Shikha Mishra

Cyclophilin D controls mitochondrial pore–dependent Ca2+ exchange, metabolic flexibility, and propensity for heart failure in mice

Cyclophilin D (which is encoded by the Ppif gene) is a mitochondrial matrix peptidyl-prolyl isomerase known to modulate opening of the mitochondrial permeability transition pore (MPTP). Apart from regulating necrotic cell death, the physiologic function of the MPTP is largely unknown. Here we have shown that Ppif(-/-) mice exhibit substantially greater cardiac hypertrophy, fibrosis, and reduction in myocardial function in response to pressure overload stimulation than control mice. In addition, Ppif(-/-) mice showed greater hypertrophy and lung edema as well as reduced survival in response to sustained exercise stimulation. Cardiomyocyte-specific transgene expression of cyclophilin D in Ppif(-/-) mice rescued the enhanced hypertrophy, reduction in cardiac function, and rapid onset of heart failure following pressure overload stimulation. Mechanistically, the maladaptive phenotype in the hearts of Ppif(-/-) mice was associated with an alteration in MPTP-mediated Ca(2+) efflux resulting in elevated levels of mitochondrial matrix Ca(2+) and enhanced activation of Ca(2+)-dependent dehydrogenases. Elevated matrix Ca(2+) led to increased glucose oxidation relative to fatty acids, thereby limiting the metabolic flexibility of the heart that is critically involved in compensation during stress. These findings suggest that the MPTP maintains homeostatic mitochondrial Ca(2+) levels to match metabolism with alterations in myocardial workload, thereby suggesting a physiologic function for the MPTP.

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.

Phospholamban ablation rescues sarcoplasmic reticulum ca2+ handling but exacerbates cardiac dysfunction in CaMKIIδC transgenic mice

Rationale: We previously showed that transgenic mice expressing Ca2+/calmodulin-dependent protein kinase II &dgr;C (CaMKII-TG) develop dilated cardiomyopathy associated with increased ryanodine receptors (RyR2) phosphorylation, enhanced sarcoplasmic reticulum (SR) Ca2+ leak and lowering of SR Ca2+ load. We hypothesized that phospholamban (PLN) ablation would restore SR Ca2+ load and prevent the decreased ventricular contractility, dilation and mortality seen in CaMKII-TG. Objective: Our objectives were to generate CaMKII-TG mice lacking PLN, determine whether the maladaptive effects of cardiac CaMKII&dgr;C expression were corrected, and establish the mechanistic basis for these changes. Methods and Results: CaMKII-TG were crossed with PLN knockout (PLN-KO) mice to generate KO/TG mice. Myocytes from wild type (WT), CaMKII-TG, PLN-KO and KO/TG were compared. The decreased SR Ca2+ load and twitch Ca2+ transients seen in CaMKII-TG were normalized in KO/TG. Surprisingly the heart failure phenotype was exacerbated, as indicated by increased left ventricular dilation, decreased ventricular function, increased apoptosis and greater mortality. In KO/TG myocytes SR Ca2+ sparks and leak were significantly increased, presumably because of the combined effects of restored SR Ca2+ load and RyR2 phosphorylation. Mitochondrial Ca2+ loading was increased in cardiomyocytes from KO/TG versus WT or CaMKII-TG mice and this was dependent on elevated SR Ca2+ sparks. Cardiomyocytes from KO/TG showed poor viability, improved by inhibiting SR Ca2+ release and mitochondrial Ca2+ loading. Conclusions: Normalizing cardiomyocyte SR Ca2+ loading in the face of elevated CaMKII and RyR2 phosphorylation leads to enhanced SR Ca2+ leak and mitochondrial Ca2+ elevation, associated with exacerbated cell death, heart failure and mortality.

Location Matters: Clarifying the Concept of Nuclear and Cytosolic CaMKII Subtypes

Rationale: Differential effects of &dgr;B and &dgr;C subtypes of Ca2+/calmodulin-dependent protein kinase (CaMKII) on cardiomyocyte Ca2+ handling and survival have been suggested to result from their respective nuclear versus cytosolic localizations. CaMKII&dgr; subtype localization and its relationship to enzyme activation and target phosphorylation have not, however, been systematically evaluated. Objective: To determine whether CaMKII&dgr; subtypes are restricted to a particular subcellular location and assess the relationship of localization to enzyme activation and function. Methods and Results: CaMKII&dgr; is highly expressed in mouse heart and cardiomyocytes and concentrated in sarcoplasmic reticulum (SR)/membrane and nuclear fractions. CaMKII&dgr;B and &dgr;C subtypes differ by a nuclear localization sequence, but both are present in nuclear and SR/membrane fractions. Nonselective subtype distribution is also seen in mice overexpressing CaMKII&dgr;B or &dgr;C, even in a CaMKII&dgr; null background. Fluorescently tagged CaMKII&dgr;B expressed in cardiomyocytes concentrates in nuclei whereas &dgr;C concentrates in cytosol, but neither localization is exclusive. Mouse hearts exposed to phenylephrine show selective CaMKII&dgr; activation in the nuclear (versus SR) compartment, whereas caffeine selectively activates CaMKII&dgr; in SR (versus nuclei), independent of subtype. Compartmentalized activation extends to functional differences in target phosphorylation at CaMKII sites: phenylephrine increases histone deacetylase 5 phosphorylation (Ser498) but not phospholamban (Thr17), whereas the converse holds for caffeine. Conclusions: These studies demonstrate that CaMKII&dgr;B and &dgr;C are not exclusively restricted to the nucleus and cytosol and that spatial and functional specificity in CaMKII&dgr; activation is elicited by mobilization of different Ca2+ stores rather than by compartmentalized subtype localization.Rationale: Differential effects of δB and δC subtypes of Ca2+/calmodulin-dependent protein kinase (CaMKII) on cardiomyocyte Ca2+ handling and survival have been suggested to result from their respective nuclear versus cytosolic localizations. CaMKIIδ subtype localization and its relationship to enzyme activation and target phosphorylation have not, however, been systematically evaluated. Objective: To determine whether CaMKIIδ subtypes are restricted to a particular subcellular location and assess the relationship of localization to enzyme activation and function. Methods and Results: CaMKIIδ is highly expressed in mouse heart and cardiomyocytes and concentrated in sarcoplasmic reticulum (SR)/membrane and nuclear fractions. CaMKIIδB and δC subtypes differ by a nuclear localization sequence, but both are present in nuclear and SR/membrane fractions. Nonselective subtype distribution is also seen in mice overexpressing CaMKIIδB or δC, even in a CaMKIIδ null background. Fluorescently tagged CaMKIIδB expressed in cardiomyocytes concentrates in nuclei whereas δC concentrates in cytosol, but neither localization is exclusive. Mouse hearts exposed to phenylephrine show selective CaMKIIδ activation in the nuclear (versus SR) compartment, whereas caffeine selectively activates CaMKIIδ in SR (versus nuclei), independent of subtype. Compartmentalized activation extends to functional differences in target phosphorylation at CaMKII sites: phenylephrine increases histone deacetylase 5 phosphorylation (Ser498) but not phospholamban (Thr17), whereas the converse holds for caffeine. Conclusions: These studies demonstrate that CaMKIIδB and δC are not exclusively restricted to the nucleus and cytosol and that spatial and functional specificity in CaMKIIδ activation is elicited by mobilization of different Ca2+ stores rather than by compartmentalized subtype localization. # Novelty and Significance {#article-title-51}

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.

SR-targeted CaMKII inhibition improves SR Ca2+ handling, but accelerates cardiac remodeling in mice overexpressing CaMKIIδC

Cardiac myocyte overexpression of CaMKIIδ(C) leads to cardiac hypertrophy and heart failure (HF) possibly caused by altered myocyte Ca(2+) handling. A central defect might be the marked CaMKII-induced increase in diastolic sarcoplasmic reticulum (SR) Ca(2+) leak which decreases SR Ca(2+) load and Ca(2+) transient amplitude. We hypothesized that inhibition of CaMKII near the SR membrane would decrease the leak, improve Ca(2+) handling and prevent the development of contractile dysfunction and HF. To test this hypothesis we crossbred CaMKIIδ(C) overexpressing mice (CaMK) with mice expressing the CaMKII-inhibitor AIP targeted to the SR via a modified phospholamban (PLB)-transmembrane-domain (SR-AIP). There was a selective decrease in the amount of activated CaMKII in the microsomal (SR/membrane) fraction prepared from these double-transgenic mice (CaMK/SR-AIP) mice. In ventricular cardiomyocytes from CaMK/SR-AIP mice, SR Ca(2+) leak, assessed both as diastolic Ca(2+) shift into SR upon tetracaine in intact myocytes or integrated Ca(2+) spark release in permeabilized myocytes, was significantly reduced. The reduced leak was accompanied by enhanced SR Ca(2+) load and twitch amplitude in double-transgenic mice (vs. CaMK), without changes in SERCA expression or NCX function. However, despite the improved myocyte Ca(2+) handling, cardiac hypertrophy and remodeling was accelerated in CaMK/SR-AIP and cardiac function worsened. We conclude that while inhibition of SR localized CaMKII in CaMK mice improves Ca(2+) handling, it does not necessarily rescue the HF phenotype. This implies that a non-SR CaMKIIδ(C) exerts SR-independent effects that contribute to hypertrophy and HF, and this CaMKII pathway may be exacerbated by the global enhancement of Ca transients.

CaMKIIδC Slows [Ca]i Decline in Cardiac Myocytes by Promoting Ca Sparks

Acute activation of calcium/calmodulin-dependent protein kinase (CaMKII) in permeabilized phospholamban knockout (PLN-KO) mouse myocytes phosphorylates ryanodine receptors (RyRs) and activates spontaneous local sarcoplasmic reticulum (SR) Ca release events (Ca sparks) even at constant SR Ca load. To assess how CaMKII regulates SR Ca release in intact myocytes (independent of SR Ca content changes or PLN effects), we compared Ca sparks in PLN-KO versus mice, which also have transgenic cardiac overexpression of CaMKIIδC in the PLN-KO background (KO/TG). Compared with PLN-KO mice, these KO/TG cardiomyocytes exhibited 1), increased twitch Ca transient and fractional release (both by ∼35%), but unaltered SR Ca load; 2), increased resting Ca spark frequency (300%) despite a lower diastolic [Ca]i, which also slowed twitch [Ca]i decline (suggesting CaMKII-dependent RyR Ca sensitization); 3), elevated Ca spark amplitude and rate of Ca release (which might indicate that more RyR channels participate in a single spark); 4), prolonged Ca spark rise time (which implies that CaMKII either delays RyR closure or prolongs the time when openings can occur); 5), more frequent repetitive sparks at single release sites. Analysis of repetitive sparks from individual Ca release sites indicates that CaMKII enhanced RyR Ca sensitivity, but did not change the time course of SR Ca refilling. These results demonstrate that there are dramatic CaMKII-mediated effects on RyR Ca release that occur via regulation of both RyR activation and termination processes.

Cardioprotective stimuli mediate phosphoinositide 3-kinase and phosphoinositide dependent kinase 1 nuclear accumulation in cardiomyocytes

The phosphoinositide 3-kinase (PI3K)/phosphoinositide dependent kinase 1 (PDK1) signaling pathway exerts cardioprotective effects in the myocardium through activation of key proteins including Akt. Activated Akt accumulates in nuclei of cardiomyocytes suggesting that biologically relevant targets are located in that subcellular compartment. Nuclear Akt activity could be potentiated in both intensity and duration by the presence of a nuclear-associated PI3K/PDK1 signaling cascade as has been described in other non-myocyte cell types. PI3K/PDK1 distribution was determined in vitro and in vivo by immunostaining and nuclear extraction of cultured rat neonatal cardiomyocytes or transgenic mouse hearts. Results show that PI3K and PDK1 are present at a basal level in cardiomyocytes nuclei and that cardioprotective stimulation with atrial natriuretic peptide (ANP) increases their nuclear localization. In comparison, overexpression of nuclear-targeted Akt does not mediate increased translocation of either PI3K or PDK1 indicating that accumulation of Akt does not drive PI3K or PDK1 into the nuclear compartment. Furthermore, PI3K and phospho-Akt(473) show parallel temporal accumulation in the nucleus following (MI) infarction challenge. These findings demonstrate the presence of a dynamically regulated nuclear-associated signaling cascade involving PI3K and PDK that presumably influences nuclear Akt activation.

Location MattersNovelty and Significance

Rationale: Differential effects of &dgr;B and &dgr;C subtypes of Ca2+/calmodulin-dependent protein kinase (CaMKII) on cardiomyocyte Ca2+ handling and survival have been suggested to result from their respective nuclear versus cytosolic localizations. CaMKII&dgr; subtype localization and its relationship to enzyme activation and target phosphorylation have not, however, been systematically evaluated. Objective: To determine whether CaMKII&dgr; subtypes are restricted to a particular subcellular location and assess the relationship of localization to enzyme activation and function. Methods and Results: CaMKII&dgr; is highly expressed in mouse heart and cardiomyocytes and concentrated in sarcoplasmic reticulum (SR)/membrane and nuclear fractions. CaMKII&dgr;B and &dgr;C subtypes differ by a nuclear localization sequence, but both are present in nuclear and SR/membrane fractions. Nonselective subtype distribution is also seen in mice overexpressing CaMKII&dgr;B or &dgr;C, even in a CaMKII&dgr; null background. Fluorescently tagged CaMKII&dgr;B expressed in cardiomyocytes concentrates in nuclei whereas &dgr;C concentrates in cytosol, but neither localization is exclusive. Mouse hearts exposed to phenylephrine show selective CaMKII&dgr; activation in the nuclear (versus SR) compartment, whereas caffeine selectively activates CaMKII&dgr; in SR (versus nuclei), independent of subtype. Compartmentalized activation extends to functional differences in target phosphorylation at CaMKII sites: phenylephrine increases histone deacetylase 5 phosphorylation (Ser498) but not phospholamban (Thr17), whereas the converse holds for caffeine. Conclusions: These studies demonstrate that CaMKII&dgr;B and &dgr;C are not exclusively restricted to the nucleus and cytosol and that spatial and functional specificity in CaMKII&dgr; activation is elicited by mobilization of different Ca2+ stores rather than by compartmentalized subtype localization.Rationale: Differential effects of δB and δC subtypes of Ca2+/calmodulin-dependent protein kinase (CaMKII) on cardiomyocyte Ca2+ handling and survival have been suggested to result from their respective nuclear versus cytosolic localizations. CaMKIIδ subtype localization and its relationship to enzyme activation and target phosphorylation have not, however, been systematically evaluated. Objective: To determine whether CaMKIIδ subtypes are restricted to a particular subcellular location and assess the relationship of localization to enzyme activation and function. Methods and Results: CaMKIIδ is highly expressed in mouse heart and cardiomyocytes and concentrated in sarcoplasmic reticulum (SR)/membrane and nuclear fractions. CaMKIIδB and δC subtypes differ by a nuclear localization sequence, but both are present in nuclear and SR/membrane fractions. Nonselective subtype distribution is also seen in mice overexpressing CaMKIIδB or δC, even in a CaMKIIδ null background. Fluorescently tagged CaMKIIδB expressed in cardiomyocytes concentrates in nuclei whereas δC concentrates in cytosol, but neither localization is exclusive. Mouse hearts exposed to phenylephrine show selective CaMKIIδ activation in the nuclear (versus SR) compartment, whereas caffeine selectively activates CaMKIIδ in SR (versus nuclei), independent of subtype. Compartmentalized activation extends to functional differences in target phosphorylation at CaMKII sites: phenylephrine increases histone deacetylase 5 phosphorylation (Ser498) but not phospholamban (Thr17), whereas the converse holds for caffeine. Conclusions: These studies demonstrate that CaMKIIδB and δC are not exclusively restricted to the nucleus and cytosol and that spatial and functional specificity in CaMKIIδ activation is elicited by mobilization of different Ca2+ stores rather than by compartmentalized subtype localization. # Novelty and Significance {#article-title-51}

Location MattersNovelty and Significance: Clarifying the Concept of Nuclear and Cytosolic CaMKII Subtypes

Rationale: Differential effects of &dgr;B and &dgr;C subtypes of Ca2+/calmodulin-dependent protein kinase (CaMKII) on cardiomyocyte Ca2+ handling and survival have been suggested to result from their respective nuclear versus cytosolic localizations. CaMKII&dgr; subtype localization and its relationship to enzyme activation and target phosphorylation have not, however, been systematically evaluated. Objective: To determine whether CaMKII&dgr; subtypes are restricted to a particular subcellular location and assess the relationship of localization to enzyme activation and function. Methods and Results: CaMKII&dgr; is highly expressed in mouse heart and cardiomyocytes and concentrated in sarcoplasmic reticulum (SR)/membrane and nuclear fractions. CaMKII&dgr;B and &dgr;C subtypes differ by a nuclear localization sequence, but both are present in nuclear and SR/membrane fractions. Nonselective subtype distribution is also seen in mice overexpressing CaMKII&dgr;B or &dgr;C, even in a CaMKII&dgr; null background. Fluorescently tagged CaMKII&dgr;B expressed in cardiomyocytes concentrates in nuclei whereas &dgr;C concentrates in cytosol, but neither localization is exclusive. Mouse hearts exposed to phenylephrine show selective CaMKII&dgr; activation in the nuclear (versus SR) compartment, whereas caffeine selectively activates CaMKII&dgr; in SR (versus nuclei), independent of subtype. Compartmentalized activation extends to functional differences in target phosphorylation at CaMKII sites: phenylephrine increases histone deacetylase 5 phosphorylation (Ser498) but not phospholamban (Thr17), whereas the converse holds for caffeine. Conclusions: These studies demonstrate that CaMKII&dgr;B and &dgr;C are not exclusively restricted to the nucleus and cytosol and that spatial and functional specificity in CaMKII&dgr; activation is elicited by mobilization of different Ca2+ stores rather than by compartmentalized subtype localization.Rationale: Differential effects of δB and δC subtypes of Ca2+/calmodulin-dependent protein kinase (CaMKII) on cardiomyocyte Ca2+ handling and survival have been suggested to result from their respective nuclear versus cytosolic localizations. CaMKIIδ subtype localization and its relationship to enzyme activation and target phosphorylation have not, however, been systematically evaluated. Objective: To determine whether CaMKIIδ subtypes are restricted to a particular subcellular location and assess the relationship of localization to enzyme activation and function. Methods and Results: CaMKIIδ is highly expressed in mouse heart and cardiomyocytes and concentrated in sarcoplasmic reticulum (SR)/membrane and nuclear fractions. CaMKIIδB and δC subtypes differ by a nuclear localization sequence, but both are present in nuclear and SR/membrane fractions. Nonselective subtype distribution is also seen in mice overexpressing CaMKIIδB or δC, even in a CaMKIIδ null background. Fluorescently tagged CaMKIIδB expressed in cardiomyocytes concentrates in nuclei whereas δC concentrates in cytosol, but neither localization is exclusive. Mouse hearts exposed to phenylephrine show selective CaMKIIδ activation in the nuclear (versus SR) compartment, whereas caffeine selectively activates CaMKIIδ in SR (versus nuclei), independent of subtype. Compartmentalized activation extends to functional differences in target phosphorylation at CaMKII sites: phenylephrine increases histone deacetylase 5 phosphorylation (Ser498) but not phospholamban (Thr17), whereas the converse holds for caffeine. Conclusions: These studies demonstrate that CaMKIIδB and δC are not exclusively restricted to the nucleus and cytosol and that spatial and functional specificity in CaMKIIδ activation is elicited by mobilization of different Ca2+ stores rather than by compartmentalized subtype localization. # Novelty and Significance {#article-title-51}