Troponin I modulation of cardiac performance: Plasticity in the survival switch.

Signaling complexes targeting the myofilament are essential in modulating cardiac performance. A central target of this signaling is cardiac troponin I (cTnI) phosphorylation. This review focuses on cTnI phosphorylation as a model for myofilament signaling, discussing key gaps and future directions towards understanding complex myofilament modulation of cardiac performance. Human heart cTnI is phosphorylated at 14 sites, giving rise to a complex modulatory network of varied functional responses. For example, while classical Ser23/24 phosphorylation mediates accelerated relaxation, protein kinase C phosphorylation of cTnI serves as a brake on contractile function. Additionally, the functional response of cTnI multi-site phosphorylation cannot necessarily be predicted from the response of individual sites alone. These complexities underscore the need for systematically evaluating single and multi-site phosphorylation on myofilament cellular and in vivo contractile function. Ultimately, a complete understanding of these multi-site responses requires work to establish site occupancy and dominance, kinase/phosphatase signaling balance, and the function of adaptive secondary phosphorylation. As cTnI phosphorylation is essential for modulating cardiac performance, future insight into the complex role of cTnI phosphorylation is important to establish sarcomere signaling in the healthy heart as well as identification of novel myofilament targets in the treatment of disease.

Functional communication between PKC-targeted cardiac troponin I phosphorylation sites.

Increased protein kinase C (PKC) activity is associated with heart failure, and can target multiple cardiac troponin I (cTnI) residues in myocytes, including S23/24, S43/45 and T144. In earlier studies, cTnI-S43D and/or -S45D augmented S23/24 and T144 phosphorylation, which suggested there is communication between clusters. This communication is now explored by evaluating the impact of phospho-mimetic cTnI S43/45D combined with S23/24D (cTnIS4D) or T144D (cTnISDTD). Gene transfer of epitope-tagged cTnIS4D and cTnISDTD into adult cardiac myocytes progressively replaced endogenous cTnI. Partial replacement with cTnISDTD or cTnIS4D accelerated the time to peak (TTP) shortening and time to 50% re-lengthening (TTR50%) on day 2, but peak shortening was only diminished by cTnIS4D. Extensive cTnIS4D replacement continued to accelerate TTP, and decrease shortening amplitude, while TTR50% returned to baseline levels on day 4. In contrast, cTnISDTD modestly reduced shortening amplitude and continued to accelerate myocyte TTP and TTR50%. These results indicate cTnIS43/45 communicates with S23/24 and T144, with S23/24 exacerbating and T144 attenuating the S43/45D-dependent functional deficit. In addition, more severe functional alterations in cTnIS4D myocytes were accompanied by higher levels of secondary phosphorylation compared to cTnISDTD. These results suggest that secondary phosphorylation helps to maintain steady-state contractile function during chronic cTnI phosphorylation at PKC sites.

Functionally conservative substitutions at cardiac troponin I S43/45.

A phospho-null Ala substitution at protein kinase C (PKC)-targeted cardiac troponin I (cTnI) S43/45 reduces myocyte and cardiac contractile function. The goal of the current study was to test whether cTnIS43/45N is an alternative, functionally conservative substitution in cardiac myocytes. Partial and more extensive endogenous cTnI replacement was similar at 2 and 4 days after gene transfer, respectively, for epitope-tagged cTnI and cTnIS43/45N. This replacement did not significantly change thin filament stoichiometry. In functional studies, there were no significant changes in the amplitude and/or rates of contractile shortening and re-lengthening after this partial (2 days) and extensive (4 days) replacement with cTnIS43/45N. The cTnIS43/45N substitution also was not associated with adaptive changes in the myocyte Ca(2+) transient or in phosphorylation of the protein kinase A and C-targeted cTnIS23/24 site. These results provide evidence that cTnIS43/45N is a functionally conservative substitution, and may be appropriate for use as a phospho-null in rodent models designed for studies on PKC modulation of cardiac performance.

Independent modulation of contractile performance by cardiac troponin I Ser43 and Ser45 in the dynamic sarcomere.

Protein kinase C (PKC) targets cardiac troponin I (cTnI) S43/45 for phosphorylation in addition to other residues. During heart failure, cTnI S43/45 phosphorylation is elevated, and yet there is ongoing debate about its functional role due, in part, to the emergence of complex phenotypes in animal models. The individual functional influences of phosphorylated S43 and S45 also are not yet known. The present study utilizes viral gene transfer of cTnI with phosphomimetic S43D and/or S45D substitutions to evaluate their individual and combined influences on function in intact adult cardiac myocytes. Partial replacement (≤40%) with either cTnIS43D or cTnIS45D reduced the amplitude of contraction, and cTnIS45D slowed contraction and relaxation rates, while there were no significant changes in function with cTnIS43/45D. More extensive replacement (≥70%) with cTnIS43D, cTnIS45D, and cTnIS43/45D each reduced the amplitude of contraction. Additional experiments also showed cTnIS45D reduced myofilament Ca(2+) sensitivity of tension. At the same time, shortening rates returned toward control values with cTnIS45D and the later stages of relaxation also became accelerated in myocytes expressing cTnIS43D and/or S45D. Further studies demonstrated this behavior coincided with adaptive changes in myofilament protein phosphorylation. Taken together, the results observed in myocytes expressing cTnIS43D and/or S45D suggest these 2 residues reduce function via independent mechanism(s). The changes in function associated with the onset of adaptive myofilament signaling suggest the sarcomere is capable of fine tuning PKC-mediated cTnIS43/45 phosphorylation and contractile performance. This modulatory behavior also provides insight into divergent phenotypes reported in animal models with cTnI S43/45 phosphomimetic substitutions.

Conserved modular domains team up to latch-open active protein kinase Cα.

Signaling proteins comprised of modular domains have evolved along with multicellularity as a method to facilitate increasing intracellular bandwidth. The effects of intramolecular interactions between modular domains within the context of native proteins have been largely unexplored. Here we examine intra- and intermolecular interactions in the multidomain signaling protein, protein kinase Cα (PKCα). We identify three interactions between two activated PKC molecules that synergistically stabilize a nanomolar affinity homodimer. Disruption of the homodimer results in a loss of PKC-mediated ERK1/2 phosphorylation, whereas disruption of the auto-inhibited state promotes the homodimer and prolongs PKC membrane localization. These observations support a novel regulatory mechanism wherein homodimerization dictates the equilibrium between the auto-inhibited and active states of PKC by sequestering auto-inhibitory interactions. Our findings underscore the physiological importance of context-dependent modular domain interactions in cell signaling.

Myocardial infarction-induced N-terminal fragment of cardiac myosin-binding protein C (cMyBP-C) impairs myofilament function in human myocardium.

Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca(2+) transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 µm) and long (2.3 µm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca(2+) sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca(2+) sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.

Myofilament incorporation and contractile function after gene transfer of cardiac troponin I Ser43/45Ala.

Phosphorylation of cardiac troponin I serines 43/45 (cTnISer43/45) by protein kinase C (PKC) is associated with cardiac dysfunction and yet there is disagreement about the role this cluster plays in modulating contractile performance. The present study evaluates the impact of phospho-null Ala substitutions at Ser43/45 (cTnISer43/45Ala) on contractile performance in intact myocytes. Viral-based gene transfer of cardiac troponin I (cTnI) or cTnISer43/45Ala resulted in time-dependent increases in expression, with 70-80% of endogenous cTnI replaced within 4days. Western analysis of intact and permeabilized myocytes along with immunohistochemistry showed each exogenous cTnI was incorporated into the sarcomere of myocytes. In contractile function studies, there were no differences in shortening and re-lengthening for cTnI and cTnISer43/45Ala-expressing myocytes 2days after gene transfer. However, more extensive replacement with cTnISer43/45Ala after 4days diminished peak shortening amplitude and accelerated re-lengthening measured as the time to 50% re-lengthening (TTR50%). A decrease in myofilament Ca(2+) sensitivity of tension also was observed in permeabilized myocytes expressing cTnISer43/45Ala and is consistent with accelerated re-lengthening observed in intact myocytes under basal conditions. Phosphorylation of cTnI Ser23/24 and the Ca(2+) transient were not changed in these myocytes. These results demonstrate extensive sarcomere expression of cTnISer43/45Ala directly modulates myofilament function under basal conditions. In further work, the accelerated re-lengthening observed in control or cTnI-expressing myocytes treated with the PKC agonist, endothelin-1 (ET, 10nM) was slowed in myocytes expressing cTnISer43/45Ala. This outcome may indicate Ser43/45 is targeted for phosphorylation by ET-activated PKC and/or influences transduction of this agonist-activated response.

Histidine button engineered into cardiac troponin I protects the ischemic and failing heart.

The myofilament protein troponin I (TnI) has a key isoform-dependent role in the development of contractile failure during acidosis and ischemia. Here we show that cardiac performance in vitro and in vivo is enhanced when a single histidine residue present in the fetal cardiac TnI isoform is substituted into the adult cardiac TnI isoform at codon 164. The most marked effects are observed under the acute challenges of acidosis, hypoxia, ischemia and ischemia-reperfusion, in chronic heart failure in transgenic mice and in myocytes from failing human hearts. In the isolated heart, histidine-modified TnI improves systolic and diastolic function and mitigates reperfusion-associated ventricular arrhythmias. Cardiac performance is markedly enhanced in transgenic hearts during reperfusion despite a high-energy phosphate content similar to that in nontransgenic hearts, providing evidence for greater energetic economy. This pH-sensitive 'histidine button' engineered in TnI produces a titratable molecular switch that 'senses' changes in the intracellular milieu of the cardiac myocyte and responds by preferentially augmenting acute and long-term function under pathophysiological conditions. Myofilament-based inotropy may represent a therapeutic avenue to improve myocardial performance in the ischemic and failing heart.

PKCßII modulation of myocyte contractile performance.

Significant up-regulation of the protein kinase Cß(II) (PKCß(II)) develops during heart failure and yet divergent functional outcomes are reported in animal models. The goal here is to investigate PKCß(II) modulation of contractile function and gain insights into downstream targets in adult cardiac myocytes. Increased PKCß(II) protein expression and phosphorylation developed after gene transfer into adult myocytes while expression remained undetectable in controls. The PKCß(II) was distributed in a peri-nuclear pattern and this expression resulted in diminished rates and amplitude of shortening and re-lengthening compared to controls and myocytes expressing dominant negative PKCß(II) (PKCßDN). Similar decreases were observed in the Ca(2+) transient and the Ca(2+) decay rate slowed in response to caffeine in PKCß(II)-expressing myocytes. Parallel phosphorylation studies indicated PKCß(II) targets phosphatase activity to reduce phospholamban (PLB) phosphorylation at residue Thr17 (pThr17-PLB). The PKCß inhibitor, LY379196 (LY) restored pThr17-PLB to control levels. In contrast, myofilament protein phosphorylation was enhanced by PKCß(II) expression, and individually, LY and the phosphatase inhibitor, calyculin A each failed to block this response. Further work showed PKCß(II) increased Ca(2+)-activated, calmodulin-dependent kinase IIδ (CaMKIIδ) expression and enhanced both CaMKIIδ and protein kinase D (PKD) phosphorylation. Phosphorylation of both signaling targets also was resistant to acute inhibition by LY. These later results provide evidence PKCß(II) modulates contractile function via intermediate downstream pathway(s) in cardiac myocytes.

An essential role for complement C5a in the pathogenesis of septic cardiac dysfunction.

Defective cardiac function during sepsis has been referred to as "cardiomyopathy of sepsis." It is known that sepsis leads to intensive activation of the complement system. In the current study, cardiac function and cardiomyocyte contractility have been evaluated in rats after cecal ligation and puncture (CLP). Significant reductions in left ventricular pressures occurred in vivo and in cardiomyocyte contractility in vitro. These defects were prevented in CLP rats given blocking antibody to C5a. Both mRNA and protein for the C5a receptor (C5aR) were constitutively expressed on cardiomyocytes; both increased as a function of time after CLP. In vitro addition of recombinant rat C5a induced dramatic contractile dysfunction in both sham and CLP cardiomyocytes, but to a consistently greater degree in cells from CLP animals. These data suggest that CLP induces C5aR on cardiomyocytes and that in vivo generation of C5a causes C5a-C5aR interaction, causing dysfunction of cardiomyocytes, resulting in compromise of cardiac performance.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes.

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Ubiquitin proteasome dysfunction in human hypertrophic and dilated cardiomyopathies.

BACKGROUND: The ubiquitin proteasome system maintains a dynamic equilibrium of proteins and prevents accumulation of damaged and misfolded proteins, yet its role in human cardiac dysfunction is not well understood. The present study evaluated ubiquitin proteasome system function in human heart failure and hypertrophic cardiomyopathy (HCM). METHODS AND RESULTS: Proteasome function was studied in human nonfailing donor hearts, explanted failing hearts, and myectomy samples from patients with HCM. Proteasome proteolytic activities were markedly reduced in failing and HCM hearts compared with nonfailing hearts (P<0.01). This activity was partially restored after mechanical unloading in failing hearts (P<0.01) and was significantly lower in HCM hearts with pathogenic sarcomere mutations than in those lacking these mutations (P<0.05). There were no changes in the protein content of ubiquitin proteasome system subunits (ie, 11S, 20S, and 19S) or in active-site labeling of the 20S proteolytic subunit beta-5 among groups to explain decreased ubiquitin proteasome system activity in HCM and failing hearts. Examination of protein oxidation revealed that total protein carbonyls, 4-hydroxynonenylated proteins, and oxidative modification to 19S ATPase subunit Rpt 5 were increased in failing compared with nonfailing hearts. CONCLUSIONS: Proteasome activity in HCM and failing human hearts is impaired in the absence of changes in proteasome protein content or availability of proteolytic active sites. These data provide strong evidence that posttranslational modifications to the proteasome may account for defective protein degradation in human cardiomyopathies.

Ca2+-independent positive molecular inotropy for failing rabbit and human cardiac muscle by alpha-myosin motor gene transfer.

Current inotropic therapies used to increase cardiac contractility of the failing heart center on increasing the amount of calcium available for contraction, but their long-term use is associated with increased mortality due to fatal arrhythmias. Thus, there is a need to develop and explore novel inotropic therapies that can act via calcium-independent mechanisms. The purpose of this study was to determine whether fast alpha-myosin molecular motor gene transfer can confer calcium-independent positive inotropy in slow beta-myosin-dominant rabbit and human failing ventricular myocytes. To this end, we generated a recombinant adenovirus (AdMYH6) to deliver the full-length human alpha-myosin gene to adult rabbit and human cardiac myocytes in vitro. Fast alpha-myosin motor expression was determined by Western blotting and immunocytochemical analysis and confocal imaging. In experiments using electrically stimulated myocytes from ischemic failing hearts, AdMYH6 increased the contractile amplitude of failing human [23.9+/-7.8 nm (n=10) vs. AdMYH6 amplitude 78.4+/-16.5 nm (n=6)] and rabbit myocytes. The intracellular calcium transient amplitude was not altered. Control experiments included the use of a green fluorescent protein or a beta-myosin heavy chain adenovirus. Our data provide evidence for a novel form of calcium-independent positive inotropy in failing cardiac myocytes by fast alpha-myosin motor protein gene transfer.

Burn-induced heart failure: lipopolysaccharide binding protein improves burn and endotoxin-induced cardiac contractility deficits.

BACKGROUND: Burn injury is frequently complicated by bacterial infection. Following burn injury, exposure to endotoxin produces a measurable decrease in cardiomyocyte sarcomere contractile function. Lipopolysaccharide-binding protein (LBP) is an acute phase protein that potentiates the recognition of lipopolysaccharide (LPS) by binding to the lipid A moiety of LPS. In this study, we sought to determine the effect of recombinant rat LBP (rLBP) on cardiomyocyte sarcomere function after burn or sham injury in the presence or absence of bacterial endotoxin. METHODS: Rats underwent a full-thickness 30% total body surface area scald or sham burn. At 24 h post-injury, cardiomyocytes were isolated, plated at 50,000 cells/well, and incubated with 50 µg/mL LPS and rLBP or chloramphenicol acetyltransferase (BVCat, an irrelevant control protein produced using the same expression system as rLBP) at concentrations by volume of 1%, 5%, 10%, and 30%. Subsets of cardiomyocytes were incubated with 5% rat serum or 30% rLBP and blocking experiments were conducted using an LBP-like synthetic peptide (LBPK95A). In vitro sarcomere function was measured using a variable rate video camera system with length detection software. RESULTS: Co-culture of burn and sham injury derived cardiomyocytes with high-dose rLBP in the presence of LPS resulted in a significant reduction to the functional impairment observed in peak sarcomere shortening following exposure to LPS alone. LBP-like peptide LBPK95A at a concentration of 20 µg/mL, in the presence of LPS, abolished the ability of 30% rLBP and 5% rat serum to restore peak sarcomere shortening of cardiomyocytes isolated following burn injury to levels of function exhibited in the absence of endotoxin exposure. CONCLUSIONS: In the setting of LPS challenge following burn injury, rLBP at high concentrations restores cardiomyocyte sarcomere contractile function in vitro. Rather than potentiating the recognition of LPS by the cellular LPS receptor complex, rLBP at high concentrations likely results in an inhibitory binding effect that minimizes the impact of endotoxin exposure on cardiomyocyte function following thermal injury.

PKC-alpha regulates cardiac contractility and propensity toward heart failure.

The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-alpha as a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-alpha into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-alpha activity affects dephosphorylation of the sarcoplasmic reticulum Ca(2+) ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca(2+) loading and the Ca(2+) transient. PKC-alpha directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-alpha on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-alpha functions as a nodal integrator of cardiac contractility by sensing intracellular Ca(2+) and signal transduction events, which can profoundly affect propensity toward heart failure.

Cardiac contractile dysfunction and protein kinase C-mediated myofilament phosphorylation in disease and aging.

Increases in protein kinase C (PKC) are associated with diminished cardiac function, but the contribution of downstream myofilament phosphorylation is debated in human and animal models of heart failure. The current experiments evaluated PKC isoform expression, downstream cardiac troponin I (cTnI) S44 phosphorylation (p-S44), and contractile function in failing (F) human myocardium, and in rat models of cardiac dysfunction caused by pressure overload and aging. In F human myocardium, elevated PKCα expression and cTnI p-S44 developed before ventricular assist device implantation. Circulatory support partially reduced PKCα expression and cTnI p-S44 levels and improved cellular contractile function. Gene transfer of dominant negative PKCα (PKCαDN) into F human myocytes also improved contractile function and reduced cTnI p-S44. Heightened cTnI phosphorylation of the analogous residue accompanied reduced myocyte contractile function in a rat model of pressure overload and in aged Fischer 344 × Brown Norway F1 rats (≥26 mo). Together, these results indicate PKC-targeted cTnI p-S44 accompanies cardiac cellular dysfunction in human and animal models. Interfering with PKCα activity reduces downstream cTnI p-S44 levels and partially restores function, suggesting cTnI p-S44 may be a useful target to improve contractile function in the future.

Tuning cardiac performance in ischemic heart disease and failure by modulating myofilament function.

The cardiac myofilaments are composed of highly ordered arrays of proteins that coordinate cardiac contraction and relaxation in response to the rhythmic waves of [Ca(2+)] during the cardiac cycle. Several cardiac disease states are associated with altered myofilament protein interactions that contribute to cardiac dysfunction. During acute myocardial ischemia, the sensitivity of the myofilaments to activating Ca(2+) is drastically reduced, largely due to the effects of intracellular acidosis on the contractile machinery. Myofilament Ca(2+) sensitivity remains compromised in post-ischemic or "stunned" myocardium even after complete restoration of blood flow and intracellular pH, likely because of covalent modifications of or proteolytic injury to contractile proteins. In contrast, myofilament Ca(2+) sensitivity can be increased in chronic heart failure, owing in part to decreased phosphorylation of troponin I, the inhibitory subunit of the troponin regulatory complex. We highlight, in this paper, the central role of the myofilaments in the pathophysiology of each of these distinct disease entities, with a particular focus on the molecular switch protein troponin I. We also discuss the beneficial effects of a genetically engineered cardiac troponin I, with a histidine button substitution at C-terminal residue 164, for a variety of pathophysiologic conditions, including hypoxia, ischemia, ischemia-reperfusion and chronic heart failure.

Secondary phosphorylation in myocytes expressing FLAG-tagged and non-tagged phospho-mimetic cardiac troponin I.

Secondary phosphorylation develops in myocytes expressing phospho-mimetic cardiac troponin I (cTnI) but it is not known whether multiple substitutions (e.g. cTnISDTD and cTnIS4D) cause preferential phosphorylation of the remaining endogenous or the phospho-mimetic cTnI in intact myocytes. Western analysis was performed to determine whether the FLAG/total cTnI ratios are similar for phosphorylated versus total cTnI in myocytes expressing phospho-mimetic cTnI with Asp(D) substitutions at S43/45 plus S23/24 (cTnIS4D) or T144 (cTnISDTD). Representative Western analysis of phosphorylated S23/24 (p-S23/24) and S150 (p-S150) are presented along with re-probes using an antibody which detects all cTnI (MAB1691 Ab). The level of p-S150 also is compared to results obtained using single S43D and/or S45D phospho-mimetic substitutions. These results are discussed in more detail in Lang et al. [1].

Mitochondrial dysfunction and apoptosis underlie the pathogenic process in alpha-B-crystallin desmin-related cardiomyopathy.

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 alpha-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.