Historically, programs of physical education and sport were housed in gymnasium buildings on academic campuses. As physical education evolved to the more scientifically focused successor departments of exercise science and kinesiology, faculty specialization developed in the physiology of exercise. With time, some faculty broadened their research to study the integrative physiology of other biological states and stressors. Through this series of events, a small group of integrative physiologists was formed in the Carlson Gymnasium at the University of Colorado Boulder during the 1990s with the goal of conducting novel biomedical research. The challenges were daunting: no contemporary core laboratory facilities, lack of temperature control, piercing external noise, pests, regular flooding, electrical power outages, and lack of funds for renovation. Despite these obstacles, the group established an innovative program of translational physiological research ranging from high-throughput molecular analyses to cell models to rodent studies to clinical trials in humans. These investigators supported their work with grant awards from the National Institutes of Health (NIH), Department of Defense, National Aeronautics and Space Administration (NASA), American Heart Association, and private research foundations totaling ∼$80 M in direct costs from the late 1980s to 2020. Collectively, the faculty and their laboratory personnel published ∼950 articles in peer-reviewed scientific journals. Over that period, 379 undergraduate students, 340 graduate students, 84 postdoctoral fellows, and dozens of junior research faculty received scientific training in Carlson, supported by >$21 M in extramural funding. What was accomplished by this handful of integrative physiologists speaks to the importance of the qualities of the investigators rather than their research facilities in determining scientific success.

Previous studies from our laboratory have shown positive benefits of linoleic acid (LA) feeding for attenuation of rat heart failure (HF). However, another research group concluded LA feeding was detrimental to cardiac function, using the American Institute of Nutrition 76A (AIN) diet as a background diet for the experimental animals only. To reconcile these conflicting results and determine whether (i) AIN has effects on cardiovascular function, and (ii) AIN reverses the positive effects of LA feeding, studies were performed using spontaneously hypertensive heart failure (SHHF) rats in both a survival study with lifetime feeding of AIN (control: Purina 5001) and a 2 × 2 factorial design for 6 weeks in young male SHHF rats with background diet and LA as variables. During a lifetime of AIN feeding, mortality from heart failure is significantly accelerated, cardiolipin altered and triglycerides increased. In young rats, 6 weeks on the AIN diet promoted increased systolic and diastolic blood pressure, increased fed and fasting blood glucose, increased serum inflammatory eicosanoids, decreased docosahexanoic acid, increased posterior wall thickness in diastole and an altered cardiolipin subspecies profile. The addition of LA to the AIN diet was able to rescue blood pressure. However, the combination increased retroperitoneal fat mass, body weight and fed blood glucose beyond the levels with the AIN diet alone. Because the AIN diet has wide ranging effects on cardiovascular parameters, our results suggest that it should not be used in animal studies involving the cardiovascular system unless induction of cardiac dysfunction is the desired outcome.

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 δ-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 κ 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.

Physical activity decreases risk for diabetes and cardiovascular disease morbidity and mortality; however, the specific impact of exercise on the diabetic vasculature is unexamined. We hypothesized that an acute, moderate exercise intervention in diabetic and hypertensive rats would induce mitochondrial biogenesis and mitochondrial antioxidant defence to improve vascular resilience. SHHF/ Mcc-fa cp lean (hypertensive) and obese (hypertensive, insulin resistant), as well as Sprague Dawley (SD) control rats were run on a treadmill for 8 days. In aortic lysates from SD rats, we observed a significant increase in subunit proteins from oxidative phosphorylation (OxPhos) complexes I–III, with no changes in the lean or obese SHHF rats. Exercise also increased the expression of mitochondrial antioxidant defence uncoupling protein 3 (UCP3) ( p < 0.05) in SHHF lean rats, whereas no changes were observed in the SD or SHHF obese rats with exercise. We evaluated upstream signalling pathways for mitochondrial biogenesis, and only peroxisome proliferators–activated receptor gamma coactivator 1α (PGC-1α) significantly decreased in SHHF lean rats ( p < 0.05) with exercise. In these experiments, we demonstrate absent mitochondrial induction with exercise exposure in models of chronic vascular disease. These findings suggest that chronic vascular stress results in decreased sensitivity of vasculature to the adaptive mitochondrial responses normally induced by exercise.

AIMS:Cardiolipin (CL) is a tetra-acyl phospholipid that provides structural and functional support to several proteins in the inner mitochondrial membrane. The majority of CL in the healthy mammalian heart contains four linoleic acid acyl chains (L(4)CL). A selective loss of L(4)CL is associated with mitochondrial dysfunction and heart failure in humans and animal models. We examined whether supplementing the diet with linoleic acid would preserve cardiac L(4)CL and attenuate mitochondrial dysfunction and contractile failure in rats with hypertensive heart failure.METHODS AND RESULTS:Male spontaneously hypertensive heart failure rats (21 months of age) were administered diets supplemented with high-linoleate safflower oil (HLSO) or lard (10% w/w; 28% kilocalorie fat) or without supplemental fat (control) for 4 weeks. HLSO preserved L(4)CL and total CL to 90% of non-failing levels (vs. 61-75% in control and lard groups), and attenuated 17-22% decreases in state 3 mitochondrial respiration observed in the control and lard groups (P < 0.05). Left ventricular fractional shortening was significantly higher in HLSO vs. control (33 ± 2 vs. 29 ± 2%, P < 0.05), while plasma insulin levels were lower (5.4 ± 1.1 vs. 9.1 ± 2.3 ng/mL; P < 0.05), with no significant effect of lard supplementation. HLSO also increased serum concentrations of several eicosanoid species compared with control and lard diets, but had no effect on plasma glucose or blood pressure.CONCLUSION:Moderate consumption of HLSO preserves CL and mitochondrial function in the failing heart and may be a useful adjuvant therapy for this condition.

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.

Frasier CR, Moore RL, Brown DA. Exercise-induced cardiac preconditioning: how exercise protects your achy-breaky heart. J Appl Physiol 111: 905–915, 2011. First published March 20, 2011; doi:10.1152/japplphysiol.00004.2011.—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.

AIMSTo determine whether myocardial resistance to ischemia/reperfusion (I/R) injury is enhanced in the senescent heart after life‐long caloric restriction, and whether AMP‐activated protein kinase (AMPK) plays a role in that phenotype.METHODS AND RESULTSHearts from ad libitum fed (AL) and life‐long calorically restricted (LCR, 40% restriction) B6D2F1 mice were examined by echocardiography, and 25/90 minute global I/R. AMPK inhibition (AraA) was applied to both groups (AL+AraA, LCR+AraA) prior to I/R. LCR hearts were smaller (AL, 270 ± 7 mg vs. LCR, 185 ± 5 mg, p < 0.001), and had thinner posterior walls (PWTd: AL, 1.07 ± 0.05 mm vs. LCR, 0.84 ± 0.05 mm, p < 0.05), but similar in vivo luminal dimension (LVIDd; LVIDs: AL, 3.8 ± 0.14; 2.0 ± 0.19 mm vs. LCR, 3.5 ± 0.18; 1.8 ± 0.17 mm, both p > 0.2). LCR hearts were protected from infarction (Infarct Size: AL, 28 ± 4 % vs. LCR, 10 ± 1 %, p < 0.01) and post‐ischemic functional deficit (LVDP recovery: AL, 65 ± 8 % vs. LCR, 93 ± 7 %, p < 0.01), and both effects were abolished by AraA (Infarct size: LCR+AraA, 22 ± 4 %; LVDP recovery: LCR+AraA, 82 ± 9 %, both p vs. AL > 0.1). LCR dramatically increased AMPKα phosphorylation (AL, 1.30 ± 0.03 vs. LCR, 1.55 ± 0.03, ratio units, p < 0.001), but not expression (AL, 1.21 ± 0.07 vs. LCR, 1.19 ± 0.01, ratio units, p = 0.8).CONCLUSIONSLife‐long caloric restriction induces powerful AMPK‐dependent I/R resistance, and involves elevated AMPKα phosphorylation.

Short-term caloric restriction (CR) protects the young myocardium against ischemia/reperfusion (I/R) injury through a mechanism involving AMP-activated protein kinase (AMPK). Here we ask whether a life-long CR intervention can extend this protection to the aged myocardium, and whether AMP-activated protein kinase (AMPK) plays a role in that protection. Hearts from ad libitum fed (AL) and life-long calorically restricted (LCR) mice were examined at 30 months of age by 25/90min global I/R, with and without AMPK inhibition (AraA). LCR hearts were protected from infarction (AL, 28±4% vs. LCR, 10±1%, p<0.01) and post-ischemic functional deficit (LVDP recovery: AL, 65±8% vs. LCR, 93±7%, p<0.01). Pre-ischemic AraA impaired both of these protective effects (Infarct size: LCR+AraA, 22±4%; LVDP recovery: LCR+AraA, 82±9%, both p vs. AL >0.1). AMPKα phosphorylation was dramatically increased in LCR hearts prior to I/R (AL, 1.18±0.01 vs. LCR, 1.68±0.04, ratio, p<0.0001), and accompanied by a more modest increase in total AMPKα (AL, 2.18±0.03 vs. LCR, 2.39±0.08 ratio, p<0.05). These results indicate that life-long caloric restriction profoundly protects the aged heart against I/R injury, and suggest that AMPK may play a role in that protection.

Peroxisome proliferator-activated receptors (PPAR) exist in three different forms, alpha (α), beta/delta (β/δ), or gamma (γ), all of which are expressed in skeletal muscle and play a critical role in the regulation of oxidative metabolism. The purpose of this investigation was to determine the mRNA expression pattern of the different PPARs and peroxisome proliferator-activated receptor alpha coactivator-1 alpha (PGC-1α) in muscles that largely rely on either glycolytic (plantaris) or oxidative (soleus) metabolism. Further, we also examined the alterations in the PPARs mRNA expression after one bout of endurance exercise or after 12 weeks of exercise training in the different muscles. Female Sprague-Dawley rats (5–8 months) were either run on the treadmill once or exercised trained for 12 weeks. The muscles were removed 24 h after the last bout of exercise. The results demonstrated with the exception of PPAR β/δ, the PPAR mRNAs are expressed to a greater extent in the soleus muscle than in the plantaris muscle in sedentary animals. PPARγ was the least abundantly expressed PPAR in either the soleus or the plantaris muscle. With respect to exercise training, only PPARγ mRNA expression increased in the soleus muscle, while PPARβ/δ and γ mRNA levels increased in the plantaris muscle. Minimal changes were detected in any of the PPARs with one bout of exercise training. These results suggest that PPARγ mRNA levels are the lowest in skeletal muscle among all of the PPARs and PPARγ mRNA is the most responsive to changes in physical activity levels.

Using neonatal rat ventricular myocytes, we previously reported that the expression of a dominant negative form of the c-Fos proto-oncogene (AFos) inhibited activator protein 1 activity and blocked the induction of the pathological gene profile stimulated by phenylephrine (PE) while leaving growth unaffected. We now extend these observations to the adult rat ventricular myocyte (ARVM) to understand the relationship between gene expression, growth, and function. Ventricular myocytes were isolated from adult rats and infected with adenovirus expressing beta-galactosidase (control) or AFos. The cells were subsequently treated with PE, and protein synthesis, gene program, calcium transients, and contractility were evaluated. As seen with the neonatal rat ventricular myocytes, in control cells PE stimulated an increase in protein synthesis, induced the pathological gene profile, and exhibited both depressed contractility and calcium transients. Although ARVMs expressing AFos still had PE-induced growth, pathological gene expression as well as contractility and calcium handling abnormalities were inhibited. To determine a possible mechanism of the preserved myocyte function in AFos-expressing cells, we examined phospholamban (PLB) and sarco(endo)plasmic reticulum calcium-ATPase proteins. Although there was no change in total PLB or sarco(endo)plasmic reticulum calcium-ATPase expression in response to PE treatment, PE decreased the phosphorylation of PLB at serine-16, an observation that was prevented in AFos-expressing cells. In conclusion, although PE-induced growth was unaffected in AFos-expressing ARVMs, the expression of the pathological gene profile was inhibited and both contractile function and calcium cycling were preserved. The inhibition of functional deterioration was, in part, due to the preservation of PLB phosphorylation.

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.

Cardiolipin (CL) is an essential phospholipid component of the inner mitochondrial membrane. In the mammalian heart, the functional form of CL is tetralinoleoyl CL [(18:2)4CL]. A decrease in (18:2)4CL content, which is believed to negatively impact mitochondrial energetics, occurs in heart failure (HF) and other mitochondrial diseases. Presumably, (18:2)4CL is generated by remodeling nascent CL in a series of deacylation-reacylation cycles; however, our overall understanding of CL remodeling is not yet complete. Herein, we present a novel cell culture method for investigating CL remodeling in myocytes isolated from Spontaneously Hypertensive HF rat hearts. Further, we use this method to examine the role of calcium-independent phospholipase A2 (iPLA2) in CL remodeling in both HF and nonHF cardiomyocytes. Our results show that 18:2 incorporation into (18:2)4CL is: a) performed singly with respect to each fatty acyl moiety, b) attenuated in HF relative to nonHF, and c) partially sensitive to iPLA2 inhibition by bromoenol lactone. These results suggest that CL remodeling occurs in a step-wise manner, that compromised 18:2 incorporation contributes to a reduction in (18:2)4CL in the failing rat heart, and that mitochondrial iPLA2 plays a role in the remodeling of CL's acyl composition.

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.