little known about the effects of nonischemic stimuli. We previously described a remote cardioprotective effect of nonischemic surgical trauma (abdominal incision) called remote preconditioning of trauma (RPCT). In the present study, we elucidate mechanisms underlying this phenomenon. Methods and Results— We used a murine model of myocardial infarction to evaluate ischemia/reperfusion injury, and either abdominal surgical incision, or application of topical capsaicin, to elicit cardioprotection. We show that the cardioprotective effect of RPCT is initiated by skin nociception, and requires neurogenic signaling involving spinal nerves and activation of cardiac sensory and sympathetic nerves. Our results demonstrate bradykinin-dependent activation and repression, respectively, of PKCϵ and PKC&dgr; in myocardium after RPCT, and we show involvement of the KATP channels in cardioprotection. Finally, we show that topical application of capsaicin, which selectively activates C sensory fibers in the skin, mimics the cardioprotective effect of RPCT against myocardial infarction. Conclusions— Nontraumatic nociceptive preconditioning represents a novel therapeutic strategy for cardioprotection with great potential clinical utility.

NF-κB as an integrator of diverse signaling pathways

NF-κB is a pleiotropic transcription factor implicated in the regulation of diverse biological phenomena, including apoptosis, cell survival, cell growth, cell division, innate immunity, cellular differentiation, and the cellular responses to stress, hypoxia, stretch and ischemia. In the heart, NF-κB has been shown to be activated in atherosclerosis, myocarditis, in association with angina, during transplant rejection, after ischemia/ reperfusion, in congestive heart failure, dilated cardiomyopathy, after ischemic and pharmacological preconditioning, heat shock, burn trauma, and in hypertrophy of isolated cardiomyocytes. Regulation of NF-κB is complicated; in addition to being activated by canonical cytokine-mediated pathways, NF-κB is activated by many of the signal transduction cascades associated with the development of cardiac hypertrophy and response to oxidative stress. Many of these signaling cascades activate NF-κB by activating the lκB kinase (IKK) complex a major component of the canonical pathway. These signaling interactions occur largely via signaling crosstalk involving the mitogen-activated protein kinase/extracellular signaling crosstalk involving the mitogen-activated protein kinase/extracellular signal-regulated kinases (MEKKs) that are components of mitogen activated protein kinase (MAPK) signaling pathways. Additionally, there are other signaling factors that act more directly to activate NF-κB via IκB or by direct phosphorylation of NF-κB subunits. Finally, there are combinatorial interactions at the level of the promoter between NF-κB, its, coativators, and other transcription factors, several of which are activated by MAPK and cytokine signaling pathways. Thus, in addition to being a major mediator of cytokine effects in the heart, NF-κB is positioned as a signaling integrator. As such, NF-κB functions as a key regulator of cardiac gene expression programs downstream of multiple signal transduction cascades in a variety of physiological and pathophysiological states. We show that genetic blockade of NF-κB reduces infarct size in the murine heart after ischemia/reperfusion (I/R), implicating NF-κB as a major determinant of cell death after I/R. These results support the concept that NF-κB may be an important therapeutic target for specific cardiovascular diseases. Furthermore, undertanding the complex signal transduction and gene regulation networks associated with NF-κB functionality will allow us to identify the discrete sets of NF-κB-dependent genes that affect specific pathophysiological phenomena. These genes may be even better therapeutic targets, allowing us to block the injurious effects while preserving the potentially beneficial effects of adaptive signaling in the heart.

Sarcoplasmic Reticulum Calcium Overloading in Junctin Deficiency Enhances Cardiac Contractility but Increases Ventricular Automaticity

Background— Abnormal sarcoplasmic reticulum calcium (Ca) cycling is increasingly recognized as an important mechanism for increased ventricular automaticity that leads to lethal ventricular arrhythmias. Previous studies have linked lethal familial arrhythmogenic disorders to mutations in the ryanodine receptor and calsequestrin genes, which interact with junctin and triadin to form a macromolecular Ca-signaling complex. The essential physiological effects of junctin and its potential regulatory roles in sarcoplasmic reticulum Ca cycling and Ca-dependent cardiac functions, such as myocyte contractility and automaticity, are unknown. Methods and Results— The junctin gene was targeted in embryonic stem cells, and a junctin-deficient mouse was generated. Ablation of junctin was associated with enhanced cardiac function in vivo, and junctin-deficient cardiomyocytes exhibited increased contractile and Ca-cycling parameters. Short-term isoproterenol stimulation elicited arrhythmias, including premature ventricular contractions, atrioventricular heart block, and ventricular tachycardia. Long-term isoproterenol infusion also induced premature ventricular contractions and atrioventricular heart block in junctin-null mice. Further examination of the electrical activity revealed a significant increase in the occurrence of delayed afterdepolarizations. Consistently, 25% of the junctin-null mice died by 3 months of age with structurally normal hearts. Conclusions— Junctin is an essential regulator of sarcoplasmic reticulum Ca release and contractility in normal hearts. Ablation of junctin is associated with aberrant Ca homeostasis, which leads to fatal arrhythmias. Thus, normal intracellular Ca cycling relies on maintenance of junctin levels and an intricate balance among the components in the sarcoplasmic reticulum quaternary Ca-signaling complex.

Blockade of Hsp20 Phosphorylation Exacerbates Cardiac Ischemia/Reperfusion Injury by Suppressed Autophagy and Increased Cell Death

Rationale: The levels of a small heat shock protein (Hsp)20 and its phosphorylation are increased on ischemic insults, and overexpression of Hsp20 protects the heart against ischemia/reperfusion injury. However, the mechanism underlying cardioprotection of Hsp20 and especially the role of its phosphorylation in regulating ischemia/reperfusion–induced autophagy, apoptosis, and necrosis remain to be clarified. Objective: Herein, we generated a cardiac-specific overexpression model, carrying nonphosphorylatable Hsp20, where serine 16 was substituted with alanine (Hsp20S16A). By subjecting this model to ischemia/reperfusion, we addressed whether: (1) the cardioprotective effects of Hsp20 are associated with serine 16 phosphorylation; (2) blockade of Hsp20 phosphorylation influences the balance between autophagy and cell death; and (3) the aggregation pattern of Hsp20 is altered by its phosphorylation. Methods and Results: Our results demonstrated that Hsp20S16A hearts were more sensitive to ischemia/reperfusion injury, evidenced by lower recovery of contractile function and increased necrosis and apoptosis, compared with non-TG hearts. Interestingly, autophagy was activated in non-TG hearts but significantly inhibited in Hsp20S16A hearts following ischemia/reperfusion. Accordingly, pretreatment of Hsp20S16A hearts with rapamycin, an activator of autophagy, resulted in improvement of functional recovery, compared with saline-treated Hsp20S16A hearts. Furthermore, on ischemia/reperfusion, the oligomerization pattern of Hsp20 appeared to shift to higher aggregates in Hsp20S16A hearts. Conclusions: Collectively, these data indicate that blockade of Ser16-Hsp20 phosphorylation attenuates the cardioprotective effects of Hsp20 against ischemia/reperfusion injury, which may be attributable to suppressed autophagy and increased cell death. Therefore, phosphorylation of Hsp20 at serine 16 may represent a potential therapeutic target in ischemic heart disease.

Gene Dosage-Dependent Effects of Cardiac-Specific Overexpression of the A3 Adenosine Receptor

We used a genetic approach to determine whether increasing the level of A3 adenosine receptors (A3ARs) expressed in the heart confers protection against ischemia without causing cardiac pathology. We generated mice carrying one (A3tg.1) or six (A3tg.6) copies of a transgene consisting of the cardiomyocyte-specific &agr;-myosin heavy chain gene promoter and the A3AR cDNA. A3tg.1 and A3tg.6 mice expressed 12.7±3.15 and 66.3±9.4 fmol/mg of the high-affinity G protein–coupled form of the A3AR in the myocardium, respectively. Extensive morphological, histological, and functional analyses demonstrated that there were no apparent abnormalities in A3tg.1 transgenic mice compared with nontransgenic mice. In contrast, A3tg.6 mice exhibited dilated hearts, expression of markers of hypertrophy, bradycardia, hypotension, and systolic dysfunction. When A3tg mice were subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion, infarct size was reduced ≈30% in A3tg.1 mice and ≈40% in A3tg.6 mice compared with nontransgenic littermates. The reduction in infarct size in the transgenic mice was not related to differences in risk region size, systemic hemodynamics, or body temperature, indicating that the cardioprotection was a result of increased A3AR signaling in the ischemic myocardium. The results demonstrate that low-level expression of A3ARs in the heart provides effective protection against ischemic injury without detectable adverse effects, whereas higher levels of A3AR expression lead to the development of a dilated cardiomyopathy.

Targeted disruption of the voltage-dependent calcium channel α2/δ-1-subunit

Cardiac L-type voltage-dependent Ca(2+) channels are heteromultimeric polypeptide complexes of alpha(1)-, alpha(2)/delta-, and beta-subunits. The alpha(2)/delta-1-subunit possesses a stereoselective, high-affinity binding site for gabapentin, widely used to treat epilepsy and postherpetic neuralgic pain as well as sleep disorders. Mutations in alpha(2)/delta-subunits of voltage-dependent Ca(2+) channels have been associated with different diseases, including epilepsy. Multiple heterologous coexpression systems have been used to study the effects of the deletion of the alpha(2)/delta-1-subunit, but attempts at a conventional knockout animal model have been ineffective. We report the development of a viable conventional knockout mouse using a construct targeting exon 2 of alpha(2)/delta-1. While the deletion of the subunit is not lethal, these animals lack high-affinity gabapentin binding sites and demonstrate a significantly decreased basal myocardial contractility and relaxation and a decreased L-type Ca(2+) current peak current amplitude. This is a novel model for studying the function of the alpha(2)/delta-1-subunit and will be of importance in the development of new pharmacological therapies.

NF‐κB mediated miR‐26a regulation in cardiac fibrosis

Micro‐RNAs (miRNAs) are a class of small non‐coding RNAs, recently emerged as a post‐transcriptional regulator having a key role in various cardiac pathologies. Among them, cardiac fibrosis that occurs as a result from an imbalance of extracellular matrix proteins turnover and is a highly debilitating process that eventually lead to organ dysfunction. An emerging theme on is that miRNAs participate in feedback loop with transcription factors that regulate their transcription. NF‐κB, a key transcription factor regulator controls a series of gene program in various cardiac diseases through positive and negative feedback mechanism. But, NF‐κB mediated miRNA regulation in cardiac fibrosis remains obscure. Bioinformatics analysis revealed that miR‐26a has targets collagen I and CTGF and possesses putative NF‐κB binding element in its promoter region. Here, we show that inhibition of NF‐κB in cardiac fibroblast restores miR‐26a expression, attenuating collagen I, and CTGF gene expression in the presence of Ang II, conferring a feedback regulatory mechanism in cardiac fibrosis. The target genes for miR‐26a were confirmed using 3′‐UTR luciferase reporter assays for collagen I and CTGF genes. Using NF‐κB reporter assays, we determine that miR‐26a overexpression inhibits NF‐κB activity. Finally, we show that miR‐26a expression is restored along with the attenuation of collagen I and CTGF genes in cardiac specific IkBa triple mutant transgenic mice (preventing NF‐κB activation) subjected to 4 weeks transverse aortic banding (TAC), compared to wild type (WT) mice. The data indicate a potential role of miR‐26a in cardiac fibrosis and, offer novel therapeutic intervention. J. Cell. Physiol. 228: 1433–1442, 2013.

Understanding radiation-induced vascular disease.

Most cardiovascular events occur >10 years after completing radiotherapy, so demonstrating causality has proven difficult (5). An estimated 50 million cancer survivors worldwide have been treated with radiation therapy; accordingly, clinicians must be aware of the potential cardiovascular risk and manage risk factors appropriately. Moreover, research into the mechanisms of radiation-induced vascular disease is paramount to understanding and potentially modifying the disease process. The study by Martin et al. (6) in this issue of the Journal is welcome, because it sheds new light on the pathogenesis of radiation-induced vascular disease in humans. Experimental studies in animals have firmly established a causal relationship between irradiation and vascular disease. Lethal total-body irradiation of atherosclerosis-prone mice followed by bone marrow transplantation noticeably altered lesion composition and stability (7,8). Nonlethal irradiation of atherosclerosis-prone mice did not change systemic indicators of inflammation or cholesterol levels but dramatically altered lesion composition long after treatment (9). There were no changes in the atherosclerotic lesions of “out-of-field” arteries, consistent with a local rather than systemic effect of radiation. Irradiated arteries 22 to 34 weeks after treatment were highly enriched with macrophages, which accounted for the majority of the lesion area. Also, intraplaque hemorrhage was restricted to and commonly observed in irradiated arteries. These studies, however, did not identify a molecular mechanism to explain the observations. Studies to address radiation-induced vascular disease in humans have largely been descriptive in nature. From the histological perspective, lesions in medium-sized to large vessels (>100 μm in diameter) exhibit typical features of atherosclerosis, including lipid accumulation, inflammation, and thrombosis (3). Increases in intimal thickness and connective tissue content are also prominent features (2). From the angiographic perspective, the lesions are longer than traditional atherosclerotic lesions, and the regions of maximal stenosis tend to be at the ends of the lesions (10). Treating these lesions via open surgical procedures is often problematic, due to extensive soft tissue scarring; hence, percutaneous approaches are usually preferred (5). How does a course of radiotherapy initiate a chronic vascular process that eventually leads to clinical events many years after treatment? Experimental studies in vitro and in vivo indicate that radiation therapy causes acute up-regulation of pro-inflammatory cytokines and adhesion molecules in endothelium that recruits inflammatory cells to sites of vascular injury (11). It is unlikely, however, that this acute insult per se is sufficient to produce long-term occlusive atherosclerotic disease. Thus, late effects of radiation therapy are more likely responsible. In this regard, induction of chronic oxidative stress is increasingly being implicated in radiation-induced late tissue injury (12). In addition to the rapid burst of free radicals produced acutely by ionization of water molecules, radiation increases chronic free radical production and oxidative stress in the affected tissues. Oxidative stress up-regulates numerous pathways pertinent to vascular disease, including matrix metalloproteinases, adhesion molecules, pro-inflammatory cytokines, and smooth muscle cell proliferation and apoptosis, while inactivating vasculoprotective nitric oxide. Considerable evidence suggests that the nuclear transcription factor NF-κB serves as a molecular link between oxidative stress and chronic inflammation (13). The nuclear factor-kappa B (NF-κB) family of transcription factors includes 5 members: p50, p52, p65, RelB, and c-Rel. The NF-κB is involved in numerous pathological and physiological conditions, including cellular function (i.e., proliferation, differentiation, and survival), tumorigenesis, and inflammation. Upon activation, NF-κB is released from its inhibitory association with the IκB proteins in the cytoplasm and translocates to the nucleus. In the nucleus, NF-κB binding to specific deoxyribonucleic acid response elements initiates robust transcriptional responses and reprograms cellular function. Depending upon the stimuli, NF-κB can activate distinct sets of downstream genes that mediate different outcomes (14). In the context of vascular biology, NF-κB is a master regulator of inflammation and leukocyte adhesion and synchronizes the expression of adhesion molecules, cytokines, and chemokines in endothelial cells. Importantly, NF-κB is controlled by redox regulation, making it a prime candidate to link chronic oxidative stress to activation of downstream inflammatory pathways in radiation injury. The study by Martin et al. (6) provides the first direct evidence that NF-κB is chronically up-regulated in human arteries after radiation exposure. The investigators examined paired arterial specimens from skin flaps of patients who had undergone irradiation therapy for head-and-neck cancer between 4 and 500 weeks previously. They directly compared irradiated versus nonirradiated arteries from the same patients, thereby avoiding confounding inter-patient variables. Differentially expressed genes in the irradiated arteries were detected with oligonucleotide microarrays, and the data were validated by real-time polymerase chain reaction. These data were used to detect clusters of altered gene expression, which demonstrated patterns consistent with up-regulated inflammation, coagulation, and angiogenesis. Importantly, the pattern of altered gene expression in the irradiated arteries suggested transcriptional regulation by NF-κB. Indeed, immunohistological studies demonstrated up-regulated NF-κB in vascular wall cells, with specific localization to macrophages. Interestingly, a group of putative NF-κB– dependent genes were found to be similarly dysregulated, regardless of the amount of time since irradiation (4 to 7 weeks vs. 20 to 500 weeks). A similar study with larger number of patients would be needed to separate early-stage from late-stage gene expression programs, which might be highly informative. The study by Martin et al. (6) provides the foundation for a molecular mechanism to explain the effects of radiation on vascular biology (Fig. 1). As with many studies that address a poorly understood phenomenon, the data generate more questions than answers. Are the results observed in the small conduit arteries in this study translatable to large arteries that produce most cardiovascular events? Does oxidative stress cause the persistent NF-κB up-regulation and reprogramming of gene expression in irradiated blood vessels? Lastly and most importantly, could modulation of NF-κB ameliorate the disease process? Several commonly used agents, such as aspirin, omega-3 fatty acids, and statins, directly or indirectly modulate NF-κB activity; whether these medications could ameliorate radiation-induced vascular disease remains to be determined. Also, inhibitors of NF-κB are being tested for a variety of inflammatory states and might eventually make their way into clinical medicine (13,14). Perhaps such therapy could be employed to treat radiation-induced vascular disease. Alternatively, the pathways responsible for up-regulated oxidative stress might be targeted. In this regard, activation of the angiotensin II-aldosterone system has been hypothesized to play a key role in propagating oxidative stress after radiation therapy (12,15). Thus, pharmacotherapy directed against this pathway could potentially be efficacious against radiation-induced vascular disease. Figure 1 Proposed Mechanism of Involvement of NF-κB in Radiation-Induced Vascular Disease In conclusion, Martin et al. (6) have made an important contribution to the field of radiation-induced vascular disease by demonstrating local and sustained up-regulation of NF-κB in irradiated human blood vessels. The expression profiles suggest that NF-κB contributes to the pathology by inducing pro-inflammatory genes. Further research is needed to determine the clinical significance of their findings and to investigate whether currently available and/or emerging therapies can modulate the disease process.