Repetitive myocardial ischemia promotes coronary growth in the adult mammalian heart

BACKGROUND: Coronary artery disease and ischemic cardiomyopathy represent the leading cause of heart failure and continue to grow at exponential rates. Despite widespread availability of coronary bypass surgery and percutaneous coronary intervention, subsequent ischemic events and progression to heart failure continue to be common occurrences. Previous studies have shown that a subgroup of patients develop collateral blood vessels that serve to connect patent and occluded arteries and restore perfusion to ischemic territories. The presence of coronary collaterals has been correlated with improved clinical outcomes; however, the molecular mechanisms governing this process remain largely unknown. METHODS AND RESULTS: To date, no mouse models of coronary arterial growth have been described. Using a closed‐chest model of myocardial ischemia, we have demonstrated that brief episodes of repetitive ischemia are sufficient to promote the growth of both large coronary arteries and the microvasculature. Induction of large coronary artery and microvascular growth resulted in improvements in myocardial perfusion after prolonged ischemia and protected from subsequent myocardial infarction. We further show that repetitive ischemia did not lead to increased expression of classic proangiogenic factors but instead resulted in activation of the innate immune system and recruitment of macrophages to growing blood vessels. CONCLUSIONS: These studies describe a novel model of coronary angiogenesis and implicate the cardiac macrophage as a potential mediator of ischemia‐driven coronary growth

Left ventricular and aortic dysfunction in cystic fibrosis mice

AbstractBackgroundLeft ventricular (LV) abnormalities have been reported in cystic fibrosis (CF); however, it remains unclear if loss of cystic fibrosis transmembrane conductance regulator (CFTR) function causes heart defects independent of lung disease.MethodsUsing gut-corrected F508del CFTR mutant mice (ΔF508), which do not develop human lung disease, we examined in vivo heart and aortic function via 2D transthoracic echocardiography and LV catheterization.ResultsΔF508 mouse hearts showed LV concentric remodeling along with enhanced inotropy (increased +dP/dt, fractional shortening, decreased isovolumetric contraction time) and greater lusitropy (−dP/dt, Tau). Aortas displayed increased stiffness and altered diastolic flow. β-adrenergic stimulation revealed diminished cardiac reserve (attenuated +dP/dt,−dP/dt, LV pressure).ConclusionsIn a mouse model of CF, CFTR mutation leads to LV remodeling with alteration of cardiac and aortic functions in the absence of lung disease. As CF patients live longer, more active lives, their risk for cardiovascular disease should be considered

Noninvasive quantitation of myocardial blood flow in human subjects with oxygen-15-labeled water and positron emission tomography

AbstractNoninvasive measurement of myocardial blood flow in absolute terms (i.e., milliliters per gram per min) has been difficult to accomplish despite the intrinsically quantitative power of positron emission tomography because of the nonphysiologic nature of tracers that have been employed conventionally as well as the limited spatial resolution of currently available instruments. It was previously demonstrated that myocardial blood flow in animals can be quantitated accurately with the diffusible tracer oxygen-15-labeled water (112[50) when the arterial input function and myocardial radiotracer concentration were measured directly. To extend the approach for completely noninvasive measurement of blood flow, a parameter estimation procedure was developed whereby effects of limited tomographic spatial resolution and cardiac motion were compensated for within the operational flow model.In validation studies in 18 dogs, myocardial blood flow measured with positron emission tomography after intravenously administered 112'S0 correlated closely with flow measured with concomitantly administered radiolabeled microspheres over the range of 0.29 to 5.04 ml/g per min (r = 0.95). Although regional ischemia was clearly identifiable tomographically, absolute flow could not be determined accurately in ischemic regions in four dogs because of poor count statistics related to wall thinning. Subsequently, myocardial blood flow was measured in 11 normal human subjects. Flow was homogeneous throughout the myocardium, averaged 0.90 t 0.22 ml/g per min at rest and increased to 3.551.15 ml/g per min after intravenous administration of dipyridamole.Therefore, positron emission tomography with H215O and the approach developed permits noninvasive measurement of myocardial blood flow in absolute terms in humans and should facilitate objective assessment of interventions designed to enhance nutritive perfusion

Autophagy is impaired in cardiac ischemia-reperfusion injury

Accumulating evidence attests to a prosurvival role for autophagy under stress, by facilitating removal of damaged proteins and organelles and recycling basic building blocks, which can be utilized for energy generation and targeted macromolecular synthesis to shore up cellular defenses. These observations are difficult to reconcile with the dichotomous prosurvival and death-inducing roles ascribed to macroautophagy in cardiac ischemia and reperfusion injury, respectively. A careful reexamination of ‘flux’ through the macroautophagy pathway reveals that autophagosome clearance is markedly impaired with reperfusion (reoxygenation) in cardiomyocytes following an ischemic (hypoxic) insult, resulting from reactive oxygen species (ROS)-mediated decline in LAMP2 and increase in BECN1 abundance. This results in impaired autophagy that is ‘ineffective’ in protecting against cell death with ischemia-reperfusion injury. Restoration of autophagosome clearance and by inference, ‘adequate’ autophagy, attenuates reoxygenation-induced cell death

Necrotic myocardial cells release damage-associated molecular patterns that provoke fibroblast activation in vitro and trigger myocardial inflammation and fibrosis in vivo

BACKGROUND: Tissue injury triggers inflammatory responses that promote tissue fibrosis; however, the mechanisms that couple tissue injury, inflammation, and fibroblast activation are not known. Given that dying cells release proinflammatory “damage-associated molecular patterns” (DAMPs), we asked whether proteins released by necrotic myocardial cells (NMCs) were sufficient to activate fibroblasts in vitro by examining fibroblast activation after stimulation with proteins released by necrotic myocardial tissue, as well as in vivo by injecting proteins released by necrotic myocardial tissue into the hearts of mice and determining the extent of myocardial inflammation and fibrosis at 72 hours. METHODS AND RESULTS: The freeze–thaw technique was used to induce myocardial necrosis in freshly excised mouse hearts. Supernatants from NMCs contained multiple DAMPs, including high mobility group box-1 (HMGB1), galectin-3, S100β, S100A8, S100A9, and interleukin-1α. NMCs provoked a significant increase in fibroblast proliferation, α–smooth muscle actin activation, and collagen 1A1 and 3A1 mRNA expression and significantly increased fibroblast motility in a cell-wounding assay in a Toll-like receptor 4 (TLR4)- and receptor for advanced glycation end products–dependent manner. NMC stimulation resulted in a significant 3- to 4-fold activation of Akt and Erk, whereas pretreatment with Akt (A6730) and Erk (U0126) inhibitors decreased NMC-induced fibroblast proliferation dose-dependently. The effects of NMCs on cell proliferation and collagen gene expression were mimicked by several recombinant DAMPs, including HMGB1 and galectin-3. Moreover, immunodepletion of HMGB1 in NMC supernatants abrogated NMC-induced cell proliferation. Finally, injection of NMC supernatants or recombinant HMGB1 into the heart provoked increased myocardial inflammation and fibrosis in wild-type mice but not in TLR4-deficient mice. CONCLUSIONS: These studies constitute the initial demonstration that DAMPs released by NMCs induce fibroblast activation in vitro, as well as myocardial inflammation and fibrosis in vivo, at least in part, through TLR4-dependent signaling

Cardiac myocyte-specific knock-out of calcium-independent phospholipase A2γ (iPLA2γ) decreases oxidized fatty acids during ischemia/reperfusion and reduces infarct size

Calcium-independent phospholipase A(2)γ (iPLA(2)γ) is a mitochondrial enzyme that produces lipid second messengers that facilitate opening of the mitochondrial permeability transition pore (mPTP) and contribute to the production of oxidized fatty acids in myocardium. To specifically identify the roles of iPLA(2)γ in cardiac myocytes, we generated cardiac myocyte-specific iPLA(2)γ knock-out (CMiPLA(2)γKO) mice by removing the exon encoding the active site serine (Ser-477). Hearts of CMiPLA(2)γKO mice exhibited normal hemodynamic function, glycerophospholipid molecular species composition, and normal rates of mitochondrial respiration and ATP production. In contrast, CMiPLA(2)γKO mice demonstrated attenuated Ca(2+)-induced mPTP opening that could be rapidly restored by the addition of palmitate and substantially reduced production of oxidized polyunsaturated fatty acids (PUFAs). Furthermore, myocardial ischemia/reperfusion (I/R) in CMiPLA(2)γKO mice (30 min of ischemia followed by 30 min of reperfusion in vivo) dramatically decreased oxidized fatty acid production in the ischemic border zones. Moreover, CMiPLA(2)γKO mice subjected to 30 min of ischemia followed by 24 h of reperfusion in vivo developed substantially less cardiac necrosis in the area-at-risk in comparison with their WT littermates. Furthermore, we found that membrane depolarization in murine heart mitochondria was sensitized to Ca(2+) by the presence of oxidized PUFAs. Because mitochondrial membrane depolarization and calcium are known to activate iPLA(2)γ, these results are consistent with salvage of myocardium after I/R by iPLA(2)γ loss of function through decreasing mPTP opening, diminishing production of proinflammatory oxidized fatty acids, and attenuating the deleterious effects of abrupt increases in calcium ion on membrane potential during reperfusion

Regulation of the transcription factor EB-PGC1α axis by beclin-1 controls mitochondrial quality and cardiomyocyte death under stress

In cardiac ischemia-reperfusion injury, reactive oxygen species (ROS) generation and upregulation of the hypoxia-inducible protein BNIP3 result in mitochondrial permeabilization, but impairment in autophagic removal of damaged mitochondria provokes programmed cardiomyocyte death. BNIP3 expression and ROS generation result in upregulation of beclin-1, a protein associated with transcriptional suppression of autophagy-lysosome proteins and reduced activation of transcription factor EB (TFEB), a master regulator of the autophagy-lysosome machinery. Partial beclin-1 knockdown transcriptionally stimulates lysosome biogenesis and autophagy via mTOR inhibition and activation of TFEB, enhancing removal of depolarized mitochondria. TFEB activation concomitantly stimulates mitochondrial biogenesis via PGC1α induction to restore normally polarized mitochondria and attenuate BNIP3- and hypoxia-reoxygenation-induced cell death. Conversely, overexpression of beclin-1 activates mTOR to inhibit TFEB, resulting in declines in lysosome numbers and suppression of PGC1α transcription. Importantly, knockdown of endogenous TFEB or PGC1α results in a complete or partial loss, respectively, of the cytoprotective effects of partial beclin-1 knockdown, indicating a critical role for both mitochondrial autophagy and biogenesis in ensuring cellular viability. These studies uncover a transcriptional feedback loop for beclin-1-mediated regulation of TFEB activation and implicate a central role for TFEB in coordinating mitochondrial autophagy with biogenesis to restore normally polarized mitochondria and prevent ischemia-reperfusion-induced cardiomyocyte death

TNF receptor-activated factor 2 mediates cardiac protection through noncanonical NF-κB signaling

To elucidate the mechanisms responsible for cytoprotective effects of TNF receptor-activated factor 2 (TRAF2) in the heart, we employed genetic gain- and loss-of-function studies ex vivo and in vivo in mice with cardiac-restricted overexpression of TRAF2 (Myh6-TRAF2LC). Crossing Myh6-TRAF2LC mice with mice lacking canonical signaling (Myh6-TRAF2LC/Myh6-IκBαΔN) abrogated the cytoprotective effects of TRAF2 ex vivo. In contrast, inhibiting the JAK/STAT pathway did not abrogate the cytoprotective effects of TRAF2. Transcriptional profiling of WT, Myh6-TRAF2LC, and Myh6-TRAF2LC/Myh6-IκBαΔN mouse hearts suggested that the noncanonical NF-κB signaling pathway was upregulated in the Myh6-TRAF2LC mouse hearts. Western blotting and ELISA for the NF-κB family proteins p50, p65, p52, and RelB on nuclear and cytoplasmic extracts from naive 12-week-old WT, Myh6-TRAF2LC, and Myh6-TRAF2LC/Myh6-IκBαΔN mouse hearts showed increased expression levels and increased DNA binding of p52 and RelB, whereas there was no increase in expression or DNA binding of the p50 and p65 subunits. Crossing Myh6-TRAF2LC mice with RelB-/+ mice (Myh6-TRAF2LC/RelB-/+) attenuated the cytoprotective effects of TRAF2 ex vivo and in vivo. Viewed together, these results suggest that crosstalk between the canonical and noncanonical NF-κB signaling pathways is required for mediating the cytoprotective effects of TRAF2