NE, acting via the ss-adrenergic pathway, stimulates apoptosis in adult rat cardiac myocytes in vitro. This effect is mediated by protein kinase A and requires calcium entry via voltage-dependent calcium channels. NE-stimulated apoptosis of cardiac myocytes may contribute to the progression of myocardial failure.

Abstract-Osteopontin (OPN), an extracellular matrix protein, is expressed in the myocardium with hypertrophy and failure. We tested the hypothesis that OPN plays a role in left ventricular (LV) remodeling after myocardial infarction (MI). Accordingly, OPN expression and LV structural and functional remodeling were determined in wild-type (WT) and OPN knockout (KO) mice 4 weeks after MI. Northern analysis showed increased OPN expression in the infarcted region, peaking 3 days after MI and gradually decreasing over the next 28 days. In the remote LV, OPN expression was biphasic, with peaks at 3 and 28 days. In situ hybridization and immunohistochemical analyses showed increased OPN mRNA and protein primarily in the interstitium. Infarct size, heart weight, and survival were similar in KO and WT mice after MI (PϭNS), whereas the lung wet weight/dry weight ratio was increased in the KO mice (PϽ0.005 versus sham-operated mice). Peak LV developed pressure was reduced to a similar degree after MI in the KO and WT mice. Key Words: extracellular matrix proteins Ⅲ osteopontin Ⅲ collagen Ⅲ myocyte slippage Ⅲ myocyte elongation T he dynamic synthesis and breakdown of extracellular matrix (ECM) proteins may play an important role in myocardial remodeling. 1,2 Recently, using spontaneously hypertensive and aortic-banded rats, we showed increased expression of osteopontin (OPN), an ECM protein, coincident with the development of heart failure. 3 Although first isolated from mineralized bone matrix, OPN has since been shown to be synthesized by several cell types, including cardiac myocytes, microvascular endothelial cells, and fibroblasts. 4 -6 OPN, an adhesive glycophosphoprotein with an arginineglycine-aspartic acid (RGD) sequence, has been shown to interact with integrins (␣ V ␤ 3 , ␣ V ␤ 1 , and ␣ V ␤ 5 ) and the CD44 receptor in an RGD-dependent manner. 4,7 OPN appears capable of mediating diverse biological functions, including cell adhesion, chemotaxis, and signaling. 4,8 OPN has also been shown to interact with fibronectin and collagen, suggesting its possible role in matrix organization and/or stability. 9 -11 Recently, using a mammary cell line, we observed that suppression of OPN synthesis leads to increased activity of matrix metalloproteinase (MMP)-2. 12 In fact, there is increased expression of OPN in several tissues in response to injury, suggesting a role in wound healing. Using a skin incision model, Liaw et al 13 observed disorganization of the matrix and alteration of collagen fibrillogenesis, leading to collagen fibrils with smaller diameters in OPN knockout (KO) mice. Similarly, OPN has been shown to play a critical role in the generation of interstitial fibrosis in the kidney after obstructive nephropathy. 14 Remodeling after myocardial infarction (MI) is associated with left ventricular (LV) dilation, decreased cardiac function, and increased mortality. 15 Early dilation of the LV is likely due to scar expansion in the infarcted region, 16 -18 followed later by progressive remodeling 19 in the noninf...

Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs – “4-way”, “7-way”, and “10-way” – each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS “physiome-on-a-chip” approaches in drug discovery.

Cardiomyocyte apoptosis is present in many cardiac disease states, including heart failure and ischemic heart disease. Apoptosis is associated with the activation of caspases that mediate the cleavage of vital and structural proteins. However, the functional contribution of apoptosis to these conditions is not known. Furthermore, in cardiac myocytes, apoptosis may not be complete, allowing the cells to persist for a prolonged period within the myocardium. Therefore, we examined whether caspase-3 cleaved cardiac myofibrillar proteins and, if so, whether it affects contractile function. The effects of caspase-3 were studied in vitro on individual components of the cardiac myofilament including ␣-actin, ␣-actinin, myosin heavy chain, myosin light chain 1͞2, tropomyosin, cardiac troponins (T, I, C), and the trimeric troponin complex. Exposure of the myofibrillar protein (listed above) to caspase-3 for 4 h resulted in the cleavage of ␣-actin and ␣-actinin, but not myosin heavy chain, myosin light chain 1͞2, and tropomyosin, into three fragments (30, 20, and 15 kDa) and one major fragment (45 kDa), respectively. When cTnT, cTnI, and cTnC were incubated individually with caspase-3, there was no detectable cleavage. However, when the recombinant troponin complex was exposed to caspase-3, cTnT was cleaved, resulting in fragments of 25 kDa. Furthermore, rat cardiac myofilaments exposed to caspase-3 exhibited similar patterns of myofibrillar protein cleavage. Treatment with the caspase inhibitor DEVD-CHO or z-VAD-fmk abolished the cleavage. Myofilaments, isolated from adult rat ventricular myocytes after induction of apoptotic pathway by using ␤-adrenergic stimulation, displayed a similar pattern of actin and TnT cleavage. Exposure of skinned fiber to caspase-3 decreased maximal Ca 2؉ -activated force and myofibrillar ATPase activity. Our results indicate that caspase-3 cleaved myofibrillar proteins, resulting in an impaired force͞Ca 2؉ relationship and myofibrillar ATPase activity. Induction of apoptosis in cardiac cells was associated with similar cleavage of myofilaments. Therefore, activation of apoptotic pathways may lead to contractile dysfunction before cell death.

In ARVMs, stimulation of beta(1)-ARs increases apoptosis via a cAMP-dependent mechanism, whereas stimulation of beta(2)-ARs inhibits apoptosis via a G(i)-coupled pathway. These findings have implications for the pathophysiology and treatment of myocardial failure.

Stimulation of beta-adrenergic receptors (betaARs) causes apoptosis in adult rat ventricular myocytes (ARVMs). The role of reactive oxygen species (ROS) in mediating betaAR-stimulated apoptosis is not known. Stimulation of betaARs with norepinephrine (10 micromol/L) in the presence of prazosin (100 nmol/L) for 24 hours increased the number of apoptotic myocytes as determined by TUNEL staining by 3.6- fold. The superoxide dismutase/catalase mimetics Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 10 micromol/L) and Euk-134 decreased betaAR-stimulated apoptosis by 89+/-6% and 76+/-10%, respectively. Infection with an adenovirus expressing catalase decreased betaAR-stimulated apoptosis by 82+/-15%. The mitochondrial permeability transition pore inhibitor bongkrekic acid (50 micromol/L) decreased betaAR-stimulated apoptosis by 76+/-8%, and the caspase inhibitor zVAD-fmk (25 micromol/L) decreased betaAR-stimulated apoptosis by 62+/-11%. betaAR-stimulated cytochrome c release was inhibited by MnTMPyP. betaAR stimulation caused c-Jun NH2-terminal kinase (JNK) activation, which was abolished by MnTMPyP. Transfection with an adenovirus expressing dominant-negative JNK inhibited betaAR-stimulated apoptosis by 81+/-12%, and the JNK inhibitor SP600125 inhibited both betaAR-stimulated apoptosis and cytochrome c release. Thus, betaAR-stimulated apoptosis in ARVMs involves ROS/JNK-dependent activation of the mitochondrial death pathway.

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Abstract-To identify genes that are differentially expressed during the transition from compensated hypertrophy to failure, myocardial mRNA from spontaneously hypertensive rats (SHR) with heart failure (SHR-F) was compared with that from age-matched SHR with compensated hypertrophy (SHR-NF) and normotensive Wistar-Kyoto rats (WKY) by differential display reverse transcriptase-polymerase chain reaction. Characterization of a transcript differentially expressed in SHR-F yielded a cDNA with homology to the extracellular matrix protein osteopontin. Northern analysis showed low levels of osteopontin mRNA in left ventricular myocardium from WKY and SHR-NF but a markedly increased (Ϸ10-fold) level in SHR-F. In myocardium from WKY and SHR-NF, in situ hybridization showed only scant osteopontin mRNA, primarily in arteriolar cells. In SHR-F, in situ hybridization revealed abundant expression of osteopontin mRNA, primarily in nonmyocytes in the interstitial and perivascular space. Similar findings for osteopontin protein were observed in the midwall region of myocardium from the SHR-F group. Consistent with the findings in SHR, osteopontin mRNA was minimally increased (Ϸ1.9-fold) in left ventricular myocardium from nonfailing aortic-banded rats with pressure-overload hypertrophy but was markedly increased (Ϸ8-fold) in banded rats with failure. Treatment with captopril starting before or after the onset of failure in the SHR reduced the increase in left ventricular osteopontin mRNA levels. Thus, osteopontin expression is markedly increased in the heart coincident with the development of heart failure. The source of osteopontin in SHR-F is primarily nonmyocytes, and its induction is inhibited by an angiotensin-converting enzyme inhibitor, suggesting a role for angiotensin II. Given the known biological activities of osteopontin, including cell adhesion and regulation of inducible nitric oxide synthase gene expression, these data suggest that it could play a role in the pathophysiology of heart failure. (Hypertension. 1999;33:663-670.)