Joshua M. Lynch

Susceptibility of the heart to ischaemia–reperfusion injury and exercise‐induced cardioprotection are sex‐dependent in the rat

The cardioprotective effects of short‐term exercise against myocardial ischaemia–reperfusion injury in male and female rats were examined. We subjected male and female rats to 0 (Sed; n= 8 males and 8 females), 1 (1 day; n= 10 males and 8 females), or 5 (5 day; n= 6 males and 6 females) days of treadmill running. Langendorff‐perfused hearts underwent 1 h of regional ischaemia and 2 h of reperfusion, and infarct size (expressed as a percentage of the zone at risk; ZAR), left ventricular pressure development, and coronary flow were measured for each heart. Preischaemic pressure development and coronary flow did not differ between the sexes nor were they influenced by exercise. Sed females had significantly smaller infarct sizes (25 ± 3%) than Sed male hearts (37 ± 3%; P < 0.001). Short‐term running significantly reduced infarct size following 1 day (27 ± 3%; P < 0.05) and 5 days (30 ± 4%; P < 0.10) of exercise in males. One day of running did not reduce infarct size in females (19 ± 3%; P= NS), but 5 day females did show a significant reduction in infarct size (13 ± 2%; P < 0.05). There was no relationship between postischaemic coronary vascular hyperaemia and infarct size across sexes or exercise training groups. Hearts from Sed females exhibited significantly higher manganese superoxide dismutase (MnSOD) protein expression than hearts from Sed males, but short‐term exercise (neither 1 nor 5 days) did not alter MnSOD protein in either sex. Increased sarcolemmal ATP‐sensitive K+ (KATP) channel subunit protein expression (SUR2A and/or Kir6.2) correlated closely with sex‐dependent and exercise‐acquired protection against myocardial infarction. These data indicate that: (1) sex‐dependent and exercise‐induced differences in the susceptibility of the heart to ischaemia–reperfusion injury are not associated with improved coronary flow or postischaemic hyperaemia; (2) increased MnSOD protein expression is not necessary for exercise‐induced protection from infarction; and (3) one possible mechanism for sex‐dependent and exercise‐mediated reductions in infarct size involves an increased protein expression of cardiac sarcolemmal KATP channels.

Cardioprotection afforded by chronic exercise is mediated by the sarcolemmal, and not the mitochondrial, isoform of the KATP channel in the rat

This study was conducted to examine the role of myocardial ATP‐sensitive potassium (KATP) channels in exercise‐induced protection from ischaemia–reperfusion (I–R) injury. Female rats were either sedentary (Sed) or exercised for 12 weeks (Tr). Hearts were excised and underwent a 1–2 h regional I–R protocol. Prior to ischaemia, hearts were subjected to pharmacological blockade of the sarcolemmal KATP channel with HMR 1098 (SedHMR and TrHMR), mitochondrial blockade with 5‐hydroxydecanoic acid (5HD; Sed5HD and Tr5HD), or perfused with buffer containing no drug (Sed and Tr). Infarct size was significantly smaller in hearts from Tr animals (35.4 ± 2.3 versus 44.7 ± 3.0% of the zone at risk for Tr and Sed, respectively). Mitochondrial KATP blockade did not abolish the training‐induced infarct size reduction (30.0 ± 3.4 versus 38.0 ± 2.6 in Tr5HD and Sed5HD, respectively); however, sarcolemmal KATP blockade completely eradicated the training‐induced cardioprotection. Infarct size was 71.2 ± 3.3 and 64.0 ± 2.4% of the zone at risk for TrHMR and Sed HMR. The role of sarcolemmal KATP channels in Tr‐induced protection was also supported by significant increases in both subunits of the sarcolemmal KATP channel following training. LV developed pressure was better preserved in hearts from Tr animals, and was not influenced by addition of HMR 1098. 5HD decreased pressure development regardless of training status, from 15 min of ischaemia through the duration of the protocol. This mechanical dysfunction was likely to be due to a 5HD‐induced increase in myocardial Ca2+ content following I–R. The major findings of the present study are: (1) unlike all other known forms of delayed cardioprotection, infarct sparing following chronic exercise was not abolished by 5HD; (2) pharmacological blockade of the sarcolemmal KATP channel nullified the cardioprotective benefits of exercise training; and (3) increased expression of sarcolemmal KATP channels was observed following chronic training.

PKC‐permitted elevation of sarcolemmal KATP concentration may explain female‐specific resistance to myocardial infarction

The female myocardium, relative to that of the male, exhibits sustained resistance to ischaemic tissue injury, a phenomenon termed sex‐specific cardioprotection (SSC). SSC is dependent upon the sarcolemmal KATP channel (sarcKATP), and protein kinase C (PKC). Here we investigate whether PKC‐mediated regulation of sarcKATP concentration can explain this endogenous form of protection. Hearts from male (M) and female (F) rats were Langendorff‐perfused for 30 min prior to either regional ischaemia–reperfusion (I/R), or global ischaemia (GISC). For both protocols, pre‐ischaemic blockade of PKC was achieved by chelerythrine (Chel) in male (M + C) and female (F + C) hearts. Additional female hearts underwent sarcKATP antagonism during I/R by HMR‐1098 (HMR), either alone or in combination with Chel (HMR + Chel). GISC hearts were fractionated to assess cellular distribution of PKCɛ and sarcKATP. Sex‐specific infarct resistance was apparent under control I/R (F, 23 ± 3%vs. M, 36 ± 4%, P < 0.05) and abolished by Chel (F + C, 36 ± 3%). Female infarct resistance was susceptible to sarcKATP blockade (Control, 16 ± 2%vs. HMR, 27 ± 3%), and PKC blockade had no additional effect (HMR + Chel, 26 ± 2%). The prevalence of Kir6.2 and SUR2 was higher in the sarcolemmal fractions of females (Kir6.2: F, 1.24 ± 0.07 vs. M, 1.02 ± 0.06; SUR2: F, 3.16 ± 0.22 vs. M, 2.45 ± 0.09; ratio units), but normalized by Chel (Kir6.2: F, 1.06 ± 0.07 vs. M, 0.99 ± 0.06; SUR2: F, 2.99 ± 0.09 vs. M, 2.82 ± 0.22, M; ratio units). Phosphorylation of sarcolemmal PKCɛ was reduced by Chel (p‐PKCɛ/PKCɛ: control, 0.43 ± 0.02; Chel, 0.29 ± 0.01; P < 0.01). We conclude that PKC‐mediated regulation of sarcKATP may account for the physiologically sustainable dependence of SSC upon both PKC and sarcKATP, and that this regulation involves PKC‐permitted enrichment of the female sarcolemma with sarcKATP. As such, the PKC‐sarcKATP axis may represent a target for sustainable prophylactic induction of cardioprotection.

Delta-6-desaturase Links Polyunsaturated Fatty Acid Metabolism With Phospholipid Remodeling and Disease Progression in Heart Failure

Background— Remodeling of myocardial phospholipids has been reported in various forms of heart failure for decades, but the mechanism and pathophysiological relevance of this phenomenon have remained unclear. We examined the hypothesis that &dgr;-6 desaturase (D6D), the rate-limiting enzyme in long-chain polyunsaturated fatty acid biosynthesis, mediates the signature pattern of fatty acid redistribution observed in myocardial phospholipids after chronic pressure overload and explored plausible links between this process and disease pathogenesis. Methods and Results— Compositional analysis of phospholipids from hearts explanted from patients with dilated cardiomyopathy revealed elevated polyunsaturated fatty acid product/precursor ratios reflective of D6D hyperactivity, manifesting primarily as lower levels of linoleic acid with reciprocally higher levels of arachidonic and docosahexaenoic acids. This pattern of remodeling was attenuated in failing hearts chronically unloaded with a left ventricular assist device. Chronic inhibition of D6D in vivo reversed similar patterns of myocardial polyunsaturated fatty acid redistribution in rat models of pressure overload and hypertensive heart disease and significantly attenuated cardiac hypertrophy, fibrosis, and contractile dysfunction in both models. D6D inhibition also attenuated myocardial elevations in pathogenic eicosanoid species, lipid peroxidation, and extracellular receptor kinase 1/2 activation; normalized cardiolipin composition in mitochondria; reduced circulating levels of inflammatory cytokines; and elicited model-specific effects on cardiac mitochondrial respiratory efficiency, nuclear factor &kgr; B activation, and caspase activities. Conclusions— These studies demonstrate a pivotal role of essential fatty acid metabolism in myocardial phospholipid remodeling induced by hemodynamic stress and reveal novel links between this phenomenon and the propagation of multiple pathogenic systems involved in maladaptive cardiac remodeling and contractile dysfunction.

Delta-6-desaturase Links PUFA Metabolism with Phospholipid Remodeling and Disease Progression in Heart Failure

Background— Remodeling of myocardial phospholipids has been reported in various forms of heart failure for decades, but the mechanism and pathophysiological relevance of this phenomenon have remained unclear. We examined the hypothesis that &dgr;-6 desaturase (D6D), the rate-limiting enzyme in long-chain polyunsaturated fatty acid biosynthesis, mediates the signature pattern of fatty acid redistribution observed in myocardial phospholipids after chronic pressure overload and explored plausible links between this process and disease pathogenesis. Methods and Results— Compositional analysis of phospholipids from hearts explanted from patients with dilated cardiomyopathy revealed elevated polyunsaturated fatty acid product/precursor ratios reflective of D6D hyperactivity, manifesting primarily as lower levels of linoleic acid with reciprocally higher levels of arachidonic and docosahexaenoic acids. This pattern of remodeling was attenuated in failing hearts chronically unloaded with a left ventricular assist device. Chronic inhibition of D6D in vivo reversed similar patterns of myocardial polyunsaturated fatty acid redistribution in rat models of pressure overload and hypertensive heart disease and significantly attenuated cardiac hypertrophy, fibrosis, and contractile dysfunction in both models. D6D inhibition also attenuated myocardial elevations in pathogenic eicosanoid species, lipid peroxidation, and extracellular receptor kinase 1/2 activation; normalized cardiolipin composition in mitochondria; reduced circulating levels of inflammatory cytokines; and elicited model-specific effects on cardiac mitochondrial respiratory efficiency, nuclear factor &kgr; B activation, and caspase activities. Conclusions— These studies demonstrate a pivotal role of essential fatty acid metabolism in myocardial phospholipid remodeling induced by hemodynamic stress and reveal novel links between this phenomenon and the propagation of multiple pathogenic systems involved in maladaptive cardiac remodeling and contractile dysfunction.

Chronic run training suppresses α-adrenergic response of rat cardiomyocytes and isovolumic left ventricle

The effects of endurance run training on α-adrenergic responsiveness of rat left ventricle (LV) were examined in cardiomyocytes and isovolumic LV. Female Sprague-Dawley rats were sedentary (Sed) or trained (Tr) for >20 wk by treadmill running. Cardiomyocyte shortening and fura 2 fluorescence ratio were recorded before and during 5-min exposure to 5 μM phenylephrine (PE) while paced at 0.5 Hz in 2 mM extracellular Ca2+ concentration at 29°C. Cardiomyocyte shortening and shortening velocity increased with PE, and these effects were more pronounced in the Sed group. The rate of cytosolic Ca2+ concentration removal was reduced by PE in the Sed cardiomyocytes, but was unaffected in the Tr. Isovolumic LV pressure was recorded immediately before and during 5-min perfusion with 5 μM PE during pacing at 280 beats/min and 37°C, and positive inotropy due to PE was more pronounced in the Sed than in the Tr. These data demonstrated that the effects of α-adrenergic stimulation on myocardial positive inotropy and calcium regulation were reduced in this rat model of run training at both the cellular and whole organ levels.