Katrina Go Yamazaki

Short- and long-term effects of (−)-epicatechin on myocardial ischemia-reperfusion injury

Epidemiological studies have shown a correlation between flavonoid-rich diets and improved cardiovascular prognosis. Cocoa contains large amounts of flavonoids, in particular flavanols (mostly catechins and epicatechins). Flavonoids possess pleiotropic properties that may confer protective effects to tissues during injury. We examined the ability of epicatechin to reduce short-and long-term ischemia-reperfusion (I/R) myocardial injury. Epicatechin (1 mg.kg(-1).day(-1)) pretreatment (Tx) was administered daily via oral gavage to male rats for 2 or 10 days. Controls received water. Ischemia was induced via a 45-min coronary occlusion. Reperfusion was allowed until 48 h or 3 wk while Tx continued. We measured infarct (MI) size (%), hemodynamics, myeloperoxidase activity, tissue oxidative stress, and matrix metalloproteinase-9 (MMP-9) activity in 48-h groups. Cardiac morphometry was also evaluated in 3-wk groups. With 2 days of Tx, no reductions in MI size occurred. After 10 days, a significant approximately 50% reduction in MI size occurred. Epicatechin rats demonstrated no significant changes in hemodynamics. Tissue oxidative stress was reduced significantly in the epicatechin group vs. controls. MMP-9 activity demonstrated limited increases in the infarct region with epicatechin. By 3 wk, a significant 32% reduction in infarct size was observed with Tx, accompanied with sustained hemodynamics and preserved chamber morphometry. In conclusion, epicatechin Tx confers cardioprotection in the setting of I/R injury. The effects are independent of changes in hemodynamics, are sustained over time, and are accompanied by reduced levels of indicators of tissue injury. Results warrant the evaluation of cocoa flavanols as possible therapeutic agents to limit ischemic injury.

A Novel Mechanism Involving Four-and-a-half LIM Domain Protein-1 and Extracellular Signal-regulated Kinase-2 Regulates Titin Phosphorylation and Mechanics

Background: Titin is critical for cardiac muscle function; however, limited knowledge exists of mechanisms important for its regulation. Results: A four-and-a-half LIM domain protein-1/extracellular signal-regulated kinase-2-associated complex modulates titin-N2B levels, phosphorylation, and mechanics. Conclusion: We reveal new mechanisms underlying titin mechano-signaling. Significance: We advance our understanding of how titin-associated complexes/mutations can impact cardiac muscle function and disease. Understanding mechanisms underlying titin regulation in cardiac muscle function is of critical importance given recent compelling evidence that highlight titin mutations as major determinants of human cardiomyopathy. We previously identified a cardiac biomechanical stress-regulated complex at the cardiac-specific N2B region of titin that includes four-and-a-half LIM domain protein-1 (Fhl1) and components of the mitogen-activated protein signaling cascade, which impacted muscle compliance in Fhl1 knock-out cardiac muscle. However, direct regulation of these molecular components in mediating titin N2B function remained unresolved. Here we identify Fhl1 as a novel negative regulator of titin N2B levels and phosphorylation-mediated mechanics. We specifically identify titin N2B as a novel substrate of extracellular signal regulated-kinase-2 (Erk2) and demonstrate that Fhl1 directly interferes with Erk2-mediated titin-N2B phosphorylation. We highlight the critical region in titin-N2B that interacts with Fhl1 and residues that are dependent on Erk2-mediated phosphorylation in situ. We also propose a potential mechanism for a known titin-N2B cardiomyopathy-causing mutation that involves this regulatory complex. These studies shed light on a novel mechanism regulating titin-N2B mechano-signaling as well as suggest that dysfunction of these pathways could be important in cardiac disease states affecting muscle compliance.

Effects of (-)-epicatechin on myocardial infarct size and left ventricular remodeling after permanent coronary occlusion.

OBJECTIVES We examined the effects of the flavanol (-)-epicatechin on short- and long-term infarct size and left ventricular (LV) structure and function after permanent coronary occlusion (PCO) and the potential involvement of the protective protein kinase B (AKT)/extracellular signal-related kinase (ERK) signaling pathways. BACKGROUND (-)-epicatechin reduces blood pressure in hypertensive patients and limits infarct size in animal models of myocardial ischemia-reperfusion injury. However, nothing is known about its effects on infarction after PCO. METHODS (-)-epicatechin (1 mg/kg daily) treatment was administered via oral gavage to 250 g male rats for 10 days before PCO and was continued afterward. The PCO controls received water. Sham animals underwent thoracotomy and treatment in the absence of PCO. Immunoblots assessed AKT/ERK involvement 2 h after PCO. The LV morphometric features and function were measured 48 h and 3 weeks after PCO. RESULTS In the 48-h group, treatment reduced infarct size by 52%. There were no differences in hemodynamics among the different groups (heart rate and aortic and LV pressures). Western blots revealed no differences in AKT or ERK phosphorylation levels. At 3 weeks, PCO control animals demonstrated significant increases in LV end-diastolic pressure, heart and body weight, and LV chamber diameter versus sham. The PCO plus (-)-epicatechin group values were comparable with those of the sham plus (-)-epicatechin group. Treatment resulted in a 33% decrease in myocardial infarction size. The LV pressure-volume curves demonstrated a right shift in control PCO animals, whereas the (-)-epicatechin curves were comparable with those of the sham group. The LV scar area strains were significantly improved with (-)-epicatechin. CONCLUSIONS These results demonstrate the unique capacity of (-)-epicatechin to confer cardioprotection in the setting of a severe form of myocardial ischemic injury. Protection is sustained over time and preserves LV structure and function. The cardioprotective mechanism(s) of (-)-epicatechin seem to be unrelated to AKT or ERK activation. (-)-epicatechin warrants further investigation as a cardioprotectant.

Changes in regional myocardial volume during the cardiac cycle: implications for transmural blood flow and cardiac structure

Although previous studies report a reduction in myocardial volume during systole, myocardial volume changes during the cardiac cycle have not been quantitatively analyzed with high spatiotemporal resolution. We studied the time course of myocardial volume in the anterior mid-left ventricular (LV) wall of normal canine heart in vivo (n = 14) during atrial or LV pacing using transmurally implanted markers and biplane cineradiography (8 ms/frame). During atrial pacing, there was a significant transmural gradient in maximum volume decrease (4.1, 6.8, and 10.3% at subepi, midwall, and subendo layer, respectively, P = 0.002). The rate of myocardial volume increase during diastole was 4.7 +/- 5.8, 6.8 +/- 6.1, and 10.8 +/- 7.7 ml.min(-1).g(-1), respectively, which is substantially larger than the average myocardial blood flow in the literature measured by the microsphere method (0.7-1.3 ml.min(-1).g(-1)). In the early activated region during LV pacing, myocardial volume began to decrease before the LV pressure upstroke. We conclude that the volume change is greater than would be estimated from the known average transmural blood flow. This implies the existence of blood-filled spaces within the myocardium, which could communicate with the ventricular lumen. Our data in the early activated region also suggest that myocardial volume change is caused not by the intramyocardial tissue pressure but by direct impingement of the contracting myocytes on the microvasculature.

Intravenous (−)-epicatechin reduces myocardial ischemic injury by protecting mitochondrial function☆

BACKGROUND Targeting the mitochondria during ischemia/reperfusion (IR) can confer cardioprotection leading to improved clinical outcomes. The cardioprotective potential of (-)-epicatechin (EPI) during IR via modulation of mitochondrial function was evaluated. METHODS AND RESULTS Ischemia was induced in rats via a 45 min occlusion of the left anterior descending coronary artery followed by 1 h, 48 h, or 3 week reperfusion. EPI (10 mg/kg) was administered IV 15 min prior to reperfusion for the single dose group and again 12 h later for the double dose group. Controls received water. Experiments also utilized cultured neonatal rat ventricular myocytes (NRVM) and myoblasts. A single dose of EPI reduced infarct size by 27% at 48 h and 28% at 3 week. Double dose treatment further decreased infarct size by 80% at 48 h, and 52% by 3 weeks. The protective effect of EPI on mitochondrial function was evident after 1h of reperfusion when mitochondria demonstrated less respiratory inhibition, lower mitochondrial Ca2+ load, and a preserved pool of NADH that correlated with higher tissue ATP levels. Mechanistic studies in NRVM revealed that EPI acutely stimulated maximal rates of respiration, an effect that was blocked by inhibitors of the mitochondrial pyruvate carrier, nitric oxide synthase, or soluble guanylyl cyclase. In myoblasts, knockdown of components of the mitochondrial pyruvate carrier blocked EPI-induced respiratory stimulation. CONCLUSIONS IV EPI confers cardioprotection via preservation of mitochondrial function potentially through enhanced substrate provision. These provocative results document a novel mechanism of a natural product with potential clinical utility.

Co-administration of the flavanol (-)-epicatechin with doxycycline synergistically reduces infarct size in a model of ischemia reperfusion injury by inhibition of mitochondrial swelling

(-)-Epicatechin (EPI) is cardioprotective in the setting of ischemia/reperfusion (IR) injury and doxycycline (DOX) is known to preserve cardiac structure/function after myocardial infarction (MI). The main objective of this study was to examine the effects of EPI and DOX co-administration on MI size after IR injury and to determine if cardioprotection may involve the mitigation of mitochondrial swelling. For this purpose, a rat model of IR was used. Animals were subjected to a temporary 45 min occlusion of the left anterior descending coronary artery. Treatment consisted of a single or double dose of EPI (10 mg/kg) combined with DOX (5 mg/kg). The first dose was given 15 min prior to reperfusion and the second 12 h post-MI. The effects of EPI +/- DOX on mitochondrial swelling (i.e. mPTP opening) were determined using isolated mitochondria exposed to calcium overload and data examined using isobolographic analysis. To ascertain for the specificity of EPI effects on mitochondrial swelling other flavonoids were also evaluated. Single dose treatment reduced MI size by ~46% at 48 h and 44% at three weeks. Double dosing evidenced a synergistic, 82% reduction at 3 weeks. EPI plus DOX also inhibited mitochondrial swelling in a synergic manner thus, possibly accounting for the cardioprotective effects whereas limited efficacy was observed with the other flavonoids. Given the apparent lack of toxicity in humans, the combination of EPI and DOX may have clinical potential for the treatment of myocardial IR injury.

Ventricular pacing-induced loss of contractile function and development of epicardial inflammation.

Perturbations in the normal sequence of ventricular activation can create regions of early and late activation, leading to dysynchronous contraction and areas of dyskinesis. Dyskinesis occurs across the left ventricular (LV) wall, and its presence may have important consequences on cardiac structure and function in normal and failing hearts. Acutely, dyskinesis can trigger inflammation and, in the long term (6 wk and above), leads to LV remodeling. The mechanisms that trigger these changes are unknown. To gain further insight, we used a canine model to evaluate transumural changes in myocardial function and inflammation induced by epicardial LV pacing. The results indicate that 4 h of LV suprathreshold pacing resulted in a 30% local loss of endocardial thickening. Assessment of neutrophil infiltration showed a significant approximately fivefold increase in myeloperoxidase activity in the epicardium versus the midwall/endocardium. Matrix metalloproteinase-9 activity increased ∼2 fold in the epicardium and ROS generation increased ∼2.5-fold compared with the midwall/endocardium. To determine the effects that electrical current alone has on these end points, a group of animals was subjected to subthreshold pacing. Significant increases were observed only in epicardial myeloperoxidase levels. Thus, the results indicate that transmural dyskinesis induced by suprathreshold epicardial LV activation triggers a localized epicardial inflammatory response, whereas subthreshold stimulation appears to solely induce the trapping of leucocytes. Suprathreshold pacing also induces a loss of endocardial function. These results may have important implications as to the nature of the mechanisms that trigger the inflammatory response and possibly long-term remodeling in the setting of dysynchrony.

Cell adhesion molecule mediation of myocardial inflammatory responses associated with ventricular pacing

Poorly synchronized activation of the ventricles can lead to impairment of normal cardiac structure/function. We reported previously that short term (4 h) left ventricular (LV) pacing-induced ventricular dyskinesis led to an inflammatory response localized to the epicardium. Results from this study demonstrated that neutrophils may play a major role in this inflammatory process. Neutrophil recruitment to a site of injury is a process that is highly dependent on an upregulation of cell adhesion molecules (CAM). The dependence of ventricular dysynchrony-induced inflammatory responses on CAM upregulation has not been explored. To gain further insight, we used a mouse model of LV pacing to evaluate the role of CAM in mediating the inflammatory response associated with ventricular dyskinesis. We first examined the effects of LV pacing in wild-type mice. Results demonstrate that 40 min of LV pacing increases ICAM-1 immunostaining as well as myeloperoxidase activity and tissue oxidative stress by twofold in early-activated myocardium. Matrix metalloproteinase-9 activity also increased in the same region by ∼3.5-fold. To determine the role of CAM, mice null for ICAM-1 or p-selectin were subjected to 40 min LV pacing. Results demonstrate that the inflammatory response seen in the wild-type mice was significantly mitigated in the ICAM-1 and p-selectin null mice. In conclusion, results demonstrate that CAM expression plays a critical role in the triggering of LV pacing-induced inflammation, thus providing evidence of a vascular mechanism underlying this response. The mechanisms that trigger an upregulation of myocardial CAM expression and, therefore, inflammation await further investigation since they suggest a specific involvement of vascular events.