Ischemic preconditioning (PC) occurs in two phases: an early phase, which lasts 2-3 h, and a late phase, which begins 12-24 h later and lasts 3-4 days. The mechanism for the late phase of PC has been the focus of intense investigation. We have recently proposed the "NO hypothesis of late PC", which postulates that NO plays a prominent role both in initiating and in mediating this cardioprotective response. The purpose of this essay is to review the evidence supporting the NO hypothesis of late PC and to discuss its implications. We propose that, on day 1, a brief ischemic stress causes increased production of NO (probably via eNOS) and .O2-, which then react to form ONOO-, ONOO-, in turn, activates the epsilon isoform of protein kinase C (PKC), either directly or via its reactive byproducts such as .OH. Both NO and secondary species derived from .O2- could also stimulate PKC epsilon independently. PKC epsilon activation triggers a complex signaling cascade that involves tyrosine kinases (among which Src and Lck appear to be involved) and probably other kinases, the transcription factor NF-kappa B, and most likely other as yet unknown components, resulting in increased transcription of the iNOS gene and increased iNOS activity on day 2, which is responsible for the protection during the second ischemic challenge. Tyrosine kinases also appear to be involved on day 2, possibly by modulating iNOS activity. According to this paradigm, NO plays two completely different roles in late PC: on day 1, it initiates the development of this response, whereas on day 2, it protects against myocardial ischemia. We propose that two different NOS isoforms are sequentially involved in late PC, with eNOS generating the NO that initiates the development of the PC response on day 1 and iNOS then generating the NO that protects against recurrent ischemia on day 2. The NO hypothesis of late PC puts forth a comprehensive paradigm that can explain both the initiation and the mediation of this complex phenomenon. Besides its pathophysiological implications, this hypothesis has potential clinical reverberations, since NO donors (i.e., nitrates) are widely used clinically and could be used to protect the ischemic myocardium in patients.

A. Targeted disruption of the voltage-dependent calcium channel ␣2/␦-1-subunit. Cardiac L-type voltage-dependent Ca 2ϩ channels are heteromultimeric polypeptide complexes of ␣1-, ␣2/␦-, and ␤-subunits. The ␣2/␦-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 ␣2/␦-subunits of voltage-dependent Ca 2ϩ 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 ␣2/␦-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 ␣2/␦-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 2ϩ current peak current amplitude. This is a novel model for studying the function of the ␣2/␦-1-subunit and will be of importance in the development of new pharmacological therapies. cardiac calcium channel; murine knockout model; gabapentin binding; myocardial contractility CARDIAC L-type voltage-dependent Ca 2ϩ channels (L-VDCCs) are heteromultimeric polypeptide complexes of ␣ 1 -, ␣ 2 /␦-, and ␤-subunits. The ␣ 1 -subunit is autoregulatory and harbors the channel pore, gating machinery, and modulatory drug binding sites (30). The accessory subunits (␣ 2 /␦ and ␤) affect channel kinetics and are involved in the trafficking and insertion of the ␣ 1 -subunit into the membrane. The ␣ 2 -subunit is closely associated with an extracellular loop of the ␣ 1 -subunit (15) and linked to a small protein called ␦ (2, 9). Both the ␣ 2 and ␦ are encoded by the same gene, separated by proteolytic cleavage, and extracellularly linked through a disulfide bridge (9). Currently, four ␣ 2 /␦-subunits, each encoded by separate genes, have been identified (4). The ␣ 2 /␦-1, originally cloned from skeletal muscle (10), is ubiquitously distributed (18), with high levels of protein expression in brain, heart, skeletal, and

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. (Circulation. 2007;115:300-309.)

NF-kappaB 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-kappaB 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-kappaB is complicated; in addition to being activated by canonical cytokine-mediated pathways, NF-kappaB 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-kappaB by activating the IkappaB 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 signalregulated kinase 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-kappaB via IkappaB or by direct phosphorylation of NF-kappaB subunits. Finally, there are combinatorial interactions at the level of the promoter between NF-kappaB, its coactivators, 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-kappaB is positioned as a signaling integrator. As such, NF-kappaB 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-kappaB reduces infarct size in the murine heart after ischemia/reperfusion (I/R), implicating NF-kappaB as a major determinant of cell death after I/R. These results support the concept that NF-kappaB may be an important therapeutic target for specific cardiovascular diseases.