Apoptosis has been shown to contribute to loss of cardiomyocytes in cardiomyopathy, progressive decline in left ventricular function, and congestive heart failure. Because the molecular mechanisms involved in apoptosis of cardiocytes are not completely understood, we studied the biochemical and ultrastructural characteristics of upstream regulators of apoptosis in hearts explanted from patients undergoing transplantation. Sixteen explanted hearts from patients undergoing heart transplantation were studied by electron microscopy or immunoblotting to detect release of mitochondrial cytochrome c and activation of caspase-3. The hearts explanted from five victims of motor vehicle accidents or myocardial ventricular tissues from three donor hearts were used as controls. Evidence of apoptosis was observed only in endstage cardiomyopathy. There was significant accumulation of cytochrome c in the cytosol, over myofibrils, and near intercalated discs of cardiomyocytes in failing hearts. The release of mitochondrial cytochrome c was associated with activation of caspase-3 and cleavage of its substrate protein kinase C ␦ but not poly(ADP-ribose) polymerase. By contrast, there was no apparent accumulation of cytosolic cytochrome c or caspase-3 activation in the hearts used as controls. The present study provides in vivo evidence of cytochrome c-dependent activation of cysteine proteases in human cardiomyopathy. Activation of proteases supports the phenomenon of apoptosis in myopathic process. Because loss of myocytes contributes to myocardial dysfunction and is a predictor of adverse outcomes in the patients with congestive heart failure, the present demonstration of an activated apoptotic cascade in cardiomyopathy could provide the basis for novel interventional strategies.

I t has been proposed that the ventricular myocardium, both right (RV) and left (LV), exists as a continuous muscle band. 1-4 The band is oriented spatially as a helix formed by basal and apical loops. We hypothesize that this unique anatomy and spatial configuration of the myocardial muscle determine the way the ventricular ejection and filling take place. 5,6 Further, knowledge of this unique morphologic and physiologic characteristic should facilitate development of more effective surgical procedures in congestive heart failure. Unwrapping of the Ventricular Myocardial Band Careful anatomic studies have established the way the cardiac band should be unrolled. 3,4 Unwrapping occurs easily (Figure 1), with least resistance, along a natural cleavage plane. Dissection of the ventricular myocardial band can be accomplished in three steps. In the first step (Figure 2, A), the basal loop is unrolled. The superficial fibers of the anterior aspect of the left ventricle are cut along the anterior interventricular sulcus (see arrow) to pull apart the RV free wall (Figure 2, B). Dissection can then proceed posteriorly following the cleavage plane. In this way the complete basal loop is extended in its full length (Figure 2, C, black and dark gray areas). In the second step (Figure 2, C and D), the aorta is dismounted, which involves separation along the cleavage plane (see arrow in Figure 2, C) defined by the two muscular strata, the fibers of the descending segment (white) and the fibers of the ascending

Cardiac fibroblasts play an essential role in the physiology of the heart. These produce extracellular matrix proteins and synthesize angiogenic and cardioprotective factors. Although fibroblasts of cardiac origin are known to be resistant to apoptosis and to remain metabolically active in situations compromising cell survival, the underlying mechanisms are unknown. Here, we report that cardiac fibroblasts were more resistant than dermal or pulmonary fibroblasts to mitochondriadependent cell death. Cytochrome c release was blocked in cardiac fibroblasts but not in dermal fibroblasts treated with staurosporine, etoposide, serum deprivation, or simulated ischemia, precluding caspase-3 activation and DNA fragmentation. Resistance to apoptosis of cardiac fibroblasts correlated with the expression of the anti-apoptotic protein Bcl-2, whereas skin and lung fibroblasts did not express detectable levels of this protein. Bcl-x L, Bax, and Bak were expressed at similar levels in cardiac, dermal, and lung fibroblasts. In addition, the death of cardiac fibroblasts during hypoxia was not associated with the cleavage of Bid but rather with Bcl-2 disappearance, suggesting the requirement of the mitochondrial apoptotic machinery to execute death receptor-induced programmed cell death. Knockdown of bcl-2 expression by siRNA in cardiac fibroblasts increased their apoptotic response to staurosporine, serum, and glucose deprivation and to simulated ischemia. Moreover, dermal fibroblasts overexpressing Bcl-2 achieved a similar level of resistance to these stimuli as cardiac fibroblasts. Thus, our data demonstrate that Bcl-2 is an important effector of heart fibroblast resistance to apoptosis and highlight a probable mechanism for promoting survival advantage in fibroblasts of cardiac origin.

Apoptosis plays a role in cardiomyocyte death in several cardiovascular disorders. Here, we show that primary postnatal cardiomyocytes did not die upon activation of the intrinsic (cytochrome c-dependent) apoptotic pathway. Release of cytochrome c from mitochondria to the cytosol occurred, but did not activate the effector phase of apoptosis. Myocardial cells did not express apoptotic protease-activating factor-1 (Apaf-1), the allosteric activator of caspase-9 acting downstream of cytochrome c release. Forced expression of Apaf-1 restored the competence to complete the cytochrome c-induced apoptotic program and this effect was prevented by overexpression of Bcl-X L . However, cardiomyocytes were able to enter the apoptotic program when it was initiated by activation of death receptors, as observed during serum deprivation and metabolic inhibition. Our results indicate that regulation of Apaf-1 expression may be a new regulatory mechanism developed in postmitotic cells in order to prevent irreversible commitment to die after release of cytochrome c.

Differentiated cardiomyocytes are resistant to caspase-dependent cell death; however, the mechanisms involved are still uncertain. We previously reported that low Apaf1 expression partially accounts for cardiomyocyte resistance to apoptosis. Here, we extend the knowledge on the molecular basis of cardiac resistance to caspase activation by showing that the whole caspase-dependent pathway is silenced during heart development. Experimental ischemia triggers caspase activation in embryonic cardiomyocytes and proliferating fibroblasts, but not in neonatal and adult cardiomyocytes. Ischemia induces the release of the proapoptotic factors cytochrome c, truncated-AIF, and EndoG from mitochondria in postnatal cardiomyocytes in the absence of caspase activation. On the one hand, lentiviral-driven knockdown of EndoG shows that this gene is essential for ischemia-induced DNA degradation in neonatal cardiomyocytes, but not in proliferating fibroblasts; on the other hand, the AIF gene is essential for high molecular DNA cleavage in fibroblasts, but not in postmitotic cardiomyocytes, where it plays a prosurvival role during reoxygenation. These results show the switch from caspase-dependent to caspase-independent death pathways after cardiac cell differentiation, and disclose the relevance of EndoG in the caspase-independent DNA processing of differentiated cardiomyocytes.

Cardiac magnetic resonance imaging (Cardiac MRI) has become a gold standard diagnostic technique for the assessment of cardiac mechanics, allowing the non-invasive calculation of left ventricular long axis longitudinal shortening (LVLS) and absolute myocardial torsion (AMT) between basal and apical left ventricular slices, a movement directly related to the helicoidal anatomic disposition of the myocardial fibers. The aim of this study is to determine AMT and LVLS behaviour and normal values from a group of healthy subjects. A group of 21 healthy volunteers (15 males) (age: 23-55 y.o., mean: 30.7 ± 7.5) were prospectively included in an observational study by cardiac MRI. Left ventricular rotation (degrees) was calculated by custom-made software (Harmonic Phase Flow) in consecutive LV short axis planes tagged cine-MRI sequences. AMT was determined from the difference between basal and apical planes LV rotations. LVLS (%) was determined from the LV longitudinal and horizontal axis cine-MRI images. All the 21 cases studied were interpretable, although in three cases the value of the LV apical rotation could not be determined. The mean rotation of the basal and apical planes at end-systole were -3.71° ± 0.84° and 6.73° ± 1.69° (n:18) respectively, resulting in a LV mean AMT of 10.48° ± 1.63° (n:18). End-systolic mean LVLS was 19.07 ± 2.71%. Cardiac MRI allows for the calculation of AMT and LVLS, fundamental functional components of the ventricular twist mechanics conditioned, in turn, by the anatomical helical layout of the myocardial fibers. These values provide complementary information about systolic ventricular function in relation to the traditional parameters used in daily practice.

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