Carol G. Au

Increased connective tissue growth factor associated with cardiac fibrosis in the mdx mouse model of dystrophic cardiomyopathy

Cardiomyopathy contributes to morbidity and mortality in Duchenne muscular dystrophy (DMD), a progressive muscle‐wasting disorder. A major feature of the hearts of DMD patients and the mdx mouse model of the disease is cardiac fibrosis. Connective tissue growth factor (CTGF) is involved in the fibrotic process in many organs. This study utilized the mdx mouse model to assess the role of CTGF and other extracellular matrix components during the development of fibrosis in the dystrophic heart. Left ventricular function of mdx and control mice at 6, 29 and 43 weeks was measured by echocardiography. Young (6 weeks old) mdx hearts had normal function and histology. At 29 weeks of age, mdx mice developed cardiac fibrosis and increased collagen expression. The onset of fibrosis was associated with increased CTGF transcript and protein expression. Increased intensity of CTGF immunostaining was localized to fibrotic areas in mdx hearts. The upregulation of CTGF was also concurrent with increased expression of tissue inhibitor of matrix metalloproteinases (TIMP‐1). These changes persisted in 43 week old mdx hearts and were combined with impaired cardiac function and increased gene expression of transforming growth factor (TGF)‐β1 and matrix metalloproteinases (MMP‐2, MMP‐9). In summary, an association was observed between cardiac fibrosis and increased CTGF expression in the mdx mouse heart. CTGF may be a key mediator of early and persistent fibrosis in dystrophic cardiomyopathy.

Integrins protect cardiomyocytes from ischemia/reperfusion injury

Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7β1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of β1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7β1D exposed to I/R had a substantial reduction in infarct size compared with that of α5β1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7β1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that β1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165-175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7β1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury.

Myocardial ischemia is more important than the effects of cardiopulmonary bypass on myocardial water handling and postoperative dysfunction: A pediatric animal model

OBJECTIVES Low cardiac output state is the principal cause of morbidity after surgical intervention for congenital heart disease. Myocardial ischemia-reperfusion injury, apoptosis, capillary leak syndrome, and myocardial edema are associated factors. We established a clinically relevant model to examine relationships between myocardial ischemia, edema, and cardiac dysfunction and to assess the role of the water transport proteins aquaporins. METHODS Sixteen lambs were studied. Seven were control animals not undergoing cardiopulmonary bypass, and 9 underwent bypass. Six had 90 minutes of aortic crossclamping with blood cardioplegia and moderate hypothermia. The remaining 3 underwent cardiopulmonary bypass without aortic crossclamping. Hemodynamic and biochemical data were recorded, and myocardial edema, apoptotic markers, and aquaporin expression were determined after death. RESULTS The group undergoing cardiopulmonary bypass with aortic crossclamping had a low cardiac output state, with early postoperative tachycardia, hypotension, increased serum lactate levels, and impaired tissue oxygen delivery (P < .05) compared with the group undergoing cardiopulmonary bypass without aortic crossclamping. The lambs undergoing cardiopulmonary bypass with aortic crossclamping had increased myocardial water (P < .05) compared with those not undergoing cardiopulmonary bypass and a 2-fold increase in aquaporin 1 mRNA expression (P < .05) compared with those not undergoing cardiopulmonary bypass and those undergoing cardiopulmonary bypass without aortic crossclamping. CONCLUSIONS A temporal association between hemodynamic dysfunction, myocardial edema, and increased aquaporin 1 expression was demonstrated. Cardiopulmonary bypass without ischemia was associated with minimal edema, negligible myocardial dysfunction, and static aquaporin expression. Ischemic reperfusion injury is the main cause of myocardial edema and myocardial dysfunction, but a causal relationship between edema and dysfunction remains to be proved.

Dysfunction induced by ischemia versus edema: Does edema matter?

OBJECTIVES Recovery from pediatric cardiac surgery is affected by ischemia-reperfusion injury, cardiac edema, and in some cases a low cardiac output syndrome. Although association has been made between the development of edema and dysfunction, modeling is confounded by intercurrent injurious stimuli that also cause cardiac edema and dysfunction. We tested whether a true causal relationship exists between edema and cardiac dysfunction. METHODS We induced either ischemia or edema alone in isolated cardiomyocytes and whole Langendorff-perfused hearts. Function was measured as shortening dynamics and developed pressure, respectively. RESULTS Ischemic injury impaired function in both cardiomyocytes and whole hearts. Isolated cells showed significant reduction in peak shortening and departure and relaxation velocities. Whole hearts displayed severely reduced developed pressures. Hyposmotic solution forced cardiomyocytes to swell to 7% greater than their normal size. No significant effect on shortening was seen. Similarly, Langendorff-perfused hearts were induced to take on 3% more water than control-perfused hearts and 9% more water than nonperfused hearts. This additional water was associated with mild dysfunction. CONCLUSIONS We demonstrate the capacity of the heart to tolerate edema greater than that seen in clinical settings without residual effect. Ischemia results in ongoing contractile dysfunction of both isolated cardiomyocytes and whole hearts. We conclude that dysfunction resulting from edema in ex vivo cardiac models is mild and suggest review of the importance given to edema-mediated dysfunction after cardiac surgery.

Myocardial membrane injury in pediatric cardiac surgery: An animal model

OBJECTIVE Reduced myocardial performance invariably follows pediatric cardiac surgery and is manifested by a low cardiac output state in its severest form. The role of myocardial membrane proteins in this setting is unknown. Dystrophin and dysferlin are involved in membrane integrity, whereas aquaporins selectively transport water. These proteins were examined in a model of pediatric cardiac surgery, together with a trial of poloxamer 188, which may reduce membrane injury. METHODS Eight lambs were randomized to saline with or without poloxamer 188. Lambs underwent 2 hours of cardiopulmonary bypass and aortic crossclamping. After a further 9 hours of monitoring, the hearts were assessed for water content, capillary leak, and protein expression. RESULTS Dystrophin expression was unaffected by ischemia/reperfusion, but dysferlin expression was reduced. Aquaporin 1 protein increased after ischemia/reperfusion. Poloxamer 188 administration was associated with supranormal levels of dystrophin, preservation of dysferlin expression, and normalization of aquaporin 1 expression. Poloxamer 188 was associated with less capillary leak, maintained colloid osmotic pressure, and less hemodilution. Poloxamer 188 was associated with an improved hemodynamic profile (higher blood pressure, higher venous saturation, and lower lactate), although the heart rate tended to be higher. CONCLUSIONS Changes in protein expression within the myocardial membrane were found in a clinically relevant model of pediatric cardiac surgery. Indicators of reduced performance, such as lower blood pressure and lower oxygen delivery, were lessened in association with the administration of the membrane protecting poloxamer 188. Poloxamer 188 was also associated with potentially beneficial changes in membrane protein expression, reduced capillary leakage, and less hemodilution.