D. Paul Thomas

The effect of pedaling frequency on glycogen depletion rates in type I and type II quadriceps muscle fibers during submaximal cycling exercise

SummaryThis study was conducted to determine whether the pedaling frequency of cycling at a constant metabolic cost contributes to the pattern of fiber-type glycogen depletion. On 2 separate days, eight men cycled for 30 min at approximately 85% of individual aerobic capacity at pedaling frequencies of either 50 or 100 rev·min−1. Muscle biopsy samples (vastus lateralis) were taken immediately prior to and after exercise. Individual fibers were classified as type I (slow twitch), or type II (fast twitch), using a myosin adenosine triphosphatase stain, and their glycogen content immediately prior to and after exercise quantified via microphotometry of periodic acid-Schiff stain. The 30-min exercise bout resulted in a 46% decrease in the mean optical density (D) of type I fibers during the 50 rev·min−1 condition [0.52 (0.07) to 0.28 (0.04)D units; mean (SEM)] which was not different (P>0.05) from the 35% decrease during the 100 rev · min−1 condition [0.48 (0.04) to 0.31 (0.05)D units]. In contrast, the meanD in type II fibers decreased 49% during the 50 rev·min−1 condition [0.53 (0.06) to 0.27 (0.04) units]. This decrease was greater (P<0.05) than the 33% decrease observed in the 100 rev·min −1 condition [0.48 (0.04) to 0.32 (0.06) units). In conclusion, cycling at the same metabolic cost at 50 rather than 100 rev·min−1 results in greater type II fiber glycogen depletion. This is attributed to the increased muscle force required to meet the higher resistance per cycle at the lower pedal frequency. These data are consistent with the view that force development as opposed to velocity of contraction determines the degree of type II fiber recruitment when the metabolic cost of exercise is held constant.

Impaired macrophage migration inhibitory factor-AMP-activated protein kinase activation and ischemic recovery in the senescent heart.

Background— Elderly patients are more sensitive than younger patients to myocardial ischemia, which results in higher mortality. We investigated how aging affects the cardioprotective AMP-activated protein kinase (AMPK) signaling pathway. Methods and Results— Ischemic AMPK activation was impaired in aged compared with young murine hearts. The expression and secretion of the AMPK upstream regulator, macrophage migration inhibitory factor (MIF), were lower in aged compared with young adult hearts. Additionally, the levels of hypoxia-inducible factor 1&agr;, a known transcriptional activator of MIF, were reduced in aged compared with young hearts. Ischemia-induced AMPK activation in MIF knockout mice was blunted, leading to greater contractile dysfunction in MIF-deficient than in wild-type hearts. Furthermore, intramyocardial injection of adenovirus encoding MIF in aged mice increased MIF expression and ischemic AMPK activation and reduced infarct size. Conclusions— An impaired MIF-AMPK activation response in senescence thus may be attributed to an aging-associated defect in hypoxia-inducible factor 1&agr;, the transcription factor for MIF. In the clinical setting, impaired cardiac hypoxia-inducible factor 1&agr; activation and consequent reduced MIF expression may play an important role in the increased susceptibility to myocardial ischemia observed in older cardiac patients.

Hypertrophic cardiomyopathy in high-fat diet-induced obesity: role of suppression of forkhead transcription factor and atrophy gene transcription

Cellular hypertrophy is regulated by coordinated pro- and antigrowth machineries. Foxo transcription factors initiate an atrophy-related gene program to counter hypertrophic growth. This study was designed to evaluate the role of Akt, the forkhead transcription factor Foxo3a, and atrophy genes muscle-specific RING finger (MuRF)-1 and atrogin-1 in cardiac hypertrophy and contractile dysfunction associated with high-fat diet-induced obesity. Mice were fed a low- or high-fat diet for 6 mo along with a food-restricted high-fat weight control group. Echocardiography revealed decreased fractional shortening and increased end-systolic diameter and cardiac hypertrophy in high-fat obese but not in weight control mice. Cardiomyocytes from high-fat obese but not from weight control mice displayed contractile and intracellular Ca2+ defects including depressed maximal velocity of shortening/relengthening, prolonged duration of shortening/relengthening, and reduced intracellular Ca2+ rise and clearance. Caspase activities were greater in high-fat obese but not in weight control mouse hearts. Western blot analysis revealed enhanced basal Akt and Foxo3a phosphorylation and reduced insulin-stimulated phosphorylation of Akt and Foxo3a without changes in total protein expression of Akt and Foxo3a in high-fat obese hearts. RT-PCR and immunoblotting results displayed reduced levels of the atrogens atrogin-1 and MuRF-1, the upregulated hypertrophic markers GATA4 and ciliary neurotrophic factor receptor-alpha, as well as the unchanged calcineurin and proteasome ubiquitin in high-fat obese mouse hearts. Transfection of H9C2 myoblast cells with dominant-negative Foxo3a adenovirus mimicked palmitic acid (0.8 mM for 24 h)-induced GATA4 upregulation without an additive effect. Dominant-negative Foxo3a-induced upregulation of pAkt and repression of phosphatase and tensin homologue were abrogated by palmitic acid. These results suggest a cardiac hypertrophic response in high-fat diet-associated obesity at least in part through inactivation of Foxo3a by the Akt pathway.

Earliest changes in the left ventricular transcriptome post-myocardial infarction

We report a genome-wide survey of early responses of the mouse heart transcriptome to acute myocardial infarction (AMI). For three regions of the left ventricle (LV), namely, ischemic/infarcted tissue (IF), the surviving LV free wall (FW), and the interventricular septum (IVS), 36,899 transcripts were assayed at six time points from 15 min to 48 h post-AMI in both AMI and sham surgery mice. For each transcript, temporal expression patterns were systematically compared between AMI and sham groups, which identified 515 AMI-responsive genes in IF tissue, 35 in the FW, 7 in the IVS, with three genes induced in all three regions. Using the literature, we assigned functional annotations to all 519 nonredundant AMI-induced genes and present two testable models for central signaling pathways induced early post-AMI. First, the early induction of 15 genes involved in assembly and activation of the activator protein-1 (AP-1) family of transcription factors implicates AP-1 as a dominant regulator of earliest post-ischemic molecular events. Second, dramatic increases in transcripts for arginase 1 (ARG1), the enzymes of polyamine biosynthesis, and protein inhibitor of nitric oxide synthase (NOS) activity indicate that NO production may be regulated, in part, by inhibition of NOS and coordinate depletion of the NOS substrate, L-arginine. ARG1 was the single-most highly induced transcript in the database (121-fold in IF region) and its induction in heart has not been previously reported.

Intra-myocardial delivery of mesenchymal stem cells ameliorates left ventricular and cardiomyocyte contractile dysfunction following myocardial infarction.

Although mesenchymal stem cells (MSCs) transplantation may improve the overall heart function, the heterogeneity of myocardial cells makes it difficult to determine the nature of cells benefited from transplantation. This study evaluated the effect of intra-myocardial MSC transplantation on myocardial function following MI. Enhanced green fluorescent protein (EGFP)-expressing donor MSCs from C57BL/6-Tg (UBC-GFP) 30Scha/J mice were transplanted into LV free wall in the region bordering an infarct in C57 recipient mice following ligation of left main coronary artery (MI+MSC group). Ten days after MI, LV function was assessed using echocardiography. Cardiomyocyte contractility and intracellular Ca(2+) transients were measured in cells from the area-at-risk surrounding the infarct scar. The EGFP donor cells were traced in the MSC recipient mice using fluorescence microscopy. TUNEL, H&E and Masson trichrome staining were used to assess apoptosis, angiogenesis and myocardial fibrosis, respectively. MI dilated LV as evidenced by increased end-diastolic and end-systolic diameters. MI significantly reduced fractional shortening, cardiomyocyte peak shortening, and maximal velocity of shortening and relengthening, all of which were attenuated or abrogated by MSC therapy. MI also reduced resting intracellular Ca(2+), intracellular Ca(2+) rise and decay rate, which were reconciled by MSC. MSC therapy attenuated MI-induced apoptosis and decreased angiogenesis but not myocardial fibrosis in the peri-infarct area. Taken together, our results demonstrated that MSC therapy significantly improved both LV and cardiomyocyte function possibly associated with its beneficial role in apoptosis and angiogenesis, indicating a key role for cardiomyocytes in stem cell tissue engineering.

Alterations in small arterioles precede changes in limb skeletal muscle after myocardial infarction

We tested the hypothesis that alterations in arterioles in locomotor skeletal muscles in rats with myocardial infarction (MI), but before development of congestive heart failure (CHF), precede structural and functional changes commonly observed in limb muscle in association with CHF. Resting diameters of third- (A3) and fourth-order arterioles (A4) in extensor digitorum longus (EDL) muscle were significantly smaller in rats with nonfailing small and medium-sized MI compared with control animals. Dilation of A4 in response to 10(-4) M adenosine was significantly attenuated in both groups (P < 0.05), whereas dilation of A3 was unaltered. Microvessels from both groups of infarcted rats constricted to all doses of acetylcholine (10(-9), 10(-8), and 10(-7) M) and showed a significantly exaggerated vasoconstrictor response to norepinephrine (10(-9), 10(-8), and 10(-7) M) compared with microvessels in control rats (P < 0.05). Peak isometric tension of combined tibialis anterior and EDL muscles and muscle fatigue (final/peak tension x 100), measured during 5-min isometric supramaximal twitch contractions at 4 Hz, were similar in control and MI rats (218 +/- 7 vs. 213 +/- 15 g/g muscle and 52 +/- 1 vs. 51 +/- 9%, respectively; n = 5 for both). There was also no difference with respect to the proportion of oxidative fibers or capillary-to-fiber ratios. Our results indicate that, in rats with left ventricular dysfunction but without failure, decreased diameter and perturbations in reactivity of small arterioles precede alterations in skeletal muscle performance often seen at a later date in association with CHF. These findings are consistent with the notion of aberrant endothelial and smooth muscle function and may contribute to the maintenance of blood pressure after MI but before CHF.We tested the hypothesis that alterations in arterioles in locomotor skeletal muscles in rats with myocardial infarction (MI), but before development of congestive heart failure (CHF), precede structural and functional changes commonly observed in limb muscle in association with CHF. Resting diameters of third- (A3) and fourth-order arterioles (A4) in extensor digitorum longus (EDL) muscle were significantly smaller in rats with nonfailing small and medium-sized MI compared with control animals. Dilation of A4 in response to 10-4 M adenosine was significantly attenuated in both groups ( P < 0.05), whereas dilation of A3 was unaltered. Microvessels from both groups of infarcted rats constricted to all doses of acetylcholine (10-9, 10-8, and 10-7 M) and showed a significantly exaggerated vasoconstrictor response to norepinephrine (10-9, 10-8, and 10-7 M) compared with microvessels in control rats ( P < 0.05). Peak isometric tension of combined tibialis anterior and EDL muscles and muscle fatigue (final/peak tension × 100), measured during 5-min isometric supramaximal twitch contractions at 4 Hz, were similar in control and MI rats (218 ± 7 vs. 213 ± 15 g/g muscle and 52 ± 1 vs. 51 ± 9%, respectively; n = 5 for both). There was also no difference with respect to the proportion of oxidative fibers or capillary-to-fiber ratios. Our results indicate that, in rats with left ventricular dysfunction but without failure, decreased diameter and perturbations in reactivity of small arterioles precede alterations in skeletal muscle performance often seen at a later date in association with CHF. These findings are consistent with the notion of aberrant endothelial and smooth muscle function and may contribute to the maintenance of blood pressure after MI but before CHF.

Endurance exercise accelerates myocardial tissue oxygenation recovery and reduces ischemia reperfusion injury in mice.

Exercise training offers cardioprotection against ischemia and reperfusion (I/R) injury. However, few essential signals have been identified to underscore the protection from injury. In the present study, we hypothesized that exercise-induced acceleration of myocardial tissue oxygenation recovery contributes to this protection. C57BL/6 mice (4 weeks old) were trained on treadmills for 45 min/day at a treading rate of 15 m/min for 8 weeks. At the end of 8-week exercise training, mice underwent 30-min left anterior descending coronary artery occlusion followed by 60-min or 24-h reperfusion. Electron paramagnetic resonance oximetry was performed to measure myocardial tissue oxygenation. Western immunoblotting analyses, gene transfection, and myography were examined. The oximetry study demonstrated that exercise markedly shortened myocardial tissue oxygenation recovery time following reperfusion. Exercise training up-regulated Kir6.1 protein expression (a subunit of ATP-sensitive K+ channel on vascular smooth muscle cells, VSMC sarc-KATP) and protected the heart from I/R injury. In vivo gene transfer of dominant negative Kir6.1AAA prolonged the recovery time and enlarged infarct size. In addition, transfection of Kir6.1AAA increased the stiffness and reduced the relaxation capacity in the vasculature. Together, our study demonstrated that exercise training up-regulated Kir6.1, improved tissue oxygenation recovery, and protected the heart against I/R injury. This exercise-induced cardioprotective mechanism may provide a potential therapeutic intervention targeting VSMC sarc-KATP channels and reperfusion recovery.

Exercise training post-MI favorably modifies heart extracellular matrix in the rat.

PURPOSE In order to assess the effect of daily exercise on extracellular matrix remodeling in the heart after myocardial infarction (MI), we measured collagen concentration (%COL) and nonreducible collagen cross-linking (hydroxylysylpyridinoline, HP) in the right ventricle (RV), and regionally within the infarcted (INF) and viable left ventricular free wall (LVF) and septum (LVS), using a rodent MI training model. METHODS Infarcts (19%-24% of LV) were surgically induced in adult rats that were assigned to either trained (MI-TR) or sedentary (MI-SED) groups and compared to sham-surgery sedentary controls (SHAM). RESULTS In LVF, 10 wk of treadmill running had no effect on the increase (P < 0.001) in %COL seen with MI (MI-SED = 7.14% ± 0.15%, MI-TR = 7.61% ± 0.19%, SHAM = 3.55% ± 0.19%). However, it normalized the increase (P < 0.05) in HP cross-linking (MI-SED = 0.43 ± 0.02, MI-TR = 0.27 ± 0.03, SHAM = 0.30 ± 0.04 mol HP·mol(-1) collagen). The INF scar in MI-SED rats showed a sevenfold increase in %COL (P < 0.001) compared to SHAM LVF myocardium, an increase that was attenuated by training (MI-SED = 26% ± 1% vs MI-TR = 21% ± 2%; P < 0.05). However, training had no effect on MI-induced increases in cross-linking in the INF scar (1.01 ± 0.22 vs 0.84 ± 0.14 mol HP·mol(-1) collagen). In LVS, although a small but significant increase in %COL was seen in both MI groups, HP cross-linking was unaltered compared to SHAM rats. Training also normalized the increase observed in cross-linking in RV after MI. CONCLUSIONS Because increased HP cross-linking in the heart is associated with decreased chamber compliance, these findings may help to explain the improved heart function seen after daily exercise in cardiac rehabilitation patients.

Frozen storage of ovine and rat tissues adversely affects lipoprotein lipase activity

Abstract The purpose of this study was to determine effects of frozen storage (frozen in liquid N, stored for 6 weeks at −80°C) of ovine adipose, and skeletal and cardiac muscle tissues on dietary induced changes in total (intra- and extracellular) lipoprotein lipase (LPL) activity previously determined in fresh tissue. Ewes were grown on either a low- (LE) or high-energy (HE) diet for 120 days (six per diet). In fresh tissue of HE ewes, LPL activity was higher (P

Caspase recruitment domain-containing protein 9 (CARD9) knockout reduces regional ischemia/reperfusion injury through an attenuated inflammatory response

Ischemic heart disease remains a leading cause of morbidity and mortality in the United States. Interventional reperfusion induces further damage to the ischemic myocardium through neutrophil infiltration and acute inflammation. As caspase recruitment domain-containing protein 9 (CARD9) plays a critical role in innate immune response and inflammation, we hypothesized that CARD9 knockout would provide protection against ischemia and reperfusion (I/R) injury through attenuation of acute inflammatory responses. C57BL/6 wild-type (WT) and CARD9-/- mice were subjected to 45 min left anterior descending (LAD) coronary artery occlusion followed by 24-h reperfusion. Area at risk (AAR) and infarct size were measured by Evans blue and triphenyltetrazolium chloride (TTC) staining. Frozen heart sections were stained with anti-mouse GR-1 antibody to detect infiltrated neutrophils. Concentrations of cytokines/chemokines TNF-α, IL-6, CXCL-1 and MCP-1 were determined in heart tissue homogenate and serum by ELISA assay. Western immunoblotting analyses were performed to measure the phosphorylation of p38 MAPK. Our results indicate that following I/R, infarct size was significantly smaller in CARD9-/- mice compared to WT. The number of infiltrated neutrophils was significantly lower in CARD9-/- mice compared to WT. Levels of TNF-α, IL-6, CXCL-1 and MCP-1 were significantly reduced in heart tissue and serum from CARD9-/- mice compared to WT. CARD9-/- mice also exhibited significantly lower levels of phosphorylated p38 MAPK. Taken together, our results suggest that CARD9 knockout protects the heart from ischemia/reperfusion (I/R) injury, possibly through reduction of neutrophil infiltration and attenuation of CARD9-associated acute inflammatory signaling.