Background-Mitochondria and sarcomeres have a well-defined architectural relation that partially depends on the integrity of the cytoskeletal network. An R120G missense mutation in the small heat shock protein ␣-B-crystallin (CryAB) causes desmin-related cardiomyopathy. Desmin-related cardiomyopathy is characterized by the formation of intracellular aggregates containing CryAB and desmin that are amyloid positive, and disease can be recapitulated in transgenic mice by cardiac-specific expression of the mutant protein. Methods and Results-To understand the resultant pathology, we explored the acute effects of R120G expression both in vitro and in vivo. In vitro, transfection of adult cardiomyocytes with R120G-expressing adenovirus resulted in altered contractile mechanics. In vivo, as the cytoskeletal network is disturbed but before deficits in organ function can be detected, alterations in mitochondrial organization and architecture occur, leading to a reduction in the maximal rate of oxygen consumption with substrates that utilize complex I activity, alterations in the permeability transition pore, and compromised inner membrane potential. Apoptotic pathways are subsequently activated, which eventually results in cardiomyocyte death, dilation, and heart failure. Conclusions-Cardiac chaperone dysfunction acutely leads to altered cardiomyocyte mechanics, perturbations in mitochondrial-sarcomere architecture, and deficits in mitochondrial function, which can result in activation of apoptosis and heart failure.

Cytoskeletal adaptor proteins serve vital functions in linking the internal cytoskeleton of cells to the cell membrane, particularly at sites of cell-cell and cell-matrix interactions. The importance of these adaptors to the structural integrity of the cell is evident from the number of clinical disease states attributable to defects in these networks. In the heart, defects in the cytoskeletal support system that surrounds and supports the myofibril result in dilated cardiomyopathy and congestive heart failure. In this study, we report the cloning and characterization of a novel cytoskeletal adaptor, obscurin-like 1 (OBSL1), which is closely related to obscurin, a giant structural protein required for sarcomere assembly. Multiple isoforms arise from alternative splicing, ranging in predicted molecular mass from 130 to 230 kDa. OBSL1 is located on human chromosome 2q35 within 100 kb of SPEG, another gene related to obscurin. It is expressed in a broad range of tissues and localizes to the intercalated discs, to the perinuclear region, and overlying the Z lines and M bands of adult rat cardiac myocytes. Further characterization of this novel cytoskeletal linker will have important implications for understanding the physical interactions that stabilize and support cell-matrix, cell-cell, and intracellular cytoskeletal connections.

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Cardiac troponin I is a phosphorylation target for endothelinactivated protein kinase C. Earlier work in cardiac myocytes expressing nonphosphorylatable slow skeletal troponin I provided evidence that protein kinase C-mediated cardiac troponin I phosphorylation accelerates relaxation. However, replacement with the slow skeletal isoform also alters the myofilament pH response and the Ca 2؉ transient, which could influence endothelin-mediated relaxation. Here, differences in the Ca 2؉ Protein kinase C (PKC)2 activation is an important pathway involved in modulating cardiac contractile function (1-5). A number of end target proteins involved in excitation-contraction coupling are phosphorylated in response to PKC activation, including the L-type Ca 2ϩ channel (6, 7) and myofilament proteins such as cardiac troponin I (cTnI) (8 -11), cardiac troponin T (10, 12), and myosin light chain 2 (MLC 2 ) (8,13,14). The contribution of each end target to the contractile response to PKC is not well understood. Our laboratory is interested in understanding the role of cTnI in the PKC-mediated contractile function response. Increased PKC expression is observed in failing human hearts (15,16), and the relationship between PKC and cTnI phosphorylation may exert an important influence on cardiac function under physiological and pathophysiological conditions (17).Earlier work demonstrated that cTnI phosphorylation is correlated with the positive inotropic response to ET (4,8,18). Accelerated relaxation also is observed in response to PKC activation by ET in myocytes expressing cTnI (4). PKC phosphorylation of purified cTnI decreases myofilament Ca 2ϩ sensitivity in reconstituted myofilaments (11), and this desensitization is expected to contribute to the accelerated relaxation. In contrast, peak shortening is diminished and relaxation is delayed rather than accelerated in response to ET when endogenous cTnI is replaced with nonphosphorylatable slow skeletal troponin I (ssTnI) in adult myocytes. These results are consistent with the idea that cTnI phosphorylation enhances peak myocyte shortening and accelerates relaxation rate in response to PKC activation by ET. However, ssTnI expression in adult myocytes also increases myofilament Ca 2ϩ sensitivity and slows the Ca 2ϩ transient (19,20). Thus, the differential response to ET observed in myocytes expressing cTnI versus ssTnI may result from the direct actions of cTnI phosphorylation on shortening and relaxation or from alterations in the Ca 2ϩ transient induced by ssTnI expression.One goal of the present study is to establish whether PKC-mediated cTnI phosphorylation acts directly to accelerate relaxation or is explained by parallel alterations in cellular Ca 2ϩ . Calcium and shortening responses are measured simultaneously during the ET response for these experiments. Stimulation frequency also alters the Ca 2ϩ transient, with accelerated sarcoplasmic reticulum Ca 2ϩ uptake observed at higher pacing frequencies (21). The relative contribution of phosphorylated cTnI to the ET-indu...