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J Thorac Cardiovasc Surg 2008;136:168-178
© 2008 The American Association for Thoracic Surgery
Evolving Technology |
a Pulmonary Hypertension Program, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
b Pulmonary Hypertension Program, Division of Cardiac Surgery, Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
c Pulmonary Hypertension Program, Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada
Received for publication July 5, 2007; revisions received December 4, 2007; accepted for publication January 29, 2008. * Address for reprints: Evangelos D. Michelakis, MD, FACC, FAHA, Canada Research Chair in Pulmonary Hypertension, Director, Pulmonary Hypertension Program, Department of Medicine (Cardiology), University of Alberta Hospitals, 2C2 WCM Health Sciences Center, Edmonton, Alberta, T6G2B7, Canada. (Email: emichela{at}cha.ab.ca).
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionConclusionsE-Supplement MethodsE-ReferencesSupplement Figure E1References |
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Methods/Results: We studied mitochondrial membrane potential, a surrogate for mitochondrial function, in human (n = 11) and rat hearts with physiologic (neonatal) and pathologic (pulmonary hypertension) right ventricular hypertrophy in vivo and in vitro. Mitochondrial membrane potential is higher in the normal left ventricle compared with the right ventricle but is highest in the hypertrophied right ventricle, both in myocardium and in isolated cardiomyocytes (P < .01). Mitochondrial membrane potential correlated positively with the degree of right ventricular hypertrophy in vivo and was recapitulated in phenylephrine-treated neonatal cardiomyocytes, an in vitro model of hypertrophy. The phenylephrine-induced mitochondrial hyperpolarization was reversed by VIVIT, an inhibitor of the nuclear factor of activated T lymphocytes, a transcription factor regulating the expression of several mitochondrial enzymes during cardiac development and hypertrophy. The clinically used drug dichloroacetate, known to increase the mitochondria-based glucose oxidation, reversed both the phenylephrine-induced mitochondrial hyperpolarization and nuclear factor of activated T lymphocytes (NFAT) activation. In Langendorff perfusions, dichloroacetate increased rat right ventricular inotropy in hypertrophied right ventricles (P < .01) but not in normal right ventricles, suggesting that mitochondrial hyperpolarization in right ventricular hypertrophy might be associated with its suboptimal performance.
Conclusions: The dynamic changes in mitochondrial membrane potential during right ventricular hypertrophy are chamber-specific, associated with activation of NFAT, and can be pharmacologically reversed leading to improved contractility. This mitochondrial remodeling might provide a framework for development of novel right ventricle–specific therapies.
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Introduction |
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In response to increased afterload (as seen in PAH), the thin RV of the normal adult heart hypertrophies but eventually quickly dilates and fails. There are, however, situations in congenital heart disease wherein the RV remains hypertrophied and compensated for years despite the development of PAH. These cases are typically seen when there is no involution of the physiologic neonatal RV hypertrophy (RVH) and the fetal morphology persists through adulthood. In contrast, the normal adult LV can develop hypertrophy and remain in a compensated state in response to an increase in its afterload (systemic hypertension) for decades. The relatively early failure of the RV in pulmonary hypertension explains largely the much worse survival of patients with PAH compared with patients with systemic hypertension; at the same time, this raises the exciting possibility that something in the neonatal hypertrophied RV (normal remodeling) offers superior function and protection compared with the acquired RVH in adults with PAH (abnormal remodeling).3 The cause of this early failure remains unknown and understudied and explains the lack of RV-specific therapies.1,2
We have recently described that phosphodiesterase 5 inhibitors, such as sildenafil, may be RV-specific inotropes.4 This theory is based on the fact that phosphodiesterase type 5 is selectively expressed in the myocardium of the hypertrophied RV but not in the LV of the same animal.4 In the search for better RV-specific therapies, as opposed to the LV, identification of differences between the two ventricles is critical. There are several studies examining the metabolism of the LV,5,6 but there is an impressive lack of studies on the metabolism of the RV. There is some evidence for differences between the metabolism of the RV and LV, at least in hypoxic animals.7 Potential differences in the metabolism or molecular biology between the two ventricles are not surprising given the recent discovery that the two ventricles have a different origin at early embryogenesis of the heart; whereas the RV develops from the anterior heart field, the LV develops from the early heart tube.8 It is therefore not appropriate to extrapolate findings or conclusions from the LV to the RV. Also, the adaptation of the RV to increased afterload may be regulated by mechanisms different from those in the LV.9 The need to specifically study RV function and failure was recently recognized by the National Institutes of Health as a priority.1
In the neonatal heart, the RV is physiologically hypertrophied, in response to the high pulmonary vascular resistance in utero. However, after birth, the thickness of the RV eventually becomes only a third of that of the LV, as the pulmonary vascular resistance gradually decreases.10 The physiologic hypertrophy in the neonatal RV might be regulated by a "fetal gene program,"11 which might be reactivated (at whole or in part) in adult disease states. Fetal and adult cardiac hypertrophy are also characterized by a predominantly glycolytic phenotype,5,6,12 which in the LV,13,14 vascular biology,15 or cancer16 is associated with a resistance to apoptosis. This has not been studied directly in the RV. The fact that metabolism and apoptosis are both directly regulated by mitochondria17 suggests that a potential mitochondrial and metabolic remodeling might be central to the regulation of RVH.
We hypothesized that there is a chamber-specific and dynamic mitochondrial remodeling during RVH, which might be associated with its suboptimal performance; reversal of this mitochondrial remodeling might be beneficial, improving RV function. We studied mitochondrial membrane potential, a surrogate for overall mitochondrial function and metabolism,15-19 in human and rat hearts. We used confocal microscopy and tetramethyl–rhodamine methyl ester (TMRM), a positively charged dye that localizes at the most negative organelles in the cell, the mitochondria.17 Mitochondrial hyperpolarization or depolarization is detected and quantified by an increase or decrease in TMRM fluorescence, respectively. We show that human and rat RVH is characterized by a dynamic increase in mitochondrial membrane potential (more hyperpolarized than that observed in the normal RV and LV) and that inhibition of this by the clinically used metabolic modulator dichloroacetate (DCA, an inhibitor of the mitochondrial pyruvate dehydrogenase kinase20) increases inotropy in the hypertrophied RV, but not in the normal RV. Our work has significant translational potential as DCA is being used in humans with mitochondrial diseases21 and has recently been shown to reverse mitochondrial hyperpolarization, increase glucose oxidation, and reverse disease phenotype in both cancer16 and PAH.15
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Methods |
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Permission from the University of Alberta committees on human ethics and animal policy and welfare was attained for all experiments on human and rat tissues, respectively.
Human Heart Tissue Samples
Human samples were acquired from patients undergoing surgery for congenital heart disease or transplantation at the University of Alberta Hospital. Excised ventricular tissue samples (free wall) were immediately placed on ice and stained with TMRM and Hoechst (a nuclear stain) for 40 minutes and visualized under confocal microscopy.15,16,19 The presence of hypertrophy was documented by the use of echocardiography for every patient (
Table 1) and confirmed macroscopically by the surgeon.
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(E-Supplement Methods).
Isolation of adult rat cardiomyocytes
Adult Sprague–Dawley rats (300–350 g) were used and cardiomyocytes were isolated from the ventricles as previously described4 (E-Supplement Methods).
Isolation and culture of neonatal rat cardiomyocytes
Neonatal Sprague–Dawley rat pups, 2 days old, were used to isolate RV and LV cardiomyocytes, which were then separated from fibroblasts and placed in culture, as previously described22 (E-Supplement Methods). Immunocytochemistry for myosin heavy chain confirmed that the studied cultured cells were cardiomyocytes (Supplement Figure E1).
Staining and confocal microscopy of cells and tissues
TMRM was made up to a concentration of 20 nmol/L in plating media along with 0.5 µmol/L of Hoechst nuclear stain. Each 35 mm x 10 mm plate of cells received 2 mL of the staining solution for a period of 30 minutes at 37°C. For ventricular rat tissue, the exposure was 40 minutes. The staining media was then removed, and each plate was rinsed and left at 37°C in another 2 mL of plating media. Staining of plates was staggered to give each plate from each ventricle the same amount of exposure to TMRM and the same amount of time before imaging.
Immunohistochemistry and confocal microscopy were performed on a Zeiss LSM 510 multiphoton confocal microscope (Carl Zeiss, Inc, Jena, Germany) using antigen retrieval and Image enhancer IT (Invitrogen Corporation, Carlsbad, Calif) for nuclear factor of activated T lymphocytes (NFATc3) and 4,6-diamino-2-phenylindole (DAPI, a nuclear stain) as previously described4,15,16,19,23 (also see E-Supplement Methods).
Isolated rat RV Langendorff perfusion
Adult rats with normal and hypertrophied heart (owing to monocrotline-induced PAH) were used. The heart were removed and perfused in a modified Langendorff preparation designed to study RV contractility, as we recently described4 (also see E-Supplement Methods).
Statistics
Comparison between LV and RV cardiomyocytes (from either neonatal or adult models) was done with a t test. Analysis of ventricular tissue (from either adult rat or human) and neonatal cultured neonatal cardiomyocytes was completed via 1-way analysis of variance with post hoc Bonferroni correction.
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Results |
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Figure 1, A). There was also increased TMRM fluorescence in RVH samples compared with the normal RV (Figure 1, B and C). Because we were able to secure only two normal hearts, direct comparisons among groups cannot be performed. However, results observed in the rat ventricular tissues were similar to the human RV and LV samples, where the normal adult rat LV had a higher mitochondrial membrane potential than the RV (
Figure 2, A; P < .01). To determine whether this was due to differences in cardiomyocytes, rather than other cells (ie, fibroblasts), we isolated rat adult RV and LV cardiomyocytes. Indeed, the differences in mitochondrial membrane potential from whole myocardial tissue persisted at the level of individual cardiomyocytes (Figure 2, B). The mean TMRM fluorescence data showed a similar increase in myocardial and cardiomyocyte mitochondrial membrane potential in the LV versus the RV, respectively (Figure 2, C). Although these results are interesting descriptive findings between the two ventricles, the possibility to exploit these differences in targeted therapeutic strategies required further assessment of the changes that occur in disease states.
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Figure 3). The worsening RVH was measured by echocardiography (free-wall thickness) and later confirmed macroscopically at autopsy (RV/LV + septum, data not shown). The increased pulmonary artery pressure was shown by measurement of the pulmonary artery acceleration time (PAAT), which we15,19 have previously shown to correlate negatively with mean pulmonary artery pressure in simultaneous right heart catheterization in rats. As hypertrophy progressed, there was a progressive increase in mitochondrial membrane potential in a "dose-dependent" manner (Figure 3).
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We looked to mimic pressure overload hypertrophy by exposing cultured neonatal cardiomyocytes to phenylephrine. In this classic model of hypertrophy, in vitro cardiomyocytes have increased levels of intracellular calcium and develop hypertrophy within 48 hours.22 Phenylephrine caused the predicted increase in cell size and a significant increase in mitochondrial membrane potential compared with vehicle-treated cardiomyocytes (
Figure 4, A). The response to phenylephrine resulted in hyperpolarization of the mitochondria similar to that seen in physiologic RVH (723 ± 41 versus 587 ± 10 FU/mm2, respectively; P < .01). Although it cannot definitely be excluded, it is unlikely that phenylephrine led to mitochondria biogenesis and an increased absolute number of mitochondria (which by itself would contribute to the increased TMRM signal), as the cells were only treated for 48 hours.
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Molecular and Metabolic Targeting of the Remodeled Mitochondria in Hypertrophy: The Role of NFAT
The increase in mitochondrial membrane potential in hypertrophy is likely multifactorial in etiology. First, there is an increase in intracellular and intramitochondrial calcium. This leads to activation of many mitochondrial enzymes that in turn cause an increase in Krebs cycle production of reducing equivalents, reactive oxygen species, and adenosine triphosphate, all of which alter mitochondrial function and mitochondrial membrane potential. Second, the increase in cytoplasmic calcium results in activation of the critical transcription factor NFAT,24 which is activated and regulates anatomic and metabolic remodeling during heart development25 and LV hypertrophy (LVH).26 NFAT regulates the expression of many mitochondrial and metabolic genes (including adenylosuccinate synthetase 1,27 pyruvate decarboxylase, heart–fatty acid binding protein, and the electron transport chain enzymes succinate dehydrogenase and cytochrome c oxidase25). NFAT is critical for heart development, as knockout of NFAT is fatal by embryonic day 10.5.25 This transcription factor is highly conserved among species with the same isoforms found in humans and mice.24 Thus, we studied whether the increase in mitochondrial membrane potential was NFAT dependent by culturing neonatal cardiomyocytes with phenylephrine and VIVIT (a selective NFAT inhibitor28). VIVIT abolished the increase in mitochondrial membrane potential caused by phenylephrine to a level similar to that of untreated control neonatal cardiomyocytes (Figure 4, A). To confirm that VIVIT inhibited NFAT, we performed immunocytochemistry on fixed neonatal cardiomyocytes for NFATc3 (the isoform that has been studied the most in the heart) and DAPI (nuclear stain). NFAT activation is associated with a translocation of NFAT into the nucleus, whereas inhibition of NFAT with VIVIT restricts NFAT to the cytoplasm.23,24 As expected, phenylephrine caused translocation of NFAT into the nucleus, whereas treatment with VIVIT inhibited this translocation and kept NFAT in the cytoplasm (Figure 4, B).
DCA inhibits the mitochondrial enzyme pyruvate dehydrogenase kinase, which in turn causes increased activity of pyruvate dehydrogenase, and thus DCA promotes the influx of pyruvate into the mitochondria, increasing glucose oxidation. Recently, DCA has been shown to reverse NFAT activation, mimicking the effects of VIVIT in cancer, increasing glucose oxidation, decreasing mitochondrial membrane potential in human cancer cell lines, and regressing tumor growth in vitro and in vivo.16 DCA mimicked VIVIT and caused a decrease in mitochondrial membrane potential and inhibited the nuclear translocation of NFAT in the cultured neonatal cardiomyocytes, despite continued exposure to phenylephrine (Figure 4, A and B). Although we did not measure metabolism directly, DCA is known to increase the coupling of glycolysis to glucose oxidation in the postischemic heart, and although it does not have significant effects in the normal LV, it improves ischemia–reperfusion recovery in mild LVH.29
To determine whether DCA and VIVIT will depolarize mitochondria from physiologically hypertrophied hearts (as in the phenylephrine-induced hypertrophy) and whether this is ventricle-specific, we harvested neonatal rat hearts and isolated cardiomyocytes from separated RV (which is hypertrophied) versus LV (which is not hypertrophied) free walls. Similar to the adult RVH myocardium from monocrotaline-induced PAH, the isolated neonatal hypertrophied cardiomyocytes from the RV free wall had more hyperpolarized mitochondria than those isolated from the LV free wall (Figure 4, C). Both DCA and VIVIT reversed this mitochondrial hyperpolarization and brought the mitochondrial membrane potential to the levels of the nonhypertrophied LV cardiomyocyte. Interestingly, DCA and VIVIT had no effect on the LV cardiomyocyte mitochondria (Figure 4, C).
These data show that in both physiologic and pathologic RVH, cardiomyocyte mitochondria are hyperpolarized, at least in part, due to NFAT activation, and that it can be reversed by DCA. Does this translate into improved RV function?
DCA Improves RV Inotropy in RVH
Since several patients with PAH or RVH owing to congenital heart disease only have affected RVs (their LVs are normal), the mitochondria-targeting DCA may selectively augment RV function in the setting of RVH. We used the ex vivo modified Langendorff perfused heart4 to measure RV contractility (
Figure 5, A). This modified model allows for real-time measurement of ex vivo developed pressures in the perfused RV, while its preload is constant (the balloon in the RV has a fixed volume) and is beating against no afterload (pulmonary artery transected). Interestingly, during perfusion of hearts 28 days after monocrotaline injection (severe RVH) compared with control animals, there was a significant and dose-dependent increase in developed pressure in the hypertrophied but not the normal RVs, whereas both had a similar increase in contractility by isoproterenol (Figure 5, B and C). Compatible with the expected DCA-induced increased glycolysis-to-glucose oxidation coupling, there was also an accompanied decrease in coronary effluent lactate only in the hearts with RVH (Figure 5, D).
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Discussion |
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and LVH26 but has not been previously studied in the RV hypertrophy (Figure 4, A and B). Furthermore, directly targeting mitochondria with DCA reversed the mitochondrial hyperpolarization in hypertrophy (Figure 4) and led to improved RV function (Figure 5). DCA did not affect the mitochondria and did not increase the contractile pressure of the normal RV. It is possible that the mitochondrial effects that we describe in RVH, both in vivo and in vitro, reflect a suboptimal mitochondrial remodeling, which may be responsible for suboptimal RV energy production and the relatively weak response of the RV to increased afterload. To the best of our knowledge, this is the first time in which the degree of hypertrophy is correlated to mitochondrial membrane potential and inotropic effects of a drug are related to the level of mitochondrial membrane potential, at least in the RV.
Although the RV and LV are currently approached and treated the same from a clinical perspective, the diversity of mitochondria between the RV and LV is not surprising given their different embryologic origins8 and the increasing evidence for significant diversity of mitochondria among different organs, such as the liver and kidney.30,31 The diversity of mitochondria extends beyond that of different organs to find diversity even within individual myocytes.32,33 We34 have previously shown that differences in mitochondrial function between the pulmonary and systemic arterial smooth muscle cells (SMC), where systemic SMC mitochondria are more hyperpolarized than pulmonary artery SMC mitochondria and account, at least in part, for the different response of the two vasculatures to hypoxia (the pulmonary arteries constrict whereas the renal arteries dilate). We subsequently demonstrated that, in PAH, the mitochondrial membrane potential in the pulmonary artery SMC is hyperpolarized compared with the SMC mitochondria from normal pulmonary artery SMC. We also demonstrated that treatment with DCA both normalized mitochondrial membrane potential and reversed PAH.15,35 It is also intriguing that the proliferative pulmonary artery SMC from PAH patients also have activated (ie, nuclear) NFAT (like the RVH cardiomyocytes), whereas the normal pulmonary artery SMC smooth do not (like the normal RV cardiomyocytes).23
The molecular basis for mitochondrial remodeling in hypertrophy is unknown, although mitochondrial diversity among other organs is associated with varying degrees of electron transport chain complex expression, which needs to be explored between the RV and LV, as well as possible changes during hypertrophy from the normal ventricles. We provide preliminary evidence that NFAT might play an important role in this mitochondrial remodeling in RVH, similar to its recently described role in PAH23 and cancer.16 Our work cannot exclude the possibility that the increase in mitochondrial membrane potential is not due to an increase in mitochondrial number. However, the fact that a short-term exposure to DCA and VIVIT normalizes the mitochondrial membrane potential and the fact that DCA acutely improves RVH function suggests that the increased mitochondrial membrane potential has a functional basis.
The profile of DCA's effects in cancer and PAH (where it selectively increases apoptosis by depolarizing mitochondria) might raise concerns, inasmuch as it might increase RV apoptosis after long-term use. However, it is remarkable that long-term use of DCA has been shown to reverse PAH and RVH and to improve functional capacity and mortality in several animal PAH models.15,16,36 It is possible that the DCA-induced mitochondrial depolarization in cardiomyocytes in the hypertrophied RV is enough to lead to improved contractile function but not enough to induce apoptosis by itself.
Furthermore, another medication that causes regression of PAH by inducing apoptosis in pulmonary artery SMC is sildenafil, which we4 recently showed also increases contractility in RVH, much like DCA. However, long-term use of sildenafil (>2 years) in patients with PAH has not resulted in any cardiovascular related deaths and has led to improvement of RV function.37 Nonetheless, this theoretical concern would need to be studied properly in the setting of a clinical trial.
The improved contractility of the hypertrophied RV with DCA is a novel finding. On the basis of the differences of the mitochondrial function between the two ventricles, we predicted that the effects of DCA would be restricted to the hypertrophied RV (and spare the normal RV and the LV). Indeed, DCA has failed to improve contractility from baseline in the LV, although there was a better recovery after ischemia.29 The lack of improvement in LV contractility with DCA has also been shown in human studies where patients with coronary artery disease or congestive heart failure did not show improved LV contractility or cardiac output with short-term administration of intravenous DCA.38,39
Our findings that DCA improved RV contractility acutely might also be relevant to the many clinical conditions in which RV-specific inotropy is needed, including patients with postcardiotomy shock who have preoperative RV dysfunction, or in the surgery of pediatric patients with congenital heart disease and RVH.
Limitations
The study of mitochondrial membrane potential as a surrogate for mitochondrial metabolism is validated and accepted in the literature35; however, there are assumptions made using an in vitro model to represent in vivo findings. Most important, the in vitro environment of isolated cardiomyocytes does not mimic in vivo conditions, and although this is a confounding factor, it is common to all isolated cells and we base our deductions from the delta membrane potential between the RV, LV, and treatment arms. Another limitation of the study is the small number of human samples obtained. However, we believe that these unselected human data are worthy of presentation because they are in agreement with our data from several in vivo and in vitro animal models, generally supporting the relevance of our hypothesis.
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; this was also supported by our finding in the ex vivo perfused hearts, in which treatment with DCA did not have significant effects on the developed pressures generated by the normal RV (Figure 5). The documented effects of DCA in PAH, and now on RVH, raise the possibility of studying this drug in humans with PAH. The ability for a combined "double-hit" mechanism, wherein pulmonary vascular remodeling and PAH are reversed and at the same time RV function is directly enhanced, will be very desirable clinically. We recently described a similar mechanism in which sildenafil dilates the pulmonary circulation in PAH and at the same time directly increases RVH inotropy.4 The fact that DCA has been used in humans with mitochondrial diseases for more than 30 years21 supports the need for such phase II studies. |
E-Supplement Methods |
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Human Heart Tissue Samples
Human samples were acquired from patients undergoing surgery for congenital heart disease or transplantation at the University of Alberta Hospital. Excised ventricular tissue samples (free wall) were immediately placed on ice and stained with TMRM and Hoechst (a nuclear stain) for 40 minutes and visualized under confocal microscopy.
E1-E3
The presence of hypertrophy was documented by the use of echocardiography for every patient (Table 1) and confirmed macroscopically by the surgeon.
Animal Model of RVH
We studied RVH using a model of experimental PAH by injecting monocrotaline, an alkaloid from Crotalaria spectabilis, a well-established model of rat PAH.
E2,E3
Monocrotaline is selectively toxic to the pulmonary arterial endothelium and causes significant RVH in 3 to 4 weeks after intraperitoneal injection. We
E2,E3
have repeatedly confirmed this using both invasive (right heart catheterization) and noninvasive (echocardiographic) methods. Here, we also confirmed the presence of PAH and RVH by echocardiography using PAAT (a parameter that correlates with mean pulmonary artery pressure and, used clinically, the shorter the PAAT the higher the pulmonary artery pressure) and RV free wall thickness in a short-axis parasternal view.
E2,E3
We further quantified RVH macroscopically at autopsy using the dry weight ratio of the RV/LV + septum.
E2,E3
Monocrotaline is activated in the liver and then absorbed in the pulmonary vascular bed without significant effects on systemic vasculature or cardiomyocytes. This concern has been examined by others, showing that all alterations to cardiomyocytes were specific to the RV owing to the developed PAH and not found in the LV of the same animals.
E4
Also, as monocrotaline requires activation by the liver, it is not possible to study the effects of monocrotaline in vitro.
Isolation of Adult Rat Cardiomyocytes
Hearts were taken from male adult Sprague–Dawley rats (300–350 g) and the aortas were cannulated and hung onto a perfusion system. Krebs buffer (NaCl, 118 mmol/L; KCl, 4.7 mmol/L; KH2PO4, 1.2 mmol/L; MgSO47H2O, 1.2 mmol/L; CaCl22H2O, 2.5 mmol/L; NaHCO3, 25 mmol/L; glucose, 5 mmol/L) at pH 7.4 (corrected to temperature = 37.5°C) was antegradely perfused through the coronary arteries for 5 minutes. Perfusate solution was then switched to a 2% w/v collagenase (Worthington Biochemical, Lakewood, NJ) supplemented Krebs buffer for 20 minutes. The hearts were then removed from the system, and the two ventricular free walls were surgically separated for myocyte isolation. After another 5-minute digestion with 2% w/v collagenase, the samples were tritrated with a glass pipette filtered through a nylon mesh and the filtrate was centrifuged to yield cardiomyocytes.
Isolation and Culture of Neonatal Rat Cardiomyocytes E5
Hearts from 2-day-old rats were excised and atria were removed. Free walls of LV and RV were isolated and kept separately in phosphate-buffered saline. Ventricular tissue was minced in ice cold phosphate-buffered saline solution and then digested in an enzymatic cocktail containing 2% w/v collagenase, 0.5% w/v deoxyribonuclease (Worthington Biochemical), and 2% w/v trypsin (Worthington Biochemical) for 20 minutes at 37°C. After digestion, administering Dulbecco modified Eagle medium (DMEM)/F12 media (Sigma, Oakville, Ontario, Canada) supplemented with 20% fetal bovine serum (Sigma) stopped enzymatic reaction. The mixture was then centrifuged at 800 rpm for 1 minute at 4°C to remove fibroblasts, red blood cells, and debris in the supernatant. The pellet was redigested 2 to 3 times for another 20 minutes and centrifuged at 800 rpm to separate cardiomyocytes into the supernatant. The collected supernatant was finally pooled and centrifuged twice for 7 minutes at 1700 rpm to yield a pellet of cardiomyocytes. Owing to the quick attachment of fibroblasts to the plates compared with the rate of attachment of cardiomyocytes, we separate any existing fibroblasts in the mixture by removing the cardiomyocyte-rich supernatant from the fibroblasts attached to the plates (differential plating) for 2 hours at 37°C. The efficacy and specificity of this procedure have been previously validated by our group.
E5
The isolated neonatal rat cardiomyocytes were plated and maintained for 2.5 days in DMEM/F12 media supplemented with 5% fetal bovine serum, 10% horse serum (Invitrogen Canada Inc, Burlington, Ontario, Canada), and 50 mg/L gentamicin (Invitrogen). Media also contained 10 nmol/L cytosine arabinoside (Sigma) to prevent fibroblast proliferation. Cultured cells were treated for 48 hours with 10 µmol/L phenylephrine to induce cardiomyocyte hypertrophy as previously described.
E5
Phenylephrine-treated cells were also treated with either 5 mmol/L DCA or 4 mmol/L 11Arg-VIVIT (a competing peptide that selectively inhibits NFAT by blocking its binding to calcineurin
E6
) (EMD Biosciences, Mississauga, Canada); the high arginine content of the peptide significantly enhances its permeability into the cell.
Staining of Cells and Tissue
TMRM was made up to a concentration of 20 nmol/L in plating media along with 0.5 µmol/L of Hoechst nuclear stain. Each 35 mm x 10 mm plate of cells received 2 mL of the staining solution for a period of 30 minutes at 37°C. For ventricular rat tissue, the exposure was 40 minutes with tissues sliced between 140 and 160 µm in thickness. The staining media was then removed, and each plate was rinsed once in media and then left at 37°C in another 2 mL of plating media. Staining of plates was staggered so as to give each plate from each ventricle the same amount of exposure to TMRM and the same amount of time before imaging. Imaging on confocal microscopy was performed at excitation of 543 nm, emission of 565 to 615 nm, and pixel scale of 0.9 mm x 0.9 mm. Background fluorescence was accounted for by usual techniques of setting the background to levels of control tissue that were not stained with TMRM. Photobleaching was not a significant factor inasmuch as regions where a short time course was obtained for quantifying TMRM fluorescence were only imaged once, avoiding the repeated imaging that causes photobleaching. Also, areas used for quantification of fluorescence were not imaged in high magnification, which also predisposes to photobleaching.
Immunohistochemistry and confocal microscopy were performed using antigen retrieval and Image enhancer IT (Invitrogen) for NFATc3 and DAPI (a nuclear stain), as previously described. E1-E3,E7,E8
Isolated Rat RV Langendorff Perfusion E8
Adult Sprague–Dawley rats (300–350 g) were anesthetized with intraperitoneal injection of 60 mg/kg pentobarbital. A midline sternotomy was performed, and within less than 30 seconds the heart was isolated and the aorta was cannulated and perfused with Krebs buffer at 12 to 13 mL/min. The hearts had a mean intrinsic rate of approximately 180 to 190 beats/min (hearts with a native rate <160 beats/min were not used). A latex balloon (Harvard Apparatus, Saint-Laurent, Quebec, Canada) was filled with water by titration to a constant volume of 30 mL and was placed in the RV via the right atrium through the tricuspid valve. The latex balloon was attached to a pressure transducer (Cobe, Richmond Hill, Ontario, Canada), and pressure traces were sampled at a rate of 1000 Hz by PowerLab data acquisition systems. Pressure readings were analyzed with Chart 5.4 software (ADInstruments Inc, Colorado Springs, Colo).
Imaging and Analysis of Data
All imaging was performed with a Zeiss LSM 510 confocal microscope. So that physiologic activity and viability of the tissue would be maintained, imaging was done on a heated platform at 37°C. Densitometry was analyzed with Zeiss Image Browser software. Fluorescence intensity of TMRM was assessed by measuring circular regions of interest (0.126 mm2 in area). A region of interest was drawn into each field of view where the circle encompassed myocardial tissue only and not coronary vessels. For cardiomyocytes, the TMRM intensity was measured in each cell excluding the area containing the nucleus (as marked by Hoechst stain in blue) and divided by the cytoplasmic area. Phenylephrine-induced hypertrophy leads to increases in area cytoplasmic area by 35% to 50% after 48 hours of treatment in neonatal cardiomyocytes (n = 400–450), whereas in freshly isolated adult myocytes from animals with RVH, there was an increase of 12% (n = 56) in cell size compared with control adult RV cardiomyocytes.
Comparison between LV and RV cardiomyocytes (from either neonatal or adult model) was done using a t test. Statistical analysis of ventricular tissue (from either adult rat or human) and neonatal cultured neonatal cardiomyocytes was completed via 1-way analysis of variance with post hoc Bonferroni correction. All mean data are presented in bar graphs with standard errors representing variance.
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Supplement Figure E1 |
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References |
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P < .01 versus RV 14 days after MCT injection. Middle third, Mean TMRM fluorescence from all rat RV myocardial samples with progression of RVH after MCT injection (n = 17 control RV, n = 5 RV 14 days after MCT injection, n = 11 RV 28 days after MCT injection; *
P < .01 vs control; 
