Noninvasive measurements of myocardial carboxymyoglobin levels (%MbCO) and oxygen tensions (PtO2) are difficult to obtain experimentally. We have developed a compartmental model which allows prediction of myocardial %MbCO levels and PtO2 for varied carbon monoxide (CO) exposures. The cardiac compartment in the model consists of vascular subcompartments which contain two tissue subcompartments varying in capillary density. Mass-balance equations for oxygen (O2) and CO are applied for all compartments. Myocardial oxygen consumption and blood flow are quantified from predictive formulas based on heart rate. Model predictions are validated with experimental data at normoxia, hypoxia, exercise and hyperoxia. CO exposures of varying concentration and time (short-high, long-low), CO rebreathing during 100% O2, and exposure during exercise is simulated. Results of the simulations demonstrate that during CO exposures and subsequent therapies, the temporal changes of %MbCO in the heart differ from those of carboxyhemoglobin levels (%HbCO). Analysis of correlation between %HbCO, %MbCO and PtO2 was done to understand myocardial injury due to CO hypoxia. This thesis demonstrates that the model is able to anticipate the uptake and distribution of CO in the human myocardium and thus can be used to estimate the extravascular burden (MbCO, PtO2 ) of CO on the human heart.

No description available.

No description available.

Titin is a giant protein spanning from the Z-disk to the M-band of the cardiac sarcomere. In the I-band titin acts as a molecular spring, contributing to passive mechanical characteristics of the myocardium throughout a heartbeat. RNA Binding Motif Protein 20 (RBM20) is required for normal titin splicing, and its absence or altered function leads to greater expression of a very large, more compliant N2BA titin isoform in Rbm20 homozygous mice (Rbm20(Delta RRm)) compared to wild-type mice (WT) that almost exclusively express the stiffer N2B titin isoform. Prior studies using Rbm20(Delta RRm) animals have shown that increased titin compliance compromises muscle ultrastructure and attenuates the Frank-Starling relationship. Although previous computational simulations of muscle contraction suggested that increasing compliance of the sarcomere slows the rate of tension development and prolongs cross-bridge attachment, none of the reported effects of Rbm20(Delta RRm) on myocardial function have been attributed to changes in cross-bridge cycling kinetics. To test the relationship between increased sarcomere compliance and cross-bridge kinetics, we used stochastic length-perturbation analysis in Ca2+-activated, skinned papillary muscle strips from Rbrn20<^>R'Rm and WT mice. We found increasing titin compliance depressed maximal tension, decreased Ca2+-sensitivity of the tension-pCa relationship, and slowed myosin detachment rate in myocardium from Rbm20(Delta RRm) vs. WT mice. As sarcomere length increased from 1.9 to 2.2 mu m, length-dependent activation of contraction was eliminated in the Rbrn20<^>R'Rm myocardium, even though myosin MgADP release rate decreased similar to 20% to prolong strong cross-bridge binding at longer sarcomere length. These data suggest that increasing N2BA expression may alter cardiac performance in a length-dependent manner, showing greater deficits in tension production and slower cross-bridge kinetics at longer sarcomere length. This study also supports the idea that passive mechanical characteristics of the myocardium influence ensemble cross-bridge behavior and maintenance of tension generation throughout the sarcomere.

O presente estudo teve por objetivo investigar os efeitos do jejum de 48 horas sobre o metabolismo do músculo cardíaco e sobre a função cardíaca. Para isso, ratos machos com 60 dias foram separados em dois grupos: jejuados por 48 h (grupo experimental) e alimentados (grupo controle). Após eutanásia, sangue e coração foram coletados. O coração foi excisado e a aurícula do átrio direito (AAD), a aurícula do átrio esquerdo (AAE), parede do ventrículo direito (PVD), septo interventricular (SIV) e parede do ventrículo esquerdo (PVE) foram separadas e analisadas individualmente. Análises de parâmetros bioquímicos plasmáticos, dosagem de metabólitos, atividade máxima de enzimas, assim como expressão gênica e proteica foram realizadas. O jejum promoveu alterações metabólicas em todas as regiões, sendo mais intensas na PVE. Registros ventriculares e hemodinâmicos também foram obtidos. O jejum diminuiu a força de contração (dP/dt+), a força de relaxamento (dP/dt-) e a frequência cardíaca (FC), aumentou o tempo de enchimento diastólico e o hematócrito. Apesar de observamos aumento do potencial oxidativo e aumento da concentração disponível de ATP, é possível que 48h de jejum comprometa a volemia e por consequência a função cardíaca. / The present study aimed to investigate the effects of 48-hour fasting on cardiac muscle metabolism and cardiac function. For this, male rats with 60 days were separated into two groups: fasted for 48 h (experimental group) and fed (control group). After euthanasia, blood and heart were collected. The heart was excised and the right atrial atrium (RAA), left atrial atrium (LAA), right ventricular wall (RVW), interventricular septum (IVS) and left ventricular wall (LVW) were separated and analyzed individually. Analyzes of plasma biochemical parameters, dosage of metabolites, maximum activity of enzymes, as well as gene and protein expression were performed. Fasting promoted metabolic alterations in all regions, being more intense in PVE. Ventricular and hemodynamic records were also obtained. Fasting decreased contraction force (dP / dt +), relaxation force (dP / dt-) and heart rate (HR), increased diastolic filling time and hematocrit. Although we observed an increase in the oxidative potential and an increase in the available ATP concentration, it is possible that 48h-fasting compromises blood volume and, consequently, cardiac function.

Action potentials of striated muscle are created through movement of ions through membrane ion channels. ATP-sensitive potassium (KATP) channels are the only known channels that are gated by the intracellular energetic level ([ATP]/[ADP] ratio). KATP channels are both effectors and indicators of cellular metabolism as part of a negative feedback system. Decreased intracellular energetic level alters the gating of KATP channels, which is reflected in alterations of the action potential morphology. These changes protect the cell from exhaustion or injury by altering energy-consuming processes that are driven by membrane potential. Assessing the effects of KATP channel activation on resting membrane potential and action potential morphology, and the relationship to cellular stress is important to the understanding of normal cellular function. To better understand how muscle cells adapt to energetic stress, the monophasic action potential (MAP) electrode and floating microelectrode were used to record action potentials in intact hearts and skeletal muscles, respectively. Intact organs provide a more physiological environment for the study of energetics and membrane electrical phenomena. Utilizing these techniques, a stress on the intracellular energetic state resulted in greater and faster shortening of the duration of cardiac action potentials, and hyperpolarization of the membrane of skeletal muscle in a KATP channel dependent manner. Motion artifacts are a limitation to studying transmembrane action potentials, but the MAP and floating microelectrode techniques uniquely allow for reading of action potential morphology uncoupled from motion artifacts. The use of the floating microelectrode in skeletal muscles is a novel approach that provides previously unavailable data on skeletal muscle membrane potentials in situ.

This thesis determined whether changes in adenosine monophosphate-activated protein kinase (AMPK) activity would influence sarcoplasmic reticulum Ca2+-ATPase (SERCA) content and function in left ventricle (LV) and skeletal muscle isolated from sedentary or exercise trained mice. The data indicate that AMPKα2 kinase dead transgenic (KD) mice, as compared to wild-type (WT) mice, were characterized by reduced SERCA1a, SERCA2a and higher phospholamban (PLN) protein levels in both cardiac and skeletal muscle. Notably, exercise-training up-regulated myocardial SERCA2a protein content by 43%, as compared to sedentary WT mice. In contrast, exercise-training did not alter myocardial SERCA2a protein content in KD mice. Even so, exercise-training up-regulated SERCA1a protein content in skeletal muscle in both WT and KD mice. Based on these data, it appears that an AMPKα2-mediated mechanism influences SERCA2a content and function in the heart and skeletal muscle, which may contribute to the pathophysiology of models characterized by impaired AMPK activity and impaired calcium-cycling.

This thesis determined whether changes in adenosine monophosphate-activated protein kinase (AMPK) activity would influence sarcoplasmic reticulum Ca2+-ATPase (SERCA) content and function in left ventricle (LV) and skeletal muscle isolated from sedentary or exercise trained mice. The data indicate that AMPKα2 kinase dead transgenic (KD) mice, as compared to wild-type (WT) mice, were characterized by reduced SERCA1a, SERCA2a and higher phospholamban (PLN) protein levels in both cardiac and skeletal muscle. Notably, exercise-training up-regulated myocardial SERCA2a protein content by 43%, as compared to sedentary WT mice. In contrast, exercise-training did not alter myocardial SERCA2a protein content in KD mice. Even so, exercise-training up-regulated SERCA1a protein content in skeletal muscle in both WT and KD mice. Based on these data, it appears that an AMPKα2-mediated mechanism influences SERCA2a content and function in the heart and skeletal muscle, which may contribute to the pathophysiology of models characterized by impaired AMPK activity and impaired calcium-cycling.

Master of Science / Department of Kinesiology / Brad J. Behnke / Prostate cancer is the most common type of non-skin cancer found in men and preliminary evidence suggests prostate cancer has atrophic effects on cardiac and left ventricle (LV) mass which are associated with reduced endurance exercise capacity in rats. Using a pre-clinical orthotopic model of prostate cancer, echocardiography was utilized to test the hypothesis that exercise training will mitigate prostate cancer induced-cardiac and skeletal muscle atrophy and improve LV function versus sedentary tumor-bearing counterparts. Methods: Dunning R-3327 AT-1 prostate cancer cells were injected orthotopically in male Copenhagen rats aged (n=39; ~5 mo. old). Animals were randomized into four groups, exercise-trained tumor-bearing (EXTB) or control (EXCON) and sedentary tumor-bearing (SEDTB), or control (SEDCON). Exercise training was performed via a rodent treadmill set at 15m/min with a 15° incline for 60 min/day for ~30 days. Animals underwent echocardiographic evaluation using the parasternal short axis view to examine ventricle dimensions pre-cancer or exercise (PRE) and 15 (Post 1) and 30 (Post 2) days post cancer cell injection and/or exercise training with tissues collected immediately after Post 2. Results: Cardiac and LV mass of SEDTB animals were significantly lower than all groups (p<0.05). Tumor mass was significantly negatively correlated with LV mass in EXTB (-0.75, p<0.02) and SEDTB animals (-0.72, p<0.02). EXCON group had significantly higher stroke volume Post 2 assessment compared to both sedentary groups (p<0.05), but not EXTB animals. Conclusion: The current investigation demonstrates prostate cancer independent of anti-cancer treatment significantly reduces cardiac mass, and LV mass as well as locomotor muscle masses. However, moderate intensity exercise training can mitigate cardiac and skeletal muscle atrophy with prostate cancer.

Nkx2.5 is a cardiac transcription factor that plays a critical role in heart development. In humans, heterozygous mutations in the NKX2.5 gene result in congenital heart defects (CHDs), but the molecular mechanisms by which these mutations cause the defects are still unknown. NKX2.5 R142C is a mutation that is found to be associated with atrial septal defect and atrioventricular block in 13 patients from one family. The R142C mutation is located within both the DNA-binding domain and the nuclear localization sequence of NKX2.5 protein. The pathogenesis of CHDs in humans with R142C point mutation is not well understood. Also, a previous study in our laboratory has identified Mypt1/PP1 as a novel interacting partner of Nkx2.5 in stem cells during cardiomyogenesis. Nkx2.5 has a PP1-binding consensus sequence RVxF located in the N-terminus of the homeodomain. Notably, the PP1-binding sequence, RVxF, is mutated from arginine to cysteine in patients with the R142C heterozygous mutation. However, the ability of the R142C mutation to bind to the Mypt1/PP1 complex has not been investigated yet. The following thesis addresses the functional deficit associated with R142C by utilizing a combination of in vitro, and in vivo models. It also addresses the interaction of Mypt1/PP1 with the R142C mutation. We have generated a heterozygous mouse embryonic stem cell (mESC) line, harboring the murine homologue (R141C) of the human mutation R142C in Nkx2.5 gene. We show reduced cardiomyogenesis and impaired subcellular localization of Nkx2.5 protein in Nkx2.5R141C/+ mESCs. Gene expression profiling of Nkx2.5R141C/+ mESCs revealed a global misregulation of genes important for heart development and identified putative direct target genes of Nkx2.5 that are affected by the R141C heterozygous mutation. We also generated a mouse model harboring the human mutation R142C. We show that the Nkx2.5R141C/R141C homozygous embryos are developmentally arrested around E10.5 with delayed heart morphogenesis. Moreover, Nkx2.5R141C/+ newborn mice are grossly normal but show variable cardiac defects and downregulation of ion channel genes that later cause AV block in adult mice. Finally, we show that the R141C mutant binds to the Mypt1/PP1 complex but is not inhibited or translocated to the perinuclear region in the presence of Mypt1/PP1 as the WT Nkx2.5 is.