The adult myocardium responds to a variety of pathologic stimuli by hypertrophic growth that frequently progresses to heart failure. The calcium͞calmodulin-dependent protein phosphatase calcineurin is a potent transducer of hypertrophic stimuli. Calcineurin dephosphorylates members of the nuclear factor of activated T cell (NFAT) family of transcription factors, which results in their translocation to the nucleus and activation of calcium-dependent genes. Glycogen synthase kinase-3 (GSK-3) phosphorylates NFAT proteins and antagonizes the actions of calcineurin by stimulating NFAT nuclear export. To determine whether activated GSK-3 can act as an antagonist of hypertrophic signaling in the adult heart in vivo, we generated transgenic mice that express a constitutively active form of GSK-3␤ under control of a cardiac-specific promoter. These mice were physiologically normal under nonstressed conditions, but their ability to mount a hypertrophic response to calcineurin activation was severely impaired. Similarly, cardiac-specific expression of activated GSK-3␤ diminished hypertrophy in response to chronic ␤-adrenergic stimulation and pressure overload. These findings reveal a role for GSK-3␤ as an inhibitor of hypertrophic signaling in the intact myocardium and suggest that elevation of cardiac GSK-3␤ activity may provide clinical benefit in the treatment of pathologic hypertrophy and heart failure.

The best understood ''fight or flight'' mechanism for increasing heart rate (HR) involves activation of a cyclic nucleotide-gated ion channel (HCN4) by ␤-adrenergic receptor (␤AR) agonist stimulation. HCN4 conducts an inward ''pacemaker'' current (If) that increases the sinoatrial nodal (SAN) cell membrane diastolic depolarization rate (DDR), leading to faster SAN action potential generation. Surprisingly, HCN4 knockout mice were recently shown to retain physiological HR increases with isoproterenol (ISO), suggesting that other I f-independent pathways are critical to SAN fight or flight responses. Here, we show that CaMKII plays a previously unanticipated but decisive role to increase SAN rates during ␤AR stimulation. Studies in hearts from mice with SAN cell CaMKII inhibition suggest that CaMKII activity is required for chronotropic responses to ISO. CaMKII is selectively engaged in SAN cells during ␤AR stimulation and leads to coordinated enhancement of SR Ca 2ϩ filling, greater diastolic SR Ca 2ϩ release, and an increased diastolic depolarization rate (DDR) to increase HRs, independent of I f . In contrast, CaMKII inhibition does not slow HRs or SAN cell action potential (AP) frequency in the absence of ␤AR stimulation or when SR Ca 2ϩ release is disabled. These studies define a novel, CaMKII-dependent cellular mechanism for SAN fight or flight physiology.

Myocardial Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) inhibition improves cardiac function following myocardial infarction (MI), but the CaMKII-dependent pathways that participate in myocardial stress responses are incompletely understood. To address this issue, we sought to determine the transcriptional consequences of myocardial CaMKII inhibition after MI. We performed gene expression profiling in mouse hearts with cardiomyocyte-delimited transgenic expression of either a CaMKII inhibitory peptide (AC3-I) or a scrambled control peptide (AC3-C) following MI. Of the 8,600 mRNAs examined, 156 were substantially modulated by MI, and nearly half of these showed markedly altered responses to MI with CaMKII inhibition. CaMKII inhibition substantially reduced the MI-triggered upregulation of a constellation of proinflammatory genes. We studied 1 of these proinflammatory genes, complement factor B (Cfb), in detail, because complement proteins secreted by cells other than cardiomyocytes can induce sarcolemmal injury during MI. CFB protein expression in cardiomyocytes was triggered by CaMKII activation of the NF-κB pathway during both MI and exposure to bacterial endotoxin. CaMKII inhibition suppressed NF-κB activity in vitro and in vivo and reduced Cfb expression and sarcolemmal injury. The Cfb -/-mice were partially protected from the adverse consequences of MI. Our findings demonstrate what we believe is a novel target for CaMKII in myocardial injury and suggest that CaMKII is broadly important for the genetic effects of MI in cardiomyocytes.

Myocardial mitochondrial Ca 2+ entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca 2+ are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca 2+ uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca 2+ entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O 2 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 Ca 2+ were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca 2+ homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca 2+ ] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca 2+ homeostasis. Mitochondrial Ca 2+ 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 Ca 2+ homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.myocardium | mitochondrial calcium uniporter | ischemia-reperfusion injury E ntry of Ca 2+ into the mitochondrial matrix is a central event for Ca 2+ homeostasis in cardiomyocytes (1) as well as for coordinating fundamental and diverse responses to physiological (2) and pathological stress (3). The paradigm for Ca 2+ as a physiological second messenger that enhances oxidative phosphorylation to enable fight-or-flight responses but in excess contributes to disease and dysfunction is well established in myocardium (4). The molecular identity of the mitochondrial Ca 2+ uniporter (MCU) was recently discovered, enabling development of new genetic models to understand the role of MCU in vivo. MCU is an ion channel protein that acts as the primary pathway for Ca 2+ entry into the mitochondrial matrix (5, 6). Recent findings in global Mcu −/− mice (7) suggest that the MCU pathway is dispensable for regulating cellular energy production, except under extreme physiological stress, and for activation of pathways leading to cell death; however, the effect of selective myocardial MCU inhibition is unknown. We developed a new transgenic mouse model with myocardial delimited dominant negative (DN)-MCU protein overexpressio...

Rationale: Parasympathetic regulation of heart rate is mediated by acetylcholine binding to G protein-coupled muscarinic M2 receptors, which activate heterotrimeric G i/o proteins to promote G protein-coupled inwardly rectifying K ؉ (GIRK) channel activation. Regulator of G protein signaling (RGS) proteins, which function to inactivate G proteins, are indispensable for normal parasympathetic control of the heart. However, it is unclear which of the more than 20 known RGS proteins function to negatively regulate and thereby ensure normal parasympathetic control of the heart.Objective: To examine the specific contribution of RGS6 as an essential regulator of parasympathetic signaling in heart. Methods and Results: We developed RGS6 knockout mice to determine the functional impact of loss of RGS6 on parasympathetic regulation of cardiac automaticity. RGS6 exhibited a uniquely robust expression in the heart, particularly in sinoatrial and atrioventricular nodal regions. Loss of RGS6 provoked dramatically exaggerated bradycardia in response to carbachol in mice and isolated perfused hearts and significantly enhanced the effect of carbachol on inhibition of spontaneous action potential firing in sinoatrial node cells. Consistent with a role of RGS6 in G protein inactivation, RGS6-deficient atrial myocytes exhibited a significant reduction in the time course of acetylcholine-activated potassium current (I KACh ) activation and deactivation, as well as the extent of I KACh desensitization.Conclusions: RGS6 is a previously unrecognized, but essential, regulator of parasympathetic activation in heart, functioning to prevent parasympathetic override and severe bradycardia. These effects likely result from actions of RGS6 as a negative regulator of G protein activation of GIRK channels. (Circ Res. 2010;107:1345-1349.) Key Words: RGS6 Ⅲ SA node Ⅲ Heart rate Ⅲ K ϩ channel Ⅲ G proteins S ince the discovery that acetylcholine (ACh) release from the vagus produces bradycardia, key proteins and mechanisms underlying this action of ACh in heart have been identified. It is now known that ACh binds to muscarinic M2 receptors (M2Rs) that activate heterotrimeric G proteins (G i/o ) in key pacemaking regions of the heart. Activation of these G proteins causes release of G␤␥ subunits that bind to and activate G proteincoupled inwardly rectifying K ϩ (GIRK) channels, which results in a large K ϩ current (acetylcholine-activated potassium current [I KACh ]) and membrane hyperpolarization. 1 RGS proteins function as GTPase-activating proteins (GAPs) for G␣ subunits, accelerating their conversion to the inactive GDP-bound form. 2 This results in their reassembly with G␤␥ to form inactive G protein heterotrimers, thereby terminating signaling by both G␣ and G␤␥ proteins. Heterologous expression of various members of the RGS protein family with GIRK channels and M2Rs are required to reconstitute the normal activation and deactivation kinetics of native atrial GIRK channels. 3 In vivo evidence for this key role of RGS proteins in controlli...