RESUMO
Aging is associated with declining cardiac contractile function as well as changes in metabolism and mitochondrial function. The relationship between age-related changes in cardiac metabolism and declining cardiac contractile function has not been determined. In order to define the role energetics play in changes in contractile function, we measured mitochondrial NADH, [NADH]m, during continuous contractions of isolated left ventricular myocytes from young (Y) and old (O) FBN rats. Second, we explored the role of metabolic disruption with rotenone and increased workload with isoproterenol (ISO) had on age-related changes in myocytes shortening. Single, intact myocytes were stimulated for 10 min of continuous contraction at either 2 Hz or 4 Hz while being perfused with Ringer's solution. Properties of shortening (peak shortening and rate of shortening) were measured at the onset (T0) and after 10 min (T10) of continuous contraction, and the decline in shortening over time (T10/T0) was determined. Although young and old myocytes had similar contractile function under resting conditions, old myocytes demonstrated decrements in [NADH]m during continuous stimulation, while young myocytes maintained constant [NADH]m over this time. In addition, old myocytes exhibited impaired contractile function to a workload (ISO) and metabolic (rotenone) stress compared to young myocytes. Taken together, these results demonstrated that old myocytes are susceptible to stress-induced contractile dysfunction which may be related to altered cellular energetics.
Assuntos
Ventrículos do Coração/crescimento & desenvolvimento , Contração Miocárdica , Miócitos Cardíacos/fisiologia , NAD/metabolismo , Animais , Células Cultivadas , Ventrículos do Coração/citologia , Masculino , Mitocôndrias Musculares/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Ratos Endogâmicos F344 , Rotenona/farmacologia , Desacopladores/farmacologiaRESUMO
Infantile-onset Pompe disease is an autosomal recessive disorder caused by the complete loss of lysosomal glycogen-hydrolyzing enzyme acid α-glucosidase (GAA) activity, which results in lysosomal glycogen accumulation and prominent cardiac and skeletal muscle pathology. The mechanism by which loss of GAA activity causes cardiomyopathy is poorly understood. We reprogrammed fibroblasts from patients with infantile-onset Pompe disease to generate induced pluripotent stem (iPS) cells that were differentiated to cardiomyocytes (iPSC-CM). Pompe iPSC-CMs had undetectable GAA activity and pathognomonic glycogen-filled lysosomes. Nonetheless, Pompe and control iPSC-CMs exhibited comparable contractile properties in engineered cardiac tissue. Impaired autophagy has been implicated in Pompe skeletal muscle; however, control and Pompe iPSC-CMs had comparable clearance rates of LC3-II-detected autophagosomes. Unexpectedly, the lysosome-associated membrane proteins, LAMP1 and LAMP2, from Pompe iPSC-CMs demonstrated higher electrophoretic mobility compared with control iPSC-CMs. Brefeldin A induced disruption of the Golgi in control iPSC-CMs reproduced the higher mobility forms of the LAMPs, suggesting that Pompe iPSC-CMs produce LAMPs lacking appropriate glycosylation. Isoelectric focusing studies revealed that LAMP2 has a more alkaline pI in Pompe compared with control iPSC-CMs due largely to hyposialylation. MALDI-TOF-MS analysis of N-linked glycans demonstrated reduced diversity of multiantennary structures and the major presence of a trimannose complex glycan precursor in Pompe iPSC-CMs. These data suggest that Pompe cardiomyopathy has a glycan processing abnormality and thus shares features with hypertrophic cardiomyopathies observed in the congenital disorders of glycosylation.
Assuntos
Doença de Depósito de Glicogênio Tipo II/metabolismo , Doença de Depósito de Glicogênio Tipo II/patologia , Complexo de Golgi/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/patologia , Western Blotting , Células Cultivadas , Genótipo , Glicosilação , Humanos , Imuno-HistoquímicaRESUMO
Skeletal muscle-derived stem cells (MDSCs) are able to differentiate into cardiomyocytes (CMs). However, it remains to be investigated whether differentiated CMs contract similar to native CMs. Here, we developed a three-dimensional collagen gel bioreactor (3DGB) that induces a working CM phenotype from MDSCs, and the contractile properties are directly measured as an engineered cardiac tissue. Neonate rat MDSCs were isolated from hind-leg muscles via the preplate technique. Isolated MDSCs were approximately 60% positive to Sca-1 and negative to CD34, CD45, or c-kit antigens. We sorted Sca-1(-) MDSCs and constructed MDSC-3DGBs by mixing MDSCs with acid soluble rat tail collagen type-I and matrix factors. MDSC-3DGB exhibited spontaneous cyclic contraction by culture day 7. MDSC-3DGB expressed cardiac-specific genes and proteins. Histological assessment revealed that cardiac-specific troponin-T and -I expressed in a typical striation pattern and connexin-43 was expressed similar to the native fetal ventricular papillary muscle. beta-Adrenergic stimulation increased MDSC-3DGB spontaneous beat frequency. MDSC-3DGB generated contractile force and intracellular calcium ion transients similar to engineered cardiac tissue from native cardiac cells. Results suggest that MDSC-3DGB induces a working CM phenotype in MDSCs and is a useful 3D culture system to directly assess the contractile properties of differentiated CMs in vitro.
Assuntos
Reatores Biológicos , Diferenciação Celular , Músculo Esquelético/citologia , Miocárdio/citologia , Células-Tronco/citologia , Animais , Animais Recém-Nascidos , Sequência de Bases , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Colágeno , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Reação em Cadeia da Polimerase , Ratos , Ratos Endogâmicos LewRESUMO
Caffeine consumption during pregnancy is reported to increase the risk of in utero growth restriction and spontaneous abortion. In the present study, we tested the hypothesis that modest maternal caffeine exposure affects in utero developing embryonic cardiovascular (CV) function and growth without altering maternal hemodynamics. Caffeine (10 mg.kg(-1).day(-1) subcutaneous) was administered daily to pregnant CD-1 mice from embryonic days (EDs) 9.5 to 18.5 of a 21-day gestation. We assessed maternal and embryonic CV function at baseline and at peak maternal serum caffeine concentration using high-resolution echocardiography on EDs 9.5, 11.5, 13.5, and 18.5. Maternal caffeine exposure did not influence maternal body weight gain, maternal CV function, or embryo resorption. However, crown-rump length and body weight were reduced in maternal caffeine treated embryos by ED 18.5 (P < 0.05). At peak maternal serum caffeine concentration, embryonic carotid artery, dorsal aorta, and umbilical artery flows transiently decreased from baseline at ED 11.5 (P < 0.05). By ED 13.5, embryonic aortic and umbilical artery flows were insensitive to the peak maternal caffeine concentration; however, the carotid artery flow remained affected. By ED 18.5, baseline embryonic carotid artery flow increased and descending aortic flow decreased versus non-caffeine-exposed embryos. Maternal treatment with the adenosine A(2A) receptor inhibitor reproduced the embryonic hemodynamic effects of maternal caffeine exposure. Adenosine A(2A) receptor gene expression levels of ED 11.5 embryo and ED 18.5 uterus were decreased. Results suggest that modest maternal caffeine exposure has adverse effects on developing embryonic CV function and growth, possibly mediated via adenosine A(2A) receptor blockade.