Michael Grimm

β-Adrenergic receptor signaling in the heart: Role of CaMKII

The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) targets a number of Ca(2+) homeostatic proteins and regulates gene transcription. Many of the substrates phosphorylated by CaMKII are also substrates for protein kinase A (PKA), the best known downstream effector of beta-adrenergic receptor (beta-AR) signaling. While PKA and CaMKII are conventionally considered to transduce signals through separate pathways, there is a body of evidence suggesting that CaMKII is activated in response to beta-AR stimulation and that some of the downstream effects of beta-AR stimulation are actually mediated by CaMKII. The signaling pathway through which beta-AR stimulation activates CaMKII, in parallel with or downstream of PKA, is not well-defined. This review considers the evidence for and mechanisms by which CaMKII is activated in response to beta-AR stimulation. In addition the potential role of CaMKII in beta-AR regulation of cardiac function is considered. Notably, although many CaMKII targets (e.g., phospholamban or the ryanodine receptor) are central to the regulation of Ca(2+) handling, and effects of CaMKII on Ca(2+) handling are detectable, inhibition or gene deletion of CaMKII has relatively little effect on the acute physiological contractile response to beta-AR. On the other hand CaMKII expression and activity are increased in heart failure, a pathophysiological condition characterized by chronic stimulation of cardiac beta-ARs. Blockade of beta-ARs is an accepted therapy for treatment of chronic heart failure although the rationale for its beneficial effects in cardiomyocytes is uncertain. There is growing evidence that inhibition or gene deletion of CaMKII also has a significant beneficial impact on the development of heart failure. The possibility that excessive beta-AR stimulation is detrimental because of its effects on CaMKII mediated Ca(2+) handling disturbances (e.g., ryanodine receptor phosphorylation and diastolic SR Ca(2+) leak) is an intriguing hypothesis that merits future consideration.

Ca2+/Calmodulin-dependent protein kinase II δ mediates myocardial ischemia/reperfusion injury through nuclear factor-κB.

Rationale: Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated as a maladaptive mediator of cardiac ischemic injury. We hypothesized that the inflammatory response associated with in vivo ischemia/reperfusion (I/R) is initiated through CaMKII signaling. Objective: To assess the contribution of CaMKII&dgr; to the development of inflammation, infarct, and ventricular dysfunction after in vivo I/R and define early cardiomyocyte–autonomous events regulated by CaMKII&dgr; using cardiac-specific knockout mice. Methods and Results: Wild-type and CaMKII&dgr; knockout mice were subjected to in vivo I/R by occlusion of the left anterior descending artery for 1 hour followed by reperfusion for various times. CaMKII&dgr; deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis, and improved functional recovery. CaMKII&dgr; deletion also attenuated I/R-induced inflammation and upregulation of nuclear factor-&kgr;B (NF-&kgr;B) target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-&kgr;B activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKII&dgr; knockout mice indicate that NF-&kgr;B activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes leads to I&kgr;B kinase phosphorylation and concomitant increases in nuclear p65. Experiments using an I&kgr;B kinase inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-&kgr;B activation. Conclusions: This is the first study demonstrating that CaMKII&dgr; mediates NF-&kgr;B activation in cardiomyocytes after in vivo I/R and suggests that CaMKII&dgr; serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.Rationale: Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) has been implicated as a maladaptive mediator of cardiac ischemic injury. We hypothesized that the inflammatory response associated with in vivo ischemia/reperfusion (I/R) is initiated through CaMKII signaling. Objective: To assess the contribution of CaMKIIδ to the development of inflammation, infarct and ventricular dysfunction following in vivo I/R and define early cardiomyocyte-autonomous events regulated by CaMKIIδ using cardiac-specific knockout (KO) mice. Methods and Results: Wild-type (WT) and CaMKIIδ KO mice were subjected to in vivo I/R by occlusion of the left anterior descending (LAD) artery for 1-hr followed by reperfusion for various times. CaMKIIδ deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis and improved functional recovery. CaMKIIδ deletion also attenuated I/R induced inflammation and upregulation of NF-κB target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-κB activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKIIδ knockout mice indicate that NF-κB activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes lead to I kappa B kinase (IKK) phosphorylation and concomitant increases in nuclear p65. Experiments using an IKK inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-κB activation. Conclusions: This is the first study demonstrating that CaMKIIδ mediates NF-κB activation in cardiomyocytes following in vivo I/R and suggests that CaMKIIδ serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.

Ca2+/Calmodulin-Dependent Protein Kinase II δ Mediates Myocardial Ischemia/Reperfusion Injury Through Nuclear Factor-κBNovelty and Significance

Rationale: Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated as a maladaptive mediator of cardiac ischemic injury. We hypothesized that the inflammatory response associated with in vivo ischemia/reperfusion (I/R) is initiated through CaMKII signaling. Objective: To assess the contribution of CaMKII&dgr; to the development of inflammation, infarct, and ventricular dysfunction after in vivo I/R and define early cardiomyocyte–autonomous events regulated by CaMKII&dgr; using cardiac-specific knockout mice. Methods and Results: Wild-type and CaMKII&dgr; knockout mice were subjected to in vivo I/R by occlusion of the left anterior descending artery for 1 hour followed by reperfusion for various times. CaMKII&dgr; deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis, and improved functional recovery. CaMKII&dgr; deletion also attenuated I/R-induced inflammation and upregulation of nuclear factor-&kgr;B (NF-&kgr;B) target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-&kgr;B activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKII&dgr; knockout mice indicate that NF-&kgr;B activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes leads to I&kgr;B kinase phosphorylation and concomitant increases in nuclear p65. Experiments using an I&kgr;B kinase inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-&kgr;B activation. Conclusions: This is the first study demonstrating that CaMKII&dgr; mediates NF-&kgr;B activation in cardiomyocytes after in vivo I/R and suggests that CaMKII&dgr; serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.Rationale: Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) has been implicated as a maladaptive mediator of cardiac ischemic injury. We hypothesized that the inflammatory response associated with in vivo ischemia/reperfusion (I/R) is initiated through CaMKII signaling. Objective: To assess the contribution of CaMKIIδ to the development of inflammation, infarct and ventricular dysfunction following in vivo I/R and define early cardiomyocyte-autonomous events regulated by CaMKIIδ using cardiac-specific knockout (KO) mice. Methods and Results: Wild-type (WT) and CaMKIIδ KO mice were subjected to in vivo I/R by occlusion of the left anterior descending (LAD) artery for 1-hr followed by reperfusion for various times. CaMKIIδ deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis and improved functional recovery. CaMKIIδ deletion also attenuated I/R induced inflammation and upregulation of NF-κB target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-κB activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKIIδ knockout mice indicate that NF-κB activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes lead to I kappa B kinase (IKK) phosphorylation and concomitant increases in nuclear p65. Experiments using an IKK inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-κB activation. Conclusions: This is the first study demonstrating that CaMKIIδ mediates NF-κB activation in cardiomyocytes following in vivo I/R and suggests that CaMKIIδ serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.

Cardiac Hypertrophy and Heart Failure Development Through Gq and Cam Kinase Ii Signaling

The molecular events associated with the development of pathological hypertrophy have been shown to be stimulated through G-protein–coupled receptors that activate Gq signaling pathways in neonatal cardiomyocytes and in transgenic (TG) and knockout mice. We demonstrated that CaMKII, a multifunctional Ca(2+)-regulated protein kinase, was activated through G-protein–coupled receptor and inositol trisphosphate–mediated Ca(2+) release and suggested that CaMKII was a downstream mediator of Gq-coupled hypertrophic signaling. This was supported by the demonstration of CaMKII activation by pressure overload [(transverse aortic constriction (TAC)] and induction of hypertrophy by TG CaMKII expression. CaMKII also phosphorylates Ca(2+) handling proteins including the ryanodine receptor (RyR2), phosphorylation of which markedly increases sarcoplasmic reticulum Ca(2+) leak. Increased RyR2 phosphorylation is associated with heart failure development in CaMKII TG mice, and mice genetically deleted for CaMKII (KO) have attenuated RyR2 phosphorylation, sarcoplasmic reticulum Ca(2+) leak, and heart failure development after long-term TAC. Genetic ablation of CaMKII also decreases development of heart failure in Gq TG mice and decreases infarct size, while improving functional recovery in mice subject to ischemia/reperfusion and preventing adverse remodeling after coronary artery occlusion. The underlying mechanisms are currently under study.

β-Adrenergic Receptor Stimulated Ncx1 Upregulation is Mediated via a CaMKII/AP-1 Signaling Pathway in Adult Cardiomyocytes

The Na(+)-Ca(2+) exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. beta-Adrenergic receptor (beta-AR) signaling plays an important role in the regulation of calcium homeostasis in the cardiomyocyte, but chronic activation in periods of cardiac stress contributes to heart failure by mechanisms which include Ncx1 upregulation. Here, using a Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIdelta(c)) null mouse, we demonstrate that beta-AR-stimulated Ncx1 upregulation is dependent on CaMKII. beta-AR-stimulated Ncx1 expression is mediated by activator protein 1 (AP-1) factors and is independent of cAMP-response element-binding protein (CREB) activation. The MAP kinases (ERK1/2, JNK and p38) are not required for AP-1 factor activation. Chromatin immunoprecipitation demonstrates that beta-AR stimulation activates the ordered recruitment of JunB homodimers, which then are replaced by c-Jun homodimers binding to the proximal AP-1 elements of the endogenous Ncx1 promoter. In conclusion, this work has provided insight into the intracellular signaling pathways and transcription factors regulating Ncx1 gene expression in a chronically beta-AR-stimulated heart.

CaMKIIδ mediates β-adrenergic effects on RyR2 phosphorylation and SR Ca2 + leak and the pathophysiological response to chronic β-adrenergic stimulation

Chronic activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the deleterious effects of β-adrenergic receptor (β-AR) signaling on the heart, in part, by enhancing RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) leak. We used CaMKIIδ knockout (CaMKIIδ-KO) mice and knock-in mice with an inactivated CaMKII site S2814 on the ryanodine receptor type 2 (S2814A) to investigate the involvement of these processes in β-AR signaling and cardiac remodeling. Langendorff-perfused hearts from CaMKIIδ-KO mice showed inotropic and chronotropic responses to isoproterenol (ISO) that were similar to those of wild type (WT) mice; however, in CaMKIIδ-KO mice, CaMKII phosphorylation of phospholamban and RyR2 was decreased and isolated myocytes from CaMKIIδ-KO mice had reduced SR Ca(2+) leak in response to isoproterenol (ISO). Chronic catecholamine stress with ISO induced comparable increases in relative heart weight and other measures of hypertrophy from day 9 through week 4 in WT and CaMKIIδ-KO mice, but the development of cardiac fibrosis was prevented in CaMKIIδ-KO animals. A 4-week challenge with ISO resulted in reduced cardiac function and pulmonary congestion in WT, but not in CaMKIIδ-KO or S2814A mice, implicating CaMKIIδ-dependent phosphorylation of RyR2-S2814 in the cardiomyopathy, independent of hypertrophy, induced by prolonged β-AR stimulation.

Crossing signals: relationships between β-adrenergic stimulation and CaMKII activation

CaMKII is a ubiquitous and pleiotropic serine threonine kinase. The predominant isoform of CaMKII in the heart is CaMKII delta (CaMKIIδ). CaMKII phosphorylates and regulates a number of cardiac proteins involved in excitation-contraction coupling and Ca++ handling including the cardiac ryanodine receptor (RyR2), phospholamban (PLN), the L-type Ca++ channel α1C- and β2a-subunits, and the Na channel (1). Studies using animal models have increasingly linked CaMKII activation and its effects on these targets to the development of heart failure and arrhythmogenesis. There is also considerable evidence that CaMKII expression and activity are increased in human heart failure (1).

Impact of AT2 receptor deficiency on postnatal cardiovascular development.

Background The angiotensin II receptor subtype 2 (AT2 receptor) is ubiquitously and highly expressed in early postnatal life. However, its role in postnatal cardiac development remained unclear. Methodology/Principal Findings Hearts from 1, 7, 14 and 56 days old wild-type (WT) and AT2 receptor-deficient (KO) mice were extracted for histomorphometrical analysis as well as analysis of cardiac signaling and gene expression. Furthermore, heart and body weights of examined animals were recorded and echocardiographic analysis of cardiac function as well as telemetric blood pressure measurements were performed. Moreover, gene expression, sarcomere shortening and calcium transients were examined in ventricular cardiomyocytes isolated from both genotypes. KO mice exhibited an accelerated body weight gain and a reduced heart to body weight ratio as compared to WT mice in the postnatal period. However, in adult KO mice the heart to body weight ratio was significantly increased most likely due to elevated systemic blood pressure. At postnatal day 7 ventricular capillarization index and the density of α-smooth muscle cell actin-positive blood vessels were higher in KO mice as compared to WT mice but normalized during adolescence. Echocardiographic assessment of cardiac systolic function at postnatal day 7 revealed decreased contractility of KO hearts in response to beta-adrenergic stimulation. Moreover, cardiomyocytes from KO mice showed a decreased sarcomere shortening and an increased peak Ca2+ transient in response to isoprenaline when stimulated concomitantly with angiotensin II. Conclusion The AT2 receptor affects postnatal cardiac growth possibly via reducing body weight gain and systemic blood pressure. Moreover, it moderately attenuates postnatal vascularization of the heart and modulates the beta adrenergic response of the neonatal heart. These AT2 receptor-mediated effects may be implicated in the physiological maturation process of the heart.

CaMKIIδ Mediates Myocardial Ischemia/Reperfusion Injury Through NF-κB

Rationale: Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated as a maladaptive mediator of cardiac ischemic injury. We hypothesized that the inflammatory response associated with in vivo ischemia/reperfusion (I/R) is initiated through CaMKII signaling. Objective: To assess the contribution of CaMKII&dgr; to the development of inflammation, infarct, and ventricular dysfunction after in vivo I/R and define early cardiomyocyte–autonomous events regulated by CaMKII&dgr; using cardiac-specific knockout mice. Methods and Results: Wild-type and CaMKII&dgr; knockout mice were subjected to in vivo I/R by occlusion of the left anterior descending artery for 1 hour followed by reperfusion for various times. CaMKII&dgr; deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis, and improved functional recovery. CaMKII&dgr; deletion also attenuated I/R-induced inflammation and upregulation of nuclear factor-&kgr;B (NF-&kgr;B) target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-&kgr;B activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKII&dgr; knockout mice indicate that NF-&kgr;B activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes leads to I&kgr;B kinase phosphorylation and concomitant increases in nuclear p65. Experiments using an I&kgr;B kinase inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-&kgr;B activation. Conclusions: This is the first study demonstrating that CaMKII&dgr; mediates NF-&kgr;B activation in cardiomyocytes after in vivo I/R and suggests that CaMKII&dgr; serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.Rationale: Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) has been implicated as a maladaptive mediator of cardiac ischemic injury. We hypothesized that the inflammatory response associated with in vivo ischemia/reperfusion (I/R) is initiated through CaMKII signaling. Objective: To assess the contribution of CaMKIIδ to the development of inflammation, infarct and ventricular dysfunction following in vivo I/R and define early cardiomyocyte-autonomous events regulated by CaMKIIδ using cardiac-specific knockout (KO) mice. Methods and Results: Wild-type (WT) and CaMKIIδ KO mice were subjected to in vivo I/R by occlusion of the left anterior descending (LAD) artery for 1-hr followed by reperfusion for various times. CaMKIIδ deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis and improved functional recovery. CaMKIIδ deletion also attenuated I/R induced inflammation and upregulation of NF-κB target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-κB activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKIIδ knockout mice indicate that NF-κB activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes lead to I kappa B kinase (IKK) phosphorylation and concomitant increases in nuclear p65. Experiments using an IKK inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-κB activation. Conclusions: This is the first study demonstrating that CaMKIIδ mediates NF-κB activation in cardiomyocytes following in vivo I/R and suggests that CaMKIIδ serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.