Cardioprotective GLP-1 metabolite prevents ischemic cardiac injury by inhibiting mitochondrial trifunctional protein-α.

Mechanisms mediating the cardioprotective actions of glucagon-like peptide 1 (GLP-1) were unknown. Here, we show in both ex vivo and in vivo models of ischemic injury that treatment with GLP-1(28-36), a neutral endopeptidase-generated (NEP-generated) metabolite of GLP-1, was as cardioprotective as GLP-1 and was abolished by scrambling its amino acid sequence. GLP-1(28-36) enters human coronary artery endothelial cells (caECs) through macropinocytosis and acts directly on mouse and human coronary artery smooth muscle cells (caSMCs) and caECs, resulting in soluble adenylyl cyclase Adcy10-dependent (sAC-dependent) increases in cAMP, activation of protein kinase A, and cytoprotection from oxidative injury. GLP-1(28-36) modulates sAC by increasing intracellular ATP levels, with accompanying cAMP accumulation lost in sAC-/- cells. We identify mitochondrial trifunctional protein-α (MTPα) as a binding partner of GLP-1(28-36) and demonstrate that the ability of GLP-1(28-36) to shift substrate utilization from oxygen-consuming fatty acid metabolism toward oxygen-sparing glycolysis and glucose oxidation and to increase cAMP levels is dependent on MTPα. NEP inhibition with sacubitril blunted the ability of GLP-1 to increase cAMP levels in coronary vascular cells in vitro. GLP-1(28-36) is a small peptide that targets novel molecular (MTPα and sAC) and cellular (caSMC and caEC) mechanisms in myocardial ischemic injury.

p53 and Mdm2 act synergistically to maintain cardiac homeostasis and mediate cardiomyocyte cell cycle arrest through a network of microRNAs.

Defining the roadblocks responsible for cell cycle arrest in adult cardiomyocytes lies at the core of developing cardiac regenerative therapies. p53 and Mdm2 are crucial mediators of cell cycle arrest in proliferative cell types, however, little is known about their function in regulating homeostasis and proliferation in terminally differentiated cell types, like cardiomyocytes. To explore this, we generated a cardiac-specific conditional deletion of p53 and Mdm2 (DKO) in adult mice. Herein we describe the development of a dilated cardiomyopathy, in the absence of cardiac hypertrophy. In addition, DKO hearts exhibited a significant increase in cardiomyocyte proliferation. Further evaluation showed that proliferation was mediated by a significant increase in Cdk2 and cyclin E with downregulation of p21Cip1 and p27Kip1. Comparison of miRNA expression profiles from DKO mouse hearts and controls revealed 11 miRNAs that were downregulated in the DKO hearts and enriched for mRNA targets involved in cell cycle regulation. Knockdown of these miRNAs in neonatal rat cardiomyocytes significantly increased cytokinesis with an upregulation in the expression of crucial cell cycle regulators. These results illustrate the importance of the cooperative activities of p53 and Mdm2 in a network of miRNAs that function to impose a barrier against aberrant cardiomyocyte cell cycle re-entry to maintain cardiac homeostasis.

Adiponectin is required for cardiac MEF2 activation during pressure overload induced hypertrophy.

Cardiomyocyte (CM) hypertrophy and increased heart mass in response to pressure overload are associated with hyper-activation of the myocyte enhancer factor-2 (MEF2) family of transcriptional regulators, and concomitant initiation of the fetal gene program. Adiponectin, an adipokine that is reduced in individuals with obesity and diabetes, has been characterized both as a negative regulator or permissive factor in cardiac hypertrophy. We therefore sought to analyze temporal regulation of MEF2 activity in response to pressure overload (PO) and changes in adiponectin status. To address this we crossed a well characterized transgenic MEF2 "sensor" mouse (MEF2-lacZ) with adiponectin null mice (Ad-KO) to create compound MEF2 lacZ/Ad-KO mice. Initially, we established that transverse aortic banding induced PO in wild-type (WT) mice increased heart mass and CM hypertrophy from 1 to 4weeks following surgery, indicated by increased CM diameter and heart weight/tibia length ratio. This was associated with cardiac dysfunction determined by echocardiography. Hypertrophic changes and dysfunction were observed in Ad-KO mice 4weeks following surgery. MEF2 lacZ activity and endogenous ANF mRNA levels, used as indicators of hypertrophic gene activation, were both robustly increased in WT mice after MTAB but attenuated in the Ad-KO background. Furthermore, activation of the pro-hypertrophic molecule p38 was increased following MTAB surgery in WT mice, but not in Ad-KO animals, and treatment of primary isolated CM with recombinant adiponectin induced p38 phosphorylation in a time dependent manner. Adiponectin also increased MEF2 activation in primary cardiomyocytes, an effect attenuated by p38 MAPK inhibition. In conclusion, our data indicate that robust hypertrophic MEF2 activation in the heart in vivo requires a background of adiponectin signaling and that adiponectin signaling in primary isolated CM directly enhances MEF2 activity through activation of p38 MAPK. We conclude that adiponectin is required for full induction of cardiomyocyte MEF2 activation, thus contributing to the myocardial hypertrophic gene expression program in response to PO.

Phosphodiesterase type 3A regulates basal myocardial contractility through interacting with sarcoplasmic reticulum calcium ATPase type 2a signaling complexes in mouse heart.

RATIONALE: cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP-mediated signaling. The role of different PDE isozymes, particularly PDE3A vs PDE3B, in the regulation of heart function remains unclear. OBJECTIVE: To determine the relative contribution of PDE3A vs PDE3B isozymes in the regulation of heart function and to dissect the molecular basis for this regulation. METHODS AND RESULTS: Compared with wild-type littermates, cardiac contractility and relaxation were enhanced in isolated hearts from PDE3A(-/-), but not PDE3B(-/-), mice. Furthermore, PDE3 inhibition had no effect on PDE3A(-/-) hearts but increased contractility in wild-type (as expected) and PDE3B(-/-) hearts to levels indistinguishable from PDE3A(-/-). The enhanced contractility in PDE3A(-/-) hearts was associated with cAMP-dependent elevations in Ca(2+) transient amplitudes and increased sarcoplasmic reticulum (SR) Ca(2+) content, without changes in L-type Ca(2+) currents of cardiomyocytes, as well as with increased SR Ca(2+)-ATPase type 2a activity, SR Ca(2+) uptake rates, and phospholamban phosphorylation in SR fractions. Consistent with these observations, PDE3 activity was reduced ≈8-fold in SR fractions from PDE3A(-/-) hearts. Coimmunoprecipitation experiments further revealed that PDE3A associates with both SR calcium ATPase type 2a and phospholamban in a complex that also contains A-kinase anchoring protein-18, protein kinase type A-RII, and protein phosphatase type 2A. CONCLUSIONS: Our data support the conclusion that PDE3A is the primary PDE3 isozyme modulating basal contractility and SR Ca(2+) content by regulating cAMP in microdomains containing macromolecular complexes of SR calcium ATPase type 2a-phospholamban-PDE3A.

Regulation of murine cardiac function by phosphodiesterases type 3 and 4.

Cyclic nucleotide phosphodiesterases (PDEs) encompass a large group of enzymes that regulate intracellular levels of two-second messengers, cAMP and cGMP, by controlling the rates of their degradation. More than 60 isoforms, subdivided into 11 gene families (PDE1-11), exist in mammals with at least six families (PDE1-5 and PDE8) identified in mammalian hearts. The two predominant families implicated in regulating contraction strength of the heart are PDE3 and PDE4. Studies using transgenic models in combination with family-specific PDE inhibitors have demonstrated that PDE3A, PDE4B, and PDE4D isoforms regulate cardiac contractility by modulating cAMP levels in various subcellular compartments. These studies have further uncovered contributions of PDE4B and PDE4D in preventing ventricular arrhythmias.

Phosphodiesterase 4D regulates baseline sarcoplasmic reticulum Ca2+ release and cardiac contractility, independently of L-type Ca2+ current.

RATIONALE: Baseline contractility of mouse hearts is modulated in a phosphatidylinositol 3-kinase-γ-dependent manner by type 4 phosphodiesterases (PDE4), which regulate cAMP levels within microdomains containing the sarcoplasmic reticulum (SR) calcium ATPase type 2a (SERCA2a). OBJECTIVE: The goal of this study was to determine whether PDE4D regulates basal cardiac contractility. METHODS AND RESULTS: At 10 to 12 weeks of age, baseline cardiac contractility in PDE4D-deficient (PDE4D(-/-)) mice was elevated mice in vivo and in Langendorff perfused hearts, whereas isolated PDE4D(-/-) cardiomyocytes showed increased whole-cell Ca2+ transient amplitudes and SR Ca2+content but unchanged L-type calcium current, compared with littermate controls (WT). The protein kinase A inhibitor R(p)-adenosine-3',5' cyclic monophosphorothioate (R(p)-cAMP) lowered whole-cell Ca2+ transient amplitudes and SR Ca2+ content in PDE4D(-/-) cardiomyocytes to WT levels. The PDE4 inhibitor rolipram had no effect on cardiac contractility, whole-cell Ca2+ transients, or SR Ca2+ content in PDE4D(-/-) preparations but increased these parameters in WT myocardium to levels indistinguishable from those in PDE4D(-/-). The functional changes in PDE4D(-/-) myocardium were associated with increased PLN phosphorylation but not cardiac ryanodine receptor phosphorylation. Rolipram increased PLN phosphorylation in WT cardiomyocytes to levels indistinguishable from those in PDE4D(-/-) cardiomyocytes. In murine and failing human hearts, PDE4D coimmunoprecipitated with SERCA2a but not with cardiac ryanodine receptor. CONCLUSIONS: PDE4D regulates basal cAMP levels in SR microdomains containing SERCA2a-PLN, but not L-type Ca2+ channels or ryanodine receptor. Because whole-cell Ca2+ transient amplitudes are reduced in failing human myocardium, these observations may have therapeutic implications for patients with heart failure.

Effects of monovalent cations on Ca2+ uptake by skeletal and cardiac muscle sarcoplasmic reticulum.

Ca(2+) transport by the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) is sensitive to monovalent cations. Possible K(+) binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca(2+) transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca(2+) transport correlated in most cases with their direct effects on SERCA. Choline(+), however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca(2+) transport. Of the monovalent cations tested, only Cs(+) significantly affected the Hill coefficient of Ca(2+) transport (n(H)). An increase in n(H) from approximately 2 in K(+) to approximately 3 in Cs(+) was seen in all of the forms of SERCA examined. The effects of Cs(+) on the maximum velocity of Ca(2+) uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K(+) binding sites of the different forms of the protein.

Inhibition of a cardiac sarcoplasmic reticulum chloride channel by tamoxifen.

Anion and cation channels present in the sarcoplasmic reticulum (SR) are believed to be necessary to maintain the electroneutrality of SR membrane during Ca(2+) uptake by the SR Ca(2+) pump (SERCA). Here we incorporated canine cardiac SR ion channels into lipid bilayers and studied the effects of tamoxifen and other antiestrogens on these channels. A Cl(-) channel was identified exhibiting multiple subconductance levels which could be divided into two primary conductance bands. Tamoxifen decreases the time the channel spends in its higher, voltage-sensitive band and the mean channel current. The lower, voltage-insensitive, conductance band is not affected by tamoxifen, nor is a K(+) channel present in the cardiac SR preparation. By examining SR Ca(2+) uptake, SERCA ATPase activity, and SR ion channels in the same preparation, we also estimated SERCA transport current, SR Cl(-) and K(+) currents, and the density of SERCA, Cl(-), and K(+) channels in cardiac SR membranes.