Cardiomiopatia hipertrófica felina

A Cardiomiopatia Hipertrófica (CMH) é uma doença é a doença cardíaca primária mais comum em gatos causada por uma mutação genética autossómica de dominância incompleta com apresentações clínicas variadas que apresenta uma grande dispersão mundial e racial. O carácter hereditário desta doença cardíaca resulta de uma mutação genética autossómica dominante com penetração incompleta, sendo mais exuberante em situações de homozigotia. A mutação mais comum ocorre em genes que codificam para a Proteína C3 de Ligação à Miosina, com alterações estruturais do mesmo e consequente hipertrofia concêntrica parcial ou total do ventrículo esquerdo. Com o aumento do septo interventricular, dos músculos papilares e/ou da parede do ventrículo esquerdo, ocorre comprometimento tanto do enchimento ventricular como do seu relaxamento, o que por sua vez contribui para a progressão da hipertrofia. A diminuição da câmara cardíaca, a diminuição do débito cardíaco, o aumento compensatório do ritmo, o aumento da pressão de enchimento diastólico e o aumento do átrio esquerdo predispõem para uma insuficiência cardíaca congestiva, formação trombos e até morte súbita. A avaliação clínica de um gato com suspeita de Cardiomiopatia Hipertrófica deve ser exaustiva, mesmo que o diagnóstico definitivo apenas possa ser confirmado através de ecocardiografia e de métodos de biologia molecular no sentido de identificar a mutação genética. A ecocardiografia pode ainda ser útil para direcionar a terapêutica em função do grau de hipertrofia estabelecer prognóstico. Atualmente, em medicina humana, existem outras formas de terapêutica, como o novo MYK 461, que se revelam bastante promissores.The Hypertrophic Cardiomyopathy is the heart disease more common in cats caused by a genetic autossomic mutation with imcomplete penetrance, having several clinic findings worldwide and inbreed. This disease assumes an hereditary character associated with a genetic mutation autossomic dominant with incomplete penetrance, being more exuberant in homozigotic. It’s a common mutation in genes that codify for the Myosin Binding Protein C (MYBPC3), with structural alteration of itself and leading to the partial or total concentric hypertrophy os left ventricule. With the increase of the interventricular septum and/or papillary muscles and/or left ventricle hall there is a compromise of the ventricular filling and it’s relaxation, which in other hand contributes to the progression of the hypertrophy. The decrease of the cardiac chamber, the decrease of cardiac output, the compensatory increase of rhythm, the increase of pressure in diastolic filling and the increase of the left atrium predispose to congestive heart failure and even sudden death. Cats clinical evaluation under the suspect of Hypertrophic Cardiomyopathy should be exhaustive, even if the definite diagnostic can only be confirmed throght Ultrasound and bio molecular methods looking for identification of genetic mutations. Ultrasound can also be useful to direct the therapeutics accordingly to the level of Hypertrophy, and better prognostics. Nowadays, there are other new therapeutic drugs, like MYK 461, that are showing promissing results

Respiratory parameters of permeabilised H9c2 cells treated with nothing (white bars), DMSO (dark grey bars), quercetin (25 μM) (grey bars) or DHS (1 μM) (light grey bars) as measured by high resolution respirometry (protocols C & D).

<p>Cells were incubated under normoxia (plain bars) or hypoxia (hatched bars) for 3 h, followed by oxygraphy with digitonin for permeabilisation. <b>(a)</b> Shows the differences in relative rate of respiration on malate + palmitate + ADP (relative to maximum respiratory rate of control permeabilised cells) prior to and following addition of cytochrome c, hence acting as a proxy for cytochrome c loss from mitochondria. <b>(b)</b> Shows the respiration of cells treated with DMSO, quercetin or DHS (following normoxia or hypoxia) on various substrates: Complex I respiration; “Complex I” (malate + palmitate + ADP + cytochrome c + pyruvate + glutamate), uncoupled respiration under complex I; “Complex I uncoupled” (“Complex I” + oligomycin +FCCP), uncoupled respiration under complex I and complex II; “Respiratory Chain” (“Complex I uncoupled” + Succ) and uncoupled complex II respiration; “Complex II uncoupled” (“Respiratory Chain” + Rotenone). Statistically significant differences from vehicle-control were denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) and from untreated control by † (p < 0.05), †† (p < 0.01) and ††† (p < 0.001).</p

Plasma membrane potential in H9c2 cells transfected with Arclight potential sensor was measured by wide field microscopy over 300 s (treat 10 s after start of experiment).

<p>Cells were treated with 5 μM FCCP (FCCP), 1 μM DHS (DHS), 1 μM ionomycin (Ionomycin) or 140 mM K⁺ (K), 10 s into the experiment <b>(a)</b>. Confocal microscopy over 600 s (treatment 30 s after start of experiment) was used to establish the response of cells to treatment with quercetin. Cells were treated with 5 μM FCCP (FCCP), 15 μM quercetin (Q), 1 μM ionomycin (Ionomycin) or 140 mM K⁺ (K), 10 s into the experiment. <b>(b)</b>. Due to the crowded nature of this figure, error bars are omitted for clarity (see supplementary data, Fig A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185691#pone.0185691.s001" target="_blank">S1 File</a>).</p

Oxygraphy protocols.

<p>This set of block diagrams details the measurements that were made in each of the seven different oxygraphy protocols used in this study. For each protocol the figure shows the final concentration of, and the duration of incubation with, each substrate, permeabilising agent and inhibitor. Prior to the start of each protocol, the polarigraphic chamber was equilibrated, and cell suspension prepared, with the appropriate medium (DMEM for protocols A and C, MiR05 for B, D, E, F and G). Each measurement was started after the cells had been injected into the polarigraphic chamber and allowed to equilibrate for 5–15 min. Protocols A and B were used to investigate the effects of hypoxia, or long term treatment with compounds of interest, on respiratory control ratios in whole cells (A) and respiratory complexes in permeabilised cells (B). Protocols C and D were used to investigate the effects of short term treatments with our compounds of interest on respiratory control ratios (C) and individual respiratory complexes (D). In these protocols “+[X]” indicates addition of quercetin or DHS. Protocols E, F and G were used to investigate mechanistic aspects of quercetin's effects, especially regarding ANT dependence and [ADP] dependence. For protocols C-G, cells did not undergo any treatment prior to trypsinisation and were treated with compounds of interest (or vehicle control) only <i>after</i> injection into the polarigraphic chamber. For protocol A, cells were either pretreated with compounds of interest (or vehicle control) for 24 h in culture (long term normoxia experiment) or subjected to 3 h of hypoxia, with or without treatments (whole cell hypoxia experiment), <i>prior to</i> trypsinisation and injection into the polarigraphic chambers. For protocol B, cells were likewise subjected to 3 h of hypoxia, with or without treatments, <i>prior to</i> trypsinisation. Protocol E was used to investigate the effect of atractyloside on quercetin's uncoupler-like effect. In each experiment, a single concentration of atractyloside (maximum of 200μM) was used. Protocols F, and G, where cells were titrated with either ADP (G) or bongkrekic acid (F), were carried out in two modes: i) to measure state III respiration, and ii) to measure oligomycin induced state IV respiration. Thus, oligomycin was only added to the chamber in these experiments when the protocols were set to measure state IV respiration. This was not applicable in protocol E, wherein a full set of inhibitor additions, and thus a full analysis, was performed for each concentration of atractyloside. Abbreviations used are as follows: “Atr”- atractyloside; “bong”- bongkrekic acid; “cyto c”- Cytochrome c; “Glut”- glutamine; “OMY”- oligomycin; “Pyr”- pyruvate; “Q”- quercetin; “ROT”- rotenone, “Succ”- succinate.</p

On the causes and consequences of the uncoupler-like effects of quercetin and dehydrosilybin in H9c2 cells - Fig 4

<p>Loss of mitochondrial membrane potential, as indicated by ratiometric fluorimetry with JC-1 following treatment with DHS <b>(a)</b> and quercetin <b>(b)</b>. Cells were incubated with JC-1 for 1 h under hypoxia or normoxia, and subsequently measurements taken immediately. The solid line represents normoxia treated cells. The dashed line represents hypoxia treated cells.</p