Russell L. Moore

Cardiac troponin T mutations result in allele-specific phenotypes in a mouse model for hypertrophic cardiomyopathy.

Multiple mutations in cardiac troponin T (cTnT) can cause familial hypertrophic cardiomyopathy (FHC). Patients with cTnT mutations generally exhibit mild or no ventricular hypertrophy, yet demonstrate a high frequency of early sudden death. To understand the functional basis of these phenotypes, we created transgenic mouse lines expressing 30%, 67%, and 92% of their total cTnT as a missense (R92Q) allele analogous to one found in FHC. Similar to a mouse FHC model expressing a truncated cTnT protein, the left ventricles of all R92Q lines are smaller than those of wild-type. In striking contrast to truncation mice, however, the R92Q hearts demonstrate significant induction of atrial natriuretic factor and beta-myosin heavy chain transcripts, interstitial fibrosis, and mitochondrial pathology. Isolated cardiac myocytes from R92Q mice have increased basal sarcomeric activation, impaired relaxation, and shorter sarcomere lengths. Isolated working heart data are consistent, showing hypercontractility and diastolic dysfunction, both of which are common findings in patients with FHC. These mice represent the first disease model to exhibit hypercontractility, as well as a unique model system for exploring the cellular pathogenesis of FHC. The distinct phenotypes of mice with different TnT alleles suggest that the clinical heterogeneity of FHC is at least partially due to allele-specific mechanisms.

A truncated cardiac troponin T molecule in transgenic mice suggests multiple cellular mechanisms for familial hypertrophic cardiomyopathy.

Mutations in multiple cardiac sarcomeric proteins including myosin heavy chain (MyHC) and cardiac troponin T (cTnT) cause a dominant genetic heart disease, familial hypertrophic cardiomyopathy (FHC). Patients with mutations in these two genes have quite distinct clinical characteristics. Those with MyHC mutations demonstrate more significant and uniform cardiac hypertrophy and a variable frequency of sudden death. Patients with cTnT mutations generally exhibit mild or no hypertrophy, but a high frequency of sudden death at an early age. To understand the basis for these distinctions and to study the pathogenesis of the disease, we have created transgenic mice expressing a truncated mouse cTnT allele analogous to one found in FHC patients. Mice expressing truncated cTnT at low (< 5%) levels develop cardiomyopathy and their hearts are significantly smaller (18-27%) than wild type. These animals also exhibit significant diastolic dysfunction and milder systolic dysfunction. Animals that express higher levels of transgene protein die within 24 h of birth. Transgenic mouse hearts demonstrate myocellular disarray and have a reduced number of cardiac myocytes that are smaller in size. These studies suggest that multiple cellular mechanisms result in the human disease, which is generally characterized by mild hypertrophy, but, also, frequent sudden death.

Loss of cardiac tetralinoleoyl cardiolipin in human and experimental heart failure

The mitochondrial phospholipid cardiolipin is required for optimal mitochondrial respiration. In this study, cardiolipin molecular species and cytochrome oxidase (COx) activity were studied in interfibrillar (IF) and subsarcolemmal (SSL) cardiac mitochondria from Spontaneously Hypertensive Heart Failure (SHHF) and Sprague-Dawley (SD) rats throughout their natural life span. Fisher Brown Norway (FBN) and young aortic-constricted SHHF rats were also studied to investigate cardiolipin alterations in aging versus pathology. Additionally, cardiolipin was analyzed in human hearts explanted from patients with dilated cardiomyopathy. A loss of tetralinoleoyl cardiolipin (L4CL), the predominant species in the healthy mammalian heart, occurred during the natural or accelerated development of heart failure in SHHF rats and humans. L4CL decreases correlated with reduced COx activity (no decrease in protein levels) in SHHF cardiac mitochondria, but with no change in citrate synthase (a matrix enzyme) activity. The fraction of cardiac cardiolipin containing L4CL became much lower with age in SHHF than in SD or FBN mitochondria. In summary, a progressive loss of cardiac L4CL, possibly attributable to decreased remodeling, occurs in response to chronic cardiac overload, but not aging alone, in both IF and SSL mitochondria. This may contribute to mitochondrial respiratory dysfunction during the pathogenesis of heart failure.

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.

Cellular adapations of the myocardium to chronic exercise

The heart responds positively to programs of chronic dynamic exercise. Hallmark adaptations of the heart include a training bradycardia, increases in end-diastolic dimension and maximal stroke volume, and a general improvement in ventricular performance and contractile function. Of considerable clinical significance are the general observations that chronic exercise renders the myocardium less susceptible to the deleterious effects of acute ischemic episodes and can effectively prevent and/or reverse many of the cardiac functional deficits that are known to occur in settings of chronic hypertension, advanced age, and myocardial infarction. In the text that follows, information gathered over the last 25 to 30 years has been reviewed in an attempt to identify cellular myocardial adaptations, both known and hypothetical, that are responsible for the observed effects of chronic dynamic exercise on the function and morphology of the heart in both normal and selected pathophysiologic settings. Finally, a variety of unresolved issues regarding the ability of chronic exercise to elicit adaptive cardiocyte responses has been identified. In so doing, it is hoped that creative thought and future work in the area will be stimulated.

Endurance training in rats with chronic heart failure induced by myocardial infarction.

The response to exercise was investigated in trained and sedentary rats with moderate compensated heart failure produced by myocardial infarction (MI) and in rats that underwent sham operations. Trained rats ran on a treadmill (10% grade at 20 m/min) for 60 min/day, 5 days/week for 10 to 12 weeks, whereas sedentary rats had only limited activity. Maximal oxygen consumption normalized for body weight (ml kg-1 min-1) was determined for each rat and found to be (1) greater in trained rats when compared with sedentary rats and (2) greater in sham-operated rats when compared with their counterparts that suffered infarction. In addition, skeletal muscle succinate dehydrogenase activities were greater and the blood lactic acid response to submaximal exercise was lower in trained rats compared with sedentary rats. Left ventricular infarct size for sedentary and trained rats with infarction was 36 +/- 3% and 34 +/- 3% of the total endocardial circumference, respectively, and resulted in (1) elevated left ventricular end-diastolic pressures at rest and during exercise, (2) lower mean arterial pressures at rest, and (3) lower maximal heart rates when compared with those in their sham-operated counterparts. However, normalization of mean arterial pressures during submaximal and maximal exercise was found along with a trend toward normalization of maximal heart rate when trained rats with infarction were compared with their sedentary counterparts. Blood flows to the kidneys, organs of the gut, and skeletal muscle during both submaximal and maximal exercise were unaffected by either myocardial infarction or training; no differences between sedentary and trained rats with infarction and sedentary and trained sham-operated rats were found. These results demonstrate that an exercise training program of moderate intensity produces beneficial hemodynamic and metabolic effects in rats with moderate compensated heart failure.

Exercise-induced cardiac preconditioning: how exercise protects your achy-breaky heart

The ability of exercise to protect the heart against ischemia-reperfusion (I/R) injury is well known in both human epidemiological studies and experimental animal models. In this review article, we describe what is currently known about the ability of exercise to precondition the heart against infarction. Just 1 day of exercise can protect the heart against ischemia/reperfusion damage, and this protection is upheld with months of exercise, making exercise one of the few sustainable preconditioning stimuli. Exercise preconditioning depends on the model and intensity of exercise, and appears to involve heightened oxidant buffering capacity, upregulated subunits of sarcolemmal ATP-sensitive potassium channels, and adaptations to cardiac mitochondria. We review the putative mechanisms involved in exercise preconditioning and point out many areas where future research is necessary to advance our understanding of how this stimulus confers resistance against I/R damage.

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

Exercise increases SOCS-3 expression in rat skeletal muscle: potential relationship to IL-6 expression

Suppressor of cytokine signalling‐3 (SOCS‐3) has been implicated in the onset of insulin resistance in non‐muscle tissue. Thus, we examined the effects of exercise training on SOCS‐3 expression and the potential role of SOCS‐3 in muscle. Female Sprague‐Dawley rats (5–8 months) were treadmill trained for 12 weeks and the muscles were removed 24 h after the last bout of exercise. Exercise training increased SOCS‐3 mRNA expression by 80% and 154% in the plantaris and soleus muscle, respectively. To mimic the effects of increased SOCS‐3 expression, SOCS‐3 cDNA was cotransfected with a NF‐kappa B (NF‐κB) luciferase construct into cultured C2C12 myotubes. SOCS‐3 overexpression increased NF‐κB transcriptional activity by 27‐fold. The proximal region of the IL‐6 gene promoter contains a NF‐κB consensus site, which contributes to increased IL‐6 expression in various tissues. SOCS‐3 cDNA was cotransfected into cultured C2C12 myotubes with either the IL‐6 luciferase construct or a mutated NF‐κB IL‐6 luciferase construct. SOCS‐3 overexpression increased IL‐6 transcriptional activity by 15‐fold, however, when the NF‐κB site was mutated SOCS‐3 failed to increase IL‐6 transcriptional activity. We subsequently found that IL‐6 mRNA expression was elevated in the plantaris and soleus muscles of the trained animals compared to the sedentary animals. Finally, exercise induced a significant reduction in IκBα and increased phosphorylation of Iκκ suggesting that NF‐κB activation was elevated after exercise training. These data suggest that training‐induced elevations in SOCS‐3 expression in skeletal muscle may contribute to the exercise‐induced increase in IL‐6 expression through alterations in the mechanisms that mediate NF‐κB activity.