The Monocrotaline Rat Model of Right Heart Disease Induced by Pulmonary Artery Hypertension.

Pulmonary artery hypertension (PAH) is characterised by increased pulmonary vascular resistance (PVR) resulting in elevated pressure in the pulmonary artery supplying the pulmonary circulation. Disease of the right ventricle (RV) often manifests as a result of PAH placing excessive pressure on the right side of the heart. Although a relatively rare disease in humans, the impact of sustained PAH is severe, with poor outcomes even in treated individuals. As PAH develops, the blood flow is restricted through the pulmonary arteries and the right ventricle hypertrophies due to the increased strain of pumping blood through the pulmonary circulation. With time, RV hypertrophy progresses to right heart failure, impacting the supply of blood to the left ventricle and systemic circulation. Although right heart failure can currently be treated, it cannot be cured. There is therefore a need for more research into the physiological changes that cause the heart to fail under pressure overload. This review aims to evaluate the monocrotaline (MCT) rat model of PAH as a means of studying the cellular mechanisms associated with the development of RV hypertrophy and right heart failure.

Increased Mitochondrial Calcium Fluxes in Hypertrophic Right Ventricular Cardiomyocytes from a Rat Model of Pulmonary Artery Hypertension.

Pulmonary artery hypertension causes right ventricular hypertrophy which rapidly progresses to heart failure with underlying cardiac mitochondrial dysfunction. Prior to failure, there are alterations in cytosolic Ca2+ handling that might impact mitochondrial function in the compensatory phase of RV hypertrophy. Our aims, therefore, were (i) to measure beat-to-beat mitochondrial Ca2+ fluxes, and (ii) to determine mitochondrial abundance and function in non-failing, hypertrophic cardiomyocytes. Male Wistar rats were injected with either saline (CON) or monocrotaline (MCT) to induce pulmonary artery hypertension and RV hypertrophy after four weeks. Cytosolic Ca2+ ([Ca2+]cyto) transients were obtained in isolated right ventricular (RV) cardiomyocytes, and mitochondrial Ca2+ ([Ca2+]mito) was recorded in separate RV cardiomyocytes. The distribution and abundance of key proteins was determined using confocal and stimulated emission depletion (STED) microscopy. The RV mitochondrial function was also assessed in RV homogenates using oxygraphy. The MCT cardiomyocytes had increased area, larger [Ca2+]cyto transients, increased Ca2+ store content, and faster trans-sarcolemmal Ca2+ extrusion relative to CON. The MCT cardiomyocytes also had larger [Ca2+]mito transients. STED images detected increased mitochondrial protein abundance (TOM20 clusters per µm2) in MCT, yet no difference was found when comparing mitochondrial respiration and membrane potential between the groups. We suggest that the larger [Ca2+]mito transients compensate to match ATP supply to the increased energy demands of hypertrophic cardiomyocytes.

Visualization of Dynamic Mitochondrial Calcium Fluxes in Isolated Cardiomyocytes.

BACKGROUND: Cardiomyocyte contraction requires a constant supply of ATP, which varies depending on work rate. Maintaining ATP supply is particularly important during excitation-contraction coupling, where cytosolic Ca2+ fluxes drive repeated cycles of contraction and relaxation. Ca2+ is one of the key regulators of ATP production, and its uptake into the mitochondrial matrix occurs via the mitochondrial calcium uniporter. Fluorescent indicators are commonly used for detecting cytosolic Ca2+ changes. However, visualizing mitochondrial Ca2+ fluxes using similar methods is more difficult, as the fluorophore must be permeable to both the sarcolemma and the inner mitochondrial membrane. Our aim was therefore to optimize a method using the fluorescent Ca2+ indicator Rhod-2 to visualize beat-to-beat mitochondrial calcium fluxes in rat cardiomyocytes. METHODS: Healthy, adult male Wistar rat hearts were isolated and enzymatically digested to yield rod-shaped, quiescent ventricular cardiomyocytes. The fluorescent Ca2+ indicator Rhod-2 was reduced to di-hydroRhod-2 and confocal microscopy was used to validate mitochondrial compartmentalization. Cardiomyocytes were subjected to various pharmacological interventions, including caffeine and ß-adrenergic stimulation. Upon confirmation of mitochondrial Rhod-2 localization, loaded myocytes were then super-fused with 1.5 mM Ca2+ Tyrodes containing 1 µM isoproterenol and 150 µM spermine. Myocytes were externally stimulated at 0.1, 0.5 and 1 Hz and whole cell recordings of both cytosolic ([Ca2+]cyto) and mitochondrial calcium ([Ca2+] mito ) transients were made. RESULTS: Myocytes loaded with di-hydroRhod-2 revealed a distinct mitochondrial pattern when visualized by confocal microscopy. Application of 20 mM caffeine revealed no change in fluorescence, confirming no sarcoplasmic reticulum compartmentalization. Myocytes loaded with di-hydroRhod-2 also showed a large increase in fluorescence within the mitochondria in response to ß-adrenergic stimulation (P < 0.05). Beat-to-beat mitochondrial Ca2+ transients were smaller in amplitude and had a slower time to peak and maximum rate of rise relative to cytosolic calcium transients at all stimulation frequencies (P < 0.001). CONCLUSION: Myocytes loaded with di-hydroRhod-2 revealed mitochondrial specific compartmentalization. Mitochondrial Ca2+ transients recorded from di-hydroRhod-2 loaded myocytes were distinct in comparison to the large and rapid Rhod-2 cytosolic transients, indicating different kinetics between [Ca2+]cyto and [Ca2+]mito transients. Overall, our results showed that di-hydroRhod-2 loading is a quick and suitable method for measuring beat-to-beat [Ca2+]mito transients in intact myocytes.

Response of non-failing hypertrophic rat hearts to prostaglandin F2α.

BACKGROUND: Prostaglandin F2α (PGF2α) has a positively inotropic effect on right ventricular (RV) trabeculae from healthy adult rat hearts, and may therefore be therapeutically useful as a non-catecholaminergic inotrope. These provide additional contractile support for the heart without the added energetic demand of increased heart rate, and are also suitable for patients with reduced ß adrenergic receptor (ß-AR) responsiveness, or impaired mitochondrial energy supply. However, the response of hypertrophied rat hearts to PGF2α has not previously been examined. Our aim was therefore to determine the effect of PGF2α on isolated perfused rat hearts with RV hypertrophy following induction of pulmonary artery hypertension. METHODS: Male Wistar rats (300 g) were injected with either 60 mg kg-1 of monocrotaline (MCT, n = 10) or sterile saline as control (CON, n = 11). Four weeks post injection; hearts were isolated and Langendorff-perfused in sinus rhythm. Measurement of left ventricular (LV) pressure and the electrocardiogram were made and the response to 0.3 µM PGF2α was determined. RESULTS: PGF2α increased LV developed pressure in CON and in 60% MCT hearts, with no change in heart rate. However, 40% of MCT hearts developed arrhythmias during the peak inotropic response. For comparison, the response to 0.03 µM isoproterenol (ISO) was also investigated. Peak LV pressure developed sooner in response to ISO compared to PGF2α in both rat groups, although the inotropic response to ISO was reduced in MCT hearts. Analysis of fixed ventricular tissue confirmed that only RV myocytes were hypertrophied in MCT hearts. Our study showed that PGF2α was positively inotropic for healthy hearts, but found it generated arrhythmias in 40% of MCT hearts at the dose investigated. However, a more physiological dose of PGF2α may be a useful alternative without the added energetic cost of catecholaminergic inotropes.