The Sarco-endoplasmic Ca2+ ATPase (SERCA2a) plays a crucial role in sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR/ER) and is an important regulator of muscle contraction and relaxation. Recent findings suggest that a novel CAMKIIα splice variant, αKAP, that plays the role of a CAMKII anchoring protein in the myocardium, also directly interacts with SERCA2a. We examined the effects of αKAP on SERCA2a activity using transfection of HEK-293T cells as well as lentiviral infection of primary neonatal mouse cardiomyocytes (NMCM). Our data showed that αKAP reduced Ca2+ ATPase activity, and downregulated SERCA2a expression in both HEK-293T cells coexpressing αKAP and SERCA2a, as well as NMCM overexpressing αKAP. Interestingly in a rat model of myocardial infarction, αKAP expression was found to be elevated, alongside elevated CaMKIIδ, and depressed SERCA2a expression. These data suggest that αKAP may be a unique regulator of SERCA2a activity and cardiac function.

Background-In cardiomyocytes from patients with hypertrophic cardiomyopathy, mechanical dysfunction and arrhythmogenicity are caused by mutation-driven changes in myofilament function combined with excitation-contraction (E-C) coupling abnormalities related to adverse remodeling. Whether myofilament or E-C coupling alterations are more relevant in disease development is unknown. Here, we aim to investigate whether the relative roles of myofilament dysfunction and E-C coupling remodeling in determining the hypertrophic cardiomyopathy phenotype are mutation specific. Methods and Results-Two hypertrophic cardiomyopathy mouse models carrying the R92Q and the E163R TNNT2 mutations were investigated. Echocardiography showed left ventricular hypertrophy, enhanced contractility, and diastolic dysfunction in both models; however, these phenotypes were more pronounced in the R92Q mice. Both E163R and R92Q trabeculae showed prolonged twitch relaxation and increased occurrence of premature beats. In E163R ventricular myofibrils or skinned trabeculae, relaxation following Ca2+ removal was prolonged; resting tension and resting ATPase were higher; and isometric ATPase at maximal Ca2+ activation, the energy cost of tension generation, and myofilament Ca2+ sensitivity were increased compared with that in wildtype mice. No sarcomeric changes were observed in R92Q versus wild-type mice, except for a large increase in myofilament Ca2+ sensitivity. In R92Q myocardium, we found a blunted response to inotropic interventions, slower decay of Ca2+ transients, reduced SERCA function, and increased Ca2+/calmodulin kinase II activity. Contrarily, secondary alterations of E-C coupling and signaling were minimal in E163R myocardium. Conclusions-In E163R models, mutation-driven myofilament abnormalities directly cause myocardial dysfunction. In R92Q, diastolic dysfunction and arrhythmogenicity are mediated by profound cardiomyocytesignaling and E-C coupling changes. Similar hypertrophic cardiomyopathy phenotypes can be generated through different pathways, implying different strategies for a precision medicine approach to treatment.

Orientadores: Maria Cláudia Gonçalves de Oliveira Fusaro, Andrea Maculano Esteves / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas / Made available in DSpace on 2018-08-27T04:11:06Z (GMT). No. of bitstreams: 1 Jorge_CarolinaOcanha_M.pdf: 1325403 bytes, checksum: 6635807ae28ce8dbb49ec5d1bcc74a75 (MD5) Previous issue date: 2015 / Resumo: A dor musculoesquelética é um importante problema de saúde mundial. Dentre todos os tipos de dor, àquela induzida pela contração isométrica sustentada está relacionada com os movimentos corporais nas atividades da vida diárias e apresenta um alto impacto socioeconômico. Apesar da sua relevância clínica, os mecanismos moleculares envolvidos no desenvolvimento da dor muscular induzida pela contração isométrica sustentada são pouco conhecidos. Portanto, o objetivo deste estudo foi avaliar o envolvimento dos receptores TRPV1 e TRPA1 na hiperalgesia muscular mecânica induzida pela contração isométrica sustentada no músculo gastrocnêmio de ratos machos, da linhagem wistar. O antagonista seletivo do receptor TRPV1, AMG9810, reduziu significativamente a hiperalgesia muscular mecânica induzida pela contração isométrica sustentada quando administrado no músculo gastrocnêmio ipsilateral, mas não no contralateral. A administração intratecal de AMG9810 apresentou a mesma resposta. Similar ao TRPV1, a administração intramuscular e intratecal do antagonista seletivo do receptor TRPA1, HC030031, reduziu significativamente a hiperalgesia muscular induzida pela contração isométrica sustentada. No entanto, não foi observado modificação da expressão proteica dos receptores TRPV1 e TRPA1 no tecido muscular após a contração isométrica sustentada. Os dados sugerem que os receptores TRPV1 e TRPA1 expressos no músculo gastrocnêmio e corno dorsal da medula espinhal estão envolvidos na hiperalgesia muscular mecânica induzida pela contração isométrica sustentada em ratos. Sugerimos, portanto, que os receptores TRPV1 e TRPA1 co-expressos nas fibras aferentes primárias trabalhem juntos para ativar os nociceptores das fibras aferentes durante a contração isométrica sustentada. Além disso, nós sugerimos que os receptores TRPV1 e TRPA1 sejam potenciais alvos para o controle da dor muscular inflamatória / Abstract: Musculoskeletal pain is an important health issue in the world. Among the kinds of muscle pain, the one induced by sustained isometric contraction is associated with body movements of the daily life and has a high socio-economic impact. Despite its clinical relevance, the molecular mechanisms involved in the development of muscle pain induced by sustained isometric contraction are poorly understood. Therefore, the aim of this study was to evaluate the involvement of TRPV1 and TRPA1 receptors in the mechanical muscle hyperalgesia induced by sustained isometric contraction of the gastrocnemius muscle of rats. The selective TRPV1 receptor antagonist AMG 9810 reduced the mechanical muscle hyperalgesia induced by sustained isometric contraction when administered in the ipsilateral but not in the contralateral gastrocnemius muscle. Also, the intratecal administration of AMG9810 reduced the same response. Similar to TRPV1, intramuscular and intrathecal administration of selective TRPA1 receptor antagonist HC030031 reduced the mechanical muscle hyperalgesia induced by sustained isometric contraction. Finally, the sustained isometric contraction did not modify the protein expression of TRPV1 and TRPA1 receptors in muscle tissue. We concluded that TRPV1 and TRPA1 receptors expressed in gastrocnemius muscle and spinal cord dorsal horn are involved with the mechanical muscle hyperalgesia induced by sustained isometric contraction in rats. We suggest that TRPV1 and TRPA1 receptors co-expressed in primary afferent fibers work together to activate nociceptive afferent fibers during sustained isometric contraction. Also, we suggest that TRPV1 and TRPA1 receptors are potential target to control inflammatory muscle pain / Mestrado / Biodinâmica do Movimento Humano e Esporte / Mestra em Ciências da Nutrição e do Esporte e Metabolismo

We investigate experimentally the turbulent flow through a two-dimensional contraction. Using a water tunnel with an active grid we generate turbulence at Taylor microscale Reynolds number Reλ ~ 250 which is advected through a 2.5:1 contraction. Volumetric and time-resolved Tomo-PIV and Shake-The-Box velocity measurements are used to characterize the evolution of coherent vortical structures at three streamwise locations upstream of, and within the contraction. We confirm the conceptual picture of coherent large-scale vortices being stretched and aligned with the mean rate of strain. This alignment of the vortices with the tunnel centerline is stronger compared to the alignment of vorticity with the large-scale strain observed in numerical simulations of homogeneous turbulence. We judge this by the peak probability magnitudes of these alignments. This result is robust and independent of the grid-rotation protocols. On the other hand, while the point-wise vorticity vector also, to a lesser extent, aligns with the mean strain, it principally remains aligned with the intermediate eigen-vector of the local instantaneous strain-rate tensor, as is known in other turbulent flows. These results persist when the distance from the grid to the entrance of the contraction is doubled, showing that modest transverse inhomogeneities do not significantly affect these vortical-orientation results.

The master´s thesis deals with research of viability and contraction measurement of cardiomyocytes. The work is divided into two main areas – theoretical and practical part. Theoretical part is aimed at electrophysiology of cardiomyocytes. Practical part includes detection of contractibility based on eccentricity in program Matlab. For research of viability, were used several media for example DMEM and MPRI.

Antibody-coated microprobes were used to determine whether immunoreactive enkephalins were released in response to fatiguing isometric contractions of the hind-limb muscles in cats anesthetized with α-chloralose. Contractions were performed by stimulating the tibial nerve via a microprocessor-controlled stimulator. Microprobes were inserted into the periaqueductal grey (P 0.5-1.0 mm) prior to, during and following fatiguing contractions. During fatiguing contractions, mean arterial blood pressure increased by 76 ± 9 mmHg above resting and recovery levels. Levels of immunoreactive enkephalins were elevated in the dorsolateral periaqueductal grey during the isometric contraction when compared to resting levels. It is possible that isometric muscle contraction causes the release of Met-enkephalin-like substances in the periaqueductal grey.

Antibody microprobes were used to study release of immunoreactive neurokinins in the dorsal horn of the anaesthetized spinal cat following sustained isometric contraction of ipsilateral hindlimb muscles. Microprobes had immobilized antibodies to neurokinin A (NKA) on their outer surfaces and bound a proportion of released molecules when inserted in the central nervous system. Bound molecules were detected in autoradiographs as zones of reduced binding of 125I-NKA in which microprobes were incubated after withdrawal from the spinal cord. The left hindlimb was immobilized using an epoxy bandage splint and isometric contraction of muscles induced by intermittent tetanic stimulation of a ventral root. A basal presence of immunoreactive neurokinins was detected and this was increased by sustained isometric muscle contraction. It is probable that ergoreceptors contain and release neurokinins.

Smooth muscle contraction regulates the size of the blood vessel lumen which directly affects the mechanical response of the vessel. Folding in arteries has been observed in arteries during excessive contraction, known as a coronary artery spasm. The interplay of muscle contraction, geometry, and material responses and their effects on stability can be understood through mathematical models. Here, we consider a three-layer cross-sectional model of a coronary artery with anisotropic properties and intimal thickening, and perform a linear stability analysis to investigate the circumferential folding patterns that emerge due to muscle contraction. Our model shows that a critical level of contractile activity yields a uniform strain distribution across the arterial wall. When the muscle is contracted above this critical level, the tissue behaves isotropically and it is more prone to circumferential instability. This theoretical framework could serve as a valuable tool to understand the relationship between arterial lumen morphology and wall contraction in health and disease.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17946号 / 医博第3830号 / 新制||医||1000(附属図書館) / 30776 / 京都大学大学院医学研究科医学専攻 / (主査)教授 稲垣 暢也, 教授 小西 郁生, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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