Haiyun Ling

Requirement for Ca2+/calmodulin–dependent kinase II in the transition from pressure overload–induced cardiac hypertrophy to heart failure in mice

Ca2+/calmodulin-dependent kinase II (CaMKII) has been implicated in cardiac hypertrophy and heart failure. We generated mice in which the predominant cardiac isoform, CaMKIIdelta, was genetically deleted (KO mice), and found that these mice showed no gross baseline changes in ventricular structure or function. In WT and KO mice, transverse aortic constriction (TAC) induced comparable increases in relative heart weight, cell size, HDAC5 phosphorylation, and hypertrophic gene expression. Strikingly, while KO mice showed preserved hypertrophy after 6-week TAC, CaMKIIdelta deficiency significantly ameliorated phenotypic changes associated with the transition to heart failure, such as chamber dilation, ventricular dysfunction, lung edema, cardiac fibrosis, and apoptosis. The ratio of IP3R2 to ryanodine receptor 2 (RyR2) and the fraction of RyR2 phosphorylated at the CaMKII site increased significantly during development of heart failure in WT mice, but not KO mice, and this was associated with enhanced Ca2+ spark frequency only in WT mice. We suggest that CaMKIIdelta contributes to cardiac decompensation by enhancing RyR2-mediated sarcoplasmic reticulum Ca2+ leak and that attenuating CaMKIIdelta activation can limit the progression to heart failure.

CaMKIIδ Isoforms Differentially Affect Calcium Handling but Similarly Regulate HDAC/MEF2 Transcriptional Responses

The δB and δC splice variants of Ca2+/calmodulin-dependent protein kinase II (CaMKII), which differ by the presence of a nuclear localization sequence, are both expressed in cardiomyocytes. We used transgenic (TG) mice and CaMKII expression in cardiomyocytes to test the hypothesis that the CaMKIIδC isoform regulates cytosolic Ca2+ handling and the δB isoform, which localizes to the nucleus, regulates gene transcription. Phosphorylation of CaMKII sites on the ryanodine receptor (RyR) and on phospholamban (PLB) were increased in CaMKIIδC TG. This was associated with markedly enhanced sarcoplasmic reticulum (SR) Ca2+ spark frequency and decreased SR Ca2+ content in cardiomyocytes. None of these parameters were altered in TG mice expressing the nuclear-targeted CaMKIIδB. In contrast, cardiac expression of either CaMKIIδB or δC induced transactivation of myocyte enhancer factor 2 (MEF2) gene expression and up-regulated hypertrophic marker genes. Studies using rat ventricular cardiomyocytes confirmed that CaMKIIδB and δC both regulate MEF2-luciferase gene expression, increase histone deacetylase 4 (HDAC4) association with 14-3-3, and induce HDAC4 translocation from nucleus to cytoplasm, indicating that either isoform can stimulate HDAC4 phosphorylation. Finally, HDAC4 kinase activity was shown to be increased in cardiac homogenates from either CaMKIIδB or δC TG mice. Thus CaMKIIδ isoforms have similar effects on hypertrophic gene expression but disparate effects on Ca2+ handling, suggesting distinct roles for CaMKIIδ isoform activation in the pathogenesis of cardiac hypertrophy versus heart failure.

RhoA protects the mouse heart against ischemia/reperfusion injury.

The small GTPase RhoA serves as a nodal point for signaling through hormones and mechanical stretch. However, the role of RhoA signaling in cardiac pathophysiology is poorly understood. To address this issue, we generated mice with cardiomyocyte-specific conditional expression of low levels of activated RhoA (CA-RhoA mice) and demonstrated that they exhibited no overt cardiomyopathy. When challenged by in vivo or ex vivo ischemia/reperfusion (I/R), however, the CA-RhoA mice exhibited strikingly increased tolerance to injury, which was manifest as reduced myocardial lactate dehydrogenase (LDH) release and infarct size and improved contractile function. PKD was robustly activated in CA-RhoA hearts. The cardioprotection afforded by RhoA was reversed by PKD inhibition. The hypothesis that activated RhoA and PKD serve protective physiological functions during I/R was supported by several lines of evidence. In WT mice, both RhoA and PKD were rapidly activated during I/R, and blocking PKD augmented I/R injury. In addition, cardiac-specific RhoA-knockout mice showed reduced PKD activation after I/R and strikingly decreased tolerance to I/R injury, as shown by increased infarct size and LDH release. Collectively, our findings provide strong support for the concept that RhoA signaling in adult cardiomyocytes promotes survival. They also reveal unexpected roles for PKD as a downstream mediator of RhoA and in cardioprotection against I/R.

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.

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.

CaMKII-dependent phosphorylation of cardiac ryanodine receptors regulates cell death in cardiac ischemia/reperfusion injury

Ca(2+)-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24h). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A) significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knock-in mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca(2+) current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage.

Mitochondrial Reprogramming Induced by CaMKIIδ Mediates Hypertrophy Decompensation

RATIONALE Sustained activation of Gαq transgenic (Gq) signaling during pressure overload causes cardiac hypertrophy that ultimately progresses to dilated cardiomyopathy. The molecular events that drive hypertrophy decompensation are incompletely understood. Ca(2+)/calmodulin-dependent protein kinase II δ (CaMKIIδ) is activated downstream of Gq, and overexpression of Gq and CaMKIIδ recapitulates hypertrophy decompensation. OBJECTIVE To determine whether CaMKIIδ contributes to hypertrophy decompensation provoked by Gq. METHODS AND RESULTS Compared with Gq mice, compound Gq/CaMKIIδ knockout mice developed a similar degree of cardiac hypertrophy but exhibited significantly improved left ventricular function, less cardiac fibrosis and cardiomyocyte apoptosis, and fewer ventricular arrhythmias. Markers of oxidative stress were elevated in mitochondria from Gq versus wild-type mice and respiratory rates were lower; these changes in mitochondrial function were restored by CaMKIIδ deletion. Gq-mediated increases in mitochondrial oxidative stress, compromised membrane potential, and cell death were recapitulated in neonatal rat ventricular myocytes infected with constitutively active Gq and attenuated by CaMKII inhibition. Deep RNA sequencing revealed altered expression of 41 mitochondrial genes in Gq hearts, with normalization of ≈40% of these genes by CaMKIIδ deletion. Uncoupling protein 3 was markedly downregulated in Gq or by Gq expression in neonatal rat ventricular myocytes and reversed by CaMKIIδ deletion or inhibition, as was peroxisome proliferator-activated receptor α. The protective effects of CaMKIIδ inhibition on reactive oxygen species generation and cell death were abrogated by knock down of uncoupling protein 3. Conversely, restoration of uncoupling protein 3 expression attenuated reactive oxygen species generation and cell death induced by CaMKIIδ. Our in vivo studies further demonstrated that pressure overload induced decreases in peroxisome proliferator-activated receptor α and uncoupling protein 3, increases in mitochondrial protein oxidation, and hypertrophy decompensation, which were attenuated by CaMKIIδ deletion. CONCLUSIONS Mitochondrial gene reprogramming induced by CaMKIIδ emerges as an important mechanism contributing to mitotoxicity in decompensating hypertrophy.

In vivo selective expression of thyroid hormone receptor α1 in endothelial cells attenuates myocardial injury in experimental myocardial infarction in mice.

Ischemic heart disease (IHD) is the single most common cause of death. New approaches to enhance myocardial perfusion are needed to improve outcomes for patients with IHD. Thyroid hormones (TH) are known to increase blood flow; however, their usefulness for increasing perfusion in IHD is limited because TH accelerates heart rate, which can be detrimental. Therefore, selective activation of TH effects is desirable. We hypothesized that cell-type-specific TH receptor (TR) expression can increase TH action in the heart, while avoiding the negative consequences of TH treatment. We generated a binary transgenic (BTG) mouse that selectively expresses TRα1 in endothelial cells in a tetracycline-inducible fashion. In BTG mice, endothelial TRα1 protein expression was increased by twofold, which, in turn, increased coronary blood flow by 77%, coronary conductance by 60%, and coronary reserve by 47% compared with wild-type mice. Systemic blood pressure was decreased by 20% in BTG mice after TRα1 expression. No effects on heart rate were observed. Endothelial TRα1 expression activated AKT/endothelial nitric oxide synthase pathway and increased A2AR adenosine receptor. Furthermore, hearts from BTG mice overexpressing TRα1 that were submitted to 20 min ischemia and 20 min reperfusion showed a 20% decline in left ventricular pressure (LVP) compared with control mice where LVP was decreased by 42%. Studies using an infarction mouse model demonstrated that endothelial overexpression of TRα1 decreased infarct size by 45%. In conclusion, selective expression of TRα1 in endothelial cells protects the heart against injury after an ischemic insult and does not result in adverse cardiac or systemic effects.