p85α deficiency alleviates ischemia-reperfusion injury by promoting cardiomyocyte survival.

Myocardial ischemia-reperfusion (I/R) injury is a prevalent cause of myocardial injury, involving a series of interconnected pathophysiological processes. However, there is currently no clinical therapy for effectively mitigating myocardial I/R injury. Here, we show that p85α protein levels increase in response to I/R injury through a comprehensive analysis of cardiac proteomics, and confirm this in the I/R-injured murine heart and failing human myocardium. Genetic inhibition of p85α in mice activates the Akt-GSK3ß/Bcl-x(L) signaling pathway and ameliorates I/R-induced cardiac dysfunction, apoptosis, inflammation, and mitochondrial dysfunction. p85α silencing in cardiomyocytes alleviates hypoxia-reoxygenation (H/R) injury through activating the Akt-GSK3ß/Bcl-x(L) signaling pathway, while its overexpression exacerbates the damage. Mechanistically, the interaction between MG53 and p85α triggers the ubiquitination and degradation of p85α, consequently enhancing Akt phosphorylation and ultimately having cardioprotective effects. Collectively, our findings reveal that substantial reduction of p85α and subsequently activated Akt signaling have a protective effect against cardiac I/R injury, representing an important therapeutic strategy for mitigating myocardial damage.

p55γ degrades RIP3 via MG53 to suppress ischaemia-induced myocardial necroptosis and mediates cardioprotection of preconditioning.

AIMS: Regulated necrosis (necroptosis) and apoptosis are important biological features of myocardial infarction, ischaemia-reperfusion (I/R) injury, and heart failure. However, the molecular mechanisms underlying myocardial necroptosis remain elusive. Ischaemic preconditioning (IPC) is the most powerful intrinsic cardioprotection against myocardial I/R injury. In this study, we aimed to determine whether IPC suppresses I/R-induced necroptosis and the underlying molecular mechanisms. METHODS AND RESULTS: We generated p55γ transgenic and knockout mice and used ligation of left anterior descending coronary artery to produce an in vivo I/R model. The effects of p55γ and its downstream molecules were subsequently identified using mass spectroscopy and co-immunoprecipitation and pulldown assays. We found that p55γ expression was down-regulated in failing human myocardium caused by coronary heart disease as well as in I/R mouse hearts. Cardiac-specific p55γ overexpression ameliorated the I/R-induced necroptosis. In striking contrast, p55γ deficiency (p55γ-/-) and cardiac-specific deletion of p55γ (p55γc-KO) worsened I/R-induced injury. IPC up-regulated p55γ expression in vitro and in vivo. Using reporter and chromatin immunoprecipitation assays, we found that Hif1α transcriptionally regulated p55γ expression and mediated the cardioprotection of IPC. IPC-mediated suppression of necroptosis was attenuated in p55γ-/- and p55γc-KO hearts. Mechanistically, p55γ overexpression decreased the protein levels of RIP3 rather than the mRNA levels, while p55γ deficiency increased the protein abundance of RIP3. IPC attenuated the I/R-induced up-regulation of RIP3, which was abolished in p55γ-deficient mice. Up-regulation of RIP3 attenuated the p55γ- or IPC-induced inhibition of necroptosis in vivo. Importantly, p55γ directly bound and degraded RIP3 in a ubiquitin-dependent manner. We identified MG53 as the E3 ligase that mediated the p55γ-induced degradation of RIP3. In addition, we also found that p55γ activated the RISK pathway during IPC. CONCLUSIONS: Our findings reveal that activation of the MG53-RIP3 signal pathway by p55γ protects the heart against I/R-induced necroptosis and underlies IPC-induced cardioprotection.

Cardiac Ischemic Preconditioning Promotes MG53 Secretion Through H2O2-Activated Protein Kinase C-δ Signaling.

BACKGROUND: Ischemic heart disease is the leading cause of morbidity and mortality worldwide. Ischemic preconditioning (IPC) is the most powerful intrinsic protection against cardiac ischemia/reperfusion injury. Previous studies have shown that a multifunctional TRIM family protein, MG53 (mitsugumin 53; also called TRIM72), not only plays an essential role in IPC-mediated cardioprotection against ischemia/reperfusion injury but also ameliorates mechanical damage. In addition to its intracellular actions, as a myokine/cardiokine, MG53 can be secreted from the heart and skeletal muscle in response to metabolic stress. However, it is unknown whether IPC-mediated cardioprotection is causally related to MG53 secretion and, if so, what the underlying mechanism is. METHODS: Using proteomic analysis in conjunction with genetic and pharmacological approaches, we examined MG53 secretion in response to IPC and explored the underlying mechanism using rodents in in vivo, isolated perfused hearts, and cultured neonatal rat ventricular cardiomyocytes. Moreover, using recombinant MG53 proteins, we investigated the potential biological function of secreted MG53 in the context of IPC and ischemia/reperfusion injury. RESULTS: We found that IPC triggered robust MG53 secretion in rodents in vivo, perfused hearts, and cultured cardiac myocytes without causing cell membrane leakage. Mechanistically, IPC promoted MG53 secretion through H2O2-evoked activation of protein kinase-C-δ. Specifically, IPC-induced myocardial MG53 secretion was mediated by H2O2-triggered phosphorylation of protein kinase-C-δ at Y311, which is necessary and sufficient to facilitate MG53 secretion. Functionally, systemic delivery of recombinant MG53 proteins to mimic elevated circulating MG53 not only restored IPC function in MG53-deficient mice but also protected rodent hearts from ischemia/reperfusion injury even in the absence of IPC. Moreover, oxidative stress by H2O2 augmented MG53 secretion, and MG53 knockdown exacerbated H2O2-induced cell injury in human embryonic stem cell-derived cardiomyocytes, despite relatively low basal expression of MG53 in human heart. CONCLUSIONS: We conclude that IPC and oxidative stress can trigger MG53 secretion from the heart via an H2O2-protein kinase-C-δ-dependent mechanism and that extracellular MG53 can participate in IPC protection against cardiac ischemia/reperfusion injury.

Glucose-Sensitive Myokine/Cardiokine MG53 Regulates Systemic Insulin Response and Metabolic Homeostasis.

BACKGROUND: Mitsugumin 53 (MG53 or TRIM72), a striated muscle-specific E3 ligase, promotes ubiquitin-dependent degradation of the insulin receptor and insulin receptor substrate-1 and subsequently induces insulin resistance, resulting in metabolic syndrome and type 2 diabetes mellitus (T2DM). However, it is unknown how MG53 from muscle regulates systemic insulin response and energy metabolism. Increasing evidence demonstrates that muscle secretes proteins as myokines or cardiokines that regulate systemic metabolic processes. We hypothesize that MG53 may act as a myokine/cardiokine, contributing to interorgan regulation of insulin sensitivity and metabolic homeostasis. METHODS: Using perfused rodent hearts or skeletal muscle, we investigated whether high glucose, high insulin, or their combination (conditions mimicking metabolic syndrome or T2DM) alters MG53 protein concentration in the perfusate. We also measured serum MG53 levels in rodents and humans in the presence or absence of metabolic diseases, particularly T2DM. The effects of circulating MG53 on multiorgan insulin response were evaluated by systemic delivery of recombinant MG53 protein to mice. Furthermore, the potential involvement of circulating MG53 in the pathogenesis of T2DM was assessed by neutralizing blood MG53 with monoclonal antibodies in diabetic db/db mice. Finally, to delineate the mechanism underlying the action of extracellular MG53 on insulin signaling, we analyzed the potential interaction of MG53 with extracellular domain of insulin receptor using coimmunoprecipitation and surface plasmon resonance assays. RESULTS: Here, we demonstrate that MG53 is a glucose-sensitive myokine/cardiokine that governs the interorgan regulation of insulin sensitivity. First, high glucose or high insulin induces MG53 secretion from isolated rodent hearts and skeletal muscle. Second, hyperglycemia is accompanied by increased circulating MG53 in humans and rodents with diabetes mellitus. Third, systemic delivery of recombinant MG53 or cardiac-specific overexpression of MG53 causes systemic insulin resistance and metabolic syndrome in mice, whereas neutralizing circulating MG53 with monoclonal antibodies has therapeutic effects in T2DM db/db mice. Mechanistically, MG53 binds to the extracellular domain of the insulin receptor and acts as an allosteric blocker. CONCLUSIONS: Thus, MG53 has dual actions as a myokine/cardiokine and an E3 ligase, synergistically inhibiting the insulin signaling pathway. Targeting circulating MG53 opens a new therapeutic avenue for T2DM and its complications.

Exploring lipid markers of the quality of coix seeds with different geographical origins using supercritical fluid chromatography mass spectrometry and chemometrics.

BACKGROUND: Lipids, a group of primary metabolites, could be used as quality markers of Traditional Chinese medicine. PURPOSE: The present study was designed to develop a research method to explore lipid markers of the quality of coix seeds with different geographical origins. STUDY DESIGN: The geographical origins of coix seeds were divided into three regions based on the latitude. A central composite design (CCD test) was used to optimize the chromatographic parameters of supercritical fluid chromatography to obtain optimal lipid profile of coix seed. METHODS: An untargeted method based on ultra-performance convergence chromatography - quadrupole/time-of-flight hybrid mass spectrometry (UPC2-QTOF) was developed. Four chromatographic parameters were optimized using CCD test, and a fusion index established by Derringer function was used to evaluate. The lipid profile of 27 batches of coix seeds were acquired and processed by Progenesis QI software, and the MS/MS spectrums were obtained to identify, simultaneously. The difference lipids were explored by orthogonal partial least squares discriminant analysis (OPLS-DA). The lipids that showed differences depending on their seeds' geographical origin were selected as markers of the quality of coix seeds from the three regions. RESULTS: A Torus 2-PIC (1.7 µm, 100 mm × 3.0 mm) was selected as the optimal column of the untargeted method which the run time was only 8 minutes. From the CCD test, the interaction of chromatographic parameters between column temperature and backpressure was founded which the optimal parameters were 55 °C and 2600 psi, respectively. Thirty-two peaks in the lipid profile of coix seed were tentatively identified, of which 20 were triglyceride, and 12 were diglyceride. Nine features that could potentially be used to distinguish the coix seeds by their geographical origin were identified, most of which were diglycerides, such as OP. CONCLUSIONS: Our findings confirm that UPC2-QTOF combined with chemometrics could be used as an efficient method for exploring potential lipid markers of the quality of herbal medicine.

Green Quantification Strategy Combined with Chemometric Analysis for Triglycerides in Seeds Used in Traditional Chinese Medicine.

Triglycerides are the primary constituents of some seed kernels used in traditional Chinese medicine. Quality control of seed kernels containing multiple components with an environmentally friendly method is indispensable for establishing their quality standards (called monographs) in pharmacopeia. Using coix seeds (Semen Coicis) as an example, a green quantification strategy was proposed by combining C8 core-shell particles with single standard to determine multicomponent technologies to quantify seven triglycerides simultaneously. A core-shell column, namely, Halo C8 (3.0 × 100 mm, 2.7 µm), was used. Methanol was used as the mobile phase at a flow rate of 0.3 mL/min, enabling UV detection of the elutes. Seven triglycerides were well separated in 20 min, and simultaneously quantified using triolein as a single standard. The conversion factor for each standard was set as 1.0 on ELSD, while for the conversion factors at 203 nm, the values increased with the reduction of linoleate. The recovery values were all in the range of 97 - 107% (RSD < 3.0%). The RSD values of precision, including intraday and intermediate precision, were < 3.0% when the total content of triglycerides was calculated. The linearity reached r ≥ 0.9990, and the limit of quantitation reached 40 - 70 ng. Forty-nine batches of coix seeds from four different places of origins and eight batches of adulterants were evaluated and differentiated using principal component analysis. In addition, the validated method was used successfully to quantity seven triglycerides in Semen Persicae, Semen Armeniacae Amarum, and Semen Pruni.

SRSF1 promotes vascular smooth muscle cell proliferation through a Δ133p53/EGR1/KLF5 pathway.

Though vascular smooth muscle cell (VSMC) proliferation underlies all cardiovascular hyperplastic disorders, our understanding of the molecular mechanisms responsible for this cellular process is still incomplete. Here we report that SRSF1 (serine/arginine-rich splicing factor 1), an essential splicing factor, promotes VSMC proliferation and injury-induced neointima formation. Vascular injury in vivo and proliferative stimuli in vitro stimulate SRSF1 expression. Mice lacking SRSF1 specifically in SMCs develop less intimal thickening after wire injury. Expression of SRSF1 in rat arteries enhances neointima formation. SRSF1 overexpression increases, while SRSF1 knockdown suppresses the proliferation and migration of cultured human aortic and coronary arterial SMCs. Mechanistically, SRSF1 favours the induction of a truncated p53 isoform, Δ133p53, which has an equal proliferative effect and in turn transcriptionally activates Krüppel-like factor 5 (KLF5) via the Δ133p53-EGR1 complex, resulting in an accelerated cell-cycle progression and increased VSMC proliferation. Our study provides a potential therapeutic target for vascular hyperplastic disease.

CaMKII is a RIP3 substrate mediating ischemia- and oxidative stress-induced myocardial necroptosis.

Regulated necrosis (necroptosis) and apoptosis are crucially involved in severe cardiac pathological conditions, including myocardial infarction, ischemia-reperfusion injury and heart failure. Whereas apoptotic signaling is well defined, the mechanisms that underlie cardiomyocyte necroptosis remain elusive. Here we show that receptor-interacting protein 3 (RIP3) triggers myocardial necroptosis, in addition to apoptosis and inflammation, through activation of Ca(2+)-calmodulin-dependent protein kinase (CaMKII) rather than through the well-established RIP3 partners RIP1 and MLKL. In mice, RIP3 deficiency or CaMKII inhibition ameliorates myocardial necroptosis and heart failure induced by ischemia-reperfusion or by doxorubicin treatment. RIP3-induced activation of CaMKII, via phosphorylation or oxidation or both, triggers opening of the mitochondrial permeability transition pore and myocardial necroptosis. These findings identify CaMKII as a new RIP3 substrate and delineate a RIP3-CaMKII-mPTP myocardial necroptosis pathway, a promising target for the treatment of ischemia- and oxidative stress-induced myocardial damage and heart failure.

p55γ functional mimetic peptide N24 blocks vascular proliferative disorders.

UNLABELLED: Proliferation and migration disorders of vascular smooth muscle cells (VSMCs) contribute to the pathogenesis of proliferative cardiovascular diseases. Although, over the past two decades, a large panel of drugs has been developed for targeting VSMC proliferation, cardiovascular disease remains the leading cause of death worldwide. Thus, there is a compelling need to identify novel signaling pathways and molecules controlling VSMC proliferation and migration, to provide not only mechanistic insights but also safe and effective therapies for the treatment of cardiovascular diseases. Our recent studies have demonstrated that p55γ, a regulatory subunit of phosphoinositide 3-kinase, functions as an endogenous brake on VSMC proliferation. Here, we demonstrate that the small peptide N24, the first 24 amino acids of the NH2 terminus of p55γ, is a functional mimetic which negatively regulates VSMC proliferation and migration. Specifically, luminal delivery of adenovirus expressing N24 or local administration of Tat transactivator protein (TAT)-tagged N24 by pluronic gel alleviates neointimal formation following balloon injury in rat carotid arteries. Enforced expression of N24 suppresses the proliferation and migration of VSMCs induced by serum- or platelet-derived growth factor-BB. Mechanistically, N24 induces cell cycle arrest via activating the p53-p21 signal pathway, without triggering cell death. N24 interacts with and stabilizes p53 by blocking its ubiquitin-dependent degradation, subsequently promotes p21 transcription, and arrests cell cycle progression. Indeed, knockdown of p21 or p53 abrogates the N24-mediated cell growth arrest. Thus, N24 is a p55γ mimetic inhibiting VSMC proliferation as well as migration, thereby conferring important therapeutic implications for anti-proliferative treatment. KEY MESSAGE: • N24 attenuates balloon injury-induced neointimal formation. • Overexpression of N24 inhibits cultured VSMC proliferation and migration. • Overexpression of N24 arrests the cell cycle at S phase. • N24 interacts with and stabilizes p53 resulting in growth suppression.

Identification of PI3K regulatory subunit p55γ as a novel inhibitor of vascular smooth muscle cell proliferation and neointimal formation.

AIMS: Phosphatidylinositol 3 kinases (PI3Ks) play a pivotal role in vascular physiology and pathophysiology. We aimed to investigate the role of p55γ, a regulatory subunit of PI3Ks, in vascular smooth muscle cell (VSMC) proliferation and neointimal formation. METHODS AND RESULTS: We identified p55γ as an important factor that suppresses VSMC proliferation and injury-evoked neointimal formation. Western blot and mRNA analyses showed that p55γ expression declined in balloon-injured rat carotid arteries and in response to PDGF-BB and serum treatment in cultured VSMCs. Overexpression of p55γ inhibited, whereas short hairpin RNA knockdown of p55γ promoted PDGF-BB- and serum-induced VSMC proliferation. Importantly, in vivo adenoviral gene transfer of p55γ into carotid arteries attenuated, while knockdown of p55γ enhanced balloon injury-induced neointimal formation. Furthermore, p55γ sequentially up-regulated p53 and p21, resulting in cell-cycle arrest in S phase; small-interfering RNA knockdown of either p53 or p21 blocked p55γ-induced VSMC growth arrest. Mechanistically, p55γ interacted with and stabilized p53 protein by blocking mouse double minute 2 homologue-mediated p53 ubiquitination and degradation, subsequently activating its target gene p21. Concurrently, p55γ up-regulated Bcl-xl expression, resulting in non-apoptotic growth arrest effect. CONCLUSION: These findings mark p55γ as a novel upstream regulator of the p53-p21 signalling pathway that negatively regulates VSMC proliferation, suggesting that malfunction of p55γ may trigger vascular proliferative disorders.

Central role of E3 ubiquitin ligase MG53 in insulin resistance and metabolic disorders.

Insulin resistance is a fundamental pathogenic factor present in various metabolic disorders including obesity and type 2 diabetes. Although skeletal muscle accounts for 70-90% of insulin-stimulated glucose disposal, the mechanism underlying muscle insulin resistance is poorly understood. Here we show in mice that muscle-specific mitsugumin 53 (MG53; also called TRIM72) mediates the degradation of the insulin receptor and insulin receptor substrate 1 (IRS1), and when upregulated, causes metabolic syndrome featuring insulin resistance, obesity, hypertension and dyslipidaemia. MG53 expression is markedly elevated in models of insulin resistance, and MG53 overexpression suffices to trigger muscle insulin resistance and metabolic syndrome sequentially. Conversely, ablation of MG53 prevents diet-induced metabolic syndrome by preserving the insulin receptor, IRS1 and insulin signalling integrity. Mechanistically, MG53 acts as an E3 ligase targeting the insulin receptor and IRS1 for ubiquitin-dependent degradation, comprising a central mechanism controlling insulin signal strength in skeletal muscle. These findings define MG53 as a novel therapeutic target for treating metabolic disorders and associated cardiovascular complications.

MG53 participates in ischaemic postconditioning through the RISK signalling pathway.

AIMS: Recent studies show that ischaemic postconditioning (PostC), similar to the well-established ischaemic preconditioning (IPC), confers cardioprotection against ischaemia/reperfusion (IR) injury, and both IPC and PostC can activate the reperfusion injury salvage kinase (RISK) pathway and the survivor activating factor enhancement (SAFE) pathway. PostC is clinically more attractive because of its therapeutic application at the predictable onset of reperfusion. Our previous studies have demonstrated that MG53 is a primary component of the IPC machinery. Here, we investigated the potential role of MG53 in PostC-mediated myocardial protection and explored the underlying mechanism. METHODS AND RESULTS: Using Langendorff perfusion, we investigated IR injury in wild-type (wt) and MG53-deficient (mg53(-/-)) mouse hearts with or without PostC. IR-induced myocardial damage was markedly exacerbated in mg53(-/-) hearts compared with wt controls. PostC protected wt hearts against IR-induced myocardial infarction, myocyte necrosis, and apoptosis, but failed to protect mg53(-/-) hearts. The loss of PostC protection in mg53(-/-) hearts was attributed to selectively impaired PostC-activated RISK signalling. Mechanistically, MG53 is required for the interaction between caveolin 3 (CaV3) and the p85 subunit of phosphoinositide 3-kinase (p85-PI3K) and PostC-mediated activation of the RISK pathway. Importantly, a structure-function study revealed that the MG53 tripartite motif (TRIM) domain (aa1-284) physically interacted with CaV3 but not p85-PI3K, whereas the MG53 SPRY domain (aa285-477) interacted with p85-PI3K but not CaV3, indicating that MG53 binds to CaV3 and p85 at its N- and C-terminus, respectively. CONCLUSIONS: We conclude that MG53 participates in PostC-mediated cardioprotection largely through tethering CaV3 and PI3K and subsequent activation of the RISK pathway.

MG53 constitutes a primary determinant of cardiac ischemic preconditioning.

BACKGROUND: Ischemic heart disease is the greatest cause of death in Western countries. The deleterious effects of cardiac ischemia are ameliorated by ischemic preconditioning (IPC), in which transient ischemia protects against subsequent severe ischemia/reperfusion injury. IPC activates multiple signaling pathways, including the reperfusion injury salvage kinase pathway (mainly PI3K-Akt-glycogen synthase kinase-3beta [GSK3beta] and ERK1/2) and the survivor activating factor enhancement pathway involving activation of the JAK-STAT3 axis. Nevertheless, the fundamental mechanism underlying IPC is poorly understood. METHODS AND RESULTS: In the present study, we define MG53, a muscle-specific TRIM-family protein, as a crucial component of cardiac IPC machinery. Ischemia/reperfusion or hypoxia/oxidative stress applied to perfused mouse hearts or neonatal rat cardiomyocytes, respectively, causes downregulation of MG53, and IPC can prevent ischemia/reperfusion-induced decrease in MG53 expression. MG53 deficiency increases myocardial vulnerability to ischemia/reperfusion injury and abolishes IPC protection. Overexpression of MG53 attenuates whereas knockdown of MG53 enhances hypoxia- and H(2)O(2)-induced cardiomyocyte death. The cardiac protective effects of MG53 are attributable to MG53-dependent interaction of caveolin-3 with phosphatidylinositol 3 kinase and subsequent activation of the reperfusion injury salvage kinase pathway without altering the survivor activating factor enhancement pathway. CONCLUSIONS: These results establish MG53 as a primary component of the cardiac IPC response, thus identifying a potentially important novel therapeutic target for the treatment of ischemic heart disease.

Receptor interacting protein 3 suppresses vascular smooth muscle cell growth by inhibition of the phosphoinositide 3-kinase-Akt axis.

Proliferation of vascular smooth muscle cells (VSMCs) is a primary mechanism underlying cardiovascular proliferative disorders. Phosphoinositide 3-kinase (PI3K)-Akt (or protein kinase B) axis has been assigned at the center of pathways that regulate cell proliferation. Here we demonstrate that enhanced PI3K-Akt signaling by mitogenic stimulation or arterial injury profoundly elevates expression of receptor interacting protein 3 (RIP3) in primary cultured rat VSMCs and in vivo and that the up-regulation of RIP3 leads to VSMC growth arrest and apoptosis via inhibiting the PI3K-Akt signaling pathway, thereby alleviating balloon injury-induced neointimal formation. Specifically, mitogenic stimulation with platelet-derived growth factor-BB or angiotensin II leads to a profound increase in RIP3 expression, which is abolished by inhibition of PI3K or Akt, and increased PI3K-Akt signaling by expression of a constitutively active PI3K mutant also elevates RIP3 expression. Importantly, adenoviral overexpression of RIP3 not only triggers apoptosis but also causes cell cycle arrest at G(1)/G(0) phases that is associated with suppressed Akt activation. In sharp contrast, RIP3 gene silencing enhances serum- and platelet-derived growth factor-induced cell proliferation and Akt activation. In vivo adenoviral gene delivery of rat RIP3 (rRIP3) increased apoptosis and reduced VSMC proliferation, thus, effectively alleviating balloon injury-induced neointimal formation. The growth-suppressive and pro-apoptotic effects are independent of rRIP3 Ser/Thr kinase activity, because overexpression of a kinase-inactive mutant of rRIP3, similar to its wild type, is sufficient to induce growth arrest and apoptosis. These findings reveal a novel growth-suppressive action of RIP3, marking RIP3 as an important factor to prevent excessive mitogenic stimulation- or injury-induced vascular smooth muscle cells hyperplasia.

Cardioprotection by CaMKII-deltaB is mediated by phosphorylation of heat shock factor 1 and subsequent expression of inducible heat shock protein 70.

RATIONALE: Ca2+/calmodulin-dependent protein kinase (CaMK)II is a multifunctional kinase involved in vital cellular processes such as Ca(2+) handling and cell fate regulation. In mammalian heart, 2 primary CaMKII isoforms, deltaB and deltaC, localize in nuclear and cytosolic compartments, respectively. Although previous studies have established an essential role of CaMKII-deltaC in cardiomyocyte apoptosis, the functional role of the more abundant isoform, CaMKII-deltaB, remains elusive. OBJECTIVE: Here, we determined the potential role of CaMKII-deltaB in regulating cardiomyocyte viability and explored the underlying mechanism. METHODS AND RESULTS: In cultured neonatal rat cardiomyocytes, the expression of CaMKII-deltaB and CaMKII-deltaC was inversely regulated in response to H2O2-induced oxidative stress with a profound reduction of the former and an increase of the later. Similarly, in vivo ischemia/reperfusion (IR) led to an opposite regulation of these CaMKII isoforms in a rat myocardial IR model. Notably, overexpression of CaMKII-deltaB protected cardiomyocytes against oxidative stress-, hypoxia-, and angiotensin II-induced apoptosis, whereas overexpression of its cytosolic counterpart promoted apoptosis. Using cDNA microarray, real-time PCR and Western blotting, we demonstrated that overexpression of CaMKII-deltaB but not CaMKII-deltaC elevated expression of heat shock protein (HSP)70 family members, including inducible (i)HSP70 and its homolog (Hst70). Moreover, overexpression of CaMKII-deltaB led to phosphorylation and activation of heat shock factor (HSF)1, the primary transcription factor responsible for HSP70 gene regulation. Importantly, gene silencing of iHSP70, but not Hst70, abolished CaMKII-deltaB-mediated protective effect, indicating that only iHSP70 was required for CaMKII-deltaB elicited antiapoptotic signaling. CONCLUSIONS: We conclude that cardiac CaMKII-deltaB and CaMKII-deltaC were inversely regulated in response to oxidative stress and IR injury, and that in contrast to CaMKII-deltaC, CaMKII-deltaB serves as a potent suppressor of cardiomyocyte apoptosis triggered by multiple death-inducing stimuli via phosphorylation of HSF1 and subsequent induction of iHSP70, marking both CaMKII-delta isoforms as promising therapeutic targets for the treatment of ischemic heart disease.

Attenuation of mitochondrial, but not cytosolic, Ca2+ overload reduces myocardial injury induced by ischemia and reperfusion.

AIM: Attenuation of mitochondrial Ca2+ ([Ca2+]m), but not cytosolic Ca2+ ([Ca2+]c), overload improves contractile recovery. We hypothesized that attenuation of [Ca2+]m, but not [Ca2+]c, overload confers cardioprotection against ischemia/reperfusion-induced injury. METHODS: Infarct size from isolated perfused rat heart, cell viability, and electrically-induced Ca2+ transient in isolated rat ventricular myocytes were measured. We determined the effects of BAPTA-AM, a Ca2+ chelator, at concentrations that abolish the overload of both [Ca2+]c and [Ca2+]m, and ruthenium red, an inhibitor of mitochondrial uniporter of Ca2+ transport, at concentrations that abolish the overload of [Ca2+]m, but not [Ca2+]c, on cardiac injury induced by ischemia/reperfusion. RESULTS: Attenuation of both [Ca2+]m and [Ca2+]c by BAPTA-AM, and attenuation of [Ca2+]m, but not [Ca2+]c, overload by ruthenium red, reduced the cardiac injury observations, indicating the importance of [Ca2+]m in cardioprotection and contractile recovery in response to ischemia/reperfusion. CONCLUSION: The study has provided unequivocal evidence using a cause-effect approach that attenuation of [Ca2+]m, but not [Ca2+]c, overload is responsible for cardioprotection against ischemia/reperfusion-induced injury. We also confirmed the previous observation that attenuation of [Ca2+]m, but not [Ca2+]c, by ruthenium red improves contractile recovery following ischemia/reperfusion.